Multidrug Resistant CTX-M-Producing Escherichia coli: A Growing Threat among HIV Patients in India

Multidrug Resistant CTX-M-Producing Escherichia coli: A Growing Threat among HIV Patients in India Kesavaram Padmavathy,1 Krishnan Padma,2 and Sikhamani Rajasekaran3 1Department of Microbiology, Research Laboratory for Oral and Systemic Health, Sree Balaji Dental College and Hospital, BIHER, Bharath University, Chennai 600100, India 2Department of Microbiology, Dr. ALM PGIBMS, University of Madras, Chennai 600113, India 3Government Hospital of Thoracic Medicine, Chennai 600047, India Received 17 December 2015; Accepted 6 March 2016 Academic Editor: Anand S. Deshmukh Copyright © 2016 Kesavaram Padmavathy et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Extended Spectrum β-Lactamases (ESBLs) confer resistance to third-generation cephalosporins and CTX-M types have emerged as the most prominent ESBLs worldwide. This study was designed to determine the prevalence of CTX-M positive ESBL-producing urinary E. coli isolates from HIV patients and to establish the association of multidrug resistance, phylogeny, and virulence profile with CTX-M production. A total of 57 ESBL producers identified among 76 E. coli strains isolated from HIV patients from South India were screened for , AmpC production, multidrug resistance, and nine virulence associated genes (VAGs), fimH, pap, afa/dra, sfa/foc, iutA, fyuA, iroN, usp, and kpsMII. The majority (70.2%) of the ESBL producers harbored and were AmpC coproducers. Among the CTX-M producers, 47.5% were found to be UPEC, 10% harbored as many as 7 VAGs, and 45% possessed kpsMII. Multidrug resistance (CIPRSXTRGENR) was significantly more common among the CTX-M producers compared to the nonproducers (70% versus 41.2%). However, 71.4% of the multidrug resistant CTX-M producers exhibited susceptibility to nitrofurantoin thereby making it an effective alternative to cephalosporins/fluoroquinolones. The emergence of CTX-M-producing highly virulent, multidrug resistant uropathogenic E. coli is of significant public health concern in countries like India with a high burden of HIV/AIDS. 1. Introduction Uropathogenic Escherichia coli (UPEC), a subset of the extraintestinal pathogenic E. coli (ExPEC), is the principal etiological agent of community onset urinary tract infection (UTI) accounting for substantial morbidity and medical costs worldwide. Recent studies have shown that ExPEC isolates have increasingly become resistant to the frontline antibiotics including the cephalosporins, fluoroquinolones, and trimethoprim-sulfamethoxazole [1]. Emergence of β-lactam resistance mediated by Extended Spectrum β-Lactamase (ESBLs, e.g., CTX-M types) and AmpC cephalosporinases (especially plasmid mediated AmpC enzymes, e.g., CMY types) is a major global problem [2]. ESBL mediated resistance has been increasingly reported among urinary Escherichia coli isolated from HIV patients in India [3, 4]. ESBL-producing isolates, especially CTX-M-producing E. coli, exhibit an alarming trend in coresistance to other classes of antibiotics [5–7]. Among strains of ExPEC, eleven serogroups (O1, O2, O4, O6, O7, O8, O16, O18, O25, O50, and O75) are associated with UTI and they generally belong to the virulent phylogroup B2 and to a lesser extent phylogroup D that elaborate an array of virulence factors including adhesins, protectins, toxins, and iron acquisition systems. Previous studies have reported that multidrug resistant E. coli strains are significantly associated with decreased virulence, non-B2 phylogenetic lineage, and host immunocompromised status [8, 9]. Also, many studies have documented a strong association between CTX-M production and decreased virulence [10–12]. Nevertheless, emergence of ESBL-producing ExPEC with a high virulence potential is of public health concern [13]. There is paucity of information on the virulence status and drug resistance profile of CTX-M-producing E. coli particularly infecting the HIV population in our geographical setting. Hence, this study was designed to assess the prevalence of CTX-M producers among the UPEC isolates from HIV patients and to establish the association of phylogeny, multidrug resistance, and virulence profile with CTX-M production. 2. Materials and Methods2.1. Bacterial Isolates A total of 76 nonrepetitive urinary E. coli isolated from HIV patients (with recent exposure to fluoroquinolones/3G cephalosporins, with CD4 count <350 cells/mm3 and not on antiretroviral therapy, with community onset UTI) from Chennai, South India, were included in the study. 2.2. Detection of ESBL and AmpC Production and Multidrug Resistance ESBL production was confirmed using cefotaxime (CTX), ceftazidime (CAZ), and cefepime (FEP) alone and in combination with clavulanic acid (CLA) as per clinical and laboratory standards institute (CLSI) guidelines [14]. PCR detection of was performed among ESBL-producing urinary E. coli isolates from HIV patients [15]. Detection of AmpC phenotype was carried out by boronic acid method [16] and confirmed by AmpC disc test [17]. Antibiotic susceptibility testing was performed as per CLSI to assess the resistance pattern to non-beta-lactam antibiotics [14]. E. coli ATCC 25922 was included as the control. CTX-M-producing isolates that were resistant to all the three tested antimicrobial classes (fluoroquinolones, ciprofloxacin; folate pathway inhibitor, cotrimoxazole; and aminoglycosides, gentamicin (CIPRSXTRGENR)) were designated as multiple drug resistant isolates. 2.3. Phylogrouping and O Serogrouping Phylogenetic grouping was done by multiplex PCR (group A (chuA−, yjaA+/−, and TspE4C2−), group B1 (chuA−, yjaA+/−, and TspE4C2+), group B2 (chuA+, yjaA+, and TspE4C2+/−), and group D (chuA+, yjaA−, and TspE4C2+/−)) [18]. O serogrouping was carried out by the traditional antiserum technique at the National Salmonella & Escherichia Centre, Central Research Institute, Kasauli, Himachal Pradesh, India. 2.4. Virulence Profiling The isolates were screened for 9 virulence associated genes linked with increased clinical severity: pap [PapA and PapC], sfa/foc [S and F1C fimbriae], afa/dra [Dr-binding afimbrial adhesins] [19], iutA [aerobactin receptor], iroN [salmochelin receptor], fyuA [aerobactin] [20], usp [uropathogenic specific protein] [21], kpsMII [group 2 capsule synthesis] [22], and FimH [type I fimbriae] [23] by PCR. Haemolysin production was assessed on sheep blood agar plates. 2.5. DNA Sequencing DNA sequencing was performed using Applied Biosystem 3130 Genetic Analyser with ABI PRISM BigDye Terminators version 3.1 (at GenOmb Biotechnologies, Pune, India) to establish positive controls and to confirm the identity of the amplicons. The nucleotide sequences were submitted to the GenBank database under the following accession numbers: (HQ284192), iroN (HQ013325), fyuA (HQ013326), iutA (HM992940), chuA (HQ284193), yj (...truncated)