Using the Traditional Ex Vivo Whole Blood Model to Discriminate Bacteria by Their Inducible Host Responses.

Whole blood models are rapid and versatile for determining immune responses to inflammatory and infectious stimuli, but they have not been used for bacterial discrimination. Staphylococcus aureus, S. epidermidis and Escherichia coli are the most common causes of invasive disease, and rapid testing strategies utilising host responses remain elusive. Currently, immune responses can only discriminate between bacterial 'domains' (fungi, bacteria and viruses), and very few studies can use immune responses to discriminate bacteria at the species and strain level. Here, whole blood was used to investigate the relationship between host responses and bacterial strains. Results confirmed unique temporal profiles for the 10 parameters studied: IL-6, MIP-1α, MIP-3α, IL-10, resistin, phagocytosis, S100A8, S100A8/A9, C5a and TF3. Pairwise analysis confirmed that IL-6, resistin, phagocytosis, C5a and S100A8/A9 could be used in a discrimination scheme to identify to the strain level. Linear discriminant analysis (LDA) confirmed that (i) IL-6, MIP-3α and TF3 could predict genera with 95% accuracy; (ii) IL-6, phagocytosis, resistin and TF3 could predict species at 90% accuracy and (iii) phagocytosis, S100A8 and IL-10 predicted strain at 40% accuracy. These data are important because they confirm the proof of concept that host biomarker panels could be used to identify bacterial pathogens.

The diversity of Campylobacter spp. throughout the poultry processing plant.

Campylobacteriosis is the leading food-borne disease in developed countries, and poultry are a major source for human infection. The diversity of Campylobacter on chicken carcasses during processing may lead to isolates that are able to survive abattoir processing. This has important implications for public health and adds a further layer to the complexity of the epidemiology of campylobacteriosis. The diversity of the Campylobacter spp. populations on broiler carcasses was studied at three different stages of processing (post-bleed, post-scald and post-chill) in three UK processing plants, using the pulsed-field gel electrophoresis (PFGE) KpnI enzyme. One hundred and sixty Campylobacter strains from 3 processing plants were identified as C. jejuni (92.3%) with 27 PFGE subtype profiles recovered from carcasses at the post-bleed point. Change in populations was identified when carcasses move towards the end of poultry processing. Seven C. jejuni genotypes were able to survive the scalding tank stage process, and 5 genotypes surviving the entire poultry process. Confirmation by PFGE gives information on the genotypic profiles of C. jejuni on chicken carcasses and how they change according to the temperatures exposed to during processing. Diversity within C. jejuni populations produces genotypes that adapt to tolerate the processing environment, and these may be capable of causing human disease. Understanding more about the genotypes that survive the processing will have important implications for public health.

Genome evolution and the emergence of pathogenicity in avian Escherichia coli.

Chickens are the most common birds on Earth and colibacillosis is among the most common diseases affecting them. This major threat to animal welfare and safe sustainable food production is difficult to combat because the etiological agent, avian pathogenic Escherichia coli (APEC), emerges from ubiquitous commensal gut bacteria, with no single virulence gene present in all disease-causing isolates. Here, we address the underlying evolutionary mechanisms of extraintestinal spread and systemic infection in poultry. Combining population scale comparative genomics and pangenome-wide association studies, we compare E. coli from commensal carriage and systemic infections. We identify phylogroup-specific and species-wide genetic elements that are enriched in APEC, including pathogenicity-associated variation in 143 genes that have diverse functions, including genes involved in metabolism, lipopolysaccharide synthesis, heat shock response, antimicrobial resistance and toxicity. We find that horizontal gene transfer spreads pathogenicity elements, allowing divergent clones to cause infection. Finally, a Random Forest model prediction of disease status (carriage vs. disease) identifies pathogenic strains in the emergent ST-117 poultry-associated lineage with 73% accuracy, demonstrating the potential for early identification of emergent APEC in healthy flocks.

Molecular and phenotypical investigation of ciprofloxacin resistance among Campylobacter jejuni strains of human origin: high prevalence of resistance in Turkey.

Campylobacteriosis is one of the most frequently reported zoonoses worldwide. The well-documented increase in the ciprofloxacin resistance has increased the importance of rapid detection of the resistance. The incidence of ciprofloxacin resistance was investigated using real-time PCR. Identification of one hundred and fifty-eight strains was performed by PCR. Minimum inhibitory concentration (MIC) of ciprofloxacin was determined by Epsilometer test. Following the confirmation of the efficiencies of singleplex real-time PCR methods using two different probes, a cytosine to thymine point mutation at codon 86 was detected by allelic discrimination. Of the 158 strains, 114 (72.2%) were determined to be resistant to ciprofloxacin. The MIC50 and the MIC90 of ciprofloxacin were found to be 8 and ≥32 mg/L, respectively. By real-time PCR, the presence of the mutation was confirmed in all, but one, resistant strains and the absence of the mutation was demonstrated in all, but one, susceptible strains. The rate of resistance is high among C. jejuni strains and ciprofloxacin should not be used in the treatment of such infections in Turkey. A cytosine to thymine mutation is the most frequently detected mechanism for the resistance. Real-time PCR can be used for the quick screening of the resistance.

