Species distribution and antimicrobial susceptibility of Burkholderia cepacia complex isolates in clinical infections: Experience from a tertiary care hospital, Southern India.

PURPOSE: Burkholderia cepacia complex (Bcc) is a diverse group of environmental bacteria associated with opportunistic infections. The identification of Bcc using conventional methods poses challenges. Bcc infections are difficult to treat due to intrinsic antibiotic resistance. The study aimed to investigate the species distribution and antimicrobial susceptibility of clinical Bcc isolates. METHODS: A total of 153 Bcc isolates obtained from clinical samples were analysed. Species identification was carried out using automated methods, including MALDI-TOF MS and VITEK2. Antimicrobial susceptibility testing was performed using the disc diffusion method. RESULTS: Burkholderia cenocepacia (70.5%) emerged as the most prevalent species, followed by Burkholderia contaminans (9.8%) and Burkholderia cepacia (7.2%). Ventilator-associated pneumonia (38.6%) was the most common infection, followed by sepsis (28.1%). Co-existence of Bcc with other pathogens in many cases suggested potential co-infection scenarios. Antimicrobial susceptibility revealed that ceftazidime, co-trimoxazole and meropenem were the most effective drugs, while levofloxacin proved to be the least effective. Moderate susceptibility was noted to minocycline, with 4.6% of isolates exhibiting multi-drug resistance. CONCLUSION: This study provides valuable insights into the prevalence, clinical associations, and antibiotic susceptibility of Bcc in India. It highlights the importance of Bcc as a nosocomial pathogen, especially in vulnerable patient populations. The findings contribute to understanding Bcc infections, their distribution, and emphasize the necessity for accurate identification methods in clinical settings.

Evaluation of different linezolid susceptibility testing methods and detection of linezolid resistance gene (cfr) in staphylococcal isolates.

Linezolid is an effective oxazolidinone antibiotic against multi resistant Gram-positive organisms. Linezolid resistance is an emerging problem and some controversy exists about the reliability of different phenotypic methods of linezolid susceptibility testing. Fifty isolates each of methicillin resistant S. aureus (MRSA) and Staphylococcus haemolyticus were tested for linezolid susceptibility using Kirby-Bauer disc diffusion, E-test, automated system VITEK2, Broth micro-dilution (reference method) and PCR for the cfr gene. Six resistant isolates were identified, three each in MRSA and S. haemolyticus, all carrying the cfr gene. E-test and VITEK2 were found to be more accurate than disc diffusion test.

Detection of vancomycin variable enterococci (VVE) among clinical isolates of Enterococcus faecium collected across India-first report from the subcontinent.

PURPOSE: Emergence of vancomycin variable enterococci (VVE) poses a challenge to empiric vancomycin therapy. Vancomycin-variable enterococci (VVE) are vanA-positive, yet phenotypically vancomycin-susceptible enterococci that can switch to a vancomycin-resistant phenotype when exposed to vancomycin. The aim of the present study was to determine the prevalence of VVE in India. METHODS: Isolates of phenotypically vancomycin susceptible Enterococcus faecium from 20 tertiary care hospitals across India were collected and tested for the presence of vanA, vanR, vanS, vanB and vanC genes by conventional PCR using previously published primers. Isolates positive for vanA gene were considered as VVE. RESULTS: The prevalence of VVE was 1.5% (5/340). Only one VVE isolate was positive for vanR and vanS, and all the isolates were negative for vanB and vanC. CONCLUSIONS: Although the prevalence is low, our finding emphasizes the importance of routinely screening for van genes in enterococci that are phenotypically susceptible. Silenced vanA able to escape detection and revert to resistance during vancomycin therapy represents a new challenge in clinical settings.

In-vitro synergistic activity of colistin and meropenem against clinical isolates of carbapenem resistant E.coli and Klebsiella pneumoniae by checkerboard method.

