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.

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.

Evaluation of the performance of nitrate reductase assay for rapid drug-susceptibility testing of mycobacterium tuberculosis in north India.

The objective of the study was to evaluate the performance of nitrate reductase assay (NRA) as a rapid, reliable and inexpensive method for drug-susceptibility testing (DST) of Mycobacterium tuberculosis against first-line antitubercular drugs, such as rifampicin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB). In total, 286 isolates were subjected to test by proportion method (PM) and NRA. By comparing the results of NRA with those of the gold standard PM, sensitivities and specificities were 98.4%, 97%, 88.5%, and 94.2% and 100%, 100%, 94%, and 99% for RIF, INH, STR, and EMB respectively. The positive predictive values were 100%, 100%, 95%, and 98% for RIF, INH, STR, and EMB respectively. The negative values were 99%, 98%, 87%, and 96% for RIF, INH, STR, and EMB respectively. The median time of obtaining results was shorter using NRA (10 days) compared to PM (28 days). An excellent agreement was observed between the two phenotypic tests with the K values of 0.98, 0.97, 0.81, and 0.93 for RIF, INH, STR, and EMB respectively. The results demonstrated that NRA is suitable for the early determination of INH and RIF resistance and has the potential to be a useful tool for rapid drug-sensitivity test of M. tuberculosis in resource-constrained settings.

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.

Compararative evaluation of rK9, rK26 and rK39 antigens in the serodiagnosis of Indian visceral leishmaniasis.

BACKGROUND: This study was designed for comparative evaluation of two relatively newer recombinant hydrophilic antigens, rK9 and rK26 of Leishmania chagasi along with rK39 (a 39-aminoacid-repetitive immunodominant B-cell epitope of kinesin-related antigen from L. chagasi) and crude soluble antigen (CSA) for the serodiagnosis of Indian visceral leishmaniasis (VL) patients by quantitative ELISA. METHODOLOGY: In the present study a total of 80 subjects comprising of 55 confirmed VL cases and 25 endemic controls (EC) were subjected to ELISA using four different antigens, namely rK9, rK26, rK39 and CSA (derived from Leishmania donovani promastigotes). RESULTS: Sensitivity was as follows: 78% (95%CI 63-100%) for rK9, 38% (95%CI 28-59%) for rK26, 100% for rK39, and 80% (95% CI 65-100%) for CSA. The specificity of rK9, rK26, rK39 and CSA was found to be 84% (95%CI 61-100%), 80% (95%CI 56-100%), 96% (95%CI 75-100%) and 72% (95%CI 49-100%), respectively. CONCLUSIONS: rK39 was observed to be the most suitable antigen as compared to rK26 and rK9 whereas rK9 performed better than rK26. Hence rK9 antigen may either be used as an adjunct to rK39 for accurate diagnosis of VL or may be used in the absence or non-availability of rK39 antigen for the serodiagnosis.

Assessment of different tests to detect methicillin resistant Staphylococcus aureus.

The heterogeneous expression of methicillin resistance in Staphylococcus aureus affects the efficiency of tests available to detect it. Not all laboratories have access to accurate molecular tests used for this purpose. This study compares the performances of four phenotypic tests used to detect methicillin resistant S. aureus (MRSA) with the mecA gene polymerase chain reaction. Two hundred thirty-seven S. aureus isolates were isolated from different patients visiting Sir Sundar Lal Hospital, Banaras Hindu University, Varanasi, India and subjected to cefoxitin and oxacillin disc diffusion tests, oxacillin minimum inhibitory concentration (MIC) test, and oxacillin screen agar test. The tests showed the following sensitivities and specificities, respectively: cefoxitin disc diffusion (98.5% and 100%), oxacillin disc diffusion (77.3% and 84.6%), oxacillin MIC (89.4% and 87.2%), and oxacillin screen agar (87.9% and 94.9%). The cefoxitin disc diffusion test can be the best method for routine detection of MRSA when molecular techniques are not available. We recommend the Clinical Laboratory Standards Institute (CLSI) cut-off point for determining cefoxitin resistance be reexamined to see if it should be revised from < or = 19 mm to < or = 20 mm.

