Antimicrobial Susceptibility Pattern of Salmonella enterica Species in Blood Culture Isolates

Enteric fever continues to be a major health problem in under developed countries including South Asian nations. It afflicts local inhabitants as well as travelers to endemic areas. Increasing multidrug resistance in Salmonella enterica serotype Typhi has been reported from various parts of the world [1-5]. Enteric fever is endemic in Nepal. S. enteric serotype Typhi and S. enteric serotype Paratyphi A have been reported as the most common culture isolates from patients with febrile illnesses needing hospital admission [6,7]. Over the past decade, increasing antibiotic resistance in S. enteric has led to a shift in the antibiotics used against this organism from chloramphenicol and ampicillin to trimethoprim–sulfamethoxazole, fluoroquinolones (ofloxacin, ciprofloxacin), and ceftriaxone. Even with the use of these antibiotics, the positive response to treatment has been only seen in the range of 16-40% in Nepal [7].


Introduction
Enteric fever continues to be a major health problem in under developed countries including South Asian nations. It afflicts local inhabitants as well as travelers to endemic areas. Increasing multidrug resistance in Salmonella enterica serotype Typhi has been reported from various parts of the world [1][2][3][4][5]. Enteric fever is endemic in Nepal. S. enteric serotype Typhi and S. enteric serotype Paratyphi A have been reported as the most common culture isolates from patients with febrile illnesses needing hospital admission [6,7]. Over the past decade, increasing antibiotic resistance in S. enteric has led to a shift in the antibiotics used against this organism from chloramphenicol and ampicillin to trimethoprim-sulfamethoxazole, fluoroquinolones (ofloxacin, ciprofloxacin), and ceftriaxone. Even with the use of these antibiotics, the positive response to treatment has been only seen in the range of 16-40% in Nepal [7].
Over the last decade, fluoroquinolones have emerged as the mainstay of therapy for enteric fever. At the same time, increasing incidence of infection with Salmonellae resistant to nalidixic acid, which usually display decreased susceptibility to fluoroquinolones, has raised considerable global concern [8]. The vast majority of nalidixic acid resistant isolates remains within the current susceptibility range for ciprofloxacin (1 µg/ml) as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). However, the probability of clinical response to fluroquinolone therapy in patients with invasive Salmonella infection is lower in those with Nalidixic acid resistant than with susceptible isolates [9]. In 1993, 23% of Salmonella enteric serotype Typhi isolates from patients in the United Kingdom exhibited decreased susceptibility to ciprofloxacin more than half of these were also resistant to Chloramphenicol, Ampicillin and Trimethoprin. Increasing numbers of treatment failures were noted. Most infections were noted in patients with a recent history of travel to India and Pakistan, Nepal, Sri Lanka, Bangladesh, and Thailand [10].

