Persistence of Campylobacter spp. in Poultry Flocks after Disinfection, Virulence, and Antimicrobial Resistance Traits of Recovered Isolates.

To investigate the persistence risk of Campylobacter spp. in poultry farms, and to study the virulence and antimicrobial resistance characteristics in the recovered strains, we collected 362 samples from breeding hen flocks, before and after disinfection. The virulence factors were investigated by targeting the genes; flaA, cadF, racR, virB11, pldA, dnaJ, cdtA, cdtB, cdtC, ciaB, wlaN, cgtB, and ceuE by PCR. Antimicrobial susceptibility was tested and genes encoding antibiotic resistance were investigated by PCR and MAMA-PCR. Among the analyzed samples, 167 (46.13%) were positive for Campylobacter. They were detected in 38.7% (38/98) and 3% (3/98) of environment samples before and after disinfection, respectively, and in 126 (75.9%) out of 166 feces samples. In total, 78 C. jejuni and 89 C. coli isolates were identified and further studied. All isolates were resistant to macrolids, tetracycline, quinolones, and chloramphenicol. However, lower rates were observed for beta-lactams [ampicillin (62.87%), amoxicillin-clavulanic acid (47.3%)] and gentamicin (0.6%). The tet(O) and the cmeB genes were detected in 90% of resistant isolates. The blaOXA-61 gene and the specific mutations in the 23S rRNA were detected in 87% and 73.5% of isolates, respectively. The A2075G and the Thr-86-Ile mutations were detected in 85% and 73.5% of macrolide and quinolone-resistant isolates, respectively. All isolates carried the flaA, cadF, CiaB, cdtA, cdtB, and cdtC genes. The virB11, pldA, and racR genes were frequent in both C. jejuni (89%, 89%, and 90%, respectively) and C. coli (89%, 84%, and 90%). Our findings highlight the high occurrence of Campylobacter strains exhibiting antimicrobial resistance with potential virulence traits in the avian environment. Thus, the improvement of biosecurity measures in poultry farms is essential to control bacterial infection persistence and to prevent the spread of virulent and resistant strains.

Relationships between Virulence Genes and Antibiotic Resistance Phenotypes/Genotypes in Campylobacter spp. Isolated from Layer Hens and Eggs in the North of Tunisia: Statistical and Computational Insights.

Globally, Campylobacter is a significant contributor to gastroenteritis. Efficient pathogens are qualified by their virulence power, resistance to antibiotics and epidemic spread. However, the correlation between antimicrobial resistance (AR) and the pathogenicity power of pathogens is complex and poorly understood. In this study, we aimed to investigate genes encoding virulence and AR mechanisms in 177 Campylobacter isolates collected from layer hens and eggs in Tunisia and to assess associations between AR and virulence characteristics. Virulotyping was determined by searching 13 virulence genes and AR-encoding genes were investigated by PCR and MAMA-PCR. The following genes were detected in C. jejuni and C. coli isolates: tet(O) (100%/100%), blaOXA-61 (18.82%/6.25%), and cmeB (100%/100%). All quinolone-resistant isolates harbored the Thr-86-Ile substitution in GyrA. Both the A2074C and A2075G mutations in 23S rRNA were found in all erythromycin-resistant isolates; however, the erm(B) gene was detected in 48.38% and 64.15% of the C. jejuni and C. coli isolates, respectively. The machine learning algorithm Random Forest was used to determine the association of virulence genes with AR phenotypes. This analysis showed that C. jejuni virulotypes with gene clusters encompassing the racR, ceuE, virB11, and pldA genes were strongly associated with the majority of phenotypic resistance. Our findings showed high rates of AR and virulence genes among poultry Campylobacter, which is a cause of concern to human health. In addition, the correlations of specific virulence genes with AR phenotypes were established by statistical analysis.

Brucella spp. Contamination in Artisanal Unpasteurized Dairy Products: An Emerging Foodborne Threat in Tunisia.

