Genotyping and antibiotic resistance of thermophilic Campylobacter isolates from Vietnam and Kenya
Thermophilic Campylobacter are well recognized as the leading cause of bacterial foodborne gastroenteritis worldwide. Campylobacter are microaerophilic growing, Gram-negative, curved corkscrew shaped and show motility. They colonize the intestine of many wild and domestic animals, particularly that of poultry. The review on Campylobacter shows specific aspects of Campylobacter/campylobacteriosis including: (i) taxonomy of the genus Campylobacter, growth and survival characteristics; (ii) detection, isolation and confirmation of Campylobacter; (iii) poultry as natural hosts for Campylobacter species as zoonotic pathogens and (iv) antimicrobial resistance in Campylobacter (Chapter 1). Knowledge on Campylobacter is limited in many developing countries, particularly in Southeast Asia. The review covering published articles from 1971 to 2016 in Cambodia, Laos and Vietnam showed them as neglected bacterial pathogens. Literature on prevalence of thermophilic Campylobacter in humans showed a prevalence up to 11% in Vietnam while it was even higher in Cambodia and Laos. Especially, children under five years of age were affected. Animals and food as source for human infections play an important role. Carriage of Campylobacter by different animal species and contamination rate of meat are generally high and can reach more than 70%. Resistance to antibiotics is of public health concern. High rates of resistance to nalidixic acid, erythromycin, tetracycline and ciprofloxacin were detected in up to 100% of isolates (Chapter 2). Nine isolates (eight C. jejuni and one C. coli) isolated from meat were identified by multiplex PCR, and tested for the presence or absence of 29 gene loci associated with virulence, lipooligosaccharide (LOS) biosynthesis and further functions. flaA typing, multilocus sequence typing and investigation by microarray assay showed a high degree of genetic diversity among these isolates. In all isolates motility genes (flaA, flaB, flhA, fliM), colonization-associated genes (cadF, docB), toxin genes (cdtA, cdtB, secD, secF), and LOS biosynthesis gene pglB were detected. Eight gene loci could not be detected by PCR. Different gene loci for ciaB (22.2 %), Cje1280 (77.8 %), docC (66.7 %), and cgtB (55.6 %) were found. iamA, cdtC, and the type 6 secretion system were present in all C. jejuni isolates but not in C. coli. flaA typing resulted in five different genotypes within C. jejuni, MLST classified the isolates into seven sequence types. The microarray assay analysis showed also high genetic diversity within Vietnamese Campylobacter isolates which resulted in eight different types for C. jejuni. Antibiotic susceptibility profiles showed that all isolates were sensitive to gentamicin and most isolates (88.8 %) were sensitive to chloramphenicol, erythromycin and streptomycin. Resistance rates to nalidixic acid, tetracycline and ciprofloxacin were 88.9 %, 77.8 % and 66.7 %, respectively. This is the first report that shows high genetic diversity and remarkable antibiotic resistance of Campylobacter strains isolated from meat in Vietnam. These strains can be considered of high public health significance (Chapter 3). Antibiotic susceptibility of thermophilic Campylobacter isolates which were collected from Kenya were tested using the broth microdilution assay. Molecular biological detection of genes associated with resistance completed the results. Thermophilic Campylobacter was identified in 53 samples by PCR (34 C. jejuni, 18 C. coli and one mix of both species) but only 35 Campylobacter isolates (31 C. jejuni and 4 C. coli) could be recultivated after transportation to Germany. C. jejuni isolates showed a high rate of resistance to nalidixic acid, tetracycline and ciprofloxacin of 77.4 %, 71.0 % and 71.0 %, respectively. Low resistance (25.8 %) was detected for gentamicin and chloramphenicol. Multidrug resistance in C. jejuni could be detected in 19 (61.3 %) isolates. Resistance patterns of C. coli isolates were comparable. Resistance to ciprofloxacin was confirmed by MAMA-PCR and PCR-RFLP in all phenotypically resistant isolates. The tet(O) gene was detected only in 54.5 % of tetracycline resistant C. jejuni isolates. The tet(A) gene, which is also responsible for tetracycline resistance, was found in 90.3% of C. jejuni and in all C. coli isolates. Thirteen phenotypically erythromycin resistant isolates could not be characterized by using PCR-RFLP and MAMA-PCR. This study showed a high level of resistance to ciprofloxacin, nalidixic acid and tetracycline but also a remarkable one to chloramphenicol and gentamicin and multidrug resistance seems to be a prevalent problem. Resistance to antibiotics is of global public health concern. In Kenya, resistance surveillance needs further attention (Chapter 4).
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