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Antibiotic susceptibility of Enteric pathogens from the Maasai community, Narok
and Kajiado Districts, Kenya.

Sang W.K1, Kariuki S.M1, Schnabel D3, Boga H.I2, Waiyaki P.G1, Wamae C. N1
1. PhD, Center for Microbiology Research Laboratory, Kenya Medical Research Institute, 54840, 00200, 2. PhD, Jomo Kenyatta University of Agriculture and Technology, 62000, 00200, Nairobi, Kenya, 3. MD, MPH, Enterics laboratory, US Army Research Unit, Kenya, 606,0621,Nairobi.

Corresponding author: Willie K. Sang, Email: wsang@wrp-nbo.org, Fax: (254) (020) 2720030 Telephone: (254)
The emergence of resistance to antimicrobial agents in bacterial pathogens is a worldwide problem that has been
associated with inappropriate use in human and veterinary medicine. Between August 2004 and July 2005 stool
samples from 380 patients were cultured for enteric pathogens and characterized by Polymerase Chain Reaction
for the presence of virulence properties. Patients were from Narok and Kajiado Districts of Kenya, mainly
populated by the Maasai community majority of who practice traditional medicine. 218 patients were from
Narok District Hospital and 62 from Entosopia Clinic in Kajiado. A total of 107 E. coli and 35 Shigella isolates
were tested.
Antibiotic susceptibility testing was done using the E-test strips containing Tetracycline, Gentamicin,
Chloramphenicol, Fosfomycin, Amoxicillin/Clavulanic acid, Trimethoprim/Sulphamethoxazole, Ticarcillin/
Clavulanic acid and Ciprofloxacin. The resistance frequencies did not differ significantly between other E. coli
and Shiga toxigenic E. coli, respectively; Gentamicin (3% vs. 3%), Chloramphenicol, (24% vs. 23%) and
ampicillin (25% vs. 23%), Tetracycline (63% vs. 68%), Fosfomycin (44% vs. 54%) and
Trimethoprim/Sulphamethoxazole (84% vs. 84%). Overall antibiotic resistance levels were at much lower levels
than those reported from the rest of Kenya, possibly due to the lower levels of exposure and usage of
antimicrobials among the Maasai community.

Introduction
likely related to the frequent unrestricted use of over- Diarrhoea is a significant health problem worldwide, the-counter drugs without medical supervision [4]. especially in the developing world where adequate The emergence and spread of antibiotic resistance in health facilities and proper sanitation are lacking [1]. bacteria is of medical importance and poses serious Globally diarrheal diseases account for an estimated constraints on the options available for the treatment of 2.2 million deaths of children below 5 years annually, many infections. This problem has been brought into [2,3]. The progressive increase in antimicrobial prominence by the recent widespread outbreaks of resistance among enteric pathogens particularly enteric diseases caused by drug resistant organisms [5, Shigella, Vibrio cholerae, Enteropathogenic E. coli, 6]. Among enteric pathogens, major epidemics of Salmonella Typhi and S. Enteriditis species is infection with antibiotic resistant Shigella have becoming a critical concern worldwide, particularly in occurred in Latin America, Asia and Africa [7, 8, 9]. In the developing world where there are high rates of Kenya, diarrheal illnesses ranked 4th after Malaria, diarrheal diseases which are associated with mortality. upper respiratory tract infections and skin infection This also affects travelers to these regions. [10]. A major outbreak of multi-drug resistant Shigella Antimicrobial resistance in developing world is most dysenteriae I, occurring concurrently with Vibrio cholerae serovar O1 Ogawa was reported along the coastline of Kenya [11]. This strain of S. dysenteriae African Journal of Health Sciences, Volume 19, Number 3-4 June-December 2011 was found to be resistant to ampicillin, Tetracycline, The strains identified as Salmonella, Shigella or E. coli Chloramphenicol, and cotrimaxazole but sensitive to by their colonial morphology and biochemical gentamycin, nalidixic acid and kanamycin, apart from properties were further serotyped using O-antigen and one strain that was resistant to kanamycin. However H-antigen antisera (Denka Seiken Co LTD, Tokyo- most