Brucellosis is a worldwide zoonotic disease transmitted to humans, predominantly by the consumption of contaminated raw milk and dairy products. This study aimed to investigate the occurrence of Brucella spp. in 200 raw milk, ricotta, and artisan fresh cheese samples, collected from individual marketing points in four districts in Tunisia. Samples were analyzed for the presence of Brucella spp. by IS711-based real-time PCR assay. Positive samples were further analyzed by qPCR for B. melitensis and B. abortus species differentiation. The DNA of Brucella spp. was detected in 75% of the samples, B. abortus was detected in 31.3%, and B. melitensis was detected in 5.3% of positive samples. A percentage of 49.3% of samples co-harbored both species, while 14% of the Brucella spp. positive samples were not identified either as B. abortus or B. melitensis. High contamination rates were found in ricotta (86.2%), cheese (69.6%), and raw milk (72.5%) samples. The study is the first in Tunisia to assess the occurrence of Brucella spp. contamination in artisanal unpasteurized dairy products and showed high contamination rates. The detection of both B. abortus and B. melitensis highlights that zoonotic high-pathogen agent control remains a challenge for food safety and consumer health protection and could represent a serious emerging foodborne disease in Tunisia.

Virulence Profiling, Multidrug Resistance and Molecular Mechanisms of Campylobacter Strains from Chicken Carcasses in Tunisia.

Antibiotic resistance in foodborne pathogens is an emergent global health concern. The objectives of this study were to assess antimicrobial resistance (AMR) in Campylobacter isolates from chicken carcasses and to investigate the AMR molecular mechanisms as well as the presence of virulence determinants. The study was performed on 257 samples collected from abattoirs and retail shops in northeastern Tunisia. Forty-eight Campylobacter isolates were recovered and identified as C. jejuni (n = 33) and C. coli (n = 15). Antibiotic resistance was tested against eight antibiotics and high resistance rates were observed against tetracycline (100%), erythromycin (97.9%), ciprofloxacin (73%), nalidixic acid (85.4%), ampicillin (83.3%), amoxicillin/clavulanic acid (22.9%), chloramphenicol (75%), and gentamicin (27.1%). All isolates were multidrug-resistant, and 22 resistance patterns were found. All isolates were screened for AMR genes (tet(O), tet(A), tet(B), tet(L), cmeB, ermB, blaOXA-61, and aphA-3), and for point mutations in gyrA (C257T substitution) and 23SrRNA (A2075G/A2074C) genes. All screened AMR genes, as well as the C257T and the A2075G mutations, were detected. The virulence genotypes were also determined, and all isolates carried the motility (flaA) and invasion (cadF) genes. Most of them also harbored the cdtA, cdtB, and cdtC genes, encoding the Campylobacter toxin. The screening of the cgtB and the wlaN genes, involved in Guillain-Barré Syndrome expression, revealed the presence of the cgtB in 21.2% of C. jejuni strains, whereas none of them carried the wlaN gene. Our findings highlight the emergence of Campylobacter strains simultaneously harboring several virulence and AMR determinants, which emphasizes the risk of transmission of MDR strains to humans via the food chain. Hence, controlling the dissemination of foodborne pathogens "from the farm to the fork" as well as restricting the use of antimicrobials in husbandry are mandatory to prevent the risk for consumers and to mitigate the dissemination of MDR pathogens.

Campylobacter spp. in Eggs and Laying Hens in the North-East of Tunisia: High Prevalence and Multidrug-Resistance Phenotypes.

Despite the importance of eggs in the human diet, and unlike other products, for which food safety risks are widely investigated, information on the occurrence of Campylobacter and antimicrobial resistance in eggs and layer hen flocks is lacking in Tunisia. This study was conducted to determine the occurrence of Campylobacter and the antimicrobial resistance in layer hens and on eggshells. Thus, 366 cloacal swabs and 86 eggshell smear samples were collected from five layer hen farms in the North-East of Tunisia. The occurrence of Campylobacter infection, and the antimicrobial resistance rates and patterns, were analyzed. The occurrence rates of Campylobacter infection in laying hens and eggshells were 42.3% and 25.6%, respectively, with a predominance of C. jejuni (68.4%, 81.9%), followed by C. coli (31.6%, 18.2%). The antimicrobial susceptibility testing revealed high resistance rates against macrolides, tetracycline, quinolones, ß-lactams, and chloramphenicol, with percentages ranging from 35.5% to 100%. All isolates were multidrug resistant (MDR) and five resistance patterns were observed. These results emphasized the risk to consumer health and the need to establish a surveillance strategy to control and prevent the emergence and the spread of resistant strains of Campylobacter in poultry and humans.

