Molecular epidemiology and antimicrobial resistance of thermophilic campylobacter infections in humans and animals in Tanzania

Abstract

PhD ThesisMembers of the genus Campylobacter are known to cause more cases of human gastrointestinal illness than any other bacterium worldwide. The organisms exist as normal flora in the intestinal tracts of domestic and wild animals, more so in avian species. Humans acquire Campylobacter infections from contaminated animal products, particularly poultry meat, either directly or through cross-contamination of other food products. Human infections are mostly attributed to Campylobacter jejuni and C. coli, the former causing a larger proportion (85-90%) of all cases reported. In addition to infections, campylobacteriosis is also associated with the emerging threat of antimicrobial resistance as evidenced in isolates derived from different sources. An accurate picture of the epidemiology of infections caused by Campylobacter and other aetiological agents is lacking in developing countries due to the absence of regular surveillance programmes. Consequently the present study was conducted in Morogoro Municipality, Eastern Tanzania, to determine the molecular epidemiology and antimicrobial resistance of thermophilic Campylobacter isolates from humans and animals. Specific objectives were; 1) To establish the prevalence of thermophilic Campylobacter infections in humans and animals, 2) To determine the genetic relatedness of chicken and human derived thermophilic Campylobacter isolates using DNA-based typing methods, 3) To evaluate the antimicrobial resistance patterns in thermophilic Campylobacter isolates derived from humans and animals; and 4) To identify risk factors for thermophilic Campylobacter infections in humans. Stool samples were collected from 1195 human subjects; and fecal samples from 1511 farm animals, 466 laboratory animals and 112 wild birds (Indian house crows). Farm animals constituted chickens (n=1267), cattle (n=98), goats (n=81), sheep (n=57), horses (n=5) and camels (n=3); whereas laboratory animals were composed of guinea pigs (Cavia porcelllus, n= 30), mice (Mus musculus, n=160), rabbits (Oryctulagusiii cuniculus, n=34) and rats (Rattus rattus, n=242). The Cape Town protocol was used for isolation of thermophilic Campylobacter from stool and fecal samples. Campylobacter isolates were identified by phenotypic and molecular techniques. The isolates were tested for resistance against several antimicrobial agents using the disc diffusion method. Risk factors for human infections with thermophilic Campylobacter were determined in an unmatched case control study. Selected human and chicken derived Campylobacter jejuni isolates were genotyped using flagellin A gene sequencing. In humans the prevalence of thermophilic Campylobacter was 11.4% (n=1195). Symptomatic (12.9%) and young individuals (16.7%) were more infected than asymptomatic (6.7%) and adults (10%), respectively. Most (84.6%) of the isolates were C. jejuni and the remaining were C. coli; and the difference was statistically significant at p≤0.05. Isolates had highest resistance (95.6%) for colistin sulphate and lowest for ciprofloxacin (22.1%). Proportions of resistant isolates for other antibiotics (azithromycin, erythromycin, tetracycline, cephalethin, gentamycin, nalidixic acid, ampicillin, amoxycillin, norfloxacin and chloramphenicol) ranged from 44.1% to 89%. Human infections with thermophilic Campylobacter were associated with young age; and consumption of chicken meat, barbecue and pre-prepared salad. In avians, thermophilic Campylobacter spp. were isolated from 44.0% and 20.5% of the sampled chickens and crows respectively. The majority of isolates from both chickens (87.6%) and crows (56.5%) were C. jejuni and the remaining were C. coli. The observed difference in proportions of C. jejuni and C. coli isolates was statistically significant (p≤0.05) in chickens but not in house crows. Chicken isolates had highest resistance to Colistin sulphate whereas crow isolates showed highest resistance to azithromycin and erythromycin. Lowest resistance was observed for gentamycin and ciprofloxacin for crow and chicken isolates respectively. Among chicken isolates significantly high proportions of C. coli were resistant to gentamycin, cephalothin, tetracycline, colistin sulphate and chloramphenical. On the other hand a high proportion C. jejuni isolates were resistant toiv nalidixic acid. Crow derived C. jejuni had significantly higher resistance to nalidixic acid, cephalothin and ciprofloxacin than C. coli isolates from the same hosts. Among farm animals thermophilic Campylobacter were detected from 18 (31.6%) sheep and 3/5 (60%) of horses. Of the isolates 12 (57%) were C. jejuni; the remaining (43%) were C. coli. Of the laboratory animals 8 (26.7%) guinea pigs and 3 (1.2%) rats were colonized with Campylobacter. Four isolates from the guinea pigs were C. jejuni and the other 4 were C. coli. From the rats two isolates were C. jejuni and one was C. coli. The isolates showed high levels of antimicrobial resistance to erythromycin, norfloxacin colistin sulphate and nalidixic acid in ascending order; whereas low levels of resistance were observed for ciprofloxacin and gentamycin. Out of 55 sequenced isolates obtained from sporadic cases of human illness and different categories of chickens, nine different flaA types (7, 36, 41, 51, 61, 62, 64, 105 and 111) were detected. Both C. jejuni isolates from humans and chickens displayed a high degree of genetic diversity thereby suggesting weak clonality among the tested isolates. Genetic relatedness of some isolates from human and avian sources was however evident as on phylogenetic analysis some clusters contained both human and chicken C. jejuni isolates. The work contained in this thesis contributes significantly to the limited, available information on epidemiology and antimicrobial resistance of human and animal Campylobacter infections in Tanzania. For the first time the occurrence of Campylobacter infections in laboratory animals, antimicrobial resistance of human derived Campylobacter isolates, risk factors for human Campylobacter infections; and the population structure and relatedness of Campylobacter jejuni isolates from humans and chickens in the country are provided. The observed clusters containing isolates from human and avian sources confirm interspecies transmission of this zoonotic pathogen. Information on antimicrobial resistance of Campylobacter isolates derived from avian species in the country is also complemented. Control measures for colonization of animals and occurrence of infections in humans with this particular bacterium species arev warranted. Similarly strategies to stem emergency and spread of antimicrobial resistant Campylobacter strains should be put in place.Danish International Development Fun