scholarly journals Epidemiology, Relative Invasive Ability, Molecular Characterization, and Competitive Performance of Campylobacter jejuni Strains in the Chicken Gut

2007 ◽  
Vol 73 (24) ◽  
pp. 7959-7966 ◽  
Author(s):  
Christopher Pope ◽  
Janet Wilson ◽  
Eduardo N. Taboada ◽  
Joanne MacKinnon ◽  
Cristiano A. Felipe Alves ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Dna Microarrays ◽  
Capsular Polysaccharide ◽  
Geographical Area ◽  
Human Infection ◽  
Poultry Meat ◽  
Vitro Assay ◽  
Real Time Quantitative Pcr ◽  
Common Time

ABSTRACT One hundred forty-one Campylobacter jejuni isolates from humans with diarrhea and 100 isolates from retailed poultry meat were differentiated by flaA typing. The bacteria were isolated in a specific geographical area (Dunedin) in New Zealand over a common time period. Twenty nine flaA types were detected, one of which (flaA restriction fragment length polymorphism type 15 [flaA-15]) predominated among isolates from humans (∼30% of isolates). This strain was of low prevalence (5% of isolates) among poultry isolates. flaA-15 strains were five to six times more invasive of HEp2 cells in an in vitro assay than a flaA type (flaA-3) that was commonly encountered on poultry meat (23% of isolates) but was seldom associated with human illness (5%). Competitive-exclusion experiments with chickens, utilizing real-time quantitative PCR to measure the population sizes of specific strains representing flaA-15 (T1016) and flaA-3 (Pstau) in digesta, were carried out. These experiments showed that T1016 always outcompeted Pstau in the chicken intestine. Genomic comparisons of T1016 and Pstau were made using DNA microarrays representing the genome of C. jejuni NCTC 11168. These comparisons revealed differences between the strains in the gene content of the Cj1417c-to-Cj1442c region of the genome, which is associated with the formation of capsular polysaccharide. The strains differed in Penner type (T1016, O42; Pstau, O53). It was concluded that poultry meat was at least one source of human infection with C. jejuni, that some Campylobacter strains detected in poultry meat are of higher virulence for humans than others, and that bacterial attributes affecting strain virulence and commensal colonization ability may be linked.

scholarly journals Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens

2005 ◽  
Vol 73 (5) ◽  
pp. 3053-3062 ◽  
Author(s):  
Manal AbuOun ◽  
Georgina Manning ◽  
Shaun A. Cawthraw ◽  
Anne Ridley ◽  
If H. Ahmed ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Human Infection ◽  
Site Directed Mutagenesis ◽  
Cytolethal Distending Toxin ◽  
Vitro Assay ◽  
Reverse Transcription Pcr ◽  
Human Sera

ABSTRACT The cytolethal distending toxin (CDT) of Campylobacter jejuni was detectable, using an in vitro assay, in most but not all of 24 strains tested. The reason for the absence of toxin activity in these naturally occurring CDT-negative C. jejuni strains was then investigated at the genetic level. CDT is encoded by three highly conserved genes, cdtA, -B, and -C. In the CDT-negative strains, two types of mutation were identified. The CDT activities of C. jejuni strains possessing both types of mutation were successfully complemented with the functional genes of C. jejuni 11168. The first type of mutation comprised a 667-bp deletion across cdtA and cdtB and considerable degeneration in the remainder of the cdt locus. Using a PCR technique to screen for this deletion, this mutation occurred in fewer than 3% of 147 human, veterinary, and environmental strains tested. The second type of mutation involved at least four nonsynonymous nucleotide changes, but only the replacement of proline with serine at CdtB position 95 was considered important for CDT activity. This was confirmed by site-directed mutagenesis. This type of mutation also occurred in fewer than 3% of strains as determined using a LightCycler biprobe assay. The detection of two CDT-negative clinical isolates raised questions about the role of CDT in some cases of human campylobacteriosis. To determine if anti-CDT antibodies are produced in human infection, a toxin neutralization assay was developed and validated using rabbit antisera. Pooled human sera from infected patients neutralized the toxin, indicating expression and immunogenicity during infection. However, no neutralizing antibodies were detected in colonized chickens despite the expression of CDT in the avian gut as indicated by reverse transcription-PCR.

scholarly journals CmeR Functions as a Pleiotropic Regulator and Is Required for Optimal Colonization of Campylobacter jejuni In Vivo

2008 ◽  
Vol 190 (6) ◽  
pp. 1879-1890 ◽  
Author(s):  
Baoqing Guo ◽  
Ying Wang ◽  
Feng Shi ◽  
Yi-Wen Barton ◽  
Paul Plummer ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Capsular Polysaccharide ◽  
Efflux Pump ◽  
Loss Of Function ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Reverse Transcription Pcr ◽  
Dicarboxylate Transport

