Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity
Helen MorcretteA. Kovacs-SimonRichard TennantJohn LoveSariqa WagleyZheng Rong YangDavid J. StudholmeOrkun S. SoyerOlivia L. ChampionClive S. ButlerRichard W. Titball
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Abstract:
The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10-3 after exposure to 100 x MIC of penicillin G for 24 hours. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this is a consequence of over-expressed redox protein activity in, or associated with, the electron transport chain. The metabolic burst in penicillin G treated C. jejuni cells could lead to limited reactive oxygen species (ROS) formation that might play a role in persister cell formation.Keywords:
Multidrug tolerance
Penicillin binding proteins
Detection and genotyping of Campylobacter jejuni and Campylobacter coli in food samples by fla-DGGE.
The aim of this study was to apply a denaturing gradient gel electrophoresis assay of the flagellin gene(fla-DGGE) for detection and genotyping of Campylobacter jejuni and Campylobacter coli in food samples without prior cμLtivation.RBB(rpeated bead beating),CTAB and acetone-chloroform method were used to extracted DNA of all the samples,and then fla-DGGE was applied.The resμLts of the three methods were the same.10 samples,8 were with Campylobacter jejuni or Campylobacter coli,which 3 samples contaminated by two or more of the Campylobacter jejuni or Campylobacter coli,or their different types.After cloning and sequencing resμLts showed that 7 samples were contaminated with the same Campylobacter jejuni,including 3 samples also have the same type of Campylobacter coli,a serious pollution samples were detected to contain one Campylobacter coli and three types of Campylobacter jejuni.Fla-DGGE method is rapid,accurate,and sensitive and could be used in food in Campylobacter jejuni and Campylobacter coli in the rapid detection and typing.
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Earlier studies have shown that the highly penicillin-resistant South African Strains of pneumococci contain altered penicillin-binding proteins (PBPs) (S. Zighelboim and A. Tomasz, Antimicrob. Agents Chemother. 17:434-442, 1980). We now describe a detailed quantitative characterization of the reaction of radioactively labeled penicillin with the PBPs of the penicillin-susceptible and penicillin-resistant pneumococci and several intermediate-resistance-level genetic transformants as well. The altered binding of the antibiotic by the PBPs of resistant cells appears to be due to a combination of two factors: lower drug affinity and change in the cellular amounts of PBPs. No alteration in the rates of deacylation of the penicilloyl-PBPs of the resistant cells was detected.
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We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 micrograms/ml; range, 0.1 to 0.7 micrograms/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, less than or equal to 0.06 micrograms/ml) for comparison. The resistant strains did not produce detectable beta-lactamase activity, otherwise modify penicillin G, or bind less total penicillin. Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3.
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This chapter explores the contribution that population studies have made to one's understanding of the biology of the Campylobacter, and the authors argue that such studies have a central role to play in understanding the epidemiology and pathogenesis of this important group of gram-negative bacteria. Campylobacter jejuni and Campylobacter coli cause the majority of human cases of Campylobacter-associated gastroenteritis; these two organisms are associated with approximately 90 and 10% of cases, respectively. This chapter discusses variation within the genus Campylobacter. Although human infection is one of the most important practical applications of studies of Campylobacter populations, in terms of Campylobacter population dynamics and evolution, infection is probably irrelevant. Understanding the population biology of Campylobacter is, however, crucial in understanding the transmission to humans and developing means for its control. It is instructive to reflect that the first study of C. jejuni and C. coli population structure by multilocus enzyme electrophoresis provided many insights that have proved to be correct and that have been extended and deepened by multilocus sequence typing (MLST) studies. Ongoing nucleotide sequence-based studies involving large numbers of isolates and improved genealogical analysis tools provide the highly attractive prospect that well within the next 10 years, the population biology of these organisms, at least insofar as it relates to human infection, will be effectively resolved.
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Campylobacter coli
Molecular Epidemiology
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Backgrounds: As zoonotic infectious agents, Campylobacter spp. are important factors causing gastroenteritis in humans. Surveys show that the three strains; Campylobacter jejuni, Campylobacter coli and Campylobacter fetus play a major role in human infections. Identification of these infectious agents is valuable for sanitary control of disease transmission through water resources.
Objectives: The aim of this study was identification and molecular diagnosis of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus in surface waters in Rasht.
Materials and Methods: This cross-sectional study was conducted on 45 samples of surface water in Rasht collected according to water health guidelines. After culture and biochemical tests on collected samples, detection and identification of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus was done using sequence-specific amplification by Multiplex PCR. The results were subjected to statistical analysis using SPSS software.
Results: Out of 45 samples tested, 6 were positive in culture, four of which were identified as Campylobacter jejuni after biochemical tests. Using Multiplex PCR, 8 samples were positive, from which 3 were Campylobacter jejuni, 1 Campylobacter coli and 4 were positive for both Campylobacter jejuni and Campylobacter coli. All the samples did not yield C. fetus.
Conclusions: Multiplex PCR is regarded a diagnostic method with higher sensitivity and specificity than compared to methods for Campylobacter. The prevalence of Campylobacter jejuni and Campylobacter coli in surface waters in Rasht is considerable. Therefore, public health measures for the control of these organisms are recommended.
