In vitro screening and in vivo colonization pilot model of Lactobacillus plantarum LP5 and Campylobacter coli DSPV 458 in mice.

The objective of this work was to determine the antibacterial effect of Lactobacillus plantarum strains of pork origin against Campylobacter coli strains, and to conduct experimental colonization pilot models in mice for both microorganisms. Inhibition assays allowed evaluation and selection of L. plantarum LP5 as the strain with the highest antagonistic activity against C. coli and with the best potential to be used in in vivo study. Adult 6-week-old female Balb/cCmedc mice were lodged in two groups. The treated group was administered with 9.4 log10CFU/2 times/wk of L. plantarum LP5. L. plantarum LP5 was recovered from the feces and cecum of the inoculated mice. However, when bacteria stopped being administered, probiotic counts decreased. Experimental colonization with C. coli was carried out in five groups of mice. All animals were treated with antibiotics in their drinking water to weaken the indigenous microbiota and to allow colonization of C. coli. Four groups were administered once with different C. coli strains (DSPV458: 8.49 log10CFU; DSPV567: 8.09 log10CFU; DSPV570: 8.46 log10CFU; DSPV541: 8.86 log10CFU, respectively). After 8 h, mice inoculated with different C. coli strains were colonized because the pathogen was detected in their feces. L. plantarum LP5 tolerated the gastrointestinal conditions of murine model without generating adverse effects on the animals. C. coli DSPV458 colonized the mice without causing infection by lodging in their digestive tract, thus generating a reproducible colonization model. Both models combined could be used as protection murine models against pathogens to test alternative control tools to antibiotics.

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates.

BACKGROUND: Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. METHODS: A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. RESULTS: Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. CONCLUSION: Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

Campylobacter coli strains from Brazil can invade phagocytic and epithelial cells and induce IL-8 secretion.

Campylobacter spp. have been a predominant cause of bacterial foodborne gastroenteritis worldwide, causing substantial costs to public healthcare systems. This study aimed to assess the invasion and pro-inflammatory cytokine production capacity of Campylobacter coli strains isolated in Brazil. A total of 50 C. coli isolated from different sources in Brazil were analyzed for their capacity of invasion in Caco-2 and U-937 cell lines. The production of pro-inflammatory cytokines was quantitatively measured in response to C. coli. All the strains studied showed invasion percentage ≥ 40% in polarized Caco-2 cells. In U-937 cells assay, 35 of 50 C. coli strains studied showed invasion percentage ≥ 50%. A significant increase in IL-8 production by infected U-937 cells was observed for 17.5% of the C. coli isolates. The high percentages of invasion in Caco-2 and U-937 cells observed for all studied strains, plus the increased production of IL-8 by U-937 cells against some strains, highlighted the pathogenic potential of the C. coli studied and bring extremely relevant data since it has never been reported for strains isolated in Brazil and there are a few data for C. coli in the literature.

Research Note: A baseline survey of thermotolerant Campylobacter in retail chicken in southern Brazil.

Chicken is a leading source of thermotolerant Campylobacter, which triggers human foodborne enteritis. This study evaluated thermotolerant Campylobacter contamination of retail chicken in southern Brazil, using qualitative and quantitative analyses. Selective enrichment in Bolton broth for 24 and 48 h after plating onto modified charcoal-cefoperazone-deoxycholate (mCCD) agar and Preston agar was assessed. The combined results of the detection and enumeration methods revealed a frequency of 70% occurrence of thermotolerant Campylobacter in chicken samples. Campylobacter was enumerated in 60% of the samples, whereas 46% of the samples were positive in the qualitative analysis. Quantitative analysis showed average counts of 3.10 ± 0.15 log10 CFU/sample. Higher numbers of Campylobacter-positive samples were found using 24-h enrichment before plating onto Preston agar (46%) than onto mCCD agar (2%). The majority of isolated strains were identified as Campylobacter jejuni, and Campylobacter coli was also found but to a lesser extent. Subtyping revealed a clear distinction between strains isolated from different chicken sources. The enriched samples plated onto mCCD agar showed extensive spreading of nonproducing extended-spectrum ß-lactamases Proteus mirabilis that hampered the identification of Campylobacter colonies. P. mirabilis strains showed resistance to cefoperazone, trimethoprim, and polymyxin B present in broth and plate media used and were inhibited by rifampicin present in Preston agar. The results underline the effect of the spread of contaminant strains on Campylobacter cultures, which might be prevented using a recently revised International Organization for Standardization method for qualitative analysis of chicken.

