J Dent Assoc S Afr, 1993 Aug, 48(8), 445 - 9 Anaerobic bacteria in orofacial abscesses; Botha SJ et al.; Obligate anaerobic bacteria were cultured from 15 orofacial abscesses . Bacteroides species constituted 43 per cent, Clostridium 21 per cent, Fusobacterium 14 per cent, Peptostreptococci 11 per cent, peptococci 7 per cent and Veillonella 4 per cent of the isolates . This study confirms the polymicrobial nature of orofacial infections . Sensitivity to antibiotics was unpredictable . Clostridium difficile, Clostridium tetani, Peptostreptococcus productus and Veillonella parvula showed resistance to some of the most frequently used antibiotics . Ampicillin and tetracycline were the most effective antibiotics and the highest resistance was shown against erythromycin. Clin Infect Dis, 1998 Feb, 26(2), 410 - 2 Isolation of a toxin B-deficient mutant strain of Clostridium difficile in a case of recurrent C . difficile-associated diarrhea; Cohen SH et al.; Clostridium difficile-associated diarrhea (CDAD) recurs in approximately 15%-20% of patients after discontinuation of metronidazole or vancomycin therapy . Most recurrences are believed to be endogenous relapses due to the persistence of spores . However, there is evidence that reinfection with a different strain is a cause of recurrence . We report the case of a patient with a history of multiple episodes of C . difficile colitis . The patient, a 56-year-old female, has had 5 years of repeated recurrences, each shortly after discontinuing vancomycin therapy . During the course of these episodes, three isolates were cultured from her stools at different times . These isolates were analyzed for the presence of toxin A and B gene sequences and genotyped by means of arbitrarily primed polymerase chain reaction (AP-PCR) . The original two isolates contained the toxin A and B genes, as determined by PCR, and were of the same AP-PCR type . During her last relapse, a C . difficile strain lacking at least a portion of the toxin B gene was isolated . AP-PCR analysis of this isolate showed a different DNA banding pattern from that of the previous isolates . A vancomycin susceptibility assay revealed a slight decrease in vancomycin activity as compared with that against the prior isolate . This case demonstrates two unique features: (1) recurrent infections can be due to reinfections and (2) toxin B mutants can possibly cause CDAD . This study also raises concerns about long-term vancomycin use and the development of resistance of C . difficile to vancomycin. Am J Infect Control, 1998 Feb, 26(1), 16 - 23 Laboratory surveillance method for nosocomial Clostridium difficile diarrhea; Mylotte JM; BACKGROUND: Clostridium difficile is the most common infectious cause of endemic nosocomial diarrhea, but traditional surveillance methods for this infection can be time-consuming . The purpose of this article is to (1) describe a laboratory surveillance method for nosocomial diarrhea and nosocomial Clostridium difficile diarrhea (CDD) that does not require chart review and (2) describe some of the epidemiology of these infections at a university-affiliated, public hospital by using this surveillance method . METHODS: The main assumption underlying the surveillance method is that all patients with nosocomial diarrhea have a C . difficile stool toxin assay performed . On the basis of this assumption, the frequency of testing stool samples for toxin is considered a surrogate for the occurrence of nosocomial diarrhea; it is also assumed that the results of the stool toxin assay distinguish between those with (positive assay) and without (negative assay) CDD . During the study period (January 1, 1993, to August 30, 1996) surveillance for nosocomial CDD was performed by monitoring results of C . difficile stool toxin assays done with the Cytoclone A and B enzyme immunoassay . Each month a list of results of all assays performed was reviewed and patients were excluded on the basis of the following criteria . First, patients with assays done within the first 4 days of admission were assumed to have community-acquired diarrhea and excluded . Among patients with assays done > 4 days after admission, patients with two or more assays done within a 7-day period were counted only once; repeated assays (positive or negative) in the 14 days after an initial positive assay (indicating nosocomial CDD) were excluded, but assays done more than 14 days after a positive or a negative assay were counted separately (representing a relapse or new episode of diarrhea) . Patients remaining on the list after all the exclusion criteria were applied represented those with nosocomial diarrhea . RESULTS: The mean (+/- SD) frequency of episodes of nosocomial diarrhea per month for each study year (1993, 1994, 1995, and first 8 months of 1996) was 52.6 +/- 16.2, 51.4 +/- 10.5, 49.2 +/- 9.3, 57.8 +/- 11.6, respectively (p = 0.48 by ANOVA); the mean frequency of nosocomial diarrhea per 1000 admissions per month was 48.4 +/- 14.5, 47.7 +/- 10.9, 44.0 +/- 9.6, and 51.6 +/- 9.3, respectively (p = 0.52); and the mean frequency of nosocomial CDD episodes per 100 episodes of nosocomial diarrhea was 24.7 +/- 8.5, 18.9 +/- 4.8, 17.4 +/- 5.7, and 12.2 +/- 7.2, respectively (p = 0.003) . The median time (days) after admission to the onset of nosocomial CDD (first positive assay) for each study year was 14.5, 13.0, 12.0, and 13.0, respectively . CONCLUSIONS: Although not all of the underlying assumptions of the method have been verified, the similarity of the findings in the present study to those of previously published studies of nosocomial CDD suggests that the method is valid . Alternatives to traditional methods of performing nosocomial infection surveillance need to be developed so that infection control practitioners can focus more of their efforts on prevention activities. Br J Surg, 1998 Feb, 85(2), 229 - 31 Timing of surgery for fulminating pseudomembranous colitis; Synnott K et al.; BACKGROUND: With increasing antibiotic usage Clostridium difficile colitis is becoming more common . Surgery for fulminating C . difficile colitis, however, is rare because of the effectiveness of specific anticlostridial chemotherapy . Surgical outcome in five patients with fulminating C . difficile colitis involved in a recent outbreak of this disease is reported . METHODS: Five of 138 patients developed fulminating C . difficile colitis unresponsive to medical therapy . All patients had antibiotics in the preceding period . Indications for operation in those who underwent surgery were systemic toxicity with a pyrexia, marked leukocytosis and abdominal signs leading to progressive organ failure, despite appropriate anticlostridial antibiotic therapy . RESULTS: At operation all patients had a markedly oedematous colon with normal serosa but with acute mucosal colitis . All underwent subtotal abdominal colectomy and ileostomy . Progressive organ failure persisted in four, leading to death, giving a mortality rate of four in five in the operated group in comparison with 3.8 per cent (five of 133 patients) in those treated medically . CONCLUSIONS: These results indicate that this increasingly common disease frequently leads to a fatal outcome in patients requiring surgery and implies that earlier surgical consultation may be necessary to improve survival in patients with fulminating C . difficile colitis unresponsive to antibiotic therapy. Appl Environ Microbiol, 1998 Mar, 64(3), 1086 - 90 Characterization of EngF from Clostridium cellulovorans and identification of a novel cellulose binding domain; Ishi A et al.; The physical and enzymatic properties of noncellulosomal endoglucanase F (EngF) from Clostridium cellulovorans were studied . Binding studies revealed that the Kd and the maximum amount of protein bound for acid-swollen cellulose were 1.8 microM and 7.1 mumol/g of cellulose, respectively . The presence of cellobiose but not glucose or maltose could dissociate EngF from cellulose . N- and C-terminally truncated enzymes showed that binding activity was located at some site between amino acid residues 356 and 557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino acids were removed from the C terminus; when 57 amino acids were removed from the C terminus, all activity was lost . EngF showed low endoglucanase activity and could hydrolyze cellotetraose and cellopentaose but not cellotriose . Activity studies suggested that EngF plays a role as an endoglucanase during cellulose degradation . Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs. Appl Environ Microbiol, 1998 Mar, 64(3), 1079 - 85 Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification; Bermejo LL et al.; A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT . Acetone production demonstrated that ace4 is expressed in E . coli and resulted in the reduction of acetic acid levels in the fermentation broth . Since different E . coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E . coli strains: ER2275, ATCC 11303, and MC1060 . Shake flask cultures of MC1060(pACT) produced ca . 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca . 40 mM acetone . Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures . External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers . In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production . Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations . These acetone titers are equal to or higher than those produced by wild-type C . acetobutylicum . This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production . In addition, acetone-producing E . coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. Appl Environ Microbiol, 1998 Mar, 64(3), 907 - 13 Detection of Clostridium proteoclasticum and closely related strains in the rumen by competitive PCR; Reilly K et al.; A competitive PCR technique was used to enumerate the proteolytic bacterium Clostridium proteoclasticum from the rumen . A PCR primer, which circumscribes this organism and several closely related strains, was designed for a variable region within their 16S rRNA genes and was used in conjunction with a universal forward primer . This primer pair was tested for specificity against 85 ruminal bacterial strains . An internal control DNA was constructed for use in competitive PCRs and was shown to amplify under the same reaction conditions and with the same amplification efficiency as the target DNA . DNA from a known number of C . proteoclasticum cells was coamplified with the internal control to construct a standard curve . Rumen samples were collected from eight dairy cows fed four diets in rotation: high nitrogen, high nitrogen supplemented with carbohydrate, low nitrogen, and low nitrogen supplemented with carbohydrate . DNA extracted from these and spiked with internal control DNA was amplified with the C . proteoclasticum primer pair . The relative intensities of the PCR products were used to quantitate the numbers of C . proteoclasticum cell equivalents from the rumen samples . The numbers ranged from 2.01 x 10(6) ml-1 to 3.12 x 10(7) ml-1 . There was no significant effect on the numbers of C . proteoclasticum detected in rumen samples among cows fed the four diets . The utility of the competitive PCR approach for quantifying ruminal bacterial populations in vivo and the occurrence of C . proteoclasticum in forage-fed dairy cows are discussed. Appl Environ Microbiol, 1998 Mar, 64(3), 836 - 42 Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate; Nishikawa S et al.; Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5'-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source . Nitrosoguanidine mutagenesis of S . paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated . The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2',3'-trihydroxy-3-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) . Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate . A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6 . The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0 . Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum. Lancet, 1998 Feb 28, 351(9103), 633 - 6 Primary symptomless colonisation by Clostridium difficile and decreased risk of subsequent diarrhoea; Shim JK et al.; BACKGROUND: Little is known about whether patients who develop Clostridium-difficile-associated diarrhoea (CDAD) are culture-positive or culture-negative before illness . The most important risk factor is antibiotic exposure . We aimed to find out whether patients identified as primary symptom-free C difficile carriers are at higher risk of developing CDAD than patients who are culture-negative . METHOD: We reviewed four longitudinal studies in which 810 patients admitted to hospital were followed up by prospective rectal-swab culture . At least two consecutive weekly cultures were obtained . We calculated the difference in risk of CDAD between colonised and non-colonised patients in each study and combined the results of the four studies in a random-effects model . FINDINGS: Of 618 non-colonised patients (mean follow-up 1.7 weeks {SD 1.3}), 22 (3.6%) developed CDAD, whereas only two (1.0%) of 192 primary symptom-free carriers (1.5 {1.5}) developed CDAD (pooled risk difference -2.3% {95% CI 0.3-4.3}, p=0.021) . Of patients who received antibiotics, the risk difference was increased: 22 (4.5%) of 491 non-colonised patients compared with two (1.1%) of 176 colonised patients developed CDAD (-3.2% {0.4-6.0}, p=0.024) . Of the primary symptom-free C difficile carriers, 95 were colonised with toxigenic strains, 76 with non-toxigenic strains, 12 with both toxigenic and non-toxigenic strains (non-concurrently), and nine with strains of undetermined toxigenicity . Nine of the 12 toxogenic strains of C difficile isolates that cause CDAD were also recovered from stools of symptom-free patients . INTERPRETATION: Primary symptomless C difficile colonisation is associated with a decreased risk of CDAD . Although the mechanism is unknown, risk reduction is found in colonisation with non-toxigenic and toxigenic strains. Gastroenterology, 1998 Mar, 114(3), 519 - 26 Stimulation of transforming growth factor beta1 by enteric bacteria in the pathogenesis of rat intestinal fibrosis; Mourelle M et al.; BACKGROUND & AIMS: Bacteria and their products stimulate inflammatory responses . Certain mediators, such as transforming growth factor beta1 (TGF-beta1), induce collagen synthesis . Excess collagen deposition results in bowel strictures . The aim of this study was to investigate the role of bacteria and TGF-beta1 in the pathogenesis of intestinal fibrosis . METHODS: In rats with colitis, the effects of bowel decontamination with antibiotics on TGF-beta1, tumor necrosis factor alpha (TNF-alpha), and collagen content in colonic tissue were studied . In normal rats, bacteria of the predominant flora were inoculated into the colonic wall . The effect of neutralizing antibody to TGF-beta1 on tissue collagen deposition was studied . RESULTS: Rats with chronic colitis showed increased levels of TGF-beta1, TNF-alpha, and collagen in the tissue and a high rate of bowel strictures . Antibiotic treatment significantly prevented the increase in TGF-beta1 and collagen and the formation of strictures . Inoculation of bacterial suspensions into the colonic wall increased tissue TGF-beta1 and collagen content . Neutralizing antibody to TGF-beta1 prevented collagen deposition . Colonic wall inoculations with single anaerobic strains (Clostridium ramosum, Bacteroides fragilis, and Bacteroides uniformis), but not with aerobes, induced collagen deposition . CONCLUSIONS: Certain strains of the common flora stimulate TGF-beta1 and induce deposition of collagen in the colonic wall. Eur J Clin Microbiol Infect Dis, 1997 Dec, 16(12), 938 - 9 Splenic abscess caused by Clostridium difficile; Kumar N et al.; Splenic abscess is a previously unreported complication of Clostridium difficile colitis . A case of Clostridium difficile splenic abscess is reported that developed after the patient had been in intensive care for five weeks . A response was seen to radiologically guided drainage and antibiotic therapy with formal laparotomy, and surgical drainage not being required. Eur J Clin Microbiol Infect Dis, 1997 Dec, 16(12), 928 - 33 Nosocomial outbreak of Clostridium difficile diarrhea in a pediatric service; Ferroni A et al.; An outbreak of nosocomial diarrhea that occurred in a pediatric orthopedic service between 1 December 1993 and 15 April 1994 is reported . A total of 37 patients (mean age, 9.6 years; range, 2 months-19.3 years) were involved in the outbreak, including six patients with bacteriologically documented Clostridium difficile infection . A multivariate analysis identified lincomycin treatment for at least three days as the only significant risk factor . Stool samples from four asymptomatic patients were also positive for Clostridium difficile and its cytotoxins . Isolates from all patients belonged to serogroup C, were highly resistant to lincomycin, and exhibited the same restriction pattern by pulsed-field gel electrophoresis . The outbreak ended after treatment with lincomycin was discontinued and hygiene control measures were implemented. Microbiology, 1998 Feb, 144 ( Pt 2), 333 - 41 Production of a non-toxic site-directed mutant of Clostridium perfringens epsilon-toxin which induces protective immunity in mice; Oyston PC et al.; A panel of ten site-directed mutants of Clostridium perfringens epsilon-toxin was generated . All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native epsilon-toxin . The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells . One mutation resulting in loss of activity in the assay was identified . This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin . Immunization of mice with the non-toxic mutated epsilon-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant epsilon-toxin. Rinsho Byori, 1998 Jan, 46(1), 26 - 32 {Genotyping of isolates of bacteria and Candida}; Fujita S; Strain differentiation of Staphylococcus species, Streptococcus pneumoniae, Escherichia coli, Clostridium difficile, and Candida species was performed by restriction endonuclease analysis (REA) of genomic DNA with HinfI or HaeIII followed by conventional agarose gel electrophoresis . REA of 19 methicillin-resistant S . aureus isolates and 19 methicillin-susceptible S . aureus isolates revealed 8, and 14 patterns, respectively . Fifty-three isolates of S . epidermidis were divided into 39 groups on the basis of REA pattern . REA patterns obtained from different strains of the same species were more similar than those from different Staphylococcus species . A total of 67 patterns were noted among 215 S . pneumoniae isolates . There was some correlation between the REA patterns and serotypes . In addition, all 61 isolates showing the pattern 6 (serotype 19) were penicillin-resistant . Sixteen isolates of E . coli O157 and 18 isolates of E . coli O25 were analyzed by REA with HaeIII . Among 14 epidemiologically unrelated E . coli O157:H7 isolates, REA patterns were either identical or differed only by a few fragment bands . REA patterns of 7 isolates of heat stable enterotoxin producing E . coli O25 from an outbreak were identical . Eight distinct REA patterns were noted among 7 toxigenic C . difficile strains, and one to two strains were included in each group . Candida strains belonging to different six Candida species were analysed by REA . All C . albicans isolates (148 strains) showed one to four bright bands ranging from 4 to 8kb in size, and these strains were divided into 37 groups according to the REA patterns . Each Candida strain showed species-specific REA patterns . Twenty-one isolates of Mycobacterium tuberculosis complex were analyzed by PCR with primer complementary to the inverted repeat of IS6110(IS6110-PCR) . The results of PCR of 19 M . tuberculosis isolates and 2 M . bovis BCG isolates revealed 15 patterns, and 1 pattern, respectively . Our results demonstrated that REA of genomic DNA with HinfI or HaeIII was a useful tool for strain differentiation . By comparing the species-specific REA patterns of reference strains, clinical isolates of Candida and Staphylococci could be identified to the species level if they could not be identified by routine biochemical methods . Because IS6110-PCR is simple and rapid to perform, it seems to be a good screening method to differentiate M . tuberculosis strains. J Protein Chem, 1998 Jan, 17(1), 53 - 60 A protease-resistant novel hemagglutinin purified from type A Clostridium botulinum; Fu FN et al.; Botulinum neurotoxin is a food poisoning agent produced by Clostridium botulinum . The neurotoxin is a 150-kDa protein that causes flaccid muscle paralysis by blocking neurotransmitter release at neuromuscular junctions . The neurotoxin is produced along with a group of neurotoxin associated proteins (NAPs), which protect it from the low pH and proteases of the gastrointestinal (GI) tract . We have isolated, for the first time, one of the major components of NAPs in a pure form . The isolated protein is a 33-kDa single polypeptide (Hn-33) that exhibits hemagglutination activity . Specific polyclonal antibodies against the Hn-33 are able to block the hemagglutination activity of the neurotoxin complex, which indicates that perhaps Hn-33 is the only strong hemagglutinating protein in the complex . The Hn-33 was found be resistant to trypsin and other protease digestion, a feature that could play a role in the protection of the neurotoxin in the GI tract during its toxicoinfection. Mol Gen Genet, 1998 Jan, 257(2), 213 - 8 Isolation and characterisation of an aryl-beta-D-glucoside uptake and utilisation system (abg) from the gram-positive ruminal Clostridium species C . longisporum; Brown GD et al.; A phosphotransferase-dependent aryl-beta-glucoside uptake and utilisation system (abg) was isolated from the ruminal Clostridium ("C . Longisporum") . The system is composed of three genes, abgG, abgF and abgA, and a number of regulatory regions, including terminator/antiterminator type stem-loop structures preceding the abgG and abgF genes . Similarity analysis of the proteins encoded by these genes indicated that they were responsible for the regulation of the abg system through antitermination (AbgG), the uptake and phosphorylation of aryl-beta-glucosides (AbgF) and the hydrolysis of the intracellular phosphorylated glycosides (AbgA) . Experimental evidence for the functions of AbgF and AbgA was obtained . Although it was not possible to demonstrate any function for AbgG, a promoter 5' to the abgG gene was identified which was responsible for expression of the downstream genes . The abg system is remarkably similar to operons from the gram negative Enterobacteriaceae, both in the coding and non-coding regulatory regions. FEBS Lett, 1998 Jan 30, 422(2), 221 - 4 Synergistic interaction of the cellulosome integrating protein (CipA) from Clostridium thermocellum with a cellulosomal endoglucanase; Ciruela A et al.; Activity of a cellulosomal endoglucanase (endoglucanase E; EGE) from Clostridium thermocellum against two crystalline forms of cellulose was enhanced by combination with the cellulosome integrating protein (CipA), but CipA did not enhance EGE activity against amorphous cellulose, even though it was able to bind to it . Similarly, CipA added in trans to genetically truncated EGE that was unable to combine with it nevertheless enhanced EGE activity against crystalline cellulose . These results indicate that the CipA cellulose binding domain does not mediate an increase in activity solely by bringing the catalytic subunits of the cellulosome complex into intimate contact with the substrate. Mol Microbiol, 1998 Feb, 27(3), 631 - 42 Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ; Crellin PK et al.; The 6.3 kb Clostridium perfringens transposon Tn4451 encodes a 50 kDa protein, TnpZ, which has amino acid sequence similarity to a group of plasmid mobilization and recombination proteins that comprise the Mob/Pre family . Members of this family interact with an upstream palindromic sequence called an RSA site, and an RSA-like sequence has been identified upstream of the tnpZ gene . In Escherichia coli, in the presence of a chromosomally integrated derivative of the broad-host-range IncP plasmid, RP4, TnpZ was able to promote plasmid mobilization in cis and was able to function in trans to enable the mobilization of a co-resident plasmid carrying an RSA site . It was also able to mediate the conjugative transfer of plasmids from E . coli to C . perfringens . Site-directed mutagenesis of two bases within the RSA site resulted in a significant reduction in mobilization frequency, demonstrating that the RSA site is required for efficient plasmid mobilization . TnpZ is the only Mob/Pre protein known to be associated with a transposable genetic element, and Tn4451 is the first mobilizable but non-self-transmissible transposon to be identified from a gram-positive bacterium. Lett Appl Microbiol, 1998 Jan, 26(1), 81 - 4 Evaluation of the use of the bioMerieux Rapid ID32 A for the identification of Clostridium botulinum; Brett MM; The neurotoxins produced by Clostridium botulinum