Distribution of sulfamethoxazole trimethoprim constin in Vibrio cholerae isolated from patients and environment in Iran

The occurrence of drug-resistant Vibrio cholerae is being reported with increasing frequency worldwide. Spread of resistant strains has been attributed, in part, sulfamethoxazole trimethoprimconstin (SXT-C). Sixty V. cholerae isolates obtained from cholera patients from different provinces in Iran during 2004 to 2006 and thirty-seven V. cholerae strains from surface water sources at 5 different locations in Tehran, Iran during 2006 were subjected to antibiotic susceptibility testing and polymerase chain reaction amplification of SXT-C. In clinical isolates the highest and the least levels of antibiotic resistance were seen to SXT, streptomycin and chloramphenicol (95, 95 and 92%, respectively) and doxycycline, gentamicin and oxytetracycline (0, 3 and 3%, respectively). PCR for SXT element of clinical and environmental isolates was positive for 95 and 19% of isolates, respectively. The results of this study showed that among the clinical and environmental V. cholerae resistance to SXT, streptomycin and chloramphenicol could be, in part, due to wide distribution of SXT-C isolates.


INTRODUCTION
Vibrio includes a wide spectrum of species causing pandemics to free-living aquatic strains. Besides morbidity and mortality associated with cholera, multiple drug-resistant Vibrio cholerae O1 has been shown as a major public health problem in Iran (Bakhshi et al., 2008a). The reports of strains resistant to commonly used antibiotics are also appearing with increasing frequency globally (Garg et al., 2000;2001). Spread of antibiotic resistance among V. cholerae occurs through mobilization of a variety of genetic elements. Antibiotic resistance determinants have been detected on sulfamethoxazole trimethoprim-constin (SXT-C), a novel conjugative transposable element, which encodes resistance to SXT, chloramphenicol and streptomycin (Glass et al., 1980). In this study we determined antibiotic susceptibility testing and distribution SXT-C among the clinical strains of V. cholerae isolates *Corresponding author. E-mail: azizlari@gmail.com. collected from cholera patients from different provinces in Iran during 2004 to 2006, and environmental isolates collected from surface water sources at 5 different locations in Tehran, Iran during 2006, where several cholera epidemics have occurred in the last several years (Bakhshi et al., 2008b;Pourshafie et al., 2007). Presence of SXT-C as an important antibiotic resistance genetic determinant among these clinical and environmental isolates was studied to understand the role of this element in emergence of drug-resistant V. cholerae isolates.  (95) SXT, sulfamethoxazole trimethoprim.

Clinical samples
Zahedan, Golestan and Qom (Table 1). V. cholerae ATCC 14035 type strain was used as a positive control throughout the study.

Collection and processing of environmental samples
Five hundred milliliters of surface water samples were collected from 5 different geographical locations in Tehran, Iran during a period of 3 months between June and August 2006. The samples were taken from local surface water at different locations in the west (labeled as sources 1 and 2), east (4 and 5) and center (3) of Tehran where there were no outlets from waste water treatment plants. Sampling from each surface water source was performed 3 times with the interval of 30 days. The transportation of each sample to the laboratory was performed in the sterile 500cc glass container on ice. Vacuum pressure of 15 to 20 lb/in2 was used to filter water samples through 0.45 mm pore size membrane after a pre-filtration through filter paper (Wattman No.1; Maidston, UK).The membranes were then cut into 8 pieces after which they were vortexed in 2 ml of 10 mM phosphate buffered saline (PBS, pH 7.4) for 3 min, followed by addition of 20 ml of alkaline peptone water (APW) containing peptone (1% wt/vol) and NaCl (1%wt/vol,pH 8.5). The tubes were then shaken (100 rpm) for 16 h at 37°C. Each sample was streaked on thiosulfate-citrate-bile-sucrose agar (TCBS) plates and incubated overnight at 37°C. All the yellow colonies on TCBS plates which were suspected of being V. cholerae were further examined by 10 biochemical assays including oxidase, motility, sucrose and lactose fermentation, growth in 0% NaCl, arginine dehydrolase, ornithine decarboxylase, methyl red, Voges-Proskauer and indole test .Confirmation of identity of isolates was performed by PCR using species-specific primers (prVC-F/ prVCM-R). A total of 37 V. cholerae strains were then isolated and subjected to further analysis.