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A large number of pneumococcal isolates (over 80 strains) from a variety of geographic locales and representing a spectrum of resistance levels from a penicillin MIC of 0.003 microgram/ml up to an MIC of 16 micrograms/ml were analyzed for their penicillin-binding protein (PBP) patterns. With a few exceptions, the great majority of strains with penicillin MICs up to about 0.05 microgram/ml contained the same set of five PBPs with molecular sizes typical of those of susceptible pneumococci. In strains with penicillin MICs of about 0.1 microgram/ml and up, virtually all isolates showed two common features: (i) all isolates showed loss of PBP 1A (98 kilodaltons) with or without a parallel appearance of a "new" PBP that ranged in molecular size between 96 and 97 kilodaltons; and (ii) in strains with penicillin MICs of 0.5 microgram/ml or more, PBP 2B could not be detected on the fluorograms even with very high concentrations of radioactive penicillin. Beyond these two common features, resistant strains with similar penicillin MICs showed a surprising variety of PBP profiles (i.e., in the number and molecular sizes of PBPs), each characteristic of a given isolate. We suggest that in pneumococci remodeling of critical PBPs in more than one way may result in comparable levels of penicillin resistance.
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Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are regarded as a major cause of human gastrointestinal disease, commonly acquired by eating undercooked chicken. We describe a PCR-ELISA for the detection of Campylobacter species and the discrimination of C. jejuni and C. coli in poultry samples. The PCR assay targets the 16S/23S ribosomal RNA intergenic spacer region of Campylobacter species with DNA oligonucleotide probes designed for the specific detection of C. jejuni, C. coli, and Campylobacter species immobilized on NucleoLink™ wells and hybridized to PCR products modified with a 5′ biotin moiety. The limit of detection of the PCR-ELISA was 100–300 fg (40–120 bacterial cells) for C. jejuni and C. coli with their respective species-specific oligonucleotide probes and 10 fg (4 bacterial cells) with the Campylobacter genus-specific probe. Testing of poultry samples, which were presumptive positive for Campylobacter following culture on the Malthus V analyzer, with the PCR-ELISA determined Campylobacter to be present in 100% of samples (n = 40) with mixed cultures of C. jejuni/C. coli in 55%. The PCR-ELISA when combined with culture pre-enrichment is able to detect the presence of Campylobacter and definitively identify C. jejuni and C. coli in culture-enriched poultry meat samples.
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1. Campylobacter Infection. 2. Taxonomy, Phylogeny, and Methods for the Identification of Campylobacter Species. 3. Population Genetics of Campylobacter jejuni. 4. Campylobacter jejuni Strain Variation. 5. Advances in Campylobacter jejuni Comparative Genomics Through Whole Genome DNA Microarrays. 6. Prevalence of Campylobacter in the Food and Water Supply: Incidence, Outbreaks, Isolation, and Detection. 7. New Methods for Epidemiological Analysis of Campylobacter jejuni. 8. Plasmids of Campylobacter jejuni 81-176. 9. Mechanisms of Antimicrobial Resistance in Campylobacter. 10. Multidrug Efflux Systems in Campylobacter. 11. Genetic Bases for the Variation in the Lipooligosaccharide Outer Core of Campylobacter jejuni and Possible Association of Glycosyltransferase Genes with Post-Infectious Neuropathies. 12. The Polysaccharide Capsule of Campylobacter jejuni. 13. Protein Glycosylation in Campylobacter. 14. Metabolism, Electron Transport and Bioenergetics of Campylobacter jejuni: Implications for Understanding Life in the Gut and Survival in the Environment. 15. Iron Transport and Regulation. 16. Campylobacter jejuni Stress Responses During Survival in the Food Chain and Colonisation.17. Motility. 18. Campylobacter Chemotaxis. 19. Invasion. 20. Cytolethal Distending Toxin. 21. Interactions of C. jejuni with Non-professional Phagocytic Cells. 22. Campylobacter jejuni Interactions with Professional Phagocytes. 23. Campylobacter spp and the Ability to Elicit Intestinal Inflammatory Responses. 24. Campylobacter jejuni Interaction with Enterocytes - Using Host Gene Expression Analysis to Unravel.
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Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C . jejuni and C . coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD- based qPCR assay to detect both C . jejuni and C . coli . The rpsKD- based qPCR assay identified 23% more C . jejuni/ C . coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C . jejuni and or C . coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.
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We report a new molecular technique for the analysis of Campylobacter jejuni and Campylobacter coli based on the separation of target fragments by denaturing high-performance liquid chromatography(DHPLC).A multiplex PCR(mPCR) assay for detection of Campylobacter jejuni and Campylobacter coli was developed using primers that specifically amplify segments of the 16S rRNA,gyrA and cdt genes.The mPCR had a detection limit of 10 pg/μL genomic DNA for Campylobacter jejuni and 1 pg/μL for Campylobacter coli.No cross-reaction was detected with non-thermo-tolerant Campylobacter and other 22 strains tested.The assay could detect 1.5 CFU/mL Campylobacter jejuni and Campylobacter coli in artificial inoculated chicken meat sample after enrichment at 42 ℃ in microaerophilic condition for 24 h.Test on 172 chicken meat samples detected 18 Campy-lobacter jejuni positive and 7 Campylobacter coli positive.These results indicated that the multiplex PCR-DHPLC assay could be used for specific detection of Campylobacter jejun and Campylobacter coli.
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