Quadruplex qPCR for detection and discrimination of C. Coli,C. fetus, and C. Jejuni from other Campylobacter species in chicken and sheep meat.

Campylobacter is gram-negative bacteria considered the predominant genera isolated from poultry samples and associated with gastroenteritis. Due to the problems in conventional cultural methods of time-consuming and technically demanding requirements, a rapid and feasible method for their identification and discrimination of the closely related spp. Including Campylobacter coli, Campylobacter fetus, and Campylobacter jejuni is needed. This study analyzes the chicken and sheep meats samples (n = 125) using culture and pre-enrichment-based Quadraplex real-time PCR by targeting OrfA, CstA, HipO, and 16 S rRNA genes of C. coli, C. fetus, C. jejuni and Campylobacter spp. Respectively. The analysis of 125 chicken and sheep meat samples by culture and real-time PCR showed high concordance between the results of the two methods. The present study show high prevalence of Campylobacter species (35% and 32% from chicken and meat respectively) of which C. jejuni were the most abundant. Reaction efficiencies were between 90 and 110%, and detect as low as 8.9 fg in C. jejuni. The need for quick detection and discrimination methods in sheep and chicken meat can be met using the described Quadraplex real-time PCR methodology.

Genomic analysis of the diversity, antimicrobial resistance and virulence potential of clinical Campylobacter jejuni and Campylobacter coli strains from Chile.

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n = 81), collected in 2017-2019 in Santiago, Chile. This culture collection accounts for more than one third of the available genome sequences from South American clinical strains. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen.

High survival rates of Campylobacter coli under different stress conditions suggest that more rigorous food control measures might be needed in Brazil.

Campylobacter spp. have been the most commonly reported gastrointestinal bacterial pathogen in many countries. Consumption of improperly prepared poultry meat has been the main transmission route of Campylobacter spp. Although Brazil is the largest exporter of poultry meat in the world, campylobacteriosis has been a neglected disease in the country. The aim of this study was to characterize 50 Campylobacter coli strains isolated from different sources in Brazil regarding the frequency of 16 virulence genes and their survival capability under five different stress conditions. All strains studied presented the cadF, flaA, and sodB genes that are considered essential for colonization. All strains grew at 4 °C and 37 °C after 24 h. High survival rates were observed when the strains were incubated in BHI with 7.5% NaCl and exposed to acid and oxidative stress. In conclusion, the pathogenic potential of the strains studied was reinforced by the presence of several important virulence genes and by the high growth and survival rates of the majority of those strains under different stress conditions. The results enabled a better understanding of strains circulating in Brazil and suggest that more rigorous control measures may be needed, given the importance of contaminated food as vehicles for Campylobacter coli.

[Multilocus Sequence Typing analysis of human Campylobacter coli in Granada (Spain)].

OBJECTIVE: Different subtypes of Campylobacter spp. have been associated with diarrhoea and a Multilocus Sequence Typing (MLST) method has been performed for subtyping. In the present work, MLST was used to analyse the genetic diversity of eight strains of Campylobacter coli. METHODS: Nineteen genetic markers were amplified for MLST analysis: AnsB, DmsA, ggt, Cj1585c, CJJ81176-1367/1371, Tlp7, cj1321-cj1326, fucP, cj0178, cj0755/cfrA, ceuE, pldA, cstII, cstIII. After comparing the obtained sequences with the Campylobacter MLST database, the allele numbers, sequence types (STs) and clonal complexes (CCs) were assigned. RESULTS: The 8 C. coli isolates yielded 4 different STs belonging to 2 CCs. Seven isolates belong to ST-828 clonal complex and only one isolate belong to ST-21. Two samples came from the same patient, but were isolated in two different periods of time. CONCLUSIONS: MLST can be useful for taxonomic characterization of C. coli isolates.

Campylobacter species isolated from poultry and humans, and their analysis using PFGE in southern Brazil.