are amongst the most potent known to man . Toxin production is detected by a mouse bioassay, which requires several days for a result and is not acceptable for routine use unless there is a high level of suspicion . The Rapid ID32 A kit produced by bioMerieux gives an identification of an isolate within 4 h . The aim of this study was to examine the efficiency of the identification of Cl . botulinum using the Rapid ID32 A . Forty-two strains of Cl . botulinum, one strain each of botulinum toxin-producing Cl . butyricum and Cl . baratii, and four strains of Cl . sporogenes, were tested . One strain of Group I Cl . botulinum gave a presumptive identification of Group II Cl . botulinum, six strains of Cl . botulinum were identified as 50-98% Cl . botulinum in some tests, and 17 strains of Cl . botulinum were identified as < 50% Cl . botulinum . Thirteen strains of Cl . botulinum were identified as > 99% Cl . sporogenes or 86% Cl . histolyticum, and five strains gave a combination of these results . All strains of Cl . sporogenes were correctly identified . These results show that some strains of Cl . botulinum may not be correctly identified using the Rapid ID32A. Lett Appl Microbiol, 1998 Jan, 26(1), 35 - 7 Conjugal transfer of transposon Tn1545 into the cellulolytic bacterium Eubacterium cellulosolvens; Anderson KL et al.; Tn1545, a self-mobilizing transposon, was introduced into the chromosome of the ruminal cellulolytic bacterium Eubacterium cellulosolvens . This was achieved by conjugal transfer of the transposon from Clostridium beijerinckii at a frequency of 1 per 10(6) recipient cells . Transconjugants of Eu . cellulosolvens were resistant to both tetracycline and erythromycin, and were able to mobilize Tn1545 back into Cl . beijerinckii . Southern blot hybridization of representative transconjugants did not reveal site-specific insertion . This potential randomness of the transposon insertion site may prove useful in the development of Tn1545 as a tool for mutagenesis of Eu . cellulosolvens. J Biol Chem, 1998 Feb 6, 273(6), 3643 - 8 A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase; Matsushita O et al.; The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3 . A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3 . In this paper we have investigated the function of the C-terminal segments using recombinant proteins . Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide . Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen . To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase . S3 as well as S2bS3 conferred collagen binding . However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity . S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b . S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase . These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure. Ann Surg, 1998 Feb, 227(2), 296 - 301 The impact of Clostridium difficile on a surgical service: a prospective study of 374 patients; Kent KC et al.; OBJECTIVE: To evaluate the epidemiology of Clostridium difficile colitis (CDC) in a subset of patients admitted specifically to a surgical service . SUMMARY BACKGROUND DATA: CDC is an increasingly prevalent nosocomial infection that can prolong hospitalization and adversely affect patient outcome . Although this disease has been investigated extensively in patients admitted to medical services, the incidence and risk factors for the development of this disease in patients admitted to a surgical service have not been studied . METHODS: Over a 5-month period, 374 patients admitted to the general, vascular, thoracic, and urologic surgery services were monitored for the development of symptomatic CDC (defined as >3 bowel movements per 24 hours and a positive cytotoxin assay or culture) . RESULTS: Twenty-one patients developed CDC (incidence, 5.6%) . Factors that independently predisposed to infection included admission from a skilled care facility, use of the antibiotic cefoxitin, and an operative procedure for bowel obstruction . Other factors associated with CDC included colectomy, treatment with any antibiotic, nasogastric tube suction, advanced age, and prior antibiotic treatment . Abdominal pain and fever were also more common in patients with CDC . Morbidity included prolonged hospitalization in all patients and urgent colectomy in one . CONCLUSIONS: CDC frequently affects surgical patients, producing morbidity ranging from mild diarrhea to life-threatening illness . A variety of factors, many of which are associated with intestinal stasis, predispose to the development of CDC. Infect Immun, 1998 Mar, 66(3), 1076 - 81 Chimeric clostridial cytotoxins: identification of the N-terminal region involved in protein substrate recognition; Hofmann F et al.; Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases . In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C . sordellii lethal toxin also glucosylates Ras subfamily proteins . By deletion analysis and construction of chimeric fusion proteins of C . sordellii lethal toxin and C . difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity . The toxin fragment of the N-terminal 546 amino acid residues of C . sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did . Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity . Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins . In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed . The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C . sordellii lethal toxin. Mol Immunol, 1997 Oct, 34(14), 1031 - 40 Immune recognition of botulinum neurotoxin type A: regions recognized by T cells and antibodies against the protective H(C) fragment (residues 855-1296) of the toxin; Oshima M et al.; Botulism toxicity is caused by botulinum neurotoxins (BoNTs), a group of protein neurotoxins produced by Clostridium botulinum . Recent studies have shown that immunization with a C-terminal fragment {H(C), residues 855-1296} of BoNT type A (BoNT/A) affords excellent protection against BoNT/A toxicity . The present work was carried out in order to map the molecular and cellular immunological recognition of H(C) . We have previously described the synthesis of 31 overlapping peptides encompassing the entire H(C)-fragment of BoNT/A . These peptides were employed in this study to localize the continuous regions recognized by T cells and by antibodies (Abs) generated in two mouse strains against H(C) . T cells from SJL that had been primed with H(C) gave a strong proliferative response to challenge in vitro with each of the six peptides spanning residues 897-985 and a lower response to peptide 1051- 1069 . While H(C)-primed T cells of BALB/c recognized three regions residing within residues 939-957, 1009-1027 and 1135-1153 (strong) . Recognition regions by Abs in SJL or BALB/c anti-H(C) antisera essentially overlapped . However, the level of Abs bound to each region differed between the two strains . These common or similar recognition regions by the two strains were: 855-915 (SJL) or 855-901 (BALB/c); 939-957; 967-1013 (BALB/c) or 981-1013 (SJL); 1051-1069; 1079-1111 (BALB/c) or 1093-1125 (SJL); 1177-1195; and 1275-1296 . In addition, BALB/c recognized region 1135-1153 . Some of these regions show considerable sequence similarity in BoNT types B and E and, therefore, H(C) of these two BoNTs might offer protection against the correlate clostridial toxins. J Physiol, 1998 Jan 1, 506 ( Pt 1), 83 - 93 Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins; Lucius C et al.; 1 . Clostridium difficile toxin B glucosylates the Ras-related low molecular mass GTPases of the Rho subfamily thereby inactivating them . In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol-induced increase in the Ca2+ sensitivity of force and myosin light chain (MLC) phosphorylation in intact intestinal smooth muscle . 2 . Small strips of the longitudinal muscle of guinea-pig small intestine were incubated in toxin B (40 ng ml-1) overnight . Carbachol-induced force and intracellular {Ca2+}, and, in a separate series, force and MLC phosphorylation, were determined . 3 . Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delayed increase in force (peak 2) . The peak of the Ca2+ signal measured with fura-2 preceded peak 1 of force and then declined to a lower suprabasal steady-state level . Peak 2 was not associated with a significant increase in {Ca2+} . Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited . Toxin B had no effect on the Ca2+ transient . 4 . In control strips, MLC phosphorylation at peak 2 was 27.7% which was significantly higher than the resting value (18.6%) . The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation . The resting MLC phosphorylation was not significantly different from that of the control strips . 5 . The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54% in the control strips . Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca2+ transient and the initial increase in force were not inhibited by toxin B . This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca2+ . 6 . These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca2+ sensitivity of MLC phosphorylation mediated by Rho proteins. J Am Geriatr Soc, 1998 Feb, 46(2), 157 - 60 Vancomycin-resistant Enterococcus faecium in a long-term care facility; Brennen C et al.; OBJECTIVE: To describe the epidemiology and natural history of colonization with vancomycin-resistant Enterococcus faecium (VREF) in a long-term care facility . DESIGN: All patients in whom VREF was isolated were followed prospectively, with rectal swab cultures at 2-week intervals, until discharge, death, or clearance of VREF . Clearance was defined as two consecutive negative cultures . In addition, three prevalence surveys were conducted of all patients in residence on one 34-bed intermediate care ward . SETTING: A 400-bed, long-term care Veterans Affairs facility . PARTICIPANTS: Thirty-six patients colonized with VREF . RESULTS: Vancomycin-resistant Enterococcus faecium was identified in 24 of the 36 patients at the time of transfer from an acute care facility . Seventeen patients had concomitant methicillin-resistant Staphylococcus aureus, and seven patients had a recent history of Clostridium difficile-associated diarrhea . VREF in these patients persisted for a median of 67 days after identification . Treatment of VREF colonization with antimicrobials was associated with prolongation of colonization . Serial surveillance of the 34-bed ward found stable rates of colonization, with only three documented instances of VREF acquisition . During 2.5 years of surveillance for infection, a single case of bacteremia occurred in a patient in whom colonization with VREF could not be demonstrated by rectal swab culture . No infections occurred in patients colonized with VREF . CONCLUSIONS: Long-term care patients have protracted carriage of VREF . Most will improve over time; however, receipt of antimicrobial therapy is associated with prolongation of VREF carriage . The risk of VREF infection is low in this population . When there are appropriate contact precautions, patient to patient transmission occurs at a low rate . These observations can be used to design a practical infection control strategy for long-term care facilities. J Am Mosq Control Assoc, 1997 Dec, 13(4), 395 - 7 A novel insecticidal serotype of Clostridium bifermentans; Seleena P et al.; A novel Clostridium bifermentans strain toxic to mosquito larvae on ingestion was isolated from a soil sample collected from secondary forest floor . This strain was designated as serovar paraiba (C . b . paraiba) according to its specific H antigen . Clostridium bifermentans paraiba is most toxic to Anopheles maculatus Theobald larvae (LC50 = 0.038 mg/liter), whereas toxicity to Aedes aegypti (Linn.) (LC50 = 0.74 mg/liter) and Culex quinquefasciatus Say (LC50 = 0.11 mg/liter) larvae was 20 and 3 times lower, respectively . The toxicity to An . maculatus larvae is as high as that of Bacillus thuringiensis serovar israelensis . C . b . paraiba was also found to exhibit significant per os insecticidal activity toward adult Musca domestica (Linn.). Rev Gastroenterol Mex, 1997 Apr-Jun, 62(2), 113 - 6 {Pseudomembranous colitis: report of four cases}; Solana de Lope J et al.; BACKGROUND: Clostridium difficile is the cause of 25-30% of cases of antibiotic-induced diarrhea . Pseudomembranous colitis is the most dramatic manifestation of C . difficile infection . METHODS: We report four cases of pseudomembranous colitis and review the literature . RESULTS: Three of the four patients, were over 80 years old and had other