Malay Ranjan Sen

Dissemination of the New Delhi metallo-β-lactamase-1 (NDM-1) among Enterobacteriaceae in a tertiary referral hospital in north India

Sir, Of the different classes of carbapenemases, class B enzymes are clinically the most significant; they possess the widest substrate hydrolysis range, including penicillins, cephalosporins and carbapenems, but not monobactams. In recent years, metallo-b-lactamase (MBL) genes have spread from Pseudomonas aeruginosa to members of the Enterobacteriaceae. An alarming report by the HPA UK in 2009 warned of a rapidly proliferating novel carbapenem-hydrolysing b-lactamase (carbapenemase) designated New Delhi MBL-1 (NDM-1) among the Enterobacteriaceae family, identified in UK hospital patients. Moreover, the common presence of these b-lactamase genes in transferable mobile elements means that these genes could reach virtually any Gram-negative bacterium and this provides an added risk of dissemination in the community. Our present work was undertaken with the objective of detecting the blaNDM-1 gene among clinical isolates of the Enterobacteriaceae family in a tertiary referral hospital in north India. A total of 780 consecutive, non-duplicate isolates of Escherichia coli (n1⁄4528), Klebsiella pneumoniae (n1⁄4126), Citrobacter species (n1⁄484), Enterobacter aerogenes (n1⁄422), Proteus mirabilis (n1⁄411) and Morganella morganii (n1⁄49) were recovered from different clinical specimens from patients who were admitted to different wards, as well as from those who attended the outpatient departments of S.S. Hospital, BHU, Varanasi, India. The study was conducted from February 2010 to July 2010. The work was approved by the Ethics Committee. An imipenem/EDTA disc potentiation test was performed for phenotypic detection of MBLs. Antimicrobial susceptibility tests were performed using the Kirby–Bauer disc diffusion method and results were interpreted according to the CLSI recommendations. For partial gene PCR amplification, primers specific for the blaNDM-1 gene were used for reaction with bacterial DNA as template. PCR was performed as described previously. Random amplified polymorphic DNA (RAPD) was performed using primer 7 (5′-GTGGATGCGA-3′) and isolates were typed according to their band patterns. Sixty-four isolates were phenotypically found to be MBL producers. On performing PCR for all of the MBL-producing isolates the presence of the gene encoding NDM-1 was confirmed among 54 isolates, consisting of E. coli (n1⁄430), Citrobacter species (n1⁄412) and K. pneumoniae (n1⁄412), with an overall occurrence of 6.9% (54/780). Similar to this study, Deshpande et al. reported 22 NDM-1-producing Enterobacteriaceae in a short span of 3 months, while in a previous multicentre study, the occurrence was reported to be 2% from this centre. Most often, NDM producers were recovered from intensive care unit patients (35.1%). The age of the patients ranged from 1 day to 85 years, with 32 male patients and 22 female patients (Table S1, available as Supplementary data at JAC Online). The presence of class 1 integrons was demonstrated in all of the NDM-1-harbouring isolates. On typing the NDM-1-harbouring isolates, 18 patterns of E. coli, 8 patterns of K. pneumoniae and 5 patterns of Citrobacter species were found by RAPD. These results suggest horizontal transmission of the gene at both the intraspecies and interspecies level. Disc diffusion susceptibility testing showed that 46 (85.1%) of the NDM-1-producing isolates were susceptible to polymyxin B and 25 (46.2%) were susceptible to tigecycline. For other antimicrobials, 51.8% showed susceptibility to piperacillin/tazobactam and 22.2%, 5.5% and 1.8% showed susceptibility to amikacin, gentamicin and tobramycin, respectively. Among 23 urinary tract isolates, 8 showed susceptibility to nitrofurantoin. As many as 27 (50%), 26 (48.1%) and 16 (29.6%) isolates were found to be susceptible to ertapenem, imipenem and meropenem, respectively. A previous study has demonstrated 32.4% similarity of NDM-1 to VIM-1/VIM-2-type MBL and it Research letters