Campylobacteriosis is a bacterial disease transmitted to humans through ingestion of contaminated food. Six hundred samples were collected, 200 from human stool samples, 200 from poultry products and 200 from poultry feces in Southern Brazil, and then inoculated on blood agar plates. A total of 58% of the poultry feces, 17% of the poultry meat, and 2% the of human stools tested positive for Campylobacter. Positive Campylobacter colonies were identified as Campylobacter jejuni or Campylobactercoli by multiplex PCR. Campylobacter isolates were analyzed using PFGE to compare different profiles according to the source. This study demonstrated that there are different Campylobacter clones distributed in different aviaries in Southern Brazil. In addition, PFGE molecular profiles suggested that broilers can be a source of contamination of poultry products. However, the human isolate studied did not show any relationship with other strains examined.

Genotyping of Campylobacter coli strains isolated in Brazil suggests possible contamination amongst environmental, human, animal and food sources.

Campylobacter coli and Campylobacter jejuni are two of the most common causative agents of food-borne gastroenteritis in numerous countries worldwide. In Brazil, campylobacteriosis is under diagnosed and under-reported, and few studies have molecularly characterized Campylobacter spp. in this country. The current study genotyped 63 C. coli strains isolated from humans (n512), animals (n521), food (n510) and the environment (n520) between 1995 and 2011 in Brazil. The strains were genotyped using pulsed-field gel electrophoresis (PFGE), sequencing the short variable region (SVR) of the flaA gene ( flaA-SVR) and high-resolution melting analysis (HRMA) of the clustered regularly interspaced short palindromic repeat (CRISPR) locus to better understand C. coli genotypic diversity and compare the suitability of these three methods for genotyping this species. Additionally, the discrimination index (DI) of each of these methods was assessed. Some C. coli strains isolated from clinical and non-clinical origins presented ≥80 % genotypic similarity by PFGE and flaA-SVR sequencing. HRMA of the CRISPR locus revealed only four different melting profiles. In total, 22 different flaA-SVR alleles were detected. Of these, seven alleles, comprising gt1647­gt1653, were classified as novel. The most frequent genotypes were gt30 and gt1647. This distribution reveals the diversity of selected Brazilian isolates in comparison with the alleles described in the PubMLST database. The DIs for PFGE, flaA­SVR sequencing and CRISPR-HRMA were 0.986, 0.916 and 0.550, respectively. PFGE and flaA-SVR sequencing were suitable for subtyping C. coli strains, in contrast to CRISPR-HRMA. The high genomic similarity amongst some C. coli strains confirms the hypothesis that environmental and food sources potentially lead to human and animal contamination in Brazil.

Detection of CDT toxin genes in Campylobacter spp. strains isolated from broiler carcasses and vegetables in São Paulo, Brazil

Campylobacteriosis is a worldwide distributed zoonosis. One of the main virulence factors related to Campylobacter spp. in animals and humans is the cytolethal distending toxin (CDT), encoded by three adjacent genes (cdtA, cdtB, cdtC). The occurrence of Campylobacter spp. in samples of vegetables has not been reported in Brazil yet, and has seldom been described in the international literature. The detection of CDT in these strains has not been reported, either. The objectives of the present study were to determine the occurrence of Campylobacter spp. strains carrying virulence factors in samples of poultry and vegetables (lettuce and spinach) from different points of sale, thus verifying if vegetables are as an important vehicle for potentially virulent Campylobacter spp. strains as poultry. Twenty four strains were identified as Campylobacter jejuni by phenotypic and genotypic methods: 22 from broiler carcasses and two from lettuce samples. Three strains were identified as Campylobacter coli: two from broiler carcasses and one from lettuce. The presence of the cdt genes were detected in 20/24 (83.3%) C. jejuni strains, and 3/3 (100%) C. coli strains. The isolation of Campylobacter spp. strains with the cdt gene cluster in lettuce samples points to a new possible source of contamination, which could have an impact in the vegetable production chain and risk to public health. Results show that potentially virulent C. jejuni and C. coli strains remain viable in samples of broiler carcasses and vegetables at the points of sale.

Detection of CDT toxin genes in Campylobacter spp. strains isolated from broiler carcasses and vegetables in São Paulo, Brazil.