Distribution of virulence and antibiotic resistance genes in Campylobacter jejuni and Campylobacter coli isolated from broiler chickens in Tunisia.

BACKGROUND: Thermo-tolerant Campylobacter species are the major cause of foodborne diseases worldwide. This study aimed to evaluate the prevalence of virulence genes and antibiotic resistance determinants in Campylobacter jejuni and Campylobacter coli isolates, and to investigate the relationship between these two traits. METHODS: A total of 132 Campylobacter isolates from poultry were tested for the presence of 13 virulence genes; flaA, cadF, racR, virB11, pldA, dnaJ, cdtA, cdtB, cdtC, ciaB, wlaN, cgtB and ceuE. The mechanisms underlying antibiotic resistance phenotypes were also studied by PCR and MAMA-PCR. RESULTS: PCR results revealed the presence of antimicrobial resistance genes in C. jejuni and C. coli as follows: cmeB (80% and 100%), tet(O) (100% and 80%), and the blaOXA-61 (81% and 93%), respectively. None of these strains harbored the aphA-3 gene. The Thr-86-Ile mutation associated with resistance to quinolones was found in 90% of C. jejuni and 80% of C. coli isolates. While the A2075G and A2074C mutations linked to the erythromycin resistance were detected in 100% of both species. Virulence genes were prevalent and ranged from 40 to 100%. A positive relationship was revealed between cadF, racR, and ciaB genes and resistance to ampicillin, amoxicillin/clavulanic acid, chloramphenicol, and nalidixic acid, in C. jejuni. However, no association was observed for C. coli isolated strains. CONCLUSION: This study provides for the first time an overview of antibiotic resistance mechanisms and pathogenic profiles of Campylobacter isolates, which emphasizes the potential risk for consumer health.

Prevalence and Antibiotic Resistance Patterns of Campylobacter spp. Isolated from Broiler Chickens in the North of Tunisia.

The aim of the current study is to assess the prevalence of Campylobacter infection in broiler chickens, raised in intensive production conditions, and to evaluate the antimicrobial susceptibility of recovered Campylobacter isolates. A total of 590 cloacal swab samples were taken from 13 broiler chicken flocks in the North East of Tunisia. All samples were tested for the presence of thermophilic Campylobacter by culture and PCR, targeting the mapA and ceuE genes, respectively. Susceptibility to antimicrobial drugs was tested against 8 antibiotics. Prevalence of Campylobacter infection, relationship with geographic origins and seasons, antimicrobial resistance rates and patterns were analyzed. Total prevalence of Campylobacter infection in broiler flocks was in the range of 22.4%, with a predominance of C. jejuni (68.9%), followed by C. coli (31.1%). Positive association was highlighted between the infection level and the season (P < 0.001), but no link was emphasized considering the geographic origin. Antimicrobial susceptibility testing revealed very high resistance rates detected against macrolide, tetracycline, quinolones, and chloramphenicol, ranging from 88.6% to 100%. Lower resistance prevalence was noticed for ß-lactams (47% and 61.4%) and gentamicin (12.9%). 17 R-type patterns were observed, and a common pattern was found in 30.3% of isolates. This study provides updates and novel data on the prevalence and the AMR of broiler campylobacters in Tunisia, revealing the occurrence of high resistance to several antibiotics and emphasizing the requirement of better surveillance and careful regulation of antimicrobials use.

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing.

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). RESULTS: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. CONCLUSION: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.