Year : 2009 | Volume
: 52 | Issue : 4 | Page : 505--508
Changing trends in antimicrobial resistance of Salmonella enterica serovar typhi and salmonella enterica serovar paratyphi A in Chennai
Padma Krishnan1, M Stalin1, S Balasubramanian2,
1 Department of Microbiology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, India
2 Kanchi Kamakoti CHILDS Trust Hospital, Nungambakkam, Chennai, India
Department of Microbiology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai - 600 113
Background and Objectives: Chloramphenicol was considered the anti-microbial gold standard for typhoid treatment but, following the increasing worldwide frequency of antibiotic resistance, ciprofloxacin has been the mainstay of therapy since 1980. Recent studies have shown a shifting of susceptibility to conventional drugs like chloramphenicol, ampicillin and cotrimoxazole. The primary objective of the study was to evaluate the in vitro activity of chloramphenicol and other first-line drugs in comparison with cephalosporins and quinolones. Materials and Methods: Fifty isolates of Salmonella obtained from blood culture were subjected to serotyping at the Central Research Institute, Kasauli. Phage typing and biotyping was performed at the National Phage Typing Centre, New Delhi. Antibiotic sensitivity testing was carried out for 10 drugs by the Kirby-Bauer disc diffusion method and minimum inhibitory concentration by broth microdilution for nalidixic acid, chloramphenicol, ciprofloxacin, ceftriaxone, cefixime and ofloxacin. Multi-drug-resistant (MDR) strains were checked for plasmid. Results: In the present study, 70 and 30% of the isolates were Salmonella enterica serovar typhi and paratyphi A, respectively. They were highly sensitive to chloramphenicol (86%), ampicillin (84%) and cotrimoxazole (88%). Highest sensitivity was seen for cephalosporins, followed by quinolones. Seventeen/21 (81%) and 100% of the Salmonella enterica serovar typhi strains belonged to E1 phage type and biotype 1, respectively. Antibiogram showed 2% of the strains to be sensitive to all the drugs tested and 12% were MDR and showed the presence of plasmids. Conclusion: The study indicates reemergence of chloramphenicol-susceptible Salmonella enterica serovar typhi and paratyphi A isolates, a significant decline in MDR strains and high resistance to nalidixic acid. E1 phage type and biotype 1 are found to be most prevalent in Chennai, India.
|How to cite this article:|
Krishnan P, Stalin M, Balasubramanian S. Changing trends in antimicrobial resistance of Salmonella enterica serovar typhi and salmonella enterica serovar paratyphi A in Chennai.Indian J Pathol Microbiol 2009;52:505-508
|How to cite this URL:|
Krishnan P, Stalin M, Balasubramanian S. Changing trends in antimicrobial resistance of Salmonella enterica serovar typhi and salmonella enterica serovar paratyphi A in Chennai. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 Nov 17 ];52:505-508
Available from: http://www.ijpmonline.org/text.asp?2009/52/4/505/56140
Typhoid fever remains a disease of the developing world, with the highest annual incidence of 33 million cases.  Enteric fever continues to be a major health problem despite the use of antibiotics and the development of newer anti-bacterial drugs. The causative organism, Salmonella enterica serovar typhi has rapidly gained resistance to antibiotics like ampicillin, chloramphenicol and cotrimoxazole and also to previously efficacious drugs like ciprofloxacin. , The last two decades have seen the emergence of multi-drug resistance against the conventional anti-typhoidal drugs (ampicillin, cotrimoxazole and chloramphenicol) among the Salmonella sp, especially in Southeast Asia. During the period between 1990 and 1992, isolates of Salmonella enterica serovar typhi were uniformly resistant to chloramphenicol, ampicillin, cotrimoxazole and tetracycline. Subsequently, during the years 1993-1997, 30-35% regained susceptibility to these drugs.  The incidence of multi-drug-resistant (MDR) Salmonella enterica serovar typhi was reported to be as high as 92%, while there are recent reports noting a decline to 22% from the same region.  These developments had left fluoroquinolones as the only choice of treatment of typhoid fever. Rampant use of ciprofloxacin not only for typhoid but also for other infections gradually led to increased minimum inhibitory concentrations (MICs) of Salmonella enterica serovar typhi to ciprofloxacin. There are reports of clinical failure after treatment with ciprofloxacin, although they showed in vitro susceptibility. Fluoroquinolone resistance is being reported with increasing frequency from all over the world. From 2000, there have been reports of high levels of chloramphenicol susceptibility from many parts of India and the Salmonella strains have regained susceptibility to this drug due to less use.  Hence, the present study was undertaken to evaluate the efficacy of chloramphenicol and other first-line drugs as against the quinolones and cephalosporins, which were introduced later for the empirical treatment of enteric fever.
