Long-term outbreak of Klebsiella pneumoniae& third generation cephalosporin use in a neonatal intensive care unit in north India.

BACKGROUND & OBJECTIVES: The indiscriminate use of third generation cephalosporin has contributed to the emergence and widespread dissemination of extended spectrum ß lactamases (ESBL) genes in Klebsiella pneumoniae. This study was undertaken to elaborate the genetic behaviour of ESBL - producing K. pneumoniae isolates in the neonatal intensive care unit (NICU) of a tertiary care hospital in north India causing successive outbreaks in context with empirical third generation cephalosporin use. METHODS: Isolates of K. pneumoniae (43 from blood, 3 from pus and endotracheal tube, 4 from environment) causing successive outbreaks in the NICU of a tertiary care university hospital were studied for two years. Antimicrobial susceptibility testing was done by disc diffusion and minimum inhibitory concentration (MIC) determination by agar dilution methods. ESBL production was determined by phenotypic and genotypic methods. Clonal relatedness among the isolates was studied by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Genetic environment of these isolates was assessed by the presence of integrons and gene cassettes. Transformation experiments were done, and plasmids of these isolates were characterized by stability testing and incompatibility testing. Subsequently, a change in the ongoing antibiotic policy was adopted, and corresponding changes in the behaviour of these isolates studied. RESULTS: During the period from August 2011 to January 2013, 46 isolates of monoclonal ESBL K. pneumoniae were obtained from different neonates and four similar environmental isolates were studied. Multidrug-resistant ESBL isolates harboured both blaCTXM-15 and bla SHV-5. The dfr and aac-6 ' resistant genes were found in gene cassettes. A 50 kb plasmid belonging to IncFIIA group was detected in all the isolates which was transferable and stable. The emergence and regression of the outbreaks coincided with antibiotic usage in the NICU, with widespread empirical use of cefotaxime being responsible for their persistence in the environment. INTERPRETATION & CONCLUSIONS: The study indicates that empirical use of third generation cephalosporins may promote the emergence, persistence, and dissemination of resistant isolates in the hospital environment. Periodic review of antibiotic policy is necessary for rationalized use of antibiotics.

Distribution of carbapenem resistant Acinetobacter baumannii with blaADC-30 and induction of ADC-30 in response to beta-lactam antibiotics.

A wide range of intrinsic Acinetobacter-derived cephalosporinases (ADC) along with other carbapenemases has now been detected in Acinetobacter baumannii leaving clinicians with few treatment options. The present study reports the spread of ADC-30 co-producing KPC-2 along with other ß-lactamases among carbapenem resistant A. baumannii strains obtained from ICU patients in two Indian hospitals. Primer extension analysis revealed higher transcript level of the ADC gene when induced with cefoxitin at 8 µg/ml (170 fold), ceftriaxone at 8 µg/ml (136 fold), ceftazidime at 4 µg/ml (65 fold), cefepime at 8 µg/ml (77 fold) and aztreonam at 8 µg/ml (21 fold) when compared with the basal level without antibiotic pressure. Slight increase in expression of blaADC-30 when induced with imipenem and meropenem at 0.25 µg/ml (3 and 6 fold) was observed and may help in conferring resistance to carbapenem. MLST analysis revealed the circulation of A. baumannii sequence types ST188, ST386, ST583 and ST390 in these hospitals.

High-level aminoglycoside resistance in Acinetobacter baumannii recovered from Intensive Care Unit patients in Northeastern India.