V. cholerae strains were sensitive to Gentamicin, Japan) by slide agglutination assays as previously Chloramphenicol and a few were resistant to Antibiotic Susceptibility Testing by MIC method ampicillin. Also multi-drug resistant enteroaggregative A total of 107 E. coli isolates of which 31 were STEC E .coli serotype O44 associated with acute and and 76 isolates from other pathogenic E. coli ( ETECs, persistent diarrhea was reported in Kenyan children Eagg, Einv and EPEC, including 35 isolates of Shigella [12]. A number of studies done at a public teaching hospital in Kenya in 1991 and 1992 [13, 14] recorded a The E-test method (AB Biodisk) was used to screen for prevalence of over 50% to ampicillin and 80% to 100% the antibiotic susceptibility patterns. The minimum resistance to Tetracycline among Salmonella and inhibitory concentration (MIC) susceptibility test was Shigella isolates causing nosocomial infections during determined in accordance with the manufacturer's the period 1986-1990. A more recent study in Kenya guidelines (AB Biodisk, Sweden). The 0.5 McFarland further attests to this trend in which about 70% of the standards isolates were inoculated onto Mueller Hinton E. coli isolated was resistant to Tetracycline, ampicillin agar plates by swabbing evenly in three directions. The and sulphamethoxazole-trimethoprim [15]. E-test strip (obtained from the refrigerator at 4oC) was applied to each plate with sterile forceps with lowest Materials and Methods
concentration toward the center of the agar plate. The After obtaining informed consent, stool samples from plates were then incubated at 30 to 35 °C for 24 hours. 380 outpatients with diarrhea (318 from Narok District The E-test MIC values were read directly from the E- Hospital and 62 from Entosopia Clinic) were collected test strip MIC scale. The following antibacterial agents: in sterile plastic containers. The specimens were Fosfomycin (Fm), Ciprofloxacin (Cip), Cefotaxime transferred into Cary-Blair transport media (MML (Ctx), Ticarcillin/Clavulanic acid (Ctl), Gentamicin Diagnostics Inc, Troutdale, Oregon, USA), labeled (Gn), trimethoprim-sulfamethoxazole (Sxt), ampicillin only with a unique study number and then placed in an (Am), Chloramphenicol (Chl) and Tetracycline (Te) insulated box with ice packs and transported to the were used as guided by CLS (2005) criteria. The Kenya Medical Research Institute Centre for concentration gradient of each antimicrobial agent on Microbiology laboratory where they were processed the E-test strips was 0.016 to 256 g/ml with the exception of Ciprofloxacin and co-trimoxazole for All stool samples were plated onto MacConkey agar, which the gradient ranged from 0.002 to 32 g/ml. Xylose-Lysine-deoxycholate agar XLD), Sorbital- MacConkey agar (for detection of O157 STEC) and Campylobacter blood-free agar. Initially selenite broth was used for enrichment purposes. The plates were The antibiotic susceptibility testing data are shown in incubated aerobically at 37oC for 18 – 24 hrs, with the Table 1. Of the 76 diarrheagenic E.coli isolates (31 exception of Campylobacter plates, which were ETEC, 20 EPEC, 14 Eagg and 11 Einv), 84% were incubated at 42oC in microaerophilic conditions for 48 resistant to SXT, 63% were resistant to TC, 44% were resistant to FM and 24% were resistant to CHL. Lower After overnight growth at 37oC one to two suspect levels of resistance were observed for gentamycin and colonies each of Shigella and Salmonella and five to ampicilin 3% and 5% respectively. All these isolates ten single colonies with typical E. coli morphology were fully sensitive to CIP, CTX and CTL. The were selected and characterized on the basis of their traditional antibiotics, including CHL, TC and STX biochemical reactions using BBL Enterotubes™ II. showed low activity against these E.coli strains (MIC (Becton Dickson Microbiology Systems Sparks, at which 90% of these isolates tested are inhibited [MIC90] of 64 mg/liter for CHL and MIC90 of 1024 African Journal of Health Sciences, Volume 19, Number 3-4 June-December 2011
Table 1: Antibiotic susceptibilities of diarrheagenic E.coli and Shigella strains.