Domestication of Campylobacter jejuni NCTC 11168.

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterized isolates among laboratories has run in parallel with research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates, identified previously to have aberrant phenotypes, varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37 °C, invasion of chicken and human cell lines, and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains. This article contains data hosted by Microreact.

The Bacterial Species Campylobacter jejuni Induce Diverse Innate Immune Responses in Human and Avian Intestinal Epithelial Cells.

Campylobacter remain the major cause of human gastroenteritis in the Developed World causing a significant burden to health services. Campylobacter are pathogens in humans and chickens, although differences in mechanistic understanding are incomplete, in part because phenotypic strain diversity creates inconsistent findings. Here, we took Campylobacter jejuni isolates (n = 100) from multi-locus sequence typed collections to assess their pathogenic diversity, through their inflammatory, cytotoxicity, adhesion, invasion and signaling responses in a high-throughput model using avian and human intestinal epithelial cells. C. jejuni induced IL-8 and CXCLi1/2 in human and avian epithelial cells, respectively, in a MAP kinase-dependent manner. In contrast, IL-10 responses in both cell types were PI 3-kinase/Akt-dependent. C. jejuni strains showed diverse levels of invasion with high invasion dependent on MAP kinase signaling in both cell lines. C. jejuni induced diverse cytotoxic responses in both cell lines with cdt-positive isolates showing significantly higher toxicity. Blockade of endocytic pathways suggested that invasion by C. jejuni was clathrin- and dynamin-dependent but caveolae- independent in both cells. In contrast, IL-8 (and CXCLi1/2) production was dependent on clathrin, dynamin, and caveolae. This study is important because of its scale, and the data produced, suggesting that avian and human epithelial cells use similar innate immune pathways where the magnitude of the response is determined by the phenotypic diversity of the Campylobacter species.

Pancreatic amylase is an environmental signal for regulation of biofilm formation and host interaction in Campylobacter jejuni.

Campylobacter jejuni is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by C. jejuni and that a secreted protease, Cj0511, is required. Exposure of C. jejuni to pancreatic amylase promotes biofilm formation in vitro, increases interaction with human epithelial cell lines, increases virulence in the Galleria mellonella infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects C. jejuni from stress conditions in vitro, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.

Validation of an HPV16-mediated carcinogenesis mouse model.

BACKGROUND/AIM: Human papillomavirus Type 16 (HPV16) infection is a necessary but alone insufficient cause of invasive cervical cancer (ICC) and likely causes other genital cancers. Individual genetic variability influences the natural history of the neoplasm. Developing a variety of animal models to investigate HPV16-mediated carcinogenesis is important to Phase 1 trials for human cancer treatments. MATERIALS AND METHODS: C57BL/6 mice expressing the HPV16-E7 transgene were treated with 100 nmoles of 7,12-dimethylbenz(a)anthracene (DMBA) on dorsal-thoracolumbar skin for ≤20 weeks. RESULTS: Transgenic-HPV16E7 mice showed more tumors (14.11±1.49 vs. 7.2±0.73) that more quickly reached maximal size (17.53±0.53 vs. 28.75±0.67 weeks) than syngeneic controls. CONCLUSION: DMBA topically-treated C57BL/6-HPV16E7 mice developed chronic inflammation as well as benign and malignant lesions, many of which ulcerated. Histology showed that the HPV16-E7 transgene more than doubled the effect of complete carcinogenesis against a C57BL/6 background alone, strongly influencing the number, size, and time-to-maximal tumor burden for DMBA-exposed transgenic-C57BL/6 mice.

Ulcerative dermatitis in C57BL/6 mice exhibits an oxidative stress response consistent with normal wound healing.

Ulcerative dermatitis (UD) is a common syndrome of unknown etiology that results in profound morbidity in C57BL/6 mice and lines on a C57BL/6 background. The lesions are due to severe pruritus-induced self-trauma, progressing from superficial excoriations to deep ulcerations. UD may be behavioral in origin, with ulcerative lesions resulting from self-mutilating behavior in response to unresolved inflammation or compulsion. Alternatively, abnormal oxidative damage may be a mechanism underlying UD. To evaluate whether UD behaves similarly to normal wounds, consistent with a secondary self-inflicted lesion, or is a distinct disorder with abnormal wound response, we evaluated expression levels of genes representing various arms of the oxidative stress response pathway UD-affected and unwounded C57BL/6J mice. No evidence indicated that UD wounds have a defect in the oxidative stress response. Our findings are consistent with an understanding of C57BL/6 UD lesions as typical rather than atypical wounds.

