World J Gastroenterol, 2005 Jan 7, 11(1), 109 - 13 Effect of Helicobacter pylori VacA on gene expression of gastric cancer cells; Wang HT et al.; AIM: To determine the effect of Helicobacter pylori VacA on gene expression of gastric cancer cells . METHODS: Gene expression profile of a gastric cancer cell line, SGC7901, after challenged by VacA+ and VacA- H pylori broth culture supernatants (BCS), was detected by the cDNA microarray technique . Cytoskeleton changes of SGC7901 and HeLa cells were observed through high-resolution laser scanning confocal microscopy . RESULTS: A total of 16,000 cDNA clones were detected . The percentage of genes with heterogeneous expression in SGC7901 cells challenged by VacA+ BCS reached 5%, compared with that challenged by VacA- BCS . There were 865 genes/EST with 2-fold differential expression levels and 198 genes/EST with 3-fold differential expression levels . Most of these genes were involved in vital cell events including signal transduction, regulation of gene expression, cytoskeleton, apoptosis, stress response and inflammation, cell cycle and tumor development . Cells co-cultured with VacA+ BCS showed collapsed and disrupted microtubular cytoarchitecture . CONCLUSION: VacA+ BCS can disrupt cytoskeletal architecture, likely through affecting the expression of cytoskeleton-associated genes, directly induce the expression of tumor promoter-related genes and inhibit the expression of tumor suppressor genes, thus favoring the development of tumors . VacA+ BCS can also alter the expression of inflammation and stress response genes . This suggests that VacA may play an important role in the pathogenicity of H pylori. Microb Pathog, 2004 Nov, 37(5), 241 - 51 The role of integrase/recombinase xerD and monofunctional biosynthesis peptidoglycan transglycosylase genes in the pathogenicity of Brucella abortus infection in vitro and in vivo; Canavessi AM et al.; Brucella abortus clones identified previously using a green fluorescence protein reporter system after 4h macrophage infection provided insight regarding possible genes involved in early host-pathogen interaction . Among identified genes were an integrase/recombinase (xerD) gene involved in cell division, and a monofunctional biosynthesis peptidoglycan transglycosylase (mtgA) gene that catalyzes the final stages of the peptidoglycan membrane synthesis . Here, we evaluate the in vitro and in vivo survival of B . abortus xerD and mtgA insertional mutants . B . abortus xerD::kan and B . abortus mtgA::kan demonstrated no significant growth defects in broth culture when compared to the parental strain, S2308 . Also, neither gene was required for B . abortus S2308 replication in RAW 264.7 macrophages . However, experimental evidence using interferon regulatory factor 1 knockout mice, a mouse strain highly susceptible to virulent Brucella, revealed that mice infected with B . abortus xerD::kan or B . abortus mtgA::kan survived longer than mice infected with S2308 . Additionally, in immunocompetent BALB/c mice, B . abortus xerD::kan had a significantly lower level of bacterial survival when compared to S2308 . Together, these results suggest that B . abortus xerD and mtgA genes play a role during the initial phase of infection in mice. J Med Microbiol, 2004 Oct, 53(Pt 10), 965 - 74 Analysis of gene expression profile in gastric cancer cells stimulated with Helicobacter pylori isogenic strains; Yuan JP et al.; To understand the biological processes within host cells induced by VacA, isogenic strains of Helicobacter pylori (NCTC 11638 or 11638-DeltavacA) were used to stimulate gastric cancer cells SGC7901, and differentially expressed genes in host cells were identified using cDNA microarray technology . More than 300 genes were found to alter their mRNA expression at different time points, among which 68 were related to the cytoskeleton, 87 were associated with cell cycle, cell death and proliferation, IL8 expression was also found to be up-regulated . Cells co-cultured with broth-culture supernatant (BCS) of NCTC 11638 showed more alteration in microtubule cytoskeleton morphology, as observed by laser scanning confocal microscopy, and a lower apoptosis rate, detected by flow cytometry, compared with those co-cultured with BCS of 11638-DeltavacA . The supernatants of cells co-cultured with NCTC 11638 showed significantly higher IL8 expression than those co-cultured with 11638-DeltavacA . It is concluded that VacA disrupts cytoskeletal architecture by influencing the expression of cytoskeleton-associated genes . VacA breaks the balance between cell proliferation and cell death by inducing the maladjustment of genes related to cell cycle . VacA is also able to induce the inflammatory response. Am J Vet Res, 2004 Jul, 65(7), 977 - 83 Stability, antigenicity, and aggregation of Moraxella bovis cytolysin after purification and storage; George LW et al.; OBJECTIVES: To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas . STUDY POPULATION: 8 isolates of M . bovis . PROCEDURE: Filter-sterilized broth culture supernatants of M . bovis were concentrated, diafiltered, and chromatographed . The endotoxin and cytolysin activities in samples were measured . Chromatographed cytolysins of M . bovis were examined by immunoblotting . Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage . Hemolysis was measured directly by use of washed bovine erythrocyte targets . Leukotoxicity was measured by use of a 51Cr release assay . RESULTS: Cytolysin was retained by a filter with 100-kd nominal molecular weight limit . Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 X 10(7) d) . Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxin-to-protein ratio . Frozen diafiltered retentate(-80 degrees C, 4 months) was cytolytic after thawing . Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd . Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd . Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd . CONCLUSIONS AND CLINICAL RELEVANCE: Diafiltered M . bovis cytolysin is aggregated with endotoxin . Antigenicity and cytolytic activities in diafiltered retentate are conserved among M . bovis isolates . Diafiltration could be useful for bulk semipurification of M . bovis cytolysin . Cytolysin-enriched vaccines of M . bovis could be contaminated by endotoxin. J Egypt Soc Parasitol, 2004 Apr, 34(1), 197 - 211 Pulmonary trichomoniasis: improved diagnosis by using polymerase chain reaction targeting Trichomonas tenax 18S rRNA gene in sputum specimens; Mahmoud MS et al.; A polymerase chain reaction (PCR) assay targeting species-specific region in 18 small subunit ribosomal RNA gene of Trichomonas tenax was used to examine sputum specimens in order to diagnose pulmonary trichomoniasis caused by T . tenax . It was compared with wet mount preparation, Giemsa-stained smear, and Kupferberg Trichononas broth culture for detection of T . tenax trophozoites in sputum . The study included 250 individuals; 100 immunocompromised patients with chest complaints (group I) and 100 patients with chronic pulmonary diseases (group II), and 50 healthy individuals as controls (group III) . 20 cases among all examined were positive in one or more method giving for pulmonary trichomniasis a total prevalence of 8%; 12 cases (12%) in group I, 8 cases (8%) in group II, and none in group III, with no significant difference between groups I & II . Pulmonary trichomoniasis was prevalent at age ranged between 31 to 50 years, and in total males (10%) than females (5.5%) with no significant difference . Among the 200 examined patients, pulmonary trichomoniasis had a prevalence of 3% by wet mount, 2.5% by Giemsa-stained smear, 7% by culture, compared to 10% by PCR . Culture was used as reference standard . All culture positive specimens were PCR positive showing a product at 0.8 Kb long by agarose gel electrophoresis, and giving a 100% sensitivity . Wet mount, Giemsa-stained smear, and culture had a sensitivity of 43%, 35.7%, and 70%, respectively . No PCR negative specimens were positive by any of the other methods . 6 specimens were culture negative PCR positive and remained PCR positive when retested 3 times . The calculated specificity of PCR was 97% . NO PCR target product was amplified with DNAs of T . vaginalis and various pulmonary pathogens . The results are discussed. New Microbiol, 2004 Jan, 27(1), 29 - 35 Population dynamics in ageing Helicobacter pylori; Cellini L et al.; The aim of this work was to characterize population changes occurring in aged broth cultures of Helicobacter pylori . Experiments were performed using clinical strains cultured immediately after isolation and after multiple subcultures in solid medium . Morphological changes in the ageing bacteria during a 7-day broth culture were analysed by optical and electron microscopy . The expression of the virulence factor, CagA, together with the presence of the cell cycle regulator, cGMP, were also assessed . The transition from bacillary to coccoid forms was the main morphological change observed in freshly isolated bacteria, together with the increase in cGMP from 1 to 2.25 nmoles/mg of proteins within the first 7 days of broth culture . A similar trend of morphological and physiological changes was observed in cells after multiple subcultures in solid medium with a major presence of large cell clusters . The cagA gene product was always expressed in all experimental conditions evaluated . These data show a significant morphological and physiological diversity in fresh, ageing and aged cultures of H . pylori. J Pediatr (Rio J), 1994 Mar-Apr, 70(2), 95 - 9 {The effect of the supernatants of enteropathogenic Escherichia coli strains on the intestinal transport of sodium in rats "in vivo"}; Fernandes F et al.; We studied the effect of the supernatants of 2 to 5 samples of one of the following serotypes of EPC: 026:H, 086:H34, 0111:H-, 0119:H6, 0126:H21, 0128ab:H35 and 0142:H-, isolated from the stools of infants with gastroenteritis . We also studied 3 samples of the serotype 0157:HNT, that produces the Shiga-like toxin . The following control solutions were selected 1-SC1 (glucose-saline solution); 2-SC2 (TSB broth culture solution); 3-SC3 (E.coli K12 711 culture supernatant, a non enterotoxigenic serotype) . These solutions were perfused in the small intestine of rats "in vivo" and sodium transport was determined . The comparison among the 3 control solutions revealed that there was always sodium absorption, but there was a difference in the intensity of the transport (SC1 >SC2 and SC3) . The serotypes 0111:H-, 026:H-, 0126:H21, and 0142:H2, as well as the serotype 0157:HNT, induced sodium secretion and these values were significantly different in comparison with the 3 control solutions . On the other hand, the serotypes 0128ab:H35 and 0119:H6 induced sodium secretion but the differences were only significant in comparison with the SC1 and SC2, and SC1, respectively . The supernatants of the serotypes investigated provoked an important derangement in sodium transport probably due to the presence of an enterotoxin. Scand J Gastroenterol, 2003 Oct, 38(10), 1023 - 30 Helicobacter pylori induces cyclooxygenase-1 and cyclooxygenase-2 expression in vascular endothelial cells; Byrne MF et al.; BACKGROUND: Helicobacter pylori induces cyclooxygenase activity in the stomach, although the COX isoform and cellular source are unclear . A potential source is the vascular endothelial cell, which plays a role in regulating mucosal blood flow and inflammatory cell infiltration . METHODS: We examined the effect of four strains (toxigenic and non-toxigenic) of H . pylori on COX isoform expression in vascular endothelial cells . Prostaglandin synthesis was measured by enzyme immunoassay and COX isozyme expression determined by Western blot and RT-PCR . Gene induction was examined using 5' deletion constructs of the COX-1 and COX-2 promoters coupled with luciferase . RESULTS: All H . pylori strains induced prostaglandin generation and expression of both COX-1 and COX-2 in HUVEC, although this was most pronounced with the highly toxigenic strain H . pylori 60190 . Treatment of the cells with selective COX inhibitors demonstrated that COX-1 was predominantly responsible for the enhanced generation of prostacyclin induced by H . pylori 60190 . Similar results were seen with H . pylori broth culture filtrates, suggesting that a secreted product was responsible . Induction of COX-2 reflected both enhanced gene expression and stabilization of the mRNA . CONCLUSIONS: H . pylori increased both COX-1 and COX-2 activity in vascular endothelial cells . This increased generation of endothelial cell prostacyclin may play a role in modulating mucosal blood flow, platelet function and inflammatory cell infiltration in response to H . pylori infection . The regulation of COX-1 at the transcriptional level by H . pylori described in this study is a novel finding and calls into question the traditional description of COX-1 as a purely constitutive, housekeeping gene. Infect Immun, 2003 Jun, 71(6), 3623 - 7 Helicobacter pylori supernatants cause epithelial cytoskeletal disruption that is bacterial strain and epithelial cell line dependent but not toxin VacA dependent; Bebb JR et al.; We show here that Helicobacter pylori broth