BACKGROUND: Acinetobacter baumannii has emerged as an important nosocomial pathogen, its ability to acquire resistance to carbapenems and aminoglycosides, has complicated their treatment regimen. The present study investigates the prevalence and diversity of aminoglycoside-modifying enzymes and 16S methyltransferases in A. baumannii isolates recovered from patients admitted in Intensive Care Unit (ICU) of a tertiary referral hospital in Northeastern India. MATERIALS AND METHODS: We investigated the high-level aminoglycoside-resistance (HLAR) (gentamicin and amikacin minimum inhibitory concentration ≥ 512 µg/ml) among 164 multidrug-resistant A. baumannii obtained from ICU. Genes encoding aminoglycoside-modifying enzymes, 16S methyltransferase and coexisting beta-lactamases were amplified. Horizontal transferability, plasmid stability and elimination assays were performed. Clonality and sequence types were evaluated by repetitive extragenic palindromic-polymerase chain reaction and multilocus sequence typing (MLST) respectively. RESULTS: A total of 130 (79.2%) isolates were found to exhibit HLAR, with acquired aminoglycoside-resistance genes in 109 (83.8%) isolates along with coexisting extended-spectrum beta-lactamases and metallo-beta-lactamases. Genes aph (3') I, aph (3') VIa and armA were predominant and horizontally transferable. Plasmids were eliminated with single sodium dodecyl sulphate treatment. Seventeen haplotypes were found responsible for the infection. MLST revealed circulation of ST583 and ST188 in ICU. CONCLUSIONS: This study reveals the presence of aminoglycoside-resistance genes in combination with blaCTXM and blaNDM, which are highly stable and not frequently reported from this geographical region. Further, the study could predict limited treatment option and need for formulating infection control strategy.

Genetic Environment of Plasmid Mediated CTX-M-15 Extended Spectrum Beta-Lactamases from Clinical and Food Borne Bacteria in North-Eastern India.

BACKGROUND: The study investigated the presence of CTX-M-15 type extended spectrum beta-lactamases (ESBL), compared their genetic arrangements and plasmid types in gram negative isolates of hospital and food origin in north-east India. From September 2013 to April 2014, a total of 252 consecutive, non-duplicate clinical isolates and 88 gram negative food isolates were selected. Phenotypic and molecular characterization of ESBL genes was performed. Presence of integrons and gene cassettes were analyzed by integrase and 59 base-element PCR respectively. The molecular environments surrounding blaCTX-M and plasmid types were investigated by PCR and PCR-based replicon typing respectively. Transformation was carried out to assess plasmid transfer. Southern blotting was conducted to localize the blaCTX-M-15 genes. DNA fingerprinting was performed by ERIC-PCR. RESULTS: Prevalence of ESBL was found to be 40.8% (103/252) in clinical and 31.8% (28/88) in food-borne isolates. Molecular characterization revealed the presence of 56.3% (58/103) and 53.5% (15/28) blaCTX-M-15 in clinical and food isolates respectively. Strains of clinical and food origin were non-clonal. Replicon typing revealed that IncI1 and IncFII plasmid were carrying blaCTX-M-15 in clinical and food isolates and were horizontally transferable. The ISEcp1 element was associated with blaCTX-M-15 in both clinical and food isolates. CONCLUSIONS: The simultaneous presence of resistance determinants in non-clonal isolates of two different groups thus suggests that the microbiota of common food products consumed may serve as a reservoir for some of the drug resistance genes prevalent in human pathogens.

Genetic acquisition of NDM gene offers sustainability among clinical isolates of Pseudomonas aeruginosa in clinical settings.

New Delhi metallo ß-lactamases are one of the most significant emerging resistance determinants towards carbapenem drugs. Their persistence and adaptability often depends on their genetic environment and linkage. This study reports a unique and novel arrangement of blaNDM-1 gene within clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital in north India. Three NDM positive clonally unrelated clinical isolates of P. aeruginosa were recovered from hospital patients. Association of integron with blaNDM-1 and presence of gene cassettes were assessed by PCR. Genetic linkage of NDM gene with ISAba125 was determined and in negative cases linkage in upstream region was mapped by inverse PCR. In which only one isolate's NDM gene was linked with ISAba125 for mobility, while other two reveals new genetic arrangement and found to be inserted within DNA directed RNA polymerase gene of the host genome detected by inverse PCR followed by sequencing analysis. In continuation significance of this novel linkage was further analyzed wherein promoter site detected by Softberry BPROM software and activity were assessed by cloning succeeding semi-quantitative RT-PCR indicating the higher expression level of NDM gene. This study concluded out that the unique genetic makeup of NDM gene with DNA-dependent-RNA-polymerase favours adaptability to the host in hospital environment against huge antibiotic pressure.

Genetic linkage of blaNDM among nosocomial isolates of Acinetobacter baumannii from a tertiary referral hospital in northern India.