Molecular typing of clinical Staphylococcus aureus isolates from northern India using coagulase gene PCR-RFLP.

Molecular typing of total 84 Staphylococcus aureus clinical isolates was performed using coagulase gene PCR. Out of 84 S. aureus strains total 33 different types of S. aureus strains were prevalent in this hospital and community. Types 2-7 and 9 were the most prevalent S. aureus strains accounting for more than 53% of total isolates. This technique is relatively inexpensive and is simple to perform and analyze.

Role of beta-lactamase inhibitors in enterobacterial isolates producing extended-spectrum beta-lactamases.

OBJECTIVES: To determine the in vitro activity of beta-lactamase inhibitors (clavulanic acid and sulbactam) in combination with third-generation cephalosporins and monobactam against extended-spectrum beta-lactamase (ESBL)-producing members of the Enterobacteriaceae family. METHODS: A total of 361 ESBL-producing enterobacterial isolates obtained from patients of a university hospital were screened for the status of co-production of AmpC beta-lactamase. These strains were further subjected to an MIC study using third-generation cephalosporins and monobactam, and reductions were observed after combining with beta-lactamase inhibitors at a fixed concentration of 4 mg/L. RESULTS: Most of the isolates showed 8-fold reduction with sulbactam when combined with ceftriaxone, cefpodoxime and cefotaxime but not with ceftazidime and aztreonam, whereas clavulanic acid showed the same result with all the cephalosporins tested. Further, both the inhibitors showed greater reduced MIC when combined with aztreonam. CONCLUSIONS: As the ability of clavulanic acid to induce AmpC production may interfere with ESBL detection, sulbactam is likely to be preferred over clavulanic acid after standardization of an appropriate concentration for ESBL detection in the scenario of increased prevalence of AmpC producers. Greater in vitro activity of these inhibitors when combined with aztreonam further indicates the need of studies to evaluate these combination antimicrobials in clinical settings as they can play a significant role for clinicians as viable alternatives to treat infections caused by such organisms.

Emergence of vancomycin resistant Staphylococcus aureus (VRSA) from a tertiary care hospital from northern part of India.

BACKGROUND: Glycopeptides such as vancomycin are frequently the antibiotics of choice for the treatment of infections caused by methicillin resistant Staphylococcus aureus (MRSA). For the last 7 years incidence of vancomycin intermediate S. aureus and vancomycin resistant S. aureus (VISA and VRSA respectively) has been increasing in various parts of the world. The present study was carried out to find out the presence of VISA and VRSA in the northern part of India. METHODS: A total 1681 staphylococcal isolates consisting of 783 S. aureus and 898 coagulase negative staphylococci (CoNS) were isolated from different clinical specimens from various outpatient departments and wards. All S. aureus and 93 CoNS were subjected to MIC testing (against vancomycin, teicolplanin and oxacillin); Brain Heart Infusion (BHI) vancomycin screen agar test; disc diffusion testing, and PCR for mecA, vanA and vanB genes detection. RESULTS: Out of 783 S. aureus two S. aureus strains were found to be vancomycin and teicoplanin resistant (one strain with MIC 32 microg/ml and the other strain with MIC 64 microg/ml); six strains of S. aureus have shown to be vancomycin intermediate (two strains with MIC 16 microg/ml and four strains with MIC 8 microg/ml); and two strains with teicoplanin intermediate (MIC 16 microg/ml). One CoNS strain was resistant to vancomycin and teicoplanin (MIC 32 microg/ml), and two CoNS strains were intermediate to vancomycin and teicoplanin (MIC 16 microg/ml). All VRSA, VISA and vancomycin resistant CoNS had shown growth on BHI vancomycin screen agar (vancomycin 6 microg/ml) and were mecA PCR positive. None of these isolates have demonstrated vanA/vanB gene by PCR. CONCLUSION: The present study reveals for the first time emergence of VISA/VRSA from this part of world and indicates the magnitude of antibiotic resistance in and around the study area. The major cause of this may be unawareness and indiscriminate use of broad-spectrum antibiotics.