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| Adv Dent Res, 1997 Apr, 11(1), 43 - 9 Phagocyte-bacteria interactions; Keisari Y et al.; Recognition and phagocytosis of micro-organisms in a serum-poor environment represent innate immunity against many extracellular pathogens . As a paradigm for such processes, we discuss the recognition of Klebsiella pneumoniae by alveolar macrophages and monocyte-derived macrophages in the absence of serum . Macrophages recognize and subsequently kill Klebsiella expressing Man-alpha 2/3-Man or Rha-alpha 2/3-Rha sequences in their capsular polysaccharides by two mechanisms: (a) recognition of the capsular structures by macrophage mannose receptors, and (b) opsonization by the lung surfactant protein A (SP-A), which binds to the capsular polysaccharides of Klebsiella and to SP-A receptors on the macrophages . Sp-A may also enhance phagocytosis by increasing the activity of macrophage mannose receptors . We conclude that a specific microbial surface structure may be a target for recognition by macrophages via several mechanisms, as exemplified in the case of Klebsiella capsular polysaccharides . Multiple recognition mechanisms of pathogens by macrophages may be essential to provide innate immunity to reduce the frequency of infections caused by a relatively less virulent bacterium in the immuno-compromised host. Biochim Biophys Acta, 1998 Mar 4, 1396(1), 8 - 14 Cloning and expression of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase: identification of the pfs gene product; Cornell KA et al.; The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) is responsible for cleavage of the glycosidic bond in both 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) . Based on amino acid sequence analysis of this enzyme from Klebsiella, we recently speculated that an open reading frame found in E . coli (designated pfs) encoded MTA/SAH nucleosidase . To explore this possibility, we amplified, cloned, and expressed the complete pfs gene from E . coli genomic DNA . The recombinant protein exhibited a molecular weight and Michaelis constants for MTA that are in agreement with those reported for native enzyme . From this biochemical evidence we confirm our original assignment of the pfs gene as encoding MTA/SAH nucleosidase. Microbios, 1997, 91(368-369), 137 - 43 Hydrophobicity and serum sensitivity of Klebsiella pneumoniae treated with sub-MICs of quinolones; Hostacka A; Cell surface hydrophobicity and serum sensitivity of Klebsiella pneumoniae, after treatment with sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin, norfloxacin and enoxacin, were studied . The quinolones tested at 1/4, 1/8 and 1/16 of the MICs decreased surface hydrophobicity . The most significant reduction of the bacterial cell surface hydrophobicity was found after treatment with antibiotics at 1/16 of the MICs (to 20.3% for both ciprofloxacin and norfloxacin, and to 23.6% for enoxacin, compared with control values) . The most significant increase in the susceptibility of the bacteria to serum bactericidal activity was seen after 180 min incubation with ciprofloxacin and enoxacin at 1/16 of their MICs . Survival of treated bacteria was 55 +/- 8% 56 +/- 10% as compared with controls without antibiotics. Kaohsiung J Med Sci, 1998 Jan, 14(1), 19 - 24 Ultrasound-guided percutaneous transhepatic drainage of gallbladder followed by cholecystectomy for acute cholecystitis--10 years' experience; Wong SR et al.; Acute cholecystitis is a common disease which may carry the risk of complications, including empyema, perforation, abscess, peritonitis and sepsis . Percutaneous transhepatic drainage of the gallbladder (PTGBD) with antibiotics can provide prompt decompression of gallbladder in acute cholecystitis and interrupt the natural history of the disease effectively . From July 1986 to June 1996, 154 patients with acute cholecystitis were reviewed retrospectively in Kaohsiung Medical College Hospital . The chief symptoms and signs were pain (98.1%), fever (57.1%) and jaundice (37.7%) . WBC count more than 10,000 was noted in 116 (75.3%) patients . Associated diseases included empyema: 42 (27.3%), septic shock: 14 (9.1%), diabetes mellitus: 13 (8.4%), pancreatitis: 10 (6.5%), perforation: 7 (4.5%), liver cirrhosis: 6 (3.9%) and respiratory failure: 1 (0.6%) . All of them underwent ultrasound-guided PTGBD immediately after the diagnosis was established . The symptoms and signs disappeared soon after this procedure . Bacterial culture was found positive in 104 (67.5%) of 154 patients in which Escherichia coli (51.9%) was the most common organism, followed by Klebsiella pneumonia (20.2%) . After acute stage, 138 patients obtained the cholangiography via PTGBD tube . Gallbladder stones were only noted in 56 (40.6%) patients, gallbladder stone concomitant with common bile duct stone in 26 (18.8%), cystic duct obstruction in 25 (18.1%), acalculous cholecystitis in 21 (15.2%), gallbladder perforation in 1 (0.7%), choledochocyst in 1 (0.7%), and cholecystocolonic fistula in 1 (0.7%) . There were 135 patients to undergo surgery after the clinical condition was stable . The operative findings included gallbladder stones only in 88 (65.2%), gallbladder stone concomitant with common bile duct stone in 34 (25.2%), acalculous cholecystitis in 13 (9.6%), choledochocyst in 1 (0.7%), and cholecysto-colonic fistula in 1 (0.7%) . The postoperative complications included wound infection 8 (5.9%), UGI bleeding 3 (2.2%), acute renal failure 1 (0.7%) and acute respiratory failure 1 (0.7%) . The postoperative mortality rate was 0.7% (1/135), which was much lower than those of previous reports, which not undergoing PTGBD initially . It led us to conclude that PTGBD, as an initial preoperative modality to treat acute cholecystitis, is effective in decreasing postoperative morbidity and mortality. Arch Surg, 1998 Mar, 133(3), 242 - 5 Pyogenic liver abscesses in patients with malignant disease: a report of 52 cases treated at a single institution; Yeh TS et al.; BACKGROUND: Prognosis of pyogenic liver abscesses in patients with malignant disease is generally considered poor . The discrepancy between the outcomes of liver abscesses caused by hepatopancreatobiliary malignant disease and those caused by other malignant diseases, however, to our knowledge has never been investigated . OBJECTIVES: To clarify the clinical course of pyogenic liver abscess in patients with different types of cancer, and to compare outcomes in abscesses caused by hepatopancreatobiliary malignant disease and other malignant disease . DESIGN: Retrospective review of case series in our experience from 1980 through 1993 . SETTING: Tertiary care university teaching hospital . PATIENTS: Fifty-two patients with pyogenic liver abscess related to the underlying cancer were divided into 2 groups . Group 1 (n=32) was composed of patients with cancer originating from the hepatic parenchyma, bile duct, and pancreas; group 2 (n=20) was composed of patients with cancer originating from other sites . INTERVENTIONS: Parenteral antibiotics, percutaneous drainage, surgical drainage, or hepatectomy, in combinations, were employed . MAIN OUTCOME MEASURES: Patient characteristics, symptoms, laboratory data, abscess characteristics, microbiological study, management, and outcome of the 2 groups were analyzed . RESULTS: Thirteen patients (41%) in group 1 and 16 patients (80%) in group 2 had undergone prior anticancer treatment . Jaundice was encountered more often in group 1 than in group 2 (29 patients {91%} vs 6 patients {30%}, respectively, P=.001), whereas nausea and vomiting were more frequently seen in group 2 than in group 1 (17 patients {52%} vs 6 patients{31%}, respectively, P=.04) . Leukocytosis, hypoalbuminemia, hyperbilirubinemia, and reversed albumin-globulin ratio were more pronounced in group 1 than in group 2 (P=.001, .02, .003, and .03, respectively) . Abscesses communicating with the intrahepatic biliary tree were more frequently encountered in group 1 than in group 2 (11 patients {34%} vs 2 patients {10%}, respectively, P=.03) . Escherichia coli and Klebsiella pneumoniae predominated in group 1, while the bacteria species in group 2 were more diverse . The hospital mortality rates of group 1 and group 2 were 28% (9 of 32 patients) vs 10% (2 of 20 patients) (P=.04), respectively . Twenty-three patients (72%) of group 1 died of uncontrolled biliary sepsis or progressive cancer or both within 6 months after the diagnosis, while 17 patients (85%) of group 2 survived longer than 1 year without relapse of the abscess and continued with anticancer treatment . CONCLUSIONS: Pyogenic liver abscess could be a presentation of hepatopancreatobiliary malignant disease at the preterminal stage, and carries a grave prognosis . Pyogenic liver abscess in patients with nonhepatopancreatobiliary malignant disease has a better chance of favorable outcome. Plasmid, 1998, 39(2), 123 - 33 Characterization of mutants of the 6'-N-acetyltransferase encoded by the multiresistance transposon Tn1331: effect of Phen171-to-Leu171 and Tyr80-to-Cys80 substitutions; Panaite DM et al.; The Klebsiella pneumoniae plasmid pJHCMW1 harbors a copy of Tn1331, a multiresistance transposon that includes the aac(6')-Ib gene which encodes a 6'-N-aminoglycoside acetyltransferase . This gene was mutagenized using the mutator Escherichia coli XL1-Red . Two plasmids with a single nucleotide mutation in aac(6')-Ib were selected for further analysis: pDP1 and pDP6 . Plasmid pDP1 codes for a mutant enzyme, AAC(6')-IbDP1, that has the Phe171 replaced by a Leu residue . This mutant derivative showed a lower specific activity than the wild-type enzyme when either kanamycin (Km) or its semisynthetic derivative amikacin (Ak) were used as substrates in enzymatic assays performed at 30 degrees C . Furthermore, AAC(6')-IbDP1 showed a change of specificity of substrate when incubated at 42 degrees C . While its acetylating activity for Km was higher at this temperature than at 30 degrees C, it had its ability to utilize Ak as substrate for acetylation considerably reduced . Accordingly, minimal inhibitory concentration assays showed that E . coli(pDP1) was resistant to Ak at 37 degrees C but susceptible at 42 degrees C . The same assays showed that E . coli(pDP1) was highly resistant to Km at either 37 degrees C or 42 degrees C . A high level of resistance to Ak was observed for E . coli(pJHCMW1) which harbors the wild-type AAC(6')-Ib at either 37 or 42 degrees C . Extension of the analyses to other aminoglycosides showed that the enzymatic activity of AAC(6')-IbDP1 as well as the E . coli(pDP1) MICs for netilmicin dropped at 42 degrees C as was the case for Ak . These results could indicate that at 42 degrees C the mutant adopts a conformation that makes it unable to efficiently acetylate aminoglycoside molecules substituted in the C-1amino group of the deoxystreptamine moiety . Plasmid pDP6 encodes the mutant AAC(6')-IbDP6 which has the Tyr80 substituted by a Cys residue . E . coli(pDP6) exhibited reduced MICs for Ak, Km, tobramycin, and netilmicin . Analysis of the acetylating activity of AAC(6')-IbDP6 showed only marginal levels of activity when either Ak, Km, tobramycin, or netilmicin were used as substrates. Alcohol Clin Exp Res, 1998 Feb, 22(1), 157 - 62 Adenoviral-mediated interferon-gamma gene therapy augments pulmonary host defense of ethanol-treated rats; Kolls JK et al.; Alcohol has long been recognized as an immunosuppressive drug and a risk factor for a spectrum of infectious diseases . Among these infections, bacterial pneumonias are most closely correlated with alcohol abuse . One potential mechanism of ethanol-induced immunosuppression is through its ability to suppress alveolar macrophage production of tumor necrosis factor (TNF-alpha) . This defect can be reversed by priming macrophages with interferon-gamma (IFN-gamma) . We hypothesized that macrophage priming in vivo in a model of acute ethanol intoxication could augment pulmonary host defenses . To test this hypothesis, we used adenoviral-mediated gene transfer of the IFN-gamma gene . This strategy resulted in prolonged expression of IFN-gamma in vivo . Moreover, in a model of acute ethanol intoxication, this vector significantly enhanced lipopolysaccharide-induced TNF-alpha responses and lung polymorphonuclear leukocyte recruitment . Furthermore, pulmonary host defenses against Klebsiella pneumoniae were significantly augmented . These enhanced host defenses were not reversed with pretreatment with a polyclonal anti-TNF-alpha antibody, suggesting that IFN-gamma's effect was through a non-TNF-alpha-dependent mechanism . These data demonstrate that ethanol-induced suppression of pulmonary host defenses can be reversed with IFN-gamma gene therapy. J Med Microbiol, 1998 Mar, 47(3), 201 - 9 Long-term investigation of the clonal dissemination of Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases in a university hospital; Branger C et al.; Seventy isolates of Klebsiella pneumoniae with extended-spectrum beta-lactamases (ESBLs) were compared . These were isolated from 51 patients on 10 separate wards in one hospital over an 18-month period between 1992 and 1994 . Antibiograms were determined and the isolates were typed by pulsed-field gel electrophoresis of their DNA digestion with XbaI . The isolates were compared to three genotypically different epidemic strains responsible for previous outbreaks at the hospital between 1988 and 1991 . Isolates from 84% of the present patients had closely related XbaI patterns, and most (74%) produced an ESBL with an iso-electric point (pI) of 7.0 . A similar pattern was found for one of the previous epidemic strains, but it produced an ESBL with a pI of 7.8; isolates with this latter enzyme variant were found only in six of the present patients . The two other previous epidemic strains had ESBLs with a pI of 6.3 and organisms related to them were found in one and two of the present patients, respectively. Epidemiol Mikrobiol Imunol, 1998 Feb, 47(1), 12 - 4 {Effectiveness of cefpirome on Klebsiella pneumoniae producing beta-lactamases with an extended spectrum of activity}; Kolar M; In the submitted paper the author investigated the effectiveness of cefpirome on Klebsiella pneumoniae strains producing beta-lactamases with an extended spectrum (ESBL) . From a total of 1348 examined strains of Klebsiella pneumoniae ESBL production was confirmed in 59 strains (4.4%) . Resistance of ESBL-positive strains of Klebsiella pneumoniae to ceftazidime was 83.1%, to cefotaxime 93.2%, while resistance to cefpirome was recorded only in 45.8%. J Antimicrob Chemother, 1998 Jan, 41(1), 123 - 5 PCR single strand conformational polymorphism can be used to detect the gene encoding SHV-7 extended-spectrum beta-lactamase and to identify different SHV genes within the same strain; M'Zali FH et al.; The PCR single strand conformational polymorphism (PCR-SSCP) technique described to identify mutants of the SHV beta-lactamases was extended to identify an SHV-7 type beta-lactamase . This was found in a strain of Klebsiella pneumoniae, the first recorded isolate in the UK to produce this type of enzyme . We also demonstrate that PCR-SSCP can be used to identify more than one SHV beta-lactamase gene in a single strain. Kansenshogaku Zasshi, 1998 Jan, 72(1), 75 - 82 Effects of cytokines and minocycline on subacute lung injuries induced by repeated injection of lipopolysaccharide; Yamaki K et al.; Pathological changes were seen in the lungs of ddY mice one week after repeated intraperitoneal injections of lipopolysaccharide (LPS) of Klebsiella pneumoniae . The infiltration of polymorphonuclear cells (PMN), mainly neutrophils, and lymphocytes into the alveolar septum, the infiltration of PMN into perivascular areas and microthrombi were recognized in this murine model . The blood levels of TNF alpha and IL-1 alpha did not rise at this time, suggesting that the most important cytokine promoting inflammation one week after LPS stimulation was neither TNF alpha nor IL-1 alpha . In the lungs of mice administered minocycline together with LPS, lymphocyte infiltration of alveoli and perivascular areas as well as microthrombi were suppressed . The blood levels of TNF alpha, IL-1 alpha, IL-4 and IL-6 were elevated in these groups, suggesting the suppression of pathological changes to be associated with the anti-inflammatory effect of IL-6 and/or persistent elevation of TNF alpha and/or IL-1 alpha levels . In conclusion, subacute pathological changes in the lung were induced by repeated intraperitoneal injections of LPS to mice . These pathological changes were suppressed by minocycline, suggesting the anti-inflammatory effects of this antibiotic to be the result of stimulating certain cytokines. Clin Infect Dis, 1998 Feb, 26(2), 468 - 74 Klebsiella endocarditis: report of two cases and review; Anderson MJ et al.; The rarity of endocarditis due to Klebsiella species limits its recognition and awareness of its often malignant course . We describe two recent cases of Klebsiella pneumoniae endocarditis and review the clinical context and outcomes of 48 other cases reported in the literature . At our hospital, endocarditis complicated only one of 86 consecutive episodes of bacteremia due to Klebsiella species . In 22 series of endocarditis that we reviewed, Klebsiella species caused < or =1.2% of cases of native valve endocarditis and up to 4.1% of cases of prosthetic valve endocarditis . Valves were replaced in 44% of these cases, and the mortality rate was 49% in cases for which outcome was specified . Valve replacement may be associated with improved survival . We conclude that Klebsiella species are a rare but ominous cause of complicated bacterial endocarditis that requires careful evaluation during the entire course of therapy. Chem Pharm Bull (Tokyo), 1998 Feb, 46(2), 319 - 23 Some properties and the inclusion behavior of three positional isomers of 6(1),6n-di-O-alpha-D-glucosyl-cyclomaltoheptaoses (beta-cyclodextrins); Okada Y et al.; Three positional isomers of 6(1),6n-di-O-alpha-D-glucosyl-cyclomaltoheptaose {1,n-(G)2-beta CDs; n = 2-4} which existed in the digests with glucoamylase of the products from cyclomaltoheptaose (beta-cyclodextrin, beta CD) and maltose with Klebsiella pneumoniae pullulanase, were purified by HPLC . The solubilities of two isomers of those doubly branched beta CDs, 1,2- and 1,3-(G)2-beta CDs, in water were much higher than those of parent non-branched beta CD and mono-branched beta CD, 6-O-alpha-D-glucosyl-beta CD (G-beta CD), while the solubility of another isomer, 1,4-(G)2-beta CD, was significantly lower than these two isomers, though it was higher than that of beta CD . On the other hand, the solubilities of 1,2- and 1,3-isomers in 10, 30, and 50% (v/v) aqueous methanol at 25 degrees C were independent of methanol concentrations and their solubilities were the same as those in water at 25 degrees C . However, that of 1,4-isomer increased with increasing methanol concentrations . The hemolytic activities of 1,n-(G)2-beta CDs on human erythrocytes in isotonic solution were lower than those of G-beta CD and beta CD, and became weaker in the order of 1,4- > 1,2- > 1,3-isomers . The complex-forming abilities of 1,n-(G)2-beta CDs for digitoxin, digoxin, fluorometholone, flurbiprofen, hydrocortisone acetate, and norfloxacin were about the same as those of beta CD and G-beta CD, whereas reserpine was more difficult to include within 1,n-(G)2-beta CDs than beta CD and G-beta CD . Nevertheless, the solubilities of those guest compounds were much more enhanced by 1,n-(G)2-beta CDs and G-beta CD than by beta CD. Methods Find Exp Clin Pharmacol, 1997 Nov, 19(9), 579 - 83 A low dose immunorestorative effect of aporphinoid alkaloid oxoglaucine on experimentally immunosuppressed and infected mice; Ivanovska N et al.; The immunoregulatory activity of aporphinoid alkaloid oxoglaucine was studied in delayed-type hypersensitivity (DTH) reaction and Klebsiella pneumoniae infection in normal and immunosuppressed mice . The alkaloid enhanced DTH reaction when applied during the developing phase and abolished the inhibitory action of cyclophosphamide . Oxoglaucine possessed a restorative effect in K . pneumoniae infection in immunosuppressed mice in combination with indomethacin, prednisolone or artemisinin, as evaluated by the number of survivors and mean survival time . These data suggest that oxoglaucine affects selected lymphocyte clones responsible for antiinfectious host resistance . Its use as a second agent in combination with another immunosuppressant might enable reduction in the dosage or time of application. Biotechnol Prog, 1998 Jan-Feb, 14(1), 116 - 25 Metabolic engineering of propanediol pathways; Cameron DC et al.; Microbial fermentation is an important technology for the conversion of renewable resources to chemicals . In this paper, we describe the application of metabolic engineering for the development of two new fermentation processes: the microbial conversion of sugars to 1,3-propanediol (1,3-PD) and 1,2-propanediol (1,2-PD) . A variety of naturally occurring organisms ferment glycerol to 1,3-PD, but no natural organisms ferment sugars directly to 1,3-PD . We first describe the fed-batch fermentation of glycerol to 1,3-PD by Klebsiella pneumoniae . We then present various approaches for the conversion of sugars to 1,3-PD, including mixed-culture fermentation, cofermentation of glycerol and glucose, and metabolic engineering of a "sugars to 1,3-PD" pathway in a single organism . Initial results are reported for the expression of genes from the K . pneumoniae 1,3-PD pathway in Saccharomyces cerevisiae . The best naturally occurring organism for the fermentation of sugars to 1,2-PD is Thermoanaerobacterium thermosaccharolyticum . We describe the fermentation of several different sugars to 1,2-PD by this organism in batch and continuous culture . We report that Escherichia coli strains engineered to express either aldose reductase or glycerol dehydrogenase convert glucose to (R)-1,2-PD . We then analyze the ultimate potential of fermentation processes for the production of propanediols . Linear optimization studies indicate that, under aerobic conditions, propanediol yields that approach the theoretical maximum are possible and CO2 is the primary coproduct . Without the need to produce acetate, final product titers in the range of 100 g/L should be possible; the high titers and low coproduct levels should make product recovery and purification straightforward . The examples given in this paper illustrate the importance of metabolic engineering for fermentation process development in general. J Bacteriol, 1998 Mar, 180(5), 1311 - 22 NasFED proteins mediate assimilatory nitrate and nitrite transport in Klebsiella oxytoca (pneumoniae) M5al; Wu Q et al.; Klebsiella oxytoca can use nitrate and nitrite as sole nitrogen sources . The enzymes required for nitrate and nitrite assimilation are encoded by the nasFEDCBA operon . We report here the complete nasFED sequence . Sequence comparisons indicate that the nasFED genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD) . The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway . The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters . To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal nasFED genes . Growth tests coupled with nitrate and nitrite uptake assays revealed that the nasFED genes are essential for nitrate transport and participate in nitrite transport as well . Interestingly, the delta nasF strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein. Eur Respir J, 1997 Dec, 10(12), 2902 - 3 A case of superior vena cava syndrome caused by Klebsiella pneumoniae; Kim JY et al.; A 27 yr old man presented with productive cough, fever and manifestations of superior vena cava syndrome . He was an alcoholic but had been in good health until 3 days prior to admission . The physical examination, the chest radiograph and the results of the sputum culture were compatible with Klebsiella pneumoniae pneumonia of the right upper lobe . The superior vena cava scintigram using technetium-99m showed near total occlusion of the superior vena cava, while sputum cytology, chest computed tomography, and bronchoscopy were all negative for malignant aetiology . Antibiotic therapy brought about slow resolution of the pneumonia and also of the superior vena caval obstruction . The follow-up scintigram showed normalized venous flow of the superior vena cava . To our knowledge, this is the first case of superior vena cava syndrome developed in probable association with Klebsiella pneumoniae pneumonia. Microbiology, 1998 Feb, 144 ( Pt 2), 555 - 9 High affinity binding of albicidin phytotoxins by the AlbA protein from Klebsiella oxytoca; Zhang L et al.; Albicidins are a family of phytotoxins and antibiotics which play an important role in the pathogenesis of sugarcane leaf scald disease . The albA gene from Klebsiella oxytoca encodes a protein which inactivates albicidin by heat-reversible binding . Albicidin ligand binding to a recombinant AlbA protein, purified by means of a glutathione S-transferase gene fusion system, is an almost instant and saturable reaction . Kinetic and stoichiometric analysis of the binding reaction indicated the presence of a single high affinity binding site with a dissociation constant of 6.4 x 10(-8) M . The AlbA-albicidin complex is stable from 4 to 40 degrees C, from pH 5 to 9 and in high salt solutions . Treatment with protein denaturants released all bound albicidin . These properties indicate that AlbA may be a useful affinity matrix for selective purification of albicidin antibiotics . AlbA does not bind to p-nitrophenyl butyrate or alpha-naphthyl butyrate, the substrates of the albicidin detoxification enzyme AlbD from Pantoea dispersa . The potential exists to pyramid genes for different mechanisms in transgenic plants to protect plastid DNA replication from inhibition by albicidins. Microbiology, 1998 Feb, 144 ( Pt 2), 343 - 52 Escherichia coli CoIV plasmid pRK100: genetic organization, stability and conjugal transfer; Ambrozic J et al.; Uropathogenic Escherichia coli strains express chromosomal and plasmid-encoded virulence-associated factors such as specific adhesins, toxins and iron-uptake systems . A CoIV plasmid (pRK100) of a uropathogenic strain and its host KS533 were studied . The host strain encodes the K1 capsule, and P and S fimbriae, but neither haemolysin nor the cytotoxic-necrotic factor CNF1, indicating that this strain does not harbour a larger pathogenicity island . A restriction map of pRK100 was constructed on the basis of hybridization experiments and nucleotide sequencing . pRK100 harbours CoIV, the conserved replication region RepFIB, the aerobactin-uptake system, a RepFIC replicon and additionally Colla as well as transposon Tn5431 . The location of the RepFIC replicon was similar to that in plasmid F . CoIV plasmids and F thus share a region spanning more than half the length of plasmid F . Even though their replication and transfer regions are homologous, CoIV plasmids are found only in E . coli strains . Among the four other species tested, conjugal transfer of pRK100 was demonstrated, with low frequency, only to Klebsiella pneumoniae, suggesting that a natural barrier effectively bars transfer . In vitro stability of the plasmid with integration into the chromosome to ensure maintenance in the presence of an incompatible plasmid was demonstrated. J Clin Apheresis, 1997, 12(4), 200 - 1 Plasmapheresis in the treatment of inadvertent intravenous infusion of an enteral feeding solution; Ong BC et al.; This 50-year-old man was on enteral full-strength Ensure through the jejunostomy tube . The enteral feed was accidentally infused through the peripheral intravenous line . He went into a septic state with hypotension, hypoxemia, and increased pulmonary shunting requiring ventilatory and inotropic support . We instituted two cycles of plasmapheresis in an attempt to remove any foreign antigen and toxins present . This resulted in improved oxygenation, rapid recovery, and discharge from intensive care . His blood culture grew Klebsiella species that was consistent with the organism grown from the culture of the Ensure feeds. Am J Perinatol, 1998 Jan, 15(1), 47 - 51 Piperacillin/tazobactam in the treatment of Klebsiella pneumoniae infections in neonates; Pillay T et al.; Nosocomial Klebsiella pneumoniae infection is associated with a high mortality in neonates and antimicrobial therapy of these infections has been complicated by the emergence of multiresistant strains . These organisms remain susceptible to only a few antimicrobial agents, and some of these are not recommended for use in children . In this study the antimicrobial agents used in the treatment of 33 neonates with Klebsiella pneumoniae (K . pneumonia) infection in our tertiary neonatal unit, during an outbreak were: piperacillin/tazobactam (13), imipenem/cilastatin (17), cefotaxime (2), and ciprofloxacin (1) . Extended-spectrum beta-lactamase production was detected in K . pneumoniae isolates from 18 of 33 (54.5%) neonates . All-cause mortality was 13 of 33 (39.4%) and there was no significant difference in mortality between neonates treated with imipenem/cilastatin (6 of 17 or 35.3%) and neonates treated with piperacillin/tazobactam (6 of 13 or 46.2%) . The duration of antimicrobial therapy and total hospital stay was similar between neonates who received imipenem/cilastatin and those that received piperacillin/tazobactam . This report suggests that piperacillin/tazobactam may be a useful antimicrobial agent in neonatal infections caused by beta-lactamase-producing organisms. Am J Gastroenterol, 1998 Feb, 93(2), 253 - 5 Cholangiocarcinoma presenting as pyogenic liver abscess: is its outcome influenced by concomitant hepatolithiasis? Jan YY, Yeh TS, Chen MF. OBJECTIVE: The etiology of pyogenic liver abscess is changing . Malignant biliary obstruction has emerged as one of the most important causes . We explored the clinical course of pyogenic liver abscess caused by cholangiocarcinoma . METHODS: The medical records of 19 patients with cholangiocarcinoma presenting as pyogenic liver abscess were reviewed . Of them, 57.8% (11 of 19) had concomitant hepatolithiasis . Escherichia coli and Klebsiella pneumoniae were the most common pathogens isolated from aspirates of the abscesses . Eight patients received percutaneous drainage, whereas 11 patients initially underwent surgical drainage because of the presence of ascites or coagulopathy or lack of awareness of the underlying cholangiocarcinoma . RESULTS: Overall, the hospital mortality rate was 36.8% (seven of 19) . Patients with hepatolithiasis had a hospital mortality rate of 54.5% (six of 11), compared with 12.5% (one of eight) in those without (p < 0.01) . Notably, 84.2% (16 of 19) of the patients died within 6 months after the diagnosis was made . CONCLUSIONS: Cholangiocarcinoma presenting as liver abscess has a dismal prognosis . Concomitant hepatolithiasis worsened the infectious process and adversely affected the survival. J Antimicrob Chemother, 1997 Dec, 40(6), 789 - 95 Characterization and amino acid sequence of the OXY-2 group beta-lactamase of pI 5.7 isolated from aztreonam-resistant Klebsiella oxytoca strain HB60; Farzaneh S et al.; Klebsiella oxytoca strain HB60 is highly resistant to cefoperazone and aztreonam (MICs = 128 mg/L) . It produces a chromosomally encoded beta-lactamase of pI 5.7 which was highly efficient against penicillins, first-generation cephalosporins and cefoperazone, a non-oxyimino third-generation cephalosporin . Aztreonam and oxyimino broad-spectrum cephalosporins were less good substrates . The beta-lactamase activity was susceptible to inhibition by clavulanic acid (IC50 = 1 microM) . The enzyme purified to homogeneity had a specific activity towards benzylpenicillin of 3670 U/mg . The 263 amino acid residues of the protein were sequenced by Edman degradation of proteolytic peptides . The beta-lactamase was shown to belong to the OXY-2 group as it had only one amino acid substitution (Asn for Asp at ABL position 197) compared with the beta-lactamase (pI 5.2) from the aztreonam-susceptible K . oxytoca strain SL911 and two substitutions (Ala223 for Val and Asp255 for Asn) compared with the beta-lactamase (pI 6.4) from the aztreonam-resistant K . oxytoca strain D488 . These three OXY-2-group enzymes behave in the same way towards beta-lactam antibiotics . The variability in the resistance of these K . oxytoca strains would thus seem to be due to variation in the level of production of the beta-lactamases rather than to structural alteration of the enzymes. J Clin Microbiol, 1998 Jan, 36(1), 266 - 8 Sporadic emergence of Klebsiella pneumoniae strains resistant to cefepime and cefpirome in Greek hospitals; Tzouvelekis LS et al.; The sporadic emergence of Klebsiella pneumoniae strains resistant to cefepime and cefpirome was observed in Greek hospitals during 1996 . Examination of six epidemiologically distinct strains and clones selected in vitro provided indications that resistance is due to the cooperation of decreased outer membrane permeability and hydrolysis of the cephalosporins by SHV-5 beta-lactamase, which was produced in large amounts. Klin Lab Diagn, 1997 Aug, (8), 38 - 41 {Methodological aspects of Klebsiella pneumoniae serotyping and its use for characterization of clinical isolates}; Morozova OT et al.; The method for serological typing of Klebsiella pneumoniae making use of Russian commercial K-sera manufactured by the Ilya Metchnikoff firm has been used to characterize 85 strains isolated from newborns at an obstetrical hospital and department of newborn diseases and from children with acute enteric infections hospitalized at the hospital for infectious diseases . The authors emphasize that their methods of serotyping are to be accurately performed, specifically, the selection of capsular forms and identification of serovars in strains which can be agglutinated by several sera . Serovars were identified by the proposed serotyping method in 89.4% of the studied strains . A wide spectrum of K-serovars typical of this or that hospital has been defined for each institution . K . pneumoniae K2 predominated in the obstetrical hospital, K . pneumoniae K24 and K25 prevailed in department for newborn diseases, and the K14 variant in the infectious diseases hospital. J Bacteriol, 1998 Feb, 180(3), 578 - 85 Two roles for the DNA recognition site of the Klebsiella aerogenes nitrogen assimilation control protein; Pomposiello PJ et al.; The nitrogen assimilation control protein (NAC) binds to a site within the promoter region of the histidine utilization operon (hutUH) of Klebsiella aerogenes, and NAC bound at this site activates transcription of hutUH . This NAC-binding site was characterized by a combination of random and directed DNA mutagenesis . Mutations that abolished or diminished in vivo transcriptional activation by NAC were found to lie within a 15-bp region contained within the 26-bp region protected by NAC from DNase I digestion . This 15-bp core has the palindromic ends ATA and TAT, and it matches the consensus for LysR family transcriptional regulators . Protein-binding experiments showed that transcriptional activation in vivo decreased with decreasing binding in vitro . In contrast to the NAC-binding site from hutUH, the NAC-binding site from the gdhA promoter failed to activate transcription from a semisynthetic promoter, and this failure was not due to weak binding or greatly distorted protein-DNA structure . Mutations in the promoter-proximal half-site of the NAC-binding site from gdhA allowed this site to activate transcription . Similar studies using the NAC-binding site from hut showed that two mutations in the promoter proximal half-site increased binding but abolished transcriptional activation . Interestingly, for symmetric mutations in the promoter-distal half-site, loss of transcriptional activation was always correlated with a decrease in binding . We conclude from these observations that if the binding in vitro reflects the binding in vivo, then binding of NAC to DNA is not sufficient for transcriptional activation and that the NAC-binding site can be functionally divided in two half-sites, with related but different functions. Arch Pathol Lab Med, 1997 Dec, 121(12), 1287 - 91 Epstein-Barr virus-associated primary central nervous system lymphoma in a child with the acquired immunodeficiency syndrome . A case report and review of the literature; Rodriguez MM et al.; A 34-month-old black boy who had contracted acquired immunodeficiency syndrome from his mother presented with fever, vomiting, and cough . He was cachectic, hypertonic, and developmentally delayed . A brain computed tomography scan revealed masses in the left frontal horn, subependymal, and periventricular regions; secondary edema; and hydrocephalus . The differential diagnosis was cerebral lymphoma versus toxoplasmosis . The patient had disseminated Mycobacterium avium-intracellulare infection, lymphoid interstitial pneumonitis, as well as Pseudomonas and Klebsiella pneumonia . He died of respiratory insufficiency 53 days after admission . The autopsy confirmed a primary cerebral B-cell lymphoma, large cell type, which was positive for Epstein-Barr virus, latent phase, by in situ hybridization . Primary central nervous system lymphomas are rare in children, in contrast to adults . To our knowledge, only five well-documented cases of primary cerebral lymphomas in infants and children with acquired immunodeficiency syndrome have been reported previously . The current study shows that these childhood lymphomas are associated with and presumably caused by Epstein-Barr virus and thus have a pathogenesis similar to that of primary central nervous system lymphomas in adults. Antimicrob Agents Chemother, 1998 Jan, 42(1), 108 - 13 A novel extended-spectrum TEM-type beta-lactamase (TEM-52) associated with decreased susceptibility to moxalactam in Klebsiella pneumoniae; Poyart C et al.; Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ) . Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX) . Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum beta-lactamase . Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element . Sequence analysis revealed that the blaT gene of pNEC865 differed from blaTEM-1 by three mutations leading to the following amino acid substitutions: Glu104-->Lys, Met182-->Thr, and Gly238-->Ser (Ambler numbering) . The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated blaTEM-52 . The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (Ki, 0.16 microM) was 10-fold higher than that of TEM-3 (Ki, 1.9 microM) . Allelic replacement analysis revealed that the combination of Lys104, Thr182, and Ser238 was responsible for the increase in the MICs of MOX for the TEM-52 producers. Antimicrob Agents Chemother, 1998 Jan, 42(1), 53 - 8 Epidemiology and successful control of a large outbreak due to Klebsiella pneumoniae producing extended-spectrum beta-lactamases; Pena C et al.; An outbreak due to extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) was detected from May 1993 to June 1995 . A total of 145 patients, particularly patients in intensive care units (ICUs) (107 patients {72%}), were colonized or infected . Infection developed in 92 (63%) patients, and primary bacteremia caused by ESBL-KP was the most frequent infection (40 of 92 patients {43%}) . A single clone of ESBL-KP was identified by pulsed-field gel electrophoresis analysis throughout the whole period, and no molecular epidemiological relationship could be found between the epidemic strain and non-ESBL-KP isolates . To determine risk factors for ESBL-KP infection weekly rectal swabs were obtained in three serial incidence surveys (470 patients); the probabilities of carriage of ESBL-KP in the digestive tract were 33% (October and November 1993), 40% (May and June 1994), and 0% (October and November 1995) at 10 days of ICU admission . A logistic regression model identified prior carriage of ESBL-KP in the digestive tract (odds ratio, 3.4; 95% confidence interval 1.1 to 10.4) as an independent variable associated with ESBL-KP infection . A statistically significant correlation was observed between the restricted use of oxyimino-beta-lactams (189 defined daily doses {DDD}/ 1,000 patient-days to 24 DDD/1,000 patient-days) and the trends of ESBL-KP infection (r = 0.7; P = 0.03). Food Chem Toxicol, 1997 Dec, 35(12), 1151 - 7 The effect of vitamin C on N-acetyltransferase activity in Klebsiella pneumoniae; Hsieh SE et al.; This study was designed to assess the effect of vitamin C on arylamine N-acetyltransferase (NAT) activity in Klebsiella pneumoniae by using HPLC to measure the acetylation of 2-aminofluorene (2-AF) with and without vitamin C . Two assay systems were performed, one with intact bacterial cell suspensions, the other with S-9 fractions (9000g supernatant) . It was found that vitamin C promoted NAT activity in K . pneumoniae in a dose-dependent manner in both systems . 