Campylobacteriosis is a worldwide distributed zoonosis. One of the main virulence factors related to Campylobacter spp. in animals and humans is the cytolethal distending toxin (CDT), encoded by three adjacent genes (cdtA, cdtB, cdtC). The occurrence of Campylobacter spp. in samples of vegetables has not been reported in Brazil yet, and has seldom been described in the international literature. The detection of CDT in these strains has not been reported, either. The objectives of the present study were to determine the occurrence of Campylobacter spp. strains carrying virulence factors in samples of poultry and vegetables (lettuce and spinach) from different points of sale, thus verifying if vegetables are as an important vehicle for potentially virulent Campylobacter spp. strains as poultry. Twenty four strains were identified as Campylobacter jejuni by phenotypic and genotypic methods: 22 from broiler carcasses and two from lettuce samples. Three strains were identified as Campylobacter coli: two from broiler carcasses and one from lettuce. The presence of the cdt genes were detected in 20/24 (83.3%) C. jejuni strains, and 3/3 (100%) C. coli strains. The isolation of Campylobacter spp. strains with the cdt gene cluster in lettuce samples points to a new possible source of contamination, which could have an impact in the vegetable production chain and risk to public health. Results show that potentially virulent C. jejuni and C. coli strains remain viable in samples of broiler carcasses and vegetables at the points of sale.

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR.

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada.

AIMS: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA-based typing methods for the characterization of these isolates. METHODS AND RESULTS: Isolates were speciated with two multiplex PCR assays and were typed with flaA-RFLP, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson's index of diversity, D=0.9061). However, the combination of flaA-RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D=0.9857). A band position tolerance of 4% in BioNumerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA-RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST-828 clonal complex. CONCLUSIONS: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry-associated Camp. coli STs by phylogenetic analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.

Molecular characterization of invasive and noninvasive Campylobacter jejuni and Campylobacter coli isolates.

Campylobacter jejuni is one of the most common causes of bacterial diarrhea worldwide and is the primary bacterial cause of food-borne illness. Adherence to and invasion of epithelial cells are the most important pathogenic mechanisms of Campylobacter diarrhea. Molecular characterization of invasive and noninvasive Campylobacter isolates from children with diarrhea and symptom-free children was performed by random amplified polymorphic DNA techniques (RAPD). A distinct RAPD profile with a DNA band of 1.6 kb was observed significantly more frequently among invasive (63%) than among noninvasive (16%) Campylobacter isolates (P = 0.000005). The 1.6-kb band was named the invasion-associated marker (IAM). Using specifically designed primers, a fragment of 518 bp of the iam locus was amplified in 85% of invasive and 20% of noninvasive strains (P = 0.0000000). Molecular typing with a PCR-restriction fragment length polymorphism assay which amplified the entire iam locus showed a HindIII restriction fragment polymorphism pattern associated mainly with invasive strains. Although cluster analysis of the RAPD fingerprinting showed genetic diversity among strains, two main clusters were identified. Cluster I comprised significantly more pathogenic and invasive isolates, while cluster II grouped the majority of nonpathogenic, noninvasive isolates. These data indicate that most of the invasive Campylobacter strains could be differentiated from noninvasive isolates by RAPD analysis and PCR using specific primers that amplify a fragment of the iam locus.

Prevalence of thermotolerant species of Campylobacter and their biotypes in children and domestic birds and dogs in southern Chile.

The prevalence of thermotolerant Campylobacter in diarrhoeic and healthy children as well as in dogs, hens, ducks and pigeons was determined in Southern Chile. Campylobacter were found in 34.5% of the faecal samples examined. The isolation rate of Campylobacter in diarrhoeic and healthy children was 16.3% and 6.4% respectively. Despite C. jejuni was always more frequent than C. coli, the latter was isolated with a high frequency (29%) from patients with diarrhoea. C. jejuni and C. coli biotypes I and II were found in healthy and diarrhoeic children and were predominant in all the animals species studied. This may point out towards the possible origin of strains infecting children.