ABSTRACT CmeR functions as a transcriptional repressor modulating the expression of the multidrug efflux pump CmeABC in Campylobacter jejuni. To determine if CmeR also regulates other genes in C. jejuni, we compared the transcriptome of the cmeR mutant with that of the wild-type strain using a DNA microarray. This comparison identified 28 genes that showed a ≥2-fold change in expression in the cmeR mutant. Independent real-time quantitative reverse transcription-PCR experiments confirmed 27 of the 28 differentially expressed genes. The CmeR-regulated genes encode membrane transporters, proteins involved in C4-dicarboxylate transport and utilization, enzymes for biosynthesis of capsular polysaccharide, and hypothetical proteins with unknown functions. Among the genes whose expression was upregulated in the cmeR mutant, Cj0561c (encoding a putative periplasmic protein) showed the greatest increase in expression. Subsequent experiments demonstrated that this gene is strongly repressed by CmeR. The presence of the known CmeR-binding site, an inverted repeat of TGTAAT, in the promoter region of Cj0561c suggests that CmeR directly inhibits the transcription of Cj0561c. Similar to expression of cmeABC, transcription of Cj0561c is strongly induced by bile compounds, which are normally present in the intestinal tracts of animals. Inactivation of Cj0561c did not affect the susceptibility of C. jejuni to antimicrobial compounds in vitro but reduced the fitness of C. jejuni in chickens. Loss-of-function mutation of cmeR severely reduced the ability of C. jejuni to colonize chickens. Together, these findings indicate that CmeR governs the expression of multiple genes with diverse functions and is required for Campylobacter adaptation in the chicken host.

scholarly journals Detection and Characterization of Autoagglutination Activity by Campylobacter jejuni

2000 ◽  
Vol 68 (11) ◽  
pp. 6168-6175 ◽  
Author(s):  
Naoaki Misawa ◽  
Martin J. Blaser
Keyword(s):  
Campylobacter Jejuni ◽  
Natural Transformation ◽  
Host Cells ◽  
Vitro Assay ◽  
Sodium Metaperiodate ◽  
Bacterial Hydrophobicity ◽  
Phosphate Buffered Saline ◽  
Common Cell

ABSTRACT In several gram-negative bacterial pathogens, autoagglutination (AAG) activity is a marker for interaction with host cells and virulence. Campylobacter jejuni strains also show AAG, but this property varies considerably among strains. To examine the characteristics of C. jejuni AAG, we developed a quantitative in vitro assay. For strain 81-176, which shows high AAG, activity was optimal for cells grown for ≤24 h, was independent of growth temperature, and was best measured for cells suspended in phosphate-buffered saline at 25°C for 24 h. AAG activity was heat labile and was abolished by pronase or acid-glycine (pH 2.2) treatment but not by lipase, DNase, or sodium metaperiodate. Strain 4182 has low AAG activity, but extraction with water increased AAG, suggesting the loss of an inhibitor. Strain 6960 has weak AAG with no effect due to water extraction. Our study with clinical isolates suggests that C. jejuni strains may be grouped into three AAG phenotypes. A variant derived from strain 81116 that is flagellate but immotile showed the strong AAG exhibited by the parent strain, suggesting that motility per se is not necessary for the AAG activity. AAG correlated with both bacterial hydrophobicity and adherence to INT407 cells. Mutants which lack flagella (flaA,flaB, and flbA) or common cell surface antigen (peb1A) were constructed in strain 81-176 by natural transformation-mediated allelic exchange. Both AAG activity and bacterial hydrophobicity were abolished in the aflagellate mutants but not the peb1A mutant. In total, these findings indicate that C. jejuni AAG is highly associated with flagellar expression.

scholarly journals Developing Innolysins Against Campylobacter jejuni Using a Novel Prophage Receptor-Binding Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Athina Zampara ◽  
Martine C. Holst Sørensen ◽  
Yilmaz Emre Gencay ◽  
Dennis Grimon ◽  
Sebastian Hougaard Kristiansen ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Receptor Binding ◽  
Capsular Polysaccharide ◽  
Binding Protein ◽  
Log Reduction ◽  
Cell Counts ◽  
Phage Receptor ◽  
Receptor Binding Protein

Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter.

scholarly journals Identification of Novel Vaccine Candidates againstCampylobacterthrough Reverse Vaccinology

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Marine Meunier ◽  
Muriel Guyard-Nicodème ◽  
Edouard Hirchaud ◽  
Alberto Parra ◽  
Marianne Chemaly ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Reverse Vaccinology ◽  
Poultry Meat ◽  
Campylobacter Coli ◽  
The European Union ◽  
Vaccine Candidates ◽  
Vaccine Antigens ◽  
Potential Vaccine

Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union. Human cases are mainly due toCampylobacter jejuniorCampylobacter coli, and contamination is associated with the handling and/or consumption of poultry meat. In fact, poultry constitutes the bacteria’s main reservoir. A promising way of decreasing the incidence of campylobacteriosis in humans would be to decrease avian colonization. Poultry vaccination is of potential for this purpose. However, despite many studies, there is currently no vaccine available on the market to reduce the intestinalCampylobacterload in chickens. It is essential to identify and characterize new vaccine antigens. This study applied the reverse vaccinology approach to detect new vaccine candidates. The main criteria used to select immune proteins were localization, antigenicity, and number of B-epitopes. Fourteen proteins were identified as potential vaccine antigens.In vitroandin vivoexperiments now need to be performed to validate the immune and protective power of these newly identified antigens.

An adapted in vitro assay to assess Campylobacter jejuni interaction with intestinal epithelial cells: Taking into stimulation with TNFα

2018 ◽  
Vol 149 ◽  
pp. 67-72 ◽  
Author(s):  
Ramila Cristiane Rodrigues ◽  
Anne-Lise Pocheron ◽  
Jean-Michel Cappelier ◽  
Odile Tresse ◽  
Nabila Haddad
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Intestinal Epithelial Cells ◽  
In Vitro Assay ◽  
Intestinal Epithelial ◽  
Vitro Assay

scholarly journals 1341. Development of a Series of High-Throughput Screens to Identify Leads for Nontuberculous Mycobacteria Drug Design

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S485-S485
Author(s):  
Sarah McGuffin ◽  
Steven Mullen ◽  
Julie Early ◽  
Tanya Parish
Keyword(s):  
Drug Discovery ◽  
Drug Development ◽  
High Throughput ◽  
Nontuberculous Mycobacteria ◽  
Human Infection ◽  
Attrition Rate ◽  
In Vitro Assays ◽  
Vitro Assay

Abstract Background Nontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex and Mycobacterium abscessus complex, cause significant morbidity and mortality in patients with impaired host immunity or pre-existing structural lung conditions. NTM infections are increasing at an alarming rate worldwide and there is a dearth of progress in regard to the development of efficacious and tolerable drugs to treat such infections. Traditional drug discovery screens do not account for the diverse physiological conditions, microenvironments, and compartments that the bacilli encounter during human infection. In order to help populate the NTM drug pipeline, and explore the disconnect between in vitro activity, in vivo activity, and clinical outcomes, we are developing a high throughput in vitro assay platform that will more closely model the unique infection-relevant conditions encountered by NTM. Methods We are developing and validating a suite of in vitro assays that screen compounds for activity against extracellular planktonic bacteria, extracellular bacteria within biofilms, intracellular bacteria, and nutrient-starved non-replicating bacteria. Results We are using both the smooth and rough morphotypes of M. abscessus and M. avium. We have validated high throughput assays to pharmaceutical standards for replicating and non-replicating M. abscessus. We have also tested a panel of 18 known anti-mycobacterial compounds. Assay development is currently underway to test compounds for activity against NTM in biofilm and inside macrophages as well. Conclusion To enhance hit identification for scaffolds to use as starting points for NTM drug development, focused libraries of compounds that have undergone significant preclinical profiling and/or compounds with known activity against M. tuberculosis (TB) will be screened. Such a “piggyback” approach usurps advances made in TB drug development and leverages them for NTM drug discovery. This will help expedite novel drug development, reduce attrition rate, and offer a shorter route to clinical use as it exploits the prior investment in medicinal chemistry, pharmacology, and toxicology. Disclosures All authors: No reported disclosures.

scholarly journals Survival of Campylobacter jejuni in Waterborne Protozoa

2005 ◽  
Vol 71 (9) ◽  
pp. 5560-5571 ◽  
Author(s):  
W. J. Snelling ◽  
J. P. McKenna ◽  
D. M. Lecky ◽  
J. S. G. Dooley
Keyword(s):  
Drinking Water ◽  
Campylobacter Jejuni ◽  
Acanthamoeba Castellanii ◽  
Tetrahymena Pyriformis ◽  
Water Systems ◽  
In Vitro Assay ◽  
Molecular Techniques ◽  
Vitro Assay ◽  
Planktonic State