4 and 8 mM vitamin C were selected for further studies in S-9 fractions and intact cell systems, respectively . Through a 4-hr time course study, vitamin C promoted the N-acetylation of 2-AF in both assay systems, but, the longer the reaction time lasted, the lower the promotion rate . In the kinetic studies, vitamin C increased the value of Km from 0.42+/-0.03 mM to 2.43+/-0.87 mM in S-9 fraction assays and from 0.54+/-0.03 mM to 0.85+/-0.18 mM in intact cell assays . Vitamin C also increased the apparent Vmax values from 3.5 +/-0.08 to 39.66+/-9.81 nmol/min/mg protein in S-9 fraction assays, and from 1.28+/-0.06 to 4.88+/-0.87 nmol/min/10(10) CFU in intact cell assays, for acetylation of 2-AF . In the presence of vitamin C, the NAT activity was increased from 0.58+/-0.06 to 1.34+/-0.02 nmol/min/mg protein in S-9 fractions, and from 0.18+/-0.02 to 0.40+/-0.02 nmol/min/10(10) CFU in intact cells, for acetylation of 2-AF . From the present study, it is concluded that vitamin C does promote the N-acetylation of 2-AF in K . pneumoniae . This is a first report suggesting that oral vitamin C may be involved in modifying the mutagenicity/carcinogenicity of ingested arylamines through enhancing the NAT activity of human enteric bacteria . This interaction should be pursued in future in vivo studies. Am J Gastroenterol, 1998 Jan, 93(1), 118 - 9 A new variant of food poisoning: enteroinvasive Klebsiella pneumoniae and Escherichia coli sepsis from a contaminated hamburger; Sabota JM et al.; For the first time, we report Klebsiella pneumoniae as an enteroinvasive food-borne pathogen transmitted from a hamburger . A 28-year-old previously healthy African-American male ingested a portion of a hamburger from a fast food chain . Symptoms of gastroenteritis rapidly deteriorated to multiorgan failure . Blood and hamburger cultures grew Escherichia coli and Klebsiella pneumoniae . Since Klebsiella had not previously been reported as enteroinvasive, the isolates were compared . Full biochemical profiles, antimicrobial sensitivity, plasmid profile, and toxin assay by DNA hybridization probe were completely concordant . The patient survived the episode of food-borne sepsis . Deliberate or inadvertent employee contamination of food products with feces may be a potential source of life-threatening food-borne illness. Zentralbl Bakteriol, 1997 Nov, 286(4), 449 - 55 Transfer of antibiotic resistance and production of extended-spectrum beta-lactamase (ESBL) in nosocomial Klebsiella pneumoniae strains from two hospitals in Czech Republic; Blahova J et al.; Six Klebsiella pneumoniae strains were collected from two hospitals in Ostrava, Czech republic . Four strains (Nos . 209, 217, 218, 222) were isolated from sputa of critically ill patients from Municipal Hospital Vitkovice-Ostrava . They were resistant to cephalothin, cefotaxime, and ceftazidime (MIC > 100 mg x l-1) . Strain No . 218 was intermediately resistant also to ofloxacin and aztreonam (MIC = 12.5 mg x l-1), strain No . 222 was resistant to aztreonam (MIC = 50 mg x l-1) . Determinants of resistance to cephalothin, cefotaxime, aztreonam and ceftazidime were transferred to recipient strains of P . mirabilis P-38 rif+ and E . coli K-12 No . 3110 rif+ by all four strains . Synergy between clavulanate-cefotaxime, clavulanate-ceftazidime and clavulanate-aztreonam indicated production of ESBLs by these strains . Two strains, No . 214 and 224, from patients of the ICU in the University Hospital were resistant to cephalothin, cefotaxime and ceftazidime (MIC > 100 mg x l-1) . Strain No . 214 was intermediately resistant to aztreonam and ofloxacin (MIC = 12.5 mg x l-1) and strain No . 224 was highly resistant to aztreonam (MIC = 50 mg x l-1) . Synergy between clavulanate and cefotaxime as well as between clavulanate and aztreonam, but not between clavulanate and ceftazidime corresponds with non-transferable ceftazidime resistance in strains No . 214 and 224 and indicates different types of ESBL in strains from each of two hospitals. J Biol Chem, 1997 Dec 19, 272(51), 32034 - 41 Characterization, sequencing, and expression of the genes encoding a reactivating factor for glycerol-inactivated adenosylcobalamin-dependent diol dehydratase; Mori K et al.; Diol dehydratase undergoes suicide inactivation by glycerol during catalysis involving irreversible cleavage of the Co-C bond of adenosylcobalamin . In permeabilized Klebsiella oxytoca and Klebsiella pneumoniae cells, the glycerol-inactivated holoenzyme or the enzyme-cyanocobalamin complex is rapidly activated by the exchange of the inactivated coenzyme or cyanocobalamin for free adenosylcobalamin in the presence of ATP and Mg2+ (Honda, S., Toraya, T., and Fukui, S . (1980) J . Bacteriol . 143, 1458-1465; Ushio, K., Honda, S., Toraya, T., and Fukui, S . (1982) J . Nutr . Sci . Vitaminol . 28, 225-236) . Permeabilized Escherichia coli cells co-expressing the diol dehydratase genes with two open reading frames in the 3'-flanking region were capable of reactivating glycerol-inactivated diol dehydratase as well as activating the enzyme-cyanocobalamin complex in situ in the presence of free adenosylcobalamin, ATP, and Mg2+ . These open reading frames, designated as ddrA and ddrB genes, were identified as the genes of a putative reactivating factor for inactivated diol dehydratase . The genes encoded polypeptides consisting of 610 and 125 amino acid residues with predicted molecular weights of 64,266 and 13,620, respectively . Co-expression of the open reading frame in the 5'-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E . coli . Thus, the product of this gene was considered not an essential component of the reactivating factor. FEMS Microbiol Lett, 1997 Dec 15, 157(2), 313 - 8 NifL of Klebsiella pneumoniae carries an N-terminally bound FAD cofactor, which is not directly required for the inhibitory function of NifL; Schmitz RA; In Klebsiella pneumoniae NifL antagonizes the action of the transcriptional activator NifA in the presence of molecular oxygen or combined nitrogen . To determine what cofactors might be involved in the oxygen sensing mechanism, we purified and analyzed fusion proteins made between the Escherichia coli maltose binding protein, MalE, and NifL . NifL synthesized and purified under strictly anaerobic conditions did not contain significant amounts of iron or acid-labile sulfur indicating the absence of an oxygen sensing iron-sulfur cluster . However, NifL protein purified in its inhibitory form contained 0.3 +/- 0.01 mol FAD and less than 0.01 mol FMN per mol NifL suggesting the presence of FAD as a cofactor . Characterization of NifL synthesized in the absence of oxygen and combined nitrogen showed that the non-inhibitory form of NifL also contained FAD (0.54 mol FAD per mol NifL) . Using fusions between MalE and different portions of NifL we localized the binding site of FAD to the N-terminal domain of NifL . These results and our previous observation that the C-terminal domain of NifL is sufficient to inhibit NifA activity indicate that the N-terminally bound FAD is not directly required for the inhibitory activity of NifL . This observation is supported by the finding that purified apoprotein of NifL was still able to inhibit transcriptional activation by NifA in vitro. Scand J Infect Dis, 1997, 29(5), 491 - 3 Dietary fish oil supplementation increases survival in mice following Klebsiella pneumoniae infection; Bjornsson S et al.; The effect of dietary fish-oil supplementation on survival of NMRI mice after Klebsiella pneumoniae infection was investigated . 30 mice in each group were fed a fish-oil enriched diet, olive-oil enriched diet or standard chow diet . After 6 weeks, the mice were injected intramuscularly with Klebsiella pneumoniae . After 120 h the survival of the mice fed fish-oil enriched diet was 40%, while the survival for mice fed standard or olive-oil enriched diets was 20% and 25%, respectively . The survival curve over 120 h was significantly improved (p = 0.0034) for mice fed a fish-oil enriched diet, compared to the survival curves for mice fed the other 2 diets . The study was repeated by comparing the survival of mice fed a fish-oil enriched diet to those given a corn-oil enriched diet . After 120 h the survival curve for mice fed the fish-oil enriched diet was significantly better compared to the survival curve for mice given the corn-oil enriched diet (p = 0.01) . A fish-oil enriched diet therefore increases survival in mice following Klebsiella pneumoniae infection, whether compared to a standard diet, olive-oil enriched diet or corn-oil enriched diet. Protein Expr Purif, 1997 Dec, 11(3), 257 - 62 Overexpression of arylsulfate sulfotransferase as fusion protein with glutathione S-transferase; Baek MC et al.; A procedure has been developed for the overexpression and purification of milligram quantities of the Klebsiella K-36 arylsulfate sulfotransferase (ASST) . The structural gene was amplified by means of a polymerase chain reaction (PCR) technique and inserted into the plasmid vector pGEX-3X . The plasmid pGEX-100, carrying the Klebsiella K-36 astA structural gene under the control of the Escherichia coli tac promoter, was transformed into the E . coli strain BL21 (DE3) . The ASST was produced in E . coli as a fusion with glutathione S-transferase . Conditions for protein production, isolation on glutathione Sepharose 4B, and Xa cleavage to generate active ASST were developed . The purification yielded approximately 0.7 mg of pure enzyme per liter of bacterial culture . Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties almost the same as those of the enzyme purified from Klebsiella K-36 cells . The purification procedure was very rapid and is suitable for obtaining considerable amounts of enzyme at a relatively high yield compared with its purifying method from the culture of the Klebsiella K-36 strain. J Appl Toxicol, 1997 Nov-Dec, 17(6), 385 - 90 Glycyrrhizic acid inhibits arylamine N-acetyltransferase activity in Klebsiella pneumoniae in vitro; Lo HH et al.; Glycyrrhizic acid, one of the proposed chemopreventive drugs, was used to inhibit arylamine N-acetyltransferase (NAT) activity in Klebsiella pneumoniae, both in cytosol and intact bacteria . The NAT activity was measured by using high-performance liquid chromatography to assay the amounts of 2-acetyl-aminofluorene and remaining 2-aminofluorene . The NAT activity in K . pneumoniae was inhibited by glycyrrhizic acid in a dose-dependent manner . The cytosol NAT activities were 0.675 +/- 0.028 nmol min(-1) mg(-1) protein for the acetylation of 2-aminofluorene without glycyrrhizic acid and 0.367 +/- 0.008 nmol min(-1) mg(-1) protein with 8 mM glycyrrhizic acid . The NAT activities measured from intact bacteria were 0.308 +/- 0.018 nmol min(-1) 10(-10) colony forming units for the acetylation of 2-aminofluorene without glycyrrhizic acid and 0.236 +/- 0.005 nmol min(-1) 10(-10) colony forming units in the presence of 8 mM glycyrrhizic acid . The inhibition of NAT activity by glycyrrhizic acid was demonstrated to remain for at least 4 h . The apparent Km and Vmax values calculated from cytosol NAT were 1.08 +/- 0.05 mM and 9.09 +/- 0.11 nmol min(-1) mg(-1) protein, respectively, for 2-aminofluorene . In the presence of 8 mM glycyrrhizic acid, the apparent Km and Vmax values were 0.15 +/- 0.01 mM and 0.95 +/- 0.11 nmol min(-1) mg(-1) protein, respectively, for 2-aminofluorene . In intact bacteria, the apparent Km and Vmax values were 1.28 +/- 0.48 mM and 4.08 +/- 1.06 nmol min(-1) 10(-10) colony forming units, respectively, for 2-aminofluorene . However, in the presence of 8 mM glycyrrhizic acid, the apparent Km and Vmax values were 0.67 +/- 0.09 mM and 1.82 +/- 0.37 nmol min(-1) 10(-10) colony forming units, respectively, for 2-aminofluorene . Taking these results together, the NAT activity in K . pneumoniae was inhibited by glycyrrhizic acid both in cytosol and intact bacteria . This study provides the first evidence to demonstrate that glycyrrhizic acid inhibits bacterial NAT activity. Minerva Chir, 1997 Jul-Aug, 52(7-8), 937 - 42 {Totally implantable venous access systems . Analysis of complications}; D'Angelo F et al.; Totally implantable central venous access devices (Port-a-Cath, PaC) allow better treatment of cancer patients, with safe administration of chemotherapeutic agents, and are well accepted by the patients . The aim of the present paper is to analyze the complications of the different implant techniques on the basis of a personal experience of 92 central venous access devices . MATERIAL AND METHODS: A total of 92 PaC (Port-a-Cath, Pharmacia: Celsite Braun) have been implanted in 88 patients between August 1992 and June 1995 for cancer treatment . Age ranged between 19 and 79 years (median 52 years), 56 were male and 32 women . PaC have been implanted by percutaneous cannulation of the subclavian vein, with Seldinger technique, in 34 cases; by venous cutdown respectively on the cephalic vein in 46 cases, the jugular vein in 7 cases, the basilar vein in 4 and the saphenous vein in 1 case . Four patients experienced a double implant . In 84 cases the implant was done under local anesthesia, while in 8 required general anesthesia, during operation for the primary neoplasm . RESULTS: A total of 7 complications were experienced (7.6%, 7/92): 4 sepsis and 3 mechanical . No cases of pnx were observed . Sepsis occurred after 29, 45, 64, 401 days of implantation respectively, and culture demonstrated S . aureus in 2 cases, and E . coli and Klebsiella oxytoca in 1 case each . Mechanical complication comprehends 2 cases of catheter dislodgement and 1 case of port rotation . No complications were noticed in case of implant during surgery for primary cancer (8 cases) . In 7 cases the procedure has been converted from cephalic vein cutdown to percutaneous cannulation of the subclavian vein due to anatomic reasons (13.2%, 7/53) . Five PaC have been explanted for complications . DISCUSSION: On the basis of the personal experience we think that PaC are of easy implant, with few complications and of good acceptance from the patients . We prefer venous cutdown on cephalic vein as implant technique because of avoidance of pnx or bleeding complications . Percutaneous puncture of subclavian vein is useful for implantation during major surgery, because less time consuming, and in case of anatomical anomalies fo the cephalic vein . Basilic vein cutdown has been utilized exclusively for esthetic reason in young people, to avoid the scar in the upper thoracic region . Alternative implant techniques has been employed in special conditions, such as catheter position in the inferior v.cava, or early in our experience (internal jugular vein) . A total of 7 complication have been reported (7.6%), 4 sepsis and 3 mechanical (2 dislodgement, 1 rotation) . Sepsis were not related to implant technique, presenting on day 29, 45, 64 and 401 respectively; all required the explant of the PaC as a treatment . Mechanical complications are related to surgical technique; all required re-exploration with 1 explant and 2 reposition of the PaC . In PaC positioning during surgery for primary cancer (8 cases) no morbidity has been reported . All but the 5 PaC explanted were functioning until patient's need; maximum length reported is 42 months. J Antimicrob Chemother, 1997 Oct, 40(4), 525 - 32 Mechanism and stability of hyperproduction of the extended-spectrum beta-lactamase SHV-5 in Klebsiella pneumoniae; Xiang X et al.; Some isolates of SHV-5 beta-lactamase-producing Klebsiella pneumoniae K2 from a single-strain outbreak of cross-infection produced approximately five-fold more beta-lactamase than others . We investigated three possible genetic mechanisms of this hyperproduction: the presence of a more powerful promoter, an increase in plasmid copy number or an amplification of the gene on a plasmid . No differences between low and high beta-lactamase producers were detected in the promoter region of the SHV-5 beta-lactamase gene, which closely resembled that of SHV-2 . SHV-5 beta-lactamase production was encoded on a low copy number plasmid, but DNA-DNA hybridization with an SHV-specific probe detected a higher gene dose in hyperproducers . The beta-lactamase hyperproduction was unstable on repeated subculture, with a reduction of about 75% after 100 generations . Hyperproducing mutants of a low-producing Klebsiella and its Escherichia coli K-12 transconjugants could be selected in vitro at a frequency of 10(-5) to 10(-6) and these variants had an increased SHV-5 beta-lactamase gene copy number on low copy number plasmids . We conclude that hyperproduction of the extended-spectrum beta-lactamase was caused by gene amplification that could be easily lost or gained in vitro . Since the change to hyperproduction occurred at a high frequency and hyperproducers showed increased resistance to many beta-lactams and beta-lactam/beta-lactamase inhibitor combinations, we suspect that these variants may readily be selected in patients during antibiotic therapy. Eur J Pediatr Surg, 1997 Oct, 7(5), 263 - 6 Characteristics of multisystem organ failure in neonates; Avanoglu A et al.; The records of 45 neonatal deaths in a four year period were reviewed retrospectively . Sixteen patients (35.5%) developed multisystem organ failure (MSOF) . The criteria for pulmonary, hepatic, renal, hematologic, cardiac and microvascular failures were established . The onset of the first organ involvement was calculated in days prior to death . The earliest organ involved was kidney (14.2 +/- 15.1) followed by microvascular (9.4 +/- 7.6), hematologic (84 . +/- 10.1), liver (6.8 +/- 6.7), lung (6.3 +/- 6.6) and cardiac (6.0 +/- 8.7) failure . Blood culture analyses revealed 5 patients with culture-positive sepsis . Yeast was the leading septic agent (n = 3) followed by Klebsiella pneumoniae (n = 2), Pseudomonas sp . (n = 1) and E . coli (n = 1) . The first organ involvement was noted at 17.6 +/- 23.2 days . We concluded that the sequence of neonatal MSOF is different from that of adults, yet the inciting events are not clear-cut . Lung, which is the first organ involved in adults, seems to be a lately involved organ in neonates. Am J Perinatol, 1997 Oct, 14(9), 519 - 21 Ilio-psoas abscess in a neonate; Andreou A et al.; A full-term, small-for-gestational-age, neonate was born 4 days after rupture of the membranes . On the 5th day of life, she developed sepsis due to Klebsiella pneumoniae . On the 18th day of life, the right hip was noted swollen with limited range of motion, but it was painless on passive movements . Ultrasonography revealed abscess of the right ilio-psoas muscle with normal appearance of the right hip joint . Surgical incision and drainage and antibiotic administration resulted in a gradual full recovery . Ultrasonography can confirm the diagnosis of this exceptional clinical entity in neonates, which is difficult to differentiate from septic arthritis of the hip. Appl Microbiol Biotechnol, 1997 Oct, 48(4), 522 - 7 Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714; Farres J et al.; Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors) . The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production . Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used . Exopolysaccharide synthesis was greatest at 30 degrees C, which was a suboptimal growth temperature . A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production . When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed . Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium. Cytometry, 1997 Nov 1, 29(3), 267 - 72 Rapid discrimination of bacterial species with different ampicillin susceptibility levels by means of flow cytometry; Walberg M et al.; An in vitro model for flow cytometric detection of heterogeneous drug response in exponentially growing Escherichia coli and Klebsiella pneumoniae was studied to evaluate the potential of this technology for rapid antibiotic susceptibility testing in polymicrobial samples . The cells, which exhibited 80-fold difference in in ampicillin susceptibility, were cultivated in the presence of ampicillin at a concentration equaling 1 MIC of the low-susceptibility strain (E . coli) . Prior to flow cytometric analysis, the cells were fixed in 70% ethanol and stained with a DNA-specific dye . After 1 h of ampicillin incubation, the light scattering and fluorescence intensities of the susceptible cells increased 4.3-fold and 5-fold, respectively, but remained about constant for the resistant cells . The corresponding cell number increase for the resistant and the susceptible cells was 9.4 and 1.7, respectively . The two strains could be distinguished in the histograms on the basis of their light scattering and fluorescence intensities and by cell number . With an incubation of up to 3 h, the light scattering and fluorescence intensities of the susceptible cells grew stronger, at the expense of cell number . By combination of histograms, the discrimination of different cell populations could be improved . The results demonstrate the ability of flow cytometry to discriminate between species in heterogeneous cultures and suggest the potential of the technique for rapid assessment of bacterial susceptibility . However, the present results are preliminary, and the application to biological liquids and clinical samples has to be demonstrated further. Gene, 1997 Oct 1, 198(1-2), 111 - 3 Evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella; Sugiyama T et al.; In order to clarify the evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella, we studied the DNA sequence of the boundary region between the rfb and his genes in a series of strains possessing mannose homopolymer as O-specific polysaccharide . All had a characteristic gene organization carrying no gene between the rfb and his genes . Further, the recombination event was suggested to occur at the same site of the hisI gene in those strains . It was suggested that there was a close evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharide in E . coli and Klebsiella. J Med Microbiol, 1997 Nov, 46(11), 921 - 6 Molecular epidemiology of a large outbreak of multiresistant Klebsiella pneumoniae; Weller TM et al.; An outbreak of multiresistant Klebsiella pneumoniae has continued in the Grampian Region of Scotland since 1992 . The organism, which generally produces an extended-spectrum beta-lactamase (ESBL), has spread to several hospitals and nursing homes . DNA from 80 possible outbreak isolates was digested with the restriction endonucleases XbaI and SpeI, and the patterns obtained by pulsed-field gel electrophoresis were compared . Restriction patterns of 79 of the isolates were found to be highly similar with both restriction enzymes, whereas one isolate was unrelated . The outbreak isolates were divided into six subtypes with SpeI and 16 subtypes with XbaI . These subtypes were independent of antibiotic susceptibility pattern, date of isolation and ward of origin, but the XbaI subtype did correlate with the SpeI subtype . It was concluded that the Klebsiella isolates of this outbreak were clonally related. Epidemiol Infect, 1997 Oct, 119(2), 135 - 42 Klebsiella meningitis in Taiwan: an overview; Tang LM et al.; Klebsiella infection has been considered to be an uncommon cause of meningitis . To determine its incidence and clinical features, we reviewed the microbiologic records of cerebrospinal fluid (CSF) and blood cultures and the medical records of patients with bacterial meningitis admitted between 1981 and 1995 . Klebsiella meningitis was diagnosed in 79 patients with 83 episodes . All patients had klebsiella isolated from CSF and/or blood and typical symptoms and signs of acute bacterial meningitis . Of these, 74 were over 16 years of age and 2 of the 5 children were infants . There was an increased prevalence rate of klebsiella meningitis after 1986 . Of the 83 episodes, only 9 occurred between 1981 and 1986, accounting for 7.8% of 115 cases with CSF and/or blood culture-proven acute bacterial meningitis, whereas in 1987-95, there were 74 episodes accounting for 17.7% of 419 bacteriologically proven cases . K . pneumoniae accounted for 69 episodes, K . oxytoca, 11 episodes and K . ozaenae, 3 episodes . Male gender, diabetes mellitus and liver cirrhosis were commonly associated with K . pneumoniae meningitis . Neurosurgical procedures were frequently associated with K . oxytoca meningitis . All three patients with K . ozaenae meningitis had a primary disease of the nasopharyngeal pathway . The mortality rate due to K . pneumoniae was 48.5%, K . oxytoca, 10% and K . ozaenae, 0% . In patients with K . pneumoniae meningitis, poor prognostic factors included age over 60 years, diabetes mellitus, bacteremia and severe neurological deficits on the first day of treatment. Biosci Biotechnol Biochem, 1997 Oct, 61(10), 1729 - 33 A protein factor is essential for in situ reactivation of glycerol-inactivated adenosylcobalamin-dependent diol dehydratase; Mori K et al.; The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme . The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E . coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+ . beta,gamma-Methylene ATP was not able to replace ATP . Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not . These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl . The in situ reactivation was also observed when an analog lacking the alpha-ribose moiety of the nucleotide loop was used as coenzyme . The results with a recombinant E . coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3'-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase. J Infect, 1997 Sep, 35(2), 185 - 8 Ruptured abdominal aorta secondary to psoas muscle abscess due to Klebsiella pneumoniae in an alcoholic; Sugawara Y et al.; An alcoholic patient with low back pain and Klebsiella pneumoniae septicaemia is reported . Computed tomography revealed abdominal aortic rupture associated with a psoas abscess . Aortic ligation above and below the rupture site and an axillo-femoral bypass were performed, but the patient died on the first postoperative day . Alcoholism is a common underlying disease in K . pneumoniae septicaemia and its septic metastasis to the psoas muscle . The prognosis of aortic infection secondary to psoas abscess is very poor once aortic rupture occurs . Prompt abscess drainage following correct diagnosis and arterial reconstruction before aortic rupture are mandatory. Diagn Microbiol Infect Dis, 1997 Sep, 29(1), 39 - 41 High rates of resistance to piperacillin/tazobactam among Escherichia coli and Klebsiella pneumoniae strains isolated in a Greek hospital; Tsakris A et al.; The activities of piperacillin/tazobactam (PTZ) and 10 other beta-lactams were evaluated by a broth microdilution method using the PASCO system in 1078 Escherichia coli and 447 Klebsiella pneumoniae strains isolated from hospitalized patients in a Greek hospital . Overall, 10.5% of the former and 62.6% of the latter strains displayed resistance to PTZ . Most of the PTZ-resistant strains (71.2%) expressed extended spectrum beta-lactamases and were also resistant to broad spectrum cephalosporins and aztreonam . The high rates of PTZ resistance in E . coli and K . pneumoniae and the extensive cross-resistance to other beta-lactams suggest that PTZ should be used with caution in our clinical setting. Acta Otolaryngol Suppl, 1997, 531, 39 - 51 Roxythromycin prevents endotoxin-induced otitis media with effusion in the guinea pig; Sugiura Y et al.; Pharmacological agents that can normalize or enhance the ciliary and mucociliary activity of the tubotympanum should also be able to break the vicious circle of chronic otitis media with effusion (OME) . Roxythromycin (RXM) has been shown to enhance the ciliary activity in vitro and also stimulate the mucociliary activity in vivo and may therefore, when clinically applied, prevent not only occurrence but also recurrence of clinical OME . The present study was designed to discuss the possible preventive effect of RXM on endotoxin-induced OME in the guinea pig . A total of 120 guinea pigs were used . The normal control group was treated with intratympanic injection of 0.1 ml of physiologic saline solution . The saline-control group was treated with oral administration of physiologic saline solution for 14 successive days . The low-dosage group and the high-dosage group were treated with oral administration of 5 and 50 mg/kg of sairei-to for 14 successive days, respectively . Then, the saline-control group, the low-dosage group and the high-dosage group were treated with intratympanic injection of 0.1 ml of lipopolysaccharide solution (100 micrograms/ml) derived from Klebsiella pneumoniae . All 10 animals in the four groups were sacrificed 1, 3, and 7 days after the intratympanic injection, to examine ciliary activity, mucociliary clearance time, and mucosal pathology of the tubotympanum . The saline-control group exhibited middle ear effusions and pathologies similar to human OME . The incidence of middle ear effusions was significantly reduced in the low-dosage and high-dosage groups . Throughout the observation period, the ciliary activity in the tubotympanum was significantly reduced in the saline-control group as compared with that of the normal control group . By contrast, the ciliary activity in the low-dosage and high-dosage groups was not so reduced in the Eustachian tube and the middle ear close to the orifice . Mucociliary clearance time in the low-dosage and high-dosage groups was not different from that in the normal control group throughout the period . The tubotympanum in the saline-control group exhibited mucosal pathologies characteristic of OME in human . By contrast, the low-dosage and high-dosage groups exhibited much milder pathological changes in the tubotympanum than those in the saline-control group . In conclusion, clinical application of RXM could be an effective measure to prevent the occurrence of OME and also the recurrence of the disease, especially in OME-prone individuals. Acta Otolaryngol Suppl, 1997, 531, 21 - 33 The herbal medicine, sairei-to, prevents endotoxin-induced otitis media with effusion in the guinea pig; Sugiura Y et al.; With current pharmacotherapy, otitis media with effusion (OME) is often recurrent and even develops to become chronic . There is now considerable experimental and clinical evidence that the cilia in the tubotympanum play an important part in the prevention of OME . A herbal medicine, sairei-to, has been shown to stimulate the ciliary activity in vitro, and oral administration of the medicine also stimulated the ciliary activity in the tubotympanum rather than physiological states . This study was designed to investigate whether oral administration of sairei-to could prevent experimental OME in the guinea pig . A total of 120 guinea pigs were used . The control group was treated with intratympanic injection of 0.1 ml of physiologic saline solution . The saline-control group was treated with oral administration of physiologic saline solution for 14 successive days . The low-dosage group and the high-dosage group were treated with oral administration of 120 and 600 mg/kg of sairei-to for 14 successive days, respectively . The saline-control group, the low-dosage group and the high-dosage group were then treated with intratympanic injection of 0.1 ml of lipopolysaccharide solution (100 micrograms/ml) derived from Klebsiella pneumoniae . All 10 animals from the 4 groups were sacrificed 1, 3, and 7 days after the intratympanic injection, to examine ciliary activity, mucociliary clearance time, and mucosal pathology of the tubotympanum . The saline-control group exhibited middle ear effusions and pathologies similar to human OME . The incidence of middle ear effusions in the low-dosage and the high-dosage groups was somewhat reduced compared with the saline-control group . The ciliary activity in the tubotympanum was significantly reduced in the saline-control and low-dosage groups compared with the normal-control group . By contrast, the magnitude of reduction in ciliary activity was much smaller in the high-dosage group . The ciliary activity especially in the Eustachian tube and the middle ear close to the tympanic orifice at 3 and 7 days in the high-dosage group was not significantly different from that in the normal-control group . Mucociliary clearance time in the high-dosage group was not different from that in the normal-control group throughout the observation period . The groups treated with sairei-to, especially the high-dosage group, exhibited much milder pathological changes in the tubotympanum than did the saline-control group . In conclusion, clinical application of sairei-to could be an effective measure to prevent the occurrence of OME and also the recurrence of the disease, especially OME-prone individuals. Arch Pediatr, 1997 Sep, 4(9), 853 - 6 {Acute pyelonephritis and subcapsular hematoma revealing nephroblastoma at the age of 15 years}; Tlili-Graiess K et al.; BACKGROUND: Nephroblastoma' the most common renal tumor in children between 1 and 5 years, occurs rarely in the oldest child . CASE REPORT: A 16-year-old teenager suffered from acute pyelonephritis caused by Klebsiella pneumoniae . Renal ultrasonography showed a left subcapsular hematoma; the CT scan confirmed the finding and also showed renal scarring . However, a second CT scan showed pulmonary nodules suggestive of metastasis, a diagnosis that was confirmed by needle biopsy of pulmonary lesions . Recovery was obtained after chemotherapy and nephrectomy with a 3-year-follow-up . CONCLUSION: This nephroblastoma was particular because its development in an adolescent, its association with acute pyelonephritis and subcapsular hemorrhage. Arch Biochem Biophys, 1997 Nov 1, 347(1), 132 - 40 Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca; Tobimatsu T et al.; Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was purified to homogeneity . The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery . Subsequent chromatography on DEAE-cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65% . The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme of K . oxytoca . The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000 . The enzyme consists of equimolar amounts of the three subunits with apparent Mr of 60,000 (alpha), 30,000 (beta), and 19,000 (gamma) . Therefore, the subunit structure of the enzyme is most likely alpha2beta2gamma2 . The recombinant enzyme was also separated into components F and S upon DEAE-cellulose chromatography in the absence of substrate . Components F and S were identified as the beta subunit and alpha2gamma2 complex, respectively . Apparent Km for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2-ethanediol were 0.83 microM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively . The three genes encoding the subunits of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli . The alpha and gamma subunits mutually required each other for correct folding forming the soluble, active alpha2gamma2 complex (component S) . Expression of the beta subunit in a soluble, active form (component F) was promoted by coexpression with both the alpha and gamma subunits, probably by coexistence with component S . These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme . Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 12145 - 50 Probing the assembly of transcription initiation complexes through changes in sigmaN protease sensitivity; Casaz P et al.; The alternative bacterial sigmaN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins . Little is known about how sigma factors respond to their ligands or how the responses lead to transcription . To examine the liganded state of sigmaN, the assembly of end-labeled Klebsiella pneumoniae sigmaN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting . V8 protease-sensitive sites in free sigmaN were identified in the acidic region II and bordering or within the minimal DNA binding domain . Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes . Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA . Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation . Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation . The sensitivity of E36 may reflect one or both of these functions . Changing patterns of protease sensitivity strongly indicate that sigmaN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation. Eur J Immunol, 1997 Sep, 27(9), 2123 - 32 Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide; Cotterill LA et al.; The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the alpha 1 and alpha 2 domains of Qa-1b and the alpha 3 domain of H-2Db . This allowed the use of a monoclonal antibody directed against H-2Db whilst retaining the peptide-binding groove of Qa-1b . By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with beta 2-microglobulin . However, at the cell surface the hybrid molecules were stably associated with beta 2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1b-presented peptide Qdm (AMAPRTLLL) . A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1b and CTL clones served to identify residues important for T cell recognition . Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition . In contrast, substitutions at position 9 resulted in loss of MHC class I binding . Mass spectrometric analysis of peptides eluted from immunopurified Qa-1b/Db molecules indicated that Qdm was the dominant peptide . The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2Dk, was also present, although it was considerably less abundant . The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues . Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms. Plasmid, 1997, 38(2), 97 - 105 Characterization of the replication and mobilization regions of the multiresistance Klebsiella pneumoniae plasmid pJHCMW1; Dery KJ et al.; A 2.4-kb EcoRI fragment including the replication and origin of transfer regions of the Klebsiella pneumoniae multiresistance plasmid pJHCMW1 has been cloned and sequenced . The isolated replication region was sufficient for stable maintenance of the plasmid and shares homology with RNA-regulated replicons . Homology was highest with the replication region of the plasmid p15A . Incompatibility experiments, however, determined that pJHCMW1 is compatible with pACYC177, a plasmid harboring the p15A replicon . Differences in their RNA I nucleotide sequences may account for their compatibility . A mobilization origin was also found in the 2.4-kb EcoRI pJHCMW1 DNA fragment analyzed . Conjugation experiments showed that although non-self-transmissible, the recombinant clone including the 2.4-kb EcoRI pJHCMW1 fragment could be mobilized in the presence of the helper plasmid pRK2073. Jpn J Antibiot, 1997 Aug, 50(8), 704 - 10 {Combination effect of fosfomycin to beta-lactam, aminoglycoside, and macrolide antibiotics against clinical isolates of Klebsiella pneumoniae}; O'Hara K et al.; It is well known that fosfomycin (FOM) shows the combination effects with some other antibiotics . Suck effects have not been known against Klebsiella pneumoniae, however . In this report, combination effects of FOM with beta-lactam, aminoglycoside, and macrolide antibiotics were investigated against clinical isolates of both FOM-susceptible and resistant Klebsiella pneumoniae . FOM had synergistic activities with beta-lactams such as ampicillin (ABPC) and cefminox (CMNX), and macrolide antibiotics as erythromycin (EM) and midecamycin (MDM) against all strains tested . Among beta-lactams, penicillin V showed synergistic actions against FOM-susceptible strain of Tf341A and additive actions against FOM-resistant 3 strains with FOM . Pheneticillin was synergistic with FOM against FOM-highly susceptible strain Tf341A and the additive or nearly synergistic effects against other strains . FOM with amoxicillin was synergistic against Tf341A, and FOM-resistant strain of Tf170B and additive against other strains . While the activities of combination of FOM with kanamycin or dibekacin against FOM-susceptible 2 strains were additive, those with amikacin were synergistic . Five different aminoglycosides tested showed antagonistic activities with FOM against 3 FOM-resistant strains . From these results, FOM appears to be clinically useful in treating FOM-susceptible and resistant strains of Klebsiella pneumoniae in combination of 4 antibiotics such as ABPC, CMNX, EM, and MDM. J Bacteriol, 1997 Oct, 179(19), 6014 - 9 Substrate recognition domains as revealed by active hybrids between the D-arabinitol and ribitol transporters from Klebsiella pneumoniae; Heuel H et al.; Two new genes, dalT and rbtT, have been cloned from the dal operon for D-arabinitol and the rbt operon for ribitol uptake and degradation, respectively, in Klebsiella pneumoniae 1033-5P14, derivative KAY2026 . Each gene codes for a specific transporter which, based on sequence data, belongs to a large family of carbohydrate transporters which constitutes 12 transmembrane helices . DalT and RbtT show an unusually high similarity (86.2% identical residues for totals of 425 and 427 amino acids, respectively) . This allowed the construction of DalT'-Rbt"T and RbtT'-Dal'T crossover hybrids by using a natural restriction site overlapping Met202 . This site is located within the large cytoplasmic loop which connects the putative helices 6 and 7 and in particular the amino- and the carboxy-terminal halves of the transporters . Both hybrids have close to normal transport activities but essentially the substrate specificities and kinetic properties of the amino-terminal half . This result localizes essential substrate binding and recognition sites to the amino-terminal halves of the proteins in this important class of carbohydrate transporters. Biochem J, 1997 Sep 15, 326 ( Pt 3), 637 - 40 Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre; Yousafzai FK et al.; We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4- . In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K . pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K . pneumoniae) . The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e . one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed . In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1 . The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per alpha2beta2 tetramer . This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom . These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex. Southeast Asian J Trop Med Public Health, 1997 Mar, 28(1), 218 - 22 Cockroaches from urban human dwellings: isolation of bacterial pathogens and control; Vythilingam I et al.; A study was carried out to determine the distribution of cockroaches in two different housing areas with central sewerage or individual septic tanks in an urban area in Kuala Lumpur, Malaysia . Six species of cockroaches were present and of these Periplaneta americana and Periplaneta brunnea were found in greater abundance . Seventeen species of bacteria were isolated and of these Escherichia coli and Klebsiella p . pneumoniae were isolated in greatest numbers . Control measures carried out using lambda cyhalothrin showed that there was no significant difference between treated and control sites. Heart Lung, 1997 Sep-Oct, 26(5), 413 - 7 Klebsiella pneumoniae pneumonia; Prince SE et al.; Klebsiella pneumoniae is an uncommon cause of community-acquired pneumonia except in alcoholics . Klebsiella may mimic pulmonary reactivation tuberculosis because it presents with hemoptysis and cavitating lesions . Klebsiella pneumoniae is a difficult infection to treat because of the organism's thick capsule . Klebsiella is best treated with third- and fourth-generation cephalosporins, quinolones, or carbapenems . Monotherapy is just as effective as a combination treatment in Klebsiella pneumoniae because newer agents are used . In the past, older agents with less anti-Klebsiella activity were needed for effective treatment . The patient we present was initially thought to have pulmonary tuberculosis, and when found to have Klebsiella pneumoniae, the suggested treatment was monotherapy with ceftriaxone . The patient was treated parenterally initially, and then was treated for 3 weeks with oral ofloxacin. Drugs, 1997, 54 Suppl 1, 33 - 6 Pharmacology of ribosomal immunotherapy; Clot J; The use of immunostimulating drugs is one way to intervene in the immune system . Many of these agents are of bacterial origin and most are able to stimulate the nonspecific immune response by acting on polymorphonuclear cells (PMNs) and macrophages . Ribosomal immunotherapy ('Ribomunyl') contains both proteoglycans from Klebsiella pneumoniae and ribosomes from 4 different bacterial strains . It can stimulate not only macrophages but also specific antibody production . 