culture supernatants disrupt the actin cytoskeleton of epithelial cell lines, leading to cell rounding and apoptosis through anoikis . We demonstrate that there are marked quantitative differences between strains and that there are different cell line sensitivities . By constructing VacA null isogenic mutants, we show that the effect is not due to the vacuolating cytotoxin. Zhonghua Kou Qiang Yi Xue Za Zhi, 2003 Jan, 38(1), 52 - 5 {Phenotypic variation in Actinobacillus actinomycetemcomitans}; Wang ZL et al.; OBJECTIVE: To investigate the colony variation in Actinobacillus actinomycetemcomitans (Aa) from rough to smooth and recognize its different morphology during laboratory translations . METHODS: Primary strains isolated from subgingival plaque of two juvenile periodontitis patients were repeatedly subcultured on agar plates and broth; for broth culture, every generation was translated in broth and on solid medium separately to observe the corresponding morphologies of Aa grow in broth . RESULTS: Three smooth strains of Aa from the broth culture were obtained . The process was about 7-8 generations: colonies changed from a small and adherence phenotype to a bigger and sediment ones and finally the culture supernatant became turbid; the corresponding morphologies grow on agar exhibiting an adherent, small rough colony phenotype which had a star-shaped internal structure converted gradually to a kind of bigger, opaque, nonadherent, smooth phenotype, then the colony extended out from the margin of the colony and finally converted to a flat, almost parent morphology and the same time the star-like inner structure converted to a simpler and smaller type and finally disappeared . We could not get completely smooth variants of Aa from agar . CONCLUSIONS: The variation in colony morphology of Aa from rough to smooth is a process, in which the colony was gradually wetter and bigger and at the same time gradually lost the inner structure . During this process three colony morphologies at least can be seen, including rough, opaque smooth and almost translucent smooth. Int J Food Microbiol, 2003 Jun 15, 83(2), 147 - 60 Influence of agitation, inoculum density, pH, and strain on the growth parameters of Escherichia coli O157:H7--relevance to risk assessment; Coleman ME et al.; Foods may differ in at least two key variables from broth culture systems typically used to measure growth kinetics of enteropathogens: initial population density of the pathogen and agitation of the culture . The present study used nine Escherichia coli O157:H7 strains isolated from beef and associated with human illness . Initial kinetic experiments with one E . coli O157:H7 strain in brain-heart infusion (BHI) broth at pH 5.5 were performed in a 2 x 2 x 3 factorial design, testing the effects of a low (ca . 1-10 colony-forming units {CFU}/ml) or high (ca . 1000 CFU/ml) initial population density, culture agitation or no culture agitation, and incubation temperatures of 10, 19, and 37 degrees C . Kinetic data were modeled using simple linear regression and the Baranyi model . Both model forms provided good statistical fit to the data (adjusted r(2)>0.95) . Significant effects of agitation and initial population density were identified at 10 degrees C but not at 19 or 37 degrees C . Similar growth patterns were observed for two additional strains tested under the same experimental design . The lag, slope, and maximum population density (MPD) parameters were significantly different by treatment . Further tests were conducted in a 96-well microtiter plate system to determine the effect of initial population density and low pH (4.6-5.5) on the growth of E . coli O157:H7 strains in BHI at 10, 19, and 37 degrees C . Strain variability was more apparent at the boundary conditions of growth of low pH and low temperature . This study demonstrates the need for growth models that are specific to food products and environments for plausible extrapolation to risk assessment models. J Huazhong Univ Sci Technolog Med Sci, 2002, 22(2), 100 - 2 The expression of VacA in BCF of Helicobacter pylori and its relationship to vacuolated effect; Shi L et al.; The vacuolated effect of Helicobacter (H . pylori) and its relationship to vacuolated cytotoxin antigen (VacA) were investigated by the method of cytotoxic test and SDS-pobyacrylamide gel electrophoresis (SDS-PAGE) . Of the 62 clinical isolates, the broth culture filter (BCF) of 43 strains caused the Vero cell intracytoplasmically vacuolated . H . pylori strains were divided into H . pylori (Toxin+) group with vacuolated effect and H . pylori (Toxin-) group without vacuolated effect . The analysis of the BCF of H . pylori (Toxin+) and that of H . pylori (Toxin-) was studied by SDS-PAGE and Scan reader . A kind of protein with 87 ku molecular weight was recognized in the BCF of 30.23% (13/43) H . pylori (Toxin+) strains but in none of that of H . pylori (Toxin-) strains, the difference was statistically significant (P < 0.05) . There was a significant and concordant relationship between OD of the protein band with 87 ku molecular weight and titer of vacuolated activity of H . pylori (Toxin+) (r = 0.67 and P < 0.05 by linear regression analysis) . H . pylori strains were divided into H . pylori (Toxin+) group with vacuolated effect and H . pylori (Toxin-) group without vacuolated effect . The vacuolated effect of H . pylori (Toxin+) was caused by the protein with 87 ku molecular weight (VacA). Mem Inst Oswaldo Cruz, 2002 Dec, 97(8), 1201 - 6 Epub 2003 Jan 20. Acute inflammatory response in the stomach of BALB/c mice challenged with coccoidal Helicobacter pylori; Rabelo-Goncalves EM et al.; An experimental murine model was used to verify the viability and pathogenicity of coccoid Helicobacter pylori . For this purpose, 27 BALB/c mice were inoculated intragastrically with 1 ml broth culture (10(8)organisms/ml) of a coccoid H . pylori clinical isolate . The animals were divided into two groups . Nine were infected on a one-time basis (GA1) and 18 were infected on two consecutive days (GA2) . Other 27 mice were inoculated with Brucella broth and divided in the same way; they composed the control group . Mice were killed at 2, 3, 7, 14 and 21 days post inoculation (pi) . Fragments of stomach and duodenum were collected, fixed with 12% formalin and stained by hematoxilin-eosin and Giemsa for histopathological examination . Until the 14th()day, only reinfected mice had mild-to-moderate inflammatory infiltrate in the stomach . The infiltration was predominantly lymphomonocytic, although plasma cells and eosinophils could be seen . However, at 21st day, severe eosinophilic infiltration was present in the lamina propria and submucosa of gastric corpus . In subgroup GA1, animals presented lymphomonocytic infiltration in the stomach from 14th()day pi . Our results showed that coccoid H . pylori was able to induce an acute inflammatory response in stomach of reinfected mice since the initial periods of infection. J Dairy Sci, 2002 Dec, 85(12), 3198 - 205 Effect of pasteurization on survival of Mycobacterium paratuberculosis in milk; Gao A et al.; Mycobacterium paratuberculosis (Mptb) is the causative agent of Johne's disease of ruminant animals including cattle, goats, and sheep . It has been suggested that this organism is associated with Crohn's disease in humans, and milk is a potential source of human exposure to this organism . A total of 18, including 7 regular batch and 11 high temperature short time (HTST) pasteurization experiments, were conducted in this study . Raw milk or ultra-high temperature pasteurized milk samples were spiked at levels of 10(3), 10(5), and 10(7) cfu of Mptb/ml . Escherichia coli and Mycobacterium bovis BCG strains at 10(7) cfu/ml were used as controls . Pasteurization experiments were conducted using time and temperature standards specified in the Canadian National Dairy Code: regular batch pasteurization method: 63 degrees C for 30 min, and HTST method: 72 degrees C for 15 s . The death curve of this organism was assessed at 63 degrees C . No survivors were detected after 15 min . Each spiked sample was cultured in Middlebrook 7H9 culture broth and Middlebrook 7H11 agar slants . Samples selected from 15 experiments were also subjected to BACTEC culture procedure . Survival of Mptb was confirmed by IS900-based PCR of colonies recovered on slants . No survivors were detected from any of the slants or broths corresponding to the seven regular batch pasteurization trials . Mptb survivors were detected in two of the 11 HTST experiments . One was by both slant and broth culture for the sample spiked to 10(7) cfu/ml of Mptb, while the other was detected by BACTEC for the sample spiked to 10(5) cfu/ml . These results indicate that Mptb may survive HTST pasteurization when present at > or = 10(5) cfu/ml in milk . A total of 710 retail milk samples collected from retail store and dairy plants in southwest Ontario were tested by nested IS900 PCR for the presence of Mptb . Fifteen percent of these samples (n = 110) were positive . However, no survivors were isolated from the broth and agar cultures of 44 PCR positive and 200 PCR negative retail milk samples . The lack of recovery of live Mptb from the retail milk samples tested may be due to either the absence of live Mptb in the retail milk samples tested or the presence of low number of viable Mptb which were undetected by the culture method used in this study. J Food Prot, 2002 Nov, 65(11), 1775 - 9 Influence of growth in a food medium on the detection of Escherichia coli O157:H7 by polymerase chain reaction; McKillip JL et al.; The effects of storage time and growth in broth culture and in a food medium on the efficiency of Escherichia coli O157: H7 DNA extraction and on the sensitivity of polymerase chain reaction (PCR) detection of E . coli O157:H7 were investigated . Detection limits were evaluated with dilution series PCR targeting the slt-II gene . The relationship between cell density and DNA yield was generally log-linear for pure cultures of E coli O157:H7 . When the bacteria were suspended in skim milk at a density of 10(6) CFU/ml . held at 4 degrees C, and sampled at 24-h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the 96-h storage period . However, when E coli O157:H7 was grown in skim milk to a final cell density of 10(6) CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E . coli O157:H7 growth was static over 96 h of storage at 4 degrees C . This result is most likely due to poor DNA purity, which was consistently observed when DNA was extracted from food matrices in which the pathogen was grown rather than stored . The results of this investigation underscore the likelihood that multiple components may drastically affect DNA extraction and PCR amplification efficiency in the detection of pathogens in the food matrix . It is clear that before nucleic acid amplification technologies are widely applied to food systems, it would be prudent to test their efficacy in multiple food matrices and under conditions in which the bacterial population is both static and actively growing. Mol Cell Probes, 2002 Aug, 16(4), 285 - 96 Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D . nodosus in sheep with footrot prior to vaccination; Dhungyel OP et al.; The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination . Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis . This process takes at least 3 to 4 weeks . This study describes the development of a simple and rapid serogroup specific PCR test for D . nodosus . A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A-I) serogroup specific reverse primers were designed from the carboxy-terminal regions of fimA of the different serogroups . To verify the specificity within D . nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups . Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D . nodosus, were used to check the specificity of these assays . They were found to be specific for D . nodosus as none of the 84 bacterial stains reacted . These primers detected 1 pg of purified chromosomal DNA, or 50-100 cells of D . nodosus in crude lysates . Sensitivity was markedly improved when an immuno-magnetic capture was employed . Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different . These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions . These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media . Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value . BMC Microbiol . 