Campylobacter jejuni is associated with, but not sufficient to cause vibrionic hepatitis in chickens.

Vibrionic hepatitis is a disease of poultry which is characterised by the presence of focal lesions in the liver, usually 1-2mm in size and greyish-white in colour. The cause of the disease remains unclear, as do the reasons for its recent re-emergence. We examined the livers of commercial broiler chickens taken during processing and found Campylobacter spp. in both normal livers and those displaying signs indicative of focal hepatitis. Livers with signs of hepatitis had significantly more Campylobacter spp. present than those without and other bacterial genera were infrequently present. We were unable to replicate the disease in a healthy host following experimental infection with a Campylobacter jejuni strain isolated from a liver showing signs of focal hepatitis. However, a significant T cell response to C. jejuni was seen in the liver of Campylobacter infected birds. We conclude that the presence of Campylobacter spp. in the liver alone is not sufficient to cause vibrionic hepatitis, but that a predisposing factor, possibly within the host is required. We also provide evidence that chickens mount an adaptive T cell response to systemic C. jejuni.

Amoxicillin therapy of poultry flocks: effect upon the selection of amoxicillin-resistant commensal Campylobacter spp.

BACKGROUND: The aim of this study was to investigate the effect of amoxicillin therapy of poultry flocks upon the persistence of commensal Campylobacter spp. and the incidence of antibiotic resistance. METHODS: Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a beta-lactamase and beta-lactamase production (nitrocefin hydrolysis) were also determined. RESULTS: Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced beta-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates. CONCLUSIONS: Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

Enrichment culture for the isolation of Campylobacter spp: Effects of incubation conditions and the inclusion of blood in selective broths.

Isolation of Campylobacter spp. using enrichment culture is time consuming and complex. Reducing the time taken to confirm the presence or absence of Campylobacter spp. would have many advantages for diagnostic, commercial and research applications. Rapid techniques such as real-time PCR can detect campylobacters from complex samples but blood in enrichment culture can inhibit the PCR reaction, if applied directly to enriched samples. The aim of this study was to investigate the effect of blood in enrichment culture on the isolation of campylobacters from chicken caeca, carcass rinses and bootsock (gauze sock walked through a broiler chicken house) samples using Bolton broth. The effect of incubation temperature (37 degrees C or 41.5 degrees C for 48 h, or 37 degrees C for 4 h then transfer to 41.5 degrees C for 44 h) and method of generating atmosphere (incubation of container in jar gassed with microaerobic atmosphere or incubation of container with small headspace and tightly screwed lid in an aerobic atmosphere) with and without blood on isolation from chicken carcass rinses and chicken faeces was also investigated. The presence of blood in enrichment culture did not improve the isolation of campylobacters from chicken faeces or bootsock samples but significantly improved recovery from chicken carcass rinse samples. There was no significant effect of the method used to generate incubation atmosphere. Isolation rates did also not depend significantly on whether broths were incubated at 37 or 41.5 degrees C for 24 or 48 h. Overall, the presence of blood in such media is not essential, although isolation can vary depending on sample type and enrichment method used.

Detection of Campylobacter jejuni and Campylobacter coli from broiler chicken-related samples using BAX PCR and conventional International Organization for Standardization culture.

In this study, the conventional International Organization for Standardization (ISO) culture method was compared with the DuPont Qualicon BAX system, a high-throughput, rapid molecular assay that can be used to detect several bacterial species, including Campylobacter jejuni and Campylobacter coli in diverse sample types. Standard enrichment culture is a time-consuming process, taking up to 6 days to obtain a confirmed result. Rapid molecular assays have been developed that provide results within 24 h. Naturally contaminated samples from the poultry production chain were examined for the presence of Campylobacter spp. Samples from broiler chicken ceca (n = 100), fresh chicken carcass rinses (n = 60), and bootsocks (gauze sock walked through a broiler chicken house; n = 50) were enriched according to the ISO 10272 method in Bolton broth specifically designed to detect Campylobacter spp. in complex sample types. Samples were enriched without blood for use with the BAX system using the Campylobacter BAX kits for the detection of C. jejuni and C. coli. Samples also were directly plated onto modified charcoal cefperazone deoxycholate agar, and results were compared with those from the enriched samples for the ability to detect Campylobacter spp. Campylobacter spp. were isolated from 49% of samples with conventional enrichment cultures, from 48% with direct culture, from 68% with the BAX system and enrichment cultures, and from 62% with the BAX system used directly with samples. Overall, the BAX system detected more positive samples than did the conventional culture method and is an effective methodology for the rapid and reliable detection of Campylobacter spp. from diverse sample types.