ANTIMICROBIAL SUSCEPTIBILITY OF PSEUDOMONAS AERUGINOSA ISOLATED FROM WOUND INFECTIONS

The primary aim of this study was to determine the prevalence of Pseudomonas aeruginosa in wound infections and its sensitivity to the commonly used antibiotics at SS Hospital, Varanasi, India. We received 940 relevant clinical specimens among, which 301 (32%) was P. aeruginosa . Antibiotic susceptibility was determined by the disc diffusion method where cefoperazone/sulbactam was found to be most effective (74%) followed by ciprofloxacin (58%) and ceftazidime (54%). Rest of the antibiotics showed a very low level of susceptibility pattern. A total of 54 (18%) isolates were resistant to all the antibiotics tested in vitro .

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

Genetic Acquisition of NDM Gene Offers Sustainability among Clinical Isolates of Pseudomonas aeruginosa in Clinical Settings

New Delhi metallo β-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 bla NDM-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 bla NDM-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.

Co-existence of Pseudomonas-derived cephalosporinase among plasmid encoded CMY-2 harbouring isolates of Pseudomonas aeruginosa in north India

CONTEXT In Pseudomonas aeruginosa, AmpC β-lactamases are often responsible for high-level resistance to β-lactam antibiotics. The co-production of plasmid-mediated AmpC along with chromosomal Pseudomonas-derived cephalosporinases thus remain a serious clinical concern owing to high resistance spectrum towards antibiotics. AIM The present study was performed to investigate the co-existence of both chromosomally-encoded and plasmid-mediated AmpC β-lactamase among clinical isolates of P. aeruginosa. SETTING AND DESIGN It is a cross-sectional study carried out in the Department of Microbiology in a tertiary referral hospital of northern India. METHODS AND METHODS A total of 329 consecutive, non-duplicate clinical isolates of P. aeruginosa, were selected for the detection of AmpC β-lactamases and confirmed for AmpC production by modified three dimensional (M3D) test. Ceftazidime -imipenem antagonism test was used to detect inducible AmpC producers. Molecular characterisation of chromosomally-encoded blaPDC and plasmid-mediated AmpC gene was studied by performing polymerase chain reaction (PCR). RESULT A total of 214 (65%) isolates were confirmed for AmpC production by M3D test. On performing multiplex PCR, 27 isolates were detected posessing blaCMY type of plasmid-mediated AmpC gene. While 48 isolates were found to harbour chromosomally-encoded blaPDC gene co-production of both chromosomal and plasmid-encoded AmpC was reported in eleven isolates. CONCLUSIONS Although these chromosomally-encoded cephalosporinases might spread more slowly than mobilised AmpC, but it is likely that in the present scenario of intense antibiotic pressure, this will become an increasing problem and may further limit our antibiotic choices.

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

Diagnostic utility of boronic acid inhibition with different cephalosporins against Escherichia coli producing AmpC β-lactamases.

Escherichia coli, one of the most important bacterial pathogens capable of causing community and nosocomial infections, may become resistant to cephamycins and oxyimino-cephalosporins by virtue of promoter and attenuator mutations or because they have acquired mobilized b-lactamases from other Gram-negative bacilli (Jacoby, 2009). The chromosomally encoded AmpC b-lactamase of E. coli is non-inducible, because of the absence of the regulatory gene ampR; the constitutive low level expression of chromosomal AmpC does not contribute to a clinically relevant level of resistance to b-lactams (Honoré et al., 1986). However, hyperproduction of these enzymes and plasmid-encoded AmpC enzyme can cause resistance to penicillins, first and second generation cephalosporins, cephamycins and to oxyiminocephalosporins (Doi & Paterson, 2007). The methods for detecting AmpC hyperproduction or plasmid-encoded AmpC enzymes among E. coli are technically demanding for clinical laboratories.

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 β 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 blaSHV-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.