Intracellular Survival and Translocation Ability of Human and Avian Campylobacter jejuni and Campylobacter coli Strains.

Campylobacter acts using complex strategies to establish and promote intestinal infections. After ingestion via contaminated foods, this bacterium invades and can survive within the intestinal cells, also inducing epithelial translocation of non-invasive intestinal bacteria. In this investigation, the ability of human and avian C. jejuni and C. coli isolates to survive within two different intestinal epithelial cells lines, Caco-2 and INT 407, as well as the intestinal translocation phenomenon, was assessed. Our data demonstrated that both C. jejuni and C. coli strains survived in Caco-2 (81.8% and 100% respectively) and INT 407 monolayers (72.7% and 100% respectively) within the first 24 h post-infection period, with a progressive reduction in the prolonged period of 48 h and 72 h post-infection. The translocation of the non-invasive E. coli 60/06 FB was remarkably increased in C. jejuni treated Caco-2 monolayers (2.36 ± 0.42 log cfu/mL) (P < 0.01) and less in those treated with C. coli (1.2 ± 0.34 log cfu/mL), compared to E. coli 60/06 FB alone (0.37 ± 0.14 log cfu/mL). Our results evidenced the ability of both human and avian strains of C. jejuni and C. coli to efficiently survive within intestinal cells and induce the translocation of a non-invasive pathogen. Overall, these findings stress how this pathogen can interact with host cells and support the hypothesis that defects in the intestinal barrier function induced by Campylobacter spp. could have potentially negative implications for human health.

Emergence of Fluoroquinolone-Resistant Campylobacter jejuni and Campylobacter coli among Australian Chickens in the Absence of Fluoroquinolone Use.

In a structured survey of all major chicken-meat producers in Australia, we investigated the antimicrobial resistance (AMR) and genomic characteristics of Campylobacter jejuni (n = 108) and C. coli (n = 96) from cecal samples of chickens at slaughter (n = 200). The majority of the C. jejuni (63%) and C. coli (86.5%) samples were susceptible to all antimicrobials. Fluoroquinolone resistance was detected among both C. jejuni (14.8%) and C. coli (5.2%), although this only included three sequence types (STs) and one ST, respectively. Multidrug resistance among strains of C. jejuni (0.9%) and C. coli (4.1%) was rare, and fluoroquinolone resistance, when present, was never accompanied by resistance to any other agent. Comparative genome analysis demonstrated that Australian isolates were found dispersed on different branches/clusters within the international collection. The major fluoroquinolone-resistant STs of C. jejuni (ST7323, ST2083, and ST2343) and C. coli (ST860) present in Australian chickens were similar to those of international isolates and have been reported previously in humans and animals overseas. The detection of a subpopulation of Campylobacter isolates exclusively resistant to fluoroquinolone was unexpected since most critically important antimicrobials such as fluoroquinolones are excluded from use in Australian livestock. A number of factors, including the low level of resistance to other antimicrobials, the absence of fluoroquinolone use, the adoption of measures for preventing spread of contagion between flocks, and particularly the genomic identities of isolates, all point to humans, pest species, or wild birds as being the most plausible source of organisms. This study also demonstrates the need for vigilance in the form of surveillance for AMR based on robust sampling to manage AMR risks in the food chain.IMPORTANCECampylobacter is one of the most common causes of gastroenteritis in humans, with infections frequently resulting from exposure to undercooked poultry products. Although human illness is typically self-limiting, a minority of cases do require antimicrobial therapy. Ensuring that Campylobacter originating from meat chickens does not acquire resistance to fluoroquinolones is therefore a valuable outcome for public health. Australia has never legalized the use of fluoroquinolones in commercial chickens and until now fluoroquinolone-resistant Campylobacter has not been detected in the Australian poultry. This structured survey of meat chickens derived from all major Australian producers describes the unexpected emergence of fluoroquinolone resistance in Campylobacter jejuni and C. coli Genetic characterization suggests that these isolates may have evolved outside the Australian poultry sector and were introduced into poultry by humans, pest species, or wild birds. The findings dramatically underline the critical role of biosecurity in the overall fight against antimicrobial resistance.

