Dhiviya Prabaa Muthuirulandi Sethuvel

Draft Genome Sequences of Three Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Isolates from Bacteremia

ABSTRACT Hypervirulent Klebsiella pneumoniae strains have been increasingly reported worldwide, and there is emergence of carbapenem resistance among them. Here, we report the genome sequences of three carbapenem-resistant hypervirulent K. pneumoniae isolates isolated from bacteremic patients at a tertiary-care center in South India.

Draft genome sequence of blaTEM-1-mediated cephalosporin-resistant Salmonella enterica serovar Typhi from bloodstream infection

Enteric fever is a major cause of concern in developing countries across the globe. The primary choice of antibiotics remains fluoroquinolones, followed by cephalosporins. Resistance to third-generation cephalosporins is rarely reported in Salmonella enterica serovar Typhi. This study reports the whole genome sequence of an S. Typhi isolate resistant to cefixime [minimum inhibitory concentration (MIC)=512μg/mL] by microbroth dilution. Interestingly, the isolate was negative for the cephalosporin resistance gene blaCTX-M by PCR, which is a known mechanism for higher cephalosporin resistance. The isolate was further subjected to next-generation sequencing that identified blaTEM-1B and blaDHA-1 genes in association with qnrB4 and sul1. blaTEM is a known gene coding for β-lactam resistance. In certain cases, overexpression of blaTEM was reported to result in cephalosporin resistance. This suggests that the high cefixime MIC would have been contributed by overexpression of blaTEM-1B. The blaTEM-1B gene was found to be associated with a promoter Px with -35 and -10 regions as TTAATA and TAAAGT, respectively. The promoter regions were unique, but the -10 region was similar to that found in Pa/Pb (previously reported promoter for blaTEM) with a single nucleotide change. In addition, an IncN plasmid was identified, which is usually reported in association with the most prevalent extended-spectrum β-lactamase (ESBL), metallo- and non-metallo-carbapenemase, and plasmid-mediated quinolone resistance (PMQR) genes. Plasmids such as IncN might possibly confer resistance and enhance spread. It is imperative to continuously monitor the drug resistance profile and evolving genetic elements.

Molecular Characterization of Intermediate Susceptible Typhoidal Salmonella to Ciprofloxacin, and its Impact

Background and ObjectiveExtensive use of ciprofloxacin to treat Salmonella typhi infections has led to the emergence of resistance, resulting in clinical failure and delayed treatment response. Interpretative breakpoints for ciprofloxacin were revised by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2012. Since the majority of S. typhi isolates fall under the category of ‘intermediate susceptible’ as per CLSI criteria, we undertook molecular characterization to better define the susceptibility of these isolates.MethodsOf 113 typhoidal Salmonella isolates collected during 2014, 33 (27 S. typhi and 6 S. paratyphi A) were randomly selected to determine the presence of chromosomal (gyrA, gyrB and parC), plasmid (qnrA, qnrB, qnrS and aac(6′)-lb-cr), and efflux-mediated fluoroquinolone resistance.ResultsTo the best of our knowledge, the parC mutation Glu(84)-Gly was observed for the first time in S. typhi in India. Of 33 isolates, only one harbored the qnrB gene, which is responsible for plasmid-mediated resistance. No significant change in efflux pump activity was observed for ciprofloxacin, except one that showed a fivefold decrease. Ninety-six percent of isolates with intermediate minimum inhibitory concentration to ciprofloxacin (CLSI) had mutations in the gyrA and parC genes, which might translate to possible/probable clinical failure in patients if treated with ciprofloxacin. In contrast, the EUCAST criteria define these isolates as resistant and may result in appropriate therapy with reduced morbidity.ConclusionIt was clear that the molecular mechanism of ciprofloxacin resistance correlates better with the EUCAST criteria than the CLSI criteria, which is also in agreement with the pefloxacin results, suggesting it as a surrogate marker for identifying fluoroquinolone susceptibility.