2002 Sep 04;2(1):24. Growth of Helicobacter pylori in a long spiral form does not alter expression of immunodominant proteins; Vinette KM et al.; BACKGROUND: We have previously reported that altered culture conditions (a broth media with shaking) could induce a strain of Helicobacter pylori to assume a long spiral morphology resembling that described for Helicobacter heilmannii . The present study was initiated to determine if other strains of H . pylori could be induced to assume that morphology and if doing so would alter the expression of immunodominant proteins . RESULTS: The six strains used in this study were American Type Culture Collection 43504, 43579, 49503, 51652, and 51653, and Sydney Strain I . Each strain was grown on solid media and in broth culture using conditions previously shown to induce the long spiral morphology in strain 43504 . DNA from each was subjected to urease gene fingerprint analysis . Results of the molecular analysis showed identical fingerprint patterns for each strain independent of culture source, indicating that only a single strain was present in each culture . Expression of immunodominant proteins was assessed by SDS polyacrylamide gel electrophoresis and Western blotting with hyperimmune rabbit anti H . pylori sera or serum from an H . pylori infected patient . Analysis of protein profiles revealed some variation between strains but no significant differences associated with morphologic alterations . CONCLUSIONS: These results indicate that growth of H . pylori in a long spiral form does not affect expression of immunodominant proteins, thus in vivo growth in the long spiral form (not documented to date) would not be distinguishable by serology. J Clin Microbiol, 2002 Sep, 40(9), 3277 - 80 Detection of Trichomonas vaginalis on modified Columbia agar in the routine laboratory; Stary A et al.; Broth culture of Trichomonas vaginalis is considered the "gold standard" for the diagnosis of trichomoniasis . Two studies were carried out to evaluate modified Columbia agar (MCA) for the isolation of T . vaginalis from clinical samples . Study I compared isolation on MCA to that on liquid medium with 889 vaginal samples . Out of 63 samples positive for T . vaginalis (7.1% of total), MCA identified 62 (98.4%) and broth identified 58 (92.1%) . In study II, trichomoniasis was diagnosed within the scope of a screening program for a total of 39,585 men and women by culture on MCA and direct microscopy . Culture on MCA detected 199 (98.5%) and Gram staining detected 163 (80.7%) of 202 positive specimens . Wet-mount preparations used for symptomatic patients identified 103 (92.8%) of 111 cases . Culture of T . vaginalis from clinical samples on MCA is highly sensitive and reliable, as well as timesaving, and therefore suitable for screening of symptomatic and asymptomatic individuals. Bioelectrochemistry, 2002 Sep, 57(2), 139 - 44 Change in broth culture is associated with significant suppression of Escherichia coli death under high magnetic field; Horiuchi S et al.; When Escherichia coli B was cultivated under an inhomogeneous magnetic field of 5.2-6.1 T, a significant 100,000-fold suppression of cell death was observed {Bioelectrochemistry 53 (2001) 149} . The limited magnetic field exposure for 12 h after logarithmic growth phase was sufficient to observe similar suppressive effects on cell death {Bioelectrochemistry 54 (2001) 101} . These results suggest some possible changes in either the medium or the cells during the magnetic field exposure . When the cell-free filtrate of the broth cultured under the magnetic field for 10 h and the cells of E . coli cultivated under the geomagnetic field for 30 h were mixed, and the mixture was subsequently cultivated under the geomagnetic field, the number of cells observed in the filtrate exposed to the high magnetic field was 20,000 times higher than that in the filtrate exposed to the geomagnetic field . When the cells cultivated under the magnetic field for 10 h and the cell-free filtrate of the broth culture exposed to the geomagnetic field were mixed, only a 50-fold difference in the number of cell between under the magnetic field and under the geomagnetic field was observed . This suggests that the filtrate of the broth culture exposed to the magnetic field is primarily responsible for the cell death suppression . It was also revealed that the small difference in pH of the filtrates of the broth culture between under the magnetic field and under the geomagnetic field was critical for the cell death suppression. Microbiology, 2002 Jul, 148(Pt 7), 1991 - 8 Influence of trehalose 6,6'-dimycolate (TDM) during mycobacterial infection of bone marrow macrophages; Indrigo J et al.; The relative role of surface lipids in the innate macrophage response to infection with mycobacteria remains unknown . Trehalose 6,6'-dimycolate (TDM), a major component of the mycobacterial cell wall, can elicit hypersensitive as well as T-cell-independent foreign body responses . The T-cell-independent contribution of TDM to the primary macrophage response to mycobacterial infection was investigated . Bone-marrow-derived macrophages isolated from C57BL/6 mice were infected with native Mycobacterium tuberculosis (MTB) or with MTB delipidated using petroleum ether extraction methods . The removal of surface lipids caused decreased bacterial survival in macrophages, but there was no loss of bacterial growth in broth culture . Bacterial survival within macrophages was restored upon reconstitution of the bacteria with purified TDM . The cytokine and chemokine parameters of the macrophage responses were also investigated . The amounts of IL-1beta, TNF-alpha, IL-6 and MIP-1alpha produced were significantly reduced following delipidation, but were restored upon reconstitution with TDM . The amount of IL-12 produced, but not the amount of IL-10 produced, was also significantly reduced upon macrophage infection with delipidated MTB . Furthermore, nitric oxide responses were not impaired upon infection with delipidated MTB, suggesting that intracellular survival and macrophage secretion of cytokines and chemokines are differentially controlled . These studies indicate that TDM is a major component contributing to the innate macrophage responses to MTB infection. Infect Immun, 2002 Jul, 70(7), 3714 - 26 Mycobacterium avium genes expressed during growth in human macrophages detected by selective capture of transcribed sequences (SCOTS); Hou JY et al.; Selective capture of transcribed sequences (SCOTS) has been employed to identify 54 cDNA molecules that represent 46 genes that are expressed by Mycobacterium avium during growth in human macrophages . Some cDNA molecules correspond to genes that are apparently expressed 48 h after infection of macrophages, while others correspond to genes expressed 110 h after infection, and still others correspond to genes expressed throughout the course of infection in our model system . Genes expressed by M . avium during growth in macrophages include genes encoding enzymes of several biosynthetic pathways (pyrimidines, mycobactin, and polyketides); genes that encode enzymes involved in intermediary metabolism, energy metabolism (tricarboxylic acid cycle, glyoxalate shunt), and nitrogen metabolism; and genes that encode regulatory proteins . A number of genes of unknown function were also identified, including genes that code for proteins similar to members of the PPE family of proteins of Mycobacterium tuberculosis and proteins similar to those encoded by the M . tuberculosis mce genes, which have been previously associated with mycobacterial virulence . The SCOTS technique, followed by enrichment for cDNA molecules that are up-regulated or are uniquely expressed by M . avium during growth in human macrophages (compared to growth in laboratory broth culture), allows recovery and identification of a greater diversity of cDNA molecules than does subtractive hybridization between cDNA mixtures from macrophage-grown and broth-grown M . avium . Data are presented demonstrating the reproducibility of recovery of a subset of cDNA molecules from cDNA mixtures purified by SCOTS on several different occasions . These results further demonstrate the beneficial utility of the SCOTS technique for identifying genes whose products are needed for successful survival and growth by an organism in a specific environment. J Gastroenterol Hepatol, 2001 Nov, 16(11), 1197 - 205 Helicobacter pylori alters n-6 fatty acid metabolism and prostaglandin E2 synthesis in rat gastric mucosal cells; Nakaya A et al.; BACKGROUND AND AIMS: Little is known about whether Helicobacter pylori infection alters fatty acid metabolism in gastric mucosal cells . By using cultured rat gastric mucosal cells (RGM-1), we investigated the effect of H . pylori broth culture filtrates on this point . Furthermore, our study aimed to find out whether n-6 long chain polyunsaturated fatty acids from linoleic acid are formed in RGM-1 cells . METHODS: Rat gastric mucosal cells were incubated with 10, 20 and 40 microg/mL of linoleic acid or medium alone . Phosphatidylcholine content extracted from whole RGM-1 cells was quantitated by using a densitometer, and its fatty acid composition was analyzed by using gas chromatography . Prostaglandin E2 concentration in the culture medium was measured by using radioimmunoassay . The expression of cyclooxygenase (COX)-1 and COX-2 was examined by using reverse transcription-polymerase chain reaction . In addition, after incubation with {1-14C} linoleic acid, radioactivities of both linoleic acid and arachidonic acid components of the PC fraction were counted . The effects of H . pylori broth culture filtrates on PC content, its fatty acid composition and prostaglandin (PG)E2 synthesis were also assessed . RESULTS: Linoleic acid addition caused an increase in the composition of arachidonic acid, as well as linoleic acid, and also in PGE2 concentration . Cyclo-oxygenase-2 expression was induced in RGM-1 cells by the addition of linoleic acid . In addition, {1-14C} linoleic acid added to the culture medium was converted to {1-14C} arachidonic acid in RGM-1 cells . Helicobacter pylori broth culture filtrates decreased linoleic acid composition and increased arachidonic acid composition . Moreover, after incubation with H . pylori broth culture filtrates, PGE2 concentrations were higher than that of the controls . CONCLUSIONS: These findings suggest the presence of fatty acid elongase and Delta5- and Delta6-desaturases synthesize arachidonic acid from linoleic acid in RGM-1 cells . Thus, H . pylori infection may enhance PGE2 synthesis and accelerate n-6 fatty acid metabolism in gastric mucosal cells, which could make the gastric mucosal barrier more fragile. Clin Microbiol Infect, 1997 Feb, 3(6), 668 - 671 Detection of Mycobacterium tuberculosis in mixed broth cultures using DNA probes; Middleton AM et al.; OBJECTIVE: To investigate the ability of a commercial acridinium ester-labeled DNA probe (AccuProbe, Gen-Probe Inc., USA) to detect the presence of Mycobacterium tuberculosis complex in a mixed culture with Mycobacterium avium complex . METHODS: The density of organisms required to produce a positive result for Mycobacterium tuberculosis complex alone in broth culture was compared with the density required to produce a positive result in the presence of Mycobacterium avium complex . RESULTS: A threshold density of 1.5 x 106 CFU/mL was required for detection of Mycobacterium tuberculosis and this threshold remained unaltered in the presence of Mycobacterium avium complex . The presence of Mycobacterium avium complex had no effect on detection of Mycobacterium tuberculosis in a mixed broth culture incubated and probed over a 21-day period . CONCLUSIONS: The findings of the study suggest that the presence of Mycobacterium avium complex has no effect on the detection of Mycobacterium tuberculosis and that the Accuprobe test is potentially capable of detecting a dual infection with organisms of both complexes. World J Gastroenterol, 1998 Oct, 4(5), 385 - 387 Helicobacter pylori acquistion of metronidazole resistance by natural transformation in vitro; Hua JS et al.; AIM:To study whether Helicobacter pylori is naturally transformable.METHODS:Transformation was performed in BHI broth supplemented with horse serum and yeast extract . Genomic DNA extracted from a metronidazole resistant H.pylori strain was added to H . pylori broth culture . The mixture was incubated at microaerophilic atmosphere . The DNA-treated cells were plated on blood agar containing 8mg/L metronidazole to select for transformants . Sterile distilled water was used as a negative DNA control.The DNA profiles of transformants were compared with that of their parent strains by randomly amplified polymorphic DNA (RAPD) fingerprinting.RESULTS:Transformation ofH . pylori with DNA from a metronidazole resistant strain as a marker was demonstrated . Out of the 12 strains of H . pylori tested, 9 (75%) strains were found to be transformable . The transformation frequencies ranged from 3.4?10(-6) to 2.4 10(-4) . By RAPD, DNA fingerprints of the transformants and their parent strains showed no change in DNA profiles though transformants were all resistant to metronidazole as compared with their metronidazole-sensitive parent strains.CONCLUSION:Helicobacter pylori is naturally transformable which might be one of the ways that H . pylori develops resistance to metronidazole. J Med Microbiol, 2001 Dec, 50(12), 1061 - 8 Isolation and characterisation of a novel spirochaete from severe virulent ovine foot rot; Demirkan I et al.; A novel spirochaete was isolated from a case of severe virulent ovine foot rot (SVOFR) by immunomagnetic separation with beads coated with polyclonal anti-treponemal antisera and prolonged anaerobic broth culture . The as yet unnamed treponeme differs considerably from the only other spirochaete isolated from ovine foot rot as regards morphology, enzymic profile and 16S rDNA sequence . On the basis of 16S rDNA, it was most