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An evaluation of escherichia coli type 1 fimbriae regulation in bacteriuria of pregnancy
FIS 98 Abstracts
Al2 RESISTANCE
TO
HELICOBACTER
PYLORZ
METROMDAZOLE IN AND ITS EFFECT ON THE EFFICACY OF ERADICATION THERAPY. P. J. Jenks, A. Labigne and R. Ferrero, UPBM, Pasteur Institute, Paris, France. The H. pylori SSl mouse model was used; 1) to characterise the development of resistance in H. pylori after treatment with metronidazole (mk), and 2) to examine the impact of prior exposure of H. pyZori to mk on the efficacy of mk-containing eradication regimens. Methods. Mice colonised with the mtz-sensitive SSl H. pylori strain were treated with various combinations of mk monotherapy and a mk-containing eradication regimen. One month after completion of therapy, the animals were sacrificed and their stomachs cultured. Susceptibility to mk was assessed by determination of the MIC. Results. After completion of treatment, 70% of mice treated with mk monotherapy harbourcd resistant strains. In all cases, these were mixed with mtz-sensitive isolates, with a ratio of resistant to sensitive isolates of 1:lOO. The MIC of resistant isolates varied from 8 to 64 mgil. H. pylori was eradicated from 25% of mice that received mk monotherapy followed by eradication therapy, compared to 70% mice not pre-treated
with this antibiotic (p < 0.01). In the former group, the ratio of Conclusions.
resistant
to sensitive isolates was 1:25. Mk monotherapy readily induces resistance in H. pylon’. Treatment of a mk-sensitive strain of H. pylon’ with mk results in the development of a mixed population of bacteria with varying degrees of susceptibility to mtz. Repeated exposure to mk in vivo selects for a resistant population of H. pylori within the stomach. Prior use of mk significantly reduces the efficacy of mk-containing eradication regimens.
AN EVALUATION OF ESCHERICHIA COLI TYPE 1 FIMBRIAE REGULATION IN BACTERIURIA OF PREGNANCY. J. Clive Graham, Julian B.S. Leathart, David L Gaily. SMIVS, University of Newcastle, Newcastle upon Tyne, UK. Forty-nine E. coli isolated from a clinic for bacteriuria of pregnancy were analysed for type 1 fimbriae, a phase variable adhesin. Presence of this adhesin increases the infectivity of E coli in animal models however an afimbriated strain is more virulent when administered by the haematogenous route. This suggests that phase variation (controlled by a short DNA segment known as the PM switch) may allow E. coli to adapt to different ecological mches. The orientation of the@ switch was determined “in viva” in a fresh mid-stream urine sample taken from women referred to the clinic. Isolates from the urine were also grown under standard laboratory conditions and thefim switch orientation determined. Analysis revealed that 8 of the 49 strams had an extra T allele, which was only seen in 2 isolates from 53 strains obtained from other patient groups (p
CD8+ OF CONSERVED, IDENTIFICATION CYTOTOXIC T CELL EPITOPES IN ESAT-6, A TUBERCULOSIS VACCINE CANDIDATE. A. Pathan’ G. Pasvo?,
R. Brookes’, A. Hill’
H. Pritchard’, R. Wilkinson*, & A. Lalvani’. ‘Nuffield
Department of Clinical Medicine, John Radcliffe Hospital, Oxford & ‘Department of Infection and Tropical Medicine, Imperial College, London. Animal models of tuberculosis point to a protective role for CD8+ cytotoxic T lymphocytes (CTL) and have identified ESAT-6 as a potential tuberculosis vaccine candidate. Since induction of HLA class I-restricted CTZ
by
vaccination
is now
feasible
in
man,
it
is
important to determine to what extent ESAT-6 is a target of CD8+ CTL in naturally infected humans. Using a sensitive ex vivo ELISPOT assay for interferon-y (IFNr), we previously identified the first HLA class Irestricted CD8+ epitope in Mycobacterium tuberculosis. Extending this approach to larger numbers of patients and contacts, we have identified further CD8+ T cell epitopes in ESAT-6. CTL lines specific for these novel epitopes displayed cytolytic activity and IFN-y secretion, while T cells freshly isolated from peripheral blood secreted IFN-y within 12-hrs of antigen contact. These circulating effector CD8+ CTL were detectable in patients and healthy contacts at frequencies of up to 11500 peripheral blood lymphocytes. The delineation of several conserved CDS+ epitopes in ESAT-6, restricted through a wide range of HLA-A and -B alleles, identifies this antigen as a major target of CD8+ CI’L in human tuberculosis and provides a rational basis for the development of a CTL-inducing subunit vaccine incorporating ESAT-6.
