Antibiotic resistance pattern of Salmonella spp. isolated from stool samples of hospitalized diarrheal patients in Bangladesh

This study was carried out to investigate the drug resistance pattern of Salmonella spp. from diarrheal patients of under 5 years of age. Collected stool samples from the hospitalized diarrheal patients were analyzed for the presence of Salmonella spp. Among 350 stool samples from diarrheal patients, 15 (4%) were positive for the Salmonella spp. Antibiotic susceptibility test of the identified Salmonella spp. was performed according to Kirby-Bauer disc diffusion method. Eight commonly used antibiotics including azithromycin (15 μg), chloramphenicol (30 μg), Sulphamethaxole-trimethoprim (25 μg), metronidazole (50 μg), tetracycline (30 μg), doxycycline (30 μg), erythromycin (15 μg) and ciprofloxacin (5 μg) used to determine drug resistance pattern of the identified Salmonella spp. Majority of the isolates were multidrug resistant showed resistance against more than three drugs. 73% of Salmonella spp. was resistant to 2-4 drugs and 20% showed resistance to more than 5 drugs. We found that 100% Salmonella showed resistance to metronidazole and 87% were resistant to erythromycin. 7% isolates were resistant to chloramphenicol and doxycycline and 20% were resistant to ciprofloxacin. Antibiotic resistance is a serious public health problem worldwide and a leading cause of mortality and morbidity. This kind of situation, leads to great socioeconomic losses from the perspective of the patient, the hospital, and the whole society. To get rid form the drug resistance problem unnecessary, over and misuse of antibiotics should immediately prohibited and policy of judicious use of antibiotics should be strengthened.


Introduction
Salmonellosis is a growing concern day by day throughout the world and over the last several decades there have been significant shift in predominant.Salmonella serovars are associated with human infections.Nontyphoidal Salmonella serovars cause as much as an estimated 1 billion cases of gastroenteritis in humans every year (Kennedy et al., 2004;Coburn et al., 2007).Furthermore, they are also responsible for some serious infections such as septicemia and endocarditis (Gill, 1979), empyema (Burney et al., 1977), and meningitis (Chusid et al., 1980), especially in immunocompromised host.Although most Salmonella infections are selflimiting, effective antimicrobial therapy is needed if spread beyond the intestine occurs.The progressive increase in antimicrobial resistance in enteric pathogens is of greatest concern in the developing world.Since 1960s, several Salmonella spp.have been showed resistance to various common antibiotics such as ampicillin, chloramphenicol, and sulfamethoxazole-trimethoprim with increasing frequency throughout the world (Smith et al., 1984).Extended-spectrum cephalosporins and fluoroquinolones have become the drugs of choice for the treatment of infections caused by multidrug-resistant Salmonella serotypes due to the increased resistance to conventional antibiotics.However, since 1991, several studies from different countries have been reporting cases of infections by extended-spectrum cephalosporins resistant salmonellae (Dunne et al., 2000;Fey et al., 2000).According to a study by Gupta et al., in the United States, from 1998 through 2001, a 5-fold increase in the prevalence of Salmonella species resistant to expanded-spectrum cephalosporins (Gupta et al., 2003).In 10 European countries a survey conducted in 2000 identified a cefotaxime resistance rate of 0.6% in Salmonella isolates recovered from human sources (Threlfall et al., 2000).In recent years, such a trend of increase in drugresistant salmonellae including resistance to quinolones has been observed in many countries, particularly in Asia (Lauderdale et al., 2006;Wang et al., 2006).The use of antimicrobials in any purpose, including disease treatment and growth promotion in domestic livestock, can potentially lead to develop drug resistant bacteria.(Gomez-Lus, 1998;Tollefson et al., 1997).Mechanisms of bacterial antimicrobial resistance includes (i) changes in bacterial cell wall permeability, (ii) removal of antimicrobials via efflux pumps mechanisms (iii) modification of the site of drug action, and (iv) destruction or inactivation of antimicrobials (Barbosa and Levy, 2000;Schwarz and Chaslus-Dancla 2001).Most often, acquired antimicrobial resistance phenotypes develop by the transfer of plasmids via conjugation process (Guerra et al., 2002;Gebreyes and Altier, 2002).Plasmids may carry class I integrons, which are mobile DNA elements, play a vital role in the proliferation of bacterial multidrug resistance (MDR) (Arduino et al., 2002;Di Conza et al., 2002).We have conducted this study on diarrheal patients of less than five years of age in a private hospital of Chittagong district of Bangladesh.

Sample Collection
A total 350 fresh watery stool samples from patients of less than 5 years exhibiting symptoms of gastroenteritis were collected from different hospitals of Chittagong, Bangladesh.Then the collected samples were taken in frozen refrigerant packs and transported to the laboratory immediately.