closely related to another unnamed spirochaete isolated from cases of bovine digital dermatitis in the USA, raising the possibility of cross-species transmission . Further information is required to establish this novel ovine spirochaete as the cause of SVOFR. J Endod, 2001 Jan, 27(1), 40 - 2 A comparative evaluation of three root-end filling materials: an in vitro leakage study using Prevotella nigrescens; Scheerer SQ et al.; This study used Prevotella nigrescens to evaluate the sealing ability of Geristore, Super-EBA, and ProRoot when used as root-end filling materials . One hundred single-canal, extracted, human teeth were cleaned and shaped with Gates-Glidden burs and .04 Taper (ProFile Series 29) rotary instruments . The root-ends were resected, and a 3-mm deep root-end preparation was made with ultrasonic tips . After steam sterilization, the teeth were randomly divided into three groups of 30 . Each group was root-end-filled with a different material . Five root-end cavities were left open and served as positive controls; another five root-end cavities filled with sticky wax and covered with two layers of nail polish acted as negative controls . The teeth were attached to presterilized snap-on plastic caps; the root-ends submerged into 12-ml vials of chopped meat carbohydrate broth and placed in an anaerobic chamber . Twice a week, a sterile micropipette was used to inoculate 0.1 ml of a broth culture of Prevotella nigrescens into the root canal of each tooth . Results after 47 days indicated there were no significant differences between the three root-end filling materials against penetration of Prevotella nigrescens. Rev Gastroenterol Mex, 2000 Oct-Dec, 65(4 Suppl 2), 25 - 33 {Importance of Helicobacter pylori in the pathogenesis of gastric cancer . Experimental models in rodents}; Barreto-Zuniga R et al.; We found that the seroprevalence in Cancer Institute of H . pylori infection was significantly more frequent in gastric cancer than in age- and gender-matched controls . This study suggested an epidemiological link between H . pylori infection and gastric cancer . H . pylori exhibits a complex system of enzymes which serve a range of functions . Toxic effects are produced by urease (UR), phospholipase (PL) and alcohol dehydrogenase (ADH) . We embarked on an exploration of the enzyme activities of H . pylori infected patients using a TLC-autoradioluminography . This method has a wide dynamic range and could offer an analytical technique for studying a radioactive compound and its enzymes in H . pylori infected mucosa . Biopsies samples taken from 21 gastric cancer patients and 95 controls were studied . Although high activity of UR indicates well the presence of H . pylori impairment, activities of ADH and PL reflects more the chronicity of mucosal damage in both groups . Clearly, the enzyme profile showed in our study reflects the "physiological" adaptations behind chronic injured mucosal changes but its relation to gastric cancer and H . pylori needs further study . There is an urgent need to understand the carcinogenesis process using animal models . We performed previous study for to explore the effect of H . pylori infection on N- methyl-N-nitrosourea-induced (MNU) gastric carcinogenesis in mice C57BL/6 mice were administered broth culture of H . pylori and given MNU in drinking water . In terms of the incidence of neoplasms development was increase in the MNU group pre-infected with H . pylori . That findings showed that C57BL/6 mice-infected model is well suited for investigating the bacteria promoter effect in the gastric carcinogenesis . Finally another rodent model study (still in process) showed rapid development of hyperplastic gastritis with gastric erosions in H . pylori-infected MTH1 knockout mice . We sought to further evaluate MTH1 knockout mice as potential test animal for carcinogenesis . CONCLUSION: It is suggested that H . pylori infection is an important risk factor for the development of gastric cancer . The possibility that this organism acts etiologically, exerting its effect over long period of time, is biologically plausible . However, the role of H . pylori per se in that process is still a matter of discussion . The various enzymes of H . pylori discussed in this paper support colonization, and are perhaps important for epithelial damage, they could contribute to the stimulation and modulation of the chronic inflammatory response, but its relation to gastric cancer and H . pylori needs further study . Finally H . pylori in C57BL/6 and knockout mice showed excellent colonization at two months and six months after infection there was adenomatous, hyperplastic and ulcerative changes . Those findings showed that both mice-infected models are well suited for investigating the bacteria promoter effect in the gastric carcinogenesis. Int J Med Microbiol, 2001 Apr, 291(1), 27 - 32 Neutralisation of cytotoxic vacuolating activity by serum antibodies of Helicobacter pylori-infected patients; Gosciniak G et al.; The study involved 196 H . pylori strains and 196 serum samples taken from the same patients . H . pylori strains were investigated for the production of vacuolating cytotoxin . Antibodies to the vacuolating cytotoxin produced by H . pylori were detected in the sera samples by neutralisation assay (on Intestine 407 cells) and ELISA . Of the 196 H . pylori strains tested, 80 (40.8%) were found to express vacuolating cytotoxic activity . The titres of cytotoxic nonconcentrated broth culture filtrates ranged from 1:2 to 1:128 . The vacuolating assay was positive in 37.1% strains isolated from children, and in 50% strains isolated from adults . Cytotoxin-positive H . pylori strains were found more frequently in duodenal ulcer (71%) than in chronic gastritis (35.2%) patients, and this difference was statistically significant p < 0.05 . Neutralising antibodies to vacuolating cytotoxin were present in 51% and 49% of the serum samples tested by neutralisation and ELISA, respectively . Duodenal ulcer patients had antibodies to vacuolating cytotoxin more frequently (p < 0.05) than chronic gastritis patients . Antibodies to cytotoxin were detected in the serum samples from patients infected by cytotoxic (100%) and noncytotoxic (18%) H . pylori strains. Gut, 2001 May, 48(5), 605 - 8 Helicobacter pylori infection induces hyperammonaemia in Mongolian gerbils with liver cirrhosis; Suto H et al.; BACKGROUND AND AIMS: We previously reported the effect of Helicobacter pylori eradication on hyperammonaemia in patients with liver cirrhosis . However, the role of H pylori as a cause of hyperammonaemia is controversial . We developed an animal model with liver cirrhosis and investigated the effect of H pylori infection on hyperammonaemia . MATERIALS AND METHODS: Five week old male Mongolian gerbils were inoculated orally with broth culture of H pylori . Forty eight gerbils were divided into four groups . Gerbils not inoculated with H pylori were fed a commercial rodent diet (group A) or a choline deficient diet (group C) . Gerbils inoculated with H pylori were fed the commercial rodent diet (group B) or the choline deficient diet (group D) . Blood ammonia levels of the femoral vein and portal vein were measured 30 weeks later . RESULTS: All gerbils fed the choline deficient diet developed liver cirrhosis with fatty metamorphosis . The survival rate of group D was significantly lower than that of the other groups . Systemic and portal blood ammonia levels in group D were significantly higher than those in the other groups . CONCLUSIONS: H pylori infection induces hyperammonaemia in gerbils with liver cirrhosis. FEMS Immunol Med Microbiol, 2001 Mar, 30(2), 103 - 8 Reduced intracellular survival of Helicobacter pylori vacA mutants in comparison with their wild-types indicates the role of VacA in pathogenesis; Petersen AM et al.; The vacuolating cytotoxin VacA of Helicobacter pylori plays an important but yet unknown role in pathogenesis . We studied the impact of the vacuolating cytotoxin on H . pylori invasion of and survival within AGS cells (human gastric cell line derived from an antral adenocarcinoma) . Isogenic vacA and cagA mutants were constructed in a wild-type clinical isolate H . pylori, AF4 . An H . pylori VacA-deficient mutant, AF4(vacA::kan), was cultured in significantly lower numbers from AGS cells after 24 h incubation with gentamicin added to the culture medium than were the type I wild-type strain AF4 (P<0.03) and an isogenic cagA mutant (P<0.01) . Complementation of the AF4 vacA mutant with broth culture supernatant from wild-type AF4 improved the intracellular survival of the vacA mutant . We conclude that H . pylori's vacuolating cytotoxin improves the intracellular survival of H . pylori within AGS cells, suggesting the role of the vacuolating cytotoxin in H . pylori pathogenesis. J Clin Microbiol, 2001 Jan, 39(1), 69 - 74 Rapid identification of laboratory contamination with Mycobacterium tuberculosis using variable number tandem repeat analysis; Gascoyne-Binzi DM et al.; Compared with solid media, broth-based mycobacterial culture systems have increased sensitivity but also have higher false-positive rates due to cross-contamination . Systematic strain typing is rarely undertaken because the techniques are technically demanding and the data are difficult to organize . Variable number tandem repeat (VNTR) analysis by PCR is rapid and reproducible . The digital profile is easily manipulated in a database . We undertook a retrospective study of Mycobacterium tuberculosis isolates collected over an 18-month period following the introduction of the BACTEC MGIT 960 system . VNTR allele profiles were determined with early positive broth cultures and entered into a database with the specimen processing date and other specimen data . We found 36 distinct VNTR profiles in cultures from 144 patients . Three common VNTR profiles accounted for 45% of true-positive cases . By combining VNTR results with specimen data, we identified nine cross-contamination incidents, six of which were previously unsuspected . These nine incidents resulted in 34 false-positive cultures for 29 patients . False-positive cultures were identified for three patients who had previously been culture positive for tuberculosis and were receiving treatment . Identification of cross-contamination incidents requires careful documentation of specimen data and good communication between clinical and laboratory staff . Automated broth culture systems should be supplemented with molecular analysis to identify cross-contamination events . VNTR analysis is reproducible and provides timely results when applied to early positive broth cultures . This method should ensure that patients are not placed on unnecessary tuberculosis therapy or that cases are not falsely identified as treatment failures . In addition, areas where existing procedures may be improved can be identified. FEMS Immunol Med Microbiol, 2000 Dec, 29(4), 263 - 70 The relationship between O-chain expression and colonisation ability of Helicobacter pylori in a mouse model; Moran AP et al.; The influence of lipopolysaccharide (LPS) O-polysaccharide chain production on the colonisation ability of Helicobacter pylori in four mouse models (NMRI, C57BL/6, CBA/Ca, and BALB/cA mice) was studied . H . pylori strains that produced smooth-form LPS (S-LPS) detectable in silver-stained electrophoretic gels colonised mice . In contrast, a laboratory-passaged strain G50 and the culture collection strain CCUG 17874 did not colonise mice; the former strain produced low amounts of O-chains only detectable in immunoblotting but not in silver-stained gels, whereas the latter produced rough-form LPS (R-LPS) without O-chains . Furthermore, a galE isogenic mutant, which produced R-LPS, did not colonise mice . However, after repeated broth culture, strains G50 and CCUG 17874 produced S-LPS detectable in silver-stained gels and were capable of colonising mice . Consistent with the production of O-chains, all colonising strains produced Lewis (Le) antigens, Le(x) and/or Le(y) . Except for low expression of Le(y) by non-colonising G50, reflecting low production of O-chains, all other non-colonising strains and the galE mutant lacked expression of Le antigens consistent with their production of R-LPS . Lectin typing of strains supported these findings, and also showed that lectin types did not differ before and after colonisation . The low level of O-chain production and Le antigen expression by the non-colonising G50 may not be sufficient to aid colonisation . Examination of protein profiles of H . pylori strains before inoculation showed that protein expression was not significantly different between colonising and non-colonising strains . These results show that S-LPS production with O-chain expression is required by H . pylori for colonisation in a number of mouse models and that care should be taken with inoculating H . pylori strains that loss of O-chains does not occur during subculturing. Carcinogenesis, 2000 Nov, 21(11), 2091 - 5 Influence of Helicobacter pylori on reactive oxygen-induced gastric epithelial cell injury; Smoot DT et al.; Risk factors for gastric cancer are receiving renewed attention in light of the recent positive association of Helicobacter