CONTEXT: The emergence of drug resistant pathogens pose major threat to hospitalized patients as well as to the community associated with increased mortality and morbidity. The treatment of carbapenem resistant enterobacteriaceae, one of the top WHO priority pathogen remains a global issue. Combination therapy with different classes of antibiotics have been tried with the aim to reduce toxicity, to increase the efficacy of the drugs and to reduce resistance. The in-vitro synergy methods have to be carried out to determine whether the combination of those antibiotics are synergistic, antagonistic or additive. AIMS: We have performed in-vitro synergy testing by checkerboard method for colistin -meropenem combination to determine whether the combination of the two antibiotics were synergistic or antagonistic. METHODS AND MATERIAL: All the consecutive twenty five blood isolates of Escherichia coli and twenty five blood isolates of Klebsiella pneumoniae which were showing resistance to carbapenems by either disc diffusion or vitek 2 were collected over a period of 6 months and checkerboard method was performed. STATISTICAL ANALYSIS USED: The reduction of MIC of colisin on combination with meropenem compared to MIC of colistin alone is analyzed by McNemar's chisquare test with the help of software Stata version 14 and p value < 0.05 is considered as significant. RESULTS: 56% of K. pneumoniae showed synergy and 44% showed additive/indifference results. For E. coli 40% showed synergy and 60% showed additive/indifference. None of the isolates of E. coli and K. pneumoniae showed antagonism. There was more than two fold reduction in MIC of colistin (significant) on combining withmeropenem. CONCLUSIONS: The study results support the combination therapy to treat infections by multi-drug-resistant Klebsiela pneumoniae and Escherichia coli by in-vitro checkerboard testing method which inturn will be helpful for clinicians for judicious use of antimicrobials.

Detection of heterogeneous vancomycin-intermediate Staphylococcus aureus: A preliminary report from south India.

Background & objectives: Although there are reports of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) across the globe, there is a lack of reliable data on hVISA in India. The present study was undertaken to determine the rate of hVISA among the methicillin-resistant Staphylococcus aureus (MRSA) isolates, and to compare the brain heart infusion agar with vancomycin 4 µg/ml (BHIV4) method with population analysis profile-area under the curve (PAP-AUC) method for the detection of hVISA and to study the distribution of mobile genetic element that carries methicillin-resistance gene SCCmec (Staphylococcal cassette chromosome mec) types among these isolates. Methods: BHIV4 and PAP-AUC methods were employed to detect hVISA among 500 clinical isolates of MRSA. SCCmec typing of these isolates was performed by multiplex polymerase chain reaction. The clinical presentation, treatment with vancomycin and outcome was documented for patients with hVISA. Results: The rate of hVISA was 12.4 per cent by PAP-AUC method. Sensitivity, specificity, PPV, NPV and kappa agreement of BHIV4 with PAP-AUC was 58.06, 93.15, 54.55, 94.01 per cent and 0.498, respectively. The isolation of hVISA was significantly (P<0.01) higher in patients admitted to intensive care units and wards than in patients attending the outpatient departments. Only 38 per cent of the patients received vancomycin as therapy. Majority of the hVISA isolates carried SCCmec type V or IV. Interpretation & conclusions: The rate of hVISA isolation in our study was 12.4 per cent. The sensitivity of the BHIV4 screening test was low, and was in moderate agreement with PAP-AUC test. SCCmec type V was the predominant type seen in half of the isolates. More studies need to be done in different parts of the country on a large number of isolates to confirm our findings.

Prevalence and genetic mechanisms of antimicrobial resistance in Staphylococcus species: A multicentre report of the indian council of medical research antimicrobial resistance surveillance network.

PURPOSE: Routine surveillance of antimicrobial resistance (AMR) is an essential component of measures aimed to tackle the growing threat of resistant microbes in public health. This study presents a 1-year multicentre report on AMR in Staphylococcus species as part of Indian Council of Medical Research-AMR surveillance network. MATERIALS AND METHODS: Staphylococcus species was routinely collected in the nodal and regional centres of the network and antimicrobial susceptibility testing was performed against a panel of antimicrobials. Minimum inhibitory concentration (MIC) values of vancomycin (VAN), daptomycin, tigecycline and linezolid (LNZ) against selected methicillin-resistant Staphylococcus aureus(MRSA) isolates were determined by E-test and MIC creep, if any, was determined. Resistant genotypes were determined by polymerase chain reaction for those isolates showing phenotypic resistance. RESULTS: The prevalence of MRSA was found to be range from moderate (21%) to high (45%) among the centres with an overall prevalence of 37.3%. High prevalence of resistance was observed with commonly used antimicrobials such as ciprofloxacin and erythromycin in all the centres. Resistance to LNZ was not encountered except for a single case. Full-blown resistance to VAN in S. aureus was not observed; however, a few VAN-intermediate S. aureus isolates were documented. The most common species of coagulase negative staphylococci (CoNS) identified was Staphylococcus haemolyticus and Staphylococcus epidermidis. Resistance among CoNS was relatively higher than S. aureus. Most phenotypically resistant organisms possessed the corresponding resistance genes. CONCLUSION: There were localised differences in the prevalence of resistance between the centres. The efficacy of the anti-MRSA antimicrobials was very high; however, almost all these antimicrobials showed evidence of creeping MIC.

