- Email: [email protected]
Random mutagenesis of the helicobacter pylori genome
FIS 98 Abstracts SMALL BOWEL MICROFLORA AND NEONATAL NECROTISING ENTEROCOLITIS. CM. Hay’, C.M. Wood’, P.M. Hawkey’ and J.W.L. Puntis’, Departments of Microbiology’ and Paediatrics2, University of Leeds
MOLECULAR PATHOGENESIS of Shigella sonnei. A.Tuanvok .and P. Shears, Centre for Tropical Medical Microbiology, University of Liverpool, Liverpool, UK
As part of a study of the microbial ecology of necrotising enterocolitis (NEC), we investigated small bowel colonisation in infants weighing 115OOg at birth. Small bowel contents were sampled by blind aspiration at routine naso-gastric tube replacement. Samples were cultured quantitatively and Enterobacteriaceae typed by REP-PCR and pulsed field gel electrophoresis. Over 17 months, 422 aspirates were collected from 122 babies. 108 samples were sterile, 150 yielded Enterobacteriaceae, 158 Gram-positive organisms and 6 yeasts. 61 infants (50%) had Enterobacteriaceae in 11 sample with counts from 53 cfu/g to 1.5 x lo* cfu/g. Molecular typing showed that infants remained colonised with a particular strain for up to 9 weeks. There was evidence of cross-colonisation within the unit with indistinguishable strains isolated from multiple babies, including an E. coli isolated from 11 infants. 23 episodes of NEC occurred during the study. Small bowel aspirates were collected up to 9 days prior to onset in 12 episodes. Enterobacteriaceae were isolated from 6 infants, 3 had strains also present in babies who did not develop NEC. We have shown that colonisation of the upper small bowel with Enterobacteriaceae is common in babies on a neonatal unit and does not appear to predispose to NEC.
As a part of a molecular epidemiological study of Shigda sonnet from the Northwest of England during 1994- 1995, the mechanisms of the pathogenicity were investigated by several molecular techniques Four different genes responsible for the virulence of Shigda sonnei were studied by polymerase chain reaction (PCR) and DNA probe hybridization, Most clinical isolates of Shigella sonnei were positive with PCR detection of the 4 genes; virF, IpaB, IpaH, and kcpA. Several oligonucleotide primers were optimally designed for the PCR reactions. IpaH and kcpA genes were found from all isolates and both were shown as conserved genes. The base sequences of the PCR products were determined and shown to represent the known sequences of part or all of these genes. PCR products of at least 500 bp were then used as the DNA probes. The DNA probe hybridization revealed that IpaH genes had multiple copies on both chromosome and on a plasmid of molecular size 120 MDa. Both virF and IpaB genes were also found on the same virulence plasmid with the IpaH gene, while the kcpA gene was found on the chromosome only. The study so far has demonstrated the utility of PCR and hybridization techniques in detecting major virulence genes of Shigellae, which may contribute to a better understanding of the eoidemiologv and pathogenicity of this enteric pathogen.
PRODUCTION OF DIARRHOEAL TOXIN BY BACILLUS SPECIES IN HOSPITAGPREPARED INFANT FEEDS N. J. Rowan.’ I. G. Anderson,’ C. G. Gemmell,2 and K. Deans.’ Department of Bioswznce & Biotechnology,’ University of Stmthclyde and Department of Bacteriology,’ University of Glasgow, Scotland. Twenty-four pastewised infant feeds prepared m a Glasgow hospital were examined micmbiologically. All exhibited a satisfactory total aerobic mesophilic count of < 1.0~10~ cfu g-’ (mean 6.3~10’ cfu g-l) within one hour of preparation. Bacillus cereus was detected in 2 infant feeds immediately afta preparation and one of these had a B. cereus count of 1.4x103 cfu g-’ exceeding the recommended safety limit of < 1.0x1 0’ cfu g-l. Subsequent storage over a 14 h period at 25°C or greater resulted in the appearance of 5. cereus in a further 8 feeds, the majority of which exceeded the safety limit of 10’ cfu g.‘. The microbiological quality of each mfant feed depended on the type and number of organisms initially present and on the temperature and duration of storage. Incubation of feeds at 5lO’C for 14 hours did not alter the micmbiological quality (P < 0.001). While b’. lichenijbmis and B. m&i/is were the predominant organisms isolated within 8 hours of incubation (45.8% and 20.8% of feeds respectively), additional storage resulted in the emergence of B. cereus I (25%) and II (20.8%) as dominant Bacillus spp. The addition of glucose polymers and other supplements to infant formulae did not affect the type and number of organisms present (7 < 0.001). Diarrhoeal entemtoxin &as detected in 3 and 1 formulations which supported the growth of B. cereus II and B. licbenifimis respectively via the BCET-RPLA system. Detection of dwnhceal enterotoxin in an infant feed containing high numbers of B. lichmifonnis (-10’ cti g-l) is surprising, as members of this genus (other than B. anfhracis and 8. cereus) are currently considered to be ‘inconsequential’ and of little clinical significance. Although the infant feeds were of similar micmbiological quality (P < O.OOl), the majority of Bacillus spp. isolated have teen recently implicated in either food-borne illnesses and/or ommxtunist infections.