PLEOMORPHIC CULTURE FORMS OR LISTERIA MONOCYTOGEA’ES (SEROVAR 4b) DIFFER IN MORPHOLOGY, ENZYMOLOGY, ANTIGEMCITY AND HEAT TOLERANCE. NJ Rowan, JG And-n, AA Candtish, K Deans, Department of Bioscience and Biotechnology, University of Stmthclyde, Glasgow Gl
lXW, Scotland. Three @ical coccobacilli cell forms (designated Smooth or S-forms) of monoqagenes semvar 4b, obtained I?om the National Collection of Type Cultures, Colindale, UK (i.e. NCTC 9663, NCTC 11994, NCTC
Listoio
4885) and reported previously
as the aetiological agents in infantile
meningitis, were examined for their ability to tolerate sublethal heating in reconstituted infant miIk formulae. We here report on the existence of atypical cell forms (designated as Rough or R-forms) consisting pmkmindy of long filamento~ chains (up to 60 )un in length -pared to 2 pm for the S-form) which were generated fkom the aforementioned parent S-forms on Tryptone Soya Yeast Extnct Agar and Listaia Selective Agar (Oxoid) within 24 h of heal treatment and plating. R-forms produced tiegdar colonies, which were uncharacteristic of typical S-forms, and were stable on repeated subculture. R-forms did not give characteristic Hemi illumination or tumbling motility tests, but retained the ability to grow at psychrotrophic temperatures, gave positive reactions for the CAMP test and
were successfully identified via the AF’I List&a test kit (API Biomerieux). Subsequent biochemical assays, using the AF’I 50 CH galleries, revealed however that the R-forms differed en~ymologically by losing their ability to produce N-acetyl+&osamidase. Immunoassay studies showed that the R-forms differed markedly in mtigenicity tiere the lower antisera dilution titre for the S and R-forms was 1116,000 and l/2,000 respectively (via indirect ELISA assays). In addition, the atypical R-forms were consistently
more themmtolenmt when subjected to heating at 56,60 and 63°C. whilst the current commercially available immunological and biochemical diagnostic tests successfully identify both pleomorphic forms of Liztetia monocyrogenes
(semvar 4b), as a collsequence of the aforementioned
changes in morphology and physiology associated with the R-form, it is likely that atypical Listeria forms may ix dismissed as ‘inconsequential contaminants’ by the medical profession and tbe food industry.
Al2 RESISTANCE
TO
HELICOBACTER
PYLORZ
METROMDAZOLE IN AND ITS EFFECT ON THE EFFICACY OF ERADICATION THERAPY. P. J. Jenks, A. Labigne and R. Ferrero, UPBM, Pasteur Institute, Paris, France. The H. pylori SSl mouse model was used; 1) to characterise the development of resistance in H. pylori after treatment with metronidazole (mk), and 2) to examine the impact of prior exposure of H. pyZori to mk on the efficacy of mk-containing eradication regimens. Methods. Mice colonised with the mtz-sensitive SSl H. pylori strain were treated with various combinations of mk monotherapy and a mk-containing eradication regimen. One month after completion of therapy, the animals were sacrificed and their stomachs cultured. Susceptibility to mk was assessed by determination of the MIC. Results. After completion of treatment, 70% of mice treated with mk monotherapy harbourcd resistant strains. In all cases, these were mixed with mtz-sensitive isolates, with a ratio of resistant to sensitive isolates of 1:lOO. The MIC of resistant isolates varied from 8 to 64 mgil. H. pylori was eradicated from 25% of mice that received mk monotherapy followed by eradication therapy, compared to 70% mice not pre-treated
with this antibiotic (p < 0.01). In the former group, the ratio of Conclusions.
resistant
to sensitive isolates was 1:25. Mk monotherapy readily induces resistance in H. pylon’. Treatment of a mk-sensitive strain of H. pylon’ with mk results in the development of a mixed population of bacteria with varying degrees of susceptibility to mtz. Repeated exposure to mk in vivo selects for a resistant population of H. pylori within the stomach. Prior use of mk significantly reduces the efficacy of mk-containing eradication regimens.