underlying illnesses . Before they developed colitis, all had received cephalosporins (cefuroxime, ceftriaxone, cefalexine) and one of them also clindamycin . All the patients had severe watery bowel movements, with mucus (one patient had also bloody stools), abdominal pain, nausea, vomit and fever . Blood tests disclosed leucocytosis with neutrophilia and increased band neutrophils in all patients . One patient had anasarca and hypo-albuminemia, suggestive of protein losing enteropathy . Sigmoidoscopy showed raised, yellow plaques covering the rectum, sigmoid and descendent colon mucosa . The response to oral metronidazole or vancomycin was good . The response to intravenous metronidazole was good in one patient and poor in another one . Two of our patients had relapses . The response of the relapses to oral metronidazole was good . One patient had two relapses eventually responding to oral metronidazole and Saccharomyces boulardii . CONCLUSIONS: Pseudomem-branous colitis has high morbility in debilitated elderly patients . Relapses are frequent in these patients . If other studies should confirm it, Saccharomyces boulardii could be useful in the prevention and treatment of relapses of this colitis. J Vet Intern Med, 1997 Nov-Dec, 11(6), 319 - 22 Prevalence and identity of translocating bacteria in healthy dogs; Dahlinger J et al.; Bacterial translocation is characterized by the passage of intestinally derived bacteria across the intestinal mucosa to local or regional tissues . This phenomenon is believed to be important in the pathogenesis of gram-negative bacteremia and septicemia; however, the pathway or route of translocation remains unclear . To define the route of translocation better, mesenteric lymph nodes from 50 apparently healthy dogs undergoing elective ovariohysterectomies were cultured aerobically and anaerobically . The aims of this study were to determine the prevalence of bacterial translocation and to quantify and identify types of organisms found in mesenteric lymph nodes . Peripheral blood and portal blood samples were similarly cultured to rule out hematogenous organisms as a source of lymph node contamination . Bacteria were isolated from mesenteric lymph nodes of 26 dogs (52%) . The number of bacteria varied from 50 to > 10(5) organisms/g of tissue . Bacteria isolated included Staphylococcus intermedius (n = 3), coagulase-negative Staphylococcus (n = 2), nonhemolytic Streptococcus (n = 4), Bacillus species (n = 5), Escherichia coli (n = 6), Salmonella species (n = 3), Pseudomonas species (n = 2), Enterococcus species (n = 2), Clostridium sordelli (n = 1), Micrococcus species (n = 1), Lactobacillus species (n = 1), and Propionibacterium acnes (n = 1) . One of 50 peripheral blood samples yielded an unidentified gram-positive coccus and a coagulase-negative Staphylococcus . No bacteria were isolated from portal blood samples of any dog . Further studies of this type on sick dogs are warranted before clinical recommendations can be made to culture mesenteric lymph nodes routinely. J Pediatr, 1998 Jan, 132(1), 177 - 9 Severe Clostridium difficile-associated colitis in young patients with cystic fibrosis; Rivlin J et al.; We report four patients with cystic fibrosis and fulminant Clostridium difficile-associated colitis: two died, and one required hemicolectomy . Three of four patients carried the N1303K mutation . Severe and fatal C . difficile colitis can occur in cystic fibrosis patients, possibly with a genotype-specific predilection (i.e., N1303K/other) . Because cystic fibrosis patients may have a wide spectrum of gastrointestinal symptoms, disease caused by C . difficile must be considered when these patients have acute abdominal pain, diarrhea, or severe leukocytosis. J Immunol, 1998 Feb 15, 160(4), 1894 - 900 RANTES activation of phospholipase D in Jurkat T cells: requirement of GTP-binding proteins ARF and RhoA; Bacon KB et al.; The chemokine RANTES is a potent agonist of T cell activation . In an investigation of signal-transduction events activated by this chemokine, we have shown that RANTES stimulates dose-dependent phospholipase D (PLD) activity in Jurkat cells . Equilibrium-binding analyses using 125I-labeled RANTES indicated the presence of a receptor for RANTES on these cells, which has a Kd of 0.1 nM, is expressed at approximately 600 sites per cell, and a binding specificity that was not comparable with that of any of the known chemokine receptors, since 125I-labeled RANTES was displaced by macrophage-inflammatory protein-1 beta (but not macrophage-inflammatory protein-1 alpha), monocyte-chemotactic protein-1 (MCP-1), MCP-3, MCP-4, and eotaxin . RANTES-induced PLD activation was augmented by GTP gamma S, but not GDP beta S, and inhibited by the protein kinase C inhibitor bisindolylmaleimide, as well as the fungal metabolite brefeldin A, and C3 exoenzyme (Clostridium botulinum), implicating the activation of RhoA . RANTES also induced GTP-GDP exchange of immunoprecipitated RhoA . RANTES-stimulated PLD activity was dependent on an ADP-ribosylation factor(s), as assessed by inhibition studies using a synthetic inhibitory peptide of the N-terminal 16 amino acids of ADP-ribosylation factor 1 . These studies indicate the potential existence of a novel receptor-mediated mechanism for activation of T cells by the chemokine RANTES. Microbiology, 1998 Jan, 144 ( Pt 1), 211 - 7 Identification of a region responsible for binding to the cell wall within the S-layer protein of Clostridium thermocellum; Lemaire M et al.; The protomer forming the S-layer of Clostridium thermocellum was identified as a 140 kDa protein which was non-covalently bound to the cell wall . Cloning and sequencing of the corresponding gene revealed an open reading frame of 3108 nucleotides encoding a polypeptide of 1036 amino acids, termed SlpA . The amino acid composition of SlpA matches the composition of a previously described exocellular glycoprotein . SlpA shared extensive similarity with the S-layer protein of Bacillus sphaericus and with the outer wall protein of Bacillus brevis . In addition, the amino-terminal region of SlpA contained a segment presenting similarities with segments termed SLH (S-layer homologous), which are found in several bacterial exoproteins . A polypeptide of 209 residues comprising this segment was shown to bind to cell walls extracted from C . thermocellum cells. Mol Microbiol, 1998 Jan, 27(1), 107 - 20 Regulated transcription of Clostridium difficile toxin genes; Dupuy B et al.; The Clostridium difficile toxA and toxB genes, encoding cytotoxic and enterotoxic proteins responsible for antibiotic-associated colitis and pseudomembranous colitis, were shown to be transcribed both from gene-specific promoters and from promoters of upstream genes . However, the gene-specific transcripts represented the majority of tox gene mRNAs . The 5' ends of these mRNAs were shown to correspond to DNA sequences that had promoter activity when fused to the Escherichia coli beta-glucuronidase (gusA) gene and introduced into C . perfringens . The appearance of tox mRNA in C . difficile was repressed during exponential growth phase but increased substantially as cells entered stationary phase . When glucose or other rapidly metabolizable sugars were present in the medium, the stationary phase-associated induction was inhibited, indicating that the toxin genes are subject to a form of catabolite repression . This glucose effect was general to many toxinogenic strains having varying levels of toxin production. FEMS Microbiol Lett, 1998 Jan 15, 158(2), 215 - 21 Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types; Kubota T et al.; The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I-III) . To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C . butyricum strain BL6340 . The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C . Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C . butyricum strain BL6340 . However, the genes of ORF-X1 (435 bp) and ORF-X2 (partially sequenced) were present just upstream of that of P47 . The orientation of these genes was in inverted direction to that of p47 . The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex. Biochem Pharmacol, 1997 Nov 15, 54(10), 1097 - 108 Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells; Kowluru A et al.; We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells . Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells . Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion . However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion . These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion . The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells . Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ({Ca2+}i), suggesting that these GTPases are involved in steps distal to elevations in {Ca2+}i . Recent findings indicate that the carboxyl methylation of Cdc42 is stimulated by only glucose, whereas that of Rap (Kowluru et al., J Clin Invest 98: 540-555, 1996) and Rac (present study) are regulated by glucose or potassium . Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e . Ca2+-induced exocytosis of insulin). Appl Environ Microbiol, 1998 Feb, 64(2), 703 - 8 Genomic analysis of Clostridium botulinum group II by pulsed-field gel electrophoresis; Hielm S et al.; Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (non-proteolytic) group II . DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation . A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results . Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes . Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation . Twenty strains yielded PFGE patterns containing 13 pulsotypes . From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C . botulinum group II was estimated to be 3.6 to 4.1 Mb (mean +/- standard deviation = 3,890 +/- 170 kb) . The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C . botulinum group II at the molecular level. Am J Physiol, 1998 Jan, 274(1 Pt 1), G196 - 202 CGRP upregulation in dorsal root ganglia and ileal mucosa during Clostridium difficile toxin A-induced enteritis; Keates AC et al.; We have previously reported that pretreatment of rats with capsaicin (an agent that ablates sensory neurons) or CP-96345 (a substance P receptor antagonist) dramatically inhibits fluid secretion and intestinal inflammation in ileal loops exposed to Clostridium difficile toxin A . The aim of this study was to determine whether calcitonin gene-related peptide (CGRP), a neuropeptide also found in sensory afferent neurons, participates in the enterotoxic effects of toxin A . Administration of toxin A was also found to increase CGRP content in dorsal root ganglia and ileal mucosa 60 min after toxin exposure . Furthermore, immunohistochemical studies demonstrated increased neuronal staining for CGRP 2 h after toxin A treatment . Pretreatment of rats with CGRP-(8-37), a specific CGRP antagonist, before instillation of toxin A into ileal loops significantly inhibited toxin-mediated fluid secretion (by 48%), mannitol permeability (by 83%), and histological damage . We conclude that CGRP, like substance P, contributes to the secretory and inflammatory effects of toxin A via increased production of this peptide from intestinal nerves, including primary sensory afferent neurons. J Clin Microbiol, 1998 Jan, 36(1), 184 - 90 Multicenter evaluation of the Clostridium difficile TOX A/B TEST; Lyerly DM et al.; Clostridium difficile, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci . The disease results from two toxins, toxins A and B, produced by this pathogen . In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B . We compared the test with the tissue culture assay, which is recognized as the "gold standard" for C . difficile testing . Evaluations were performed in-house at TechLab, Inc . (Blacksburg, Va.) and off-site at four clinical laboratories . Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests . The sensitivity and specificity were 92.2 and 100%, respectively . The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8% . When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively . The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0% . There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture . Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture . Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing . There were no indeterminate results, since the test does not have an indeterminant zone . In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs . Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA-/toxB+) isolates were identified . One specimen was from a patient with a clinical history consistent with C . difficile infection . Isolates obtained from these specimens by selective culture on solid media and in broth tested toxA-/toxB+ when grown in brain heart infusion dialysis flasks, which stimulate in vitro production of both toxins . Our findings show that the TOX A/B TEST is suitable as a diagnostic aid for C . difficile disease because it correlates well with tissue culture and detects isolates that may be missed with toxin A-specific EIAs. J Clin Microbiol, 1998 Jan, 36(1), 30 - 6 Evidence that the enterotoxin gene can be episomal in Clostridium perfringens isolates associated with non-food-borne human gastrointestinal diseases; Collie RE et al.; Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of human C . perfringens type A food poisoning . CPE-producing C . perfringens isolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea . The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43 cpe-positive C . perfringens isolates obtained from diverse sources . All North American and European food-poisoning isolates examined in this study were found to carry a chromosomal cpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry their cpe on an episome . Collectively, these results provide the first evidence that distinct subpopulations of cpe-positive C . perfringens isolates may be responsible for C . perfringens type A food poisoning versus CPE-associated non-food-borne human GI diseases . If these putative associations are confirmed in additional surveys, cpe RFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations of cpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases. Gut, 1997 Sep, 41(3), 366 - 70 Intravenous immunoglobulin therapy for severe Clostridium difficile colitis; Salcedo J et al.; BACKGROUND: Many individuals have serum antibodies against Clostridium difficile toxins . Those with an impaired antitoxin response may be susceptible to recurrent, prolonged, or severe C difficile diarrhoea and colitis . AIMS: To examine whether treatment with intravenous immunoglobulin might be effective in patients with severe pseudomembranous colitis unresponsive to standard antimicrobial therapy . PATIENTS: Two patients with pseudomembranous colitis not responding to metronidazole and vancomycin were given normal pooled human immunoglobulin intravenously (200-300 mg/kg) . METHODS: Antibodies against C difficile toxins were measured in nine immunoglobulin preparations by ELISA and by cytotoxin neutralisation assay . RESULTS: Both patients responded quickly as shown by resolution of diarrhoea, abdominal tenderness, and distension . All immunoglobulin preparations tested contained IgG against C difficile toxins A and B by ELISA and neutralised the cytotoxic activity of C difficile toxins in vitro at IgG concentrations of 0.4-1.6 mg/ml . CONCLUSION: Passive immunotherapy with intravenous immunoglobulin may be a useful addition to antibiotic therapy for severe, refractory C difficile colitis . IgG antitoxin is present in standard immunoglobulin preparations and C difficile toxin neutralising activity is evident at IgG concentrations which are readily achieved in the serum by intravenous immunoglobulin administration. Dtsch Med Wochenschr, 1997 Oct 17, 122(42), 1281 - 4 {Clostridium sordellii sepsis with intravascular haemolysis}; Hungerland E et al.; HISTORY AND CLINICAL FINDINGS: A 37-year-old chronic alcoholic had increasingly frequent haematemesis and recently had developed upper abdominal pain . His general condition had clearly deteriorated and he was finally admitted as an emergency . There were no previous illnesses other than alcoholism . He had a fever of 39.0 degrees C on admission, his sclerae were icteric and his abdomen was hard and resistant to palpation . He was slightly drowsy . INVESTIGATIONS: Laboratory tests indicated haemolysis with a haemoglobin of 10 g/dl, lactate dehydrogenase concentration of 330 U/l, urobilinogenuria and a raised white blood cell count of 11,500/microliter . Sonography revealed liver cirrhosis and ascites . TREATMENT AND COURSE: Because of the haematemesis a gastroscopy was performed, during which the patient went into acute shock from which he died . At autopsy the cause of the bleeding was found to be an ulcer at the oesophagocardiac junction . The gastrointestinal tract was filled with blood . He also had alcoholic liver cirrhosis with portal hypertension, as well as splenomegaly as sign of a septicaemia . Histologically masses of rod-shaped bacteria were found, especially in the heart, but also in liver, kidneys and peritoneum, which bacteriologically were identified as Clostridium sordellii . CONCLUSION: In patients with impaired immunological resistance Clostridium sordellii may cause an acute and quickly fatal septicaemia with intravascular haemolysis. Anasthesiol Intensivmed Notfallmed Schmerzther, 1997 Aug, 32(8), 518 - 21 {Complicated amebic liver abscess--course and therapy}; Nierhaus A et al.; We report on a case of an amoebic liver abscess acquired during a holiday in Bali . Transdiaphragmatic migration and consecutive atelectasis of the right lung caused respiratory insufficiency, requiring immediate surgical intervention . Complications consisted of massive bleeding into the colon concomitant with a reactivated CMV-infection . In addition, toxins of Clostridium difficile and enterohaemorrhagic E . coli were seen in the faeces . In contrast to the majority of uncomplicated cases of amoebic liver abscess, usually treated with amoebicidal drugs only, surgical intervention was clearly indicated in our patient. Oral Dis, 1997 May, 3 Suppl 1, S141 - 8 Epidemiology and diagnosis of HIV-associated periodontal diseases; Lamster IB et al.; A review of periodontal disease as a manifestation of HIV infection suggests a shift in emphasis over the past 5 years . Initially the focus was on newly described forms of periodontal disease (i.e., HIV-associated gingivitis or linear gingival erythema (LGE); HIV-associated periodontitis or necrotizing ulcerative periodontitis (NUP) . While the clinical definition of LGE varies from study to study, an association between LGE and Candida infection has been described . Furthermore, the prevalence of NUP is quite low and this disorder is associated with severe immunosuppression . In contrast, the focus today is on the accelerated rate of chronic adult periodontitis occurring in seropositive patients . While the organisms that characterize adult periodontitis in seronegative individuals are present in subgingival plaque from seropositive individuals, reports suggest that atypical pathogens are also present (i.e., Mycoplasma salivarium, Enterobacter cloacae) . Recent studies from our laboratory have identified a novel strain of Clostridium isolated from the subgingival plaque of injecting drug users that has pathologic potential . This organism, however, was found in both seropositive and seronegative individuals in this cohort, suggesting an association with lifestyle rather than serostatus . In addition, data has been published examining the local host response in periodontitis in seropositive individuals . Distinctly elevated levels of IgG in gingival crevicular fluid (GCF) have been observed in seropositive patients . Furthermore, data from our laboratory examining inflammatory mediators in GCF (polymorphonuclear leukocyte lysosomal enzyme beta-glucuronidase and the pro-inflammatory cytokine interleukin-1 beta) suggests an altered response in patients with HIV infection . The alteration manifests as the absence of the expected strong correlation between polymorphonuclear leukocyte activity in the gingival crevice and clinical measures of existing periodontal disease, as well as elevated levels of interleukin-1 beta in sites with deeper probing depths . Therefore, it can be concluded that the progression of periodontal disease in the presence of HIV infection is dependent upon the immunologic competency of the host as well as the local inflammatory response to typical and atypical subgingival microorganisms. Glycobiology, 1997 Dec, 7(8), 1215 - 27 Structural characterization of the disialogangliosides of murine peritoneal macrophages; Yohe HC et al.; Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events . Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component . Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods . All detectable components of the polysialo fraction were determined to be disialogangliosides . Treatment of the polysialo fraction with Clostridium perfringens sialidase produced mostly the sialidase-resistant monosialoganglioside, GM1a, and a minor amount of asialoGM1 . Periodate oxidation and mass spectrometry analyses demonstrated the lack of tandem disialo moieties which indicated the absence of GD1b or GD1c (GD1) entities . The combined data showed the major disialogangliosides consisted of GD1a entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer, IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, and IV3-NeuGc,II3NeuGc-GgOse4Cer . Minor components consisted of GD1alpha entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGc-GgOse4Cer, and also positional isomer(s) of GD1alpha(NeuAc, NeuGc) . These isomeric components were identified by collision analysis and tandem mass spectrometry . Consistent with previous analyses, the ceramide portion of all polysialo (disialo) gangliosides contained solely C18 sphingosine with C16 and C24 fatty acid moieties . These results, combined with the previous characterization of macrophage monosialogangliosides, indicate normal murine macrophage ganglioside biosynthesis proceeds along the "a" ganglioside pathway, e.g., GM3-->GM2-->GM1a-->GD1a, and the proposed asialoganglioside or "alpha" pathway, asialoGM1-->GM1b-->GD1alpha . The presence of totally sialidase-sensitive gangliosides appears to be characteristic of functional murine peritoneal macrophages while they are reduced in genetically impaired cells. Clin Diagn Lab Immunol, 1998 Jan, 5(1), 87 - 90 HEp-2 cell-adherent Escherichia coli and intestinal secretory immune response to human immunodeficiency virus (HIV) in outpatients with HIV-associated diarrhea; Mathewson JJ et al.; HEp-2 cell-adherent Escherichia coli and the human immunodeficiency virus (HIV) itself have recently been incriminated as causes of chronic HIV-associated diarrhea . This study sought to determine the prevalence of these two agents among HIV-infected patients with diarrhea in an outpatient setting in the United States and to compare their prevalence to that of other commonly recognized enteropathogens known to be present in this population . HEp-2 cell-adherent E . coli was found in 20 of 83 (24.1%) patients with diarrhea . A diffuse pattern of adherence was the most common, found in 14 of 20 (70%) patients, followed by a localized adherence pattern (6 of 20; 30%) . An intestinal secretory immune response against the p24 antigen of HIV was found in 9 of 34 (27.5%) patients with HIV-associated diarrhea . The following pathogens or products were also detected in lower frequencies: Cryptosporidium spp . (10.8%), Clostridium difficile toxin (8.8%), microsporidia (6%), Isospora belli (3.6%), Blastocystis hominis (2.4%), Giardia spp . (1.2%), Salmonella spp . (1.2%), and Mycobacterium spp . (1.2%) . The role of HEp-2 cell-adherent E . coli and HIV enteric infections in patients with HIV-associated diarrhea deserves further study. Clin Infect Dis, 1998 Jan, 26(1), 141 - 5 A prospective nationwide study of Clostridium difficile-associated diarrhea in Sweden . The Swedish C . difficile Study Group; Karlstrom O et al.; Clostridium difficile-associated diarrhea (CDAD) is regarded as an emerging nosocomial infection . All patients positive for C . difficile in Sweden were recorded during 1995, including primary care patients . Those positive for toxin in feces were defined as CDAD cases . A total of 5,133 CDAD cases were recorded (58 per 100,000 inhabitants per year), as compared with 86 cases diagnosed in 1978 and 553 in 1983 . CDAD was almost twice as prevalent as all (combined) diagnosed domestic cases of reportable bacterial and protozoal diarrhea . The age-specific incidence was little affected by gender but increased > 10-fold over the age range of 60-98 years . The differences in overall CDAD incidence were sixfold between counties and threefold between major hospitals . Among hospitalized patients the incidences were highest in geriatric/rehabilitation wards, followed by infectious diseases and internal medicine wards; 28% of all cases