Association of biofilm production with multidrug resistance among clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa from intensive care unit.

BACKGROUND AND AIMS: Given choice, bacteria prefer a community-based, surface-bound colony to an individual existence. The inclination for bacteria to become surface bound is so ubiquitous in diverse ecosystems that it suggests a strong survival strategy and selective advantage for surface dwellers over their free-ranging counterparts. Virtually any surface, biotic or abiotic (animal, mineral, or vegetable) is suitable for bacterial colonization and biofilm formation. Thus, a biofilm is "a functional consortium of microorganisms organized within an extensive exopolymeric matrix." MATERIALS AND METHODS: The present study was undertaken to detect biofilm production from the repertoire stocks of Acinetobacter baumannii (A. baumannii) and Pseudomonas aeruginosa (P. aeruginosa) obtained from clinical specimens. The tube method was performed to qualitatively detect biofilm production. RESULTS: A total of 109 isolates of both organisms were included in the study, out of which 42% (46/109) isolates showed biofilm detection. Among the biofilm producers, 57% of P. aeruginosa and 73% of A. baumannii showed multidrug resistance (MDR) pattern which was statistically significant in comparison to nonbiofilm producers (P < 0.001). CONCLUSION: To the best of our knowledge, this is the only study to have tested the biofilm production in both P. aeruginosa and A. baumannii in a single study. Biofilm production and MDR pattern were found to be significantly higher in A. baumannii than P. aeruginosa. Antibiotic resistance was significantly higher among biofilm producing P. aeruginosa than non producers. Similarly, antibiotic resistance was significantly higher among biofilm producing A. baumannii than non producers.

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.

Real-time multiplex polymerase chain reaction with high-resolution melting-curve analysis for the diagnosis of enteric infections associated with diarrheagenic Escherichia coli.

INTRODUCTION: Although diarrheagenic Escherichia coli (DEC) strains are important bacterial causative agents of diarrhoea, they are not routinely sought as stool pathogens in clinical laboratories as conventional microbiological testing are unable to distinguish between normal flora and pathogenic strains of E. coli. This study was undertaken to determine the prevalence of DEC pathotypes amongst children with and without diarrhoea and to detect specific virulent genes present in different DEC pathotypes, using real-time multiplex polymerase chain reaction (PCR) with high-resolution melting (HRM) technology. MATERIALS AND METHODS: Stool samples were obtained from cases and controls. Using a set of conventional biochemical tests, E. coli strains were identified. Further, these isolates were subjected to multiplex PCR system for the detection of virulence genes of different pathotypes of DEC. Real-time multiplex PCR was performed for the detection of specific virulent genes of DEC pathotypes, using Rotor-Gene Q instrument (Qiagen) having High-resolution Melt analyser using Type-it HRM PCR kit (Qiagen) containing EvaGreen fluorescent intercalating dye. RESULTS: In this study, we had successfully standardised two multiplex PCR assays which were found to be effective for direct detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC). A total of 42 DEC strains were detected at an overall rate of 19.3% (n = 42), from the total 217 E. coli isolates recovered from the cases (n = 39, 17.9%) and control (n = 3, 3.8%) groups. Amongst the 42 DEC pathotypes (39 from cases and 3 from controls), EPEC (10%), EAEC (8.82%), ETEC (2.94%) and EIEC (1.18%) were found in children with diarrhoea (cases) and in children without diarrhoea (control) only EAEC (2.13%) and EPEC (4.26%) were detected. Age distribution, gender variation, seasonal variation and clinical features were also analysed. CONCLUSION: This study helped evaluate the prevalence of DEC amongst children (<18 years of age) with and without diarrhoea using multiplex real-time PCR with HRM analysis.

Inducible and constitutive clindamycin resistance in Staphylococcus aureus in a northeastern Indian tertiary care hospital.

INTRODUCTION: Staphylococcus aureus is one of the most common pyogenic bacteria. They are notorious for developing prompt resistance to newer antimicrobials. With increasing incidence of methicillin-resistant S. aureus (MRSA) isolates, the treatment options are also becoming limited. Clindamycin is an excellent drug for skin and soft tissue infections, but resistance mediated by the inducible phenotype (iMLS(B)) leads to in vivo therapeutic failure even though there may be in vitro susceptibility. The double disk approximation test (D-test) can reliably detect the presence of such isolates. This study was aimed to detect and report the prevalence of the iMLS(B) phenotype in NEIGRIHMS, a tertiary care center in Northeast India. METHODOLOGY: A total of 243 consecutive isolates were subjected to routine identification tests followed by antimicrobial sensitivity testing. Erythromycin-resistant isolates were tested for inducible resistance phenotype by the D-test. RESULTS: Among strains tested, 95 (39%) were erythromycin resistant. Twenty-six (10.7%) isolates were D-test positive (iMLS(B) phenotype), 41 (16.88%) were constitutively resistant (cMLS(B) phenotype), and 28 isolates (11.52%) were found to be negative by D-test. The incidence of both inducible and constitutive phenotypes was higher in MRSA isolates compared to methicillin-sensitive S. aureus (MSSA) isolates. CONCLUSIONS: This study revealed a moderate prevalence of the inducible clindamycin phenotype in the staphylococcal isolates tested. Clinical microbiology laboratories in areas of high MRSA prevalence should consider performing the D-test routinely. This will help prevent prescription of drug(s) whose therapeutic efficacy is doubtful.