Np Singh

Changing trends in bacteriology of burns in the burns unit, Delhi, India

A retrospective study of bacterial isolates from the wounds of patients admitted to burns unit, was undertaken at Guru Tegh Bahadur Hospital, University College of Medical Sciences, Delhi, India, over a period of 5 years between July 1997 and April 2002. The study compared the results obtained with the previous 5 years data (June 1993-June 1997) from the same unit to determine the changing patterns and emerging trends of bacterial isolates and their antimicrobial susceptibilities. Pseudomonas species (31%) and Staphylococcus aureus (22%) were the most common pathogens followed by Klebsiella species (19%). Multi-drug resistant (MDR) Acinetobacter species (9%) have emerged as an important nosocomial pathogen in our burn unit. Most of the gram-negative isolates obtained were found to be multi-drug resistant and 61% of the tested isolates were extended spectrum beta-lactamase (ESBL) producers. Concomitant resistance to penicillin and aminoglycosides was observed in 61% of Enterococcus fecalis isolates. When compared with the results of previous 5 years (June 1993-June 1997) Pseudomonas spp. was still the commonest pathogen in the burns unit. However, isolation of this organism and other gram-negative organisms has decreased in comparison to previous years. The incidence of antimicrobial resistance has markedly increased over the past years resulting in limitation of therapeutic options.

Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

PURPOSE The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates. MATERIALS AND METHODS Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. RESULTS TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12-17) cycles (mean Cp value 14), indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108. CONCLUSIONS The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.

Antimicrobial resistance in invasive and colonising Streptococcus pneumoniae in North India

The present study was done to detect the antibiotic resistance in S. pneumoniae. One hundred twenty S. pneumoniae isolates from clinical specimens and 50 from nasopharyngeal sites were subjected to antimicrobial susceptibility testing by Kirby Bauer disk diffusion method and minimum inhibitory concentration (MIC) determination for penicillin and cefotaxime non-susceptible isolates. A total of 22 isolates (18.3%) from clinical sites and eight (16%) from nasopharyngeal sites showed decreased susceptibility to penicillin by oxacillin disk diffusion test. MICs of 26 of these resistant strains ranged from 0.12-1 microg/mL (intermediate resistance) by broth dilution and E test. Only four isolates, two from sputum and two from nasopharyngeal swabs, showed MIC of 2 microg/mL (complete resistance). However, MIC of two cefotaxime resistant isolates (by disk diffusion) was in the susceptible range (0.5 microg/mL). Highest antimicrobial resistance was seen to cotrimoxazole (55.2%) and tetracycline (61.2%). Antimicrobial resistance to cotrimoxazole and tetracycline was much more in clinical isolates than colonizing isolates. Multi-drug resistant phenotype was detected in 76.9% (20 of 26) of isolates that were intermediately sensitive to penicillin and 50% (2 of 4) of penicillin resistant isolates (co-resistant to tetracycline and cotrimoxazole). Routine screening for antibiotic susceptibility is recommended for clinical isolates of pneumococci. Strains with reduced susceptibility to penicillin should be subjected to MIC determination to detect relative resistance or true resistance as such strains are associated with increased virulence. The choice of antibiotics should be guided by the prevalence of local resistance patterns of pneumococci.

Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii.

PURPOSE Carbapenem resistance in Acinetobacter baumannii has become highly rampant, which has been ascribed to the presence of multiple carbapenemases. The objective of the present study was to prospectively investigate the presence of multiple carbapenemase encoding genes in clinical isolates of A. baumannii. MATERIALS AND METHODS A total of 30 imipenem resistant, consecutive non-repeat clinical isolates A. baumannii from a Tertiary Care Centre of Delhi were subjected to antimicrobial susceptibility testing (AST), screening for carbapenemase production by modified Hodge test (MHT) and determination of minimum inhibitory concentration for imipenem by E-Test® . These were subjected to Real time PCR for blaIMP-1 and 2 , blaVIM-1 and 2, blaOXA23, 24, 51 and 58 using SYBR green-I. These were grouped together on the basis of their genotype as each isolate harboured multiple carbapenemases and correlated with their AST profile. Detection of the novel carbapenemase blaNDM-1 was performed by real time PCR using TaqMan probes on 14 isolates. RESULTS Colistin appeared to be the most effective drug in vitro, followed by tetracycline and beta lactam/beta lactamase inhibitor combinations. All, but one isolate were positive for the MHT. All 30 isolates were positive for blaOXA-51 like gene as well as blaIMP-1 and blaVIM-1 genes. blaOXA 24 and 58 were not detected in any of the isolates. blaIMP-2 , blaVIM-2 , blaOXA-23 were present in 15, 6 and 14 isolates respectively. Grouping based on the genotypic profile did not correlate with susceptibility pattern. Nine among the 14 isolates also harboured the novel blaNDM-1 gene. CONCLUSIONS This is the first study from North India, which comprehensively detected the presence of multiple carbapenemases as well the blaNDM-1 gene. The presence of the novel gene blaNDM-1 indicated ability of A. baumannii to acquire new carbapenemase genes despite the existence of multiple carbapenemase genes. The present study confirmed the presence of multiple genetic mechanisms for carbapenemases production among the clinical isolates of A. baumannii in north India.

Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae

PURPOSE The emergence and spread of zinc-dependent carbapenem resistance has become a diagnostic challenge for clinical microbiologists. The objective of the present study was to screen zinc-dependent carbapenemase activity in clinical isolates of family Enterobacteriaceae. MATERIALS AND METHODS A total of 102 multidrug-resistant organisms (MDROs), non-repeat clinical isolates of family Enterobacteriaceae from two tertiary care centres in Delhi, were screened for carbapenemase production by a modified Hodge test (MHT) and additionally by a re-modified Hodge test, EDTA double disc synergy test, and combined disc test (or disc enhancement test) to determine zinc dependence of carbapenemases harbouring bacteria. RESULTS Of the total 102 clinical isolates (June through November 2010), 91 were from urine and 11 were from blood specimens. The isolates were obtained from patients visiting the outpatient department (18 isolates), admitted in non-ICU inpatient care units (74 isolates) and patients admitted in ICUs (4 isolates). MHT identified 92 (90.2%) isolates as carbapenemases producers. Among those found negative for MHT (n=10), metallo-beta-lactamases (MBLs) activity was demonstrated through the EDTA disc diffusion synergy test and the combined disc test in 8 and 9 isolates respectively. A total of 63 (61.7%) isolates demonstrated MBL activity despite in vitro sensitivity to Imipenem. CONCLUSIONS The study demonstrated that supplementing the MHT with at least one of the screening methods increases the likelihood of picking up such isolates that may be missed by the MHT. The study also demonstrates the wide-spread presence of MBLs in Enterobacteriaceae members from patients visiting hospitals in east Delhi.

Liver abscess caused by Edwardsiella tarda biogroup 1 and identification of its epidemiological triad by ribotyping.

Two clinical isolates and an environmental isolate of Edwardsiella tarda biogroup 1 (ETB1), recovered from liver pus, the stool specimen and from the pond water of the village of the patient, diagnosed to have liver abscess, were found to be identical by protein fingerprinting and ribotyping. It can be construed that the pond water served as the source of infection. The epidemiological triad of the agent (ETB1), host (the patient) and environment (pond water) was thus established. This is the first report in which the triad for extraintestinal Edwardsiellosis caused by ETB1 has been identified. This also constitutes the first report of typing of ETB1 strains by SDS-PAGE and ribotyping.

Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: rapid and inexpensive method for low-resource settings.

The aim of this study was to evaluate a nitrate reductase assay (NRA) for the direct detection of multidrug resistance (MDR) in Mycobacterium tuberculosis from 100 smear-positive sputum samples. The NRA results were compared with the reference proportion method for 100 sputum specimens for which comparable results were available. NRA results were obtained at day 7 for 61 specimens, results for 26 specimens were obtained at day 10, and the results for 13 specimens were obtained at day 14. Thus, 87% of NRA results were obtained in 10 days. NRA is a rapid, accurate, and cost-effective method for the detection of MDR in M. tuberculosis isolates as compared to the proportion method, which is time consuming. Therefore, NRA constitutes a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities due to its low-cost, ease of performance and lack of requirement of sophisticated equipment.