Investigating the antimicrobial and antibiofilm effects of cinnamaldehyde against Campylobacter spp. using cell surface characteristics.

Campylobacter species are known as biofilm-forming bacteria in food systems. The aim of this study was to evaluate the antimicrobial and antibiofilm effects of cinnamaldehyde against Campylobacter jejuni and Campylobacter coli isolated from chicken meat. The biofilm-forming C. jejuni and C. coli strains from chicken meat were investigated using minimum inhibitory concentration (MIC) and Campylobacter spp. characteristics. The MIC value was 31.25 µg/mL for the Campylobacter strains tested. Cinnamaldehyde had an inhibition and degradation effect on Campylobacter biofilms at concentrations > 15.63 µg/mL. Campylobacter strains treated with 15.63 µg/mL CA exhibited significantly decreased autoaggregation, motility, exopolysaccharide production, and soluble protein. In addition, Campylobacter biofilms formed on stainless steel were degraded following cinnamaldehyde treatment, as determined by scanning electron microscopy. Taken together, these results suggest that cinnamaldehyde constitutes a potential natural preservative against Campylobacter and a nontoxic biofilm remover that could be applied to control food poisoning in the poultry manufacturing-related food industry. PRACTICAL APPLICATION: Cinnamaldehyde was able to effectively remove the biofilm of Campylobacter in the small crack of stainless steel. Cinnamaldehyde has a potential to replace the synthetic antimicrobial and/or antibiofilm agent as well as has a positive influence on consumer concern for the food safety issues of the poultry industries.

Strain-Specific Differences in Survival of Campylobacter spp. in Naturally Contaminated Turkey Feces and Water.

Campylobacter jejuni and Campylobacter coli are leading causes of human foodborne illness, with poultry as a major vehicle. Turkeys are frequently colonized with Campylobacter, but little is known about Campylobacter survival in turkey feces, even though fecal droppings are major vehicles for Campylobacter within-flock transmission as well as for environmental dissemination. Our objective was to examine survival of Campylobacter, including different strains, in freshly excreted feces from naturally colonized commercial turkey flocks and in suspensions of turkey feces in water from the turkey house. Fecal and water suspensions were stored at 4°C, and Campylobacter populations were enumerated on selective media at 48-h intervals. C. jejuni and C. coli isolates were characterized for resistance to a panel of antibiotics, and a subset was subtyped using multilocus sequence typing. Campylobacter was recovered from feces and water for up to 16 days. Analysis of 548 isolates (218 C. jejuni and 330 C. coli) revealed that C. jejuni survived longer than C. coli in feces (P = 0.0005), while the reverse was observed in water (P < 0.0001). Strain-specific differences in survival were noted. Multidrug-resistant C. jejuni isolates of sequence type 1839 (ST-1839) and the related ST-2935 were among the longest-surviving isolates in feces, being recovered for up to 10 to 16 days, while multidrug-resistant C. coli isolates of ST-1101 were recovered from feces for only up to 4 days. Data on Campylobacter survival upon excretion from the birds can contribute to further understanding of the transmission dynamics of this pathogen in the poultry production ecosystem.IMPORTANCECampylobacter jejuni and Campylobacter coli are leading foodborne pathogens, with poultry as a major reservoir. Due to their growth requirements, these Campylobacter spp. may be unable to replicate once excreted by their avian hosts, but their survival in feces and the environment is critical for transmission in the farm ecosystem. Reducing the prevalence of Campylobacter-positive flocks can have major impacts in controlling both contamination of poultry products and environmental dissemination of the pathogens. However, understanding the capacity of these pathogens to survive in transmission-relevant vehicles such as feces and farmhouse water remains poorly understood, and little information is available on species- and strain-associated differences in survival. Here, we employed model conditions to investigate the survival of C. jejuni and C. coli from naturally colonized turkey flocks, and with diverse genotypes and antimicrobial resistance profiles, in turkey feces and in farmhouse water.