African Journal of Health Sciences, Volume 19, Number 3-4 June-December 2011 Out of 35 STEC strains, 31 were tested for all the in the developed countries found low levels of antibiotics. The resistance frequencies did not differ resistance of E. coli isolates to Ciprofloxacin ranging significantly between other E. coli pathotypes and from 1% to 5% [20, 21]. The use of antibiotics to treat STEC respectively (p>0.05), that is gentamycin (3% patients with STEC infections has been quite vs. 3%), and Chloramphenicol, (24% vs. 23%) with the controversial. Most clinicians experienced in the exception of ampicillin (5% vs. 23%). The frequencies management of STEC infections in the United States were slightly higher for Tetracycline (63% vs. 68%), and Canada have found that antimicrobial agents at best have little clinical effect and at worst causes harm, Trimethoprim/Sulphamethoxazole (84% vs. 84%). for example increase the chances of acquiring Hemolytic Uremic Syndrome (HUS) [22, 23] Thirty five Shigella strains tested showed that high All Shigella isolates were fully sensitive to levels of resistance for TC and SXT 77% and 91% Ciprofloxacin and Gentamycin. Moreover, as respectively. TC and SXT showed very low activity compared to E.coli isolates the resistance of Shigella against Shigella strains with MIC90’s of 192 and 32 isolates was much higher to antimicrobials which mg/liter, respectively. All Shigella strains were 100% ranged from 29% resistant to chloramphenical and to sensitive to CIP and Gen. All Shigella strains were not 91% resistance to trimethoprim-sulphamethoxazole. The increase in resistance of Shigella species to trimethoprim- sulphamethoxazole and to ampicillin has Discussion
been reported world wide [21, 24]. Also in Kenya, a There are several reports on the multiple antimicrobial study by Kariuki et al reported the resistance rate of resistance among strains of pathogenic E. coli in Kenya Shigella spp. to trimethoprim/sulphamethoxazole and [12, 18, 15]. However, the resistance rates observed in ampicillin to be as high as 100% for both drugs [25]. In this study are much lower than those that have been this study, resistance rates of Shigella spp. to these reported from the rest of Kenya. As compared to our antibiotics were not as high as those reported in study which showed lower prevalence of resistance to Burundi [26], Zimbabwe [27] and in the coastal AM, CHL, TC for STEC and non STEC strains, a study by Kariuki et al in 1997 recorded multiple The level of antimicrobial resistance were much lower resistance rates in E. coli as high as 86% to compared with those reported elsewhere [28, 29, 30]. Tetracycline and 89% resistance to ampicillin [18]. The results of STEC strains from our study could be Study results showed moderate activity against E.coli different in that the Maasai people have not been strains for AM and CHL. The overall occurrence of grossly exposed to antibiotics compared to other STEC drug resistant isolates range from 3% resistance Trimethoprim/Sulphamethoxazole for both STEC and Conclusion
none STEC. The MIC of STEC isolates showed almost We observed low levels of antibiotic resistance among similar patterns as those of non STEC with an the Maasai communities who may not have been exception of Ampicilin drug where non-STEC showed grossly exposed to antibiotics and still practice the use lower levels of resistance at 3% as compared to 23% of traditional medicine. The antibiotics that have developed resistance in the rest of Kenya can still be Variation in resistance frequencies of STEC and non administered to the patients in the Maasai STEC strains agree with findings of a similar study on antibiotic resistance (8% vs. 26%), [19]. The The overall antibiotic resistance levels were at much observation that all STEC and non STEC strains were lower levels than those reported from the rest of fully sensitive to Ciprofloxacin is important Kenya, possibly due to the lower levels of exposure considering that fluoroquinolones are used to treat a and usage of antimicrobials among the Maasai people. range of E. coli infections in humans. Other researchers
Acknowledgements
We gratefully thank the Walter Reed- GEIS Program through the director Dr. Sam Martin and Dr. Rodney Coldren and Dr. Sheryl Bedno for PHD sponsorship award and for providing research materials to successfully complete the research project. We are also grateful to the Director KEMRI for his support. African Journal of Health Sciences, Volume 19, Number 3-4 June-December 2011 References
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