ABSTRACT The failure to reduce the Campylobacter contamination of intensively reared poultry may be partially due to Campylobacter resisting disinfection in water after their internalization by waterborne protozoa. Campylobacter jejuni and a variety of waterborne protozoa, including ciliates, flagellates, and alveolates, were detected in the drinking water of intensively reared poultry by a combination of culture and molecular techniques. An in vitro assay showed that C. jejuni remained viable when internalized by Tetrahymena pyriformis and Acanthamoeba castellanii for significantly longer (up to 36 h) than when they were in purely a planktonic state. The internalized Campylobacter were also significantly more resistant to disinfection than planktonic organisms. Collectively, our results strongly suggest that protozoa in broiler drinking water systems can delay the decline of Campylobacter viability and increase Campylobacter disinfection resistance, thus increasing the potential of Campylobacter to colonize broilers.

scholarly journals Campylobacter jejuni Cocultured with Epithelial Cells Reduces Surface Capsular Polysaccharide Expression

2009 ◽  
Vol 77 (5) ◽  
pp. 1959-1967 ◽  
Author(s):  
N. Corcionivoschi ◽  
M. Clyne ◽  
A. Lyons ◽  
A. Elmi ◽  
O. Gundogdu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Capsular Polysaccharide ◽  
Bacterial Species ◽  
Molecular Techniques ◽  
Conditioned Media ◽  
Host Cells ◽  
Proteinase K ◽  
Surface Polysaccharides

ABSTRACT The host cell environment can alter bacterial pathogenicity. We employed a combination of cellular and molecular techniques to study the expression of Campylobacter jejuni polysaccharides cocultured with HCT-8 epithelial cells. After two passages, the amount of membrane-bound high-molecular-weight polysaccharide was considerably reduced. Microarray profiling confirmed significant downregulation of capsular polysaccharide (CPS) locus genes. Experiments using conditioned media showed that sugar depletion occurred only when the bacterial and epithelial cells were cocultured. CPS depletion occurred when C. jejuni organisms were exposed to conditioned media from a different C. jejuni strain but not when exposed to conditioned media from other bacterial species. Proteinase K or heat treatment of conditioned media under coculture conditions abrogated the effect on the sugars, as did formaldehyde fixation and cycloheximide treatment of host cells or chloramphenicol treatment of the bacteria. However, sugar depletion was not affected in flagellar export (fliQ) and quorum-sensing (luxS) gene mutants. Passaged C. jejuni showed reduced invasiveness and increased serum sensitivity in vitro. C. jejuni alters its surface polysaccharides when cocultured with epithelial cells, suggesting the existence of a cross talk mechanism that modulates CPS expression during infection.

scholarly journals Identification ofIn Vivo-Induced Bacterial Proteins during Human Infection with Salmonella enterica Serotype Paratyphi A

2013 ◽  
Vol 20 (5) ◽  
pp. 712-719 ◽  
Author(s):  
Mohammad Murshid Alam ◽  
Lillian L. Tsai ◽  
Sean M. Rollins ◽  
Alaullah Sheikh ◽  
Farhana Khanam ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Human Infection ◽  
Mrna Levels ◽  
Oxidoreductase Activity ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Bacterial Proteins ◽  
Structure Adaptation

ABSTRACTSalmonella entericaserotype Paratyphi A is a human-restricted pathogen and the cause of paratyphoid A fever. Using a high-throughput immunoscreening technique,in vivo-induced antigen technology (IVIAT), we identified 20 immunogenic bacterial proteins expressed in humans who were bacteremic withS. Paratyphi A but not those expressed inS. Paratyphi A grown under standard laboratory conditions. The majority of these proteins have known or potential roles in the pathogenesis ofS. enterica. These include proteins implicated in cell adhesion, fimbrial structure, adaptation to atypical conditions, oxidoreductase activity, proteolysis, antimicrobial resistance, and ion transport. Of particular interest among thesein vivo-expressed proteins wereS. Paratyphi A (SPA)2397, SPA2612, and SPA1604. SPA2397 and SPA2612 are prophage related, and SPA1604 is inSalmonellapathogenicity island 11 (SPI-11). Using real-time quantitative PCR (RT-qPCR), we confirmed increased levels of mRNA expressed by genes identified by IVIAT in a comparison of mRNA levels in organisms in the blood of bacteremic patients to those inin vitrocultures. Comparing convalescent- to acute-phase samples, we also detected a significant increase in the reaction of convalescent-phase antibodies with two proteins identified by IVIAT: SPA2397 and SPA0489. SPA2397 is a phage-related lysozyme, Gp19, and SPA0489 encodes a protein containing NlpC/P60 and cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains. In a previous study utilizing a different approach, we found that transcripts for 11 and 7 of the genes identified by IVIAT were detectable in organisms in the blood of humans in Bangladesh who were bacteremic withS. Paratyphi A andSalmonella entericaserovar Typhi, respectively.S.Paratyphi A antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis and might have diagnostic, therapeutic, or preventive relevance.

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