Isolation and identification of Salmonella spp.
Collected samples were inoculated in Luria-Bertani (LB) broth for the enrichment of bacteria and incubated at 37 ∘ C for 24 hours.After overnigt of enrichment, cultures were aseptically inoculated onto Hektoen enteric agar and Salmonella-Shigella (SS) agar (Oxoid, Hampshire, England) plates and incubated at 37˚C for 24 h.Then the Hektoen agar plates were examined for characteristic green or blue-green colonies (with or without black centre), and distinct colorless colonies onto Salmonella-Shigella (SS) agar plates were primarily selected as species of Salmonella.After primary confirmation each isolates were finally confirmed as Salmonella by conducting several physiological and biochemical tests.Motile isolates from primary selection with negative indole tests where red violet colored ring in the top of the broth cultures after adding kovac's solution didn't formed, positive citrate tests where blue green color of the media formed and, provided red alkaline slant and yellow acidic butt with black colored H 2 S production were confirmed as Salmonella.

Antimicrobial susceptibility test
Antibiotics susceptibility test was performed by Kirby-Bauer disc diffusion method (Hudzicki, 2009).Bacterial suspensions were prepared from fresh culture grown overnight onto nutrient agar plates by using sterile normal saline and the turbidity of the suspension was adjusted to 0.5 McFarland Standard that corresponds to approximately 1x10 8 CFU/ml of suspension.A sterile cotton swab was dipped into the inoculum then streaked on the Mueller-Hinton agar plate properly.Then antibiotic discs impregnated with azithromycin (15 µg), chloramphenicol (30 µg), Sulphamethaxole-trimethoprim (25 µg), metronidazole (50 µg), tetracycline (30 µg), doxycycline (30 µg), erythromycin (15 µg), ciprofloxacin (5 µg) (Oxoid, UK) were dispensed onto the dried agar surface using a sterile forceps.The plates were incubated overnight at 37°C.After incubation period, the resulted zone of inhibition was compared with that of CLSI guideline (CLSI, 2010) for the interpretation of the data and categorization of the test strains as intermediate, sensitive, or resistant.

Results and Discussion
Among 350 collected samples from diarrheal patients, 4% (15) of them were positive for Salmonella spp.Results of antibiotic resistance pattern of the isolated Salmonella spp.are shown in Table 1 and Figure 1.Majority of the isolates were multidrug resistant having resistance pattern against at least three antibiotics.We found that 100% (15) of Salmonella spp.showed resistance to metronidazole and 87% (13) were resistant to erythromycin.7% (1) isolates were resistant to chloramphenicol and doxycycline and 20% (3) were resistant to ciprofloxacin.40% (6) of the total isolates were resistant to azithromycin and trimethoprim-sulfamethoxazole and 33% (4) were resistant to tetracycline.In developing countries multidrug resistance in bacteria is a common problem including South Asia.This problem is most likely related to the frequent use of antibiotics without proper medical supervision (Sack et al., 1997).The goal of this study was to investigate drug resistance patterns of the identified Salmonella spp. in Bangladesh.This study results revealed that 20% Salmonella were resistant to more than 5 drugs and 73% were resistant to 2-4 drugs.In another result we found that 20% Salmonella was resistant to ciprofloxacin which is similar to the study conducted by Xia et al., 2009.Reports from other countries have found an increasing frequency of ciprofloxacin-resistant isolates (Cailhol et al., 2006;Davis et al., 1999).Ciprofloxacin-resistant isolates are involved with increased morbidity and mortality (Varma et al., 2005;Helms et al., 2004) and high frequency makes it difficult to use fluoroquinolones for treatment of Salmonella infections in humans.Due to the increasing rate of antimicrobial resistance among salmonellae, broad-spectrum agents such as imipenem or cefepime are now more likely to be used for treatment.These antibiotics are usually more expensive and toxic, and more harmful to the commensurate microflora.This kind of situation, leads to great socioeconomic losses from the perspective of the patient, the hospital, and the whole society.A recent report stated that, they have isolated a Salmonella strain that was resistant to imipenem, which is a most fearful indication for future (Miriagou et al., 2003).

Conclusions
Drug resistant Salmonella have constituted a serious threat to public health.To overcome the challenge of drug resistance of Salmonellae, overuse and misuse of antimicrobial agents in food animals should be stopped (McEwen et al., 2002;Swartz et al., 2002).Continued surveillance for resistance pattern for salmonellae is mandatory and ongoing surveillance should be expanded and well standardized.It is however expected that this study will open a new window in exploring the true condition of antibiotic resistance patterns of Salmonella in Bangladesh.