Emergence of bla TEM Type Extended-Spectrum β-Lactamase Producing Salmonella spp. in the Urban Area of Bangladesh

Salmonellosis, an acute invasive enteric infection, is endemic in Bangladesh. We analyzed 128,312 stool samples of diarrheal patients to identify Salmonella spp. during 2005–2013. A total of 2120 (1.7%) Salmonella spp. were isolated and the prevalence of Salmonella spp. decreased significantly over time (2→1%, P < 0.001). Among the typhoidal Salmonella (TS) serogroups, S. Typhi was predominant (404, [65.1%]) followed by S. Paratyphi B (139, [22.4%]) and S. Paratyphi A (78, [12.6%]). Of the nontyphoidal Salmonella (NTS) isolates, the serogroup C1 (560, [37%]) was predominant followed by B (379, [25%]), C2 (203, [14%]), E (127, [9%]), and D (94, [6%]). Most of the resistance was found towards nalidixic acid (40%), ampicillin (36%), cotrimoxazole (20%), chloramphenicol (13%), ciprofloxacin (4%), and ceftriaxone (4%). Interestingly, 32% of the isolates showed reduced susceptibility to Cip. Multiantibiotic resistance (MAR, ≥3 drugs) was more common among TS than NTS strains (P < 0.001). Among the representative ceftriaxone-resistant isolates, bla TEM gene was detected among 88% (7/8) of the strains, whereas only one strain of S. Typhi was positive for both bla TEM and bla CTX-M genes. The study reflects higher prevalence of MAR Salmonella spp. and is the first to report the bla TEM gene mediated ESBL production among Salmonellae in Bangladesh. Emergence of MAR Salmonella spp. in particular ESBL strains should be considered a public health concern.


Introduction
Salmonellosis due to nontyphoidal Salmonella (NTS) infection is a global public health concern, particularly in Salmonella endemic low and middle income countries (LMICs). Treatment is critical for persons with severe disease, particularly children and immune compromised people. Treatment with an appropriate antibiotic can shorten the duration of symptoms, significantly reduce severity of disease and the risk of transmission, and prevent potentially lethal complications. Emergence of resistance to first-line therapy like ampicillin, chloramphenicol, and cotrimoxazole including ciprofloxacin among Salmonella spp. during the last decades has complicated the situation [1,2]. For treatment of Salmonella infection resistant to these drugs, extended-spectrum cephalosporins (ESCs) are considered as an alternative therapeutic choice. With the increased use of -lactam antibiotics to treat enteric infection, Salmonella spp. had acquired resistant to third generation cephalosporin antibiotics in different parts of the world and had been associated with clinical treatment failure [3,4].
Extended-spectrum beta-lactamases (ESBL) are usually encoded by large plasmids (≥100 kb) that are transferable from strain to strain and between bacterial species [5][6][7]. Resistance to ESCs is mediated primarily by production of class A ESBLs, which can hydrolyze oxyimino cephalosporins but are not active against cephamycins and carbapenems. The plasmid-encoded CTX-M type ESBLs production was initially identified in 1983 in Germany [8]. The CTX-M family enzymes, which confer high levels of resistance to ESCs, have similar substrate specificities and inhibitor profiles to TEM. The CTX-M type ESBLs have been reported to be found worldwide in different members of Enterobacteriaceae isolated from human and other animal sources [9]. TEM type ESBL producer S. Typhi in one-year-old child with recurrent high-grade fever [10]. In recent years there have been several reports indicating the emergence of resistance to -lactam antibiotics among Salmonella species [2]. Here, we present TEM gene mediated ESBL production among Salmonella spp. isolated from stool specimen of patients with diarrhea in an urban setting of Bangladesh.

Material and Methods
As part of a microbiological analysis of stool sample received at Dhaka Treatment Centre of International Centre for Diarrheal Diseases Research, Bangladesh (icddr,b) during 2005-2013, we have analyzed 128,312 fecal specimen following standard microbiological method to identify Salmonella spp. Antibiotic susceptibility test was done by Kirby-Bauer disk diffusion test. The putative extended-spectrum betalactamase (ESBL) producing strains were tested by the double-disc synergy method and detection of -lactamase genes ( CTX-M , TEM , SHV , and OXA genes) was performed by PCR as describe elsewhere [10].

Result and Discussion
Overall, the annual incidence of Salmonella infection showed a decreasing trend and the proportion came down significantly from 2% in 2005 to 1 Around 40% of the Salmonella isolates showed resistance to nalidixic acid (Na) followed by 36% to ampicillin (Amp), 20% to cotrimoxazole (Sxt), 4% to ciprofloxacin (Cip), 13% to chloramphenicol (C), and 4% to ceftriaxone (Cro). Interestingly, 32% of the isolates were with reduced susceptibility to ciprofloxacin. Resistance to ≥3 antibiotic classes was more common among TS strains than NTS counterpart (36.6% versus 19.8%, < 0.001). Among the representative Salmonella isolates with unusual resistant phenotype (AmpCCipCroNaSxt) R , first identified in 2011, TEM gene was detected in 88% (7/8) of the strains (Figure 1). Interestingly, one S. Typhi was positive for both TEM and CTX-M genes which is a very rare phenomenon. In our earlier report, we noticed that this same phenomenon was observed in one S. Typhi isolated from blood of a typhoid patient [10]. These findings suggest that multi-ESBL producer strains are circulating in Bangladesh. However, to the best of our knowledge, this is the first report of ESBL production among Salmonella isolated from stool specimen of diarrheal patients in urban Dhaka, Bangladesh. Detailed molecular characterization including sequencing is necessary for further geno-and subtyping of these strains.

Conclusion
The study finding reflects the higher prevalence of MAR Salmonella spp. among children aged <5 years and TEM gene mediated ESBL production among Salmonella spp. isolated from stool sample of diarrheal patient in urban Bangladesh. Therefore, it is important to establish a surveillance program to understand actual disease burden due to Salmonella as well as promote specific and actual line of therapy for Salmonellosis.