Antimicrobial susceptibility testing of environmental samples
A total of 12 antimicrobial resistance profiles were observed among the total isolates (Table 4). Profile 1 was the predominant pattern (51%) and showed sensitivity to all 10 antimicrobials tested. The profile 2 constituted 11% of the isolates and represented resistance to only SXT. Among the isolates, the highest level of resistance was observed with ampicillin (27%), followed by SXT (22%) and streptomycin (16%). No resistance to gentamicin or ciprofloxacin was observed. Five isolates (13.5%) showed resistance to more than 3 antimicrobials tested.

Distribution
of SXT-C among clinical and environmental samples The majority of 60 clinical strains showed significant

DISCUSSION
In this study, we analyzed one important genetic determinant responsible for drug susceptibility pattern of clinical V. cholerae strains isolated from four different provinces in Iran over a 3 year period between 2004 and 2006 and environmental V. cholerae strains from surface water sources at 5 different locations in Tehran, Iran during 2006. The pattern of antibiotic resistance among clinical isolates was similar throughout the 3 years period, observing that the time of sample collection had no effect on the drug susceptibility. During the 3 years period only a few isolates, obtained during 2005, were resistant to ciprofloxacin, gentamicin, oxytetracycline and tetracycline. This trend of antibiotic sensitivity, however, was changed, and clinical isolates become sensitive during 2006. The results showed that the trend of ampicillin resistance had significantly increased from 2004 to 2006, whereas erythromycin resistance was decreased. Such change could be an indication of the antibiotic usage by the public in Iran, which further signifies the need to further investigate the genetic mechanisms involved. PCR assay for SXTint revealed that 95% of our total clinical isolates in 3 years contained SXT-C responsible for resistance to cotrimoxazole (95%), streptomycin (95%) and chloramphenicol (92%). The appearance of SXT-C has been reported by Iwanaga et al. (2004) to have occurred in El TorO1 strains isolated in Laos after 1997, which has replaced the class I integron with an aadA1 gene cassette in pre-1997 V. cholerae isolates. Similarly, Amita et al. (2003) have reported that class I integron with an aadA1 gene cassette was widely distributed among clinical O1 strains before emergence of pre-O139 in India, which eventually disappeared and replaced with SXT-C. The results may suggest that the SXT-C has turn into the dominant antibiotic-resistant element among clinical V. cholerae O1 isolates in Iran.
Increasing V. cholerae antibiotic resistant strains of clinical origin poses the need for assessing mobile nature of resistance gene content of the environmental isolates (Sack et al., 2003). Among the antibiotic resistance profiles, significant resistance to SXT and streptomycin was seen in comparison to other antibiotics among our clinical isolates. We previously found resistance to same antibiotics in the clinical isolates obtained in Iran (Pourshafie et al., 2007), which suggests the presence of the resistant genetic determinants in the environmental isolates even during non-endemic cholera periods. Presence of the SXT element (19%) among our environmental isolates supports this supposition. Two of our isolates (5.4%) were found to be resistant to 8 antibiotics. Among the SXTint environmental isolates (19% of the total isolates), 57 and 71% were resistant to cotrimoxazole and streptomycin, respectively. The resistant genes for these two antibiotics are known to be encoded by SXT element. The reason that not all SXTint isolates were resistant to SXT and streptomycin could be the finding that the SXT element may acquire the resistance genes through horizontal transfer . Therefore, the presence of SXT element should be monitored even in drug susceptible V. cholerae isolates. Miyazato et al. (2004) have indicated that no mobile genetic elements such as plasmid, class I integron or SXT element was detected in 22 environmental non-O1, non-O139 strains which were isolated from 13 distant sampling sites in Vietnam. In conclusion, the data presented in this study showed that the SXT-C element intrinsically harboring several different antibiotic resistance gene cassettes is widely distributed among different clinical and in acceptable number, in environmental V. cholerae in Iran. Although the resistance element found in this study may be responsible for the antibiotic resistance seen in this study, further global investigations are needed to determine the importance of SXT element in particular in the horizontal acquisition and dissemination of antibiotic resistance genes among V. cholerae and other microbes. Also the limited information on the epidemiological linkage between the clinical and environmental isolates of V. cholerae pose the need to further monitor the environmental isolates.