uring the past decade, multidrug-resistant (MDR) Salmonella typhi have increased substantially in most part of the world especially in developing countries. The prevalence of this bacterium increased from 1% of human isolates in 1998 to 21% in 2003. S. typhi is resistant to at least chloramphenicol, streptomycin, sulfamethoxazole, cefoxitin, tetracycline, amoxicillin-clavulanic acid, and ampicillin. This phenotype exhibits decreased susceptibility to Ceftriaxon-Author : Department of Sciences Technology, Microbiology Unit, Federal Polytechnic, P.M.B. 53513, Ado -Ekiti, Nigeria. E-mail : [email protected] e (CDC, 2006), a critically important antimicrobial agent for treating invasive Salmonellosis in children (Guerrant et al., 2001). Members of the bacterial genus Salmonella are among the major pathogens that cause Salmonellosis in humans. Most human Salmonella infections are thought to be associated with food borne transmission from contaminated animal-derived meat and dairy products (Voetsch et al., 2004). Human infections are commonly caused by ingestion of food that has been contaminated by animal feces (Herikstad et al., 2002). Although >2,500 serovars of S. typhihavebeen identified, most human infections are caused by a limited number of serovars. S. serovar typhimuriumand enteritidis are the most common causes of humanSalmonellosis worldwide, although other serovars havebeen reported to be more prevalent in some regions (Haldet al., 2007).Adequate sanitary measures have led to a decrease in cases of typhoidal Salmonella infections in developedcountries such as the USA where the incidence is low. However, in these countries, non-typhoidal Salmonellosis is common, and most of these cases are associated with outbreaks from contaminated meat, dairy products or by crosscontamination from foods contaminated with Salmonella (, Glynn,et al., 2005). In Nigeria especially in Ekiti state, morbidityassociated with illness due to Salmonella continues to be on the increase and in some cases, resulting in death (Ajibade et al., 2010).

Although, the primary cause of Salmonellosis is consumption of contaminated foods, there is the potential for secondary spread, from person-topersonand also to other foods. Person-to-person spread with family groups is often associated with poor personal hygiene but there is the opportunity of airborne andsurface-to-surface spread within the toilet and the bathroom, especially during the diarrhea phase (Ajibadeet al., 2010; Ajibade, 2012). Despite improved public health, serious infections with Salmonella remain a major clinical and public health concern in Nigeria and worldwide.S. enterica are a leading cause of food borne disease worldwide (Hald et al., 2007). In the United States, as many as 1.4 million cases of S.entericaassociated disease occur annually (Humphery, 2000;Voetsch et al., 2004 persons. However, antimicrobialdrug resistance has become increasingly common in S. typhi, which has complicated therapy (Herikstad et al., 2002). Antimicrobial agents such as fluoroquinolone and thirdgeneration cephalosporin's are commonly used to treat severe human Salmonella infections (Lee et al., 2009). Resistance to these and other antimicrobial drugs, as well as multidrug resistance, has increased over the last several decades, probably as a consequence of antimicrobial agents in use at intensive animal husbandry and medicine (WHO, 2002). Antimicrobial resistant strains of Salmonella species are now widespread all over the world. In developed countries, it is becoming more and more accepted that a majority of resistant strains are of zoonotic origin and have acquired their resistance in an animal host before being transmitted to human through the food chain (Helms et al., 2004). The emergence of antimicrobial drug resistance is a matter of concern. This work intends to determine the prevalence of resistance of Sal typhi isolated from major towns in Ekiti Stateto conventional antimicrobial drugs.

Stool samples were collected from patients who visited hospital in Ado Ekiti, Ido Ekiti, Ikere Ekiti, Ikole Ekiti, Ifaki Ekiti between February 2009 and September 2011. Fifty (50) samples each were collected from the hospital.

Samples were cultured on Salmonella-Shigella agar, incubated at 37 o C for 24hrs. Smooth colonies withblack centre were sub cultured on to MacConkey agar (Oxoid), incubated at 37 o C for 18-24hr. Pale colonieswere isolated and confirmatory biochemical test (Gram's reaction, urease reaction, citrate utilization andmotility) were conducted. Salmonella enterica was identified by using standard biochemical tests andcommercial typing antiserum (Statens Serum Institute, Copenhagen, Denmark) according to the manufacturer's instructions.

The susceptibility profile of samples from which Sal typhi (44) was isolated was determined. Minimum inhibitory concentration (MIC's) of ten (10) antimicrobial drugs: Phenols (chloramphenicol), Penicillins (Ampicillin), Cephalosporin (cefepime, cefotaxime), Tetracycline (tetracycline), Aminoglycosides (Gentamycin, streptomycin), Sulfonamides (trimethoprin-sulfamethoxazole), Quinolones and Fluoroquinolone (nalixidic acid, ciprofloxacin) were determined by usingthe brothmicrodilution method; susceptibility to streptomycin was measured by using the disk-diffusionmethod as recommended by the Clinical and Laboratory Standards Institute(CLSI,2012).

All Salmonellae isolates were analyzed for genetic relatedness by PFGE by using Xba1 according to the CDC PulseNet protocol (Ribopt et al., 2006). Electrophoresis was performed with a CHEF-DR111 system in the Science Technology Research Lab, Federal Polytechnic, Ado-Ekiti by using 1% Seakem agarose in 0.5x Tris-borate-EDTA at 180V. Running conditions consisted of 1 phase from 2.2 to 63.8s for a run of 22h.