First report of blaOXA-181-mediated carbapenem resistance in Aeromonas caviae in association with pKP3-A: Threat for rapid dissemination

OBJECTIVES Carbapenemase-producing Aeromonas spp. are of great concern in healthcare settings and are also known to acquire clinically relevant resistance genes. In this study, carbapenem-non-susceptible Aeromonas isolates were characterised for their molecular mechanisms of resistance. METHODS Among 180 Aeromonas isolates, 10 carbapenem-non-susceptible isolates were selected based on their antimicrobial susceptibility profile. Carbapenemase production was investigated by the CarbaNP test. ESBL-, AmpC- and carbapenemase-encoding genes were screened by PCR. Isolates VBF557 and VBF856 with high MICs for imipenem were selected for whole-genome sequencing (WGS). Conjugation experiments were performed to determine the transmissibility of resistance. RESULTS WGS remarkably revealed the presence of class D β-lactamases (AmpS/AmpH), class C β-lactamases and class B2 metallo-β-lactamase (cphA3) in VBF557. In contrast, VBF856 had multiple resistance genes coding for aminoglycoside, sulphonamide, carbapenem (blaOXA-181 class D β-lactamase), macrolide, fluoroquinolone, rifampicin, phenicol, tetracycline and trimethoprim resistance. This is the first global report of blaOXA-181 in Aeromonas spp. Interestingly, blaOXA-181 was identified in association with transposon Tn2013 in plasmid pKP3-A. Additionally, an IncQ2 plasmid with qnrS2 was identified. Among the tested isolates, VBF1116 and VBF888 possessed blaNDM and blaVEB, respectively, by PCR. None of the other isolates harboured any tested β-lactamase genes. The resistance gene was transmissible in the presence of imipenem. CONCLUSIONS Presence of such resistance genes in plasmids further adds complexity for control of spread of carbapenem resistance. This study reveals the emergence of carbapenem resistance among Aeromonas spp. and the importance of mobile genetic elements such as plasmids in interchanging resistance determinants between species.

Molecular diagnosis of non-serotypeable Shigella spp.: Problems and Prospects

It is not always possible to identify Shigella serogroups/serotypes by biochemical properties alone. Specific identification requires serotyping. Occasionally, isolates that resemble Shigella spp. biochemically, but are non-agglutinable with available antisera, have been observed. Several mechanisms have been reported to limit the efficiency of the serotyping assay. Serotype conversion is a major mechanism in Shigella spp. to escape protective host immune responses. This easy conversion through significant modification of the O-antigen backbone results in different serotypes, which makes laboratory identification difficult. Furthermore, members of the family Enterobacteriaceae are closely related and there is antigenic cross-over (intra- and inter-specific cross-reaction) which affects the agglutination reaction. The performance of the available methods for identification of non-serotypeable Shigella is discussed here, and reveals them to be non-reliable. This shows a need for an alternative method for identification and typing of Shigella spp.

Pefloxacin as a Surrogate Marker for Fluoroquinolone Susceptibility for Salmonella typhi: Problems and Prospects.