Impact of Skip-a-Day and Every-Day Feeding Programs for Broiler Breeder Pullets on the Recovery of Salmonella and Campylobacter following challenge.

The impact of restrictive feeding programs on Salmonella and Campylobacter colonization and persistence after challenge was investigated for broiler breeder pullets housed in an experimental rearing facility. Pullet-chicks were placed on litter in 3 feeding program rooms and each room contained 2 replicate pens. The feeding programs were: (1) Skip-a-day in trough feeders (SAD); (2) Every-day in trough feeders (EDT); (3) Every-day on the pen litter (EDL). On d 1, an additional group of hatchmate chicks were housed in a separate room and gavaged with Salmonella Typhimurium, to later serve as seeder chicks. After seeders were confirmed Salmonella-positive at wk 4, at wk 5 seeders were placed into each feeding program pen to commingle with 135 penmates. At 7, 9, 11, 17, 18, and 20 wk the litter surface in each pen was sampled using intermittently stepped-on drag-swabs. At 8, 12, 16, and 20 wk of age the ceca were sampled from 10 penmates/pen and 2 pooled spleen samples/pen were collected. SAD litter remained Salmonella-positive through 20 wk of age while EDL and EDT pens had no detectible litter Salmonella recovery by 18 and 20 wk. EDL fed pens had no direct (<102 cfu/mL) litter Salmonella recovery during the entirety of the experiment. Salmonella prevalence for ceca from SAD pullets was significantly (P < 0.05) higher at 8 wk (70%) compared to EDT (40%) and EDL (30%). At wk 12, SAD pullets for both on and off-feed sampling days had significantly higher Salmonella recovery (40%), compared to EDT and EDL (both at 5% recovery). By 16 and 20 wk, only the SAD pullets on the on-feed day (48 h without feed) had recovery of Salmonella at 20%. Salmonella recovery in pooled spleen samples did not appear associated with feeding treatments (22% positive). The remaining pullets challenged with Campylobacter at 21 wk produced similar trends as was seen for Salmonella. SAD program pullets had significantly higher Campylobacter from ceca (80 to 100%) compared to pullets on EDL (30 to 60%) or EDT (40 to 95%). These results suggest that using a Skip-a-Day feeding program for broiler breeder pullets contributes to persistently higher Salmonella and Campylobacter ceca colonization and litter prevalence.

New colorimetric aptasensor for rapid on-site detection of Campylobacter jejuni and Campylobacter coli in chicken carcass samples.

Campylobacter is the most common cause of infectious intestinal disease, with nearly all cases caused by two species: C. jejuni and C. coli. We recently reported a gold nanoparticle-based two-stage aptasensing platform, which was improved in the present study for the rapid and on-site detection of both C. jejuni and C. coli in food samples. Compared to the previous platform, the improved platform yielded a more obvious colour change from red to purple due to the aggregation of gold nanoparticles, and does not require additional time or a pH optimization step for the aptamers to be adsorbed onto the gold nanoparticles. Using a highly specific aptamer that binds to live C. jejuni and C. coli, the improved aptasensor was highly effective for testing pure culture samples. The accuracy of the newly developed platform was comparable (p = 0.688) to that of the gold-standard detection method of tazobactam-supplemented culture, whereas it was superior to the official agar-based detection method (p = 0.016) in a validation study with 50 naturally contaminated chicken carcass samples. This is the first study on a colorimetric sensor that targets both live C. coli and C. jejuni in naturally contaminated samples. In addition, we provide the first evidence that both morphological status and the amount of Campylobacter present play key roles in the effectiveness of colorimetric detection. Thus, suitable selection of an antibody or aptamer with consideration of the morphological status of pathogens in samples is essential for direct detection without enrichment. Our data suggest that the sensor developed in this study can provide an excellent screening method, with a reduction in the detection time from 48 h to 30 min after enrichment, thus saving time, labour, and cost.