Prevalence of MRSA Nasal Carriage in Patients Admitted to a Tertiary Care Hospital in Southern India.

INTRODUCTION: Infections with MRSA, both community and hospital acquired, are well established and the source of infection is often a carrier. There are very few studies showing the magnitude of MRSA nasal colonization among healthy persons from the community. This study was conducted to detect the prevalence of MRSA nasal carriage in patients who did not have any known risk factors associated with HA- MRSA colonization, admitted to a tertiary care centre in Kerala. MATERIALS AND METHODS: Nasal swabs were collected from patients within 24 hours of admission. Specimen were inoculated on chromogenic agar (HiCrome MeReSa agar-HiMedia) for MRSA screening. Isolates were then subjected to antibiotic sensitivity tests, SCCmec typing and PVL gene detection. RESULTS: Out of 683 patients, 16 carried MRSA in their nares (2.3%). Of the 16 strains 13 (81.25 %) strain were SCCmec type III and one belonged to SCCmec type IV (6.25 %). Two strains failed to amplify SCCmec genes. Three strains carried genes for PVL toxin (18.75%). CONCLUSION: With a better understanding of the complex epidemiology of MRSA it is increasingly apparent that demarcations between the HA and CA phenotypes are not as clear cut as previously thought. In this study of nasal carriage of MRSA in the community we have demonstrated prevalence consistent with published data. Most isolates however were shown to belong to the type conventionally assigned to HA-MRSA.

Serodiagnosis of Scrub Typhus at a Tertiary Care Hospital from Southern India.

INTRODUCTION: Scrub typhus, a zoonotic disease is one of the most covert emerging and re-emerging Rickettsial infections. There is an upsurge in the incidence of the disease worldwide with ever-changing habitat. Clinical diagnosis of scrub typhus is challenging as the signs and symptoms of scrub typhus are similar to other febrile illnesses. In developing countries, among the various laboratory tests to diagnose scrub typhus, Weil-Felix test is commonly performed despite its low sensitivity. The Immunofluorescence antibody (IFA) test has its limitations in terms of cost and expertise required. The present study was conducted to determine the seropositivity of IgM ELISA for scrub typhus in clinically suspected cases. MATERIALS AND METHODS: Weil-Felix test and IgM ELISA were performed using clinically suspected cases of scrub typhus using commercially available kits. RESULTS: Out of 482 samples tested, 109 were positive by both Weil-Felix test and IgM ELISA. One hundred and sixteen samples which were negative by Weil-Felix test reacted positive by IgM ELISA. Fourteen samples which were positive by Weil-Felix test were negative by ELISA. CONCLUSION: Owing to the limitations of the Weil-Felix test and IFA, commercially available recombinant IgM ELISA which has a good sensitivity and specificity may be an alternative in laboratories with moderate set up.

Fecal carriage rates of extended-spectrum ß-lactamase-producing Escherichia coli among antibiotic naive healthy human volunteers.

INTRODUCTION: Higher prevalence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli fecal carriage has been reported in the nosocomial setting than in the community. We tried to determine the fecal carriage of ESBL-producing E. coli among healthy volunteers in a relatively isolated community. MATERIALS AND METHODS: This study was conducted on 115 healthy adult volunteers from whom one fecal sample was collected and was plated on selective media. Each morphotypes were identified, characterized, and ESBL phenotype was confirmed by double-disk potentiation method. Molecular characterization of ESBL gene was done using multiplex polymerase chain reaction and pulse-field gel electrophoresis (PFGE) was done to identify their clonal relation. RESULTS: ESBL-producing E. coli had a prevalence of 19% (22/115) among the healthy volunteers in the community. CTX-M was the predominant type, showed a presence 95.5% (21/22), TEM 63%, SHV 9%, and both TEM and CTX-M were present in 63.6% (14/22), all three present in 4.5% (1/22). The lineage using PFGE showed a single clone in 17 isolates. Seven isolates were type A (all TEM & CTX-M), six were type A1 (all TEM & CTX-M except 2), four were type A2 (all CTX-M), and three belonged to types B, C, and D respectively Conclusion: High prevalence rate of 19% in the community indicated by this study implies the possibility of sustained ESBL carriage even among isolated population, which could serve as a reservoir for enriching the ESBL pool in the hospital. Clonal relations also indicate a possible epidemiological source that needs to be evaluated.