involved no recent hospitalization and were defined as community-acquired CDAD. Naunyn Schmiedebergs Arch Pharmacol, 1997 Dec, 356(6), 769 - 76 Identification of G protein-coupled receptors potently stimulating migration of human transitional-cell carcinoma cells; Lummen G et al.; The expression of G protein-coupled receptors inducing calcium mobilization and stimulating cell migration was examined in human transitional-cell carcinoma (J82) cells . Measurements of cytoplasmic Ca2+ concentration ({Ca2+}i) and phospholipase C activity indicated that these cells express several calcium-mobilizing receptors, including those for lysophosphatidic acid (LPA), thrombin, bradykinin, bombesin and histamine, of which only the LPA response was sensitive (approximately 50%) to pertussis toxin (PTX) . Migration of J82 cells was strongly stimulated by LPA and thrombin, by 5- to 20-fold, whereas bradykinin, bombesin and histamine were ineffective . Migration induced by either LPA or thrombin was inhibited by the actin cytoskeleton-disrupting agent, cytochalasin B, by the Rho protein-inactivating Clostridium difficile toxin B, by preventing {Ca2+}i transients with an intracellular calcium-chelating agent, and by the phorbol ester, phorbol 12-myristate 13-acetate, which also blocked the LPA- and thrombin-induced {Ca2+}i increases . On the other hand, ADP-ribosylation of Gi type G proteins by PTX abrogated the migratory response to LPA, without affecting the thrombin effect . Similarly, raising cAMP levels inhibited, by about 50%, the LPA- but not the thrombin-induced J82 cell migration . In conclusion, human transitional-cell carcinoma (J82) cells express various G protein-coupled, calcium-mobilizing receptors, out of which only those for LPA and thrombin stimulate cell migration, indicating that phospholipase C-derived second messengers per se are not sufficient for initiating this response . The complex signal transduction processes leading to LPA- and thrombin-stimulated motility of these human carcinoma cells apparently involve several common, essential factors, such as {Ca2+}i changes and Rho protein-regulated reorganization of the cytoskeleton, as well as some distinct components, most notably distinct subtypes of heterotrimeric G proteins and apparently also distinct cAMP-sensitive targets. FEMS Microbiol Lett, 1998 Jan 1, 158(1), 17 - 23 Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis; Steinthorsdottir V et al.; Recombinant beta-toxin has been expressed and secreted from Bacillus subtilis . Biological activity was tested in vivo and in vitro . The lethal dose in mice was determined . Hemolysis of rabbit and sheep erythrocytes was tested but no effect was observed . Seven mutant proteins were produced . Targets for mutagenesis were mostly selected on the basis of the similarity between beta-toxin and alpha-toxin from Staphylococcus aureus, a pore-forming toxin . Mutations of two amino acids affected the lethal dose in mice . Both residues have counterparts in the membrane binding region of alpha-toxin . Alteration of the single cysteine residue did not affect protein function, contrary to previous suggestions. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14291 - 3 Visualization of a peripheral stalk in V-type ATPase: evidence for the stator structure essential to rotational catalysis; Boekema EJ et al.; F- and V-type ATPases are central enzymes in energy metabolism that couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes . They consist of a soluble headpiece that contains the catalytic sites and an integral membrane-bound part that conducts the ion flow . Energy coupling is thought to occur through the physical rotation of a stalk that connects the two parts of the enzyme complex . This mechanism implies that a stator-like structure prevents the rotation of the headpiece relative to the membrane-bound part . Such a structure has not been observed to date . Here, we report the projected structure of the V-type Na+-ATPase of Clostridium fervidus as determined by electron microscopy . Besides the central stalk, a second stalk of 130 A in length is observed that connects the headpiece and membrane-bound part in the periphery of the complex . This additional stalk is likely to be the stator. Can J Vet Res, 1998 Jan, 62(1), 56 - 62 Characterization of the interaction between VP8 of bovine rotavirus C486 and cellular components on MA-104 cells and erythrocytes; Lee J et al.; Rotavirus VP8*, the N-terminal trypsin cleavage product of VP4, has been shown to bind to MA-104 cells and human O type erythrocytes . To examine whether bacterially expressed VP8* binds to cellular components of MA-104 cells, the VP8* (aa 1-247) was expressed in E . coli and radiolabelled with 35S-methionine . The radiolabelled rVP8* was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV) . The rVP8* was found to bind to MA-104 cells and its binding was competed by BRV . To study the interaction between VP8* and receptors of erythrocytes, hemagglutination (HA) and hemagglutination inhibition (HI) assays were carried out using solubilized rVP8* . rVP8* showed HA which could be inhibited by antiserum to BRV . This interaction was also inhibited by gangliosides, demonstrating a sialic acid dependent interaction . To study the contribution of the C-terminal region of VP8* to HA, a number of approaches were used . First, a peptide spanning aa 230-247 was synthesized and antisera was raised against the peptide to see whether it could inhibit HA of rVP8* . Second, a truncated form of VP8* (tVP8*: aa 1-229) was expressed to examine its hemagglutinating activity . Third, the dimerization of rVP8* and tVP8* was compared by Western-blotting following electrophoresis using native SDS-PAGE . The results indicated that antibody to aa 230-247 inhibits hemagglutination by preventing dimerization of VP8* which in turn allows the molecule to cause HA . To characterize the interaction between the HA domain and sialic acid receptors, erythrocytes were treated with sialidases of different specificities . Arthrobacter ureafaciens, Clostridium perfringens and alpha 2-8 linkage-specific neuraminidase destroyed the ability of sialic acid of erythrocytes to interact with rVP8*, indicating that bovine rotavirus C486 binding requires an alpha 2-8 linkage but acetylation of the sialic acid is not necessary. Dig Dis Sci, 1997 Dec, 42(12), 2577 - 80 Nitrate-reducing bacteria in diversion colitis: a clue to inflammation? Neut C, Guillemot F, Colombel JF. A pathogenic role of nitric oxide has been suggested in acute and chronic intestinal inflammation . We took the opportunity offered by studies in patients with excluded colon, which represents a model of chronic intestinal inflammation with no exogenous nitrite or nitrate supply, to evaluate the quantity and the quality of nitrate reducers in diversion colitis . Thirty patients (17 men, 13 women, mean age 45 years) having an excluded colon for various reasons were sampled by rectal swabs and compared to 30 healthy controls (11 men, 19 women, mean age 28 years) . The percentage of nitrate-reducers among the total count of subcultured bacteria was 46 +/- 41% (mean +/- SD) in patients with diversion colitis as compared to 19 +/- 24% in healthy controls . This difference was significant (P < 0.05) despite great heterogeneity in individual values . In patients with diversion colitis, 75/254 (29.5%) different isolated bacterial strains were nitrate-reducers as compared to 61/294 (21%) (P < 0.05) in controls . Among the 75 nitrate-reducing strains isolated from patients with diversion colitis, 55 were aerobes . Pseudomonas species were only encountered in this population . The predominant group was enterobacteria with a high isolation rate of species belonging to the genera Proteus, Providencia, and Morganella . In healthy controls nitrate-reducing anaerobes were nearly as frequent as aerobes . The most frequent species was Eubacterium lentum, followed by Clostridium perfringens . It could be suggested that nitric oxide synthase might produce a bacterial substrate increasing the growth of bacteria with a high pathogenic potential, creating conditions for chronic inflammation and infection in patients with excluded colon. Biosci Biotechnol Biochem, 1997 Dec, 61(12), 2004 - 9 Amino acid sequence and stereoselective hydrolytic reaction of an endo-1,4-beta-glucanase from a Bacillus strain; Hitomi J et al.; Bacillus sp . KSM-522 produces three different extracellular endo-1,4-beta-glucanases {EGs; Okoshi et al., Agric . Biol . Chem., 54, 83-89 (1990)} . Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction . The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2 . The molecular mass of the extracellular mature enzyme (Ser26 through Lys636) was calculated to be 69,076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis . The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50 degrees C, respectively . By 1H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl beta-D-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration. Biochemistry, 1997 Dec 23, 36(51), 16328 - 37 Neuromedin B receptor activation causes tyrosine phosphorylation of p125FAK by a phospholipase C independent mechanism which requires p21rho and integrity of the actin cytoskeleton; Tsuda T et al.; Recent studies show that tyrosine phosphorylation by a number of neuropeptides may be an important intracellular pathway in mediating changes in cell function, particularly related to growth . Neuromedin B (NMB), a mammalian bombesin related peptide, functions through a distinct receptor, the neuromedin B receptor (NMB-R), of which little is known about its cellular basis of action . In the present study we explored the ability of NMB-R activation to cause tyrosine phosphorylation of focal adhesion kinase (p125(FAK)), an important substrate for tyrosine phosphorylation by other neuropeptides . NMB caused rapid increases in p125(FAK) phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B receptor (rNMR-R) transfected BALB 3T3 cells . NMB had a half-maximal effect was at 0.4 nM and was 30-fold more potent than gastrin-releasing peptide (GRP) . The stoichiometric relationships between increased p125(FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells . TPA (1 microM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125(FAK) seen with NMB . A23187 potentiated the effect of TPA . Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) tyrosine phosphorylation, but it had no effect on the NMB stimulation . Pretreatment with thapsigargin completely inhibited NMB-stimulated increases in {Ca2+}i, but had no effect on NMB-stimulation of p125(FAK) phosphorylation either alone or with GF109203X . The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125(FAK) by 52% . However, tyrphostin A25 did not inhibit NMB-stimulated increases in {3H}inositol phosphates . Cytochalasin D, an agent which disrupts actin microfilaments, inhibited BN- and TPA-induced tyrosine phosphorylation of p125(FAK) completely . In contrast, colchicine, an agent which disrupts microtubules, had no effect . Pretreatment with Clostridium botulinum C3 exoenzyme which inactivates the small GTP-binding protein rho p21, also inhibited tyrosine phosphorylation of p125(FAK) by 55% . These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK) . NMB-induced tyrosine phosphorylation of p125(FAK) is independent of NMB-induced changes in {Ca2+}i or PKC . The integrity of the actin cytoskeleton but not of microtubules is necessary for NMB-stimulated phosphorylation of p125(FAK) . The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine phosphorylation of p125(FAK) . These results suggest that similar to some other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growth. Biochemistry, 1997 Dec 23, 36(51), 16065 - 73 Atomic resolution (0.94 A) structure of Clostridium acidurici ferredoxin . Detailed geometry of {4Fe-4S} clusters in a protein; Dauter Z et al.; The crystal structure of the 2{4Fe-4S} ferredoxin from Clostridium acidurici has been solved using X-ray diffraction data extending to atomic resolution, 0.94 A, recorded at 100 K . The model was refined with anisotropic representation of atomic displacement parameters for all non-hydrogen atoms and with hydrogens riding on their parent atoms . Stereochemical restraints were applied to the protein chain but not to the iron-sulfur clusters . The final R factor is 10.03 % for all data . Inversion of the final least-squares matrix allowed direct estimation of the errors of individual parameters . The estimated errors in positions for protein main chain atoms