'Ribomunyl' has been shown to stimulate many of the functions of PMNs, specifically the formation of oxygenated free radicals, chemotaxis and adhesion . The effect of 'Ribomunyl' immunostimulant on the properties of macrophages is of special interest, as these cells participate in both the nonspecific immune response (phagocytosis, proinflammatory cytokine production) and the specific immune response (antigen processing and presentation, lymphocyte proliferation) . 'Ribomunyl' has been shown to increase the production of many cytokines {interleukin (IL)-1, IL-6, IL-8, tumour necrosis factor-alpha and colony-stimulating factor}, leading to the activation of the cytokine network . 'Ribomunyl' was also able to stimulate natural killer cells involved in viral immunity . Because of the presence of ribosomes from 4 frequently encountered bacterial strains, 'Ribomunyl' has specific immunostimulant properties . This has been clearly demonstrated in animals and humans, where specific antibody-forming B cells were found in the tonsils after oral administration . However, specific T-cell response has not been reported, suggesting that 'Ribomunyl' could act directly on B cells such as T-cell-independent bacterial antigens. Curr Microbiol, 1997 Oct, 35(4), 195 - 200 Ibuprofen inhibits arylamine N-acetyltransferase activity in the bacteria Klebsiella pneumoniae; Chung JG et al.; Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo . The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo . In vitro, the NAT activity was 0.675 +/- 0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene . In the presence of 8 mM ibuprofen, the NAT activity was 0.506 +/- 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene . In vivo, the NAT activity was 0.279 +/- 0.016 nmol/min/10(10) colony forming units (CFU) for the acetylation of 2-aminofluorene . In the presence of 8 mM ibuprofen, the NAT activity was 0.228 +/- 0.008 nmol/min/10(10) CFU for the acetylation of 2-aminofluorene . The inhibition of NAT activity by ibuprofen was shown to persist for at least 4 h . For in vitro examination, the values of apparent Km and Vmax were 1.08 +/- 0.05 mM and 9.17 +/- 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene . However, when 8 mM of ibuprofen was added to the reaction mixtures, the values of apparent Km and Vmax were 1.19 +/- 0.01 mM and 6.67 +/- 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene . For in vivo examination, the values of apparent Km and Vmax were 1.24 +/- 0.48 mM and 4.18 +/- 1.06 nmol/min/10 x 10(10) CFU, respectively, for 2-aminofluorene . However, when 8 mM of ibuprofen was added to the culture, the values of apparent Km and Vmax were 0.95 +/- 0.29 mM and 2.77 +/- 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively . This report is the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae. J Biotechnol, 1997 Aug 11, 56(2), 135 - 42 Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae; Menzel K et al.; Stoichiometric analysis of pathways involved in anaerobic bioconversion of glycerol by Klebsiella pneumoniae revealed that enzyme(s) in addition to pyruvate formate-lyase (PFL) must be involved in pyruvate decarboxylation . In this work, enzymatic evidence is presented that confirmed a simultaneous involvement of pyruvate dehydrogenase complex (PDH) and excluded the presence of pyruvate:ferredoxin oxidoreductase in this anaerobic bioprocess . The in vitro PDH activity of cell extract from continuous culture was found to be strongly affected by the substrate (glycerol) concentration in medium and cell growth rate (dilution rate) . It increases with increasing glycerol concentration and correlates well with the specific substrate uptake rate at different dilution rates in a kind of saturation function . At a similar substrate uptake rate, it decreases with cell growth rate . The in vitro activity of PDH is much higher than its in vivo activity calculated from the pathway stoichiometry but comparable to the calculated in vivo activity of PFL. Zh Mikrobiol Epidemiol Immunobiol, 1997 May-Jun, (3), 73 - 6 {The fractional composition of hydroxylamine preparations and of the lipopolysaccharide of a vaccinal strain of Klebsiella pneumoniae}; Miriasova LV et al.; The fractional composition of the vaccine preparation obtained by the hydroxylamine treatment of K . pneumoniae 204 cells was studied with the use of gel chromatography in sepharose CL-6B . The preparation was shown to form 2 peaks, only the first high molecular peak being serologically active . The method for the treatment of the culture, obtained directly from a fermenter and stored in a frozen state before treatment, was proposed . In the technology of the manufacture of Klebsiella vaccine preparation the possibility of replacing dialysis with gel chromatography was shown. Clin Diagn Lab Immunol, 1997 Sep, 4(5), 550 - 5 O-antigen seroepidemiology of Klebsiella clinical isolates and implications for immunoprophylaxis of Klebsiella infections; Trautmann M et al.; To provide a database for the development of an O-antigen-polysaccharide-containing vaccine against Klebsiella spp., we examined the O-antigen seroepidemiology of 378 Klebsiella clinical isolates collected prospectively in two university centers . Strains were typed by competitive enzyme-linked immunosorbent assay with rabbit antisera specific for serogroups O1 to O12 and monoclonal antibodies (MAbs) specific for serogroups O1, O2ab, O2ac, and the genus-specific core antigen . The numbers of isolates (percentages) of individual O serogroups were as follows: 148 (39.2) for serogroup O1, 40 (10.6) for serogroup O2ab, 4 (1.1) for serogroup O2ac, 89 (23.6) for serogroup O3, 2 (0.5) for serogroup O4, 32 (8.5) for serogroup O5, none for serogroups O7, O9, and O12, and 21 (5.6) for serogroup O11 . Forty-two (11.1) of the strains were non-O-typeable . O-serogroup distributions were virtually identical between isolates from invasive infections and those from noninvasive infections or colonizations . A vaccine containing the O-specific polysaccharides of serogroups O1, O2ab, O3, and O5 would cover 82% of clinically occurring O-antigen specificities . Three hundred thirty-eight of 378 isolates (89.4%) reacted with the genus-specific MAb V/9-5, which recognizes an epitope of the outer core region of Klebsiella lipopolysaccharide . Antibodies directed against this epitope may represent a further alternative for O-antigen-targeted immunoprophylaxis of Klebsiella infections . These data support further experimental investigations on the protective potential of O-antigen-based vaccines and/or hyperimmune globulins in Klebsiella infection. Eur J Surg, 1997 Aug, 163(8), 591 - 6 Comparative trial of four antibiotic combinations for perforated appendicitis in children; Ciftci AO et al.; OBJECTIVE: To compare the therapeutic efficacy of four antibiotic regimens: penicillin, tobramycin, and clindamycin; penicillin, tobramycin, and ornidazole; piperacillin alone; and ceftriaxone and ornidazole in the treatment of children operated on for perforated appendicitis . DESIGN: Prospective randomised study . SETTING: Teaching hospital, Turkey . SUBJECTS: 200 patients aged between 1 and 16 years treated from December 1991 to December 1995 who were randomly assigned to one of four groups each consisting of 50 patients . INTERVENTIONS: Preoperative antibiotics given intravenously, peritoneal drainage by Penrose drains without irrigation, appendicectomy with the inversion of the stump by a purse string, taking peritoneal swabs, and primary skin closure . MAIN OUTCOME MEASURES: Comparability of the groups, duration of fever, leucocytosis, antibiotic treatment, stay in hospital, nasogastric intubation, and drainage, as well as results of cultures and complications . RESULTS: There were no significant differences between the groups for any variable studied . The predominant bacterial species were Escherichia coli, Klebsiella spp, Pseudomonas spp, Fusobacteria, and Peptostreptococci which were appropriately covered by all the antibiotic regimens . Fourteen patients had complications including wound infections (n = 10), prolonged ileus (n = 2) and intra-abdominal abscess (n = 2) all of which were treated conservatively . There was no mortality and no major complications . All regimens had the same clinical and bacteriological efficacy . CONCLUSION: There is no gold standard for antimicrobial chemotherapy in perforated appendicitis . Different antibiotic combinations or a single broad spectrum antibiotic, which include both aerobic and anaerobic coverage, can safely be used in children with perforated appendicitis. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1997 Jul-Aug, 38(4), 282 - 7 Clinical experience with early enteral feeding in very-low-birth-weight infants; Wang LY et al.; The primary objective of this study was to evaluate the safety and benefit of early enteral feeding in very-low-birth-weight (VLBW) infants without parenteral nutrition . Weight gain, feeding intolerance, nosocomial infection rate and a postnatal growth curve were recorded for 61 VLBW premature infants who were admitted to the Neonatal Intensive Care Unit of Mackay Memorial Hospital from September 1, 1995 to February 28, 1997 . Nine infants were unable to complete the study and three were excluded because of severe bronchopulmonary dysplasia; therefore only 49 infants could be evaluated . They were divided into two groups based on birth weight: 1001 gm to 1250 gm (Group A, mean birth weight 1153 +/- 64 gm, mean gestational age 29.0 +/- 2.2 weeks), and less than or equal to 1000 gm (Group B, mean birth weight 911 +/- 82 gm, mean gestational age 27.1 +/- 1.5 weeks) . They received breast milk or premature formula by intermittent nasogastric or continuous nasogastric feeding . Growth was followed over the first 30 postnatal days . Group A reached 100 kcal/kg/day of enteral feeding at a mean age of 17 days as compared with a mean age of 20 days for group B . Infants regained their birth weight at 20 and 25 days in Groups A and B, respectively . By the 30th postnatal day, weight gain exceeded birth weight by 218.2 +/- 143.1 gm and 95.3 +/- 81.5 gm in groups A and B respectively . No definite episodes of necrotizing enterocolitis (NEC) developed . Two cases of Escherichia coli sepsis and one of Klebsiella sepsis occurred . The conclusion was that early enteral feeding in very-low-birth-weight infants does not increase the risk of NEC . It was also demonstrated that enteral feeding alone can produce biphasic postnatal growth curves in very-low-birth-weight infants . Although early enteral feeding was well tolerated in the study infants, the occurrence of feeding intolerance in some (36%) would suggest that additional parenteral nutrition may benefit some infants until full enteral feeding can be achieved. Pathol Biol (Paris), 1997 May, 45(5), 347 - 56 {In vivo pharmacokinetic of amikacin and its pharmacodynamic in combination with cefepime, cefpirome and meropenem in an in vitro/ex vivo micropig model}; Elkhaili H et al.; Three female Yucatan micropigs were included and received a single dose of amikacin (15 mg/kg) by short infusion (30 min) combined either with a single dose of cefepime or cefpirome (30 mg/kg/12 h) or meropenem (7 mg/kg/8 h) . The beta-lactams were administered either by intravenous intermittent injection or by continuous infusion . The mean elimination half-life and clearance value of amikacin were 1.88 h and 2.15 ml/min.kg-1 respectively . These pharmacokinetic parameters were similar to those obtained in man (t1/2 = 2,42 h et Cl = 1,61 ml/min kg-1) . Furthermore, they were not affected by coadministration of cefepime, cefpirome and to meropenem . While resistant to cefepime, cefpirome and amikacin, Klebsiella pneumoniae producing ESBL was susceptible to combination of these cephalosporins with amikacin in an in vitro/ex vivo micropig model . For the six dosage regimens used in this study, the killing activities were similar and resulted in at least 4 log decrease at 6 h after drug exposure . For antimicrobial combination consisting of bolus dosing of amikacin plus continuous infusion of cefepime or cefpirome, the 12 h serum bactericidal titers (SBTs) were 1:8 for cefepime and 1:2 for cefpirome dosage regimen . When each drug administered intermittently, the 12 h SBTs were 1:4 for cefepime and 1:2 for cefpirome . The 8 h SBTs for dosing schedule containing meropenem combined with amikacin were 1:4 and 1:16 after 30 min short infusion and continuous infusion respectively . In conclusion, our study showed that the micropig model is a reliable model for pharmacokinetic investigation of amikacin . It was concluded that beta-lactam antibiotics tested with amikacin may be coadministered by using the standard recommended dosing regimen of amikacin . Continuous infusion of beta-lactams combined with once dosing of amikacin seems to be as or more effective than intermittent injection of each drug. J Clin Microbiol, 1997 Sep, 35(9), 2365 - 9 Identification of clinical isolates of indole-positive Klebsiella spp., including Klebsiella planticola, and a genetic and molecular analysis of their beta-lactamases; Liu Y et al.; In a collection of 43 indole-positive Klebsiella clinical isolates, which were initially identified as Klebsiella oxytoca, there were 18 isolates which exhibited a pattern characteristic of extended-spectrum beta-lactamase (ESBL) resistance . This study aimed to confirm their identity by biochemical tests and by PCR and to determine the genetic basis for their resistance to the beta-lactams and broad-spectrum cephalosporins . Chromosomal beta-lactamase genes were analyzed by PCR, and plasmid-mediated beta-lactamase genes were analyzed by conjugation and transformation . There were 39 isolates which grew on melezitose but failed to grow on 3-hydroxybutyrate, confirming them as K . oxytoca . PCR analysis of their beta-lactamase genes divided these isolates into two groups, the bla(OXY-1) group and the bla(OXY-2) group . Each group had beta-lactamases with different isoelectric points; the bla(OXY-1) group had beta-lactamases with isoelectric points at 7.2, 7.8, 8.2, and 8.8, and the more common bla(OXY-2) group had beta-lactamases with pIs at 5.2, 5.4 (TEM-1), 5.7, 5.9, 6.4, and 6.8 . A pI of 5.2 was the most frequently detected and accounted for 59% of all the bla(OXY-2) beta-lactamases . Hyperproduction of clavulanate-inhibited chromosomal beta-lactamases was detected in 17 K . oxytoca isolates, resulting in an ESBL phenotype . K . oxytoca isolates having a plasmid-mediated genetic basis for their ESBL phenotype were not found, confirming that, in K . oxytoca, plasmids are rarely involved in conferring resistance to the newer cephalosporins . Four isolates proved to be isolates of K . planticola in which the beta-lactamase genes failed to react with the primers used in the PCR . One K . planticola isolate contained a transferable plasmid harboring the SHV-5 beta-lactamase gene and showed an ESBL phenotype, while the other non-ESBL K . planticola isolates contained chromosomal beta-lactamases with isoelectric points at 7.2, 7.7, and 7.9 plus 7.2. J Clin Microbiol, 1997 Sep, 35(9), 2191 - 7 Comparison of screening methods for detection of extended-spectrum beta-lactamases and their prevalence among blood isolates of Escherichia coli and Klebsiella spp . in a Belgian teaching hospital; Vercauteren E et al.; Using a set of 33 well-defined extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli and Klebsiella pneumoniae, we compared three screening methods for ESBL detection: (i) a double-disk synergy test, (ii) a three-dimensional test (both the double-disk synergy test and the three-dimensional test were performed with ceftriaxone, ceftazidime, aztreonam, and cefepime), and (iii) the Etest ESBL screen (AB Biodisk, Solna, Sweden), based on the recognition of a reduction in the ceftazidime MIC in the presence of clavulanic acid . In the double-disk test, all four indicator antibiotics scored equally and 31 of the 33 reference strains were recognized . In the three-dimensional test, ceftriaxone was the only satisfactory indicator and 30 ESBL-positive strains were detected by this antibiotic . Both systems produced two false-positive results with cefepime . With the Etest ESBL screen, 15 of 16 TEM-related and 11 of 16 SHV-related ESBL-producing strains scored positive . In 10 cases the clavulanic acid on one end of the strip interfered with the MIC determination for ceftazidime, which was read on the opposite end . This MIC had to be determined with an extra ceftazidime-only strip . No false-positive results were noted . Eighty-six blood isolates of E . coli and Klebsiella species were screened for ESBL expression by the double-disk and three-dimensional tests, both with ceftriaxone . Six strains with suspicious antibiogram phenotypes also gave positive results by the double-disk test . One E . coli strain remained undetected by the three-dimensional test . Identification of the enzymes suspected of being ESBLs by isoelectric focusing (all strains) and DNA sequencing (1 strain) confirmed the screening test results except for one Klebsiella oxytoca strain, which proved to be a hyperproducer of its chromosomal enzyme and which also had a negative Etest score . The five true ESBL producers were all confirmed by the Etest ESBL screen . Pulsed-field gel electrophoresis proved that the E . coli strains were unrelated, but that two of the three K . pneumoniae strains were closely related. J Mol Biol, 1997 Aug 22, 271(3), 342 - 8 KpnAI, a new type I restriction-modification system in Klebsiella pneumoniae; Lee NS et al.; The KpnAI restriction-modification (R-M) system has been identified in Klebsiella pneumoniae strain M5a1 . The restriction gene of KpnAI was first cloned into pBR322 using an r-m+ M5a1 derivative and phage SBS for screening . Subsequently, an adjacent DNA fragment showing modification activity was cloned into pUC19 . A total of 7.2 kb DNA sequencing data revealed three open reading frames, corresponding to hsdR, hsdM and hsdS genes of type I R-M systems . The predicted hsdR, hsdM and hsdS-coded peptides shared 95%, 98% and 44% identity, respectively, with the corresponding peptides of the recently identified StySBLI system, a prototype of the type ID family . This high homology suggests that KpnAI is also a member of the type ID family . The KpnAI system seems to be the first type I system identified in Klebsiella species. J Appl Microbiol, 1997 Aug, 83(2), 166 - 74 Formate and ethanol are the major products of glycerol fermentation produced by a Klebsiella planticola strain isolated from red deer; Jarvis GN et al.; The rumen contents of red deer (Cervus elaphus) were used to isolate bacterial capable of fermenting glycerol . The biochemistry, physiology, morphology and phylogeny of one isolate were studied in detail . The isolate (DR3) was tentatively identified as a strain of the species Klebsiella planticola as based on phenotypic characterization . The data obtained from 16S rRNA sequence analysis showed that the deer rumen isolate DR3 was 99.7% similar to the type strain of Kl . planticola (DSM 3069T), thus confirming the results of the phenotypic characterization . During cell growth, it was established that glycerol dissimilation by Kl . planticola DR3 led to the production of formate and ethanol at equimolar levels of 32 mmol 1(-1) and 30 mmol 1(-1), respectively . As a result of the data obtained, a closed carbon balance was constructed for Kl . planticola DR3 . This finding represented the first report of the complete end-product profile for glycerol dissimilation by a strain of Kl . planticola isolated from cervine rumen contents. Microbiology, 1997 Aug, 143 ( Pt 8), 2673 - 83 The Klebsiella pneumoniae cytochrome bd' terminal oxidase complex and its role in microaerobic nitrogen fixation; Juty NS et al.; Cytochrome bd' has been implicated in having an important role in microaerobic nitrogen fixation in the enteric bacterium Klebsiella pneumoniae, where it is expressed under all conditions that permit diazotrophy . In this paper the sequence of the genes encoding this terminal oxidase (cydAB) of Klebsiella pneumoniae and the characterization of a cyd mutant are reported . The deduced amino acid sequences support the proposal that His 19, His 186 and Met 393 provide three of the four axial ligands to the Fe of the three haems in the oxidase complex . The nitrogen-fixing ability of the mutant was severely impaired in the presence of low concentrations of oxygen compared with the wild-type bacterium . Only the wild-type organism was capable of microaerobic nitrogenase activity supported by fermentation products . It is proposed that formate dehydrogenase-O may be involved in supplying electrons to a respiratory chain terminated by the bd-type oxidase, which would remove inhibitory oxygen and supply ATP for nitrogenase activity. Microbiology, 1997 Aug, 143 ( Pt 8), 2521 - 30 Cadmium-specific formation of metal sulfide 'Q-particles' by Klebsiella pneumoniae; Holmes JD et al.; Klebsiella pneumoniae overcomes cadmium toxicity through the 'biotrans-formation' of cadmium ions into photoactive, nanometre-sized CdS particles deposited on the cell surface . The kinetics of particle formation during batch culture growth was monitored by electron microscopy (EM), energy-dispersive X-ray analysis and electronic absorption spectroscopy (EAS) . During the deceleration phase of bacterial growth, the presence of CdS particles on the outer cell wall of K . pneumoniae (> or = 5 nm in diameter) was detected by EM . The size of these electron-dense particles continued to increase throughout the stationary phase of growth, with some of the particles reaching a diameter > 200 nm . The formation of the extracellular CdS particles contributed to around 3-4% of the total cell biomass . EAS undertaken on these extracellular 'bio-CdS' particles suggested that the large 'superparticles' observed by EM, e.g . 200 nm, were aggregates of smaller particles termed 'Q-particles', approximately 4 nm in diameter . Metal sulfide particles were not formed in batch cultures of K . pneumoniae grown in the presence of lead, zinc, mercury, copper or silver ions . Growth in the presence of lead ions resulted in the formation of extracellular electron-dense particles containing lead but not sulfide or phosphate . Intracellular phosphorus-containing electron-opaque particles were formed during growth in the presence of copper and mercury . Intracellular electron-dense particles were formed in the presence of zinc ions but these did not contain phosphorus . From these results it was thought that metal sulfide formation in K . pneumoniae showed some cadmium-specificity . When cadmium and zinc ions were both added to the growth medium, metal sulfide particles were formed that were predominantly composed of cadmium, e.g . 48.6% cadmium and 0.04% zinc . Similarly, when cadmium and lead ions were both present during growth only CdS particles formed . In both cases analysis of the cells by EAS confirmed the presence of CdS only . These observations suggest that the mechanism of CdS formation is unlikely to occur simply through a cadmium-induced release of hydrogen sulfide by the cells into the external environment . If hydrogen sulfide production was the mechanism of sulfide formation then metal sulfide particles containing lead and zinc ions in addition to cadmium ions should have been produced. Antimicrob Agents Chemother, 1997 Aug, 41(8), 1830 - 1 Comparative in vitro activities of carbapenem L-749,345 and other antimicrobials against multiresistant gram-negative clinical pathogens; Jacoby G et al.; Carbapenems L-749,345 and imipenem had the lowest MICs at which 90% of isolates were inhibited (0.5 microg/ml) of 14 antimicrobial agents tested against 76 multiresistant gram-negative clinical isolates with TEM- or SHV-type extended-spectrum beta-lactamases and chromosomal or plasmid-determined AmpC beta-lactamases, but the MIC of L-749,345 for one isolate of Klebsiella pneumoniae was 16 microg/ml. Antimicrob Agents Chemother, 1997 Aug, 41(8), 1641 - 8 Variability of chromosomally encoded beta-lactamases from Klebsiella oxytoca; Fournier B et al.; The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2) . The two beta-lactamase groups were each represented by beta-lactamases with four different pIs . In each group, one form of beta-lactamase is more frequent than the others combined . The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 {OXY-2}, 7.1, 8.2, and 8.8 {OXY-1}) was cloned and sequenced . The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did . Comparison of K . oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis . In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions . The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group . This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration). J Bacteriol, 1997 Aug, 179(15), 4789 - 94 Cloning and expression in Escherichia coli of genetic determinants for production of and immunity to microcin E492 from Klebsiella pneumoniae; Wilkens M et al.; Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492 . The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K . pneumoniae RYC492 . The microcin E492 expressed in E . coli had the same properties as that of K . pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties . Microcin E492 expression in E . coli, like that in K . pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase . The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase . This phenomenon is not dependent on rpoS, the stationary-phase sigma factor. Gene, 1997 Jul 31, 194(2), 235 - 40 Transcriptional regulation of a second flavodoxin gene from Klebsiella pneumoniae; Achenbach LA et al.; A second flavodoxin gene, distinct from the nifF gene encoding nitrogenase flavodoxin, has been isolated from Klebsiella pneumoniae . This flavodoxin gene is a homologue of the E . coli fldA gene and is located 286 bp upstream of the K . pneumoniae fur gene . Primer extension analysis revealed an unusual promoter region upstream of the K . pneumoniae fldA gene that does not match the -35/-10 consensus sequence . Transcriptional analyses using a Fur titration assay and fldA gene fusions demonstrated that, unlike E . coli fldA, the K . pneumoniae fldA gene is not constitutively expressed . Rather, the fldA gene of K . pneumoniae is repressed in high iron conditions by the ferric uptake regulator (Fur) protein . Expression of K . pneumoniae fldA is also induced by heat shock but not by salt stress. FEMS Microbiol Lett, 1997 Jul 15, 152(2), 195 - 204 Regulation of nitrogen fixation in Azospirillum brasilense; Zhang Y et al.; The regulation of nitrogen fixation in Azospirillum brasilense is very complicated, and it responds to exogenous fixed nitrogen or a change of oxygen concentration . This regulation occurs at both transcriptional and posttranslational levels . Unlike regulation seen in Klebsiella pneumoniae, transcription of nifA does not require NTRB/NTRC in A . brasilense and the expression of nifHDK is controlled by posttranslational regulation of NIFA activity . Addition of NH4+ or a shift from microaerobic to anaerobic conditions also causes a rapid loss of nitrogenase activity in A . brasilense . This posttranslational regulation of nitrogenase activity involves the DRAT/DRAG regulatory system, which is similar to that of Rhodospirillum rubrum . Both DRAT and DRAG activities are regulated in vivo, but the mechanisms for their regulation are unknown. Carbohydr Res, 1997 Jul 11, 302(1-2), 79 - 84 The structure of the capsular polysaccharide from Klebsiella type 52, using the computerised approach CASPER and NMR spectroscopy; Stenutz R et al.; The structure of the capsular polysaccharide from Klebsiella type 52 has been elucidated using an improved and extended version of the computerised approach CASPER and NMR spectroscopy as principal methods . A previous suggestion to the structure but without the anomeric prefixes, could be shown correct {H . Bjorndal et al., Carbohydr . Res., 31 (1973) 93-100} . The polysaccharide has a hexasaccharide repeat with the following structure: {formula: see text} Br J Rheumatol, 1997 Jul, 36(7), 758 - 62 Macrophage targeting with 99mTc-labelled J001 for scintigraphic assessment of experimental osteoarthritis in the rabbit; Goupille P et al.; The potential of scintigraphy with technetium 99m-labelled J001 (99mTc-J001) to detect synovitis was studied in 15 rabbits with osteoarthritis (OA) of the right knee (section of cruciate ligaments), in five sham-operated rabbits and in four non-operated rabbits . J001 is a non-pyrogenic, acylated poly (1,3) galactoside isolated from the membrane of a non-pathogenic strain of Klebsiella pneumoniae which is able to bind selectively to macrophages via the binding to CD11b and CD14 molecules . The results of 99mTc-J001 scintigraphy were compared with those of scintigraphy with 99mTc-labelled methylene diphosphonate (99mTc-MDP) and GC-APG (a derivative of J001 unable to bind macrophages in vitro) . The mean scintigraphic ratios (diseased healthy knee) of 99mTc-J001 were significantly higher in OA rabbits than in sham- and non-operated rabbits, from as early as day 18 until day 90 . 99mTc-J001 scintigraphy demonstrated earlier increased uptake than 99mTc-MDP scintigraphy . The mean scintigraphic ratios of 99mTc-J001 were significantly higher than those of 99mTc-GC-APG (which remained normal) in OA rabbits . The normal scintigraphic ratios of 99mTc-J001 in sham-operated and non-operated rabbits, as well as of 99mTc-GC-APG in OA rabbits, suggested that the increased uptake demonstrated with 99mTc-J001 in OA rabbits, as early as day 18 corresponded to imaging of synovitis via elective macrophage targeting . These results showed that 99mTc-J001 scintigraphy should be a specific method of detecting synovitis in OA. Infect Control Hosp Epidemiol, 1997 Jul, 18(7), 492 - 8 Evidence of interhospital transmission of extended-spectrum beta-lactam-resistant Klebsiella pneumoniae in the United States, 1986 to 1993 . The National Nosocomial Infections Surveillance System; Monnet DL et al.; BACKGROUND: In addition to single-hospital outbreaks, interhospital transmission of extended-spectrum beta-lactam-resistant (ESBLR) Klebsiella pneumoniae has been suspected in some reports . However, these studies lacked sufficient epidemiological information to confirm such an occurrence . METHODS: We reviewed the surveillance data reported to the National Nosocomial Infections Surveillance (NNIS) System during 1986 to 1993 for K pneumoniae isolates and their susceptibility to either ceftazidime, cefotaxime, ceftriaxone, or aztreonam . Pulsed-field gel electrophoresis (PFGE) was used to study available ESBLR K pneumoniae isolates . RESULTS: Among 8,319 K pneumoniae isolates associated with nosocomial infections, 727 (8.7%) were resistant or had intermediate-level resistance to at least one of these antibiotics . One hospital (hospital A) accounted for 321 isolates (44.2%) of ESBLR K pneumoniae . During 1986 to 1993, the percentage of K pneumoniae isolates that were ESBLR increased from 0 to 57.7% in hospital A, from 0 to 35.6% in NNIS hospitals 0 to 20 miles from hospital A (area B), and from 1.6 to 7.3% in NNIS hospitals more than 20 miles from hospital A, including hospitals located throughout the United States . Analysis of PFGE restriction profiles showed a genetic relationship between a cluster of isolates from hospital A and some isolates from one hospital in area B, and consecutive admission in these two hospitals was confirmed for two patients from whom isolates were available . CONCLUSIONS: These data provide evidence of interhospital transmission of ESBLR K pneumoniae in one region of the United States and stress the interrelationship between hospitals when trying to control antimicrobial resistance. FEMS Microbiol Lett, 1997 Jul 1, 152(1), 163 - 7 Substitution of alanine for aspartate at position 179 in the SHV-6 extended-spectrum beta-lactamase; Arlet G et al.; SHV-6 was previously identified by its susceptibility pattern and biochemical criteria in a clinical isolate of Klebsiella pneumoniae which was resistant to ceftazidime . It contains only a single point difference with the beta laSHV-1 gene as determined by PCR amplification and nucleotide sequencing . This is the result of a single amino acid substitution, Ala for Asp, at position 179 . Directed mutagenesis experiments have shown this substitution to confer selective resistance to ceftazidime in the TEM family. J Bacteriol, 1997 Jul, 179(14), 4623 - 6 Evidence for two possible glnB-type genes in Herbaspirillum seropedicae; Benelli EM et al.; Two glnB-like genes have been isolated from Herbaspirillum seropedicae by complementation of the Klebsiella pneumoniae glnB502 mutant for growth on nitrate . One of these glnB-like genes has been sequenced and shows strong identity with GlnB proteins derived from other organisms . A Tn5-20 mutation of this glnB was Nif negative. J Bacteriol, 1997 Jul, 179(13), 4081 - 6 Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease; Moncrief MB et al.; In vivo urease metallocenter assembly in Klebsiella aerogenes requires the presence of several accessory proteins (UreD, UreF, and UreG) and is further facilitated by UreE . In this study, UreG was isolated and shown to be a monomer with an Mr of 21,814 +/- 20 based on gel filtration chromatography and mass spectrometric results . Although it contains a P-loop motif typically found in nucleotide-binding proteins, UreG did not bind or hydrolyze ATP or GTP, and it exhibited no affinity for ATP- and GTP-linked agarose resins . Site-directed mutagenesis of ureG allowed the substitution of Ala for Lys-20 or Thr-21 in the P-loop motif and resulted in the production of inactive urease in cells grown in the presence of nickel; hence, an intact P-loop may be essential for UreG to function in vivo . These mutant cells were unable to synthesize the UreD-UreF-UreG-urease apoprotein species that are thought to be the key urease activation complexes in the cell . An insoluble protein species containing UreD, UreF, and UreG (termed the DFG complex) was detected in cells carrying deletions in ureE and the urease structural genes . The DFG complex was solubilized in 0.5% Triton X-100 detergent, shown to bind to an ATP-linked agarose resin, and found to elute from the resin in the presence of Mg-ATP . In cells containing a UreG P-loop variant, the DFG complex was formed but did not bind to the nucleotide-linked resin . These results suggest that the UreG P-loop motif may be essential for nucleotide binding by the DFG complex and support the hypothesis that nucleotide hydrolysis is required for in vivo urease metallocenter assembly. Biochemistry, 1997 Jul 1, 36(26), 8164 - 72 Structures of Cys319 variants and acetohydroxamate-inhibited Klebsiella aerogenes urease; Pearson MA et al.; Cys319 is located on a mobile flap covering the active site of Klebsiella aerogenes urease but does not play an essential role in catalysis . Four urease variants altered at position C319 range from having high activity (C319A) to no measurable activity (C319Y), indicating Cys is not required at this position, but its presence is highly influential {Martin, P . R., & Hausinger, R . P . (1992) J . Biol . Chem . 