Identification of differentially temperature regulated virulence factors of Campylobacter jejuni and tested in Galleria mellonella

Background Campylobacter jejuni is the major cause of bacterial gastroenteritis in man, while it is generally regarded as a commensal of the avian gut. Consumption and handling of contaminated poultry meat products are a major risk factor for human infection. The body temperature in man 37°C and chickens 42°C differ markedly, and differential gene regulation and protein expression at different temperatures may in part explain the behaviour in the two hosts. Results We performed proteomics analyses with C. jejuni cells grown at 37°C and 42°C. Q tof analysis was carried out after samples were digested with the Fasp method and peptides were fractionated by strong anion exchanges. Differentially regulated proteins were identified by Mascot and Scaffold analyses. QQQ analysis confirmed that a total of 33 proteins were differentially regulated between 37°C and 42°C. Several upregulated proteins were selected for their corresponding gene knock-out mutants to be tested for their virulence in the Galleria mellonella model. To correlate with other tissue/animal models, the GADH mutant was selected for its reduced ability to colonize chickens. At 37°C, the mutants of Omp50 and GroEL significantly increased virulence; while at 42°C, the mutants of YceI, Omp50, and GADH reduced virulence against Galleria mellonella compared with the wild type strains. Conclusion The results of current and previous studies indicate that GADH is a virulent factor in G. mellonella and a colonization factor in chickens. The workflow of this study may prove a new way to identify stress related virulent factors. The implications of these findings are discussed for pathogenesis in the model and other hosts

Correlation of Anti-Salmonella Antibodies Between Serum and Saliva Samples Collected From Finisher Pigs

Saliva samples obtained by using absorptive devices, can provide an alternative diagnostic matrix to serum for monitoring disease status in pigs. The aim of this study was to investigate the correlation of anti-Salmonella antibodies between serum and saliva samples collected from pigs. Twenty individual paired serum and saliva samples were collected from a single farm. Anti-Salmonella IgG was detected in individual serum samples using a commercial Salmonella ELISA kit, validated for sera. The same kit was used with a protocol modified by extending incubation time and increasing temperature to test individual saliva samples. Anti-Salmonella IgG antibodies in pig saliva were always detected at a lower level than in the matching serum samples. A correlation (rho = 0.66; p = 0.002) and a moderate agreement (K > 0.62 p = 0.003) was found between individual Salmonella IgG in serum and saliva samples. Both correlation and the agreement levels are moderate. The size of this investigation was small, and further studies are necessary to further confirm these findings. The results of this work provide some evidence that saliva samples have the potential to be used for the diagnosis of Salmonella infection in pig farms

Determining antimicrobial susceptibility in Salmonella enterica serovar Typhimurium through whole genome sequencing: a comparison against multiple phenotypic susceptibility testing methods

Background : UK public health organisations perform routine antimicrobial susceptibility tests (ASTs) to characterise the potential for antimicrobial resistance in Salmonella enterica serovars. Genetic determinants of these resistance mechanisms are detectable by whole genome sequencing (WGS), however the viability of WGS-based genotyping as an alternative resistance screening tool remains uncertain. We compared WGS-based genotyping, disk diffusion and agar dilution to the broth microdilution reference AST for 102 Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates across 11 antimicrobial compounds. Results : Genotyping concordance, interpreted using epidemiological cut-offs (ECOFFs), was 89.8% (1007/1122) with 0.83 sensitivity and 0.96 specificity. For seven antimicrobials interpreted using Salmonella clinical breakpoints, genotyping produced 0.84 sensitivity and 0.88 specificity. Although less accurate than disk diffusion (0.94 sensitivity, 0.93 specificity) and agar dilution (0.83 sensitivity, 0.98 specificity), genotyping performance improved to 0.89 sensitivity and 0.97 specificity when two antimicrobials with relatively high very major error rates were excluded (streptomycin and sulfamethoxazole). Conclusions : An 89.8% concordance from WGS-based AST predictions using ECOFF interpretations suggest that WGS would serve as an effective screening tool for the tracking of antimicrobial resistance mechanisms in S. Typhimurium. For use as a standalone clinical diagnostic screen, further work is required to reduce the error rates for specific antimicrobials

A sensitive method for the recovery of Escherichia coli serogroup O55 including Shiga toxin-producing variants for potential use in outbreaks

AIM: Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrhoea, kidney failure and occasionally death. However, identifying the source of infection caused by STEC other than serogroup O157 is hampered by availability of sensitive methods for detecting these pathogens. In this study we developed novel tools for detecting E. coli O55 that is potentially associated with human outbreaks. METHOD AND RESULTS: Overall specificity of immuno-magnetic separation (IMS) beads coated with anti-O55 serum was good with exception of cross reactivity with E. coli O22 and O23, which was eliminated using an O55 specific PCR. Limit of detection for E. coli O55 using O55-IMS-beads in spiked cattle faeces was on average 50 CFU ml-1 (range 1-90), and improved to <10 CFU ml-1 using the O55 specific PCR, following IMS on samples enriched for 2h with E. coli O55. Application of these tools to test cattle faeces collected on-farm allowed the isolation of O55:H19, which through whole genome sequencing was compared to STEC O55:H7 human outbreak strains. CONCLUSION: These tools provide a sensitive method which could be used to screen samples for STEC O55, whether environmental or human clinical. SIGNIFICANCE AND IMPACT OF THE STUDY: Several human outbreaks reported in England were caused by STEC O55:H7. Tools developed here could assist in identification of the environmental source for these isolates, which has not yet been established. This article is protected by copyright. All rights reserved