Genetic characterisation of virulence genes associated with adherence, invasion and cytotoxicity in Campylobacter spp. isolated from commercial chickens and human clinical cases.

Virulence-associated genes have been recognised and detected in Campylobacter species. The majority of them have been proven to be associated with pathogenicity. This study aimed to detect the presence of virulence genes associated with pathogenicity and responsible for invasion, expression of adherence, colonisation and production of the cytolethal distending toxin (cdt) in Campylobacter jejuni and Campylobacter coli. Commercial chicken faecal samples were randomly sampled from chicken farms within the Durban metropolitan area in South Africa. Furthermore, human clinical Campylobacter spp. isolates were randomly sampled from a private pathology laboratory in South Africa. Out of a total of 100 chicken faecal samples, 78% (n = 78) were positive for Campylobacter growth on modified charcoal cefoperazone deoxycholate and from the random laboratory collection of 100 human clinical isolates, 83% (n = 83) demonstrated positive Campylobacter spp. growth following culturing methods. These samples were screened for the presence of the following virulence genes: cadF, hipO, asp, ciaB, dnaJ, pldA, cdtA, cdtB and cdtC. As expected, the cadF gene was present in 100% of poultry (n = 78) and human clinical isolates (n = 83). Campylobacter jejuni was the main species detected in both poultry and human clinical isolates, whilst C. coli were detected at a significantly lower percentage (p < 0.05). Eight per cent of the C. jejuni from human clinical isolates had all virulence genes that were investigated. Only one C. coli isolate demonstrated the presence of all the virulence genes investigated; however, the pldA virulence gene was detected in 100% of the C. coli isolates in poultry and a high percentage (71%) in human clinical C. coli isolates as well. The detection of cdt genes was found at higher frequency in poultry than human clinical isolates. The high prevalence rates of virulence genes detected in poultry and human clinical isolates demonstrate their significance in the pathogenicity of Campylobacter species.

Adherence Reduction of Campylobacter jejuni and Campylobacter coli Strains to HEp-2 Cells by Mannan Oligosaccharides and a High-Molecular-Weight Component of Cranberry Extract.

Campylobacter infections are a leading cause of human bacterial gastroenteritis in the United States and are a major cause of diarrheal disease throughout the world. Colonization and subsequent infection and invasion of Campylobacter require that the bacteria adhere to the surface of host cells. Agents that inhibit adherence could be used prophylactically to reduce Campylobacter carriage and infection. Mannan oligosaccharides (MOS) have been used as a feed supplement in livestock animals to improve performance and to replace growth-promoting antibiotics. However, MOS and other nondigestible oligosaccharides may also prevent pathogen colonization by inhibiting adherence in the gastrointestinal tract. In addition, plant extracts, including those derived from cranberries, have been shown to have antiadherence activity against pathogens. The goal of this study was to assess the ability of MOS and cranberry fractions to serve as antiadherence agents against strains of Campylobacter jejuni and Campylobacter coli. Adherence experiments were performed using HEp-2 cells. Significant reductions in adherence of C. jejuni 29438, C. jejuni 700819, C. jejuni 3329, and C. coli 43485 were observed in the presence of MOS (up to 40 mg/ml) and with a high-molecular-weight fraction of cranberry extract (up to 3 mg/ml). However, none of the tested materials reduced adherence of C. coli BAA-1061. No additive effect in adherence inhibition was observed for an MOS-cranberry blend. These results suggest that both components, MOS and cranberry, could be used to reduce Campylobacter colonization and carriage in livestock animals and potentially limit human exposure to this pathogen.

The evolution of Campylobacter jejuni and Campylobacter coli.

The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure.

Development of transient phage resistance in Campylobacter coli against the group II phage CP84.

