Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013 Shanta Dutta 1 *, Surojit Das 1 , Utpala Mitra 1 , Priyanka Jain 1 , Indranil Roy 2 , Shelley S. Ganguly 3 , Ujjwayini Ray 4 , Phalguni Dutta 1 , Dilip Kumar Paul 5 1 National Institute of Cholera and Enteric Diseases, Kolkata, India, 2 Calcutta Medical Research Institute, Kolkata, India, 3 Advance Medical Research Institute, Salt Lake, Kolkata, India, 4 Apollo Gleneagles Hospitals, Kolkata, India, 5 Dr. B. C. Roy Post Graduate Institute of Pediatric Sciences, Kolkata, India Abstract Enteric fever, caused by Salmonella enterica, remains an unresolved public health problem in India and antimicrobial therapy is the main mode of treatment. The objective of this study was to characterize the Salmonella enterica isolates from Kolkata with respect to their antimicrobial resistance (AMR), virulence profiles and molecular subtypes. Salmonella enterica blood isolates were collected from clinically suspected enteric fever patients attending various hospitals in Kolkata, India from January 2009 to June 2013 and were tested for AMR profiles by standard protocols; for resistance gene transfer by conjugation; for resistance and virulence genes profiles by PCR; and for molecular subtypes by Pulsed Field Gel Electrophoresis (PFGE). A total of 77 Salmonella enterica serovar Typhi (S. Typhi) and 25 Salmonella enterica serovar Paratyphi A(S. Paratyphi A) from Kolkata were included in this study. Although multidrug resistance (resistance to chloramphenicol, ampicillin, co-trimoxazole) was decreasing in S. Typhi (18.2%) and absent in S. Paratyphi A, increased resistance to fluoroquinolone, the current drug of choice, caused growing concern for typhoid treatment. A single, non-conjugative non- IncHI1 plasmid of 180 kb was found in 71.4% multidrug resistant (MDR) S. Typhi; the remaining 28.6% isolates were without plasmid. Various AMR markers (bla TEM-1 , catA, sul1, sul2, dfrA15, strA-strB) and class 1 integron with dfrA7 gene were detected in MDR S. Typhi by PCR and sequencing. Most of the study isolates were likely to be virulent due to the presence of virulence markers. Major diversity was not noticed among S. Typhi and S. Paratyphi A from Kolkata by PFGE. The observed association between AMR profiles and S. Typhi pulsotypes might be useful in controlling the spread of the organism by appropriate intervention. The study reiterated the importance of continuous monitoring of AMR and molecular subtypes of Salmonella isolates from endemic regions for better understanding of the disease epidemiology. Citation: Dutta S, Das S, Mitra U, Jain P, Roy I, et al. (2014) Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013. PLoS ONE 9(8): e101347. doi:10.1371/journal.pone.0101347 Editor: Nicholas J. Mantis, New York State Dept. Health, United States of America Received November 19, 2013; Accepted June 5, 2014; Published August 6, 2014 Copyright: ß 2014 Dutta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was funded by the Indian Council of Medical Research (ICMR) intramural fund. Senior Research Fellowships from Indian Council of Medical Research to Mr. S. Das and Ms. P. Jain are gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish or preparation of manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: shanta1232001@yahoo.co.in Introduction Enteric fever caused by Salmonella enterica serovar Typhi (S. Typhi) and Salmonella enterica serovar Paratyphi A (S. Paratyphi A) still continues to be a major public health problem in developing countries like India. Typhoid fever was estimated to have caused 21.6 million illnesses and 216,500 deaths globally, and the less severe paratyphoid fever caused an estimated 5.4 million illnesses in 2000 [1]. The incidence of typhoid fever was estimated as 214.2 per 100,000 people/year during 2003–2004 in a population-based study conducted at different urban slums of Kolkata [2]. Although S. Typhi was observed to be the predominant serovar worldwide, a shift in the most prevalent Salmonella serotype from S. Typhi to S. Paratyphi A has been reported recently by many researchers [3–6]. Antimicrobial therapy is the mainstay for treatment of enteric fever and typhoid fever may become fatal in 30% cases due to complications in absence of appropriate antibiotics. Since the late eighties, increased isolation of multidrug resistant (MDR, resistant to ampicillin, chloramphenicol and co-trimoxazole with or without tetracycline) S. Typhi was reported from different parts of India limiting the use of these drugs in the treatment of typhoid [7,8]. Subsequently ciprofloxacin was used as the drug of choice in adults. But frequent use of ciprofloxacin led to the global emergence of nalidixic acid resistant S. Typhi and S. Paratyphi A associated with decreased susceptibility to ciprofloxacin, causing increased treatment failure cases [6,9–12]. Thereafter emergence of ciprofloxacin resistant S. Typhi and S. Paratyphi A further decreased the number of treatment options [10,13,14]. Sporadic reports of Salmonella enterica isolates resistant to higher gener- ation cephalosporins like ceftazidime, cefotaxime, cefuroxime erased even the last option for treatment of enteric fever [15– 17]. Azithromycin has been used as an effective alternative in PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e101347