Materials and Methods
A total of 50 Salmonella isolates from the blood culture of patients presenting with enteric fever from tertiary care hospitals of Chennai during 2006-2007 were included in the study. The isolates were identified by standard procedures  and confirmed by high-titer antiserum (King Institute of Preventive Medicine, Chennai). Further serological confirmation was performed at the National Salmonella and Escherichiae Centre, Central Research Institute (CRI), Kasauli. Antibiotic Susceptibility testing was performed by the Kirby-Bauer disk diffusion method for the following antibiotics (Hi Media Laboratories Ltd., Mumbai, India): chloramphenicol (30 μg), ampicillin (10 μg), cotrimoxazole (25 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), gatifloxacin (5 μg), ofloxacin (5 μg), ceftriaxone (30 μg), cefixime (5 μg) and cefotaxime (30 μg). The disk strength and zone size interpretation was in accordance with the Clinical Laboratory Standard Institute (CLSI).  MICs of ciprofloxacin, ofloxacin, nalidixic acid, ceftriaxone, cefixime and chloramphenicol were determined by broth microdilution performed in accordance with the CLSI guidelines. Phage typing was carried out at the National Salmonella Phage Typing Centre, Lady Hardinge Medical College, New Delhi. Plasmid DNA was extracted by the alkaline lysis method using a plasmid isolation kit (Eppendorf India Limited, Chennai, India). The plasmid DNA was qualitatively analyzed by agarose gel electrophoresis on a 0.8% agarose gel with standard DNA markers to check for the presence of plasmid DNA and to determine the size of plasmid DNA among MDR strains.
In the present study, of the 50 isolates, only two serotypes were encountered. Thirty-five (70%) were identified as Salmonella enterica serovar typhi and 15 (30%) were identified as Salmonella enterica serovar paratyphi A. When the Salmonella enterica serovar typhi was subjected to phage typing, 17/21 (81%) belonged to E1 phage type while the others were non-typable. Two of the isolates were found to be Vi negative. All the Salmonella enterica serovar typhi strains belonged to biotype I. Antibiogram of Salmonella enterica serovar typhi showed [Table 1] one (2%) strain to be sensitive to all the drugs tested. Resistance pattern varied from resistance to single to six of the anti-microbial agents. one (2%) isolate showed resistance to ampicillin alone, 32 (64%) to nalidixic acid alone (which formed the predominant pattern prevalent in Chennai during the study period), eight (16%) to two antibiotics, two (4%) to three antibiotics and six (12%) were MDR. Among MDR, the following patterns were seen: three were ACCoNA, one was ACoNACiCe, one was ACNACfOf and one was ACNAOfCfG.
The anti-microbial sensitivity against various drugs tested showed highest sensitivity to cephalosporins, followed by quinolones [Table 2]. Forty-three (86%) of the isolates were sensitive to chloramphenicol. Sensitivity index (SI =%sensitivity/%resistance) indicated highest values for cephalosporins, followed by quinolones. The SI values for chloramphenicol and other first-line drugs indicated the suitability of these drugs as therapeutic agents for typhoid fever.
When tested for susceptibility to nalidixic acid by disc diffusion and MIC [Table 3], 48 (96%) of the Salmonella enterica serovar typhi were nalidixic acid-resistant (NAR) strains and two (4%) were nalidixic acid-sensitive strains. Among the NAR strain, 73% were sensitive, 25% were moderately sensitive and 2% were resistant to ciprofloxacin. There was a positive correlation between NAR and decreased susceptibility to ciprofloxacin.
Comparison of MIC with median zone diameter [Table 4] showed that chloramphenicol had the lowest MIC value permissible and a correspondingly larger median zone diameter than breakpoint, whereas median zone diameter for ciprofloxacin was the breakpoint value and it also showed correspondingly higher MIC 50 . There was a good correlation between median zone diameter and MIC 50 for the antibiotics tested. Highest MIC value was seen for nalidixic acid. There was an overall increase in MIC 50 values for the quinolones and cephalosporins tested. All the MDR strains carried a plasmid of >33,500 bp.