Genes Presence of qnr genes was determined by using PCR with primers QP1 and QP2 for qnrA, FQ1 and FQ2 for qnrB, and 5'-ATGGAAACCTACAATCATAC-3' and 5'-AAAAACACCTCGACTTAAGT-3' for qnrS. The qnrB allele was determined by amplification and sequencing with primers FQ1 and FQ2. Screening for aac(6')-1b-cr was performed as as described in Park et al. (2006). Primer pairs used for amplification of ?-lactamase genes were: bla cmy (5'-ATGATGAAA AAATCGTTATGC-3') and

(5'TTGCAGCTTTTCAAG AATGCGC-3'), bla OXA-1 (5'AATGGCACCAGATTCAACTT-3') and (5'-CTTGGCTTTTATGCTTGATG-3'); and bla SHV (5'-GGTTATGCGTTATATTCGCC-3') and (5'-TTAGCGTT GCCAGTGCTC-3'). bla CTX-M genes were screened by using a multiplex PCR assay (Woodford et al.,2006).

The prevalence of Saltyphi in the various locations in Ekiti state over the years of 2009 -2011 is shown in table 1. The highestnumber of the isolates from 50 samples was found at Ikere-Ekiti, Ikole-Ekiti and Ifaki Ekiti at a total number of 131 respectively, this was followed by Ado-Ekiti with an isolate number of 130. The lowest number of isolate was recorded at Ido Ekiti with 78 isolates. The lowest number of isolates was recorded in the year 2009 and this increased in both 2010 and 2011.The susceptibility patterns reported in 2009 in the different locations are shown in table 2. It was observed that resistance was indicated in streptomycin, chloramphenicol, cefepime, nalidixic acid tetracycline and trimethoprim-sulfamethoxazole. This same trend of resistance was repeated in 2010 and 2011 (tables 3 and 4). The notable change was a significant increase in the resistance to ampicillin from 20% to 100% in Ado Ekiti and from 39% to 100% at Ikole Ekiti and also Gentamycin from 18% at Ikere -Ekiti in 2009, to 86% and 84% in 2010 and 2011 respectively.

The difference in demographic factors in the different locations could be a convincing factor in the prevalence of Sal typhi in the towns. Ado-Ekiti been a capital city with high urbanization is prone to a lot of condition and the inhabitants especially in the access to good medical condition.

The increasing prevalence of multidrug-resistant Salmonella sp is a global health concern. In Ekiti State, colonization of infection with Saltyphi has been a reportable condition for ? 20 years. From 2009 to 2011, the agents for which a major change in susceptibility was observed were Tetracycline, Trimethoprimsulfamethoxazole, nalidixic acid, ampicillin, cefotaxime and streptomycin. With PFGE, a total of thirty-five 35 (79%) patterns in the locations were observed and thirtysix 36(72%) of eachisolates had the 3 most common patterns. The isolates with these patterns were found to show high resistance to Streptomycin, Chloramphenicol, Cefepime, Ampicilin and nalidixic acid (SCCAN). All isolates that are resistant to the antibiotics mentioned earlier contain qnrB2, 35(70%) isolates contained bla CMY-2' ; 39 (78%) isolates contained bla CMY-23' the mechanism for extended-spectrum cephalosporin, Aminoglycosides and Quinolone resistance. The genes that code for this resistance have proven to be remarkably mobile and widely distributed within and between species. Integrons are widely distributed among Saltyphi and are potentially capable of transmitting drug resistance (Mary et al., 2009). These findings correspond to that of Karen et al. (2010) where it was reported that most Salmonella isolates in South Africa are resistant to at least ampicillin (A), Chloramphenicol (C), Streptomycin(S), Sulfonamide (Su) and Tetracycline (T) (ACSSuT). They have been described as been found in a multidrug resistance (MDR) region that is located on the chromosome in a region termed Salmonella genome island 1 (SGI1) (Boyd et al., 2001).The spread of resistance has repeatedly been shown to be associated with antimicrobial drug use, which stresses the importance of the prudent use of these drugs; a notion reinforced by the observation that resistance can be revised (Marie et al., 2012). Plasmid mediated Fluoroquinolone resistance have been reported in South Africa to have implications for cons transference of resistance to the major antimicrobial agents used to treat typhoid fever (Karen et al.,2010). Multidrug resistance is evidenced in this study and this can be ascribed to the wide spread use of antibiotics both inside and outside of medicine, selling of the antibiotics over the counter without prescription, misuse and over use of antibiotics by doctors as well as patients and inappropriate prescription. The emergence of Sal typhi strains that are resistant to commonly used antibiotics is important to clinicians, microbiologists and those responsible for the control of communicable diseases.

Because use of antimicrobial agents contributes to increasing resistance and facilitates transmission of multidrug-resistant Salmonellae, promoting guidelines aimed at improving appropriate use of antimicrobial agents may help prevent transmission of multidrugresistant Sal typhi infections in the environment. In addition, the avoidance of unnecessary antibiotics usage and good hygiene at all stages in food production chain and adequate treatment of sewage, supply and drinking of portable water is of paramount importance in reducing the incidence and preventing multi-drug resistance.

IV.

In conclusion, this study underscores the magnitude of the problem of antimicrobial drug resistance in low resource settings and the urgent need for surveillance and control of this phenomenon is necessary. The state has seen a persistently high incidence of multidrug-resistant Sal typhi because ma vigilant screening and decolonization program has not been implemented. From this work it was observed that for the past three (3) years, growth of multidrug resistant strain of Sal typhi has been exponential, rural and metropolitan rates apparently stabilized. The findings of this study can be used to assist in the direction of policy and interventions; to conduct other analyzes like the evaluation of economic cost of these diseases while attributing it to various environmental conditions. Endemic persistence of the bacterium and the measures that should be undertaken to control or eradicate it from the state are likely to remain topical subjects.