The aim of this study was to evaluate the emergence of blaNDM-1 among clinical isolates of Acinetobacter baumannii isolated from a north Indian tertiary care hospital and to assess the gene cassettes and resistance determinants located within them. In total, 74 A. baumannii were screened for MBL production by the imipenem-EDTA method and were characterised for antibiotic sensitivity. PCR was performed to detect the presence of blaNDM and the co-existence of ESBL and AmpC genes. NDM-producing isolates were typed by ERIC-PCR, and the association of integrons with blaNDM-1 and the presence of gene cassettes were determined using specific primers. The genetic location of blaNDM in ISAba125 was also determined. Transformation was performed using a heat-shock method. Three isolates were found to harbour blaNDM, all of which co-produced blaEBC, blaDHA and blaCIT AmpC ß-lactamases. All MBL-producers showed resistance to cephalosporins, carbapenems, aminoglycosides, fluoroquinolones and tigecycline but were susceptible to polymyxin B. Presence of class 1 integrons was demonstrated in all three blaNDM-harbouring isolates, whilst linkage between the integron and blaNDM could not be established. Detection of gene cassettes revealed the presence of dihydrofolate reductase (dhfr) and aminoglycoside 6'-N-acetyltransferase [aac(6')] genes. Presence of blaNDM in ISAba125 was also observed. These findings suggest that ISAba125 appears to be the main genetic component for dissemination of blaNDM in A. baumannii. The association of blaNDM-1 with ISAba125 and the mobility of other multiresistance region (gene cassette)-carrying integrons provide an easy way to cross species barriers and reach a level that places the patients at risk.

Diagnostic utility of combination of inducer and inhibitor based assay in detection of Pseudomonas aeruginosa producing AmpC ß-lactamase.

The diagnostic utility of inducer and inhibitor based assays among 214 AmpC positive isolates of Pseudomonas aeruginosa were evaluated. Of different methods, combination of ceftazidime-imipenem antagonism and boronic acid inhibition tests came up with maximum sensitivity (76%) and specificity (100%). This combination showed reliability for both inducible and non-inducible AmpC producers.

Presence of different beta-lactamase classes among clinical isolates of Pseudomonas aeruginosa expressing AmpC beta-lactamase enzyme.

INTRODUCTION: Infections caused by Pseudomonas aeruginosa are difficult to treat as the majority of isolates exhibit varying degrees of beta-lactamase mediated resistance to most of the beta-lactam antibiotics. It is also not unusual to find a single isolate that expresses multiple beta-lactamase enzymes, further complicating the treatment options. Thus the present study was designed to investigate the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa. METHODOLOGY: A total of 202 clinical isolates of P. aeruginosa were tested for the presence of AmpC beta-lactamase, extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) enzyme. Detection of AmpC beta-lactamase was performed by disk antagonism test and a modified three-dimensional method, whereas detection of ESBL was done by the combined disk diffusion method per Clinical and Laboratory Standards Institute (CLSI) guidelines and MBL were detected by the Imipenem EDTA disk potentiation test. RESULTS: A total of 120 (59.4%) isolates were confirmed to be positive for AmpC beta-lactamase. Among them, 14 strains (7%) were inducible AmpC producers. Co-production of AmpC along with extended spectrum beta-lactamase and metallo beta-lactamase was reported in 3.3% and 46.6% isolates respectively. CONCLUSION: The study emphasizes the high prevalence of multidrug resistant P. aeruginosa producing beta-lactamase enzymes of diverse mechanisms. Thus proper antibiotic policy and measures to restrict the indiscriminative use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta-lactamase producing pathogens.

Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study.

BACKGROUND: Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-beta-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK. METHODS: Enterobacteriaceae isolates were studied from two major centres in India--Chennai (south India), Haryana (north India)--and those referred to the UK's national reference laboratory. Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene bla(NDM-1) was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing. Case data for UK patients were reviewed for evidence of travel and recent admission to hospitals in India or Pakistan. FINDINGS: We identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those from UK and Chennai were readily transferable whereas those from Haryana were not conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within the past year, or had links with these countries. INTERPRETATION: The potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated international surveillance is needed.