Carriage prevalence of carbapenem-resistant Enterobacteriaceae in stool samples: A surveillance study

BACKGROUND With more people being exposed to antibiotics, intestinal microflora faces constant pressure of antibiotic selection, which has resulted in the emergence of multidrug resistant strains. This may pose a severe problem as intestinal Enterobacteriaceae members are commonly implicated in human infections. AIMS This surveillance study was undertaken to investigate the carriage of carbapenem-resistant Enterobacteriaceae (CRE) in the gastrointestinal tract among patients attending the outpatient clinic in a tertiary care center of East Delhi, India. METHOD We performed a prospective surveillance study to screen 242 Enterobacteriaceae isolates for carbapenemase production from the stool samples of 123 outpatients attending a tertiary care hospital in East Delhi over a four-month period. RESULTS Twenty-four (9.9 per cent) isolates demonstrated carbapenemase activity among 242 screened Enterobacteriaceae isolates. Four stool samples had two isolates of different species, both eliciting this feature and therefore indicating presence of multiple carbapenem-resistant Enterobacteriaceae (CRE) isolates in a single sample. CONCLUSION Screening for carriage of CRE in stools of patients undergoing elective or emergency gastrointestinal surgical procedures, with haematological malignancies taking chemotherapy, or those planned for bone marrow transplantation can guide clinicians about gut colonisation of multidrug-resistant Enterobacteriaceae as these groups of patients are at risk of possible endogenous infection.

Changing trends in antimicrobial susceptibility pattern of bacterial isolates in a burn unit

INTRODUCTION Emerging antimicrobial resistance in nosocomial bacterial isolates, limits the available treatment options for burn wound infections, among them multi-drug resistant Gram negative bacteria and methicillin-resistant Staphylococcus aureus (MRSA) are major contributors to the increase in morbidity and mortality rates. MATERIAL AND METHODS A retrospective cross-sectional study was done in the Department of Microbiology, University College of Medical Sciences & Guru Teg Bahadur Hospital, Delhi. A total of 818 wound samples from patients admitted in the burn wards and Intensive Care Units (ICUs) examined between 2010-2014 (5 years period). Pseudomonas aeruginosa was found as the most common isolate (37%) followed by Klebsiella pneumoniae (15%) and Acinetobacter baumanii (12%) among Gram negative organisms while S. aureus (12%) remained the major isolates among Gram positive organisms. A significant decrease in incidence of Gram positive organisms was observed in comparison with previous study. However, resistance to ceftazidime and aminoglycosides were increased significantly in Gram negative organisms. Multi-drug resistant P. aeruginosa (MDR PA) accounted for 15.2%, multi-drug resistant A. baumanii (MDR AB) was prevalent in 13.8% and MRSA in 77.4% of burn wound infections. DISCUSSION AND CONCLUSION Emerging bacterial drug resistance has both clinical and financial implications for the therapy of infected burn patients. Spectrum of bacterial drug resistance in an institution is important for epidemiological as well as clinical purposes. Rising frequency of MDR strains in burn patients is alarming for clinicians as it downgrades the treatment efficacy.

Current status of multidrug resistant tuberculosis in a tertiary care hospital of East Delhi

BACKGROUND AND OBJECTIVE Multidrug resistant tuberculosis (MDR-TB) is caused by infection due to Mycobacterium tuberculosis which is resistant to both isoniazid (INH) and rifampicin (RIF). It is caused by selection of resistant mutant strains due to inadequate treatment and poor compliance. MDR-TB is a major public health problem as the treatment is complicated, cure rates are well below those for drug susceptible tuberculosis and patient remains infectious for months despite receiving the best available therapy. The drug susceptibility pattern of M. tuberculosis is essential for proper control of MDR-TB in every health care setting, hence the study was initiated with the aim of studying the prevalence of MDR-TB in patients attending a tertiary care hospital in east Delhi. MATERIALS AND METHODS Five hundred and forty-three pulmonary and extrapulmonary samples from suspected cases of tuberculosis received in the mycobacteriology laboratory from November 2009 through October 2010 were investigated for M. tuberculosis. All the samples were subjected to direct microscopic examination for demonstration of acid fast bacilli followed by culture on Lowenstein-Jensen (LJ) medium to isolate M. tuberculosis. Identification was done by conventional biochemical methods. Drug susceptibility of isolated M. tuberculosis strains was done by conventional 1% proportion method followed by sequencing of RIF resistant isolates to detect mutations to confirm resistance. RESULTS AND CONCLUSIONS M. tuberculosis was isolated from 75 out of 543 suspected cases of pulmonary/extrapulmonary TB. Three of the total 75 M. tuberculosis isolates (4%) showed resistance to any one of the first line drugs. Prevalence of MDR-TB was 1.3%. The sequencing of single MDR strain showed mutations at codons 516, 517, and 518. Amplification of rpoB and sequential analysis of the amplicon is a better way of detection of mutation and the evidence of new mutation in this study indicate that mutations continue to arise, probably due to the ability of M. tuberculosis to adapt to drug exposure.