pylori infection with gastric cancer . The effect of H.pylori on the balance between oxidants and antioxidants in the stomach is not well known . In this study, we investigated if exposure of gastric cells to H . pylori increases oxidant-associated gastric epithelial cell injury . A human gastric epithelial cell line (AGS) was grown on 96-well clusters, then exposed overnight to either live H.pylori (four cagA(+) and four cagA(-)) or broth culture supernatant from an isogenic H.pylori cagA(+) strain with and without vacA activity . Incubation of AGS cells with cagA(+) and cagA(-) H.pylori strains before exposure to reactive oxygen species (ROS) reduced cell viability on average to 73.7% and 39.5% of controls, respectively . The percent viability of cells exposed to ROS after incubation with control broth, vacA(-) broth and vacA(+) broth was 97.7%, 70.5% and 63.5%, respectively . Experiments were then performed to evaluate the effects of H.pylori exposure on the activities of ROS-scavenging enzymes {catalase, glutathione peroxidase and superoxide dismutase (SOD)} and formation of 8-hydroxy-2-deoxyguanosine (8-OH-dG) adducts in AGS cells . Overnight exposure to cagA(-) strains reduced catalase activity by 42%; in contrast, exposure to cagA(+) H.pylori strains increased catalase activity by 51% . Glutathione peroxidase activity increased with exposure to both cagA(-) and cagA(+) strains by 95% and 240%, respectively . Total SOD activity increased 156% after exposure to cagA(+) strains and was marginally increased (52%) with exposure to cagA(-) strains . CuZn-SOD protein levels, assayed by enzyme-linked immunosorbent assay, were not significantly altered by exposure to H.pylori strains; however, Mn-SOD concentrations were significantly increased (P: < 0.02) after exposure to both cagA(-) and cagA(+) H.pylori strains . Exposure of AGS cells to cagA(+) and cagA(-) H.pylori was associated with, on average, 44.5 and 99.0 8-OH-dG/10(6) dG, respectively . The increase in catalase, glutathione peroxidase and SOD activity is associated with fewer 8-OH-dG DNA adducts and reduced susceptibility of AGS cells to lethal injury from ROS after exposure to cagA(+) H.pylori strains when compared with exposure to cagA(-) H.pylori strains . Alteration in the activity of ROS-scavenging enzymes by the presence of H . pylori may in part be responsible for the increased risk of gastric cancer in persons infected with H.pylori. J Infect, 2000 Sep, 41(2), 184 - 7 Comparison of fitness of two isolates of Mycobacterium tuberculosis, one of which had developed multi-drug resistance during the course of treatment; Davies AP et al.; OBJECTIVES: We report the cases of two patients, brother and sister, both with pulmonary tuberculosis . Both patients complied poorly with treatment . One developed multi-drug resistant disease, whilst the other did not . We aimed to show that the two infecting strains were the same, and then to compare the fitness of the resistant strain to that of the sensitive strain . METHODS: The isolates were typed by RFLP . The fitness of the multi-drug resistant tuberculosis strain was determined by calculating the ratio of generation produced by the drug-resistant and a drug-susceptible strain in a mixed culture . The number of bacteria present in this broth culture was estimated using the Miles and Misra technique . The number of drug-resistant bacteria present was determined by inoculating aliquots of broth onto Middlebrook 7H10 agar with 5mg/l rifampicin . RESULTS: The infecting strain of Mycobacterium tuberculosis was shown to be the same on RFLP typing in both cases . It was found that the multi-drug resistant organism had decreased fitness compared to the sensitive organism . CONCLUSION: The decreased relative fitness of the resistant strain implies a physiologic cal cost for the development of drug resistance. Infect Immun, 2000 Sep, 68(9), 5225 - 33 Effect of Helicobacter pylori on polymorphonuclear leukocyte migration across polarized T84 epithelial cell monolayers: role of vacuolating toxin VacA and cag pathogenicity island; Hofman V et al.; Helicobacter pylori infection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis . The mechanisms underlying this process are poorly documented . The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced by H . pylori . In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains of H . pylori . We have evaluated the role of the vacuolating cytotoxin VacA and of the cag pathogenicity island (PAI) of H . pylori in PMNL migration via their effects on T84 epithelial cells . We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positive H . pylori strains . In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells with cag PAI-positive and cagE-positive H . pylori strains . Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL . These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that the cag PAI, with a pivotal role for the cagE gene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response. Indian J Exp Biol, 2000 Mar, 38(3), 293 - 6 Protease from Sporosarcina sp . RRLJ 1; Boruah HP et al.; Protease was isolated from Sporosarcina RRLJ1 which was collected from acid tea (Camellia sinensis) plantations . It showed potential for production of the enzyme for commercial purposes . The study revealed that optimum pH for growth of the organism was 6.5-7 and supplement of casein (1%) in the medium was required for production of protease . Enzyme production and enzyme activity was maximum in 72 hr old broth culture . Maximum activity of the enzyme was found at pH 6.5. Vaccine, 2000 Jun 15, 18(25), 2825 - 31 Cytological and immunological changes in bronchoalveolar lavage fluid and histological observation of lung lesions in pigs immunized with Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture supernate; Okada M et al.; We have compared the cytology of bronchoalveolar lavage fluid (BALF) and the pathology of lung lesions in pigs immunized with/without Mycoplasma hyopneumoniae inactivated vaccine prepared from broth culture supernate on experimental infection . Numbers of total cells, macrophages, neutrophils and lymphocytes have decreased in BALF of vaccinated pigs following infection . The mean percentage of lung lesions, inflammatory cell infiltration into the airways and T cells accumulation around the bronchi were reduced in vaccinated pigs . The levels of tumor necrosis factor (TNF)-alpha also decreased in vaccinated pigs . These results suggest that the vaccination may contribute to decrease TNF-alpha production, and therefore, inflammatory cell responses in the lung due to M . hyopneumoniae infection were suppressed, resulting in fewer lung lesions. J Clin Microbiol, 2000 Mar, 38(3), 1063 - 5 Evaluation of the Oricult-N dipslide for laboratory diagnosis of vaginal candidiasis; Carlson P et al.; The Oricult-N semiquantitative dipslide (Orion Diagnostica, Espoo, Finland) was evaluated for the laboratory diagnosis of vaginal candidiasis . It was compared with broth culture (Vagicult; Orion Diagnostica) . Oricult-N was positive for 14.5% of 124 symptomatic patients and 12% of 50 asymptomatic controls . The results for broth cultures were 17 and 22%, respectively . Thus, the test group and the control group did not differ significantly by either method . High vaginal yeast counts (>/=10(5) CFU/ml) were detected by Oricult-N in 7% of patients and in 0% of controls, but both groups harbored low numbers of yeasts . An accurate quantitative cutoff point separating a level of yeast associated with infection from vaginal yeast carriage could not be defined in the study . Nevertheless, the easy semiquantitation allowed by the Oricult-N method could be helpful because, especially in low-count carriers of Candida, other potential causes of vaginal symptoms should be considered . The Oricult-N method was technically simple and could be applied in primary health care . Further studies are required, however, before Oricult-N can be recommended as a routine diagnostic tool. J Clin Pathol, 1999 Aug, 52(8), 616 - 9 A comparison of lysis centrifugation, pour plate, and conventional blood culture methods in the diagnosis of septicaemic melioidosis; Simpson AJ et al.; AIMS: To determine whether quantitative blood culture methods could improve the diagnosis of septicaemic melioidosis . METHODS: A comparison of conventional broth based blood cultures, a pour plate method, and a commercial lysis centrifugation (Isolator 10) blood culture system was conducted in 71 Thai patients with severe melioidosis . The time to identification of B pseudomallei was recorded for each method . RESULTS: 42 patients (59%) were septicaemic . Compared with conventional blood culture, the Isolator and pour plate methods had sensitivities of 81% and 61%, respectively . The median times to a positive culture were: Isolator 39.3 hours, pour plates 45.5 hours, broth culture 61.8 hours (p < 0.001 Isolator v broth) . There was a significant inverse correlation between Isolator tube or pour plate quantitative counts and time to detection (r = -0.44 and -0.57, respectively) . Mortality was higher in patients who were septicaemic . CONCLUSIONS: Routine use of one of these quantitative methods, in addition to conventional broth culture, may lead to earlier diagnosis of septicaemic melioidosis. J Vet Med Sci, 1999 Oct, 61(10), 1131 - 5 Evaluation of Mycoplasma hyopneumoniae inactivated vaccine in pigs under field conditions; Okada M et al.; An inactivated vaccine prepared from broth culture supernatant of Mycoplasma hyopneumoniae with an aluminum adjuvant was evaluated in three herds (herd A: specific pathogen-free herd, herd B: high health status herd with no clinical signs of respiratory infection, herd C: low health status herd with serious epidemiological and economical problems) . A total of 212 pigs from the three herds were divided into two groups . One group was injected twice with the vaccine at 4-week intervals and the other was a control group . No adverse reactions were noted following the vaccinations either systematically or locally in any of the vaccinated pigs from any of the herds . In herd A, the vaccination provided antibody response within 4 weeks after the second vaccination and antibody responses continued for more than 12 weeks . In herds B and C, the number of pigs with lung lesions, mean percentage of lung lesions, and the numbers of M . hyopneumoniae recovered from pigs at slaughter in the vaccinated group were significantly (P < 0.05) reduced compared to the control group . Furthermore, vaccination resulted in improved average daily weight gain (ADG), improved feed conversion ratio (FCR), and improved days to market weight in herd C, whereas no difference in growth performance was shown in herd B . It is suggested that the inactivated vaccine prepared from broth culture supernatant of M . hyopneumoniae is effective in reducing clinical signs and lung lesions . Also, vaccination resulted in improved growth performance in herds where clinical signs and economic losses were significant. Infect Immun, 1999 Oct, 67(10), 5247 - 52 Kinetics and mechanisms of extracellular protein release by Helicobacter pylori; Schraw W et al.; To investigate the kinetics and mechanisms of extracellular protein release by Helicobacter pylori, we analyzed the entry of metabolically radiolabeled bacterial proteins into broth culture supernatant . At early time points, vacuolating cytotoxin (VacA) constituted a major extracellular protein . Subsequently, culture supernatants accumulated many proteins that were components of intact bacterial cells . This nonselective release of proteins was associated with a decreasing turbidity of cultures and loss of bacterial viability, indicative of an autolytic process . The rates of VacA secretion and autolysis were each influenced by medium composition, and therefore these may be regulated phenomena . Extracellular release of proteins by H . pylori may be an important adaptation that facilitates the persistence of H . pylori in the human gastric mucus layer . Moreover, entry of proinflammatory proteins into the gastric mucosa may contribute to the induction of a mucosal inflammatory response. Dis Aquat Organ, 1999 Jun 23, 37(1), 43 - 52 Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum; Wiens GD et al.; Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish . Biological activities of secreted p57 include agglutination of salmonid leucocytes and rabbit erythrocytes . We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4C11, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a monomer that retains salmonid leucocyte agglutinating activity . The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57 . When combinations of the MAbs were used in an antigen capture ELISA, the epitopes recognized by the 3 MAbs were shown to be sterically separate . However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed . These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer . Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed . Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity . A model in which p57 is released from R . salmoninarum as a biologically active monomer during infection of salmonid fish is proposed. J Clin Microbiol, 1999 May, 37(5), 1602 - 5 Use of nucleic acid probes for identification of Mycobacterium tuberculosis directly from MB/BacT bottles; Badak FZ et al.; The feasibility of using nucleic acid probes directly from positive MB/BacT broth to identify mycobacteria was determined in this study . A total number of 2,727 specimens were cultured into the MB/BacT (Organon Teknika) automated system and on conventional Loweinstein-Jensen (LJ) slants . The Gen-Probe AccuProbe culture identification tests (DNA probes) were used on samples from bottles which were identified as positive for mycobacteria by MB/BacT . Samples of positive MB/BacT broth (0.1 ml) were used directly in the broth culture method for the DNA probes as published by Gen-Probe . Centrifugation of the contents of the bottle was not done prior to probe testing . The number of mycobacteria detected by MB/BacT and LJ was 253 (221 isolates of M . tuberculosis and 32 isolates of mycobacteria other than M . tuberculosis {MOTT}) . A total of 96.4% (213 of 221) of the bottles growing M . tuberculosis produced a positive direct DNA probe result for M . tuberculosis complex . One hundred percent (16 of 16) of the bottles growing M . gordonae produced a positive direct DNA probe result for M . gordonae . A total of 3.6% (8 of 221) of the bottles growing M . tuberculosis did not yield a positive direct DNA probe result for M . tuberculosis complex . The testing of subcultures made onto solid media from the positive bottles by AccuProbe identified six of these eight M . tuberculosis isolates . Two (0.9%) M . tuberculosis isolates gave a negative result for the M . tuberculosis probe test applied on the MB/BacT broth and its subculture . The rest of the positive MB/BacT bottles growing MOTT (16 of 32) were negative for M . gordonae, M . avium, M . intracellulare, and M . kansasii probes . The sensitivity and specificity of AccuProbe for the identification of M . tuberculosis and M . gordonae directly from MB/BacT broth were 96.4 and 100% for M . tuberculosis and 100 and 100% for M . gordonae, respectively . The direct testing of positive MB/BacT broth by AccuProbe, without prior centrifugation, allows for the accurate and rapid identification of M . tuberculosis and M . gordonae. J Clin Microbiol, 1999 Apr, 37(4), 1045 - 8 Helicobacter pylori can be induced to assume the morphology of Helicobacter heilmannii; Fawcett PT et al.; Cultures of Helicobacter pylori obtained from the American Type Culture Collection (strain 43504) were grown as isolated colonies or lawns on blood agar plates and in broth culture with constant shaking . Examination of bacterial growth with Gram-stained fixed preparation and differential interference contrast microscopy on wet preparations revealed that bacteria grown on blood agar plates had a morphology consistent with that normally reported for H . pylori whereas bacteria from broth cultures had the morphologic appearance of Helicobacter heilmannii . Bacteria harvested from blood agar plates assumed an H . heilmannii-like morphology when transferred to broth cultures, and bacteria from broth cultures grew with morphology typical of H . pylori when grown on blood agar plates . Analysis by PCR of bacteria isolated from blood agar plates and broth cultures indicated that a single strain of bacteria (H . pylori) was responsible for both morphologies. Infect Immun, 1998 Dec, 66(12), 5785 - 91 Helicobacter pylori alters exogenous antigen absorption and processing in a digestive tract epithelial cell line model; Matysiak-Budnik T et al.; To study the influence of Helicobacter pylori on epithelial barrier function, bacteria, bacterial sonicates, or broth culture supernatants were incubated for 24 h with HT29-19A intestinal cells grown as monolayers . Subsequently, the monolayers were mounted in Ussing chambers, and electrical resistance (R), fluxes of 22Na (JNa) and 14C-mannitol (JMan) (markers of the paracellular pathway), and fluxes of horseradish peroxidase (HRP) in total (J3H-HRP), intact (JHRPi), and degraded forms were measured . H . pylori did not induce any modification of the paracellular pathway (R = 148 +/- 10 versus 174 +/- 16 Omega . cm2; JNa = 4.16 +/- 0.44 versus 3.51 +/- 0.41 microEq/h . cm2; JMan = 0.081 +/- 0.01 versus 0.058 +/- 0.009 micromol/h . cm2), nor did it modify J3H-HRP (2,201 +/- 255 versus 2, 110 +/- 210 ng/h . cm2 for H . pylori-infected and control cells, respectively) . However, in the presence of H . pylori, we observed a significant increase in JHRPi (520 +/- 146 versus 171 +/- 88 ng/h . cm2) . This effect was not dependent of the cag status of the strain and was not reproduced by the sonicates or the culture supernatants . It was related to the presence of urease, since a urease-negative mutant of H . pylori did not induce this effect . Ammonia and bafilomycin A1, two agents known to increase the endolysosomal pH, reproduced the increase in JHRPi . In conclusion, H . pylori does not affect directly the integrity of intercellular junctions of epithelial cells in vitro, but it increases the passage of intact HRP, probably by inhibition of the intralysosomal degradation due to the release of ammonia . The increased transport of intact macromolecules may contribute to the induction and maintenance of gastric inflammation by H . pylori. J Biol Chem, 1998 Oct 30, 273(44), 28560 - 3 Helicobacter pylori up-regulates cyclooxygenase-2 mRNA expression and prostaglandin E2 synthesis in MKN 28 gastric mucosal cells in vitro; Romano M et al.; Helicobacter pylori has been suggested to play a role in the development of gastric carcinoma in humans . Also, mounting evidence indicates that cyclooxygenase-2 overexpression is associated with gastrointestinal carcinogenesis . We studied the effect of H . pylori on the expression and activity of cyclooxygenase-1 and cyclooxygenase-2 in MKN 28 gastric mucosal cells . H . pylori did not affect cyclooxygenase-1 expression, whereas cyclooxygenase-2 mRNA levels increased by 5-fold at 24 h after incubation of MKN 28 cells with broth culture filtrates or bacterial suspensions from wild-type H . pylori strain . Also, H . pylori caused a 3-fold increase in the release of prostaglandin E2, the main product of cyclooxygenase activity . This effect was specifically related to H . pylori because it was not observed with Escherichia coli and was independent of VacA, CagA, or ammonia . H . pylori isogenic mutants specifically lacking picA or picB, which are responsible for cytokine production by gastric cells, were less effective in the up-regulation of cyclooxygenase-2 mRNA expression and in the stimulation of prostaglandin E2 release compared with the parental wild-type strain . This study suggests that development of gastric carcinoma associated with H . pylori infection may depend on the activation of cyclooxygenase-2-related events. Am J Physiol, 1998 Oct, 275(4 Pt 1), G681 - 8 Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells; Sommi P et al.; The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium . Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H . pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell . The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting . We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than approximately 90 kDa . Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity. J Immunol Methods, 1998 Apr 1, 213(1), 19 - 30 A novel flow cytometric assay for quantitating adherence of Helicobacter pylori to gastric epithelial cells; Logan RP et al.; Adherence may be an important virulence factor for Helicobacter pylori . Current methods available for quantitation of adherence are time consuming and liable to observer error . A new direct technique for fluorescent labelling of bacteria has been developed to quantitate adherence of H . pylori to epithelial cells by fluorescence activated cell sorting (FACS) . Type strains of H . pylori, H . mustelae, H . cinaedi and H . fennelliae were grown microaerobically in broth culture for 24 h and fluorescently labelled by incubation with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37 degrees C . After washing to remove excess CFDA-SE, bacteria were co-incubated (ratio 10:1) with gastric epithelial cells at 37 degrees C for up to 24 h . After washing to remove non-adherent bacteria, epithelial cells were detached with EDTA (2 mM) and fixed with formaldehyde for flow cytometry . Adherence was quantitated both in terms of the proportion of cells with adherent H . pylori and as the mean number of adherent bacteria per cell . All H . pylori strains adhered to gastric-type epithelial cells . The proportion of cells with bound bacteria varied from 40-99% and the number of bacteria per cell from 1-50, both of which correlated with microscopy (r = 0.6, and r = 0.8 respectively, n = 35) . Time course studies demonstrated saturation of binding by H . pylori within 90 min . For H . mustelae, H . cinaedi and H . fennelliae the proportion of cells with bound bacteria varied from 5-15% and the mean number of bacteria per cell was < 4 . Binding of H . pylori to epithelial cells could be partly blocked by pre-incubation with polyclonal anti-sera or using oligosaccharides against potential binding epitopes of gastric mucus . Fluorescent labelling of H . pylori with CFDA-SE in combination with flow cytometry provides a quick, specific, and sensitive method to quantitate in vitro the adherence of H . pylori. J Hosp Infect, 1998 Jun, 39(2), 149 - 57 Detection of persistent vegetative bacteria and amplified viral nucleic acid from in-use testing of gastrointestinal endoscopes; Deva AK et al.; Hospital-acquired infection attributed to inadequate decontamination of gastrointestinal endoscopes prompted an in use evaluation of recommended procedures . Specimens were obtained from the internal channels of 123 endoscopes before, during and after decontamination by flushing with saline and brushing with a sterile brush, and examined for vegetative bacteria by broth and plate culture . Four endoscopy units were tested; the chemical disinfectants used were: 2% glutaraldehyde in Centres 1 and 2 (automated) and Centre 3 (manual); peracetic acid in Centre 4 (automated) . Samples from patients in Centre 1 with known chronic hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1) infection were also examined for viral nucleic acid by ultracentrifugation, nucleic acid extraction, reverse transcription (for RNA) and polymerase chain reaction (PCR) . No persistent vegetative bacteria were found following standard manual cleaning and disinfection for 20 min in 2% glutaraldehyde in Centres 2 and 3 (N = 37) . At Centre 1, while plate culture yielded no growth, 34% of samples (10/29) grew vegetative bacteria in broth culture after cleaning and disinfection for 20 min in 2% glutaraldehyde . Investigation revealed an error in manual cleaning; no bacteria were detected in 37 samples taken after this was corrected . At Centre 4, despite the use of peracetic acid as a sterilant, three out of 20 (15%) of post decontamination samples grew bacteria; one contained persistent bacteria . HBV and HCV PCR analysis detected viral nucleic acid in three out of four and four out of six samples from viraemic patients undergoing endoscopy in Centre 1 during the period of improper manual washing . After proper cleaning was instituted, samples from nine out of nine HCV viraemic patients were negative . HIV RNA was detected in five of 14 samples taken from endoscopes after use on HIV positive patients but all post decontamination samples were negative . Detection of bacteria in washes from endoscope channels is a useful warning of a breakdown in decontamination practice . Inadequate brushing of internal channels may result in persistent HCV and HBV viral nucleic acid, the significance of which is not clear . These results reinforce the importance of adequate manual cleaning of endoscopes before chemical disinfection. Infect Immun, 1998 Jul, 66(7), 3088 - 94 Heterogeneity in levels of vacuolating cytotoxin gene (vacA) transcription among Helicobacter pylori strains; Forsyth MH et al.; Broth culture supernatants from Tox+ Helicobacter pylori strains induce vacuolation of HeLa cells in vitro and contain VacA in concentrations that are higher than those found in supernatants from Tox- H . pylori strains . To investigate the basis for this phenomenon, we analyzed the transcription of the vacuolating cytotoxin gene (vacA) in eight Tox+ strains (each with a type s1/m1 vacA genotype) and nine Tox- strains (each with a type s2/m2 vacA genotype) . Most of the Tox+ and Tox- strains tested used the same vacA transcriptional start point, but Tox+ strains yielded significantly stronger primer extension signal intensities than did Tox- strains (mean densitometry values of 15.8 +/- 1.9 versus 8.9 +/- 1.7, P = 0 . 