Materials and Methods
The study was conducted from February to August 2013, at Civil Service Hospital at Kathmandu, Nepal. Blood samples were obtained from patients suspected with enteric fever. The samples to be tested were inoculated in Bactec® culture bottle vial with soybean-casein digested broth which was inserted into the BACTEC 9050® for incubation and periodic reading. Each culture vial contains a chemical sensor which can detect Co 2 produced by the growth of microorganism. The sensor was monitored by the instrument every 10 minutes for an increase in its fluorescence, which was proportional to the amount of present. A positive reading indicated the presumptive presence of viable microorganism in the vial [11]. From the positive vial subculture was done on Blood agar and MacConkey agar, Identification of bacteria was done using standard microbiological techniques [12][13][14]. Serotyping of Salmonella enterica was done by done using polyvalent O-antisera (Denka Seiken, Japan), Salmonella 9-O, Salmonella Vi(Remel Europe, UK). Antimicrobial susceptibility for Salmonella enterica was performed by Kirby-Bauer disk diffusion method on Mueller Hinton Agar following National Committee for Clinical Laboratory Standards (NCCLS) recommendations [12][13][14][15]. The antibiotics tested were: Amoxycillin (10 μg)and Azithromycin (15 μg) with zone of inhibition (ZOI)≥18 mm, Ceftriaxone (30 μg)and Ciprofloxacin (5 μg) ZOI≥21 mm, Chloramphenicol (30 μg) ZOI≥18 mm, Co-trimoxazole (25 μg) ZOI≥mm, Nalidixic acid (30 μg) ZOI≥19 mm and Tetracycline (30 μg) ZOI ≥15 mm(Hi Media Laboratory Ltd., Mumbai, India) [15]. The disk strength and zone-size interpretation were in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) [15]. MDR was categorized if they were resistant to at least two classes of firstline agents including Amoxycillin, Chloramphenicol, Trimethoprimsulfamethoxazole, Fluoroquinolones (Ciprofloxacin and Ofloxacin), and Cephalosporins (Ceftriaxone).
Statistical comparisons of prevalence rates between the two serotypes and differences in resistance rates against the antibiotics were done by Fisher's exact tests and Chi-square tests and Fisher's exact test (where the sample size was less than 5) using SPSS software version 22.0 (SPSS Inc., Chicago, USA).    Pearson Chi-Square=109.564, P value =0.000 In the other hand 7.18% of Salmonella enterica serotype typhi isolates were resistant to Azithromycin and Nalidixic acid whereas in case of Salmonella enterica serotype paratyphi A, the resistant pattern has increased up to 11.7%.

Discussion
The result of this study showed that a prevalence rate of 11.8% for the S. enteric in the study area, a similar trend has been reported in a recent study done in Nepal [15]. Chloramphenicol, Ampicillin, and Trimethoprim-sulfamethoxazole were the antibiotics widely used as the primary for the treatment of treatment of enteric fever. In our study 48% of isolates were resistant to two or more drugs where 81.2% were S. enteric serotypes Typhi and 18.2% were Paratyphi A and only 0.5% were Paratyphi B ( Table 3). All of these multidrug resistant isolates showed reduced susceptibility to fluoroquinolones ( Table 2). A similarresults show a high sensitivity of both Salmonella enterica serovar typhi (96%) and Salmonella enterica serovar paratyphi A (100%) to chloramphenicol. Sensitivity to ciprofloxacin and amikacin was 88% and 84% respectively ( Table 1). Sensitivity of Salmonella enterica serovar paratyphi A was 100% to Chloramphenicol, Ciprofloxacin, Ofloxacin, Nalidixic acid and Ceftriaxone, 95% to Amikacin and 30% to ampicillin [16]. Our result (Table 1 and 4) showed different comparable view (i.e. Chloramphenicol 100% susceptibility) than that of others studies reporting Chloramphenicol resistant in S.enterica [17][18][19]. In 1970s the isolates resistant to Chloramphenicol were reported in various countries like UK, India, Mexico, Greece, Israel, and Pakistan [2,4]. While In the 1990s, the patter showing resistance of Chloramphenicol suggested to the use of fluoroquinolones (Ciprofloxacin and Ofloxacin) [20], which werefound to be highly resistant in this study which accounts as 40%. Similarly a different view was reported on Pakistan reporting Salmonella enteric serotype paratyphi-A were sensitive to Ciprofloxacin, Ofloxacin and Nalidixic acid and 62.7% isolates were resistant to firstline antibiotics (Ampicillin, Chloramphenicol and Co-Trimoxazole) on disc diffusion testing [21]. According to Thonget et al. [5] The MDR S. typhi isolates were resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Analysis by PFGE showed that 50 MDR isolates of S. typhi had a single, homogenous PFGE profile, which was distinctly different from that of 50 antibiotic-sensitive isolates obtained in the same time frame from the same area [22].

Conclusion
Our study has further accentuated concern about the sensitivity pattern and the status of multi drug resistant Salmonella enterica spp. This study suggests Chloramphenicol as a drug of choice for enteric fever and further monitoring of efficacy of older and newer antibiotics are desirable.