Detection of OXA-1 ß-lactamase gene of Klebsiella pneumoniae from blood stream infections (BSI) by conventional PCR and in-silico analysis to understand the mechanism of OXA mediated resistance.

Klebsiella pneumoniae strains producing extended-spectrum ß-lactamases (ESBL) exhibit resistance to antibiotic classes. The production of ESBLs (TEM-1, TEM-2, SHV-1, OXA-1) results in resistance to ampicillin, ticarcillin, piperacillin and cephalosporins. High levels of ß-lactamases leads to development of resistance to ß-lactamase inhibitors. The present study deals with characterizing antimicrobial resistance pattern among septicemia causing K. pneumoniae and the prevalence of inhibitor resistant OXA-1 ß-lactamase genes among them. Of 151 study isolates, 59 were resistant to piperacillin/tazobactam and these isolates were further selected for blaOXA-1 screening. Amplification of ß-lactamases genes by conventional PCR showed the presence of blaOXA-1 genes among 12 K. pneumoniae (20.3%) isolates. OXA-1 ß-lactamase producing strains were found to be resistant to piperacillin/tazobactam(100%), levofloxacin (91.6%), amikacin (75%), cefoxitin (50%), ertapenem (25%), imipenem (16.6%) and meropenem (16.6%); all were susceptible to tigecycline. 3D models of OXA-1 ß-lactamase were generated and docking was performed with various ß-lactam antibiotics. Molecular docking (MD) revealed the molecular basis of drug sensitivity. MD simulation results clearly confirmed the notable loss in stability for tigecycline-blaOXA-1 complex. Findings of the present study will provide useful insights for understanding the mechanism of resistance and help with strategies for the development of new antibiotics. The conventional PCR assay designed in this study can be routinely used in clinical microbiology laboratories to determine the blaOXA-1 genes.

Detection of oxacillin-susceptible mecA-positive Staphylococcus aureus isolates by use of chromogenic medium MRSA ID.

Reports of oxacillin-susceptible mecA-positive Staphylococcus aureus strains are on the rise. Because of their susceptibility to oxacillin and cefoxitin, it is very difficult to detect them by using routine phenotypic methods. We describe two such isolates that were detected by chromogenic medium and confirmed by characterization of the mecA gene element.

Phenotypic & molecular characterization of AmpC ß-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from five Indian Medical Centers.

BACKGROUND & OBJECTIVES: AmpC ß-lactamases which are often plasmid mediated hydrolyze all ß-lactam antibiotics except cefepime and carbapenems. We evaluated the presence of AmpC ß-lactamases among Enterobacteriaceae strains recovered prospectively from patients at five Indian tertiary care centres. METHODS: The study included 909 consecutive Gram-negative isolates recovered from clinically significant specimens during June 2007 - May 2008 as part of an ICMR-ESBL study. Among the study isolates, 312 were found to be cefoxitin resistant by disc diffusion test (DDT). Minimum inhibitory concentration (MIC) determination by E test was done against amikacin, levofloxacin, impinem, meropenem, ertapenem, tigecycline and piperacillin-tazobactam. Combined DDT using phenyl boronic acid as inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. The common Amp C genotypes ACC, FOX, MOX, DHA, CIT and EBC were detected by multiplex PCR. RESULTS: Plasmid mediated Amp C phenotype was confirmed in 114 of the 312 (36.5%) cefoxitin resistant isolates with 255 (81.7%) showing multidrug resistance. Susceptibility to tigecycline was highest (99%) followed by imipenem, meropenem (97%), ertapenem (89%), amikacin (85%), and piperacillin-tazobactam (74.6%). Levofloxacin resistance was 82 per cent. ESBL co carriage was observed among 92 per cent of Amp C producers. Among 114 Amp C producers, 48 could be assigned a genotype, this included CIT- FOX (n = 25), EBC (n = 10), FOX (n = 4), CIT (n = 3), EBC-ACC (n = 2) and one each of DHA, EBC-DHA, FOX -DHA and FOX-EBC-DHA. INTERPRETATION & CONCLUSIONS: Overall, AmpC phenotypes were found in 12.5 per cent isolates, multidrug resistance and ESBL co-carriage among them was high suggesting plasmid mediated spread. The study results have implications in rational antimicrobial therapy and continued surveillance of mechanisms of resistance among nosocomial pathogens.