0016) . Correspondingly, when we introduced vacA::xylE transcriptional fusions into the chromosomes of a Tox+ strain (60190) and a Tox- strain (86-313), the level of XylE activity in 60190 vacA::xylE was about 30-fold higher than that in 86-313 vacA::xylE . Sequence analysis and promoter exchange experiments indicated that the different levels of vacA transcription in these two strains cannot be explained solely by a difference in promoter strength . These data indicate that Tox+ and Tox- H . pylori strains typically differ not only in the VacA amino acid sequence but also in the level of vacA transcription. Am J Pathol, 1998 Jun, 152(6), 1617 - 24 Helicobacter pylori culture supernatant interferes with epidermal growth factor-activated signal transduction in human gastric KATO III cells; Pai R et al.; The mechanisms by which Helicobacter pylori infection leads to gastroduodenal ulceration remain poorly understood . Previous studies have shown that H . pylori vacuolating cytotoxin (VacA) inhibits proliferation of gastric epithelial cells, which suggests that H pylori may interfere with gastric mucosal repair mechanisms . In this study, we investigated the effects of H . pylori broth culture supernatants on epidermal growth factor (EGF)-mediated signal transduction pathways in a gastric carcinoma cell line (KATO III) . Exposure of these cells to EGF resulted in increased expression and phosphorylation of the EGF receptor (EGF-R), increased ERK2 activity and phosphorylation, and increased c-fos protein levels . Preincubation of cells with broth culture supernatant from VacA (+) H . pylori strain 60190 inhibited the capacity of EGF to induce each of these effects . In contrast, preincubation of cells with broth culture supernatant from an isogenic VacA-mutant strain (H . pylori 60190-v1) failed to inhibit the effects of EGF . These results suggest that the H . pylori vacuolating cytotoxin interferes with EGF-activated signal transduction pathways, which are known to be essential for cell proliferation and ulcer healing. Infect Immun, 1998 Jun, 66(6), 2984 - 6 Extracellular release of antigenic proteins by Helicobacter pylori; Cao P et al.; Screening a Helicobacter pylori genomic library with antisera raised against H . pylori broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA) . H . pylori antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation. J Clin Invest, 1998 Apr 15, 101(8), 1604 - 13 Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells; Romano M et al.; Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro . EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing . We therefore studied the acute effect of H . pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma . Exposure of MKN 28 cells to H . pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha . This effect was specifically related to H . pylori since it was not observed with E . coli, and was independent of VacA, CagA, PicA, PicB, or ammonia . Moreover, H . pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells . AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H . pylori filtrate, but had no effect in the presence of H . pylori broth culture filtrates . Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H . pylori broth culture filtrates . Increased expression of AR and HB-EGF were mediated by an H . pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size . We conclude that H . pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H . pylori on cell growth . The inhibitory effect of H . pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H . pylori-induced gastroduodenal injury. DNA Cell Biol, 1998 Apr, 17(4), 343 - 8 Inhibitory effect of lipopolysaccharide on immune response after DNA immunization is route dependent; Boyle JS et al.; The DNA prepared from E . coli contained high levels of lipopolysaccharide (LPS) . When antigen-encoding DNA was injected into mice, toxicity and increased IgM responses were observed . A method for purifying high yields of DNA (up to 12 mg/L of broth culture) with very low levels of LPS (0.05 ng/mg) was developed . When this purified DNA was used for immunization studies, the toxicity and increased IgM responses were abrogated . Thus, LPS was added to DNA in order to examine its influence on the IgG and cytotoxic T lymphocyte (CTL) response after intramuscular (i.m.) or intradermal (i.d.) DNA immunization . The IgG response to DNA-encoded antigen was inhibited in a dose-dependent manner by the i.d., but not the i.m., route of immunization . Surprisingly, no effect on the CTL response was observed . Therefore, the ability to produce high yields of plasmid DNA with very low levels of endotoxin contamination is advantageous for DNA immunization studies, not only for toxicologic but also for immunologic considerations . Furthermore, these results provide further evidence that immune induction occurs via different mechanisms after i.m . and i.d . DNA immunization. Mol Microbiol, 1998 Feb, 27(3), 531 - 9 Histoplasma acquisition of calcium and expression of CBP1 during intracellular parasitism; Batanghari JW et al.; A highly adapted parasite of macrophages, the yeast phase of Histoplasma capsulatum, survives and proliferates within phagolysosomes, while the mycelial phase exists only as a saprophyte in the soil . We have shown previously that these two phases of Histoplasma differ in their calcium requirements for growth and in the production of a released calcium-binding protein (CBP) . Cloning and sequencing the CBP1 gene revealed two introns, a putative signal peptide and potential calcium-binding sites . We also evaluated CBP1 expression by reverse transcription-polymerase chain reaction (RT-PCR) of yeasts grown in broth culture and within two host cell types, a macrophage-like cell line and respiratory epithelial cells . H . capsulatum yeasts expressed CBP1 in all of these settings . Splenocytes from mice immunized with H . capsulatum yeasts responded to purified CBP in proliferation assays, providing evidence for the production of CBP during the infection of mammalian hosts . In addition, after H . capsulatum yeasts were subjected to a calcium-free shock, exogenously added CBP allowed yeasts to incorporate more calcium than yeasts incubated without added CBP . These results suggest that CBP may function to provide yeasts with calcium when they are in a low-calcium environment, such as the phagolysosomal compartment within macrophages. Comp Immunol Microbiol Infect Dis, 1997 Sep, 20(4), 319 - 33 Immunoblot examination of humoral response of chickens infected with Mycoplasma gallisepticum at various ages; Ellakany H et al.; Mycoplasma gallisepticum- and Mycoplasma synoviae-free chickens were infected with 0.2 ml broth culture of M . gallisepticum strain 1226 intra air sac at 3, 14, 18, 28, 42, 49 and 65 days of age . Blood samples were taken 0-5 weeks before infection and 1-6 weeks after infection (depending on age of infection) . The antibody response was examined by Western blot . As a control of infection, serum plate agglutination test (SPA), pathological lesions, and presence of Mycoplasma in air sacs were used . Antibodies to p64-67 kDa appeared in all groups of birds on the first week post-infection . Antibodies to p56 were detected from the second week post-challenge if infection was performed at 3 or 14 days of age, while on first week if challenge was done at 18, 28, 42, 49 or 65 days of age . Antibodies to p200, p120, p98, p80, p75, p72, p60, p50, p45, p40, p35, p33, p31, p28, p26, p24 and p22 were also detected. Cancer Lett, 1998 Jan 16, 123(1), 63 - 9 Helicobacter pylori promotes development of pepsinogen-altered pyloric glands, a preneoplastic lesion of glandular stomach of BALB/c mice pretreated with N-methyl-N-nitrosourea; Shimizu N et al.; H . pylori is thought to be a stomach carcinogen . Since no experimental model has hitherto been established to clarify the relationship between H . pylori and stomach carcinogenesis, the effects of infection with the bacteria on experimental carcinogenesis in the glandular stomach of mice were investigated . BALB/c mice were given salty diet or N-methyl-N-nitrosourea (MNU) and administered broth culture of H . pylori . The incidence of pepsinogen-altered pyloric glands, considered as precancerous lesions, was increased in the H . pylori inoculated group pre-treated with MNU . The findings provide the new experimental model demonstrating the relationship between stomach cancer and H . pylori. J Clin Microbiol, 1998 Jan, 36(1), 86 - 9 Use of an enrichment broth cultivation-PCR combination assay for rapid diagnosis of swine erysipelas; Shimoji Y et al.; We have previously described the creation by Tn916 mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas . In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E . rhusiopathiae . According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E . rhusiopathiae chromosome by PCR . The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrix species and 27 strains of other genera different from Erysipelothrix . A 937-bp DNA fragment could be amplified from all E . rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested . To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E . rhusiopathiae in the specimens from swine with arthritis . After samples were enriched in selective broth culture, detection of E . rhusiopathiae was tested by either conventional methods or the PCR . Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR . The detection limit of the PCR was 10(3) CFU per reaction mixture for the PCR-positive samples . These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E . rhusiopathiae in specimens. J Appl Microbiol, 1997 Dec, 83(6), 712 - 7 Identification of entero-aggregative Escherichia coli based on surface properties; Chart H et al.; Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a 'stacked brick' pattern (EAggEC), and four nonadherent control strains, were examined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA) HEp-2 cell adhesion phenotypes . These strains were also tested for the ability to express an 18 kDa membrane-associated outer-membrane protein (MAP), to agglutinate erythrocytes, and to produce a pellicle during broth culture . Thirteen of the 29 HEp-2 adherent strains of E . coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outer membrane protein (OMP) which was antigenically related to the MAP expressed by strains of E . coli O126:H27 . The strains that did not carry the additional DA genes did not express an 18 kDa OMP . Although strains of EAggEC share the ability to adhere to HEp-2 cells with a stacked brick pattern, these strains exhibit a diverse range of physical and biochemical properties . From the results of this study, it was concluded that currently, the possession of EAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain the only reliable means of identifying EAggEC. J Physiol Pharmacol, 1997 Sep, 48(3), 415 - 21 Relationship between antibody to cytotoxin and Helicobacter pylori infection; Gosciniak G et al.; Broth culture supernatants from 14 (34%) out of the 41 H . pylori strains tested, induced vacuolization in Intestine 407 cells in titers ranging from 1:2 to 1:64 . 20% of H . pylori strains isolated from children and 42% of strains isolated from adults expressed vacuolating activity . Serum antibody to cytotoxin produced by H . pylori was detected with a neutralization assay . Anticytotoxic antibodies were present in all sera from patients infected with cytotoxic H . pylori strains . The toxin-neutralizing activity of sera from individuals infected with H . pylori suggests that the cytotoxin is produced in vivo. Mol Cell Probes, 1997 Aug, 11(4), 251 - 8 Cloning of the antigen 85A from Mycobacterium gordonae and its use for the specific PCR identification of these mycobacteria; Dumonceaux M et al.; The complete nucleotide sequence of 85A antigen of Mycobacterium gordonae was determined . This gene encodes 339 amino acids, including 43 amino acids for the signal peptide, followed by a mature protein of 296 amino acids . A polymerase chain reaction (PCR) assay for the rapid detection of M . gordonae DNA using two pairs of oligonucleotide primers, derived from our sequence, is described . This one-step PCR has been used successfully to amplify 38 strains of M . gordonae . Conversely, the primers did not amplify DNA from any of the 25 mycobacterial species tested . The results suggest that this PCR assay could be a good alternative to existing commercial assays for the specific identification of M . gordonae on early culture on solid medium or on early BACTEC broth culture. Poult Sci, 1997 Jun, 76(6), 791 - 7 Variation in toe-web response of turkey poults to phytohemagglutinin-P and their resistance to Escherichia coli challenge; Bayyari GR et al.; One thousand 5-wk-old male turkeys from each of two commercial strains (A and B) were grouped into low, medium, and high responders based on the cutaneous basophil hypersensitivity (CBH) response obtained 24 h after toe-web inoculation with 100 micrograms of phytohemagglutinin-P (PHA-P) . The CBH response for Strain A was higher than strain B (P = 0.00001) and ranged from 0 to 1.95 mm, with a mean of 0.66, whereas the CBH response for Strain B ranged from 0 to 1.67 mm with a mean of 0.38 . At 6 wk of age, 36 birds from each of the six response groups were inoculated into the left thoracic air sac with 1.5 x 10(7) cfu of an early log phase broth culture of Escherichia coli . Samples of 5 or 10 birds were necropsied from each of the six groups at 7, 14, 28, and 42 d postinfection (PI) . Birds were scored for air-sacculitis/pericarditis (AS) and turkey osteomyelitis complex (TOC) . Overall