Phenotypic and Genotypic Characterization of Salmonella Enterica Isolates in Ibadan, Nigeria.
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos.
Salmonella is an increasingly important foodborne pathogen with widespread resistance to antibiotics. Control measures entail identification of serovars of public health importance, institution of measures against sources and antibiotic surveillance to limit spread. There is scarce information about Salmonella serovars, sources and antibiotic resistant profiles in Nigeria. Data about medically important Salmonella serovars and their sources are necessary for targeted control measures to be put in place in the country to safeguard public health. This study was designed to determine the sources and clinical burden of Salmonella enterica coupled with serovar evaluation, determination of antibiotic resistance profiles and molecular determinants. Samples collected and analysed by culture for Salmonella isolation included 984 blood samples from febrile patients and 2,910 faecal samples from healthy people. Faecal samples were also collected from food animals: 1,201 from chickens in poultry farms, 500 from cattle, 500 from goats, 560 from pigs and 500 from sheep and 18 environmental samples. Serotyping by slide agglunitination was used to identify Salmonella serovars. Isolates were tested for antibiotic susceptibility by determination of minimum inhibitory concentration using the broth micro-dilution method. Resistance genes and integrons were screened by polymerase chain reaction and DNA sequencing. Molecular subtyping of some of the isolates to determine clonal relatedness was done by pulsed-field gel electrophoresis. Salmonella enterica accounted for 23% of bacteremia cases in patients with a prevalence of 5.0% and overall mortality of 12 %. Majority (92%) of the Salmonella bacteremic cases were found in children under 15 years of age. The prevalence rate in male patients (6.0%) was higher than in the females (3.8%) but this was statiscally insignificant (P=0.17; at P < 0.5%). The prevalence in healthy people was 8.6%. The prevalence rates in the food animals ranged from 6% (cattle) to 15% (goat) and all the environmental samples yielded Salmonella. The nine serovars found in the bacteremic patients were different from the nine serovars found in chickens. The serovars from the patients consisted of 82% non-typhoidal and 18% typhoidal; predominant serovars were S. Enteritidis (33%), S. Dublin (18%) and S. Typhimurium (18%). The predominant serovars in chicken were S. Virchow (44%) and S. Kentucky (39%). Resistance to chloramphenicol, ampicillin and cotrimoxazole ranged from 28.6% to 46.9% in the patient isolates. The isolates from chicken showed high frequency of resistance to ciprofloxacin (30%) with a high proportion also showing reduced susceptibility to ciprofloxacin (37%). All the ciprofloxacin resistant strains were S. Kentucky; the reduced susceptibility to ciprofloxacin was observed in seven of the nine serovars. There was also high resistance to nalidixic acid (64.8%), gentamicin (35.2%) and tetracycline (66.1%). Among the animals, chicken harbored most of the resistant strains. Majority of the isolates in both patients (71%) and chickens (72%) showed multi-drug resistance (resistance to ≥ 2 antibiotics). βeta-lactamase enzymes encoded by TEM gene were the main determinant mediating resistance to the β-lactam antibiotics in the Salmonella isolates. There was also high prevalence of class 1 integrons in resistant strains: 75% (patients) and 77% (chickens), with no isolates habouring multiple integrons. Sequencing of an integron amplicon from S. Kentucky revealed antibiotic resistance gene cassette aaCA5 that mediates resistance to gentamicin. Pulsed-field gel electrophoresis analysis of the ciprofloxacin-resistant isolates revealed limited genetic diversity with 40 (91%) of them constituting six different clusters; each cluster encompassed isolates from chicken fecal samples from more than one poultry farm thus indicating dissemination of the same strains in the poultry farms. This study highlights the involvement of several serovars of Salmonella in bacteremia predominated by S. Enteritidis; the involvement of rarely isolated serovars previously associated with reptiles and S. Dublin is noteworthy. Chickens are sources of Salmonella serovars of potential public health importance which may not be associated with bacteremia. The high rates of resistance to antibiotics, particularly to critical antibiotics like ciprofloxacin and gentamicin in chicken, is a public health concern. The sources of bacteremic serovars need to be identified and appropriate control measures initiated against them and in the poultry industry to prevent spread.