RANDOM MUTAGENESIS OF THE HELZCOBACTER GENOME. P. J. Jenks and A. Labigne, Unite de Pathogtnie Bact&ienne des Muqueuses, Pasteur Institute, Paris, France. PYLORI
The aim of this study was to establish and validate a random mutagenesis system for H. pylon’. Methods. 2kb fragments of H. pylon’ chromosomal DNA were cloned and transformed into Escherichia coli. Shuttle mutagenesis was performed in 96-well format using the MiniTn3Km transposable element. Pooled constructs were transformed into H. pylori for homologous recombination. Resultant kanamycin-resistant colonies were screened for urease-negative and metronidazole-resistant phenotypes. Results. 600 mutants were constructed. Southern hybridisation with a Km probe confirmed the random nature of Tn3Km insertion. Three urease-negative and six metronidazole-resistant colonies were obtained. The urease-negative mutants were identified as containing mutations in the weE (2) and urel genes. Confirmation of the role of the genes conferring metronidazole resistance is currently in process. Conclusion. Random mutagenesis allows the determination of the function of unknown genes in the H. pylon’ genome. Novel genes Involved in resistance to metronidazole have been identified. The system is currently being adapted for the analysis of pooled mutants in an H. pylori mouse model.
All
MOLECULAR PATHOGENESIS of Shigella sonnei. A.Tuanvok .and P. Shears, Centre for Tropical Medical Microbiology, University of Liverpool, Liverpool, UK
As part of a study of the microbial ecology of necrotising enterocolitis (NEC), we investigated small bowel colonisation in infants weighing 115OOg at birth. Small bowel contents were sampled by blind aspiration at routine naso-gastric tube replacement. Samples were cultured quantitatively and Enterobacteriaceae typed by REP-PCR and pulsed field gel electrophoresis. Over 17 months, 422 aspirates were collected from 122 babies. 108 samples were sterile, 150 yielded Enterobacteriaceae, 158 Gram-positive organisms and 6 yeasts. 61 infants (50%) had Enterobacteriaceae in 11 sample with counts from 53 cfu/g to 1.5 x lo* cfu/g. Molecular typing showed that infants remained colonised with a particular strain for up to 9 weeks. There was evidence of cross-colonisation within the unit with indistinguishable strains isolated from multiple babies, including an E. coli isolated from 11 infants. 23 episodes of NEC occurred during the study. Small bowel aspirates were collected up to 9 days prior to onset in 12 episodes. Enterobacteriaceae were isolated from 6 infants, 3 had strains also present in babies who did not develop NEC. We have shown that colonisation of the upper small bowel with Enterobacteriaceae is common in babies on a neonatal unit and does not appear to predispose to NEC.