Host-specific differences in the contribution of an extended spectrum β-lactamase (ESBL) IncI1 plasmid to intestinal colonisation by Escherichia coli O104:H4

Objectives. To assess stability and contribution of a large extended spectrum β-lactamase (ESBL)-containing IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts. Methods. Specific-pathogen-free 3-day old New Zealand White rabbits and conventionally-reared 6-week-old weaned lambs were orally infected with wild-type E. coli O104:H4 or the ESBL-plasmid cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time. Results. Carriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonisation of the two experimental hosts. The plasmid cured strain was recovered at significantly higher levels than wild type during late-stage colonization of rabbits, but at lower levels than wildtype in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined. Conclusions. These findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon the animal species it encounters and demonstrates that, as for E. coli O157:H7, ruminants could represent a potential transmission reservoir.</p

Contrasting long-term dynamics of antimicrobial resistance and virulence plasmids in Salmonella Typhimurium from animals

Plasmids are mobile elements that can carry genes encoding traits of clinical concern, including antimicrobial resistance (AMR) and virulence. Population-level studies of Enterobacterales, including Escherichia coli, Shigella and Klebsiella, indicate that plasmids are important drivers of lineage expansions and dissemination of AMR genes. Salmonella Typhimurium is the second most common cause of salmonellosis in humans and livestock in the UK and Europe. The long-term dynamics of plasmids between S. Typhimurium were investigated using isolates collected through national surveillance of animals in England and Wales over a 25-year period. The population structure of S. Typhimurium and its virulence plasmid (where present) were inferred through phylogenetic analyses using whole-genome sequence data for 496 isolates. Antimicrobial resistance genes and plasmid markers were detected in silico. Phenotypic plasmid characterization, using the Kado and Liu method, was used to confirm the number and size of plasmids. The differences in AMR and plasmids between clades were striking, with livestock clades more likely to carry one or more AMR plasmid and be multi-drug-resistant compared to clades associated with wildlife and companion animals. Multiple small non-AMR plasmids were distributed across clades. However, all hybrid AMR-virulence plasmids and most AMR plasmids were highly clade-associated and persisted over decades, with minimal evidence of horizontal transfer between clades. This contrasts with the role of plasmids in the short-term dissemination of AMR between diverse strains in other Enterobacterales in high-antimicrobial-use settings, with implications for predicting plasmid dissemination amongst S. Typhimurium

Development of a multiplex bead assay to detect serological responses to Brucella species in domestic pigs and wild boar with the potential to overcome cross-reactivity with Yersinia enterocolitica O:9

This article belongs to the Special Issue Emerging Themes in Brucella and Brucellosis.The aim of this study was to develop a multiplex bead assay using a Brucella rLPS antigen, a Brucella suis smooth antigen, and a Yersinia enterocolitica O:9 antigen that not only discriminates Brucella-infected from Brucella-uninfected pigs and wild boar, but also overcomes the cross reactivity with Y. enterocolitica O:9. Sera from 126 domestic pigs were tested: 29 pigs were Brucella infected, 80 were non-infected and 17 were confirmed to be false positive serological reactors (FPSR). Sera from 49 wild boar were tested: 18 were positive and 31 were negative. Using the rLPS antigen, 26/29 Brucella-infected domestic pigs and 15/18 seropositive wild boar were positive, while 75/80 non-Brucella infected domestic pigs, all FPSR, and all seronegative wild boar were negative. Using the smooth B. suis 1330 antigen, all Brucella-infected domestic pigs, 9/17 FPSR and all seropositive wild boar were positive, while all non-infected pigs and 30/31 seronegative wild boar were negative. The ratio of the readouts from the smooth B. suis antigen and Y. enterocolitica O:9 antigen enabled discriminating all Brucella infected individuals from the FPSR domestic pigs. These results demonstrate the potential of this assay for use in the surveillance of brucellosis, overcoming the cross-reactivity with Y. enterocolitica.We thankfully acknowledge the financial support of the European Union Seventh Framework Programme (2007–2013) under grant agreement no. 222633 (WildTech) entitled “Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife”.Peer reviewe

Development of a multiplex bead assay for simultaneous serodiagnosis of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis in wild boar

This article belongs to the Special Issue Farm Animal and Wildlife Zoonotic Microorganisms.The aim of this study was to evaluate the diagnostic performance of a multiplex bead assay for the simultaneous detection of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis. Sera from Eurasian wild boar of known serological status for TB (64 seropositive, 106 seronegative), Brucella (30 seropositive, 39 seronegative), and Trichinella (21 seropositive, 97 seronegative) were used for the development and evaluation of the assay. Magnetic beads coated with recombinant MPB83 antigen (TB), a whole-cell B. suis 1330 antigen, and an E/S T. spiralis antigen were used for the detection of specific antibodies using Bio-Rad Bio-Plex technology. The sensitivities (Se) and specificities (Sp) of the multiplex assay were, for M. bovis, 0.98 and 0.86; for B. suis, 1.00 and 0.97; and for T. spiralis, 0.90 and 0.99 (Se and Sp, respectively). The results show the diagnostic potential of this assay for the simultaneous detection of antibodies against M. bovis, B. suis, and T. spiralis in wild boar.We thankfully acknowledge the financial support of the European Union Seventh Framework Programme (2007–2013) under grant agreement no. 222633 (WildTech) titled “Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife”.Peer reviewe

Human and chicken immune responses to Campylobacter jejuni

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