Sir, The rise of MDR Salmonella typhi promoted the use of ciprofloxacin as the first line therapy since 2000 for enteric fever [1,2]. Due to selective pressure by extensive usage, there had been an emergence of resistance to ciprofloxacin. As per Clinical and Laboratory Standards Institute (CLSI), strains of Salmonella that test non-susceptible (intermediate), especially to ciprofloxacin, levofloxacin, ofloxacin, pefloxacin, or nalidixic acid may be associated with clinical failure or delayed response in fluoroquinolone-treated patients with salmonellosis [3]. Tests with nalidixic acid 30 μg and ciprofloxacin 5 μg disc will not reliably detect low-level resistance in Salmonella spp. Recently in 2015, CLSI recommended the use of 5 μg pefloxacin disc diffusion as a surrogate marker for identification of fluoroquinolone resistance in S.typhi [3]. This study was undertaken to evaluate the effectiveness of pefloxacin disc diffusion with ciprofloxacin disc diffusion and MIC breakpoints and highlights the problem and prospects of pefloxacin as surrogate marker. Prospects of Pefloxacin Earlier in 2012 the interpretative breakpoints for ciprofloxacin had been revised, where the susceptibility cut off using disc diffusion was raised from 21 to 31 mm and the MIC value was lowered from 1 to 0.06 μg/mL. In 2013, the disc diffusion interpretative criterion of levofloxacin and ofloxacin for S.typhi was removed. Meanwhile, the MIC interpretative criteria for levofloxacin and ofloxacin have been lowered to ≤0.12 μg/mL susceptible, 0.25-1 μg/mL intermediate and ≥2 μg/mL resistant. It is noteworthy that the interpretative criteria for ciprofloxacin, levofloxacin and ofloxacin have been changed only for typhoidal Salmonella in the Enterobacteriaceae family [4]. Recently (in 2015), CLSI and The European Committee on Antimicrobial Susceptibility Testing (EUCAST) have recommended the use of 5 μg pefloxacin disc diffusion test as reliable surrogate marker to identify the fluoroquinolone susceptibility to S.typhi [3,5]. Pefloxacin is understood to identify chromosomal (gyrA, gyrB, parC and parE); plasmid (qnrA, qnrB, qnrS and aac(6’)-lb-cr) mediated fluoroquinolone resistance better than nalidixic acid and ciprofloxacin (see [Table/Fig-1]). In addition, using pefloxacin can avoid the testing of ciprofloxacin and nalidixic acid by disc diffusion and MIC determination of levofloxacin, ofloxacin and ciprofloxacin [3]. [Table/Fig-1]: Genotypic, phenotypic and quinolone correlation of resistance mechanism in Salmonella typhi. Pefloxacin, in Our Observation Overall, 282 S.typhi isolates from community acquired blood stream infection were collected from January 2012 to December 2014 at Christian Medical College, Vellore, Tamil Nadu, India. All the isolates were tested for antimicrobial susceptibility by Kirby-Bauer disc diffusion method using ciprofloxacin (5 μg) and pefloxacin (5 μg), and E-test for ciprofloxacin. The results were interpreted according to the CLSI 2015 breakpoints and analysed. Among the total 282, 4.3% (n = 12), 80.5% (n = 227) and 15.2% (n = 43) isolates were susceptible, intermediate and resistant respectively to ciprofloxacin by disc diffusion method. Similarly, 4.3% (n = 12), 80.5% (n = 227) and 15.2% (n = 43) isolates were susceptible, intermediate and resistant respectively to ciprofloxacin MIC breakpoints. Interestingly, 4.6% (n = 13) and 95.4% (n = 271) isolates were susceptible and resistant respectively to pefloxacin by disc diffusion. According to CLSI interpretative criteria 80% (n = 225) of the isolates, intermediate to ciprofloxacin MIC fell under the category of resistant as per pefloxacin disc diffusion test. This observation perfectly matches with EUCAST ciprofloxacin MIC breakpoints as well. Further, a representative of 25 pefloxacin resistant isolates {ciprofloxacin MIC resistant (n = 14) and ciprofloxacin MIC intermediate (n = 11)} and two pefloxacin susceptible isolates were tested for gene mutations in gyrA, gyrB and parC genes. All the tested pefloxacin resistant isolates (n = 25) were observed to harbour gyrA and parC mutations (unpublished data). Problems with Pefloxacin The interpretative breakpoints defined both by CLSI and EUCAST for pefloxacin were narrow. The interpretative breakpoints for pefloxacin disc diffusion as per CLSI are ≤23 resistant and ≥24 susceptible, and <24 resistant and ≥24 susceptible by EUCAST. The chances for error are high with ± 1 mm difference in zone of inhibition by manual methods. Since there is no gold standard to compare for pefloxacin disc diffusion testing, we have included a stringent quality control (QC) to avoid interpretation errors to a great extent. QC range for E. coli ATCC 25922 should be 25-31 mm, with a target of 28 mm and mean value of repeated tests for pefloxacin should be within 27-29 mm (target ± 1 mm) [6]. Also, one should be aware that the presence of inner colonies in pefloxacin disc diffusion testing suggests resistance [7]. In addition, pefloxacin cannot be used to detect the resistance mediated by aac(6’)-lb-cr, as this plasmid mediated mechanism is specific for fluoroquinolones possessing a piperazinyl secondary amine (ciprofloxacin and norfloxacin) [8]. The remarkable observation of 80% of the isolates, intermediate to ciprofloxacin MIC falls under the category of resistant as per peflo-xacin disc diffusion test (confirmed by molecular characterization). This clearly indicates that the patients with intermediate ciprofloxacin MIC if treated with high dose of ciprofloxacin will lead to treatment failure or delayed response. Conversely, if we consider pefloxacin as a surrogate marker for fluoroquinolone resistance, this will lead to appropriate interpretation and therapeutic success rate.

Association of blaCTX-M-15 and qnr genes in multidrug-resistant Salmonella Typhimurium and Shigella spp from India.