are below 0.02 A and about 0.003 A for the heavier {4Fe-4S} cluster atoms . Significant differences between the stereochemistry of the two clusters and distortion of both of them from ideal Td tetrahedral symmetry can be defined in detail at this level of accuracy . Regions of alternative conformations include not only protein side chains but also two regions of the main chain . One such region is the loop of residues 25-29, which was highly disordered in the room temperature structure. Eur J Cell Biol, 1997 Dec, 74(4), 329 - 35 Subcellular localization of the GTP-binding protein Rho in the sea urchin sperm; Castellano LE et al.; The Rho proteins are small G-proteins that belong to the Ras superfamily and play an essential role in the organization of the actin cytoskeleton . They are characteristically ADP-ribosylated by the exoenzyme C3 from Clostridium botulinum . Sea urchin sperm contain multiple small G proteins (28-24 kDa) whose identity and function are unknown . Here, we examined whether some of these proteins corresponded to the Rho subfamily . A sperm homogenate incubated with C3 and {32P}NAD revealed, by electrophoresis and autoradiography, a single radiolabeled band with a molecular mass of 25 kDa; this size was identical, under the same conditions, to that displayed by RhoA from human platelets . In flagellar fractions, the 25 kDa protein ADP-ribosylated by C3 localized in the cytosol and in the plasma membrane . In the sperm head, the 25 kDa protein was detected in a membrane preparation enriched in acrosomal and plasma membranes . Separation of these head membranes through a continuous density gradient revealed that both the 25 kDa protein {32P}ADP-ribosylated by C3 and actin had the same localization as bindin, the adhesive protein characteristic of the acrosome . An antibody against RhoB cross-reacted by immunoblotting with the 25 kDa protein and it revealed by both immunofluorescence and immunogold the presence of Rho in the acrosomal region, the middle piece of the head, and in the flagellum . Thus, the results indicate that the G-protein of 25 kDa previously detected in sea urchin sperm is Rho, likely the type B . Based on its cellular localization, Rho may participate in regulating motility and the actin polymerization that accompanies the acrosome reaction. Immunol Lett, 1997 Jun, 58(1), 25 - 8 Quantification of antigen-specific immunoglobulin A after oral booster immunization with ovalbumin in mice mono-associated with segmented filamentous bacteria or Clostridium innocuum; Snel J et al.; Segmented filamentous bacteria (SFB) are known to stimulate the mucosal immune system . Here, the effect of SFB on oral booster immunization with ovalbumin was investigated . Mice mono-associated with SFB or Clostridium innocuum were sensitized by intraperitoneal administration of 100 micrograms ovalbumin with Freunds complete adjuvant . After 4 weeks, mice received 80 mg ovalbumin orally . A maximum IgA response was found 5 days after this booster immunization . Comparison of mice with SFB and mice with C . innocuum revealed a much higher level of IgA in the gut lumen and more IgA secreting cells in the lamina propria of the SFB-associated mice . However, no differences between both groups of animals were found in specific levels of IgA secreting cells or luminal IgA against ovalbumin . It is concluded that there is no enhancing effect of SFB after booster immunization when mice are primed intraperitoneally with ovalbumin. Am J Physiol, 1997 Dec, 273(6 Pt 1), G1333 - 40 IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes; Linevsky JK et al.; Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis . This study examines whether monocyte activation by C . difficile toxins is instrumental in initiating neutrophil activation and recruitment . Human monocytes were exposed to low concentrations of highly purified C . difficile toxins, and the conditioned media were harvested for cytokine and functional assays . Monocytes exposed to C . difficile toxin A (10(-10) M) or toxin B (10(-12) M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8) . Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure . Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively) . This effect was completely blocked by IL-8 antiserum . These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression . C . difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration . Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C . difficile colitis. Biochim Biophys Acta, 1998 Jan 7, 1395(1), 21 - 7 Molecular characterization of type E Clostridium botulinum and comparison to other types of Clostridium botulinum; Li B et al.; Determination of nucleotide sequence upstream to the neurotoxin binding protein (NBP) gene of type E Clostridium botulinum has revealed an open reading frame whose stop codon is only 18 bp apart from the start codon of the NBP gene . Amino acid sequence derived from the corresponding nucleotide sequence suggested the existence of the open reading frame as a 47.8 kDa protein (P-48) . Protein data bank search revealed that the 47.8 kDa protein has 80% sequence identity to P-47 of type F C . botulinum . The gene organization of type E . Clostridium botulinum was predicted and compared to other types of C . botulinum . In type E C . botulinum, genes for the P-48, the neurotoxin binding protein and the neurotoxin form an operon which was similar to that of type F C . botulinum . However, type E C . botulinum has a P-18 gene instead of P-21 gene observed in type F C . botulinum, both located upstream to their respective P-48/P-47 gene. Mol Microbiol, 1997 Dec, 26(5), 867 - 76 The carboxy-terminal C2-like domain of the alpha-toxin from Clostridium perfringens mediates calcium-dependent membrane recognition; Guillouard I et al.; The lethal, cytolytic alpha-toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N-terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C-terminal domain . Calcium ions are essential for the binding of alpha-toxin to lipid films . Sixteen alpha-toxin variants with single amino acid substitutions in the C-terminal region were obtained using site-directed mutagenesis and T7 expression technology . Five of these variants showed reduced phospholipase C activity and were considerably less active than native alpha-toxin under calcium-limiting conditions . Replacement of Thr-272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface-exposed conformational epitope . The results of sequence comparisons and molecular modelling indicate that the C-terminal region probably belongs to the growing family of C2 beta-barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain . The combined findings suggest that the C-terminal region of alpha-toxin mediates interactions with membrane phospholipids in a calcium-dependent manner . Mutations to this domain may account for the natural lack of toxicity of the alpha-toxin homologue, phospholipase C of Clostridium bifermentans. Gene, 1997 Dec 5, 203(1), 65 - 73 Beta2 toxin, a novel toxin produced by Clostridium perfringens; Gibert M et al.; A novel toxin (Beta2) and its gene were characterized from a Clostridium perfringens strain isolated from a piglet with necrotic enteritis . At the amino-acid level, Beta2 toxin (27670 Da) has no significant homology with the previously identified Beta toxin (called Beta1) (34861 kDa) from C . perfringens type B NCTC8533 ( Hunter, S.E.C., Brown, J.E., Oyston, P.C.F., Sakurai, J., Titball, R.W., 1993 . Molecular genetic analysis of beta-toxin of Clostridium perfringens reveals sequence homology with alpha-toxin, gamma-toxin, and leukocidin of Staphylococcus aureus . Infect . Immun . 61, 3958-3965) . Both Beta1 and Beta2 toxins were lethal for mice and cytotoxic for the cell line 1407, inducing cell rounding and lysis without affecting the actin cytoskeleton . The genes encoding Beta1 and Beta2 toxins have been localized in unlinked loci in large plasmids of C . perfringens . In addition, Beta2 toxin-producing C . perfringens strains were found to be associated with animal diseases such as necrotic enteritis in piglets and enterocolitis in horses. J Bacteriol, 1998 Jan, 180(1), 136 - 42 Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens; Zhao Y et al.; Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringens enterotoxin, a common cause of food poisoning in humans and animals, were identified . Nested and internal deletions of the cpe promoter region were made to narrow down the location of promoter elements . To measure the effects of the deletions on the expression of cpe, translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli, which codes for beta-glucuronidase; E . coli-C . perfringens shuttle vectors carrying the fusions were introduced into C . perfringens by electroporation . In addition, in vitro transcription assays were performed with the cpe promoter region as the DNA template for extracts made from sporulating cells . DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters . SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis, the same compartment in which enterotoxin is synthesized in C . perfringens. Microbiology, 1997 Dec, 143 ( Pt 12), 3841 - 7 The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin; Fujinaga Y et al.; Binding of the purified type C 7S (neurotoxin), 12S and 16S botulinum toxins to epithelial cells of ligated small intestine or colon of the guinea pig (in vivo test) and to pre-fixed gastrointestinal tissue sections (in vitro test) was analysed . The 16S toxin bound intensely to the microvilli of epithelial cells of the small intestine in both in vivo and in vitro tests, but did not bind to cells of the stomach or colon . The neurotoxin and 12S toxin did not bind to epithelial cells of the small intestine or to cells of the stomach or colon . Absorption of the toxins was assessed by determining the toxin titre in the sera of guinea pigs 6-8 h after the intra-intestinal administration of the toxins . When the 16S toxin {1 x 10(5) minimum lethal dose (MLD)} was injected, 200-660 MLD ml-1 was detected in the sera, whereas when the 12S toxin (2 x 10(5) MLD) or 7S toxin (2 x 10(5) MLD) was injected, little toxin activity was detected in the sera . Therefore, the haemagglutinin of type C 16S toxin is apparently very important in the binding and absorption of botulinum toxin in the small intestine. J Antimicrob Chemother, 1997 Nov, 40(5), 707 - 11 Successful control of Clostridium difficile infection in an elderly care unit through use of a restrictive antibiotic policy; McNulty C et al.; Toxin-producing Clostridium difficile is the commonest bacterial cause of nosocomial diarrhoea and is a well recognized cause of hospital outbreaks in elderly care units . High C . difficile disease rates have been associated with the use of broad-spectrum antibiotics, especially cephalosporins . An outbreak of C . difficile infection in the elderly care unit at Gloucestershire Royal NHS Trust continued despite increased ward cleaning and strict implementation of infection control measures . A restrictive antibiotic policy that would maintain colonization resistance in the gastrointestinal tract was introduced throughout this unit . Patients admitted with suspected infection were prescribed intravenous (i.v.) benzylpenicillin 1.2-1.8 g every 6 h to cover streptococcal infections and i.v . trimethoprim 200 mg twice daily to cover urinary tract pathogens and Haemophilus influenzae . If the patient had septic shock a single iv dose of gentamicin was given (120-180 mg) to cover more resistant gram-negative bacilli . The following were monitored before and after the policy change . The number of cases of C . difficile toxin-positive diarrhoea; cefuroxime and total antibiotic use on the elderly care wards; patient mortality rates; and length of hospital stay: two hundred and fifty-two and 234 patients respectively with a discharge diagnosis of infection were admitted before and after the antibiotic policy change . Mortality rates and length of hospital stay were unchanged . Cefuroxime prescribing and total antibiotic prescribing costs fell by 5150 pounds sterling and 8622 pounds sterling respectively in the 7 month period after the change . Thirty-seven cases of C . difficile diarrhoea occurred in the period before and 16 in the period after the policy change . The incidence of C . difficile diarrhoea and of cefuroxime use has remained low since then . The use of narrow-spectrum antibiotics for hospital treatment of community-acquired infections in the elderly should be encouraged . Outbreaks of C . difficile diarrhoea