In the present study, Salmonella enterica serovar typhi and paratyphi A showed significant increase in sensitivity to chloramphenicol (86%), ampicillin (84%) and cotrimoxazole (88%), the first-line drugs for enteric fever. A study from Kolkata has indicated similar sensitivities (85.6% for ampicillin, 83.6% for chloramphenicol, 83.1% for cotrimoxazole).  Dutta et al. have also reported a remarkable reversal in the resistance pattern of Salmonella enterica serovar typhi in Kolkata.  Multi-drug resistance is still common in Salmonella enterica serovar typhi, although it is declining with increased use of fluroquinolones and cephalosporin for the treatment. There was a significant decline in the MDR strains (12%) found in our study compared with earlier reports, indicating up to 92% of multi-drug resistance. , The median zone diameter in the present study showed a positive correlation with the MIC.
Despite reasonable %S and low %R populations, there is unnoticed %I and correspondingly low values of SI for various antibiotics. Based on SI values, cephalosporins with the highest SI values (cefixime 100%, cefotaxime and ceftriaxone 96 and 94%, respectively) could be considered to be the best therapeutic options followed by quinolones. The first-line drugs show sensitivity index values high enough to be suitable options for empirical therapy.
The quinolone group of drugs emerged as useful drugs for the treatment of MDR cases of typhoid infection. Previous study has shown resistance to ciprofloxacin in 21.4% of the cases  and 18.1% of the cases.  Nalidixic acid susceptibility has been validated as a screening test for reduced susceptibility to ciprofloxacin and nalidixic acid is associated with a high MIC of ciprofloxacin, which is associated with treatment failure. , In the present study, 48 of 50 isolates were NAR and were associated with decreased susceptibility and increased resistance to ciprofloxacin. In our study, about 2% of the isolates were resistant to ciprofloxacin and 24% showed decreased susceptibility to ciprofloxacin. Whereas the study showed a much higher nalidixic acid resistance and a higher MIC for ciprofloxacin compared with disc diffusion breakpoint results, strains showing decreased susceptibility to ciprofloxacin by disc diffusion and with increased MIC are now endemic in India.  The quinolone resistance is due to altered DNA gyrase sub-unit but, recently, plasmid-mediated quinolone resistance has also been reported  and so is significant in transmission of resistance. Currently, there is emergence of high level of MIC not only to ciprofloxacin but also to ofloxacin and ceftriaxone in Salmonella enterica serovar typhi and Salmonella enterica serovar paratyphi A. 
In the recent past, cephalosporins have gained importance for the treatment of enteric infection. Third-generation cephalosporins in particular are effective in the treatment of typhoid fever. Ceftriaxone, administered either intra-venously or intra-muscularly, and cefixime, which is administered orally, are both effective in typhoid fever, even for NAR infection.  Among the drugs tested, cefixime alone gave 100% SI, followed by other cephalosporins. Although there was no resistance to cephalosporins, intermediate resistance was seen toward cefotaxime and ceftriaxone and also a corresponding increase in the MIC values. This has also been observed by other workers. 
In our plasmid study, the MDR strains carried a plasmid of >33,500 bp. Previous reports have indicated plasmids of molecular weight 25.4 and 62.5 kb to be more frequent among strains with the following resistant pattern: ACoCT, ACTCoS, ACTCe and ACTS. 
Indiscriminate use of drugs is one of the important factors leading to drug resistance and, in case of ciprofloxacin, moderate cost, advantage of oral route, tolerability and convenient dosage schedule have contributed toward its indiscriminate use. The present study encourages the re-introduction of chloramphenicol in the treatment of typhoid fever.
We would like to acknowledge the financial assistance of Piramal Healthcare Limited for carrying out the above study.
We would like to thank the National Salmonella and Escherichia Serotyping Centre, CRI, Kasauli, for carrying out the serotyping of the isolates and the National Phage Typing Centre, Lady Hardinge Medical College, for carrying out the phage typing. The help of Orchid Chemicals and Pharmaceuticals Ltd. in providing the cephalosporin powder is duly acknowledged.
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