As a part of a molecular epidemiological study of Shigda sonnet from the Northwest of England during 1994- 1995, the mechanisms of the pathogenicity were investigated by several molecular techniques Four different genes responsible for the virulence of Shigda sonnei were studied by polymerase chain reaction (PCR) and DNA probe hybridization, Most clinical isolates of Shigella sonnei were positive with PCR detection of the 4 genes; virF, IpaB, IpaH, and kcpA. Several oligonucleotide primers were optimally designed for the PCR reactions. IpaH and kcpA genes were found from all isolates and both were shown as conserved genes. The base sequences of the PCR products were determined and shown to represent the known sequences of part or all of these genes. PCR products of at least 500 bp were then used as the DNA probes. The DNA probe hybridization revealed that IpaH genes had multiple copies on both chromosome and on a plasmid of molecular size 120 MDa. Both virF and IpaB genes were also found on the same virulence plasmid with the IpaH gene, while the kcpA gene was found on the chromosome only. The study so far has demonstrated the utility of PCR and hybridization techniques in detecting major virulence genes of Shigellae, which may contribute to a better understanding of the eoidemiologv and pathogenicity of this enteric pathogen.
PRODUCTION OF DIARRHOEAL TOXIN BY BACILLUS SPECIES IN HOSPITAGPREPARED INFANT FEEDS N. J. Rowan.’ I. G. Anderson,’ C. G. Gemmell,2 and K. Deans.’ Department of Bioswznce & Biotechnology,’ University of Stmthclyde and Department of Bacteriology,’ University of Glasgow, Scotland. Twenty-four pastewised infant feeds prepared m a Glasgow hospital were examined micmbiologically. All exhibited a satisfactory total aerobic mesophilic count of < 1.0~10~ cfu g-’ (mean 6.3~10’ cfu g-l) within one hour of preparation. Bacillus cereus was detected in 2 infant feeds immediately afta preparation and one of these had a B. cereus count of 1.4x103 cfu g-’ exceeding the recommended safety limit of < 1.0x1 0’ cfu g-l. Subsequent storage over a 14 h period at 25°C or greater resulted in the appearance of 5. cereus in a further 8 feeds, the majority of which exceeded the safety limit of 10’ cfu g.‘. The microbiological quality of each mfant feed depended on the type and number of organisms initially present and on the temperature and duration of storage. Incubation of feeds at 5lO’C for 14 hours did not alter the micmbiological quality (P < 0.001). While b’. lichenijbmis and B. m&i/is were the predominant organisms isolated within 8 hours of incubation (45.8% and 20.8% of feeds respectively), additional storage resulted in the emergence of B. cereus I (25%) and II (20.8%) as dominant Bacillus spp. The addition of glucose polymers and other supplements to infant formulae did not affect the type and number of organisms present (7 < 0.001). Diarrhoeal entemtoxin &as detected in 3 and 1 formulations which supported the growth of B. cereus II and B. licbenifimis respectively via the BCET-RPLA system. Detection of dwnhceal enterotoxin in an infant feed containing high numbers of B. lichmifonnis (-10’ cti g-l) is surprising, as members of this genus (other than B. anfhracis and 8. cereus) are currently considered to be ‘inconsequential’ and of little clinical significance. Although the infant feeds were of similar micmbiological quality (P < O.OOl), the majority of Bacillus spp. isolated have teen recently implicated in either food-borne illnesses and/or ommxtunist infections.
RANDOM MUTAGENESIS OF THE HELZCOBACTER GENOME. P. J. Jenks and A. Labigne, Unite de Pathogtnie Bact&ienne des Muqueuses, Pasteur Institute, Paris, France. PYLORI
The aim of this study was to establish and validate a random mutagenesis system for H. pylon’. Methods. 2kb fragments of H. pylon’ chromosomal DNA were cloned and transformed into Escherichia coli. Shuttle mutagenesis was performed in 96-well format using the MiniTn3Km transposable element. Pooled constructs were transformed into H. pylori for homologous recombination. Resultant kanamycin-resistant colonies were screened for urease-negative and metronidazole-resistant phenotypes. Results. 600 mutants were constructed. Southern hybridisation with a Km probe confirmed the random nature of Tn3Km insertion. Three urease-negative and six metronidazole-resistant colonies were obtained. The urease-negative mutants were identified as containing mutations in the weE (2) and urel genes. Confirmation of the role of the genes conferring metronidazole resistance is currently in process. Conclusion. Random mutagenesis allows the determination of the function of unknown genes in the H. pylon’ genome. Novel genes Involved in resistance to metronidazole have been identified. The system is currently being adapted for the analysis of pooled mutants in an H. pylori mouse model.
All