Campylobacteriosis is the most commonly reported food-borne zoonosis in the European Union (EU) since 2005. The main source of infection is the consumption of chicken meat contaminated with Campylobacter (mainly C. jejuni and C. coli). The high prevalence of Campylobacter in broilers on farm represents a high risk of contamination of the final product and thereby the risk of infection for the consumer. Therefore, it is a priority for the EU to prevent and reduce the number of Campylobacter positive batches at slaughter age. For this, it is necessary to gain insight into the behaviour and epidemiology of this pathogen. Thus, within the framework of this PhD thesis we performed a number of studies: (i) we studied the diversity and dynamics of Campylobacter strains from broilers on farm; (ii) we characterized the isolates in terms of resistance against a panel of antimicrobials and the presence of virulence-associated genes; (iii) we characterized in depth a selection of isolates by whole genome sequencing, analysing the mechanisms for resistance, the distribution of virulence-associated genes, the genotypes and their phylogenetic relationship. The isolates of these studies came from a two-year longitudinal study, performed in five different farms from Catalonia. From the Campylobacter-positive crops, randomly selected isolates from different birds along the rearing cycles were preserved. In order to study in depth the diversity, seasonality and dynamics of Campylobacter strains circulating in farms, molecular typing of the isolates was performed using Pulsed Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). By PFGE and using two restriction enzymes, a total of 343 isolates were analysed (C. jejuni n = 254 and C. coli n = 89) and overall 92 macrorestriction profiles (between 12 and 24 profiles per farm) were identified. Overall, a large genotypic diversity was detected, identifying mostly between 1 and 2 different genotypes per crop, although not isolated from successive crops. On the other hand, it was observed persistent genotypes within the same crop along the rearing cycle. When comparing the isolates from different farms, common C. jejuni genotypes among different farms were found. A total of 127 isolates were selected (93 C. jejuni and 34 C. coli) from the different PFGE genotypes, which were analysed by MLST, and high genetic diversity was observed as well. The C. jejuni isolates were grouped in 15 different clonal complexes (CC) distributed in 34 sequence types (ST), whereas the C. coli isolates were all grouped within the CC828, distributed in 12 STs. In addition, we identified five novel STs of C. jejuni and two of C. coli. Certain seasonality was observed for some CC. The CC21 and CC206 were found along the year, except during the coldest trimester, being more commonly found during the warmer months. In the second study, we evaluated the susceptibility of the 344 isolates against a panel of 12 antimicrobials of different families using the disc diffusion method. Resistance to quinolones (nalidixic acid and ciprofloxacin) was the most frequent, followed by tetracyclines. This represents a major public health problem, given that these are the drugs of choice for treating human enteric infections. In contrast, resistance against erythromycin and gentamycin were the less prevalent. All the analysed isolates were sensitive to chloramphenicol, amoxicillin + clavulanate, imipenem and meropenem. A 62,2% of all the isolates were multidrug resistant, being nalidixic acid, ciprofloxacin, enrofloxacin, tetracycline, doxicycline and ampicillin, the most frequent profile. On the other hand, 14 virulence genes involved in colonization and infection were analysed, and a heterogeneous distribution in the analysed isolates was observed. It is worth noting the detection of the wlaN gene in 19% of the isolates; this gene is involved in the Guillain-Barré syndrome, a serious neurological syndrome that may develop after Campylobacter infection. In the last study, whole genome sequencing using the Illumina platform was performed in 16 C. jejuni and C. coli isolates for their in-depth characterization. High throughput sequencing has become a powerful, fast and accessible tool for characterizing pathogens such as Campylobacter in epidemiological studies. Data obtained in this PhD thesis confirms the genetic diversity of Campylobacter in broilers and the great complexity of strain dynamics on farm. This may be the main factor that hampers effective control strategies of this bacterium on farm. Likewise, the high frequency of resistant and multiresistant strains isolated from broilers highlight the importance of making the producers an d/or managers of intensive poultry production aware about the prudent use of antimicrobials. Effective biosecurity measures to control Campylobacter in the farm, not only would reduce the high prevalence of positive batches at slaughter age, but indirectly would also improve the overall sanitary status of the farms. This would lower the need for the therapeutic use of antibiotics and would probably lead to a reduction in resistant strains.
|Date of Award||25 Jul 2017|
|Supervisor||Marta Cerdà-Cuéllar (Director)|