Molecule characterization of antibiotic resistance in SHIGELLA species isolated from diarrhoeic stool samples of patients attending Kwale District hospital, Kenya

Abstract

Acute diarrheal diseases are a major cause of high morbidity and mortality and shigellosis is a major cause of this problem. Shigella boydii, S. sonnei, S. dysenteriae and 8.flexneri are implicated in causing shigellosis. Shigellosis caused by multidrug-resistant (MDR) Shigella strains constitutes an increasing problem due to limited therapeutic options. In Kenya, shigellosis is mainly due to S. jlexneri. Despite the observed increase in resistance among Shigella strains to commonly used antibiotics, data on the occurrence and the role of plasmids and integrons in the emergence and spread of MDR Shigella in Kenya is scanty.

This study used molecular methods to analyse Shigella plasmids and class 1 and 2 integrons known to harbor resistance to antibiotics. Stool samples from diarrheal patients attending K wale District hospital in Kenya were cultured for Shigella and antimicrobial susceptibility tests performed. Conjugations, integrons and plasmid profile analysis were carried out to determine the molecular basis for transmissible drug resistance in Shigella. An overall Shigella incidence rate of 8.1 % was found. The age group 1-5 years had the highest incidence rate at 18.3% and statistically significant differences was shown between incidence of Shigella and age (x.2 = 8.684, df = 4, p = 0.003). As high as 96.6% of the Shigella isolates had resistance to one or more antibiotics with 86.2% showing MDR characteristics.

A total of 15 MDR profiles were identified with the sulfamethoxazole-trimethoprim(Sxt), streptomycin(Str), ampicillin(Amp) and tetracycline(Tet) being the most common resistance pattern at 53%. All the isolates were susceptible to ciprofloxacin and cephalosporin antibiotics tested. 96.6% of the isolates screened contained one or more plasm ids ranging from 1-6. Majority were small plasmids comprising of 1-2kb, 3-4kb and 5-9kb occurring in 86.9%, 78.3% and 47.5% of the isolates respectively. Species specific plasmids of 3.2kb, 9.0kb and 3.8kb plasm ids were found in S. flexneri, S. dysenteriae and S. sonnei respectively. The large plasm ids and middle range plasmids were found in 34.5% and 79.3% of the isolates respectively.

In all species, no precise correlations were found between specific plasmid profiles and antibiotic resistance patterns but significant statistical associations were shown between harboring middle range plasm ids (48kb and 80kb) and MDR phenotypes among the isolates (Fishers Exact Test, p=0.002). Of all MDR Shigella isolates, 32% were able to transfer plasmids to E. coli J53 strain. These plasm ids specified resistance to Sxt, Amp, Str and Tet. Class 1 and 2 integrons were detected in 6.9% and 82.8% of the isolates respectively and significant statistical association between carriage of class 2 integron and MDR phenotype shown(Fisher's Exact Test, p = 0.0004). None of the non-MDR strains carried integrons. A statistical association was shown between presence of class 2 integron and the resistance profile, Sxt, Amp, Str, Tet (Fisher's Exact Test, p = 0.0001). 28.5%, of Class 2 integrons were self transferable.

This study showed that shigellosis is endemic at Kwale and that children were at a higher risk of shigellosis. The findings also show that Sxt, Str, Amp may not be used empirically as the first line drugs in treatment of shigellosis in the region. Self transferable plasmids and class 2 integrons carrying drug resistance gene cassettes are widespread among MDR Shigella spp indicating the active role of these elements in the spread of multidrug resistance. The study recommends continuous surveillance of drug resistance pattern changes in Shigella for consideration in treatment of shigellosis and adoption of ciprofloxacin and/or cephalosporin antibiotics as first line therapeutic options for empirical treatment of shigellosis.