mortality of birds inoculated with E . coli was 31% . There were no mortalities in unchallenged controls . Strain A had significantly higher Week 1 mortality, marginally higher overall mortality (P = 0.1), and higher AS scores than Strain B . There were no TOC lesions detected until 7 d PI, after which all mortalities had TOC lesions in multiple sites . The differences in CBH response within each strain were not clearly correlated to E . coli susceptibility . However, these data suggest that air sac inoculation of E . coli can provide a useful model for the study of TOC . The greater incidence of disease in Strain A indicates that an enhanced inflammatory response may increase susceptibility to E . coli septicemia. Int J Syst Bacteriol, 1997 Apr, 47(2), 363 - 8 Characterization of smooth and rough morphotypes of Peptostreptococcus micros; Kremer BH et al.; Isolation of the smooth (Sm) morphotype of Peptostreptococcus micros, a suspected oral pathogen, is sometimes accompanied by isolation of a rough (Rg) morphotype of P . micros . The Rg type readily changes to a Sm-like variant (RgSm) in broth culture . Sm and Rg isolates and RgSm variants were compared to determine whether these three types are the result of phase variation . The RgSm variants resembled the Sm morphotype in colony morphology; furthermore, the Sm type and the RgSm type did not have the fibrillar surface structures characteristic of the Rg type, and the Sm and RgSm types were more hydrophobic than the Rg type . However, when we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell proteins, serotyping data, pyrolysis mass spectrometry data, 16S ribosomal DNA sequences, and hemolytic activities, the RgSm variants and the Rg isolates were very similar and were clearly distinct from the Sm isolates . These results suggest that the Rg and RgSm types form a cluster distinct from the Sm type and thus provide evidence that P . micros can be differentiated into two groups, one consisting of the Sm type and the other consisting of the Rg and RgSm types. Adv Exp Med Biol, 1997, 412, 363 - 6 Interaction of Escherichia coli producing cytotoxic necrotizing factor with HeLa epithelial cells; De Rycke J et al.; Cytotoxic necrotizing factors (CNF) constitute a group a cell-associated proteic toxins of 110-115 kDa produced by some clinical isolates of Escherichia coli from man and animals . Purified CNFs are known to exacerbate actin polymerization in exposed cells, a property that has been ascribed to their ability to modify rho a small GTP-protein involved in the regulation of the cytoskeleton . We speculated that, in spite of their lack of excretion in broth culture supernatants, CNF might be expressed upon direct interaction of organisms with infected cells . To test this hypothesis, we set up a model of interaction using epithelial cell line HeLa and the CNF1-producing strain BM2-1, which is adherent to Hela cells . An interaction of four hour duration triggers the progressive development of a dose-dependent cytopathic effect (CPE) with following characteristics: (1) intense cell enlargement with formation of a dense network of stress fibers, (2) inhibition of cell mitosis due to an irreversible block in G2/M transition phase, (3) nucleus swelling and fragmentation, and (4) cell death starting five days after infection . The three last features clearly differentiate CPE from the effect produced by CNF1 alone . In addition CPE, was not produced by cell-free culture supernatants nor abolished by an antiserum neutralizing CNF1 . Tn5::PhoA insertion in the 3' end of cnf1 structural gene abolished CPE, which was not restored by trans complementation with cloned cnf1 . These results demonstrate that CNF1-producing E . coli exert a specific pathogenic effect in HeLa cells, which is determined by cnf1 and at least one additional gene, located downstream cnf1. Gut, 1996 Dec, 39(96), 795 - 9 Effects of Helicobacter pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells; Smoot DT et al.; BACKGROUND: Many Helicobacter pylori strains produce a cytotoxin that induces cytoplasmic vacuolation in various types of eukaryotic cells . In contrast with the marked cell vacuolation that occurs in vitro in response to this cytotoxin, comparatively little epithelial vacuolation has been observed in the gastric mucosa of H pylori infected persons . AIMS: Experiments were performed to determine the susceptibility of human gastric epithelial cells in vitro to H pylori vacuolating cytotoxin activity . METHODS: Human gastric epithelial cells, harvested from upper gastrointestinal endoscopic biopsy specimens, were incubated overnight with broth culture supernatants from either a wild type cytotoxin producing (tox+) H pylori strain or an isogenic mutant strain that lacks cytotoxin activity . RESULTS: Prominent cytoplasmic vacuolation occurred in response to tox+ supernatant, but not supernatant from the isogenic mutant strain . Primary human gastric epithelial cells were significantly more sensitive to H pylori vacuolating cytotoxin activity than were either HeLa or AGS cells . Exposure of human gastric epithelial cells to high concentrations of tox+ supernatant for 48 hours caused lethal cell injury . CONCLUSIONS: These studies indicate that primary human gastric epithelial cells are highly sensitive to H pylori vacuolating cytotoxin activity. Infect Immun, 1996 Nov, 64(11), 4501 - 7 Effect of growth phase and acid shock on Helicobacter pylori cagA expression; Karita M et al.; Helicobacter pylori strains possessing cagA are associated with peptic ulceration . To understand the regulation of expression of cagA, picB, associated with interleukin-8 induction, and ureA, encoding the small urease subunit, we created gene fusions of cagA, ureA, and picB of strain 3401, using a promoterless reporter (xylE) . Expression of XylE after growth in broth culture revealed that basal levels of expression of cagA and urea in H . pylori were substantially greater than for picB . For cagA expression in stationary-phase cells, brief exposure to acid pH caused a significant increase in xylE expression compared with neutral pH . In contrast, expression of xylE in urea or picB decreased after parallel exposure to acid pH (pH 7 > 6 > 5 > 4), regardless of the growth phase . Expression of the CagA protein varied with growth phase and pH exposure in parallel with the observed transcriptional variation . The concentration of CagA in a cell membrane-enriched fraction after growth at pH 6 was significantly higher than after growth at pH 5 or 7 . We conclude that the promoterless reporter xylE is useful for studying the regulation of gene expression in H . pylori and that regulation of CagA production occurs mainly at the transcriptional level. Clin Lab Med, 1996 Sep, 16(3), 551 - 67 Primary processing of specimens and isolation and cultivation of mycobacteria; Hall GS; Processing of specimens for mycobacteria need to proceed as rapidly as possible to provide 24 hour turnaround for smears and 21-day turnaround for the identification of Mycobacterium tuberculosis . If these goals are to be accomplished, a broth culture or microcolony plate method must be part of the media and in the planting process . Use of conventional solid media alone does not provide adequate turnaround time. Avian Dis, 1996 Jul-Sep, 40(3), 654 - 60 Standardized method of aerosol challenge for testing the efficacy of Mycoplasma gallisepticum vaccines; Whithear KG et al.; A special chamber was constructed with the goal of controlling the process of aerosol infection of chickens with Mycoplasma gallisepticum (MG) . The virulent Australian MG field strain Ap3AS was used in each of three experiments . The response to infection of layer-strain pullets was measured serologically, by the incidence and severity of gross lesions in tracheas and air sacs, and by the relative numbers of MG isolated from tracheas and air sacs 2 wk after challenge . In two of the experiments tracheal sections were assessed microscopically . Exposure to a nebulized, undiluted broth culture of MG for 10, 20, or 30 min produced uniformly severe lesions and serological responses . By contrast, results were less severe and less consistent when doses of up to 10(8) color-changing units (CCU) were injected directly into the abdominal air sacs . Gross air sac lesions were consistently produced in almost all pullets by exposure to an infectious aerosol containing 10(2)-10(3) CCU/liter of air for 10 min and an air flow rate of 40 liters/min . This can be achieved by nebulizing a 10(-3) dilution of a fresh, early stationary phase culture of MG strain Ap3AS . However, under the conditions of these experiments, this dose did not produce significant gross or histologic lesions in the trachea. Infect Immun, 1996 Jul, 64(7), 2829 - 33 Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA; Ricci V et al.; Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage . In this study, we sought to test the hypothesis that two H . pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo . Human gastric epithelial cells (MKN 28) were incubated with undialyzed or dialyzed broth culture filtrates from wild-type H . pylori strains or isogenic mutants defective in production of VacA, CagA, or both products . We found that (i) VacA specifically inhibited cell proliferation without affecting cell migration, (ii) CagA exerted no effect on either cell migration or proliferation, and (iii) undialyzed H . pylori broth culture filtrates inhibited both cell migration and proliferation through a VacA- and CagA-independent mechanism . These findings demonstrate that, in addition to damaging the gastric mucosa, H . pylori products may also impair physiological processes required for mucosal repair. Digestion, 1996, 57(5), 299 - 304 Significance of ammonia in the genesis of gastric epithelial lesions induced by Helicobacter pylori: an in vitro study with different bacterial strains and urea concentrations; Sommi P et al.; Two Helicobacter pylori products cause cell damage both in vivo and in vitro: ammonia, from bacterial urease activity, and a vacuolating toxin named VacA . In this in vitro study, the vacuolating effect of H . pylori broth culture filtrate from a VacA-positive/urease-positive strain is compared with that of a VacA-negative/urease-positive strain and a VacA-negative/urease-negative strain . The effect of VacA and ammonia was evaluated with and without addition of 10 mM urea, a physiological concentration for the human stomach, and with and without addition of 0.5 mg/ml acetohydroxamic and (AHA), an urease inhibitor . Our data show that: (1) both urease-positive H . pylori strains caused cell vacuolation in the absence of urea, the VacA-positive strain being approximatively twice as potent as the VacA-negative strain; (2) addition of urea to the culture medium caused an approximatively 3-fold increase in the vacuolating activity of both urease-positive strains; (3) a VacA-negative/urease-negative strain did not exert any vacuolating effect, either in the presence or in the absence of urea; (4) the ratio between cell vacuolation induced by VacA-positive and VacA-negative strains was enhanced by the presence of AHA: ratio was about 2 in the absence of AHA and about 6 in the presence of AHA, either with or without urea added . The increment of vacuolation is likely due to an interaction between AHA and VacA . In conclusion, a VacA-negative/urease-positive strain becomes highly cytotoxic when physiological levels of urea are present in the incubation medium . This finding suggests that all urease-positive H . pylori strains, both with and without VacA expression, should be considered as potentially cytotoxic for the human gastric mucosa, although VacA enhances the severity of cell damage. Eur J Clin Microbiol Infect Dis, 1996 Jan, 15(1), 79 - 82 The value of cerebrospinal fluid enrichment culture in the diagnosis of acute bacterial meningitis; Lessing MP et al.; The records of cerebrospinal fluid (CSF) examinations for the three-year period ending 30 September 1991 were studied retrospectively . From 3,161 examinations, 66 pathogens were detected, 64 of which were from primary agar plate culture . Enrichment broth culture yielded 219 isolates of which 217 were likely contaminants . Only two pathogens were isolated by enrichment, and in both samples the CSF leucocyte counts were abnormal . The use of enrichment broth culture should be confined to CSF specimens with abnormal leucocyte counts and/or Gram stains. Microb Pathog, 1995 Nov, 19(5), 227 - 84 A Brucella melitensis high temperature requirement A (htrA) deletion mutant demonstrates a stress response defective phenotype in vitro and transient attenuation in the BALB/c mouse model; Phillips RW et al.; Bacterial stress response proteins of the high temperature requirement A (HtrA) family are serine proteases which appear to play an important role in scavenging oxidatively damaged proteins from the cell before they reach toxic levels . An isogenic htrA deletion mutant, designated RWP5, was constructed from virulent Brucella melitensis 16M via gene replacement to determine whether the B . melitensis HtrA protein functions as a stress response protein, and to evaluate the contribution of this protein to virulence .