Recently, there is a growing interest in the use of bacteriophages for pre- and post-harvest applications to reduce foodborne pathogens (including Campylobacter) along the food chain. Quantitative Campylobacter reductions of up to three log10 units have been achieved by phage application. However, possible phage resistance might limit this approach. In Campylobacter (C.) jejuni, phage resistance mechanisms have been described in detail but data on these mechanisms in C. coli are still missing. To study phage resistance in C. coli, strain NCTC 12668 was infected with the lytic phage CP84, belonging to group II of Campylobacter phages. Resistant and sensitive clones were analysed using phenotypic and genotypic assays. C. coli clones acquired only transient resistance against CP84. The resistance led to cross-protection to one out of five other group II phages tested. Phage resistance was apparently neither caused by large genomic rearrangements nor by a CRISPR system. Binding assays demonstrated that CP84 could not adsorb to resistant C. coli clones suggesting a bacterial phage receptor to be involved in resistance. However, phage resistant C. coli clones did not reveal an altered motility or modified flaA sequence. Considering the loss of binding capacity and the reversion to a phage sensitive phenotype we hypothesize that acquired resistance depends on temporal phase variable switch-off modifications of the phage receptor genes, even though the resistance mechanism could not be elucidated in detail. We further speculate that even closely related phages of the same group use different bacterial receptors for binding on C. coli.

Genotypes and antibiotic resistance of bovine Campylobacter and their contribution to human campylobacteriosis.

Campylobacter jejuni and Campylobacter coli are the most important bacterial causes of human gastroenteritis. Chicken has been recognized as a major source for human infection, whereas cattle might also contribute to a lesser extent. However, there is a paucity of information available regarding Campylobacter in Swiss cattle and their role for human campylobacteriosis. To gain more information on genotypes and antibiotic resistance of bovine C. jejuni and C. coli and on their contribution to human disease, 97 cattle isolates were analysed. Multilocus sequence typing (MLST) and flaB typing were applied and the gyrA and 23S rRNA genes were screened for point mutations responsible for quinolone and macrolide resistance, respectively. A total of 37 sequence types (STs) and 44 flaB types were identified, including two sequence types and five flaB types not previously described. Most common sequence types were ST21 (21%), ST61 (12%) and ST48 (11%). Only one isolate was macrolide resistant while 31% (n = 30) were quinolone resistant. Source attribution indicated chicken as the main source of human infection with cattle being second. In conclusion, cattle should not be underestimated as a potential source of human campylobacteriosis.

Investigating the host specificity of Campylobacter jejuni and Campylobacter coli by sequencing gyrase subunit A.

BACKGROUND: Surveillance and field investigations of Campylobacter infections require molecular tools with genetic markers appropriate for tracing purposes, i.e. based on the principle that some Campylobacter lineages acquire a host signature under adaptive selection pressure. We developed a sequence-based method targeting the quinolone resistance determining region within the subunit A of DNA gyrase (gyrA). Host specificity was evaluated by characterizing two collections of Campylobacter jejuni (N = 430) and Campylobacter coli (N = 302) originating from surface waters, domestic mammals and poultry. RESULTS: Based on nucleotide identity, a total of 80 gyrA alleles were observed. Thirty nine alleles assigned to C. coli encoding two peptides fell into three clades: two associated with surface waters and one associated with domestic mammals and poultry. The variability in GC content generated by synonymous mutations suggested that surface waters isolates originated from two distinct ecological niches. A total of 42 alleles were recorded from C. jejuni strains and encoded 8 peptides including one lying in a distinct lineage associated with wildlife. Seven of the 23 alleles encoding peptide #1 displayed the synonymous mutation G408A not identified in poultry isolates. By contrast, the substitution Ser22Gly observed in 4 different peptide groups was significantly associated with domestic birds (P = 0.001). The change in amino acid sequences Thr86Ile conferring resistance to quinolones was significantly associated with poultry (P < 0.001) in both C. jejuni and C. coli with 38.7% and 67.9% of quinolone-resistant strains, respectively. CONCLUSIONS: The gyrA typing method presented here is an informative tool as sequences appear to be predictive of particular ecological niches. Combined with multi-locus sequence typing, it could increase the resolution of source attribution, and combined with porA/flaA typing it could be suitable for detecting temporal clusters of human cases. All gyrA alleles identified were deposited in the freely accessible online database http://pubmlst.org/campylobacter.