AN EVALUATION OF ESCHERICHIA COLI TYPE 1 FIMBRIAE REGULATION IN BACTERIURIA OF PREGNANCY. J. Clive Graham, Julian B.S. Leathart, David L Gaily. SMIVS, University of Newcastle, Newcastle upon Tyne, UK. Forty-nine E. coli isolated from a clinic for bacteriuria of pregnancy were analysed for type 1 fimbriae, a phase variable adhesin. Presence of this adhesin increases the infectivity of E coli in animal models however an afimbriated strain is more virulent when administered by the haematogenous route. This suggests that phase variation (controlled by a short DNA segment known as the PM switch) may allow E. coli to adapt to different ecological mches. The orientation of the@ switch was determined “in viva” in a fresh mid-stream urine sample taken from women referred to the clinic. Isolates from the urine were also grown under standard laboratory conditions and thefim switch orientation determined. Analysis revealed that 8 of the 49 strams had an extra T allele, which was only seen in 2 isolates from 53 strains obtained from other patient groups (p
CD8+ OF CONSERVED, IDENTIFICATION CYTOTOXIC T CELL EPITOPES IN ESAT-6, A TUBERCULOSIS VACCINE CANDIDATE. A. Pathan’ G. Pasvo?,
R. Brookes’, A. Hill’
H. Pritchard’, R. Wilkinson*, & A. Lalvani’. ‘Nuffield
Department of Clinical Medicine, John Radcliffe Hospital, Oxford & ‘Department of Infection and Tropical Medicine, Imperial College, London. Animal models of tuberculosis point to a protective role for CD8+ cytotoxic T lymphocytes (CTL) and have identified ESAT-6 as a potential tuberculosis vaccine candidate. Since induction of HLA class I-restricted CTZ
by
vaccination
is now
feasible
in
man,
it
is
important to determine to what extent ESAT-6 is a target of CD8+ CTL in naturally infected humans. Using a sensitive ex vivo ELISPOT assay for interferon-y (IFNr), we previously identified the first HLA class Irestricted CD8+ epitope in Mycobacterium tuberculosis. Extending this approach to larger numbers of patients and contacts, we have identified further CD8+ T cell epitopes in ESAT-6. CTL lines specific for these novel epitopes displayed cytolytic activity and IFN-y secretion, while T cells freshly isolated from peripheral blood secreted IFN-y within 12-hrs of antigen contact. These circulating effector CD8+ CTL were detectable in patients and healthy contacts at frequencies of up to 11500 peripheral blood lymphocytes. The delineation of several conserved CDS+ epitopes in ESAT-6, restricted through a wide range of HLA-A and -B alleles, identifies this antigen as a major target of CD8+ CI’L in human tuberculosis and provides a rational basis for the development of a CTL-inducing subunit vaccine incorporating ESAT-6.
PLEOMORPHIC CULTURE FORMS OR LISTERIA MONOCYTOGEA’ES (SEROVAR 4b) DIFFER IN MORPHOLOGY, ENZYMOLOGY, ANTIGEMCITY AND HEAT TOLERANCE. NJ Rowan, JG And-n, AA Candtish, K Deans, Department of Bioscience and Biotechnology, University of Stmthclyde, Glasgow Gl
lXW, Scotland. Three @ical coccobacilli cell forms (designated Smooth or S-forms) of monoqagenes semvar 4b, obtained I?om the National Collection of Type Cultures, Colindale, UK (i.e. NCTC 9663, NCTC 11994, NCTC
Listoio
4885) and reported previously
as the aetiological agents in infantile
meningitis, were examined for their ability to tolerate sublethal heating in reconstituted infant miIk formulae. We here report on the existence of atypical cell forms (designated as Rough or R-forms) consisting pmkmindy of long filamento~ chains (up to 60 )un in length -pared to 2 pm for the S-form) which were generated fkom the aforementioned parent S-forms on Tryptone Soya Yeast Extnct Agar and Listaia Selective Agar (Oxoid) within 24 h of heal treatment and plating. R-forms produced tiegdar colonies, which were uncharacteristic of typical S-forms, and were stable on repeated subculture. R-forms did not give characteristic Hemi illumination or tumbling motility tests, but retained the ability to grow at psychrotrophic temperatures, gave positive reactions for the CAMP test and
were successfully identified via the AF’I List&a test kit (API Biomerieux). Subsequent biochemical assays, using the AF’I 50 CH galleries, revealed however that the R-forms differed en~ymologically by losing their ability to produce N-acetyl+&osamidase. Immunoassay studies showed that the R-forms differed markedly in mtigenicity tiere the lower antisera dilution titre for the S and R-forms was 1116,000 and l/2,000 respectively (via indirect ELISA assays). In addition, the atypical R-forms were consistently
more themmtolenmt when subjected to heating at 56,60 and 63°C. whilst the current commercially available immunological and biochemical diagnostic tests successfully identify both pleomorphic forms of Liztetia monocyrogenes
(semvar 4b), as a collsequence of the aforementioned
changes in morphology and physiology associated with the R-form, it is likely that atypical Listeria forms may ix dismissed as ‘inconsequential contaminants’ by the medical profession and tbe food industry.