Dear Editor, Acute infectious diarrhoea continues to be a significant cause of morbidity and mortality in children in low income countries. Among bacterial isolates, Shigella spp and Salmonella spp are known to contribute to the high burden of this illness in children. Recent literature survey reveals a sharp declining of prevalence of S. Typhi, while non-typhoidal Salmonella (NTS) and Shigella flexneri are increasing [1]. Among the Shigella isolates, > 90% are resistant to ampicillin and sulfonamides [2], 4%–10% of isolates are resistant to at least one of the thirdgeneration cephalosporins (ceftriaxone/cefotaxime) [3,2], and 7%–33% to fluoroquinolones [2]. Whereas in NTS, 86% are resistant to ampicillin, 33%–48% to sulfonamides [4,5], 34% to third-generation cephalosporins [3], and 24%-35% to fluoroquinolones [4,5]. Monitoring of cephalosporins and fluoroquinolones resistance is particularly important because these antibiotics are among the few therapeutic options commonly used for moderate to severe Salmonella and Shigella infections. The objective of this study was to determine the co-existence of qnr and extended-spectrum β lactamase (ESBL) genes in clinical isolates of multidrug-resistant (MDR) Shigella spp and Salmonella spp. (defined here as isolates resistant to ≥ 3 drugs)

IncFII plasmid carrying antimicrobial resistance genes in Shigella flexneri: Vehicle for dissemination

OBJECTIVES Plasmids harbouring antimicrobial resistance determinants in clinical strains are a significant public-health concern worldwide. The present study investigated such plasmids in clinical isolates of Shigella flexneri. METHODS A total of 162 Shigella isolates were obtained from stool specimens in the year 2015. Among the 70 multidrug-resistant (MDR) Shigella spp., 27 S. flexneri isolates were randomly selected for further characterisation. Antimicrobial resistance genes (ARGs) and plasmid incompatibility (Inc) types were analysed. RESULTS IncFII plasmids were found in 63% (17/27) of the studied S. flexneri isolates. ARGs such as dhfr1a (81%), sulII (74%), blaOXA (74%), blaTEM (33%), blaAmpC (30%), qnrS (15%) and qnrB (4%) were identified by PCR, whereas blaCTX-M was not detected. Next-generation sequencing of a representative S. flexneri IncFII-type plasmid (pSF470) revealed the presence of blaTEM1-B, blaDHA-1, qnrB10, mphA, sulI, sulII, strA, strB and tetR ARGs along with the intI1 integrase gene. In addition, pMLST analysis showed that the replicon belonged to F2:A-:B- type. CONCLUSIONS This study helps to know the prevalent plasmid types in MDR Shigella isolates and will improve our understanding of resistance dissemination among enteric bacteria. ARGs in plasmids further highlight the importance of such studies in enteric bacteria.

First report on whole-genome shotgun sequences of 23 biochemically indistinguishable clinical Shigella isolates

Shigella spp. are a major diarrhoeal disease pathogen worldwide and can cause considerable morbidity and mortality. Notably, limited genome data are available for serogroups/sub-serogroups of Shigella. Here we report the whole-genome shotgun sequences of 23 non-typeable Shigella from stool specimens that biochemically resembled Shigella spp. but were non-typeable with Shigella-specific antisera.

Methicillin-Susceptible Teicoplanin-Resistant Staphylococcus haemolyticus Isolate from a Bloodstream Infection with Novel Mutations in the tcaRAB Teicoplanin Resistance Operon

Staphylococcus haemolyticus is a coagulase-negative staphylococcus that is frequently isolated from blood cultures. Here, we report a case of methicillin-susceptible S. haemolyticus that is resistant to teicoplanin (TEC) and heteroresistant to vancomycin (VAN). The isolate was susceptible to cefoxitin and resistant to TEC by Etest. Population analysis profile-area under the curve analysis confirmed the presence of a VAN heteroresistant subpopulation. Next-generation sequencing analysis of the genome revealed the presence of blaZ and msr(A), which encode cross-resistance to macrolide, lincosamide, and streptogramin B, and the quinolone resistance-conferring gene norA. In addition, several amino acid substitutions were observed in the TEC resistance operon tcaRAB, including I3N, I390N, and L450I in tcaA and L44V, G52V, and S87P in tcaR, as well as in the transpeptidase encoding gene walK (D336Y, R375L, and V404A) and L315 and P316 in graS. We hypothesized that this combination of mutations could confer TEC resistance and reduced VAN susceptibility.