should be managed with the combined approach of infection control and strict antibiotic policies. J Antimicrob Chemother, 1997 Nov, 40(5), 631 - 7 Comparative in-vitro and in-vivo activity of AM-1155 against anaerobic bacteria; Kato N et al.; The in-vitro activity of AM-1155, a 6-fluoro-8-methoxy quinolone, was compared with those of temafloxacin, sparfloxacin, tosufloxacin, ciprofloxacin, ofloxacin and cefmetazole, a cephamycin, against a variety of anaerobic bacteria . Although AM-1155 demonstrated only modest activity against the Bacteroides fragilis group and Prevotella bivia (MIC90s > or =3.13 mg/mL), 76% of the B . fragilis strains tested were inhibited at AM-1155 concentrations of 0.78 mg/L . AM-1155 was highly active against Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium spp., Clostridium perfringens and Mobiluncus spp . (MIC90s < or =0.39 mg/L) . An in-vivo study using a mixed infection with AM-1155- and tosufloxacin-susceptible B . fragilis and Escherichia coli strains in rat granuloma pouch was performed . AM-1155 was effective against both organisms whereas tosufloxacin was effective only against E . coli . These results correlated well to the higher pouch levels of AM-1155 than those of tosufloxacin . Clostridium difficile overgrowth was found in the caecum of mice treated with ampicillin both 1 and 7 days after 5 days dosing, but not in AM-1155-treated mice . These results suggest that the clinical efficacy of AM-1155 against infections involving most anaerobic bacteria except for the B . fragilis group and P . bivia should be evaluated further. Lett Appl Microbiol, 1997 Nov, 25(5), 339 - 44 PCR detection of Clostridium perfringens producing different toxins in faeces of goats; Uzal FA et al.; A polymerase chain reaction (PCR) was used to identify the genes encoding the major toxins of Clostridium perfringens in faeces of goats . When pure cultures of Cl . perfringens types A, B, C, D and E were used as templates in the PCR, amplicons were observed on the agarose gel as bands at approximately the 247 (alpha primers), 1025 (beta primers), 403 (epsilon primers) and 298 (iota primers) bp level of the DNA marker . When used to identify different types of Cl . perfringens in samples artificially spiked with these micro-organisms, the PCR detected as few as 1-1.5 x 10(2) cfu g-1 of the five types of Cl . perfringens tested . The PCR technique allowed the identification and typing of Cl . perfringens strains in faeces of goats, without recourse to other techniques such as the mouse neutralization test. Anasthesiol Intensivmed Notfallmed Schmerzther, 1997 Sep, 32(9), 583 - 8 {Therapeutic problems in tetanus--presented via a case report}; Gross H; This is the case presentation of a forty-year old female patient, who had incurred a tetanus infection as a result of intravenous drug abuse . Clostridium tetani could be detected repeatedly in abscesses caused by injections . The patient had to be put on continuous relaxation, sedation and artificial respiration for 42 days . Besides the usual intensive care regimen, a high-dose antitoxin therapy was initiated . The areas of abscesses had to be eradicated surgically several times . With the exception of a thrombus of the vena cava superior (without haemodynamic consequences) and a pneumonia, the further course was without any other serious complications . After seven months of hospitalisation the patient could be dismissed at "restitutio ad integrum" . The known immunosuppressive effect of a high dosed tetanus antitoxin therapy could be confirmed by the patient's antitoxin titre course . Repeated active immunisation attempts to produce a sufficient endogenous antitoxin titre failed . The existing therapeutic uncertainties regarding the dosage of the tetanus antitoxin therapy, the titre control and the proper antibiotic treatment are described. Ann Pharmacother, 1997 Dec, 31(12), 1507 - 13 Tetanus: pathophysiology and management; Ernst ME et al.; OBJECTIVE: To review the epidemiology, pathophysiology, clinical manifestations, diagnosis, and management of tetanus and its complications . DATA SOURCES: MEDLINE and Iowa Drug Information Services databases were searched for literature pertaining to tetanus . Additional literature was obtained from the references of selected articles identified in the search . Information from all articles was considered for inclusion in the manuscript . STUDY SELECTION AND DATA EXTRACTION: Articles selected were those considered by the authors to assist in providing the reader an understanding of the epidemiology, pathophysiology, clinical manifestations, diagnosis, and management of tetanus . DATA SYNTHESIS: While the number of tetanus cases has decreased markedly since data reporting for the disease began in 1947, mortality among those who acquire the disease remains high . Elderly patients are particularly susceptible to tetanus and its complications . Prevention of tetanus focuses on primary immunization and scheduled boosters . Management of tetanus involves initial stabilization of the patient and protection of the airway, prevention of tetanospasmin absorption by administration of human tetanus immune globulin 3000-6000 IU, and eradication of Clostridium tetani with antimicrobial therapy (metronidazole 500 mg q8h) . Supportive measures include the administration of neuromuscular blocking agents such as pancuronium in patients requiring artificial ventilation, as well as benzodiazepines (midazolam 5-15 mg/h) for sedation and muscle relaxation . Autonomic dysfunction should be managed with beta-adrenergic blockers such as propranolol or labetalol . CONCLUSIONS: Despite the relative infrequency of tetanus cases, mortality among untreated patients remains significantly high . Clinicians should become knowledgeable in the pathophysiology, clinical manifestations, and management of this potentially fatal disease. Bone Marrow Transplant, 1997 Nov, 20(9), 711 - 4 Octreotide (SMS 201-995) for hematopoietic support-dependent high-dose chemotherapy (HSD-HDC)-related diarrhoea: dose finding study and evaluation of efficacy; Wasserman E et al.; Emphasis has been put on the intensification of chemotherapy programs through high-dose chemotherapy regimens . While their myelosuppression is managed through the use of colony-stimulating factors and/or infusion of autologous peripheral blood progenitor cell transfusions (PBPCT), extramedullary dose-limiting toxicities, including gastrointestinal mucosal injury, are a treatment-limiting factor and their management is a critical issue in HSD-HDC . Octreotide is effective in the control of diarrhoea induced by fluoropyrimidines . We have studied its effect on hematopoietic support-dependent high-dose chemotherapy (HSD-HDC) related diarrhoea . HSD-HDC-treated patients were included in the study when they had > or =4 loose stools per day . Diagnostic work-up included physical examination, stool culture, Clostridium difficile toxin assay, abdominal plain films, complete blood counts, liver and renal function tests . Patients were treated with 0.1 mg octreotide, q 8 h, subcutaneously for 48 h . Responding patients (< or =2 loose stools per day) continued treatment at the same dose for an additional 24 h . Lack of response (> or =3 loose stools per day), led to dose escalation by 0.1 mg increments, up to a 0.5 mg/dose and the latter dose was maintained for 24 h . Patients not responding at 0.5 mg/8 h were considered failures . A consecutive cohort of 24 HSD-HDC treated patients was studied . Fourteen (n = 14) (58.33%) patients developed severe diarrhoea with a median number of 7.5 loose stools per day (range, 4-11) . Diarrhoea started at a median of 8 days (2-18) from day 0 of the infusion of HSD-HDC . Seven patients (50%) had less than 500 ANC/mm3 (grade 4 neutropenia) simultaneously with the diarrhoea . Twelve of 14 patients (86%) had their diarrhoea controlled, seven of them (50%) at the starting dose level of octreotide . In five of the responding patients (35.7%), octreotide had to be increased to 0.2 mg (one patient), 0.3 mg (two patients) and 0.5 mg (two patients) . No toxicity was observed, while one patient had a subcutaneous hematoma at the injection site . We have concluded that octreotide appears to be safe and effective in controlling the diarrhoea induced by HSD-HDC . Prospective controlled trials are needed to confirm its value. Gut, 1997 Nov, 41(5), 642 - 5 Antisecretory factor suppresses intestinal inflammation and hypersecretion; Johansson E et al.; BACKGROUND: Antisecretory factor (AF) is a recently identified regulatory protein which inhibits the intestinal fluid secretion induced by cholera toxin . AIMS: To test the effect of AF on: (a) inflammation and hypersecretion induced by toxin A from Clostridium difficile; and (b) morphological changes and hypersecretion induced by okadaic acid (the blue mussel toxin) in rat intestinal mucosa . METHODS: Morphological changes and fluid accumulation were observed in intestinal loops challenged with 1 microgram of toxin A or 3 micrograms of okadaic acid administered before or after injection of 0.1 microgram of recombinant AF (rAF) . RESULTS: The cytotoxic and inflammatory reaction caused by toxin A was abolished after treatment with rAF given either intraveneously or intraluminally prior to the toxin or one hour after the toxin . The intestinal fluid response induced by toxin A and okadaic acid was reduced 55-80% by rAF . However, the characteristic increase in goblet cells at the tips of villi in the okadaic acid treated mucosa was not inhibited by rAF . CONCLUSION: Results suggest that AF might be involved in protection against inflammation and in counteracting dehydration caused by enterotoxins . Both effects are probably mediated via the enteric nervous system. Gene, 1997 Nov 12, 201(1-2), 159 - 68 Physical map of the Clostridium difficile chromosome; Norwood DA Jr et al.; Clostridium difficile is a causative agent in antibiotically induced diarrhea and pseudomembraneous colitis . The ability of strains of C . difficile to cause disease depends upon the presence of two toxin genes and their corresponding proteins, designated toxin A and toxin B . Previous studies conducted in this laboratory indicated that toxigenic strains of C . difficile possess both toxin genes, whereas non-toxigenic strains do not . Likewise, the studies showed that toxigenic and non-toxigenic strains of C . difficile differ significantly in chromosomal organization by ribotype analysis . Therefore, the chromosomal organization of a reference strain was investigated . Pulsed field gel electrophoresis was utilized to generate a physical map of the chromosome of the toxigenic Clostridium difficile strain ATCC 43594 . Restriction digestions of whole chromosomes with the enzymes NruI and SacII generated consistent macrofragment profiles . NruI digestion resulted in 14 discernible bands containing 16 fragments of DNA . SacII digestions resulted in 14 discernible bands containing 15 fragments of DNA . Restriction digestions with both SacII and NruI resulted in 21 discernible bands containing 31 fragments of DNA . Probing of single and double digests with an extensive set of NruI and SacII single- and double-digest bands clarified the location of individual fragments in relation to one another, resulting in a restriction map of the chromosome . PCR-generated probes of five loci of C . difficile were used to map the location of seven genes on the chromosome . Finally, the addition of all fragments from NruI, SacII and NruI/SacII digestions resulted in an approximate chromosome size of 4.4 Mb. Proteins, 1997 Dec, 29(4), 517 - 27 Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: prediction of specificity determinants of the dockerin domain; Pages S et al.; The cross-species specificity of the cohesin-dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated . Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species . In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species . Thus, in the case of these two bacteria, the cohesin-dockerin interaction seems to be species-specific . Based on intra- and cross-species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins . The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain . According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface. Neuropediatrics, 1997 Oct, 28(5), 287 - 8 Infant botulism . The first culture-confirmed Danish case; Balslev T et al.; Infant botulism is caused by intestinal colonization by Clostridium botulinum, C . barati or C . butyricum . Infant botulism has only rarely been reported outside the U