267, 20024-20027} . Here, we present 2.0 A resolution crystal structures of C319A, C319S, C319D, and C319Y proteins and the C319A variant inhibited by acetohydroxamic acid . These structures show changes in the hydration of the active site nickel ions and in the position and flexibility of the active site flap . The C319Y protein exhibits an alternate conformation of the flap, explaining its lack of activity . The changes in hydration and conformation suggest that there are suboptimal protein-solvent and protein-protein interactions in the empty urease active site which contribute to urease catalysis . Specifically, we hypothesize that the suboptimal interactions may provide a significant source of substrate binding energy, and such hidden energy may be a common phenomenon for enzymes that contain mobile active site loops and undergo an induced fit . The acetohydroxamic acid-bound structure reveals a chelate interaction similar to those seen in other metalloenzymes and in a small molecule nickel complex . The inhibitor binding mode supports the proposed mode of urea binding . We complement these structural studies with extended functional studies of C319A urease to show that it has enhanced stability and resistance to inhibition by buffers containing nickel ions . The near wild-type activity and enhanced stability of the C319A variant make it useful for further studies of urease structure-function relationships. J Mol Biol, 1997 Jun 27, 269(5), 719 - 31 In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A + T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniae; Meyer M et al.; The genes specifically required for citrate fermentation in Klebsiella pneumoniae form a cluster on the chromosome consisting of two divergently transcribed groups, citCDEFG and citS-oadGAB-citAB . Northern blot analyses described here and elsewhere indicate that each group forms an operon . The transcriptional start sites of citC and citS, which were mapped in this work by primer extension, are separated by a stretch of 193 bp with an extraordinary high A + T content of 67% . Expression of the citrate fermentation genes was recently shown to be positively controlled by a two-component signal transduction system encoded by the promoter-distal genes of the citS operon, citA (sensor kinase) and citB (response regulator) . As a first step towards the functional characterization of CitB, we analysed its DNA-binding properties . To this end, the entire CitB, its N-terminal receiver domain (CitBN), and its C-terminal output domain (CitBC), all modified by a (His)6-tag, were purified . CitB(His) and CitBN(His) could be phosphorylated either with acetylphosphate or with ATP plus MalE-CitAC . The latter protein contains the kinase domain of CitA fused to the C terminus of the maltose-binding protein . Upon phosphorylation, CitB(His) became more resistant towards limited proteolysis by trypsin, reflecting substantial changes in tertiary structure . In gel retardation assays, CitB(His) bound specifically to the citC-citS intergenic region . The retardation pattern changed significantly upon phosphorylation and the apparent binding affinity increased 10 to 100-fold . Depending on the protein concentration, four different phospho-CitB(His)-DNA complexes could be resolved, suggesting the presence of multiple binding sites between citC and citS . DNase I footprints revealed two protected regions extending maximally from -55 to -89 relative to the citS transcription start and from -50 to -96 relative to the citC transcription start . Gel retardation and DNase I footprint assays with CitBC(His) showed that the C-terminal domain is sufficient for specific DNA binding . Since its properties were similar to that of unphosphorylated CitB(His), an essential role of the N-terminal receiver domain in high-affinity DNA binding was indicated . The positions of the binding sites for CitB and of putative recognition sequences for the cAMP receptor protein suggested a model for the interaction of these activators with RNA polymerase. Indian J Med Res, 1997 Jun, 105, 262 - 5 Anaerobic flora in endodontic infections; Chaudhry R et al.; Microbiological and clinical data from 56 patients with endodontic infections were evaluated . Samples were collected using autoclaved paper points . Specimens were processed for isolation of aerobic and anaerobic bacteria . Antimicrobial sensitivity and resistance profiles of the recovered isolates was also performed . Forty nine positive cultures (87.5%) were obtained from the 56 consecutive necrotic root canal systems which were sampled . A total of 69 aerobic bacteria and 21 anaerobic bacteria were recovered . Aerobic bacteria were isolated from 35 patients (72%), anaerobic bacteria from 3 (6%) and mixed aerobic and anaerobic bacteria from 11 patients (22%) . The most common aerobic isolate was Klebsiella pneumoniae . The predominant anaerobic isolate was Bacteroides species . One isolate was recovered from 25 patients (51%) whereas in the remaining 24 patients (49%) more than 1 isolate were recovered . These data illustrate the polymicrobial nature of endodontic infections in half the patients studied and the role of anaerobic bacteria in a quarter of them. J Antimicrob Chemother, 1997 Jun, 39(6), 737 - 45 Rarity of transferable beta-lactamase production by Klebsiella species; Leung M et al.; We report a survey of beta-lactamases and their transferability in Klebsiella spp . isolated from blood during 1992-95 . beta-Lactamases were characterized by determination of isoelectric point (pI), by hybridization of plasmid DNA preparations with probes for SHV and TEM sequences and by PCR with SHV- or TEM-specific primers . There were 80 isolates of Klebsiella pneumoniae and 22 isolates of Klebsiella oxytoca . Most isolates of K . pneumoniae had a chromosomally encoded SHV-1 beta-lactamase (or a closely related enzyme); K . oxytoca also produced chromosomal beta-lactamases, but these were distinct from SHV-1 . Plasmid-encoded beta-lactamases were rare in Klebsiella spp., being found in six (7.5%) isolates of K . pneumoniae and in none of the K . oxytoca . beta-Lactamase activities were relatively low (< 100 nmoles nitrocefin hydrolysed per minute per mg of protein) and ampicillin MICs were < or = 128 mg/L for most isolates of both species . However, all isolates of K . pneumoniae with plasmid-encoded beta-lactamases, three other isolates of K . pneumoniae and three isolates of K . oxytoca had high beta-lactamase activities (> 100 nmoles/mg/min) and very high ampicillin MICs (> or = 1024 mg/L). Eur J Biochem, 1997 Jun 1, 246(2), 530 - 8 Sequence of a gene cluster from Klebsiella pneumoniae encoding malonate decarboxylase and expression of the enzyme in Escherichia coli; Hoenke S et al.; Malonate decarboxylase of Klebsiella pneumoniae consists of four different subunits and catalyzes the conversion of malonate plus H+ to acetate and CO2 . The catalysis proceeds via acetyl and malonyl thioester residues with the phosphribosyl-dephospho-CoA prosthetic group of the acyl carrier protein (ACP) subunit . From a cosmid library of K . pneumoniae, a gene cluster of 9 kb has been isolated and sequenced that included the structural genes for the malonate decarboxylase . The cluster consisted of the eight consecutive genes mdcABCDEFGH and the divergently oriented mdcR gene . The intergenic regions were short (usually < 17 bp, 136 bp between mdcE and mdcF) and ribosome binding sites were found 4-10 bp before each gene . According to N-terminal protein sequencing, the mdcA, C, D and E genes encoded subunits alpha, delta, beta and gamma of malonate decarboxylase . Data bank searches for related proteins with known function revealed that MdcA represents the ACP-transferase and that MdcD and E together probably function as malonyl-S-ACP decarboxylase . MdcC is the (apo) ACP subunit . MdcB and MdcG could be involved in the synthesis and attachment of the prosthetic group . MdcH is similar to various malonyl-CoA:ACP-SH transacylases and therefore probably involved in the initial activation of the enzyme by malonylation . MdcF is a membrane protein that could function as a malonate carrier . The mdcR gene encodes a protein of the LysR regulator family . Malonate decarboxylase was functionally expressed in Escherichia coli from plasmids harbouring the entire gene cluster including mdcR . As partial deletion of the mdcR gene impaired growth of the transformants on malonate, MdcR is probably a transcriptional regulator of the mdc genes. Eur J Biochem, 1997 Jun 1, 246(2), 311 - 9 Comparative in-vivo and in-vitro 99Mo-time-differential-perturbed-angular-correlation studies on the nitrogenase MoFe protein and on other Mo species of different N2-fixing bacteria; Muller A et al.; Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing 99MoO4(2-) . The distribution of 99Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion-exchange (DEAE-Sephacel) chromatography . Cells of K . pneumoniae took up MoO4(2-) only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kp1) as the predominant Mo-containing species . In the case of A . vinelandii, under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Av1) . However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo-storage protein . In the presence of 20 mM NH4+, conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo-storage protein exclusively . This protein proved to be unstable towards DEAE-Sephacel, apparently releasing all the molybdenum in form of MoO4(2-) during the fractionation procedure . R . capsulatus contained, in addition to the MoFe protein (Rc1), significant amounts of other not-yet-identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase . The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process 99Mo(beta-)99Tc using time differential perturbed angular correlation spectroscopy . The quadrupole coupling constant (v(Q)) determined for the Mo center in MoFe proteins was consistently in the range 66-81 MHz . The values of the coupling constants determined with intact cells and with the isolated, partially purified, MoFe proteins were in very good agreement . For the Mo-storage protein of A . vinelandii, a quadrupole coupling constant of approximately 180 MHz was determined by measurements performed with nitrogenase-repressed cells as well as with gel-filtered cell-free extracts . Our work proves that the relevant study of hyperfine interactions allows the identification of the MoFe protein and also other Mo proteins in vivo as well as in vitro. J Clin Microbiol, 1997 Jun, 35(6), 1499 - 503 Relationship between adhesion to intestinal Caco-2 cells and multidrug resistance in Klebsiella pneumoniae clinical isolates; Di Martino P et al.; Klebsiella pneumoniae is an opportunistic gram-negative pathogen involved in outbreaks of nosocomial infections in intensive care units . Strains are resistant to multiple antibiotics, and 15 to 30% of them are also resistant to the broad-spectrum cephalosporins by the production of R plasmid-encoded extended-spectrum beta-lactamases . Because the gastrointestinal tracts of patients have been shown to be the reservoir for nosocomial strains of K . pneumoniae, we looked for a correlation between antibiotic resistance and adhesion of K . pneumoniae strains to intestinal cells . We investigated adhesion to the human intestinal epithelial Caco-2 cell line of 61 clinical K . pneumoniae strains isolated in hospitals in Clermont-Ferrand, France . None of the strains tested expressed the previously described adhesive factors CF29K and KPF-28 . Adhesive properties were found for 42.6% of the strains tested (26 strains) . Just 7.7% (2 strains) of the 26 strains producing only the chromosomally encoded SHV-1 beta-lactamase adhered to the Caco-2 cell line, whereas 68.5% (24 strains) of the 35 strains producing a plasmid-encoded beta-lactamase were adherent . All the adherent strains, and even the two strains producing only the SHV-1 enzyme, harbored at least one self-transmissible R plasmid . At variance for CAZ-1/TEM-5 or CAZ-5/SHV-4 beta-lactamase-producing K . pneumoniae strains, curing and mating experiments demonstrated that the self-transmissible R plasmids encoding the TEM-1, CTX-1/TEM-3, CAZ-2/TEM-8, CAZ-6/TEM-24, or CAZ-7/TEM-16 beta-lactamase were not involved in the adhesion of K . pneumoniae strains to intestinal epithelial cells . Nevertheless, there was an association of multiple antibiotic resistance, including resistance to extended-spectrum cephalosporins, and adhesive properties in K . pneumoniae clinical isolates. Arch Biochem Biophys, 1997 May 15, 341(2), 201 - 6 Purification and characterization of a phytase from Klebsiella terrigena; Greiner R et al.; A cytoplasmatic phytase was purified about 410-fold to apparent homogeneity with a recovery of 28% . The enzyme is induceable under carbon limitation in the presence of phytate . It behaves as a monomeric protein of a molecular mass of about 40 kDa . The phytase is rather specific for phytate and exhibits optimal conditions for phytate degradation at pH 5.0 and 58 degrees C . Kinetic parameters for the hydrolysis of Na phytate are KM 300 microM and kcat 180 s-1 at 35 degrees C and pH 5.0 . Phytate is hydrolyzed in a stepwise manner; the penta- and tetrakisphosphate were identified as I(1,2,4,5,6)P5 and I(1,2,5,6)P4 . Consequently, this enzyme is a 3-phytase (EC 3.1.3.8). Biosci Biotechnol Biochem, 1997 May, 61(5), 768 - 71 Oxygen sensitivity of NifA protein of Azospirillum lipoferum FS as suggested by gene cloning and expression in Escherichia coli; Shigematsu T et al.; We cloned and sequenced a 2.8-kb SalI fragment of Azospirillum lipoferum FS as a homologue of the Klebsiella oxytoca nifA gene . The amino acid sequence deduced from an open reading frame of 1872 bases showed 91% identity to that of the A . brasilense NifA, and the putative central sigma54 interaction domain was conserved as well as the C-terminal DNA-binding domain . The NifA function on the nifH promoter was examined in Escherichia coli using a combination of a nifA driver plasmid and a nifH-lacZ reporter plasmid, in which the transcriptional activation of the nifH promoter by the NifA was evaluated with the beta-galactosidase activity . The A . lipoferum NifA activated the nifH promoter solely under microaerobic conditions, while the K . oxytoca NifA activated it irrespective of the oxygen condition . These observations suggest that oxygen sensitivity is an intrinsic property of the A . lipoferum NifA. Clin Exp Rheumatol, 1997 May-Jun, 15(3), 269 - 74 Macrophage targeting with 99mTc-labelled J001 for scintigraphy of joint inflammation in ovalbumin-induced arthritis in rabbits; Goupille P et al.; BACKGROUND AND OBJECTIVE: J001 scintigraphy is a new approach, based on macrophage targeting, developed for tumor and inflammation imaging . J001, a non-pyrogenic acylated poly(1,3)galactoside purified from the membrane of a non-encapsulated strain of Klebsiella pneumoniae associates selectively with macrophages via binding to CD11b and CD14 molecules . Since macrophages play a primary role in inflammatory arthritis processes, J001 labeled with 99mTc appeared to be of interest for the scintigraphic imaging of inflammatory lesions . The purpose of this study was to assess the potential of J001 scintigraphy for imaging inflammatory arthritis in the model of ovalbumin-induced arthritis in rabbits . MATERIAL AND METHODS: Ovalbumin-induced arthritis was developed in 17 rabbits . 99mTc-J001 scintigraphy was performed 4 weeks after arthritis induction in 17 rabbits and was repeated at 6 and 8 weeks in 8 rabbits . 99mTc-J001 and 99mTc-MDP scintigraphy were performed before and 2.5 months after radionuclide synovectomy with the intra-articular injection of a high energy beta-emitting radionuclide (186Re) in 3 rabbits and 186Re (first subjected to a complete decrease of radioactivity) in 3 rabbits . RESULTS: 99mTc-J001 scintigraphy was able to image inflammatory arthritis 4 weeks after induction . J001 scintigraphy demonstrated an increased uptake earlier than MDP, which was maintained at week 8 . After radionuclide synovectomy, a clear decrease in the J001 scintigraphy ratio occurred, whereas the MDP scintigraphy ratio was stable . After the intra-articular injection of inactive 186Re, no changes in MDP and J001 scintigraphy ratio appeared . CONCLUSION: 99mTc-J001 scintigraphy is able to image joint inflammation and to assess the response to anti-inflammatory treatment in an experimental model of arthritis. J Hosp Infect, 1997 May, 36(1), 23 - 36 Epidemiological typing of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates responsible for five outbreaks in a university hospital; Branger C et al.; Thirty-seven isolates of extended-spectrum beta-lactamase-producing (ESBL) Klebsiella pneumoniae implicated in five nosocomial outbreaks (I-V) on three distinct wards of our hospital were compared using capsular typing, biotyping, antibiotyping, enzyme electrophoresis typing and DNA macrorestriction analysis with Xba I resolved by pulsed-field gel electrophoresis . The isolates from each outbreak had common phenotypic and genotypic characteristics indicating that they were related epidemiologically . Isolates from outbreaks I (four patients) and V (13 patients), although they occurred in two different wards (neurology and surgery) and three years apart, produced the same ESBL with a pI of 7.8 (SHV-4) and were of serotype K25 . The Xba I patterns were closely related . The isolates of outbreaks II (seven patients), III (four patients) and IV (seven patients), which occurred in a single surgical intensive care unit, produced an ESBL with a pI of 6.3 (TEM-3) . Isolates from outbreaks III and IV, which occurred six months apart, were of serotype K68 and had similar Xba I patterns suggesting that the two outbreaks were due to a single strain which persisted endemically in the ward . The isolates from outbreak II were of serotype K62, and had distinct characteristics from the two later outbreaks . The Xba I patterns of the isolates from outbreaks "I and V', II and "III and IV' had Dice similarity coefficients under 40% showing that the three groups were genetically distant . DNA macrorestriction analysis was a useful complement to phenotypic methods for identifying K . pneumoniae strains responsible for outbreaks harbouring a common ESBL. Antimicrob Agents Chemother, 1997 May, 41(5), 1053 - 7 Efficacies of piperacillin-tazobactam and cefepime in rats with experimental intra-abdominal abscesses due to an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae; Thauvin-Eliopoulos C et al.; The in vivo activities of piperacillin-tazobactam and cefepime were compared with those of ticarcillin-clavulanate, ceftazidime, cefotaxime, and imipenem in a rat model of intra-abdominal abscess with a strain of Klebsiella pneumoniae elaborating an extended-spectrum beta-lactamase (TEM-26) . With the exception of ceftazidime, all of the antimicrobial agents significantly reduced bacterial counts within abscesses at the end of therapy compared with those in untreated controls . Residual viable cell counts (mean +/- standard deviation in log10 CFU/gram) were as follows: control, 8.76 +/- 0.97; ceftazidime, 8.00 +/- 0.76; piperacillin-tazobactam, 3.87 +/- 1.72; ticarcillin-clavulanate, 3.74 +/- 1.34; cefepime, 3.15 +/- 1.19; cefotaxime, 2.61 +/- 0.77; imipenem, 2.41 +/- 0.93 . Imipenem was more effective than either of the inhibitor combinations (P < 0.05) . Cefotaxime was unexpectedly effective given its poor in vivo activity against this organism in our earlier studies, which used a different dose and total duration of therapy (L . B . Rice, J . D . C . Yao, K . Klimm, G . M . Eliopoulos, and R . C . Moellering, Jr., Antimicrob . Agents Chemother . 35:1243-1244, 1991) . These observations suggest that the effectiveness of cephalosporins in the treatment of experimental infections caused by extended-spectrum beta-lactamase-producing K . pneumoniae may be highly dependent on dosing regimens, even for a specific organism and site of infection. Infect Immun, 1997 May, 65(5), 1870 - 5 Nitric oxide is required for effective innate immunity against Klebsiella pneumoniae; Tsai WC et al.; Nitric oxide (NO) has been associated with protection against various parasitic and viral infections and may play a similar role in bacterial infections . We studied the role of NO in host defense against Klebsiella pneumoniae infection in the lung . Initial studies demonstrated a time-dependent increase in NO production of the lungs of CBA/J mice following the intratracheal administration of K . pneumoniae (7 x 10(2) CFU) . To assess the role of NO in Klebsiella pneumonia, mice were treated intraperitoneally with either L-NAME (N-omega-nitro-L-arginine methylester), a competitive inhibitor of NO synthesis, or D-NAME, an inert enantiomer . The treatment of Klebsiella-infected mice with L-NAME resulted in a 10- and 46-fold increase in K . pneumoniae CFU in lungs and blood, respectively, at 48 h post-K . pneumoniae inoculation compared to treatment of mice with D-NAME . In addition, a greater-than-twofold increase in mortality was evident in L-NAME-treated mice compared to the mortality in control animals . No significant difference in bronchoalveolar lavage inflammatory cell profiles was noted between L-NAME- and D-NAME-treated mice with Klebsiella pneumonia . Interestingly, increased levels of tumor necrosis factor, gamma interferon, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-2 mRNA and protein were noted in infected mice treated with L-NAME compared to the levels in mice treated with D-NAME . Importantly, the in vitro incubation of murine alveolar macrophages with L-NAME, but not with D-NAME, resulted in a significant impairment in both the phagocytosis and killing of K . pneumoniae . In total, these results suggest that NO plays a critical role in antibacterial host defense against K . pneumoniae, in part by regulating macrophage phagocytic and microbicidal activity. Infect Immun, 1997 May, 65(5), 1754 - 60 Protective effect of antilipopolysaccharide monoclonal antibody in experimental Klebsiella infection; Rukavina T et al.; An O-antigen-specific murine monoclonal antibody (MAb) directed against an immunodominant epitope expressed on Klebsiella O1, O6, and O8 lipopolysaccharides (LPS) was examined with respect to its binding to nonencapsulated and encapsulated bacterial cells and its ability to protect against lethal murine Klebsiella sepsis . While the MAb (clone Ru-O1, mouse immunoglobulin G2b) bound well to nonencapsulated organisms of the O1 serogroup, binding was significantly, but not completely, abolished by the presence of the K2 capsule . In a model of experimental Klebsiella peritonitis and sepsis induced by a virulent O1:K2 serogroup strain, higher doses of anti-LPS MAb Ru-O1 than of a previously described anticapsular MAb specific for the K2 capsular polysaccharide were needed to provide protection . However, high-dose (40 microg/g of body weight) pretreatment with anti-LPS MAb Ru-O1 significantly reduced bacterial dissemination to various organs as well as macroscopic and histologic pulmonary alterations . Thus, since the number of Klebsiella capsular antigens occurring in clinical material is too large to be completely "covered" by a K-antigen-specific hyperimmunoglobulin preparation, O-antigen-specific antibodies may supplement K-antigen-specific immunoprophylaxis and -therapy of clinical Klebsiella infection. Proc Natl Sci Counc Repub China B, 1997 Apr, 21(2), 37 - 42 The role of nitrogenase in a cyanide-degrading Klebsiella oxytoca strain; Liu JK et al.; It is well known that the major function of nitrogenase is to fix atmospheric nitrogen . However, cyanide can also serve as a subtrate for nitrogenase and can be reduced to CH4 and NH4+ . A cyanide-degrading Klebsiella oxytoca strain was isolated from cyanide contaminated water . This isolate was also found to have a nitrogen-fixation capability . Nitrogenase activities in this organism could be induced by KCN . However, there was no significant difference of the induction effect between 1 mM KCN and 5 mM KCN . It was found that the cyanide-degrading ability of this isolate could be inhibited by multicopy hybrid pGR112 nif-containing plasmids . Comparing the wild type K . oxytoca strain with the pGR112 plasmid transformed strain, a typical diauxic growth of the wild type strain was observed in a medium containing NH4Cl and KCN . Although the nif plasmid transformed strain also exhibited diauxic growth in the same medium, a much longer second lag phase was noted . In addition, methane, the nitrogenase reduction end product of cyanide, could be detected on cyanide-containing growth cultures . Ammonium chloride, a repressor of nitrogenase gene expression, was consumed prior to KCN in both strains . Again, the degradation of KCN in the pGR112 transformed strain occurred only under loose control of the nitrogenase gene . These findings strongly suggest that nitrogenase may be the sole cyanide-degrading enzyme in this organism. Pharmacol Res, 1997 Apr, 35(4), 267 - 72 Immunopharmacological activity of aporphinoid alkaloid oxoglaucine; Ivanovska N et al.; The ability of aporphinoid alkaloid oxoglaucine to influence T- and B-cell immune response was studied in mice models . The substance inhibited in vitro mitogen-induced lymphocyte proliferation and suppressed antibody response to sheep red blood cells (SRBC) and lipopolysaccharide (LPS) in vivo effectively . The action depended on the relative timing of antigen and oxoglaucine administration . The substance manifested stimulatory effect in popliteal lymph node (PLN) reaction and LPS-induced B-cell activation . In the chronic inflammatory model of adjuvant arthritis oxoglaucine exhibited stimulatory or suppressive action related to the kinetics of the process . At low doses (1 or 2 mg kg-1) oxoglaucine improved the outcome of Klebsiella pneumoniae infection, while at higher doses (10 or 20 mg kg-1) the substance caused an impairment of host resistance to infectious agent . The comparison with cyclophosphamide in some tests showed that oxoglaucine was effective in manifold lower doses . In conclusion, oxoglaucine exerted immunomodulatory effects in vivo in a dose-dependent and protocol-dependent manner . Yet, its overall action might be attributed to the different sensitivity of the cells involved in the developing immune response. J Clin Pathol, 1997 Apr, 50(4), 352 - 3 Inflammatory pseudotumour of the liver; Kafeel G et al.; Inflammatory pseudotumour is not a common lesion . The first series of 12 cases was described in 1986, to which 37 more cases have now been added . The histology, differential diagnosis, and prognosis of this lesion have been described in detail, but the aetiology is unknown and the mode of treatment remains controversial . A new case is presented and compared with the previously reported cases . Fine needle aspirate yielded a growth of klebsiella organisms . The possibility of this infection as an aetiological agent is considered. Indian J Med Res, 1997 Apr, 105, 158 - 61 Extended spectrum beta-lactamase mediated resistance to third generation cephalosporins in Klebsiella pneumoniae in Nagpur, central India; Hansotia JB et al.; Out of 66 clinical isolates of Klebsiella pneumoniae, 17 showed resistance or decreased susceptibility to third generation cephalosporins (17 to cefotaxime, 16 to ceftriaxone, and 9 to ceftazidime) while the remaining 49 were sensitive by the disc diffusion method . The minimum inhibitory concentrations (MICs) of the third generation cephalosporins (3GC) for the strains ranged from 2-128 micrograms/ml by agar dilution method . Their sensitive phenotypes had zone diameters smaller (mean difference 3 . 1 mm for ceftriaxone, and 6.5 mm for ceftazidime), and MICs > 10 fold higher than the corresponding values in the fully sensitive isolates . Resistance to cefotaxime was transferred to recipient Escherichia coli K12 strain in 15 isolates . All the resistant isolates were sensitive to imipenem but were variably sensitive to aminoglycosides, and quinolones . In all 17 resistant isolates extended spectrum beta-lactamase (ES beta L) was detected . The sensitivity testing systems may fail to recognise the potential ES beta L mediated resistance to 3GC . Hence ES beta L detection should be routinely undertaken. Infect Immun, 1997 Apr, 65(4), 1139 - 46 Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival; Broug-Holub E et al.; To study the in vivo role of alveolar macrophages (AM) in gram-negative bacterial pneumonia in mice, AM were eliminated by the intratracheal (i.t.) administration of dichloromethylene diphosphonate encapsulated liposomes . Subsequently, the AM-depleted mice were infected i.t . with 100 CFU of Klebsiella pneumoniae, and the effects of AM depletion on survival, bacterial clearance, and neutrophil (polymorphonuclear leukocyte {PMN}) recruitment were assessed . It was shown that depletion of AM decreases survival dramatically, with 100% lethality at day 3 postinfection, versus 100% long-term survival in the control group . This increased mortality was accompanied by 20- to 27- and 3- to 10-fold increases in the number of K . pneumoniae CFU in lung and plasma, respectively, compared to those in nondepleted animals . This decreased bacterial clearance was not due to an impaired PMN recruitment; on the contrary, the K . pneumoniae-induced PMN recruitment in AM-depleted lungs was sevenfold greater 48 h postinfection than that in control infected lungs . Together with an increased PMN infiltration, 3- and 10-fold increases in lung homogenate tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) levels, respectively, were measured . Neutralization of TNF-alpha or MIP-2, 2 h before infection, reduced the numbers of infiltrating PMN by 41.6 and 64.2%, respectively, indicating that these cytokines mediate PMN influx in infected lungs, rather then just being produced by the recruited PMN themselves . Our studies demonstrate, for the first time, the relative importance of the AM in the containment and clearance of bacteria in the setting of Klebsiella pneumonia. Biochem J, 1997 Mar 15, 322 ( Pt 3), 737 - 44 Covalent modification of nitrogenase MoFe protein by ADP; Miller RW et al.; MgADP- reacted with the nitrogenase molybdenum-iron (MoFe) protein of Klebsiella pneumoniae (Kp1) over a period of 2 h to yield a stable, catalytically active conjugate . The isolated protein exhibited a new, broad 31P NMR resonance at -1 p.p.m . lacking phosphorus J coupling . The adenine ring of {8-14C}ADP remained associated with the conjugate . A covalently bound nucleotide was identified as AMP by NMR and TLC . Extended dialysis of Kp1 against MgADP- resulted in further AMP binding at the protein surface . ADP was initially bound tightly to Kp1 at a site distinct from the AMP sites . ATP did not replace ADP . The time course of the formation of the Kp1-AMP was altered by the nitrogenase iron protein (Kp2) and was dependent on redox potential . Kp1-AMP was stable to concentration and oxidation with ferricyanide ion at -350 mV . Slow hydrolysis of Kp1-AMP over a period of 6 h yielded AMP and unaltered Kp1 . The adenine ring of ADP exchanged with adenine of MgATP2- during reductant-limited turnover of nitrogenase under N2, indicating reversibility of ATP hydrolysis at 15 degrees C . {32P}Pi exchanged with the terminal phosphate group of both ADP and ATP on incubation with Kp1 . 32P exchange and the catalytic activity of Kp1 were inhibited by a 20-fold molar excess of the lysine-modifying reagent, o-phthalaldehyde (OPT) . Preincubation with MgADP- protected against OPT inactivation . Two potentially reactive lysine residues on the alpha chain of the MoFe protein near a putative hydrophobic docking site for the nitrogenase Fe protein are proposed as sites of OPT and nucleotide binding . Azotobacter vinelandii MoFe protein (Av1) also formed an AMP adduct but Kp2 did not . Catalase did not interact with ADP . The reactions of the nitrogenase MoFe protein with adenine nucleotides have no counterpart in known protein-nucleotide interactions. J Mol Biol, 1997 Mar 7, 266(4), 642 - 8 The first glimpse of a complex of nitrogenase component proteins by solution X-ray scattering: conformation of the electron transfer transition state complex of Klebsiella pneumoniae nitrogenase; Grossman JG et al.; An essential feature of the mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, is the formation of a transient electron transfer complex between the MoFe protein containing the active site at which N2 is reduced, and the Fe protein, which functions as a specific electron donor to the MoFe protein . We have obtained high quality solution X-ray scattering data using synchrotron X-rays of a stable putative electron transfer complex, (MoFe-protein)(Fe-protein.ADP.AIF4)2, of Klebsiella pneumoniae and used the model-independent approach based on the multipole expansion method to provide a stable and unique shape restoration at approximately 15 A resolution . The biological significance of this first molecular structure of a nitrogenase complex is discussed. Arch Microbiol, 1997 Mar 7, 167(2/3), 160 - 6 Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae Gibello A, Suarez M, Allende JL, Martin M. The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced . K . pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses . HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da . HpaH, with a molecular mass of 18,680 Da, seems to be a "helper" protein required for productive hydroxylation of the substrate . The hpa genes were expressed and the hydroxylase was active in Escherichia coli . Comparison of the enzyme with other monooxygenases indicates that K . pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases. Burns, 1997 Mar, 23(2), 166 - 9 Controlled mechanical hypoventilation in a paediatric burn patient as treatment of acute respiratory distress syndrome; Trop M et al.; The paediatric patient we are describing suffered a scald injury covering 83 per cent of the total body surface area (TBSA) . This injury was complicated by Klebsiella pneumoniae septicaemia resulting in multiorgan failure (MOF) . Acute respiratory distress syndrome (ARDS), gastrointestinal insufficiency, hepathopathy and wound conversion to full thickness posed the main problems . The boy was ventilated with pressure-controlled mechanical ventilation . The concept of permissive hypercapnia (PHC) resulted in a complete resolution of ARDS within 4 weeks . From our experience, further lung injury among infants and children suffering from severe ARDS can be avoided by using controlled mechanical hypoventilation . It is a simple and safe technique that allows adequate oxygenation. Can J Physiol Pharmacol, 1997 Mar, 75(3), 205 - 7 Citrulline malate limits increase in muscle fatigue induced by bacterial endotoxins; Goubel F et al.; Citrulline malate is known to improve performance in weakened muscles . The present experiment was designed to test the hypothesis that citrulline malate can limit the effect of endotoxins on muscle fatigability . Endotoxemia was induced in rats by injection of lipopolysaccharides from Klebsiella pneumoniae . Resistance to fatigue was quantified by measuring tension production during repetitive electrical stimulation of the isolated epitrochlearis muscle . Oral treatment by citrulline malate was found to increase resistance to fatigue in infected rats, whereas twitch tension was not modified . This demonstrates the efficacy of citrulline malate for limiting an increase in muscle fatigue elicited with bacterial endotoxins. Epidemiol Mikrobiol Imunol, 1997 Mar, 46(1), 18 - 22 {Subspecific classification of 11 clinical strains of Klebsiella pneumoniae based on their biochemical, antibiotic and plasmid profiles}; Seman M et al.; The paper summarizes at a subspecific level 11 clinical strains of K . pneumoniae . The objective of the work was to determine in a simple and effective way differences between different strains of the mentioned taxon . Biochemical characteristics, antibiogram and part of the plasmid spectrum were used for assessment of inter-species differences between different strains and at the same time their use as simple markers of epidemiological analyses is presented. Chemotherapy, 1997 Mar-Apr, 43(2), 132 - 6 Binding of complement C3 to Klebsiella pneumoniae treated with sub-MIC cefodizime and chemiluminescence response of human polymorphonuclear leukocytes; Nomura S et al.; The binding of complement C3 to the cell surface of Klebsiella pneumoniae exposed to human serum complement after treatment with or without sub-MIC of antibiotics was examined by double diffusion immunoprecipitation against anti-human complement C3, and the production of oxygen-derived radicals by human polymorphonuclear leukocytes stimulated by complement-opsonized K . pneumoniae after treatment with or without sub-MIC of antibiotics was measured using the chemiluminescence (CL) assay . Complement C3 bound to the cell surface of K . pneumoniae treated with cefodizime was detected after exposure to human serum complement . The CL response induced by complement-opsonized bacteria after treatment with cefodizime was much higher than the response induced by nontreated bacteria or complement-opsonized bacteria after treatment with other antibiotics . These findings indicate that treatment with sub-MIC cefodizime make K . pneumoniae more susceptible to opsonization by complement and promotes the specific phagocytosis mediated by complement receptors. Antimicrob Agents Chemother, 1997 Mar, 41(3), 699 - 701 Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae; Deguchi T et al.; We determined a partial sequence of the Klebsiella pneumoniae parC gene, including the region analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene, and examined 26 clinical strains of K . pneumoniae for an association of alterations in GyrA and ParC with susceptibilities to quinolones . The study suggests that in K . pneumoniae DNA gyrase is a primary target of quinolones and that ParC alterations play a complementary role in the development of higher-level fluoroquinolone resistance. J Bacteriol, 1997 Mar, 179(5), 1497 - 504 Identification and characterization of acoK, a regulatory gene of the Klebsiella pneumoniae acoABCD operon; Peng HL et al.; By using transposon insertional mutagenesis and deletion analyses, a recombinant clone containing the region upstream of the acoABCD operon of Klebsiella pneumoniae was found to be required for acetoin-inducible expression of the operon in Escherichia coli . The nucleotide sequence of the region was determined, and it displayed an open reading frame of 2,763 bp that is transcribed divergently to the acoABCD operon . This gene, designated acoK, is capable of encoding a protein with an overall 58.4% amino acid identity with MalT, the transcriptional activator of the E . coli maltose regulon . A conserved sequence for nucleotide binding at the N-terminal region, as well as a helix-turn-helix motif belonging to the LuxR family of transcriptional regulators at the C terminus, was also identified . Primer extension analysis identified two transcription initiation sites, S1 and S2, located 319 and 267 bp, respectively, upstream of the putative start codon of acoK . Several copies of NtrC recognition sequence {CAC-(N11 to N18)-GTG} were found in the promoter regions of both the acoK gene and the acoABCD operon . Acetoin-dependent expression of the acoABCD operon could be restored in the E . coli acoK mutants by supplying a plasmid carrying an intact acoK, suggesting a transactivating function of the gene product . The AcoK protein overproduced in E . coli was approximately 100 kDa, which is in good agreement with the molecular mass deduced from the nucleotide sequence . A specific DNA binding property and an ATPase activity of the purified AcoK were also demonstrated. J Biol Chem, 1997 Feb 14, 272(7), 4121 - 8 UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO1 gene; Koplin R et al.; The O-side-chain polysaccharide in the lipopolysaccharide of Klebsiella pneumoniae O1 is based on a backbone structure of repeat units of {-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->}; this structure is termed D-galactan I . The rfb (O-antigen biosynthesis) gene cluster directs the synthesis of D-galactan I and consists of six genes termed rfbA-FKPO1 . In this paper we show that rfbDKPO1 encodes a UDP-galactopyranose mutase (NAD(P)H-requiring) (EC 5.4.99 . 9), which forms uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactofuranosyl ester (UDP-Galf), the biosynthetic precursor of galactofuranosyl residues . The deduced amino acid sequence of rfbDKPO1 shows 85% and 37.5% identity to the rfbDKPO8 gene of K . pneumoniae serotype O8 and the glf gene of Escherichia coli, respectively . The molecular mass of the purified RfbDKPO1 enzyme is 45 kDa as determined by SDS-polyacrylamide gel electrophoresis, while gel filtration revealed a molecular mass of 92 kDa, suggesting a dimeric structure for the native protein . The rfbDKPO1 gene product interconverts uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactopyranosyl ester (UDP-Galp) and UDP-Galf . Unlike Glf, RfbDKPO1 showed a requirement for NADH or NADPH, which could not be replaced by NAD or NADP . RfbDKPO1 was used to synthesize milligram quantities of UDP-Galf, allowing this compound to be purified and fully characterized in an intact form for the first time . The structure of UDP-Galf was proven by NMR spectroscopy. Gene, 1997 Feb 7, 185(2), 201 - 7 The fur gene from Klebsiella pneumoniae: characterization, genomic organization and phylogenetic analysis; Achenbach LA et al.; The Fur (ferric uptake regulator) protein controls the expression of a number of bacterial virulence determinants including those involved in iron uptake . The fur gene was cloned and characterized from Klebsiella pneumoniae . The gene is preceded by a single autoregulated promoter whose -10 region overlaps the putative Fur binding site . The autoregulated nature of the K . pneumoniae fur gene and functionality of the encoded Fur repressor were tested in Fur titration and complementation assays . A partial open reading frame upstream from the fur gene was identified as a flavodoxin (fldA) gene . An open reading frame located 50 bases downstream from the fur stop codon appears to be a truncated citA gene that, if functional, would encode only the carboxy terminus of a citrate utilization protein . The fldA-fur arrangement is also present in Escherichia coli . However, the fur-citA arrangement found in K . pneumoniae is novel . It appears that the chromosomal region downstream from the fur gene is unstable and, thus, variable even in closely related bacterial lineages . To assess of the ability of the Fur protein sequence to reflect organismal phylogeny, the Fur protein tree was compared to the tree of 16S rRNA (ribosomal RNA) . The Fur dataset comprises almost an order of magnitude fewer characters than the 16S rRNA but is nonetheless able to track the phylogenetic signal reasonably well, suggesting that the fur gene, like the 16S rDNA, may not be subject to horizontal gene transfer in these bacteria. Arch Microbiol, 1997 Feb-Mar, 167(2-3), 160 - 6 Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae; Gibello A et al.; The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced . K . pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses . HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da . HpaH, with a molecular mass of 18,680 Da, seems to be a "helper" protein required for productive hydroxylation of the substrate . The hpa genes were expressed and the hydroxylase was active in Escherichia coli . Comparison of the enzyme with other monooxygenases indicates that K . pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases. Am J Physiol, 1997 Feb, 272(2 Pt 1), L344 - 52 SP-A enhances phagocytosis of Klebsiella by interaction with capsular polysaccharides and alveolar macrophages; Kabha K et al.; We found that surfactant protein A (SP-A) enhances phagocytosis of Klebsiella pneumoniae K21a but not of K2 serotypes by alveolar macrophages . SP-A interacted with the capsule of K21a (containing Man alpha1 Man sequences) as shown by SP-A-induced agglutination of the bacteria, by binding of SP-A-coated particles onto the bacterial surface, and by binding of SP-A to immobilized parent K21a strain and recombinant strains that switched their capsule from K2 to K21a . In contrast, only marginal binding of SP-A to K2 parent strain (lacking this sequence) could be detected . Furthermore, binding of capsular polysaccharide of K21a to immobilized SP-A was inhibited by mannan but not by lipopolysaccharide and K2 capsular polysaccharide . SP-A-treated macrophages bound increased numbers of parent K21a strain and recombinant strains of K21a capsule type but considerably less parent K2 strain . SP-A also enhanced killing of K21a strains by macrophages . The enhanced binding of K21a by macrophages pretreated with SP-A was inhibited by mannan, suggesting that binding is mediated by the mannose receptor on macrophages . We conclude that SP-A increases phagocytosis of the Klebsiella by two mechanisms, one of which is by serving as an opsonin, which binds to the capsular polysaccharides of the bacteria and potentially to SP-A receptors on the macrophages, and the other by activating the macrophages, resulting in increased activity of the mannose receptor. J Formos Med Assoc, 1997 Feb, 96(2), 134 - 6 Fulminating gas-forming psoas muscle abscess due to Klebsiella pneumoniae following a deep neck infection; Jang TN et al.; Psoas muscle abscess due to Klebsiella pneumoniae infection is rare . We report a 55-year-old diabetic man who presented with progressive back pain of 1 month's duration . The patient had undergone surgical drainage for a deep neck infection with K . pneumoniae 43 days previously . On the present admission, physical examination revealed tenderness over the anterior upper aspect of both thighs, and computed tomography showed pneumoretroperitoneum dissecting the bilateral iliopsoas muscles . Parenteral administration of antibiotics was started immediately . Due to the patient's poor health status, we opted for repeated computed tomographic and sonographic-guided percutaneous drainage rather than surgical drainage . Blood and pus cultures revealed only K . pneumoniae . The patient recovered without significant sequelae . This report stresses the risk of metastatic infections caused by K . pneumoniae, especially in diabetic patients . Our experience suggests that repeated percutaneous drainage is feasible in cases of severe iliopsoas abscess, especially when risks associated with surgery are high. Ophthalmic Surg Lasers, 1997 Feb, 28(2), 147 - 50 Vitrectomy for endogenous Klebsiella pneumoniae endophthalmitis with massive subretinal abscess; Yarng SS et al.; A 39-year-old man with pyogenic liver abscess had bilateral endogenous Klebsiella pneumoniae endophthalmitis . At presentation, the left eye had a localized subretinal abscess . Despite repeated intravitreal amikacin and dexamethasone injections, a subretinal abscess spread and detached all of the retina . Pars plana vitrectomy with drainage of the subretinal abscess was performed . The retina was reattached, and the patient had 5/200 vision 5 months postoperatively . Early vitrectomy with drainage of the subretinal abscess may save some eyes with endogenous Klebsiella pneumoniae endophthalmitis. Infect Immun, 1997 Feb, 65(2), 609 - 16 The central variable V2 region of the CS31A major subunit is involved in the receptor-binding domain; Di Martino P et al.; CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines . In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure . We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies . The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A . Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells . Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion . Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen . These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region. Folia Microbiol (Praha), 1997, 42(3), 184 - 92 Pullulanase: model protein substrate for the general secretory pathway of gram-negative bacteria; Pugsley AP et al.; Pullulanase of Klebsiella oxytoca is one of a wide variety of extracellular proteins that are secreted by Gram-negative bacteria by the complex main terminal branch (MTB) of the general secretory pathway . The roles of some of the 14 components of the MTB are now becoming clear . In this review it is proposed that most of these proteins form a complex, the secretion, that spans the cell envelope to control the opening and closing of channel in the outer membrane . Progress toward the goal of testing this model is reviewed. Zh Mikrobiol Epidemiol Immunobiol, 1997 Jan-Feb, (1), 43 - 7 {The effect of the processes of Klebsiella pneumoniae cultivation on the antigenic activity of vaccinal preparations for peroral use}; Aleksakhina NN et al.; Whole-cell preparations, obtained from the microbial mass of K . pneumoniae grown by batch and continuous cultivation at a dilution rate of 0.24 and 0.41 hr-1 and used in animal experiments for oral administration, were shown to have different serological activity . The preparation obtained from biomass grown by continuous cultivation at a dilution rate of 0.41 hr-1 proved to be most active regarding the level of hemagglutinating antibodies to K . pneumoniae LPS . At the same time the 360-fold rise of the level of anti-LPS antibodies in rabbit immune sera was observed . On day 258 oral revaccination was made; after that the twofold rise of the level of anti-LPS antibodies in the sera of the animals was observed . These antibody levels exceeded 40-fold those registered before primary immunization, and sufficiently high antibody levels were retained for 4 more months (the term of observation). Zh Mikrobiol Epidemiol Immunobiol, 1997 Jan-Feb, (1), 39 - 42 {The isolation amd study of lipopolysaccharide from Klebsiella pneumoniae vaccinal strain 204}; Stukalova NV et al.; K . pneumoniae lipopolysaccharide (LPS) was isolated from biomass grown under the conditions of controlled multicycle and continuous cultivation . A considerable yield of LPS containing the minimal amount of protein and nucleic acid admixtures was obtained from biomass grown by continuous cultivation with the concentration of glucose in the medium equal to 20 g/l and the content of dissolved oxygen at a level of 0% of complete saturation . Erythrocyte diagnosticum prepared on the basis of this LPS was found to have a sensitizing dose of 100 micrograms per ml of solid erythrocytic precipitate and high specificity, studied in the passive hemagglutination test with the use of rabbit sera. Plasmid, 1997, 37(2), 105 - 18 The resistance and integrase genes of pACM1, a conjugative multiple-resistance plasmid, from Klebsiella oxytoca; Preston KE et al.; pACM1 is an 85-kb conjugative plasmid from a clinical isolate of Klebsiella oxytoca that encodes resistance to beta-lactams (mediated by SHV-5 extended spectrum beta-lactamase), trimethoprim, sulfonamides, tetracycline, aminoglycosides, and mercuric chloride . The expression of the aminoglycoside resistance is difficult to detect, which could have clinical implications . A region of pACM1 containing five resistance genes and two putative integrons was characterized by restriction mapping and partial DNA sequencing . One integron appears to be class I (sull type); the second lacks a recognizable 3' conserved segment . Neither integron has the BamHI site predicted for the 5' conserved segment . Plasmids encoding SHV-5 from other bacterial strains appear to be closely related to pACM1 by restriction enzyme analysis, but have resistance/ integron regions that vary in size and content from that of pACM1 . Integrase-mediated recombination might be responsible for genetic divergence in a widely distributed family of pACM1-like plasmids. J Clin Lab Anal, 1997, 11(3), 175 - 8 Effect of glucan on murine lupus evolution and on host resistance to Klebsiella pneumoniae; Harima HA et al.; Glucan is a polysaccharide from the yeast Saccharomyces cerevisiae that stimulates the mononuclear phagocytic system (MPS) . NZB/NZW F1 mice were divided into two groups: one group received a subcutaneous injection of 0.5 mg glucan/animal for 1 week, and the other received the same dose for 3 months . No changes were observed in those animals submitted to short-term glucan treatment, whereas animals with active lupus and submitted to long-term glucan administration presented early death, with significant differences in accumulated mortality rates over 33-37 weeks, when compared to controls . No deaths were observed in lupus mice treated with glucan 24 hours before the induction of septic shock by Klebsiella pneumoniae, in contrast to mortality of 95.3% in the control group during the follow-up period of 12 days . We conclude that although glucan is able to exacerbate lupus activity, it enhances resistance to infection in lupus mice. Zhonghua Yi Xue Za Zhi (Taipei), 1997 Jan, 59(1), 59 - 64 Graves' disease and diabetes mellitus associated with acute suppurative thyroiditis: a case report; Li CC et al.; Acute suppurative thyroiditis (AST) is a rare disorder . The rarity of AST is a result of the resistance of the thyroid gland to local infection . Thyroid function tests are usually normal in AST . In a review of the literature from 1966 to 1995, only three cases of AST associated with thyrotoxicosis have been convincingly demonstrated . The thyrotoxicosis in these cases was caused by diffuse inflammation of the thyroid gland and to the disruption of follicles with the release of pre-formed thyroid hormone into the circulation . Thus, the thyrotoxicosis in these cases was transient . With successful therapy, nearly all patients showed complete recovery of thyroid function within two to three months . The patient in the case here was a diabetic woman with Graves' disease in whom thyrotoxicosis occurred after Klebsiella pneumoniae thyroiditis, with relapse nine months after discharge . Thus, the patient's thyrotoxicosis might not have been caused simply from thyroid tissue destruction by AST, but also as a result of enhancing autoimmune activity . No previous case has ever been reported in the English literature . In diabetics, the impairment of chemotaxis and phagocytosis has been noted . Therefore, diabetes mellitus (DM) might have been the precipitating factor for this patient's acquiring this unusual infection . Thyrotoxicosis and AST will increase insulin requirements and thus aggravate diabetes . In addition, poor control of blood sugar will enhance the severity of AST. J Perinatol, 1997 Jan-Feb, 17(1), 29 - 32 Early hospital discharge of preterm very low birth weight infants; Cruz H et al.; OBJECTIVE: The objective of this study was to investigate whether early discharge from the hospital was feasible for selected very low birth weight (VLBW) infants . STUDY DESIGN: A randomized clinical trial of discharge of VLBW infants from the neonatal intensive care unit at 1300 gm versus 1800 gm was done comparing weight gain and incidence of infection . Forty-three VLBW infants treated in the neonatal intensive care unit and follow-up clinics of the Hospital Universitario del Valle, Cali, Colombia, were entered into the study at 1300 to 1350 gm when they met behavioral criteria for discharge and the family home was approved . RESULTS: There were no differences in weight gain or incidence of infection in the home group compared with the hospital group . A significant saving in hospital days and hospital costs was realized for the home group . Family cooperation was heightened in the home group . CONCLUSIONS: Early discharge from the hospital at weights as low as 1300 to 1350 gm is safe for the VLBW infant when properly selected on the basis of behavioral criteria and environmental approval . The potential savings in hospital costs should be considered when resources are allocated for continued support for these infantsPIP: It is traditional policy to delay the discharge of preterm infants from hospital nurseries until a predetermined weight (2000 g or more) has been achieved . However, prolonged hospitalization is associated with numerous adverse effects, including delayed mother-child bonding, reduced staff time for sick infants, an increased risk of nosocomial infections, and high costs . This study investigated the hypothesis that early hospital discharge is safe and feasible for very-low-birth-weight infants when behavioral and parental criteria, rather than achieved weight, serve as discharge indicators . Preterm infants from the Hospital Universitario del Valle in Calle, Colombia, were enrolled in the study at 1300-1350 g if they met the following criteria: maintenance of normal body temperature in an open crib, nippled feedings of at least 120 cal/kg/day, consistent weight gain for at least 3 days, asymptomatic and free of medications for at least 3 days, and a social worker-approved home environment (e.g., single-family home with utilities and phone, access to transportation, and parental willingness to participate in follow-up) . 43 infants met these criteria; 27 were discharged and 16 were kept in the hospital . The duration of the hospital stay was 23.5 days for the home group and 42.5 days for the hospital group . 2 infants in the home group were readmitted to the hospital, 1 with diarrhea and 1 with pneumonia; 1 infant in the hospital group developed nosocomial Klebsiella aerobacter meningitis . There were no differences in weight gain or incidence of infection between the 2 groups and no infant deaths in the study period (up to 40 weeks of age) . The parents of discharged infants kept all clinic appointments . These findings confirm the feasibility of this strategy and suggest that, in some very-low-birth-weight infants, behavioral development may be advanced even at weights as low as 1300-1400 g . Eur J Epidemiol, 1997 Jan, 13(1), 103 - 7 Diversity of beta-lactamases among Klebsiella pneumoniae strains isolated in a university hospital in Greece; Tsakris A et al.; Among 48 clinical strains of Klebsiella pneumoniae isolated in a university hospital in Northern Greece, 29 (60.4%) exhibited resistance to third generation cephalosporins (3GC) and aztreonam . Thirty-two (66.7%) of the isolates were found resistant to the combination of ampicillin/sulbactam and six (12.5%) exhibited resistance to all the above antibodies plus cefoxitin . Resistance to 3GC was related mostly with the presence of a beta-lactamase exhibiting pI 8.2 and substrate profile of an SHV-5 type enzyme and rarely (in two of the cefoxitin resistant strains) with the presence of plasmid-mediated class C cephalosporinases . Resistance to the ampicillin/sulbactam combination was associated with the presence of a beta-lactamase with pI 5.4, presumably representing a TEM-1 beta-lactamase . These findings record a diversity of beta-lactamases and explain, at least partly, the various beta-lactam resistance patterns observed in our K . pneumoniae sample. Zentralbl Bakteriol, 1997 Jan, 285(2), 305 - 10 Applications of pyrolysis mass spectrometry in studies on the mode of action of antimicrobial agents; Magee JT et al.; Changes in overall cell composition during exposure to antimicrobial agents were investigated by pyrolysis mass spectrometry (Py-MS) in parental (O+K+), K antigen deficient (O+K-), and O and K antigen deficient (O-K-) strains of Klebsiella aerogenes NCTC 5055 . Changes followed distinct patterns that correlated with mode of action: penicillin binding protein (PBP) 3 blockade (ceftazidime, piperacillin); PBP 2 blockade (imipenem); membrane disruption (colistin); bacteriostatic (chloramphenicol), and bactericidal (gentamicin) protein synthesis inhibition; and DNA synthesis inhibition (ciprofloxacin) . Changes were pronounced after exposure for 1 hour at 10 x MIC . In general, the O-K- strain showed the largest compositional shifts, and the parental O+K+ strain the least shifts, correlating with bactericidal kinetic studies . Py-MS may be useful in studies of the mode of action of antimicrobial agents, particularly in early screening of new compounds, and in rapid detection of the effects of antimicrobial agents on micro-organisms. Zentralbl Bakteriol, 1997 Jan, 285(2), 252 - 7 Epidemiological typing of Klebsiella pneumoniae by pyrolysis mass spectrometry; Jackson RM et al.; Thirteen isolates of ceftazidime-resistant Klebsiella pneumoniae from a suspected cross-infection outbreak involving patients on an intensive care unit and a haematology ward were examined in pyrolysis-mass spectrometry (Py-MS), along with eight concurrent non-outbreak-associated clinical isolates of klebsiellae as controls . Py-MS showed tight clustering of the suspected outbreak isolates, suggesting cross-infection with a single strain . Non-outbreak isolates were clearly distinct from one another and from the outbreak strain . The results confirm that Py-MS is a powerful tool for rapid strain comparison in investigations of cross-infection incidents. J Leukoc Biol, 1997 Jan, 61(1), 17 - 23 Alteration of dealcylated lipopolysaccharide antagonistic properties by interaction with plasma factors; Mey A et al.; The acyl poly(1,3)galactoside (APG) from Klebsiella pneumoniae is a bis-acylated lipopolysaccharide (LPS) devoid of ester-linked fatty acids . APG interacts with CD14 and CD11b/CD18 on monocytes . This study addressed the role of serum proteins in the binding and functional properties of APG as a candidate LPS antagonist . In the absence of serum, APG did not induce tumor necrosis factor alpha (TNF-alpha) synthesis by human mononuclear cells and dose-dependently inhibited their activation induced by different LPS . Conversely, in the presence of 5% autologous plasma, APG activated cells and did not antagonize LPS . Serum decreased APG but not LPS binding to monocytes . Binding competition experiments indicated that APG and LPS competed for the same receptors in serum-free conditions but bound to different receptors in the presence of plasma . The data indicate that serum-dependent LPS receptors do contribute to LPS activation of monocytes but do not recognize deacylated LPS analogues. FEMS Microbiol Lett, 1997 Jan 1, 146(1), 85 - 9 On the amine oxidases of Klebsiella aerogenes strain W70; Cooper RA; Klebsiella aerogenes W70 was reported previously to produce a membrane-associated tyramine oxidase (TynA) that did not act on 2-phenylethylamine . Subsequently, a gene cloned from K . aerogenes W70 produced a soluble amine oxidase (MaoA) that acted readily on 2-phenylethylamine and tyramine . This enzyme appeared to be equivalent to a 2-phenylethylamine oxidase of Escherichia coli K-12 (MaoA) but was assumed to be the originally described K . aerogenes W70 tyramine oxidase (TynA) . However, as described here, whole cells and cell-free extracts of K . aerogenes W70 showed only the tyramine oxidase (TynA) that is inactive against 2-phenylethylamine and not the maoA gene product . It seems that the organism has two amine oxidase genes, tynA and maoA, but only tynA is expressed . Hence, data relating to the expression of the K . aerogenes W70 tynA gene cannot be assumed to apply to the maoA gene of E . coli K-12 because they encode different enzymes. J Immunol, 1996 Dec 15, 157(12), 5221 - 4 Leukotriene-deficient mice manifest enhanced lethality from Klebsiella pneumonia in association with decreased alveolar macrophage phagocytic and bactericidal activities; Bailie MB et al.; Leukotrienes (LTs) are potent mediators of inflammation derived from the 5-lipoxygenase pathway of arachidonic acid metabolism . Although they are known to enhance leukocyte recruitment and function, their role in antimicrobial host defense has not been established . To determine the role of endogenous LTs in the host response to pulmonary infection, wild-type mice and mice rendered LT-deficient by targeted disruption of the 5-lipoxygenase gene (knockout mice) were studied following intratracheal challenge with Klebsiella pneumoniae . Wild-type mice demonstrated a marked increase in lung LT levels and neutrophil numbers following bacterial challenge . As compared with wild-type animals, knockout animals manifested a greater degree of lethality as well as bacteremia following challenge . Interestingly, they displayed no defect in neutrophil recruitment to the lung . However, alveolar macrophages from knockout animals exhibited impairments in bacterial phagocytosis and killing, and these defects were overcome by in vitro addition of exogenous LTB4 . We conclude that endogenous LTs play a critical role in the defense against bacterial pneumonia in this murine model. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 635 - 42 Spectroscopic and voltammetric characterisation of the bacterioferritin-associated ferredoxin of Escherichia coli; Quail MA et al.; The bacterioferritin-associated ferredoxin (Bfd) of Escherichia coli is a 64-residue polypeptide encoded by the bfd gene located upstream of the gene (bfr) encoding the iron-storage haemoprotein, bacterioferritin . The Bfd sequence resembles those of the approximately 60-residue domains found in NifU proteins (required for metallocluster assembly), nitrite reductases, and Klebsiella pneumoniae nitrate reductase . These related-domains contain four well-conserved cysteine residues, which are thought to function as ligands to a {2Fe-2S} cluster . The Bfd protein was over-produced, purified, and characterised . Bfd was found to be a positively-charged monomer containing two iron atoms and two labile sulphides . Ultraviolet-visible, EPR, variable-temperature magnetic-circular dichroism and resonance Raman spectroscopies, together with cyclic voltogram measurements, revealed the presence of a {2Fe-2S}2+,+ centre (E1/2 = -254 mV) having remarkably similar properties to the Fe-S cluster of NifU . Bfd may thus be a 2Fe ferredoxin participating either in release/delivery of iron from/to bacterioferritin (or other iron complexes), or in iron-dependent regulation of bfr expression. Antimicrob Agents Chemother, 1996 Dec, 40(12), 2848 - 53 Resistance to amikacin and isepamicin in rabbits with experimental endocarditis of an aac(6')-Ib-bearing strain of Klebsiella pneumoniae susceptible in vitro; Caulin E et al.; The effect of production of the aminoglycoside 6'-N-acetyltransferase {AAC(6')-IB} in Klebsiella pneumoniae on the outcome of amikacin and isepamicin treatment of rabbits with experimental endocarditis was assessed . Isogenic high-level (Hi) and low-level (Lo) AAC(6')-Ib-producing transconjugants (T) were constructed from clinical isolates with plasmid-borne resistance determinants . The MICs of amikacin and isepamicin, their bactericidal effects, and AAC(6')-Ib production appeared to be well correlated among the clinical isolates and the transconjugants . The susceptibility data determined in vitro, with MICs (in micrograms per milliliter) of amikacin and isepamicin for LoT and HiT of 4 and 0.5 and 32 and 8, respectively, were, however, not predictive of the in vivo efficacies of the drugs . While amikacin and isepamicin caused reductions in bacterial densities (log10 CFU per gram of cardiac vegetation) of 5.1 and 4.8 of the fully susceptible recipient strain (MICs of amikacin and isepamicin, 0.5 and 0.25, respectively), the reductions in density of both LoT and HiT caused by the two drugs (2.7 and 2.4 and 2.9 and 2.2, respectively) were only marginally significant, if at all . There was no significant difference (P > 0.05) when the reductions in density of LoT and HiT by either drug were compared or when the efficacies of the two drugs in reducing the density of any strain {non-AAC(6')-producing, LoT, or HiT} were compared (P > 0.5) . It is concluded that AAC(6')-Ib in K.pneumoniae, even when produced at a low level and not conferring resistance to amikacin and isepamicin in vitro, compromises the efficacies of both drugs in vivo and possibly does so beyond the experimental model studied here. J Antimicrob Chemother, 1996 Dec, 38(6), 1013 - 22 Potential effects of amoxycillin/clavulanic acid and ticarcillin/clavulanic acid on human granulocyte activity: a comparative study; Cuffini AM et al.; The efficacy of an antimicrobial agent in the therapy of infection depends upon the interaction of bacteria, antibiotic and phagocytes . The influence of both ticarcillin/clavulanic acid and amoxycillin/clavulanic acid on the phagocytic and bactericidal activity of human PMNs in vitro was investigated . The results show that clavulanic acid, with its beta-lactamase inhibitory properties, potentiated the efficacy of both ticarcillin and amoxycillin, resulting in a significantly increased susceptibility of a beta-lactamase producing strain of Klebsiella pneumoniae to phagocytic and microbicidal activities of human PMNs . Overall, amoxycillin/clavulanic acid showed greater enhancement of the killing of K . pneumoniae by human PMNs. Mol Microbiol, 1996 Dec, 22(5), 779 - 88 Effector-induced self-association and conformational changes in the enhancer-binding protein NTRC; Farez-Vidal ME et al.; The Klebsiella pneumoniae nitrogen regulatory protein NTRC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of sigma N-dependent enhancer-binding proteins . Using limited trypsin digestion, two domains of NTRC were detected and conformational changes within the protein in response to the binding of ligands were also observed . In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively . Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain . Analysis of the dependence of this insensitivity on ATP gamma S concentration suggested an apparent dissociation constant (Kd) for ATP gamma S of 150 microM . In the presence of DNA, both the central and C-terminal domains of NTRC were insensitive to proteolytic cleavage, indicative of a further conformational change . NTRC S160F, a mutant form of NTRC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein . This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NTRC . The involvement of ligands in self-association was studied using sedimentation equilibrium analysis . In the absence of ligand, wild-type NTRC displayed a monomer-dimer equilibrium with a Kd of 6 microM . In the presence of ATP gamma S the equilibrium was shifted towards the dimer form (Kd = 0.8 microM) . A similar dissociation constant for the monomer-dimer interaction was observed with NTRC S160F in the absence of ATP gamma S (Kd = 0.5 microM) . The addition of ATP gamma S induced a significant association of NTRC S160F to higher-order states with a dimer-octamer model producing a slightly, but not significantly better fit to the data than a dimer-hexamer model . We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins. FEMS Microbiol Lett, 1996 Dec 1, 145(2), 273 - 9 An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila; Jain B et al.; Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells . We characterised a Tn5-mutant of the L . pneumophila Corby strain and were able to identify the insertion site of the transposon . It is localised within an open reading frame which shows high homology to the alpha-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae . The OadA homologous protein of L . pneumophila was detected in the wild-type strain by Western blotting . Since the intracellular multiplication of the oadA- mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oadA gene product has an effect on the intracellular survival of L . pneumophila. J Bacteriol, 1996 Dec, 178(23), 6817 - 23 Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae; Govantes F et al.; The nifLA operon of Klebsiella pneumoniae codes for the two antagonistic regulatory proteins which control expression of all other nitrogen fixation genes . NifA is a transcriptional activator, and NifL inhibits NifA . The importance of a correct NifL-NifA stoichiometry for efficient regulation of nitrogen fixation genes has been investigated by constructing a strain with an altered nifL-nifA gene dosage ratio, resulting from the integration of an extra copy of nifA . Results showed that a balanced synthesis of both gene products is essential for correct regulation . Effects of mutations provoking translation termination of nifL upstream or downstream of its natural stop codon, combined with overproduction of both proteins when the genes are transcribed and translated from signals of the phi10 gene of the phage T7, showed that, in addition to the previously reported transcriptional polarity, there is translational coupling between nifL and nifA . In spite of the apparently efficient ribosome binding site of nifA, its rate of independent translation is very low . This is due to a secondary structure masking the Shine-Dalgarno sequence of nifA, which could be melted by ribosomes translating nifL . Mutational analysis confirmed the functional significance of the secondary structure in preventing independent translation of nifA . Translational coupling between the two cistrons is proposed as an efficient mechanism to prevent production of an excess of NifA, which would affect the normal regulation of nitrogen fixation genes. Infect Immun, 1996 Dec, 64(12), 5211 - 8 Tumor necrosis factor mediates lung antibacterial host defense in murine Klebsiella pneumonia; Laichalk LL et al.; Tumor necrosis factor (TNF) is a proinflammatory cytokine which has recently been shown to have beneficial effects in the setting of acquired host immunity . However, the role of TNF in innate immune responses, as in the setting of bacterial pneumonia, has been incompletely characterized . To determine the role of TNF in gram-negative bacterial pneumonia, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally, resulting in the time-dependent expression of TNF MRNA and protein within the lung . Passive immunization of animals with a soluble TNF receptor-immunoglobulin (Ig) construct (sTNFR:Fc) intraperitoneally 2 h prior to K . pneumoniae inoculation resulted in a significant reduction in bronchoalveolar lavage neutrophils, but not macrophages, at 48 h, as compared with animals receiving control IgG1 . Furthermore, treatment with sTNFR:Fc resulted in 19.6- and 13.5-fold increases in K . pneumoniae CFU in lung homogenates and plasma, respectively, as compared with animals receiving control IgG1 . Finally, treatment of Klebsiella-infected mice with sTNFR:Fc markedly decreased both short- and long-term survival of these animals . In conclusion, our studies indicate that endogenous TNF is a critical component of antibacterial host defense in murine Klebsiella pneumonia. Clin Rheumatol, 1996 Nov, 15(6), 573 - 6 Ig A antibodies to klebsiella in ankylosing spondylitis; Ardicoglu O et al.; In this study anti-klebsiella Ig A values were compared in 40 patients with definite diagnosis of ankylosing spondylitis and a control group of 40 healthy subjects . Anti-Klebsiella Ig A antibody values were significantly higher in patients with ankylosing spondylitis as compared to the control group (p < 0.001) . Correlation between these antibodies and erythrocyte sedimentation rate, CRP, serum Ig A, HLA B 27, age, sex and disease duration was searched, but no correlation was found . In our opinion, these results support the suggestion that inflammatory response in ankylosing spondylitis is triggered by Klebsiella but is insufficient to prove the causal relationship between ankylosing spondylitis and Klebsiella. Br J Pharmacol, 1996 Nov, 119(6), 1145 - 8 Pulmonary surfactant as vehicle for intratracheally instilled tobramycin in mice infected with Klebsiella pneumoniae; van't Veen A et al.; 1 . The use of pulmonary surfactant has been proposed as a vehicle for antibiotic delivery to the alveolar compartment of the lung . This study investigated survival rates of mice with a respiratory Klebsiella pneumoniae infection treated intratracheally with tobramycin using a natural exogenous surfactant preparation as vehicle . 2 . At day 1 after infection, animals were injected intratracheally with 20 microliters of the following solutions: (1) a mixture of surfactant (500 micrograms) and tobramycin (250 micrograms); (2) tobramycin (250 micrograms) alone; (3) surfactant (500 micrograms) alone; and (4) NaHCO3 buffer (control, sham-treatment) . A fifth group received no treatment (control) . Deaths were registered every 12 h for 8 consecutive days . 3 . The results show an increased survival in the group receiving the surfactant-tobramycin mixture compared to the group receiving tobramycin alone (P < 0.05), the group receiving surfactant alone (P < 0.01) and the control groups (P < 0.01) . It is concluded that intratracheal instillation of surfactant-tobramycin is superior to tobramycin alone in protecting animals from death due to a respiratory Klebsiella pneumoniae infection. Infect Control Hosp Epidemiol, 1996 Nov, 17(11), 743 - 5 Investigation of an apparent cluster of Klebsiella pneumoniae bacteremias using random amplified polymorphic DNA analysis; Hurley JC et al.; A cluster of bacteremia episodes with Klebsiella pneumoniae was noted in patients in a hematology-oncology ward during a 3-week period . Random amplified polymorphic DNA (RAPD) analysis, a novel technique for generating chromosomal fingerprints from bacterial isolates, was used as an aid to the epidemiological investigation of this cluster . For each of the two patients from whom multiple isolates had been obtained, identical RAPD patterns were observed in the serial isolates, even for a patient where the isolates had different biotypes . Isolates from different patients gave distinct patterns . Random amplified polymorphic DNA was found to be a useful typing technique for this cluster of K pneumonia bacteremias. Infect Immun, 1996 Nov, 64(11), 4726 - 32 Analysis of complement C3 deposition and degradation on Klebsiella pneumoniae; Alberti S et al.; The majority of Klebsiella pneumoniae serum-resistant strains activate complement and bind C3b, the opsonic fragment of C3, without C5b-9 formation and bacterial killing . The mechanisms leading to C3b deposition without cell death were studied, and the results indicate that serum-resistant strains activate principally the alternative pathway and that serum-sensitive strains activate both the alternative and classical pathways . Bacterial molecules implicated in C3b deposition are the outer membrane porin proteins and smooth and rough lipopolysaccharides . Porins activate both complement pathways, and the rough lipopolysaccharide activates the classical pathway, causing deposition of C3b in serum-sensitive strains . The smooth lipopolysaccharide of serum-resistant strains activates only the alternative pathway, impeding the binding of C1q to porins (S . Alberti, G . Marques, S . Camprubi, S . Merino, J . M . Tomas, F . Vivanco, and V . J . Benedi, Infect . Immun . 61:852-860, 1993; S . Alberti, F . Rodriguez-Quinones, T . Schirmer, G . Rummel, J . M . Tomas, J . P . Rosenbusch, and V . J . Benedi, Infect . Immun . 63:903-910, 1995) and rough lipopolysaccharide molecules and thereby preventing activation of the classical pathway . After its deposition, C3b is quickly degraded to iC3b on both types of strains, but the higher-level deposition of C3b on serum-sensitive strains, resulting from activation of both the alternative and classical complement pathways, supports further complement activation and killing of serum-sensitive strains. Infect Immun, 1996 Nov, 64(11), 4719 - 25 Interaction between complement subcomponent C1q and the Klebsiella pneumoniae porin OmpK36; Alberti S et al.; The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiella pneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments . We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K . pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin . Scatchard analysis of the binding of {125I}C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM . The decrease of {125I}C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon . In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks. Nucleic Acids Res, 1996 Oct 15, 24(20), 4096 - 7 Replicon rescue: a novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA; McMahon TL et al.; A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions . After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously . Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion . Replicon rescue was used successfully firstly in a test system to clone the chromosomal orl from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis-deficient mutant of Dictyostelium. Eur J Biochem, 1996 Oct 15, 241(2), 602 - 10 Complete NMR structural elucidation of the capsular polysaccharide adjuvant from Klebsiella I-714; Adam O et al.; The capsular polysaccharide (CPS) produced by the non-pathogenic Klebsiella strain I-714, selected for its immunomodulating activity, has been purified and almost completely detoxified in a previous study {Adam, O., Vercellone, A., Paul, F., Monsan, P . F . & Puzo, G . (1995) Anal . Biochem . 225, 321-327} . The present report concerns the structural elucidation of this CPS by several one-dimensional and two-dimensional 1H-NMR and 13C-NMR experiments performed on the native molecule . It was found to be a high molecular-mass branched polymer constituted by a hexasaccharide repeating unit of following structure: 4)-alpha-L-Rhap-(1-->3)-beta-D-Galp-(1-->2)-alpha-L-Rhap-(1- ->4)-beta-D- GlcpA-(1-->3)- inverted question markalpha-L-Rhap-(1-->2)- inverted question mark-alpha-D-Galp-(1 . The presence of two glycosidic substitutions on the Galp residue resulted in a strong overlapping of its proton signals, preventing direct assignment of both proton and carbon resonances . However, assignment could be achieved using the two-bond and three-bond 1H-13C heteronuclear coupling observed in the 1H-13C heteronuclear multiple-bond correlation (HMBC) spectrum. Biochem J, 1996 Oct 15, 319 ( Pt 2), 575 - 83 Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases; Chavagnat F et al.; A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced . It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor . P . alginovora Aly has been overproduced in E . coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided . His-tagged P . alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase . It can be purified in a one-step procedure by affinity chromatography on Ni(2+)-nitriloacetate resin . The yield is of 5 mg of enzyme per litre of culture . The amplification factor is 12.5 compared with the level of production by wild-type P . alginovora . The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P . alginovora Aly and Klebsiella pneumoniae Aly. Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 41 - 6 Kinetic analysis and comparison of models of xylose metabolism by Klebsiella planticola; Bastianoni S et al.; A model for the degradation of xylose and ethanol production by Klebsiella planticola is proposed and compared with the exponential and Michaelis-Menten approaches . This model is based on the energy system diagrams and it is a simplified version of a previous model developed for the glucose and ethanol kinetics of the yeast Saccharomices cerevisiae . In this model the dynamics of the substrate and of the final product are strictly related by means of the cellular activity . This model shows superior performances with respect to the two alternatives, behaving better along the whole dynamics. Lijec Vjesn, 1996 Oct, 118(10), 244 - 8 {Extended spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae}; Bedenic B; Resistance of Klebsiella pneumoniae isolates to broad spectrum cephalosporins is mainly attributable to the production of extended-spectrum beta-lactamases . They are plasmid-mediated beta-lactamases derived from TEM or SHV enzymes by mutations that alter the configuration of the active site to expand their spectrum of activity . These enzymes are ineffective against cephamycins with an alpha-methoxy substituent in the C7 position such as cefoxitin, cefotetan and moxalactam . They can be distinguished by determination of isoelectric point, substrate profile, sensitivity to inhibitors, molecular weight and amino acid sequence . Extended spectrum beta-lactamases are divided into four groups: TEM, SHV, ampC derivates and unclassified beta-lactamases . Multiple resistance to beta-lactams is often accompanied by resistance to aminoglycosides, sulphonamides, tetracyclines, chloramphenicol and trimethoprim . Besides plasmid-mediated beta-lactamases hyperproduction of chromosomal beta-lactamases can be responsible for the resistance to the new cephalosporins in some strains of Klebsiella pneumoniae . Chromosomal resistance is intrinsic and is not transferable. New Microbiol, 1996 Oct, 19(4), 293 - 300 Molecular typing of Klebsiella pneumoniae isolates from a neonatal intensive care unit; Marranzano M et al.; The epidemiological features of 60 multiresistant K . pneumoniae strains isolated from 1991 to 1995 in a neonatal ward are described . Antibiotic . Susceptibility testing and plasmid profile analysis were used as subtyping procedures . Antibiotic susceptibility typing was not informative enough since discrimination among isolates was typically poor . Plasmid profile analysis demonstrated that 58 out of 60 strains harboured one or more plasmid DNA bands, of different molecular weights ranging between 1.8 and 150 Mda . Small plasmids were best visualized after the alkaline lysis procedure, while large plasmids by the Kado and Liu method . A combination of plasmid patterns obtained by the two extraction procedures was used to define the final plasmid profile for each strain . Thirteen different plasmid profiles were identified among the collection of K . pneumoniae isolates from newborn patients of the same intensive care unit . The investigation showed that the strains were not responsible for a single outbreak. Mol Microbiol, 1996 Oct, 22(1), 1 - 7 Identification of two regions of Klebsiella oxytoca pullulanase that together are capable of promoting beta-lactamase secretion by the general secretory pathway; Sauvonnet N et al.; Pullulanase (PulA) is a 116 kDa amylolytic lipoprotein secreted by the Gram-negative bacterium Klebsiella oxytoca via the general secretory pathway . A deletion strategy was used in an attempt to determine the nature and the location of the secretion signal(s) in PulA presumed to be necessary for its specific secretion . The starting material was a gene fusion coding for an efficiently secreted PulA-beta-lactamase hybrid protein . Successive series of exonuclease III-generated deletions were used to remove internal segments of PulA from this hybrid . A simple plate test allowed the identification of truncated hybrids that retained beta-lactamase activity and that were secreted . Two non-adjacent regions, A and B (78 and 80 amino acids, respectively), were together necessary and sufficient to promote beta-lactamase translocation across the outer membrane . Secretion of PulA itself was markedly reduced when either of these regions was deleted, and was completely abolished when both regions were eliminated. Diabetes Metab, 1996 Oct, 22(5), 341 - 8 Prevention of diabetes in the nonobese diabetic mouse by oral immunological treatments . Comparative efficiency of human insulin and two bacterial antigens, lipopolysacharide from Escherichia coli and glycoprotein extract from Klebsiella pneumoniae; Sai P et al.; As oral administration of insulin reduces the incidence of diabetes in NOD mice, and to achieve a better approximation of oral insulin trials being developed for human studies which will use human insulin, we attempted to determine the preventive efficacy of oral administration of human insulin rather than resorting to the animal insulins used in previous studies . As the strength of prevention obtained by oral insulin has not been adequately demonstrated, we determined whether the protection persisted after the oral treatment was discontinued and whether it was resistant to a diabetogenic injection of cyclophosphamide (CY) . We also determined whether the effect of insulin could be increased by oral administration of lipopolysaccharide from Escherichia coli (LPS) or another immunostimulant (glycoprotein extracts from Klebsiella pneumoniae, GEKP) which may be more feasible for human application . Female NOD mice were fed once a week (from 35 to 300 days of age) with insulin, LPS, GEKP, insulin plus LPS, insulin plus GEKP, or PBS . A decreased incidence of diabetes were observed in animals fed human insulin (p < 0.01 incidence of diabetes at 300 days of age: 31% in mice fed with insulin and 65% in those fed PBS) . Prevention by insulin was not enhanced by oral LPS or GEKP . Yet unexpectedly, mice fed with LPS alone or GEKP alone displayed decreases in diabetes incidence (p < 0.01) . The severity of insulitis was reduced in animals fed insulin, LPS, GEKP or combinations of insulin and either immunostimulant (p < 0.02) . Although the oral treatments were stopped at 300 days of age, the incidence of diabetes at 360 days remained lower in mice previously fed insulin, LPS, GEKP or combinations of insulin and either immunostimulant (p < 0.01) . In mice previously fed PBS, CY injection (60 days after withdrawal of the oral treatment) led to a final incidence of diabetes of 90% (sum of the incidence during the initial 360 days and the further CY-induced incidence) . Previous feedings with insulin, LPS, GEKP or combinations of insulin and either immunostimulant did not protect against CY-induced diabetes since incidences reached the final control incidence . T splenocytes from animals fed insulin, LPS, or GEKP, similarly reduced the capacity of T cells from diabetic mice to transfer the disease (p < 0.01) . It is concluded that oral treatment with human insulin to be used in human trials reduces the incidence of diabetes in NOD mice . Equivalent preventive efficacy was obtained through feedings with LPS or GEKP (even though no cumulative efficiency was observed with insulin) . The latter results suggest that it would be advisable to evaluate the efficiency of oral bacterial antigens for the prevention of human Type 1 diabetes . The protection afforded by oral treatments with insulin or bacterial antigens may be attributed to cellular suppression, persists for some time after treatments are stopped, but is not resistant to major immune stimulation such as injection of CY. J Clin Microbiol, 1996 Oct, 34(10), 2448 - 53 Comparison of pulsed-field gel electrophoresis and randomly amplified DNA polymorphism analysis for typing extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae; Gori A et al.; The incidence and transmission patterns of extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients admitted to the intensive care unit (ICU) of a university hospital were investigated over a 3-year period . K . pneumoniae isolates were characterized by antibiotic susceptibility, capsular serotyping, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with XbaI, and the results were compared with those obtained by typing with the randomly amplified polymorphic DNA (RAPD) patterns . The discriminatory power of RAPD typing was evaluated for three primers . The incidence of isolation of ESBL-producing K . pneumoniae was 2.5 cases per 1,000 admissions to the ICU versus 0.35 cases per 1,000 admissions to other units (relative risk, 7.03; 95% confidence interval, 3.89 to 12.69) . Infection developed in 53% of evaluable patients . Thirty-six percent of the cases were possibly acquired in other institutions . Isolates from ICU patients were subdivided into six capsular serotypes and into four clonal groups based on antibiotype, plasmid content, and PFGE and RAPD patterns . Two clones were associated with clusters of cross-infection, involving 5 and 12 patients, respectively . Following implementation of contact isolation precautions, the incidence of nosocomial acquisition of ESBL-producing K . pneumoniae decreased from 0.55 to 0.26 cases per 1,000 admissions (P = 0.03) . PFGE and RAPD analysis showed concordant results and comparable discrimination for differentiation between groups of epidemiologically related strains of ESBL-producing K . pneumoniae . More subclonal variants were determined among epidemic clones by PFGE analysis than by RAPD analysis . Both methods are useful for typing K . pneumoniae strains in epidemiological investigations, although RAPD analysis is more efficient. J Immunol, 1996 Oct 1, 157(7), 3006 - 12 IL-12 gene therapy protects mice in lethal Klebsiella pneumonia; Greenberger MJ et al.; IL-12 is a proinflammatory cytokine that has recently been shown to have beneficial effects in the setting of acquired host immunity . To determine the role of IL-12 in innate immunity against Gram-negative bacterial organisms, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally (i.t.), resulting in the time-dependent expression of IL-12 mRNA (p35 and p40) and protein within the lung . Passive immunization of animals with anti-IL-12 serum i.p . at the time of K . pneumoniae inoculation resulted in a 12-fold increase in K . pneumoniae CFU in lung homogenates at 48 h, as compared with animals receiving control serum . In addition, treatment of Klebsiella-infected mice with anti-IL-12 Abs significantly decreased both short and long term survival . To assess the effect of compartmentalized IL-12 overexpression on outcome in Klebsiella pneumonia, animals were treated i.t . with 5 x 10(8) PFU of a nonreplicating adenoviral vector containing a human cytomegalovirus promoter and cDNAs coding for the p35 and p40 subunits of IL-12 inserted into the E1 and E3 domains (Ad5mIL-12), respectively . In vivo transfection with Ad5mIL-12 resulted in 45% long term survival in Klebsiella pneumonia, whereas no animals with Klebsiella pneumonia receiving control adenovirus survived . Moreover, treatment with anti-IFN-gamma Abs or soluble TNF receptor:Ig construct partially and completely attenuated survival benefits observed in animals receiving Ad5mIL-12, respectively . In conclusion, endogenous IL-12 is a critical component of antibacterial host defense, and the compartmentalized overexpression of IL-12 using recombinant adenoviral gene therapy represents a safe and effective approach to deliver IL-12 to the lung in the setting of murine Klebsiella pneumonia. FEMS Microbiol Lett, 1996 Sep 15, 143(1), 89 - 95 Purification and biochemical characterization of gentisate 1,2-dioxygenase from Klebsiella pneumoniae M5a1; Suarez M et al.; Gentisate 1,2-dioxygenase (E.C.1.14.13) was purified to homogeneity from Klebsiella pneumoniae M5a1, a soil bacterium able to degrade a great variety of aromatic compounds . The molecular mass of the purified holoenzyme was 159 kDa and its structure was deduced to be a tetramer with 38 kDa per subunit . Gentisate 1,2-dioxygenase appears to contain Fe2+ in its active site . The optimum temperature for enzyme activity was estimated to be 30 degrees C, the optimum pH values varied between 8 and 9 and the isoelectric point was 4.7 . Gentisate dioxygenase exhibited typical saturation kinetics and had an apparent K(m) of 52 microM for gentisate . Its amino acid content was determined to be very similar to that of the enzyme from Pseudomonas acidovorans. J Biol Chem, 1996 Sep 13, 271(37), 22352 - 7 Cloning, sequencing, and high level expression of the genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae; Tobimatsu T et al.; The gld genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae were cloned by cross-hybridization with a DNA fragment of Klebsiella oxytoca diol dehydrase genes . Since the Escherichia coli clones isolated did not show appreciable enzyme activity, plasmids for high level expression of cloned genes were constructed . The enzyme expressed in E . coli was indistinguishable from the wild-type glycerol dehydrase of K . pneumoniae by the criteria of polyacrylamide gel electrophoretic, immunochemical, and catalytic properties . It was also shown that the recombinant functional enzyme consists of Mr 61,000, 22,000, and 16, 000 subunits . Sequence analysis of the genes revealed four open reading frames separated by 2-12 bases . The sequential three open reading frames from the first to the third (gldA, gldB, and gldC genes) encoded polypeptides of 555, 194, and 141 amino acid residues with predicted molecular weights of 60,659(alpha), 21,355(beta), and 16,104(gamma), respectively . High level expression of these three genes in E . coli produced more than 14-fold higher level of fully active apoenzyme than that in K . pneumoniae . It was thus concluded that these are the genes encoding the subunits of glycerol dehydrase . The deduced amino acid sequences of the three subunits were 71, 58, and 54% identical with those of the alpha, beta, and gamma subunits of diol dehydrase, respectively, but failed to show any apparent homology with other proteins. Jpn Circ J, 1996 Sep, 60(9), 703 - 6 Klebsiella pneumoniae-induced mycotic aneurysm of the abdominal aorta complicated by bloody pleural effusion--a case report; Lai CP et al.; A 68-year-old diabetic male who suffered from recurrent severe lumbago and high fever was found to have mycotic abdominal aneurysm . His symptoms did not improve after maximum-dose antibiotic therapy . Bloody pleural effusion on the left side was noticed hours before he expired . Klebsiella pneumoniae alone was isolated from blood from cellulitis-related bacteremia, when aneurysm formation was complete and later from bloody pleural effusion . To our knowledge, this is the first report of mycotic abdominal aneurysm of solely Klebsiella pneumoniae complicated by bloody pleural effusion. J Antimicrob Chemother, 1996 Sep, 38(3), 409 - 24 Antibiotic resistance and production of extended-spectrum beta-lactamases amongst Klebsiella spp . from intensive care units in Europe; Livermore DM et al.; Consecutive klebsiellae were collected from ICU patients at 35 centres in Western and Southern Europe . Of 966 isolates obtained, 716 were Klebsiella pneumoniae, 248 were Klebsiella oxytoca and two were Klebsiella ozaenae . Most were from Belgium, France, Germany, Holland, Italy, Portugal, Spain, Turkey and a few from Greece and the UK . Production of extended-spectrum beta-lactamases (ESBLs) was inferred in 220 isolates on the basis of synergy between ceftazidime and clavulanate . Putative ESBL producers were received from 23 centres, including 20 of the 27 that contributed more than 10 klebsiellae . Over 88% of putative ESBL producers were resistant to ceftazidime 2 mg/L, ceftriaxone 1 mg/L and aztreonam 1 mg/L, whereas, amongst ESBL-negative isolates, more than 98% of K . pneumoniae and 87% of K . oxytoca were susceptible to these concentrations . Putative ESBL producers wre also more resistant to cefuroxime and cefoxitin than non-producers, but not to biapenem . MIC distributions of ciprofloxacin, piperacillin/tazobactam and aminoglycosides were bimodal for ESBL producers, with some isolates highly sensitive and others very resistant . For example, 70% of putative ESBL producers were susceptible to piperacillin/tazobactam 16 + 4 mg/L, but 30% were resistant, some highly so . Resistance to this combination, and to ciprofloxacin, was clustered in certain centres . Two other groups of cephalosporin-resistant isolates were identified besides ESBL producers, viz . (i) nine isolates, from three centres, with AmpC beta-lactamases and (ii) 20 K . oxytoca, from 15 centres, that hyperproduced K1 enzyme . Examination of the hospitals' own susceptibility data indicated that up to 33% of putative ESBL producers had been reported susceptible to third-generation cephalosporins or monobactams . This is disturbing, since ESBLs have been associated with clinical failure even when only low-level resistance was apparent in vitro. Antimicrob Agents Chemother, 1996 Sep, 40(9), 1988 - 94 Molecular aspects of high-level resistance to sulbactam-cefoperazone in Klebsiella oxytoca clinical isolates; Kimura K et al.; Nine Klebsiella oxytoca strains which demonstrated resistance to the combination of sulbactam and cefoperazone were isolated from geographically separate hospitals in Japan in 1995 . Among them, K . oxytoca SB23 showed high-level resistance to sulbactam-cefoperazone (MIC > 128 micrograms/ml) and aztreonam (MIC, 128 micrograms/ml) . The sulbactam-cefoperazone resistance was not transferred from strain SB23 to Escherichia coli CSH2 by conjugation, beta-Lactamase RbiA, produced by strain SB23, was purified, and the molecular mass was estimated to be 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Kinetic parameters for RbiA revealed that cefoperazone and aztreonam were hydrolyzed efficiently by this enzyme . Moreover, ceftazidime and imipenem were also hydrolyzed weakly by RbiA, although strain SB23 did not show any resistance to these agents . Clavulanate, sulbactam, and tazobactam failed to block the hydrolysis of cefoperazone by RbiA . The structural gene of RbiA (blaRBI) was cloned and sequenced, and the deduced amino acid sequence of RbiA demonstrated high-level similarities to those of the beta-lactamases found in K . oxytoca D488, E23004, and plasmid-mediated MEN-1, which have been classified into Bush functional group 2be . Although RbiA demonstrates high-level molecular similarity to the enzymes in group 2be, from an enzymological point of view, this enzyme might be differentiated from the enzymes in that group . Hybridization analysis revealed that beta-lactamase genes highly similar to blaRBI were generally encoded on the chromosome of the sulbactam-cefoperazone-resistant clinical isolates of K . oxytoca tested in the study, despite their different derivations . This observation suggests that sulbactam-cefoperazone-resistant A . oxytoca strains which produce RbiA-type beta-lactamases have been proliferating in many hospitals in Japan. J Bacteriol, 1996 Sep, 178(18), 5417 - 21 Purification and activation properties of UreD-UreF-urease apoprotein complexes; Moncrief MB et al.; In vivo assembly of the Klebsiella aerogenes urease nickel metallocenter requires the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE . Prior studies had shown that urease apoprotein exists in an uncomplexed form as well as in a series of UreD-urease (I.-S . Park, M.B . Carr, and R.P . Hausinger, Proc . Natl . Acad . Sci . USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S . Park and R.P . Hausinger, J . Bacteriol . 177:1947-1951, 1995) apoprotein complexes . This study demonstrates the existence of a distinct series of complexes consisting of UreD, UreF, and urease apoprotein . These novel complexes exhibited activation properties that were distinct from urease and UreD-urease apoprotein complexes . Unlike the previously described species, the UreD-UreF-urease apoprotein complexes were resistant to inactivation by NiCl2 . The bicarbonate concentration dependence for UreD-UreF-urease apoenzyme activation was significantly decreased compared with that of the urease and UreD-urease apoproteins . Western blot (immunoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF . We propose that the binding of UreF modulates the UreD-urease apoprotein activation properties by excluding nickel ions from binding to the active site until after formation of the carbamylated lysine metallocenter ligand. J Bacteriol, 1996 Sep, 178(18), 5410 - 6 Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus; Brayman TG et al.; Klebsiella aerogenes UreE, one of four accessory proteins involved in urease metallocenter assembly, contains a histidine-rich C terminus (10 of the last 15 residues) that is likely to participate in metal ion coordination by this nickel-binding protein . To study the function of the histidine-rich region in urease activation, ureE in the urease gene cluster was mutated to result in synthesis of a truncated peptide, H144* UreE, lacking the final 15 residues . Urease activity in cells containing H144* UreE approached the activities for cells possessing the wild-type protein at nickel ion concentrations ranging from 0 to 1 mM in both nutrient-rich and minimal media . In contrast, clear reductions in urease activities were observed when two ureE deletion mutant strains were examined, especially at lower nickel ion concentrations . Surprisingly, the H144* UreE, like the wild-type protein, was readily purified with a nickel-nitrilotriacetic acid resin . Denaturing polyacrylamide gel electrophoretic analysis and N-terminal sequencing confirmed that the protein was a truncated UreE . Size exclusion chromatography indicated that the H144* UreE peptide associated into a homodimer, as known for the wild-type protein . The truncated protein was shown to cooperatively bind 1.9 +/- 0.2 Ni(II) ions as assessed by equilibrium dialysis measurements, compared with the 6.05 +/- 0.25 Ni ions per dimer reported previously for the native protein . These results demonstrate that the histidine-rich motif is not essential to UreE function and is not solely responsible for UreE nickel-binding ability . Rather, we propose that internal nickel binding sites of UreE participate in urease metallocenter assembly. J Infect Dis, 1996 Sep, 174(3), 529 - 36 Ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli bloodstream infection: a case-control and molecular epidemiologic investigation; Schiappa DA et al.; In a molecular, microbiologic, and case-control study to describe the epidemiology of ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli bloodstream infection, 32 unique isolates were recovered over 31 months from the blood of patients hospitalized in a 900-bed hospital in Chicago . Multivariate analysis revealed cases occurred more frequently in debilitated nursing home patients with central venous catheters than in younger, healthier patients . Mortality rates were similar for cases and controls . Case-patients were less likely to die if they received appropriate antibiotic treatment within 3 days of bacteremia onset (P = .02) . Pulsed-field gel electrophoresis analysis indicated a polyclonal outbreak, with strain-specific temporal and geographic clustering . Isoelectric focusing results suggested that a predominant enzyme, TEM-10, was responsible for the ceftazidime resistance . The resistance gene was usually carried on a large conjugative plasmid . The polyclonality of the resistant strains suggests that ceftazidime resistance due to TEM-10 is now endemic in Chicago. J Bacteriol, 1996 Sep, 178(17), 5205 - 14 Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species; Kelly RF et al.; Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I . Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries . In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked . The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8 . By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I . The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core . Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable . Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization . However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions . The clonal groups cannot be predicted by features of the O-antigen structure . To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype . The nucleotide sequences for the six rfb genes showed variations in moles percent G+C values and in the values for nucleotide sequence identity, which ranged from 66.9 to 79.7% . The predicted polypeptides ranged from 64.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity) . The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb genes through recent lateral transfer events. Biochemistry, 1996 Aug 20, 35(33), 10616 - 26 Structures of the Klebsiella aerogenes urease apoenzyme and two active-site mutants; Jabri E et al.; Urease from Klebsiella aerogenes {Jabri et al . (1995) Science 268, 998-1004} is an (alpha beta gamma)3 trimer with each alpha-subunit having an (alpha beta)8-barrel domain containing a binickel active center . Here we examine structure-function relations for urease in more detail through structural analysis of the urease apoenzyme at 2.3 A resolution and mutants of two key catalytic residues (H219A and H320A) at 2.5 A resolution . With the exception of the active site, in which a water molecule takes the place of the missing carbamate and nickel atoms, the structure of the apoenzyme is nearly identical to that of the holoenzyme, suggesting a high degree of preorganization which helps explain the tight binding of nickel . In the structure of H219A, the major change involves a conformational shift and ordering of the active site flap, but a small shift in the side chain of Asp alpha 221 could contribute to the lower activity of H219A . In the H320A structure, the catalytic water, primarily a Ni-2 ligand in the holoenzyme, shifts into a bridging position . This shift shows that the nickel ligation is rather sensitive to the environment and the change in ligation may contribute to the 10(5)-fold lower activity of H320A . In addition, these results show that urease is resilient to the loss of nickel ions and mutations . Analysis of the urease tertiary/quaternary structure suggests that the stability of this enzyme may be largely due to its burial of an unusually large fraction of its residues: 50% in the gamma-subunit, 30% in the beta-subunit, and 60% in the alpha-subunit. Biochem J, 1996 Aug 15, 318 ( Pt 1), 111 - 8 Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P-clusters are replaced by the non-coordinating amino acid alanine; Yousafzai FK et al.; Nitrogenase MoFe protein (Kp1) from the mutant strain pHK17 or Klebsiella pneumoniae has been purified to give three catalytically active fractions . In this mutant, each of the two bridging cysteine ligands to the P-clusters, alpha-Cys-89 and beta-Cys-94, has been replaced by a non-coordinating residue, alanine . SDS/PAGE and earlier native gels showed that the three fractions retained the normal alpha 2 beta 2 tetrameric form of wild-type Kp1; therefore we conclude that in each of the fractions the subunits are folded differently, thus resulting in different surface charges and allowing separation of the fractions on ion-exchange chronatography . Earlier EPR and magnetic CD data had shown that the mutant fractions contain P-clusters, and thus the mutated residues are not as essential for maintaining the integrity of the P-clusters as they appear from the X-ray structure . The specific activity of each of the three fractions was less than that of wild-type Kp1, the most active fraction having only 50% of wild-type activity . No change in substrate specificity or in the relative distribution of electrons to various substrates was found . The relationship between ATP hydrolysis and substrate-reducing activity, the EPR spectra of the S = 3/2 spin state of the iron-molybdenum cofactor (FeMoco) and the pH profile of acetylene-reduction activities of the three fractions did not differ significantly from those exhibited by wild-type Kp1 . The specific activities of the three mutant fractions and of wild-type Kp1 were linearly proportional to the intensity of the S = 3/2 EPR signal from the FeMoco centres . This implies that those molecules of the three mutant fractions and the wild-type protein that contain EPR-active FeMoco are fully active, i.e . that the Cys to Ala substitution of the P-cluster ligands does not affect the specific activity of the protein . This in turn implies that the P-clusters are not directly associated with the rate-limiting step in enzyme turnover . We conclude that the lower specific activities of the mutant fractions are observed because the fractions are mixtures of species containing a full complement of FeMoco and P-clusters and species lacking some or all of these clusters . On the basis of the Mo contents and EPR spectroscopy of the mutant fractions, we propose that the loss of the P-clusters causes (i) the physical loss or inhibition of binding of some FeMoco; (ii) the EPR and catalytic inactivation of some FeMoco; and/or (iii) the incorporation of a FeMoco-like species into the FeMoco site of the mutant molecules. J Biol Chem, 1996 Aug 2, 271(31), 18632 - 7 Characterization of the mononickel metallocenter in H134A mutant urease; Park IS et al.; A mutant form of Klebsiella aerogenes urease possessing Ala instead of His at position 134 (H134A) is inactive and binds approximately half the normal complement of nickel (Park, I.-S., and Hausinger, R . P.(1993) Protein Sci . 2, 1034-1041) . The crystal structure of the H134A protein was obtained at 2.0-A resolution, and it confirms that only Ni-1 of the two nickel ions found in the native enzyme is present . In contrast to the pseudotetrahedral geometry observed for Ni-1 in native urease (where it is liganded by His-246, His-272, one oxygen atom of carbamylated Lys-217, and a water molecule at partial occupancy), the mononickel metallocenter in the H134A protein was found to possess octahedral geometry and was coordinated by the above protein ligands plus three water molecules . The nickel site of H134A urease was probed by UV-visible, variable temperature magnetic circular dichroism, and x-ray absorption spectroscopies . The spectroscopic data are consistent with the presence of Ni(II) in octahedral geometry coordinated by two histidylimidazoles and additional oxygen and/or nitrogen donors . These data underscore the requirement of Ni-2 for formation of active urease and demonstrate the important role of Ni-2 in establishing the proper Ni-1 coordination geometry. Int J Immunopharmacol, 1996 Aug-Sep, 18(8-9), 515 - 9 Correlation between inhibited alternative complement activity and the protective effect induced by Nocardia lysozyme digest (NLD) during Klebsiella pneumoniae infection in mice; Ivanovska N et al.; Nocardia lysozyme digest (NLD) was administered intraperitoneally (i.p.) at a dose of 500 micrograms/kg to normal and immunosuppressed mice for 3 consecutive days prior to inoculation with Klebsiella pneumoniae . A protective effect was observed when the pathogen was injected subcutaneously and intravenously, as opposed to an aggravating effect obtained in the case of intraperitoneal inoculation The i.p . administration of NLD partially restored the immunosuppression caused by cyclophosphamide but did not change cobra venom-induced deterioration of the infection . The results obtained could be regarded as a consequence of the lowered alternative pathway serum complement activity and the crucial role of the diminished level of complement in the peritoneal cavity. Microbiology, 1996 Aug, 142 ( Pt 8), 2115 - 20 In vitro formation of a catabolic plasmid carrying Klebsiella pneumoniae DNA that allows growth of Escherichia coli K-12 on 3-hydroxybenzoate; Robson ND et al.; The four enzymes needed to convert 3-hydroxybenzoate to pyruvate and fumarate via the gentisate pathway, as well as a putative positive regulator protein, were encoded on an 8 kb Sphl fragment of Klebsiella pneumoniae DNA . The five genes were clustered in the order regulator-gentisate dioxygenase-fumarylpyruvate hydrolase-3-hydroxybenzoate monooxygenase-maleylpyruvate isomerase (mhbRDHMI), with the catabolic genes transcribed in the dioxygenase to isomerase direction . 2-Hydroxybenzoate was found to be a non-metabolizable inducer analogue for the mhb genes, supporting the view that gentisate rather than maleylpyruvate was the physiological inducer . The plasmid pNDR20 encoding the full gentisate catabolic pathway endowed Escherichia coli with the ability to grow on 3-hydroxybenzoate but the host cell appeared to be responsible for substrate uptake. J Bacteriol, 1996 Aug, 178(15), 4679 - 87 Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae; Schmitz RA et al.; In Klebsiella pneumoniae, products of the nitrogen fixation nifLA operon regulate transcription of the other nif operons . NifA activates transcription by sigma54-holoenzyme . In vivo, NifL antagonizes the action of NifA under aerobic conditions or in the presence of combined nitrogen . In contrast to a previous report, we show that depletion of iron (Fe) from the growth medium with the chelating agent o-phenanthroline (20 microM) mimics aerobiosis or combined nitrogen in giving rise to inhibition of NifA activity even under anaerobic, nitrogen-limiting conditions . Adding back Fe in only twofold molar excess over phenanthroline restores NifA activity, whereas adding other metals fails to do so . By using strains that lack NifL, we showed that NifA activity itself does not require Fe and is not directly affected by phenanthroline . Hence, Fe is required to relieve the inhibition of NifA activity by NifL in vivo . Despite the Fe requirement in vivo, we have found no evidence that NifL contains Fe or an iron-sulfur (Fe-S) cluster . Determination of the molecular mass of an inhibitory form of NifL overproduced under aerobic conditions indicated that it was not posttranslationally modified . When NifL was synthesized in vitro, it inhibited transcriptional activation by NifA even when it was synthesized under anaerobic conditions in the presence of a high Fe concentration or of superoxide dismutase, which is known to protect some Fe-S clusters . Moreover, overproduction of superoxide dismutase in vivo did not relieve NifL, inhibition under aerobic conditions, and attempts to relieve NifL inhibition in vitro by reconstituting Fe-S clusters with the NifS enzyme (Azotobacter vinelandii) were unsuccessful . Since we obtained no evidence that Fe acts directly on NifL or NifA, we postulate that an additional Fe-containing protein, not yet identified, may be required to relieve NifL inhibition under anaerobic, nitrogen-limiting conditions. Zh Mikrobiol Epidemiol Immunobiol, 1996 Jul-Aug, (4), 3 - 6 {The adhesive activity of Klebsiella pneumoniae controlled by plasmid 55 MD}; Bondarenko VM et al.; In K . pneumoniae clinical strains a nonconjugative plasmid, denoted as pAdh-55 and responsible for their adhesive activity with respect to HEp-2 cells, was detected . The comparison of genetically related pairs of K . pneumoniae strains differing in the presence of pAdh-55 revealed that the loss of this plasmid by bacteria was accompanied by a decrease, and its acquisition by a considerable increase, in the number of Klebsiella-affected cells in the monolayer . The correlation between the presence of pAdh-55 in K . pneumoniae strains and their adhesive activity was established both in experiments aimed at the morphological study of preparations stained with azure eosin and in experiments with isotope-labeled bacteria. Biol Signals, 1996 Jul-Aug, 5(4), 203 - 8 Expression and regulation of chemokines in acute bacterial pneumonia; Standiford TJ et al.; Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which are dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines . In this study, we have demonstrated that both C-X-C and C-C chemokines are integral components of antibacterial host defense . Specifically, studies in vitro indicate that macrophage inflammatory protein-2 (MIP-2) and MIP-1 alpha augment the ability of PMN and alveolar macrophages, respectively, to phagocytose and kill Escherichia coli . In addition, MIP-2 and MIP-1 alpha are expressed within the lung in response to the intratracheal instillation of Klebsiella pneumoniae, and the inhibition of MIP-2 bioactivity in vivo results in decreases in lung PMN influx, bacterial clearance, and early survival . Finally, the anti-inflammatory cytokine interleukin-10 (IL-10) is also expressed within the lung during the evolution of Klebsiella pneumonia, and neutralization of IL-10 results in enhanced proinflammatory cytokine production, bacterial clearance, and increases in both short- and long-term survival . In conclusion, our studies indicate that specific chemokines are important mediators of leukocyte recruitment and/or activation in bacterial pneumonia, and that the expression of these chemokines is regulated by endogenously produced IL-10. Ren Fail, 1996 Jul, 18(4), 601 - 5 Mortality in elderly patients with acute renal failure; Santacruz F et al.; In a retrospective study, we identified 55 elderly patients with acute renal failure (ARF) admitted to our hospital during an 8-year period from 1985 to 1993 . Information about the etiology, complications, laboratory data, and treatment course were obtained from the clinical history . Of the 200 patients with ARF admitted to the hospital during this period, 28% were patients more than 60 years old (41 male and 14 female) with an average age of 68.5 +/- 7 years . The main causes of ARF were sepsis, volume depletion, low cardiac output, arterial hypotension, nephrotoxicity by antibiotics, and obstructive uropathy . The global mortality of elderly patients with ARF was 53% . The mortality rate of the different types of the ARF were: prerenal 35%, intrinsic 64% (oliguric 76%, nonoliguric 50%), and postrenal 40% . Mortality as a result of sepsis occurred in 18 patients (62%), by cardiovascular disease in 4 patients (13%), by acute respiratory failure in 2 patients (7%), and by other causes in 5 patients (18%) . In the cases of sepsis, Pseudomonas was detected in 7 cases (39%), Escherichia coli in 2 cases (11%), Gram-negative nonspecific in 3 cases (17%), Klebsiella in 1 case (5%), and in 5 cases (16%), the hemoculture was negative . The patient survival rate was 47% (26 of 55 patients) . Of these patients, 19 recovered their normal renal function (73%), but 7 patients remained with renal failure (27%) . In conclusion, the global mortality in the elderly patients without considering the types of ARF was 53% . The oliguric form had the highest mortality rate with 76% . The main causes for mortality were sepsis with 62%, cardiovascular disease with 13%, and other causes 18%. Mol Microbiol, 1996 Jul, 21(2), 233 - 45 Purification and activities of the Rhodobacter capsulatus RpoN (sigma N) protein; Cannon W et al.; The rpoN-encoded sigma factors (sigma N) are a distinct class of bacterial sigma factors, with no obvious homology to the major sigma 70 class . The sigma N-containing RNA polymerase holoenzyme functions in enhancer-dependent transcription to allow expression of positively controlled genes . We have purified the Rhodobacter capsulatus sigma N protein, which is distinctive in lacking an acidic region implicated in the melting of promoter DNA by the Escherichia coll sigma N holoenzyme, and may represent a minor subclass of sigma N proteins . Assays of promoter recognition and holoenzyme formation and function showed that the purified R . capsulatus sigma N protein is distinct in activity compared to the enteric proteins, but retains the broad functions described for these proteins . As first described for the Klebsiella pneumoniae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R . capsulatus sigma N protein and its response to core RNA polymerase was clearly different from that determined for the K . pneumoniae and E . coli proteins . Results are discussed in the context of a requirement to modulate the activity of the DNA-binding surfaces of sigma N to regulate sigma N function . Circular dichroism was used to evaluate the structure of the R . capsulatus protein and revealed differences in the tertiary signals as compared to the K . pneumoniae protein, some of which are attributable to the DNA-binding domain of sigma N. J Antimicrob Chemother, 1996 Jul, 38 Suppl A, 107 - 15 Antibiotic susceptibility patterns of respiratory isolates of Klebsiella pneumoniae in Europe and the USA in 1992 and 1993 . The Alexander Project Collaborative Group; Bauernfeind A; The overall antibiotic susceptibility of Klebsiella pneumoniae isolates collected in 1992 (n = 35) and 1993 (n = 85) was highly variable ranging from 0.8% for amoxycillin to 95.8% for ceftriaxone . On a weight for weight basis, the activity of the compounds decreased in the following sequence: ceftriaxone, cefixime, co-trimoxazole, ciprofloxacin, ofloxacin, cefaclor, amoxycillin/clavulanate, doxycycline, cefuroxime, chloramphenicol, amoxycillin . A small percentage of resistant strains was found for each of the compounds (between 0.8% for ceftriaxone and 12.5% for chloramphenicol) with the exception of amoxycillin which was almost uniformly resistant . There was a trend for the percentage of resistant strains to increase between the two study years for all compounds except for chloramphenicol . Resistance to beta-lactams in France and the USA is likely to have been due to strains producing extended spectrum beta-lactamases. Int J Urol, 1996 Jul, 3(4), 301 - 3 Infected renal cystic mass associated with bacterial meningitis: a case report; Fuse H et al.; We report a case of an infected renal cystic mass associated with bacterial meningitis in a 70-year-old woman who had had poorly-controlled diabetes mellitus for approximately 30 years . She suffered from bacterial meningitis due to Klebsiella pneumoniae, which was successfully treated with antimicrobial chemotherapy for 1 month . Approximately 2 weeks later she developed left flank pain and a high fever . A CT scan and an ultrasonogram revealed a left renal cystic mass, which was considered to be an infected renal cyst . Turbid and thick fluid was obtained by percutaneous aspiration which contained numerous white blood cells . Culture of this fluid yielded K . pneumoniae . The bacterial meningitis was considered to be a secondary infection of the septicemia which resulted from the infected renal cystic mass. Zentralbl Bakteriol, 1996 Jul, 284(2-3), 372 - 7 Adhesive properties of P-like fimbriae in Klebsiella-species; Przondo-Mordarska A et al.; Clinical isolates of three encapsulated Klebsiella strains with type 1 (mannose-sensitive, MS+MR-), type 3 (mannose-resistant, MS-MR+), type 1.3 (MS+MR+) fimbriae and facultatively coexpressing P-like fimbria were investigated for their ability to adhere to uroepithelial cells (UECs) and tracheal epithelial cells (TECs) . Irrespective of the type of epithelial cells, adhesion of the MS+MR+ (type 1.3) fimbriated Klebsiella strain was significantly stronger than adhesion of strains carrying only type 1 (MS+MR-) or type 3 (MS-MR+) fimbriae . The coexpression of P-like fimbriae increased the adhesive properties of Klebsiella strains to UECs but not to TECs . Adhesion of P-like fimbriated Klebsiella strains to UECs was significantly inhibited in the presence of the P+ fimbriae-specific Gal alpha-4-Gal beta (galabiose) . Such adhesion was unrelated to the coexpression of type 1, type 3 or type 1.3 fimbriae . However, adhesion to TECs was only moderately inhibited. J Cell Sci, 1996 Jul, 109 ( Pt 7), 1825 - 35 Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants; Stoeckelhuber M et al.; Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner . To further characterize this interaction we isolated hisactophilin overexpression (hisII+) and hisactophilin minus (his-) mutants . D . discoideum contains two hisactophilin isoforms; both genes are independently transcribed and carry a short intron at the same position of the coding region . The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of a total 118 amino acids . After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+) . Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two . Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression . The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette . The properties of the hisII+ and his- cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions . However, the hisII+ transformants revealed a significant difference to wild-type cells and his- cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor . HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his- cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin . Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane . The fact that cells deficient in hisactophilin again lose resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration. Clin Infect Dis, 1996 Jul, 23(1), 179 - 81 Immunogenicity of a 24-valent Klebsiella capsular polysaccharide vaccine and an eight-valent Pseudomonas O-polysaccharide conjugate vaccine administered to victims of acute trauma; Campbell WN et al.; We measured the antibody response in 10 victims of acute blunt trauma and penetrating trauma who were immunized against Klebsiella pneumoniae and Pseudomonas species within 72 hours of injury . The two vaccines, which were previously shown to be safe and immunogenic in uninjured humans, were a 24-valent K . pneumoniae capsular polysaccharide vaccine and an eight-valent Pseudomonas O-polysaccharide-toxin A conjugate vaccine . The patients were between 18 and 44 years of age, had Injury Severity Scores that ranged between 9 and 34, and did not have chronic infections or malignancies . On days 14 and 28 after immunization, all patients had a response of greater than fourfold to at least six of the nine Pseudomonas vaccine antigens . Half of the patients responded to eight of the nine antigens . Nine patients responded to at least 18 of 24 Klebsiella antigens, and seven patients responded to 22 of the 24 antigens . No important side effects were attributed to the vaccines . The results of this preliminary study indicate that active immunization against potential pathogens is possible in victims of acute trauma. Clin Infect Dis, 1996 Jul, 23(1), 118 - 24 Ceftazidime-resistant Klebsiella pneumoniae isolates recovered at the Cleveland Department of Veterans Affairs Medical Center; Rice LB et al.; The rate of ceftazidime resistance among Klebsiella pneumoniae isolates recovered from patients at the Cleveland Department of Veterans Affairs Medical Center increased from 6% in the first quarter of 1993 to 28% in the first quarter of 1994 . The outbreak was hospitalwide, with the highest rates of resistance occurring on wards where ceftazidime was administered most frequently . Although many plasmid patterns were observed in the clinical isolates, molecular epidemiological analysis with use of pulsed field gel electrophoresis revealed substantial similarities between the strains; this finding suggested that most of the strains-if not all of them-were derived from the original clone . The addition of piperacillin/tazobactam to the hospital formulary and educational efforts focused on minimizing the administration of ceftazidime were associated with a marked decrease in the drug's use and a concomitant decrease in the percentage of ceftazidime-resistant isolates . We have not yet observed a significant rise in the rate of resistance to piperacillin/tazobactam among clinical isolates of K . pneumoniae. Biochem J, 1996 Jul 1, 317 ( Pt 1), 285 - 90 Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae {corrected}; Cornell KA et al.; Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiella pneumoniae . Chromatography using a novel 5'-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form . The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes . Purified MTR kinase exhibits an apparent molecular mass of 46-50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 microM . Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 microM . Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins . However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase. J Bacteriol, 1996 Jul, 178(13), 3949 - 52 Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus; Gennaro G et al.; The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF . nifF was found to not be contained in the previously described nif regions I, II, and III . Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae . Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at the protein level . Insertional mutagen esis showed that NifF contributes significantly to nitrogenase activity under normal nitrogen-fixing conditions and that it is absolutely required for nitrogen fixation under iron limitation. J Am Coll Surg, 1996 Jul, 183(1), 56 - 60 Intrahepatic bile duct stenosis causing intrahepatic calculi formation following excision of a choledochal cyst; Ando H et al.; BACKGROUND: Formation of intrahepatic calculi is one of the major late complications after excision of a choledochal cyst . There are few studies, however, that have examined this complication . Generally, an anastomotic stricture is believed to be the main cause of intrahepatic calculi . We report our experience with eight patients who had intrahepatic calculi after excision of a choledochal cyst . STUDY DESIGN: To determine what caused the intrahepatic calculi to form, seven patients underwent cholangioscopy and direct visual inspection during the operation, and one patient underwent percutaneous transhepatic cholangioscopy . Intrahepatic bile was cultured, and calculi were analyzed . RESULTS: Two types of stenoses (membranous and septal) were demonstrated near the hepatic hilum in all patients . Calculi were always located on the hepatic side of the stenoses . No anastomotic strictures were found in the region of the hepaticojejunostomy . The calculi contained mainly calcium bilirubinate . Escherichia coli and Klebsiella pneumoniae were cultured from the bile in all patients . CONCLUSIONS: Stenoses of the intrahepatic bile ducts were demonstrated in all eight patients . The stenoses were considered to be the primary cause of intrahepatic calculi formation after excision of the choledochal cysts. Enzyme Microb Technol, 1996 Jul, 19(1), 68 - 73 Heterologous expression of full-length and truncated forms of the recombinant guluronate-specific alginate lyase of Klebsiella pneumoniae; Hicks SJ et al.; The alyA gene, which encodes a guluronate-specific alginate lyase from Klebsiella pneumoniae, was cloned into the plasmid pCT54 to facilitate heterologous expression of the enzyme in Escherichia coli . The greatest enzyme specific activity was observed when the complete gene and flanking regions of DNA (contained within a 1.95 kb HindIII fragment) were cloned in the correct orientation relative to the vector-encoded trp promoter . Heterologous expression of the intact gene complete with flanking regions resulted in a 20-fold increase in enzyme yield compared to the original construct, pRC5 . PCR mutagenesis and/or restriction endonuclease digestion was used to generate a series of fragments containing either the whole or truncated versions of the gene . Active enzyme was produced from constructs in which the region encoding the signal peptide had been deleted, although the recombinant protein was retained within the bacterial cells . An internal methionine (position 74) could be used as a start site for translation but resulted in the accumulation of inactive protein within inclusion bodies. J Biochem (Tokyo), 1996 Jun, 119(6), 1118 - 23 Identification and characterization of the acoD gene encoding a dihydrolipoamide dehydrogenase of the Klebsiella pneumoniae acetoin dehydrogenase system; Peng HL et al.; The acoD gene, which encodes a dihydrolipoamide dehydrogenase component of the acetoin dehydrogenase enzyme system of Klebsiella pneumoniae was isolated and the nucleotide sequence determined . The gene is capable of encoding a protein of 465 amino acid residues with conserved binding domains for NAD and FAD, and two redox-active cysteine residues . The acoD gene product exhibited a Michaelis constant of 170 microM for NAD, while NADP can not be used as a substrate . The purified enzyme appeared to be a dimer of the acoD gene product . It did not associate tightly with the E1 and E2 components of either acetoin dehydrogenase or 2-oxoglutarate dehydrogenase to form an active multi-enzyme complex. Mol Microbiol, 1996 Jun, 20(6), 1235 - 45 Multimers of the precursor of a type IV pilin-like component of the general secretory pathway are unrelated to pili; Pugsley AP; Both the mature and precursor forms of PulG, a type IV pilin-like component of the general secretory pathway of Klebsiella oxytoca, can be chemically cross-linked into multimers similar to those obtained by cross-linking the components of type IV pili . To explore the possibility that the PulG precursor could form a pilus-like structure, the PulG sequence was altered in a variety of ways, including (i) replacement of the characteristic hydrophobic region, which is required for the assembly of type IV pilins by the MalE signal peptide, or (ii) fusion of beta-lactamase (beta laM) to the C-terminus . Neither of these changes affected multimerization . PulG precursor could be post-translationally processed by prepilin peptidase (PulO), indicating that the N-terminus of prePulG remains on the cytoplasmic side of the cytoplasmic membrane where it is accessible to the catalytic site of this enzyme . Finally, precursor and mature forms of PulG could be efficiently cross-linked in a mixed dimer, indicating that at least a subpopulation of the two forms of the protein are probably located in clusters in the cytoplasmic membrane . These results provide further evidence that the cross-linked multimers of the precursor form of PulG are unrelated to type IV pilus-like structures . It is still unclear whether a subpopulation of processed PulG can be assembled into a rudimentary pilus-like structure. Infect Immun, 1996 Jun, 64(6), 2266 - 73 A new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae strains involved in nosocomial infections; Di Martino P et al.; We purified and characterized a new fimbria termed KPF-28 (Klebsiella pneumoniae fimbria with a fimbrin molecular mass of 28 kDa) involved in K . pneumoniae adherence to the human carcinoma cell line Caco-2 . Electron microscopy of bacterial surface protein preparations and immunogold labeling of bacterial cells showed that KPF-28 was a long, thin, and flexible fimbria about 4 to 5 nm in diameter and 0.5 to 2 microm long . The N-terminal amino acid sequence of the KPF-28 major fimbrial subunit showed no homology with type 1 and type 3 pili of K . pneumoniae but showed 61.7% identity with residues 6 to 19 of the N-terminal amino acid sequence of PapA, the Pap major pilus subunit expressed by uropathogenic Escherichia coli strains . Total amino acid content determination showed that the KPF-28 major subunit composition was close to that of the GVVPQ fimbrial family major subunits expressed by pathogenic E . coli strains . The study of the prevalence of KPF-28 among K . pneumoniae strains involved in nosocomial infections revealed that KPF-28 was found in the great majority of the K . pneumoniae strains producing the CAZ-5/SHV-4 extended-spectrum beta-lactamase . As shown by curing and mating experiments, the R plasmid encoding the CAZ-5/SHV-4 enzyme was found to be involved in but not solely responsible for KPF-28 expression . Hybridization experiments using an oligonucleotide probe corresponding to the N-terminal part of the 28-kDa protein revealed that the structural gene encoding the KPF-28 major subunit was localized on this R plasmid . KPF-28 is a putative colonization factor of the human gut, since the ceftazidine-sensitive derivative strain CF914-1C no longer adhered and since the Fab fragments of antibodies raised against KPF-28 inhibited adhesion of K . pneumoniae CF914-1 to the Caco-2 cell line. J Med Microbiol, 1996 Jun, 44(6), 444 - 52 Analysis of the fim cluster of an avian O2 strain of Escherichia coli: serogroup-specific sites within fimA and nucleotide sequence of fimI; Marc D et al.; Escherichia coli MT78, an avian pathogenic strain of serogroup 02, produces a variant form of type 1 fimbriae with distinct antigenic properties and apparent mol . wt of the major subunit . The fim gene cluster of strain MT78 was cloned and its sequence was determined in a region spanning upstream of fimB to the beginning of fimD . Whereas most genes were well conserved relative to fim genes previously described, comparison of the fimA gene from strain MT78 with homologous sequences from other strains of E . coli and Klebsiella pneumoniae revealed that most differences were clustered in four well defined regions . A PCR assay, based upon these variable sequences, allowed amplification of a fragment of gene fimA which is specific for most 02 strains . In addition, the sequence of the previously uncharacterised gene fimI, which is located between genes fimA and fimC, was determined. Microb Pathog, 1996 May, 20(5), 255 - 61 Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43; Chang HY et al.; Non-mucoid mutants of a Klebsiella pneumoniae wild type strain CG43 were generated by transposon mutagenesis . One of the mutants was incapable of fermenting galactose and was designated CG43-17 . Alterations of the bacterial surface including capsule, lipopolysaccharides, and several species of outer membrane proteins were noted . The mutant was avirulent to mice and became highly sensitive to human serum . The defects could not be complemented by the gaIETK operon . Diminished activity of UDP-glucose pyrophosphorylase in CG43-17 suggested that it is a gaIU mutant . This possibility was confirmed because the parental phenotypes could be fully restored in the mutant by transforming it with a human liver cDNA encoding UDP-glucose pyrophosphorylase. Plasmid, 1996 May, 35(3), 204 - 10 A novel plasmid gene involved in bacteriophage PRD1 infection and conjugative host-range; Holcik M et al.; PRD1 infects bacteria carrying IncN plasmids by binding to their conjugative pili . Mutations in a plasmid locus kikA close to the pilus region result in PRD1 resistance and reduced conjugation proficiency to Klebsiella but not to Escherichia coli . One of the two genes of kikA is sufficient to restore both normal phenotypes . PRD1 binds to cells carrying the mutant plasmid but fails to inject its genome. Plasmid, 1996 May, 35(3), 189 - 203 Structure and mode of action of kikA, a genetic region lethal to Klebsiella oxytoca and associated with conjugative antibiotic-resistance plasmids of the IncN group; Holcik M et al.; Transmission of conjugative plasmids of the IncN group into Klebsiella oxytoca, but not into Escherichia coli, results in the marked reduction of viability of the recipients . In the plasmid pCU1 a 500-bp locus called kikA has the major role in determining this phenotype . Expression of two open reading frames (orf104 and orf70) is required for the Kik+ phenotype and they can function in cis or in trans . orf104 encodes a 8.9-kDa soluble protein which is translocated into the periplasm . orf70 encodes a 7.6-kDa soluble protein which is found in the cytoplasm . The expression of the kikA region from its natural promoter(s) is positively regulated at the level of transcription by an additional plasmid locus which is located within the tra region . Further experiments show that the action of kikA causes reversible growth inhibition but does not affect cellular respiration and does not induce any morphological changes of the host cell. Zh Mikrobiol Epidemiol Immunobiol, 1996 May-Jun, (3), 106 - 9 {A mathematical model of operative analysis and prognosis in outbreaks of hospital infections in newborn infants (exemplified by Klebsiella infection)}; Boev BV et al.; The results of investigations on the development of a mathematical model (computer program) for the study (imitation) of outbreaks of hospital infections among newborns, caused by Klebsiella pneumoniae . The mathematical model, when "saturated" with real data of the outbreak, opens wide possibilities for the operative analysis and prognosis of morbidity and mortality levels among newborns at the intensive therapy departments of maternity clinics and hospitals . The possibilities of the model (computer program) are illustrated by the prognostic calculations of 3 outbreaks (hypothetical) of hospital infections at the intensive therapy department of the university clinic of Universitario del Valle, Cali (Colombia), resulting from violations of sanitary regulations by medical staff members. J Antimicrob Chemother, 1996 May, 37(5), 931 - 42 In-vitro susceptibility of Klebsiella oxytoca strains to 13 beta-lactams in the presence and absence of beta-lactamase inhibitors; Fournier B et al.; The susceptibility of Klebsiella oxytoca isolates was tested by an agar diffusion method (167 strains collected in six countries) and an agar dilution method (38 strains) . Multivariate analysis of inhibition zone diameters by principal component analysis clearly individualised four susceptibility patterns, including the phenotype of strains overproducing beta-lactamase and resistant to penicillins, first-generation cephalosporins, cefuroxime and aztreonam, but susceptible to ceftazidime . This phenotype was different from that conferred by plasmid-mediated extended-spectrum beta-lactamases; strains expressing these enzymes were also resistant to ceftazidime and cefotaxime . The bla(oxy) gene from K . oxytoca was introduced into Escherichia coli and K . oxytoca recipients and conferred increased resistance to beta-lactams in the recipient cells . Clavulanic acid was effective in association with piperacillin (MIC decreased 36-fold), ceftriaxone (35-fold) and aztreonam (19-fold) against overproducing strains, in spite of a relatively high IC50 (0.3 microM) . Sulbactam (IC50, 400 microM) was ineffective in this context when combined with piperacillin (MIC decreased 1.5-fold), ceftriaxone (1.6-fold) and aztreonam (1.6-fold) . The inhibitory activity of tazobactam (IC50, 8.2 microM) was heterogeneous depending on the strain and the beta-lactam with which it was combined . When combined with piperacillin or ceftriaxone little potentiation in antibiotic activity occurred (MIC decreased 3.9-fold and 4.5-fold, respectively); however, tazobactam plus aztreonam resulted in a 50-fold decrease in MIC of antibiotic. Mol Microbiol, 1996 May, 20(3), 559 - 68 Identification and sequence of a nifJ-like gene in Rhodospirillum rubrum: partial characterization of a mutant unaffected in nitrogen fixation; Lindblad A et al.; A nifJ-like gene was identified in the photosynthetic purple non-sulphur bacterium Rhodospirillum rubrum . A DNA segment hybridizing to Klebsiella pneumoniae nifJ was isolated, the gene was inactivated, and a mutant strain, SNJ-1, was constructed by allele replacement . The mutation was confirmed by DNA sequencing . Northern blotting and by the lack of pyruvate oxidoreductase activity . This is the first report of a nifJ-like gene in photosynthetic bacteria . Unexpectedly, SNJ-1 was capable of nitrogen fixation, and growth was similar to the wild-type strain under all conditions investigated . Therefore, this is also the first demonstration that a nifJ homologue, when present, is not essential for nitrogen fixation in a diazotroph . The nifJ-like gene was sequenced and found to have considerable similarity to published nifJ gene sequences from other organisms . By primer extension, the initiation site for transcription was located, and a typical sigma 54 promoter sequence was identified. Eur J Biochem, 1996 May 1, 237(3), 584 - 91 Cloning of the maoA gene that encodes aromatic amine oxidase of Escherichia coli W3350 and characterization of the overexpressed enzyme; Steinebach V et al.; The mao operon of Escherichia coli W3350, which comprises the genes maoC and maoA, was cloned and appeared to be similar to that of Klebsiella aerogenes {Sugino, H., Sasaki, M., Azakami, H., Yamashita, M . & Murooka, Y . (1992) J . Bacteriol . 174, 2485-2492} . The gene that encodes aromatic amine oxidase (maoA) was isolated, sequenced, and expressed in E . coli TG2 . The purified enzyme exhibited properties characteristic of a copper/topaquinone(TPQ)-containing amine oxidase with respect to the optical absorption and EPR spectra, the size of the subunits, and the optical absorption spectra obtained upon derivatization with hydrazines . However, high-resolution anion-exchange chromatography revealed that the preparation was heterogeneous . The enzyme preparation appeared to consist of at least four enzyme species with different specific activities, A474nm/A340nm ratios and TPQ/subunit ratios . Since the overall properties of the overexpressed enzyme and the authentic enzyme were similar and the separated enzyme species had identical N-terminal amino acid sequences, the heterogeneity does not seem to be caused by improper expression of the gene in the recombinant strain but by factors that interfere with the processing of the specific tyrosine in the precursor enzyme to functional TPQ . Although other causes cannot be excluded, the spectral data and TPQ/subunit ratios reported in the literature for other amine oxidases suggest that suboptimal synthesis of functional TPQ also occurs in other organisms. J Bacteriol, 1996 May, 178(10), 2975 - 7 Perturbation of nifT expression in Klebsiella pneumoniae has limited effect on nitrogen fixation; Simon HM et al.; In the nitrogenase system of Klebsiella pneumoniae, nifT is located between nifDK, the structural genes for dinitrogenase, and nifY, whose product is involved in nitrogenase maturation . It is, therefore, a reasonable hypothesis that the NifT protein might also have a role in the maturation of nitrogenase . However, the phenotypic characterization of nifT and nifT-overexpressing strains for effects on the regulation, maturation, and activity of nitrogenase identified no properties that were distinct from those of the wild type . We conclude that the K . pneumoniae NifT protein is not essential for nitrogen fixation under the conditions examined. Infect Immun, 1996 May, 64(5), 1720 - 3 ADP-ribosylation of an approximately 70-kilodalton protein of Klebsiella pneumoniae; Geipel U et al.; An approximately 70-kDa protein in the culture supernatant of a human pathogenic strain of Klebsiella pneumoniae was labeled in the presence of {32P-adenylate}NAD . Labeling was significantly increased by the addition of dithiothreitol ( > 1 mM) but prevented by treatment of the culture supernatant for 3 min at 56 degrees C . The addition of unlabeled NAD, but not of ADP-ribose, blocked labeling of the approximately 70-kDa protein . The radioactive label was released by formic acid but not by HgCl2 (1 mM) or neutral hydroxylamine (0.5 M) . The addition of homogenates of human platelets, human neutrophils, rat brain, rat lung, or rat spleen tissues to the culture supernatant did not induce labeling of eukaryotic proteins . The data indicate that the K . pneumoniae strain produces ADP-ribosyltransferase which modifies an endogenous protein. Biochemistry, 1996 Apr 23, 35(16), 5345 - 52 Metal ion interaction with urease and UreD-urease apoproteins; Park IS et al.; Klebsiella aerogenes urease in a Ni-containing enzyme (two Ni per alpha beta gamma unit) that is purified as an apoprotein from cells grown in Ni-free medium . Partial activation of urease and UreD-urease apoproteins is achieved in vitro by incubation in the presence of Ni(II) and CO2, whereas incubation of these proteins with Ni alone leads to the formation of inactive species {Park, I.-S., & Hausinger, R . P . (1995) Science 267, 1156-1158} . Here we determined the kinetics of these inhibitory reactions and demonstrated the presence of two Ni ions per alpha beta gamma unit in the inactive proteins . Although metal-substituted urease has never been purified from Ni-deprived cell, several other metal ions were shown to bind to the urease apoproteins . Divalent Zn, C, Co, and Mn all inhibited Ni- and Co2-promoted urease activation at concentrations below that of Ni, whereas Mg and Ca ions did not inhibit this process . Ni-inhibited species recovered their ability to be partially activated after EDTA treatment . In contrast, samples that were exposed to Co or Cu ions were irreversibly inactivated, and EDTA treatment of Zn- or Mn-inhibited samples led to reduced levels of activation competence . Mn-substituted urease, generated from urease apoprotein samples in a Mn- and Co2-dependent manner, was shown to be active, whereas other metal-substituted forms if urease lacked activity . The Mn-protein possessed only 2% of the activity of Ni-activated apoprotein { approximately 8.0 vs approximately 400 mumol min-1 (mg protein)-1}, but its KM value was only moderately altered from that of the native enzyme (3.86 +/- 0.15 mM vs 0.2 mM) . Unlike the Ni-containing enzyme, Mn-urease was inhibited by EDTA . Given the evidence that urease apoprotein binds numerous metal ions, we speculate on possible roles for the UreD, UreF, and UreG accessory proteins in urease activation. Int J Cardiol, 1996 Apr 19, 54(1), 69 - 72 Afebrile spontaneous pneumopyopericardium; Tsai WC et al.; A 77-year-old woman had spontaneous pneumopyopericardium caused by Klebsiella pneumoniae . Chest X-ray revealed a horizontal air-fluid level in the cardiac shadow . Echocardiogram showed characteristic spontaneous contrast echoes in the pericardial effusion . She received pericardiectomy and remained in good condition postoperatively. Gene, 1996 Apr 17, 170(1), 101 - 6 Cloning, sequencing and transcriptional regulation of the draT and draG genes of Azospirillum lipoferum FS; Inoue A et al.; From Azospirillum lipoferum (Al) FS, a nitrogen-fixing bacterium isolated from the rhizosphere of rice, we cloned and sequenced draT, encoding dinitrogenase reductase ADP-ribosyltransferase, and draG, encoding dinitrogenase reductase-activating glycohydrolase . The nucleotide sequences of draTG showed extensive similarity to the same genes from Azospirillum brasilense, Rhodospirillum rubrum and Rhodobacter capsulatus, and they are assumed to be co-transcribed as a single operon . When this draTG operon was introduced into Klebsiella oxytoca, this organism acquired the ability to respond to extracellular NH(+4) ions with reversible inhibition of nitrogenase activity, similar to that seen in Al FS . We constructed a plasmid containing a draT::lacZ gene fusion and found that beta-galactosidase activity was detected under microaerobic conditions, regardless of NH(+4) concentration, but not under aerobic conditions . This indicates that the transcription of draTG responds to the level of oxygen, but not to that of NH(+4) ions. Biochemistry, 1996 Apr 16, 35(15), 4689 - 96 The acyl carrier protein of malonate decarboxylase of Malonomonas rubra contains 2'-(5"-phosphoribosyl)-3'-dephosphocoenzyme A as a prosthetic group; Berg M et al.; Malonate decarboxylase of Malonomonas rubra is composed of soluble and membrane-bound components and contains an acetyl residue that is essential for catalytic activity . Upon incubation with hydroxylamine, the acetyl residue is removed, forming an inactive thiol enzyme, which is reactivated by acetylation with ATP, acetate, and a specific ligase . After incubation of the thiol enzyme with iodoacetate in the presence of excess dithioerythritol, the prosthetic group thiol residue was carboxymethylated and reactivation by acetylation was impaired . Radioactive labeling with {1-14C} iodoacetate revealed the site of carboxymethyation on a distinct cytoplasmic protein with the apparent molecular mass of 14 000 Da . The same protein was specifically labeled by enzymic acetylation of the thiol enzyme with {1-14C}acetate and ATP . Malonate decarboxlyation by {14C}acetyl malonate decarboxlyation resulted in the release of the radioactive acetyl residue from the enzyme,indicating that this acetyl residue is exchanged for a malonyl residue during catalysis . The acyl carrier protein has been purified as its {14C}carboxymethylated derivative to apparent homogeneity . The prosthetic group of the acyl carrier protein was isolated after alkaline hydrolysis, and its chemical structure was identified by high-performance liquid chromatography (HPLC) with the corresponding compound from citrate lyase from Klebsiella pneumoniae as reference and by mass spectrometry . Malonate decarboxylase was found to carry the same prosthetic group as citrate lyase, i.e . 2'-(5"-phosphoribosyl)-3'-dephospho-CoA. EMBO J, 1996 Apr 15, 15(8), 1842 - 9 Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump; Di Berardino M et al.; We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae . The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane . The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate . The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis . Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities . Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme . Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated . In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane . The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation . Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction . The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model. Rev Latinoam Microbiol, 1996 Apr-Jun, 38(2), 121 - 7 Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase; Parra-Campos V et al.; In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K . pneumoniae whole bacterial extracts . An initial screening for nitrogenase producing K . pneumoniae strains was performed in 31 clinical isolates . The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations . Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located . On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status . Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress. Microbiology, 1996 Apr, 142 ( Pt 4), 1005 - 12 Bacterial production of transdihydroxycyclohexadiene carboxylates by metabolic pathway engineering; Muller R et al.; Homochiral-cis-cyclohexa-3,5-diene-1,2-diols are important synthons . We found a way to produce trans-configured homochiral diols using recombinant Klebsiella pneumoniae 62-1 . Transformation of this mutant (Phe- Trp- Tyr-) with plasmids carrying genes involved in chorismic and isochorismic acid metabolism leads to the production of either (+)-trans-(2S,3S)-2,3-dihydroxycyclohexa-4,6-dienecarboxylic acid or (-)-trans-(3R,4R)-3,4-dihydroxycyclohexa-1,5-dienecarboxylic acid, with a yield of 70 or 90 mg (1 culture broth)-1, respectively . The metabolic shift from one diene to the other is caused by a change in activity of isochorismate hydroxymutase and/or isochorismatase which in turn results from growth under iron deficiency or overexpression of genes (entC and/or entB) involved in chrismate metabolism. Antimicrob Agents Chemother, 1996 Apr, 40(4), 1027 - 9 Survey of Klebsiella pneumoniae producing extended-spectrum beta-lactamases: prevalence of TEM-3 and first identification of TEM-26 in France; Soilleux MJ et al.; Crude extracts from 115 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates were analyzed biochemically . The TEM-3 type was encountered 108 times, SHV types were encountered 7 times, and the TEM-26 type was encountered only once . For the last one, the gene was identified; an adenine was detected at position 925, as in blaTEM-26B not in blaTEM-26. J Clin Microbiol, 1996 Apr, 34(4), 908 - 11 Detection of extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli; Jacoby GA et al.; Forty clinical isolates of Escherichia coli and 141 isolates of Klebsiella pneumoniae that either transferred ceftazidime resistance or showed sulbactam enhancement of oxyimino-beta-lactam susceptibility were tested by disk diffusion methodology for susceptibility to aztreonam, cefotaxime, ceftazidime, and cefoxitin . With standard 30 micrograms antibiotic disks, the fraction of these extended-spectrum beta-lactamase (ESBL)-producing isolates testing resistant by National Committee for Clinical Laboratory Standards criteria was lowest (24%) with cefotaxime disks . Forty percent of the E . coli and 29% of the K . pneumoniae isolates appeared susceptible with at least one oxyimino-beta-lactam disk . Ceftazidime and aztreonam disks were equivalent in differentiating ESBL production, and both were superior to cefotaxime disks . Over half the E . Coli and 29% of the K . pneumoniae isolates tested cefoxitin resistant . In 30 isolates, cefoxitin resistance was transmissible and due to a plasmid-mediated AmpC-type beta-lactamase . With a 5-micrograms ceftazidime disk, a breakpoint could be chosen with high sensitivity and specificity for ESBL-producing organisms . Present disk diffusion criteria underestimate the prevalence of ESBL-producing strains. Eur J Biochem, 1996 Apr 1, 237(1), 272 - 8 Characterization of lipopolysaccharides of polymyxin-resistant and polymyxin-sensitive Klebsiella pneumoniae O3; Helander IM et al.; Lipopolysaccharides isolated from the polymyxin-resistant Klebsiella pneumoniae O3 mutant OM-5 and its polymyxin-sensitive parent LEN-1 were analyzed for chemical composition, and their lipid A portions were structurally characterized . The lipopolysaccharide of OM-5 contained approximately five times more 4-amino-4-deoxy-L-arabinopyranose than that of LEN-1 . Other saccharide and phosphate components exhibited no significant differences . Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A . In the OM-5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4-amino-4-deoxy-L-arabinopyranose . In the sensitive-type LEN-1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate . Phosphate in these K . pneumoniae lipopolysaccharides was almost exclusively found in lipid A . These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity . OM-5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN-1. Eur J Biochem, 1996 Apr 1, 237(1), 221 - 8 Malonate decarboxylase of Klebsiella pneumoniae catalyses the turnover of acetyl and malonyl thioester residues on a coenzyme-A-like prosthetic group; Schmid M et al.; During aerobic growth of Klebsiella pneumoniae on malonate, a soluble malonate decarboxylase is induced . Malonate decarboxylation consumes a proton (not H2O) and forms acetate and CO2 (not HCO3-) as products . The enzyme was purified 56-fold to apparent homogeneity . It has a native molecular mass of 142 kDa and consists of four subunits alpha, beta, gamma and delta with molecular masses of 65, 34, 30, and 12 kDa, respectively . Two different forms of the enzyme were recognised: a catalytically inactive SH-enzyme and the catalytically active acetyl-S-enzyme which is formed by post-translational acetylation of the SH-enzyme with ATP, acetate and a specific ligase . The acetyl-S-enzyme was converted into the SH-enzyme by incubation with hydroxylamine or dithioerythritol . Chemical reacylation of the SH-enzyme, which restores catalytic activity, was achieved with acetic anhydride or more efficiently with malonyl-CoA . This acylation of the SH group was prevented after incubation with various thiol-specific reagents . After incubation of the SH-enzyme with iodo{1-14C}acetate, the delta subunit became specifically labelled . This subunit was also labelled after incubation of the acetyl-S-enzyme with {2-14C}malonate . The radioactivity was completely liberated from the protein upon malonate addition . These results indicate that the delta subunit is the acyl-carrier protein of the complex and that malonate decarboxylation proceeds in two steps: the acetyl residue on the ACP is first replaced by a malonyl residue which subsequently undergoes decarboxylation thereby regenerating the acetyl-S-ACP . The binding site for the acyl residues on the acyl-carrier protein was shown to be 2'-(5"-phosphoribosyl)-3'-dephospho-CoA after alkaline cleavage of this prosthetic group from the enzyme and chromatographic as well as mass spectroscopic analyses. J Bacteriol, 1996 Apr, 178(7), 2154 - 7 Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1; Springer AL et al.; Methylobacterium extorquens AM1 pqqEF are genes required for synthesis of pyrroloquinoline quinone (PQQ) . The nucleotide sequence of these genes indicates PqqE belongs to an endopeptidase family, including PqqF of Klebsiella pneumoniae, and M . extorquens AM1 PqqF has low identity with the same endopeptidase family . M . extorquens AM1 pqqE complemented a K . pneumoniae pqqF mutant. J Clin Invest, 1996 Mar 15, 97(6), 1366 - 72 Low-density lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections; Netea MG et al.; Lipoproteins can bind lipopolysaccharide (LPS) and decrease the LPS-stimulated production of pro-inflammatory cytokines . We investigated the effect of increased plasma concentrations of low-density-lipoproteins (LDL) on survival and cytokine production after a lethal challenge with either LPS or live Gram-negative bacteria in LDL receptor deficient mice (LDLR-/-) . The LDLR-/- mice challenged with LPS had an eightfold increased LD50 when compared with the wild type controls (C57Bl/6J), while tumor necrosis factor alpha (TNFalpha) and interleukin-1 alpha (IL-1 alpha) plasma concentrations were decreased twofold . LDLR-/- mice had significantly lower and delayed mortality than control mice after infection with Klebsiella pneumoniae . No differences in the outgrowth of bacteria in the organs were present between the two groups, while circulating cytokine concentrations were decreased twofold in LDLR-/- mice . In contrast, the LPS-stimulated in vitro production of cytokines by peritoneal macrophages of LDLR-/- mice was significantly increased compared with controls . This increase was associated with enhanced specific binding of LPS to the macrophages of LDLR-/- mice . In conclusion, endogenous LDL can protect against the lethal effects of endotoxin and Gram-negative infection . At least part of this protection is achieved through decreased in vivo production of pro-inflammatory cytokines, in spite of increased cytokine production capacity. Mol Gen Genet, 1996 Mar 7, 250(4), 447 - 54 Transcription termination within the regulatory nifLA operon of Klebsiella pneumoniae; Govantes F et al.; The effect of premature stop codons in the nifL gene on the expression of nifA-lacZ operon and protein fusions in Klebsiella pneumoniae was analysed in detail . Our results revealed transcriptional polarity in this operon . By dissecting the operon, intragenic regions containing Rho-dependent transcription terminators have been identified . As shown for other Rho-dependent terminators, their cytosine content is much higher than the incidence of guanines . However, other regions of the operon that have this feature did not show termination activity, suggesting that, contrary to previous reports, a correlation between these parameters cannot readily be established . Some of our results alos suggested that, in addition to polarity, other mechanisms may prevent expression of nifA when translation of nifL is altered . Their importance for efficient regulation of nitrogen fixation genes is discussed. Southeast Asian J Trop Med Public Health, 1996 Mar, 27(1), 102 - 6 Risk factors for neonatal Klebsiella septicemia in Srinagarind Hospital; Pengsaa K et al.; Three years' data were analysed to assess the risk factors for neonatal Klebsiella septicemia in Srinagarind Hospital . The incidence of Klebsiella septicemia was 4.1 per 1,000 livebirths or 5.2 per 100 discharged infants . Eighty-two per cent of infected cases were low birth weight infants and 67.7% were born prematurely . From multivariate analysis, the risk factors were endotracheal intubation (OR 31.57, 95% CI 289-343.82) and central venous catheterization (OR 16.99, 95% CI1.15-250.37) . The overall mortality rate was 67.7% . Periodic review and continuous reinforcement of infection control policies in the neonatal unit are of paramount importance to decrease the incidence of nosocomial infection and successful control of outbreaks as well. FEMS Microbiol Lett, 1996 Mar 1, 136(3), 297 - 303 The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist; Orellana C et al.; Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae . Different growth conditions of the producer strain affect microcin activity . The production of a microcin antagonist is responsible for the changes in microcin activity . The microcin antagonist is induced when cells are iron-deprived, resulting in a low microcin activity . The microcin antagonist was purified using a procedure developed for the isolation of a catechol-type siderophore, and its activity was titrated using purified microcin . The inhibitory effect of the microcin antagonist is not observed when this compound is forming a complex with iron . The same inhibitory effect on microcin activity was obtained using purified enterochelin from Escherichia coli . The microcin antagonist was identified as enterochelin through thin-layer chromatography. Eur J Clin Microbiol Infect Dis, 1996 Mar, 15(3), 245 - 8 Detection of SHV-5 extended-spectrum beta-lactamase in Klebsiella pneumoniae strains isolated in Italy; Marchese A et al.; Thirty-five Klebsiella pneumoniae strains isolated during 1993-1994 in intensive care units of a large Italian hospital were examined for the presence of extended-spectrum beta-lactamases . Five strains showed a high level of simultaneous resistance to beta-lactam agents, including ceftazidime and aztreonam, conferred by a large (130 kb) self-transferable plasmid (in 4 of 5 strains) . Isoelectrofocusing and hybridisation studies suggest that these enzymes can be identified as SHV-5 extended-spectrum beta-lactamases . Pulsed-field get electrophoresis analysis showed three different genomic fingerprinting profiles, while plasmid restriction enzyme digestion revealed three different patterns, demonstrating that the diffusion of SHV-5 beta-lactamase is not the result of a single strain or plasmid dissemination. Pediatr Hematol Oncol, 1996 Mar-Apr, 13(2), 143 - 50 Infection-associated hemophagocytic syndrome complicated by infectious lymphoproliferation: a case report; Syruckova Z et al.; The case of a 7-year-old boy with virus-associated hemophagocytic syndrome (VAHS) and serologically proven parvovirus B-19 infection is described . The patient with VAHS presented with fever, hepatosplenomegaly, pancytopenia, and hyperlipidemia type IV . After induction therapy with VP-16 and prednisone, partial remission was achieved . Despite maintenance therapy, reinductions, and the addition of cyclosporine A for 3 months, several relapses occurred . The therapy was stopped because of life-threatening complications (Klebsiella sepsis, neutropenic enterocolitis, and stercoral peritonitis) . The complications were treated successfully . The patient status was stabilized after splenectomy . However, hepatomegaly progressed slowly and the hyperlipidemia endured . Ten months after the diagnosis leukocytosis with absolute T lymphocytosis appeared . Reactivation of VAHS was suspected and intravenous immunoglobin and then antilymphocyte immunoglobulin ALG therapy were started . The resultant decrease in leukocytosis was prompt, but lymphopenia did not occur . Virostatic treatment with foscarnet was introduced based on human herpesvirus-6 seroconversion . Twenty-six months after the diagnosis, the patient is well, without any sign of VAHS or lymphoproliferation. EMBO J, 1996 Mar 1, 15(5), 978 - 88 Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein; Hardie KR et al.; Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane . PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment . The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS . Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene . We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane . PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed. J Mol Biol, 1996 Mar 1, 256(3), 423 - 35 Nitrate and nitrite-mediated transcription antitermination control of nasF (nitrate assimilation) operon expression in Klebsiella pheumoniae M5al; Lin JT et al.; Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth . Nitrate is converted through nitrite to ammonium by assimilatory nitrate and nitrite reductase, respectively . Enzymes required for nitrate assimilation are encoded by the nasFEDCBA operon of K . pneumoniae; nasF operon expression is subject to both general nitrogen control and pathway-specific nitrate/nitrite induction, mediated by the NtrC and NasR proteins, respectively . Sequence inspection revealed a presumptive sigmaN (sigma54)-dependent promoter as well as two presumptive upstream NtrC protein binding sites . Site-specific mutational and primer extension analyses confirmed the identity of the sigmaN-dependent promoter . Deletions removing the apparent NtrC protein binding sites greatly reduced NtrC-dependent regulation, indicating that these sites are involved in general nitrogen control . However, deletions removing most of the sequence upstream of the promoter had little effect on nitrate/nitrite regulation, suggesting that the nasF leader region is involved in nitrate/nitrite regulation . The 119 nucleotide long transcribed leader region contains an apparent factor-independent transcription terminator . Promoter replacement experiments demonstrated that the leader region is involved in nitrate/nitrite regulation of nasF operon expression . Deletions removing the transcription terminator structure resulted in a nitrate-blind constitutive phenotype, indicating that the transcription terminator structure serves a negative function . Other deletions, removing proximal portions of the leader region, resulted in an uninducible phenotype, indicating that this region serves a positive function . These results indicate that nitrate/nitrite regulation of nasF operon expression is determined by a transcription attenuation mechanism . We hypothesize that in the absence of nitrate or nitrite, the terminator structure abrogates transcription readthrough into the nasF operon . In the presence of nitrate or nitrite, the NasR protein mediates transcription antitermination, thereby allowing transcription to proceed into the nasF operon. Arch Microbiol, 1996 Mar, 165(3), 206 - 12 Metabolism of cyclodextrins by Klebsiella oxytoca m5a1: purification and characterisation of a cytoplasmically located cyclodextrinase; Feederle R et al.; It has been shown previously that the products of 11 genes are required for metabolism of starch by Klebsiella oxytoca via a novel pathway . An extracellular cyclodextrin glucanotransferase first degrades starch into alpha- and beta-cyclodextrins; evidence then has been presented that the cyclodextrins are transported into the cytoplasma via a specific system and that they are metabolised inside the cell . To provide support for this model, we have analysed whether Klebsiella oxytoca possesses a cytoplasmic enzyme able to linearise cyclodextrins . A possible candidate was the product of the cymH gene since it displays sequence similarity with cyclodextrinases from other organisms . The cymH gene was overexpressed, and the CymH protein was purified . CymH is a monomer of 69 kDa molecular mass and hydrolysed cyclodextrins at an optimum pH of 7.0 and an optimum temperature of 23 degrees C, respectively . The apparent Km increased with increasing size of the cyclodextrins, but the reaction velocity decreased . Linear malto-oligosaccharides were also accepted as substrates, but were hydrolysed with a lower efficiency . Final products in each case were maltose and maltotriose . It was demonstrated by immunoblotting that CymH is located in the cytoplasm and that no signal peptide was cleaved off . Since cymH mutants were no longer able to grow on cyclodextrins, these results prove that cyclodextrins are degraded inside the cell, and they support the contention of the existence of a specific transport system. J Mol Biol, 1996 Feb 23, 256(2), 279 - 91 Genetics of a novel starch utilisation pathway present in Klebsiella oxytoca; Fiedler G et al.; A 14.3 kb DNA fragment from Klebsiella oxytoca M5a1 has been cloned and shown to provide Escherichia coli with the capacity for growth on alpha- and beta-cyclodextrins . This fragment is located immediately upstream of the previously identified cgt gene coding for cyclodextrin glycosyltransferase . It contains ten genes (cym) organised in two divergently oriented clusters separated by a non-coding region of 419 bp . Four of the genes code for products homologous to the maltose and linear maltodextrin uptake system, another one for a putative cytoplasmic cyclodextrinase . The cym genes of K . oxytoca are distinct and different from the mal genes; cym mutations do not affect maltose catabolism . On the other hand, whereas mutations in the maltose/maltodextrin-uptake genes do not influence cyclodextrin metabolism, a mutation inactivating the malPQ genes coding for maltodextrin phosphorylase and amylomaltase does . Cyclodextrin catabolism is independent of the presence of a functional cyclodextrin glycosyltransferase but degradation of starch and gamma-cyclodextrins requires the activity of this enzyme . The results indicate the existence of a novel starch degradation pathway which involves the extracellular conversion of starch into cyclodextrins by cyclodextrin glycosyltransferase, uptake of the cyclodextrins by a specific uptake system and intracellular linearisation by a cyclodextrinase . The malto-oligosaccharides produced are then channelled into the maltodextrin-degradation route involving the activity of maltodextrin phosphorylase and amylomaltase. FEMS Microbiol Lett, 1996 Feb 1, 136(1), 31 - 7 Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli; Di Berardino M et al.; The genes oadGAB encoding the oxaloacetate decarboxylase gamma, alpha and beta-subunits from Klebsiella pneumoniae were expressed in Escherichia coli . Using different expression vectors, the entire enzyme or its individual subunits were synthesised . The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase . The expressed alpha-subunit or a combination of alpha and beta-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a gammaalpha-complex were located mostly in the cytoplasmic membrane . Interestingly, overexpression of the gammaalpha-complex or the entire oxaloacetate decarboxylase in E . coli led to a significant immunogold labelling in the cytoplasm, indicating that the alpha-subunit was not completely complexed to the membrane-bound gamma or betagamma-subunits. Antimicrob Agents Chemother, 1996 Feb, 40(2), 509 - 13 Sequences of beta-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other beta-lactamases; Bauernfeind A et al.; Amino acid sequences determined either by protein sequencing or by DNA sequencing are identical for cefotaximases CTX-M-1 and MEN-1, whereas CTX-M-2 is 84% identical to CTX-M-1/MEN-1 . Both beta-lactamases are distantly related to other plasmidic class A enzymes (homology to TEM-1 is 38.1% for CTX-M-1/MEN-1 and 36.5% for CTX-M-2); the closest relationship was with the chromosomal beta-lactamase of Klebsiella oxytoca E23004 (homologies of 74.5% for CTX-M-1/MEN-1 and 77.9% for CTX-M-2) . The cefotaximases CTX-M-1/MEN-1 and CTX-M-2 represent two members of a new subgroup of plasmidic class A beta-lactamases. Antimicrob Agents Chemother, 1996 Feb, 40(2), 460 - 3 beta-lactamase gene promoters of 71 clinical strains of Klebsiella oxytoca; Fournier B et al.; beta-Lactamase gene promoters of 45 clinical Klebsiella oxytoca isolates resistant to beta-lactams and exhibiting beta-lactamase hyperproduction differed from those in 26 susceptible strains . Direct sequencing revealed one mutation in either the -10 or -35 conserved sequences: a G-to-A transition of the fifth base (67%) or a G-to-T transversion of the first base of the -10 sequence (27%) or a T-to-A transversion in the fourth base in the -35 sequence (4%) . One strain carried both the -10 transition and the -35 transversion. Antimicrob Agents Chemother, 1996 Feb, 40(2), 342 - 8 In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum-cephalosporins; Martinez-Martinez L et al.; Four Klebsiella pneumoniae isolates (LB1, LB2, LB3, and LB4) with increased antimicrobial resistance were obtained from the same patient . The four isolates were indistinguishable in biotype, plasmid content, lipopolysaccharide, and DNA analysis by pulse-field gel electrophoresis . Isolate LB1 made TEM-1 and SHV-1 beta-lactamases . Isolates LB2, LB3, and LB4 produced SHV-5 in addition to TEM-1 and SHV-1 . MICs of cefoxitin, ceftazidime, and cefotaxime against LB1 were 4, 1, and 0.06 micrograms/ml, respectively . MICs of ceftazidime against K . pneumoniae LB2, LB3, and LB4 were > 256 micrograms/ml, and those of cefotaxime were 2, 4, and 64 micrograms/ml, respectively . MICs of cefoxitin against K . pneumoniae LB2 and LB3 were 4 micrograms/ml, but that against K . pneumoniae LB4 was 128 micrgrams/ml . K . pneumoniae LB4 could transfer resistance to ceftazidime and cefotaxime, but not that to cefoxitin, to Escherichia coli . Isolate LB4 and cefoxitin-resistant laboratory mutants lacked an outer membrane protein of about 35 kDa whose molecular mass, mode of isolation, resistance to proteases, and reaction with a porin-specific antiserum suggested that it was a porin . MICs of cefoxitin and cefotaxime reverted to 4 and 2 micrograms/ml, respectively, when isolate LB4 was transformed with a gene coding for the K . pneumoniae porin OmpK36 . We conclude that the increased resistance to cefoxitin and expanded-spectrum cephalosporins of isolate LB4 was due to loss of a porin channel for antibiotic uptake. Antimicrob Agents Chemother, 1996 Feb, 40(2), 325 - 30 Assessment of two penicillins plus beta-lactamase inhibitors versus cefotaxime in treatment of murine Klebsiella pneumoniae infections; Fournier JL et al.; The in vivo efficacies of piperacillin, piperacillin plus tazobactam, ticarcillin, ticarcillin plus clavulanic acid, piperacillin plus clavulanic acid, and cefotaxime were compared in a mouse model of pneumonia induced by the SHV-1 beta-lactamase-producer Klebsiella pneumoniae . Each antibiotic was injected either once intraperitoneally at 24 h postinfection or at repeated times during 24 h . The efficacies of the drugs and therapeutic protocols were assessed by counting viable bacteria recovered from the lungs of mice sacrificed at selected times . No emergence of beta-lactam-resistant organisms was detected . Ticarcillin at 300 mg/kg was ineffective . Repeated injections of piperacillin at 300 mg/kg, either alone or in combination with tazobactam (8:1), led to a significant decrease in bacterial counts, but this was followed by bacterial regrowth . The pharmacokinetic analysis demonstrated that this short-lasting antibacterial effect was not due to a failure of piperacillin and/or tazobactam to penetrate the lungs . The combinations of ticarcillin at 300 mg/kg plus clavulanic acid (15:1) and piperacillin at 300 mg/kg plus tazobactam (4:1) were proven to be effective in that they decreased the bacterial burden in the lungs from 10(5) to < 10(3) CFU . This dose effect of tazobactam can be explained by its dose-dependent penetration in the lungs . Cefotaxime at 100 mg/kg and the combination of piperacillin (slightly hydrolyzed by SHV-1) at 300 mg/kg plus clavulanic acid (15:1) led to the best efficacy . Both of these treatments induced a decrease in bacterial counts of nearly 4 log10 units . The survival rates correlated with the quantitative measurements of in vivo bacterial killing . These experimental results obtained from the restricted animal model used here may help in the design of further protocols for clinical trials. J Clin Microbiol, 1996 Feb, 34(2), 358 - 63 Hospital outbreak of Klebsiella pneumoniae resistant to broad-spectrum cephalosporins and beta-lactam-beta-lactamase inhibitor combinations by hyperproduction of SHV-5 beta-lactamase; French GL et al.; An aminoglycoside- and ceftazidime-resistant strain of Klebsiella pneumoniae K2 producing the extended-spectrum beta-lactamase SHV-5 infected or colonized 14 pediatric patients at Guy's Hospital . The patients were mostly neonates recovering from cardiac surgery for congenital defects . The organism was also isolated from a nurse and from the father of one of the children . Four patients had septicemia, and two septicemic neonates with postoperative renal failure died . Aminoglycoside and cephalosporin resistance transferred to Escherichia coli in vitro on a 160-kb plasmid, and a similar resistant E . coli strain was isolated from the stools of one of the affected children . The epidemic organism colonized the bowel and skin and was probably transmitted via staff hands . Five wards were involved because of extensive patient movements . The outbreak was controlled by patient isolation and attention to handwashing . All of the isolates of the outbreak strain were identical by phage typing, ribotyping, plasmid profiling, and biochemical and serological testing, but they varied in their production of SHV-5 . Some isolates produced normal amounts of SHV-5 and were susceptible to beta-lactam-beta-lactamase inhibitor combinations . Others, including the single isolate of multiresistant E . coli, produced up to five times as much enzyme as "normal" isolates . This hyperproduction resulted in increased resistance to several penicillins and cephalosporins and to the beta-lactam-beta-lactamase inhibitor combinations amoxicillin-clavulanic acid, ampicillin-sulbactam, piperacillin-tazobactam, and ceftazidime-clavulanic acid . The hyperproduction of SHV-5 by K . pneumoniae and E . coli seen in this outbreak suggests that beta-lactam-beta-lactamase inhibitor combinations may be unreliable for the treatment of organisms producing extended-spectrum beta-lactamases. Eur J Pediatr, 1996 Feb, 155(2), 102 - 5 Nosocomial neonatal septic arthritis; Abuekteish F et al.; Between August 1993 and August 1994, 17 cases of neonatal septic arthritis occurred at the intensive care baby unit of Princess Badia' Teaching Hospital in Northern Jordan . Klebsiella species was the causative pathogen in 10 patients (59%), which indicates a nosocomial acquired infection . The hip was the main joint involved in 94% of cases . An epidemiological survey showed that the spread of Klebsiella occurred via contaminated covered sheets of both delivery and resuscitation tables . Control measures resulted in a decrease in cross contamination and a dramatic slowing of the outbreak . The clinical features, risk factors, outcome and bacteriology are also discussed. Prep Biochem Biotechnol, 1996 Feb, 26(1), 67 - 76 Small scale biosynthesis and purification of gram quantities of chorismic acid; Rieger CE et al.; This paper details improvements in the biosynthetic method of purification of chorismic acid using Klebsiella pneumoniae 62-1 . New growth and accumulation conditions yielded 4 L of accumulation medium containing approximately 0.5 g/L of chorismate from 2 L of bacterial growth . Improvements in the handling and extraction procedures produced yields of approximately 2 g of 75 to 90% chorismic acid . A new recrystallization procedure yielded chorismic acid 97% pure by weight (using epsilon 275 = 2630 M-1 cm-1), and 99.8% pure by enzymatic conversion . These results represent a three- to five-fold increase in yield over average published values. Am J Perinatol, 1996 Feb, 13(2), 99 - 102 An outbreak of antibiotic multiresistant Klebsiella at the Neonatal Intensive Care Unit, Kaplan Hospital, Rehovot, Israel, November 1991 to April 1992; Flidel-Rimon O et al.; From November 1991 through April 1992, 8 infants developed systemic infections due to antibiotic multiple resistant Klebsiellaa (MRK) . All were premature and 6 of the 8 weighed less than 1100 g; 7 of the 8 had received previous antibiotic therapy . Five infections occurred during the first week of life . MRK were isolated from blood (8 cases), tracheal secretions (TS-6), stool (3), and CSF (1) . All Klebsiella blood isolates were resistant to ampicillin, mezlocillin, and cefotaxime, 7 of 8 to ceftazidime and amikacin, and 4 of 7 to aztreonam; all isolates were sensitive to quinolones and imipenem . Four infants died . In all 4 of the isolates, they were sensitive only to quinolones and imipenem, and the empiric therapy used for suspected sepsis proved to be inappropriate . The outbreak was terminated by temporary closure of NICU in May 1992 . Strict hand washing practices were reemphasized, and the previous empiric antibiotic protocol used for suspected sepsis (mezlocillin plus amikacin, and lately ceftazidime plus amikacin) was changed to imipenem and amikacin in the risk population . At closure, 5 additional infants had MRK in stools and/or tracheal suction specimens . Development of MRK organisms should dictate a rational use of empiric antibiotics for neonatal infections in NICU. Br J Rheumatol, 1996 Feb, 35(2), 125 - 8 Similarly increased serum IgA1 and IgA2 subclass antibody levels against Klebsiella pneumoniae bacteria in ankylosing spondylitis patients with/without extra-articular features; Maki-Ikola O et al.; IgA1 and IgA2 subclass serum antibodies against whole Klebsiella pneumoniae bacteria were studied earlier in the sera of 98 patients with ankylosing spondylitis (AS) and in 100 healthy blood donors by enzyme immunoassay . In this study, the patients were divided into groups according to the clinical picture, i.e., the presence or absence of iritis and enthesitis . The previous findings of increased IgA1 and IgA2 subclass antibody levels against K . pneumoniae in AS patients when compared to the healthy controls were not specifically associated with any single AS patient group in the present study, but instead were similarly seen in all patient groups with/without extra-articular features . This is in line with the previous studies suggesting a role for K . pneumoniae in the pathogenesis of AS. Infect Immun, 1996 Feb, 64(2), 528 - 34 Bacterial enzymes can add galactose alpha 1,3 to human erythrocytes and creates a senescence-associated epitope; Hamadeh RM et al.; Humans have abundant circulating anti-alpha (1,3-di)-galactosyl (alpha Gal) antibodies (anti-Gal) . Anti-Gal has been implicated in the clearance of senescent human erythrocytes (RBCs) . The nature of the anti-Gal-binding RBC epitope has defied explanation, given that humans repress expression of the alpha 1,3 galactosyltransferase (alpha 1,3 GT) enzyme . This study explored whether alpha Gal epitopes on human RBCs might be synthesized by alpha 1,3 GTs of bacterial origin that are translocated into the circulation during commensal colonization of the gut by gram-negative bacteria . We found that an acellular Klebsiella pneumoniae sonicate could add 3H-UDP-Gal to human RBCs in the alpha configuration at 37 degrees C in the presence of 6 mM MnCl2 (pH 7.6) . Gradient anion-exchange chromatography of the Klebsiella sonicate yielded four fractions that could catalyze the addition of 3H-Gal to human RBCs . Size-exclusion chromatography of these anion-exchange fractions yielded peaks of high GT activity for each, but only those derived from the first, third, and last anion-exchange fractions incorporated Gal such that the RBCs bound anti-Gal by fluorescence-activated cell sorter, suggesting that these three GTs are alpha 1,3 GTs . Thus, Klebsiella spp . make at least four GTs that can add an alpha Gal to human cell surface acceptor structures . Three of these GTs can form alpha 1,3 Gal structures on human RBCs that bind anti-Gal, thereby creating "autoimmune" senescence-associated RBC epitopes. Biochemistry, 1996 Jan 23, 35(3), 1018 - 26 Functional properties of the purified Na(+)-dependent citrate carrier of Klebsiella pneumoniae: evidence for asymmetric orientation of the carrier protein in proteoliposomes; Pos KM et al.; The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified and reconstituted into liposomes to investigate the properties of this transport system without interference from other proteins . Citrate uptake was an electroneutral process, where delta pNa+ and/or delta pH are driving forces . Delta psi was unable to stimulate citrate transport, either alone or in addition to the other driving forces . Sodium ions on the inside of the proteoliposomes stimulated the uptake of citrate, indicating that Na+ ions recycle during the transport of citrate . CitS also performed Na+ counterflow in the absence of citrate . The citrate carrier performed citrate/citrate counterflow but no heterologous antiport of citrate with one of the end products arising from the anaerobic citrate fermentation pathway (acetate, formate, or bicarbonate) in K . pneumoniae . Citrate counterflow kinetics revealed that CitS transports citrate according to a simultaneous type of mechanism . The Km and Ki values revealed two binding sites for citrate: one with low and one with high affinity . This transport mode is in accord with an asymmetric organization of the carrier protein in proteoliposomes. Microbiol Immunol, 1996, 40(5), 339 - 44 Electron microscopic observation of the antibody-induced capsular swelling phenomenon in Klebsiella pneumoniae; Meno Y et al.; The capsular swelling phenomenon of Klebsiella pneumoniae strain 277 was examined morphologically using the electron microscopy techniques of freeze-substitution . The capsules of strain 277 measured about 52 nm in thickness, and were composed of a number of fine fibers . After treating the bacteria with anti-capsular serum, the size of the capsules increased to about twice the normal size and they lost their electron density . The capsular fibers that are tightly packed in normal cells became loose and thus the identification of the individual capsular fibers was difficult in the swollen capsules . Capsule swelling was induced by washing the cells with phosphate-buffered saline . The removal of either divalent cations or some other materials might thus be important for maintaining the normal capsule structure . the mechanism of the swelling phenomenon was also discussed. Zentralbl Gynakol, 1996, 118(9), 530 - 2 {Multiple liver abscesses in long-term intrauterine device}; Klare P et al.; We report on two cases of multiple hepatic abscesses caused by sever pelvic inflammatory disease secondary to intrauterine device . Such complication have previously been reported only twice . The course of the diseases were non-specifically for a long time . As causative agent we isolated Klebsiella pneumoniae in the first case and in the other Fusobacterium necroforum . The patients were cured with combined operative and medical therapy. Microbiol Immunol, 1996, 40(8), 531 - 7 Cloning and sequencing of the Klebsiella K-36 astA gene, encoding an arylsulfate sulfotransferase; Baek MC et al.; A gene-encoding arylsulfate sulfotransferase (ASST) was cloned from a Klebsiella K-36 genomic library . ASST transfers a sulfate group from phenolic sulfate esters to a phenolic acceptor substrate . The gene, designated astA, was subcloned into vector pGEM3Zf(-) and sequenced . Recombinant clone-harbouring astA was directly identified using a fluorescent product . The nucleotide sequencing revealed an open reading frame (ORF) of 2,082 bp encoding a protein of 694 amino acids with a secretory signal sequence . A protein of similar size was visualized after in vitro transcription and translation using a plasmid carrying the cloned 3.1-kb fragment as a template . The N-terminal amino acid sequence of the purified processed protein was found to be identical to that predicted from the gene sequence . When searching the database for astA nucleotide or its deduced amino acid sequence, no significant homology to any sequence was found. Microbiol Immunol, 1996, 40(6), 407 - 13 Crystallization of an R-form lipopolysaccharide from Klebsiella pneumoniae; Kato N et al.; An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (03-:K1-) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 x 0.8 microns, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl2 and kept at 4 C for as long as 870 days . K . pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS . Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an alpha axis of 4.62 A and c axis of 79.8 +/- 2.6 A . The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl2 at 4 C. Rev Argent Microbiol, 1996 Jan-Mar, 28(1), 39 - 44 {Biodegradation of a commercial mixture of polychlorinated biphenyls}; Casasco PV et al.; A bacterial strain capable of growing on a commercial mixture of polychlorinated biphenyls (PCBs) was isolated from PCBs-contaminated soil . The isolate was identified as a Klebsiella oxytoca strain, able to grow on 6-chloroquinoline and chlorobenzene . The above mentioned PCBs mixture contains several congeners of bi-, tri- and tetra-chloro biphenyls . Xenobiotic consumption could be measured by an assay in glass-stoppered flask . After three days PCBs were almost completely degraded with the production of phenolic and/or acidic products, that were consumed after seven days. Res Immunol, 1996 Jan, 147(1), 39 - 48 Effects of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) on haematopoietic regeneration and resistance to infection of sublethally irradiated mice; Neway T et al.; The influence of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) treatment on the reversal of irradiation-induced leukopenia (granulocytopenia, monocytopenia) and thrombocytopenia and its ability to protect mice against lethal infections were investigated in this study . The administration of pGPL-Mc to irradiated mice significantly accelerated the recovery of leukocyte and thrombocyte numbers in the peripheral blood . Granulocytes and monocytes were the principal cells of the leukocyte population that responded to the potent stimulus of this product . The reversal of granulocytopenia and monocytopenia in treated mice was achieved on day 14 and reached a peak value on day 20 . Responses in mice receiving 100 mg/kg of pGPL-Mc was about 40-fold compared to controls and about 4-fold compared to the rhG-CSF-treated group . Normal levels of thrombocytes were reached by day 17 in mice treated with 100 mg/kg and by day 20 in those receiving 25 mg/kg of pGPL-Mc . The administration of pGPL-Mc to mice with irradiation-induced granulocytopenia was characterized by highly significant protection of these animals against lethal Klebsiella pneumoniae or Escherichia coli infections . Therefore, pGPL-Mc appears to possess a considerable potential for improvement of the outcome of radiotherapy and may contribute to the successful avoidance of irradiation-induced toxicities. Int J Immunopharmacol, 1996 Jan, 18(1), 69 - 74 RU 41 740 (Biostim) and IL-4, or IL-13, have opposite effects on CD14, CD23, HLA-DR and HLA-DQ on monocytes; Garin L et al.; RU 41 740 (Biostim) is a glycoprotein extract obtained from Klebsiella pneumoniae . Its immunostimulating properties on monocytes have been established in vivo and in vitro . To confirm its spectrum of action at molecular level we studied its role on the modulation of four molecules involved in antigen presentation (HLA-DR, HLA-DQ), uptake of endotoxin (CD14) and activation (CD23) . These four molecules are known to be modulated by interleukins IL-4 and IL-13 . We found that HLA-DR, HLA-DQ, CD14 and CD23 were differentially regulated by biostim and IL-4 or IL-13 . Surprisingly, Biostim inhibited the IL-4 or IL-13-induced expression of CD23, HLA-DQ and HLA-DR, while it did not have any action on these molecules by itself . We therefore hypothesize that Biostim, through the action on its receptor, could interact with the IL-4 receptor and IL-13 receptor and/or inhibit the IL-4 and IL-13 receptor transducing signal. Vasa, 1996, 25(2), 184 - 7 Mycotic aneurysm of the internal iliac artery caused by Klebsiella pneumoniae; Tatebe S et al.; Mycotic aneurysms are rarely caused by Klebsiella species . We describe a male patient with diabetes mellitus and alcoholism who developed a mycotic aneurysm of the right internal iliac artery complicated by the formation of a false aneurysm . Klebsiella pneumoniae was cultured from arterial blood . An extra-anatomic bypass (left external iliac artery to right common femoral artery) was performed with a successful excision of the aneurysm. Life Sci, 1996, 58(9), PL153 - 8 K1 and K3 capsular antigens of Klebsiella induce tumor necrosis factor activities; Choy YM et al.; Capsular polysaccharide antigens isolated from Klebsiella pneumoniae sero-type 1 (K1) and sero-type 3 (K3) could induce tumor necrosis factor-alpha in ICR mice . K1 and K3 capsular antigens were found to be non-toxic by brine shrimp bioassay . When injected into Ehrlich ascites tumor-bearing mice, both K1 and K3 capsular antigens exhibited significant suppression in the growth of tumor cells . The significance of these observations is discussed. Eur J Nucl Med, 1996 Jan, 23(1), 61 - 8 Macrophage targeting with technetium-99m labelled J001 acylated poly-galactoside for scintigraphy of inflammation: optimization and assessment of imaging specificity in experimental arthritis; Miot-Noirault E et al.; J001, an acylated poly-(1,3)-galactoside purified from the membrane of Klebsiella pneumoniae, associates selectively with macrophages via the binding to CD11b and CD14 molecules . Inflammatory foci known to recruit macrophages could thus be imaged with technetium-99m labelled J001 . This study aims to define the optimal scintigraphic protocol for 99mTc-J001 imaging and to evaluate the specificity of J001 scans . A dose range study was conducted in rabbits with immunological arthritis using six different specific activities ranging from 370 to 11840 MBq·mg-1 while the intravenously injected activity was constant (37 MBq) Radiochemical purity for each preparation was documented together with the in vivo stability of the 99mTc-J001 complex using exclusion-diffusion radioHPLC of serum collected 1 h after radiopharmaceutical administration . Scintigraphic images were recorded at 2, 3 and 4h and analysed using indexes calculated from regions of interest . Specificity of the macrophage imaging was assessed by comparison with scans obtained after administration of 99mTcO4(- )or 99mTc-albumin nanocolloids . A protocol of plasma transfusion was also used to inject 99mTc-J001 after complete removal of radioactive colloids likely to be generated during the labelling . For the higher specific activities (5920 and 11840 MBq.mg-1), radiochemical purity degradation and in vitro 99mTc transchelation were noted . To prevent transchelation and 99mTc bond hydrolysis likely to impair imaging specificity, 1480 MBq.mg-1 corresponding to 25microg injected J001 was found to be the optimal usable specific activity . Results obtained with the various tracers support the hypothesis that macrophage targeting is the main factor involved in the J001 imaging of arthritis. J Leukoc Biol, 1996 Jan, 59(1), 24 - 8 Expression and regulation of chemokines in bacterial pneumonia; Standiford TJ et al.; Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells which is dependent on the coordinated expression of both pro and anti-inflammatory cytokines . In this review, we present evidence indicating that both C-X-C and C-C chemokines are integral components of antibacterial host defense . Specifically, in vitro studies indicate that C-X-C chemokines {interleukin-8 (IL-8) and macrophage inflammatory protein 2 (MIP-2) and the C-C chemokine macrophage inflammatory protein 1 alpha (MIP-1 alpha) augment the ability of polymorphonuclear leukocytes (PMNs) and alveolar macrophages, respectively, to phagocytose and kill Escherichia coli . In addition, the intratracheal instillation of Klebsiella pneumoniae in CD-1 mice results in time-dependent production of MIP-2 and MIP-1 alpha and the inhibition of MIP-2 bioactivity in vivo results in decreases in lung PMN influx, impaired bacterial clearance, and early mortality . Finally, the anti-inflammatory cytokine interleukin-10 (IL-10) is also expressed within the lung during the evolution of Klebsiella pneumonia, and neutralization of IL-10 in vivo results in enhanced proinflammatory cytokine production, bacterial clearance, and increases in both short- and long-term survival . In conclusion, our studies indicate that specific chemokines are important mediators of leukocyte recruitment and/or activation in bacterial pneumonia and that the expression of these chemokines is regulated by endogenously produced IL-10. J Med Microbiol, 1996 Jan, 44(1), 44 - 51 Evaluation of a competitive ELISA method for the determination of Klebsiella O antigens; Trautmann M et al.; Strains of Klebsiella spp . are often inagglutinable by O-specific antisera because of the copious capsule produced by most isolates . A competitive ELISA method based on the observation that bacterial supernates containing homologous O antigen specifically inhibited the reaction of type-specific antisera with purified LPS coated on ELISA plates was used to examine the O antigen of 82 isolates of different Klebsiella species and subspecies . The O antigens O1/2ab (19 isolates), O2ab (13 isolates), O2ac (11 isolates) and O3 (16 isolates) were found to account for > 70% of the O antigenic types . Overall, 65 (79%) of the strains could be assigned to a specific O serogroup . The method is suitable for examining the role of individual O antigens in systemic klebsiella infections such as nosocomial septicaemia and pneumonia. J Am Coll Surg, 1996 Jan, 182(1), 33 - 6 Endogenous endophthalmitis associated with pyogenic hepatic abscess; Chou FF et al.; BACKGROUND: Endogenous endophthalmitis has been associated with pyogenic hepatic abscess in several recent anecdotal reports . The purpose of this study was to determine the incidence of endophthalmitis associated with pyogenic hepatic abscess, identify the degree of association with Klebsiella pneumoniae as a causative organism, and determine the outcome of treatment . STUDY DESIGN: A retrospective study was performed of 352 consecutive patients with a clinical diagnosis of pyogenic hepatic abscess who had been admitted to Chang Gung Memorial Hospital in Kaohsiung between 1986 and 1993 . Findings from complete ophthalmologic evaluations and treatment results were recorded . RESULTS: Eleven patients (3.1 percent) with endogenous endophthalmitis (monocular in eight and binocular in three) were found among the 352 cases of pyogenic hepatic abscess . Seven of the patients had diabetes mellitus and their blood glucose was poorly controlled . Only one patient had an intrahepatic stone as the cause of hepatic abscess, the other abscesses were of cryptogenic origin . The causative organism was mainly K . pneumoniae and the diagnosis was made by blood culture in ten patients, hepatic aspirate culture in seven, and vitreous contents culture in three . Systemic antibiotics were given in all patients with endogenous endophthalmitis . Percutaneous catheter drainage for hepatic abscess under echo guidance was performed in seven patients, medical treatment only was performed in three patients, and percutaneous tapping of abscess was done in one patient . All 11 patients were alive at the time of writing . Intravitreous culture followed by injection of antibiotics and steroids was immediately undertaken if septic endophthalmitis was suspected, except in two patients, who lost vision before any treatment was given . In five patients, cefamezin and gentamicin were given, and in four patients vancomycin, amikacin, and dexamethasone were given every three days if necessary . Finally, among the total of 14 eyes, there was blindness in ten, three of these had no light perception initially . In seven patients there had been a delay of treatment longer than one day . In one eye there was "counting fingers" vision and in three eyes there remained some vision . CONCLUSIONS: Physicians should be alert to the development of endogenous endophthalmitis when a patient with pyogenic hepatic abscess or bacteremia complains of ocular symptoms . Prompt diagnosis and vigorous treatment with intravitreous injections of vancomycin, amikacin, and dexamethasone within 24 hours can save the patient's eyes and vision. J Infect Dis, 1996 Jan, 173(1), 159 - 65 Neutralization of macrophage inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia; Greenberger MJ et al.; The role of macrophage inflammatory protein-2 (MIP-2) in bacterial pneumonia was characterized . Mice were challenged with Klebsiella pneumoniae intratracheally, and organs were harvested at 8, 24, and 48 h . Inoculation with K . pneumoniae resulted in the time-dependent expression of MIP-2 mRNA and protein within the lung, which was maximal 48 h after inoculation . Mice were then passively immunized with rabbit anti-murine MIP-2 serum intraperitoneally 2 h before administration of K . pneumoniae . Treatment with anti-MIP-2 serum resulted in a 60% decrease in lung neutrophil (PMNL) influx and a significant increase in K . pneumoniae colony-forming units in both lung and liver homogenates . Finally, treatment with anti-MIP-2 serum decreased early (48-72 h) but not late (after 72 h) survival in animals with Klebsiella pneumonia . This study indicates that MIP-2 is produced during Klebsiella pneumonia and inhibition of MIP-2 bioactivity in vivo results in decreased PMNL influx and lung bacterial clearance in murine Klebsiella pneumonia . MIP-2 is produced during Klebsiella pneumonia and inhibition of MIP-2 bioactivity in vivo results in decreased PMNL influx and lung bacterial clearance in murine Klebsiella pneumonia. J Infect Dis, 1996 Jan, 173(1), 151 - 8 Molecular genetics of resistance to both ceftazidime and beta-lactam-beta-lactamase inhibitor combinations in Klebsiella pneumoniae and in vivo response to beta-lactam therapy; Rice LB et al.; The molecular basis of ceftazidime resistance in 2 isolates of Klebsiella pneumoniae was studied . The first (21300) expressed resistance to ceftazidime and piperacillin-tazobactam . The second (26139) expressed resistance to ceftazidime but remained susceptible to piperacillin-tazobactam . The 2 strains harbored similar large plasmids that hybridized to TEM- and SHV-related beta-lactamase genes . An Escherichia coli strain harboring the plasmid conferring resistance to both compounds (pLRM7) produced beta-lactamases of pI 5.9 (TEM-6) and pI 7.6 (SHV-1) . E . coli harboring the other plasmid (pLRM8) expressed only the TEM enzyme because of insertion of IS15 within blaSHV-1 . In vivo studies suggested that resistance to beta-lactam-beta-lactamase inhibitor combinations conferred by pLRM7 will be clinically important . Clinical resistance to both extended-spectrum cephalosporins and beta-lactam-beta-lactamase inhibitor combinations is achievable via the production of two enzymes, with only one possessing an extended spectrum of activity. Gene, 1995 Dec 29, 167(1-2), 59 - 62 A new restriction-modification system, KpnBI, recognized in Klebsiella pneumoniae; Valinluck B et al.; A unique DNA restriction-modification (R-M) system has been identified in the GM236 strain of Klebsiella pneumoniae using the newly isolated phage, SBS . The system was designated KpnBI . The gene (hsdRKpnBI) complementing the restriction activity of the KpnBI system was cloned in pBR322 . The nucleotide sequence of the cloned DNA revealed one open reading frame (ORF) of 3035 bp . Analysis of the deduced amino-acid sequence shows seven helicase motifs which are common to the restriction (R) subunit of both type-I and type-III R-M systems . Computer analysis (Dendrogram) of the R polypeptide of KpnBI suggests a closer relationship to EcoR124/3I, a member of type-IC family, than to other representative type-I and type-III systems. Arch Biochem Biophys, 1995 Dec 20, 324(2), 317 - 24 The concentration of cellular nitrogenase proteins in Azotobacter vinelandii whole cells as determined by activity measurements and electron paramagnetic resonance spectroscopy; Jacobs D et al.; The concentration of MoFe protein (Av1) in Azotobacter vinelandii whole-cell crude extract was measured by electron paramagnetic resonance spectroscopy at g = 3.7 resonance . The Av1 concentration was also measured from the activity of crude extract to which increasing amounts of purified Av1 and Av2 were added . The Av2 concentration was determined by fitting activity measurements of crude extract and crude extract to which purified Av2 was added . The Av1 concentration was found to be 26-28 microM and that for Av2 was 42-45 microM in whole cells, with a Av2/Av1 ratio of 1.6 . In vitro activity measurements carried out as a function of Av1 concentration at Av2/Av1 ratios of 1 and 4 showed a dilution effect below 0.08 microM, a factor of 2 below that observed for nitrogenase reactivity for Klebsiella pneumoniae . No deviations from linearity were observed up to 26 microM for the Av1-Av2 interaction . The flavoprotein (AvFlp) was shown to enhance nitrogenase reactivity at low Av2/Av1 ratios, a result attributed to decreasing the Km for Av2-Av1 interaction . Direct reduction of bound Av2 is possibly the source of this kinetic enhancement . The kinetic results are considered in terms of the Thorneley and Lowe scheme. FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 203 - 8 Molecular characterisation by PCR-restriction fragment length polymorphism of TEM beta-lactamases; Arlet G et al.; To rapidly characterise TEM-derived extended-spectrum beta-lactamases a fast and easy method using polymerase chain reaction-restriction fragment length polymorphism was developed . This method was validated with ten reference TEM-type extended-spectrum beta-lactamases . The mutations involved in TEM-20 and TEM-21, which were previously reported only with biochemical analysis, were then characterised . TEM-20 differed from TEM-19 by a silent mutation at position 925 (A for G), and TEM-21 differed from TEM-3 and TEM-14 by a single mutation (G for A) in an unreported position 660 . beta-lactamase conferring low resistance to ceftazidime (TEM-29), was described . TEM-29 derived from TEM-1, with an amino acid substitution, his-164 . Finally, the combination of polymerase chain reaction-restriction fragment length polymorphism and plasmid analysis allowed us to investigate nosocomial outbreaks due to clinical isolates of multi-resistant Klebsiella pneumoniae in three hospitals.
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