Molecular Epidemiology of enterotoxigenic escherichia coli and other enteric pathogenic bacteria isolated in Machakos District Hospital Machakos County.

Abstract

Diarrhoeal disease is a worldwide public health problem mostly affecting children, the immunocompromised and elderly in developing countries. Among children in developing countries, Enterotoxigenic Escherichia coli (ETEC) alone is responsible for more than 650 million diarrhoeal episodes and 2,000,000 deaths annually. However, in most developing countries including Kenya, there is a paucity of data on prevalence and characterictics of specific bacterial etiologies of diarrhoeal illness. Diarrhoea outbreaks have been common on yearly basis in Kenya. This study was carried out from September 2006 to March 2008 in Machakos District Hospital, a referral hospital serving most of the lower part of the former Eastern Province. Its catchment includes the neighbouring divisional and distict hospitals in Kitui, Mwingi and Mbooni. It has a bed capacity of 250. During the study, a total of 301 participants were enrolled following specific inclusion criteria. The participants ranged in age from 4 months to 85 years with a median age of 14 years and 7 months. The study was designed to detect bacterial pathogens associated with diarrhoea and in particular ETEC. Feacal specimens were collected and cultured in selective and differential media and E. coli phenoptypes identified using standard biochemical methods. A mixture of eight specific primers were used in a single PCR (Multiplex Polymerase Chain Reaction; mPCR) to detect diarrhoeagenic E. coli pathogotypes. Ninety eight isolates from thirty three out of 300 participants (11%) identified as ETEC positive were subcultured on Colonising Factor Antigen Agar (CFA) and analysed for hydrophobicity as presumptive indication of CFA presence. This gave a presumptive confirmation for CFA and Coli Surface Antigens (CS) which were then analysed using another set of mPCR with specific primers. Other bacterial agents were biochemically identified. They included S. dysenteriae (8/300; 2.7%), S. flexneri (13/300; 4.3%), S. sonnei (1/300; 0.3%), S. boydii (1/300; 0.3%), Untypable Shigella (44/300;14.7%) Salmonella spp (1/300;0.3%), Serratia spp (1/300; 0.3%), Pseudomonas spp (1/300; 0.3%), and Klebsiella spp (37/300; 12.3%). Shigella and Salmonella were then serotyped using specific polyvalent and monovalent antisera from Denka Seiken, Japan. Antibiotic susceptibility tests were performed on all isolates using the disc diffusion and minimum inhibitory concentration methods. ETECs as well as other Enteric bacteria were resistant to more than four antibiotics (Chloramphenical (28%), Cotrimoxazole (78%), Co-amoxilav (70%) Erythromycin (98%) Ciprofloxacin (5%), Cefotoxime (18%) and Tetracyclin (56%). Those found resiatant to third generation cephalosporins were tested for Extended Spectrum Beta Lactamases (ESBL) while those resistant to quinolones were tested for Gyrase resistance genes (Gyrase A, B and Topoisomerases). Sequencing was done on all Gyrase and topoisomerase positive isolates for detection of mutations. From the study, ETEC was detected significantly more often in children less than five years old (25/300) than in those aged above six years (8/300) (X2 = 4.2; P<0.05). Among the ETECs isolated, CFAI, CS1, CS2, CS3, CS5, CS6, CS14 and CS19 were detected. Other than ETEC which was the main focus of the study, Atypical enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and enteroraggregative E. coli (EAEC) were also detected though with slightly lower frequencies in both children and adults (16.0%, 8% and 36.4%, respectively). Enterohaemorrhagic E. coli (EHEC) was only isolated in children and adults with bloody diarrhoea (2.1%). Enteroaggregative E. coli was the most prevalent pathotype (83/228 ;36.4%). Shigella spp (67/300; 22.3%) were isolated in patients of all age groups. The drug susceptibility results revealed multidrug resistance in these isolates. ETECs were resistant to Chloramphenical (28%), Cotrimoxazole (78%), Augmentin (70%) Erythromycin (98%) Ciprofloxacin (5%) and Tetracycline (56%). The quinolone resistance gene (gyrA) was detected in 10% of quinolone resistant isolates. Sequence analysis of strains with decreased susceptibilities or total resistance to fluoroquinolones showed a single mutation at either Ser-83 or Asp-87 codon. No mutations were detected in the gyrB, and topoisomerase genes. Extended – Spectrum Beta Lactamase (ESBL) was detected in (18/300; 6%). ETEC, other E. coli pathotypes and Shigella spp were shown to be heterogenous by Pulsed Field Gel Eletrophoresis. The main etiologies of diarrhoea found were ETEC and other E. coli pathotypes, Shigella spp and Salmonella spp while the main CFs detected were CFAI (25/98), CS3 (1/98), CS6 (13/98), CS7 (2/98), CS14(12/98) and CS19 (12/98). The establishment of the existence of ETECs carrying varied CFA / CSs and multidrug resistance (MDR) phenotypes in this study is of great public health concern. There is a possibility of a reservoir in the population in the study area and the possibility of the reservoir spreading is real. This may lead to difficulties in the management of diarrhoeal disease. It is possible CS19 was detected for the first time in this country. The CS19 together with CS6 which are persistent adhesins can be utilised in the formulation of a novel vaccine for the prevention of diarrhoea due to ETEC.