Validation according to ISO 16140:2003 of a commercial real-time PCR-based method for detecting Campylobacter jejuni, C. coli, and C. lari in foods.

Campylobacteriosis was the most frequently reported zoonosis in the European Union (EU) in 2010, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari as the most frequently reported species in foodborne outbreaks (FBOs). Relatively sensitive to environmental factors, these species may be present in low numbers. In line with EU policy for food control and FBO detection and in view of the need to reduce response time, we validated an alternative molecular method according to ISO 16140:2003 which establishes the general principle and technical protocol for the validation of alternative methods in the microbiological analysis of food. We used a qualitative real-time PCR commercial kit for the detection of C. jejuni, C. coli, and C. lari in two food categories "fruit and vegetable-based products" and "dairy products". The validation protocol comprises two phases: the first is a method comparison study of the alternative method against the reference method, and the second is an interlaboratory study of each of the two methods. In the first step, ISO 16140:2003 validation examines the following parameters: limit of detection (LOD); relative accuracy, relative specificity and sensitivity; relative detection level (RDL); and inclusivity and exclusivity. Except for LOD, inclusivity and exclusivity, the other steps were performed against the reference method (ISO 10272:2006). The LOD of the real-time PCR method was set at 4CFU/25g or mL for both food categories. Relative accuracy (98.33%), specificity (96.77%), and sensitivity (100%) were recorded for the food category "fruit and vegetable-based products" and 93.3%, 88.24%, 100%, respectively, for "dairy products". The RDL according to Fisher's exact test was p=1 for both food categories, for each level, and each food/strain combination. The interlaboratory study results showed correct identification of all 24 blind samples with both methods by all the participating laboratories. The results show that this commercial kit is suitable for the rapid detection of C. jejuni, C. coli, and C. lari on fruit, vegetables and dairy products and may aid in official controls. In conclusion, the use of alternative methods is recommended for the rapid identification of positive samples and the identification of the possible bacterial source in a FBO within 48 h.

Comparative analysis and limitations of ethidium monoazide and propidium monoazide treatments for the differentiation of viable and nonviable campylobacter cells.

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R(2)) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 µg/ml) and PMA (51.10 µg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10(4)) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.

Campylobacter infection in wild artiodactyl species from southern Spain: occurrence, risk factors and antimicrobial susceptibility.

A cross-sectional study was performed to assess the occurrence of Campylobacter species and to identify potential associated risk factors for wild artiodactyl species in southern Spain. Campylobacter species were isolated in 55 of 363 (15.2%) faecal samples. Campylobacter was identified in faeces from wild boar (49/126; 38.9%), red deer (5/179; 2.8%) and mouflon (1/13; 7.7%) but not from fallow deer (0/45). The isolated Campylobacter species were identified as C. jejuni (2 isolates; 3.6%), C. coli (11 isolates; 20.0%) and C. lanienae (37 isolates; 67.3%). Five isolates (9.1%) could not be identified at the species level. This report is the first to describe C. lanienae infection in wild ruminant species. Resistance to erythromycin (4.8%), ciprofloxacin (37.5%), tetracycline (52.9%) and streptomycin (55%) were detected. C. lanienae presented a significantly higher number of susceptible isolates to ciprofloxacin and tetracycline than C. coli. Due to the low number of positive wild ruminants, a Generalised Estimating Equations model was only carried out for wild boar. The model indicated that the risk factors associated with Campylobacter infection were the density of wild boar (>10/100ha) (OR: 3.05; CI95%: 2.2-4.3), the presence of artificial waterholes (OR: 3.67; CI95%: 1.3-10.5) and the winter season (OR: 3.30; CI95%: 1.9-5.8). Campylobacter infection is widespread in wild boar populations in southern Spain. These findings suggest that wild artiodactyls, particularly wild boar, constitute a reservoir of Campylobacter species, including resistant and multi-resistant strains, which may be of public health concern.

Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by qPCR.

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.