BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resi... more BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resistant Klebsiella pneumoniae. Resistance to carbapenems in K. pneumoniae is mainly due to metallo-beta-lactamases (NDM, IMP, and VIM) and class D oxacillinase (OXA-48-like). This study was undertaken to detect the genes encoding for carbapenemase in K. pneumoniae and to determine the clonal relatedness of selected isolates of K. pneumoniae producing NDM and OXA-48 by pulsed-field gel electrophoresis method (PFGE). The isolates were collected over a period of 1 year. A total of 370 clinically significant, nonduplicate isolates of K. pneumoniae were included in this study. Phenotypic tests for the detection of carbapenemases were performed for all the isolates. Polymerase chain reaction (PCR) was carried out for the detection of carbapenemase genes such as bla KPC , bla IMP , bla VIM , bla NDM , and bla OXA-48 . PFGE was performed, and the PFGE profiles were analyzed and compared using BioNumerics version 7.6. Of the 370 isolates of K. pneumoniae, carbapenemase genes were detected in 13.78% (51/370). bla OXA-48 was the prevalent gene detected followed by bla NDM and bla KPC . Thirty strains of K. pneumoniae selected by PFGE analysis were divided into five clusters (A, B, C, D, and E). Cluster C was the major type detected carrying bla NDM and bla OXA-48 genes. CONCLUSION: bla OXA-48 was the most prevalent gene detected in this study. PCR is useful in detecting carbapenemase genes, especially bla NDM , which may show false susceptibility to carbapenems. There was no direct correlation detected between PFGE profiles and antibiotic susceptibility pattern. PFGE has revealed the genomic diversity among isolates, thereby suggesting heterogeneity in strain circulation within intensive care unit and wards of the hospital. Monitoring and molecular typing is essential to curtail the spread of multidrug-resistant strains and control the outbreaks of infection.
Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminogl... more Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminoglycoside by producing aminoglycoside modifying enzymes (AMEs). In enterococci, high level resistance to aminoglycosides is mediated by acquisition of plasmid mediated genes encoding for aminoglycoside modifying enzymes (AMEs). High level gentamicin resistance (MIC ! 500μg /mL) is predominantly mediated by aac(6′)-Ie-aph(2″)-Ia, encoding the bifunctional aminoglycoside modifying enzyme AAC(6′)-APH(2″). This enzyme eliminates the synergistic activity of gentamicin when combined with a cell wall active agent. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id and ant(4′)-1a have also been detected in enterococci. Objective This study was carried out to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of Enterococci. Materials and Methods A total number of 150 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion. Minimum Inhibitory Concentration (MIC) was ascertained by agar dilution method. Polymerase chain reaction was done to screen the following AMEs (aac(6′)-Ie-aph(2″)-Ia; aph(2″)-Ib; aph(2″)-Ic; aph(2″)-Id and aph(3′)-IIIa genes). Results 51.3% of the study isolates exhibited high level gentamicin resistance. Polymerase chain reaction revealed that aph(3′)-111a is the most prevalent AME, followed by aac(6′)-1e-aph(2″)-1a. The combination of both the genes were detected in 44.1% of the study isolates. The rest of the AMEs and their combinations were not encountered in this study. 8.6% of the study isolates did not harbour any AME genes screened for, but was phenotypically resistant to gentamicin. In contrast 31.3% anchored the AME genes but phenotypically appeared susceptible to gentamicin. Conclusion This study indicates the high-level aminoglycoside resistance disseminated among Enterococci in our geographical region. It also emphasizes the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbour one or more AMEs genes leading to therapeutic failure.
BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the em... more BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the emergence of multidrug resistance in these organisms limits the treatment options for serious infections caused by them. K. pneumoniae carbapenemase (KPC) is one of the clinically significant Class A beta-lactamases. This study was aimed to detect the KPC and its coexistence with other beta-lactamases in K. pneumoniae. A total of 370 isolates, collected over a period of 1 year, were included in this study. The source of these isolates were urine (n = 170), exudative specimens (n = 132), respiratory secretions such as bronchial wash, endotracheal aspirate, and pleural fluid (n = 38), and blood (n = 30). For all the isolates, antibiotic susceptibility tests by disc diffusion, modified Hodge test, and KPC screening test were done. Polymerase chain reaction (PCR) was performed for the detection of KPC and the copresence of other beta-lactamases genes. RESULTS: Among the 370 isolates, 41 were resistant to the carbapenem by disc diffusion and minimum inhibitory concentration tests. Screen test using ertapenem and the boronic acid disk was positive in 14 isolates. Only one isolate harbored KPC gene by PCR, and it was co-produced with SHV-12 and CTX-M-15. CONCLUSION: PCR remains the gold standard for detection of KPC compared with any other phenotypic methods. Early detection of these genes helps in initiating proper antibiotic treatment.
Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens in health... more Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens in health care-associated infections. Fluoroquinolone resistance has emerged in these pathogens. In this study, we aimed to determine the occurrence of plasmid-mediated quinolone resistance (PMQR) determinants (qnrA, qnrB, qnrS, aac(6′)-Ib-cr, oqxAB, and qepA) by polymerase chain reaction (PCR) and the transmissibility of plasmid-borne resistance determinants in clinical isolates of P. aeruginosa and A. baumannii. The study included P. aeruginosa (85) and A. baumannii (45) which were nonduplicate, clinically significant, and ciprofloxacin resistant. Antibiotic susceptibility testing was done by disk diffusion method for other antimicrobial agents, namely amikacin, ceftazidime, piperacillin/tazobactam, ofloxacin, levofloxacin, and imipenem. Minimum inhibitory concentration of ciprofloxacin was determined. Efflux pump activity was evaluated using carbonyl-cyanide m-chlorophenylhydrazone (CCCP). The presence of PMQR genes was screened by PCR amplification. Transferability of PMQR genes was determined by conjugation experiment, and plasmid-based replicon typing was performed. Results Resistance to other classes of antimicrobial agents was as follows: ceftazidime (86.9%), piperacillin/tazobactam (73.8%), imipenem (69.2%), and amikacin (63.8%). The minimal inhibitory concentration (MIC)50 and MIC90 for ciprofloxacin were 64 and greater than or equal to 256 µg/mL, respectively. There was a reduction in MIC for 37 (28.4%) isolates with CCCP. In P. aeruginosa, 12 (14.1%) isolates harbored qnrB, 12 (14.1%) qnrS, 9 (10.5%) both qnrB and qnrS, 66 (77.6%) aac(6′)-Ib-cr, and 3 (3.5%) oqxAB gene. In A. baumannii, qnrB was detected in 2 (4.4%), 1 (2.2%) harbored both the qnrA and qnrS, 1 isolate harbored qnrB and qnrS, 21 (46.6%) aac(6′)-Ib-cr, and 1 (2.2%) isolate harbored oqxAB gene. Notably, qepA gene was not detected in any of the study isolates. Conjugation experiments revealed that 12 (9.2%) were transferable. Of the transconjugants, seven (58.3%) belonged to IncFII type plasmid replicon, followed by four (33.3%) IncA/C and one (8.3%) IncFIC type. Conclusion The plasmid-mediated resistance aac(6′)-Ib-cr gene is primarily responsible for mediating fluoroquinolone resistance in clinical isolates of P. aeruginosa and A. baumannii. The predominant plasmid type is IncFII.
Introduction Fluoroquinolones are widely used broad-spectrum antibiotics. Recently, increased rat... more Introduction Fluoroquinolones are widely used broad-spectrum antibiotics. Recently, increased rate of resistance to this antibiotic has been observed in Klebsiella pneumoniae. The aim of the present study was to determine the presence of quinolone resistance determining regions (QRDR) mutation genes and plasmid-mediated quinolone resistance (PMQR) determinants in clinical isolates of ciprofloxacin-resistant K. pneumoniae. The study included 110 nonduplicate ciprofloxacin-resistant K. pneumoniae clinical isolates. Antibiotic susceptibility testing by disk diffusion method and minimum inhibitory concentration (MIC) by agar dilution methods for ciprofloxacin was performed according to the recommendations of Clinical Laboratory Standards Institute. The presence of QRDR genes and PMQR genes was screened by polymerase chain reaction (PCR) amplification. Result All 110 isolates were resistance to ciprofloxacin, levofloxacin, and ofloxacin. As much as 88% of the isolates exhibited high-level of MIC to ciprofloxacin. Among the 110 isolates, 94(85%) harbored gyrA and 85 (77%) gyrB. The parC and parE genes were detected in 88 (80%) and 64 (58%) isolates. qnrB was detected in 13 (12%) isolates and qnrS in 5 (4.5%) isolates. Two (1.8%) isolates carried both qnrB and qnrS genes. The acc (6')-Ib-cr gene was found in 98 (89%) isolates and oqxAB was detected in 7 (6.3%) isolates. One (0.9%) isolate carried qnrB, acc(6')-Ib-cr and oqxAB genes. Conclusion The prevalence of acc (6')-Ib-cr gene is high among PMQR determinants, followed by qnrB, oqxAB and qnrS.
BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hyp... more BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hypervirulent K. pneumoniae (hvKP), new variant of K. pneumoniae, can cause invasive infections in young healthy individuals as well as in the immunocompromised population. Hypervirulent strains frequently belong to capsular serotypes K1 or K2. Emergence of antimicrobial resistance in hvKP is a cause for concern. The present study was done to detect the K1 and K2 serotypes among clinical isolates of K. pneumoniae, spectrum of infections caused by them and presence of common beta-lactamases encoding genes in them. A total of 370 isolates of K. pneumoniae, isolated from various clinical samples over a period of 1 year was included in this study. Antibiotic susceptibility testing to various classes of antimicrobials was done as per Clinical and Laboratory Standard Institute guidelines. The presence of K2A (specific to serotype K2), magA (specific to serotype K1), and rmpA genes was detected by multiplex polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (TEM, SHV, and CTX-M), plasmid-mediated AmpCs (MOX, CIT, DHA, ACC, EBC, and FOX), and carbapenemase genes (IMP, VIM, NDM, KPC, and OXA-48) were also determined by PCR. RESULTS: Among the 370 isolates, 8 harbored K2A gene and one harbored magA. rmpA gene was detected in three isolates along with K1 or K2 serotypes. Seven K2A-positive isolates were resistant to one or more classes of antimicrobials. The studied ESBL genes were present in four isolates. Two isolates harbored carbapenemase genes (NDM-1, OXA-48) along with ESBLs. CONCLUSION: K2 serotype is more prevalent among hvKP isolates. They can harbor ESBLs and Carbapenemase genes. K1 serotype is rather uncommon in K. pneumoniae. Acquisition of multidrug-resistant genes by these strains adds to their virulence and limits the treatment options.
Background Methicillin-resistant Staphylococcus aureus (MRSA) is a widely recognized multidrug-re... more Background Methicillin-resistant Staphylococcus aureus (MRSA) is a widely recognized multidrug-resistant bacteria presenting a major therapeutic challenge to clinicians. Staphylococcus aureus possesses a number of pathogenicity factors that attribute to the severity of infections. This study was undertaken to investigate the common virulence genes in clinical isolates of Staphylococcus aureus, determine their antimicrobial susceptibility profile, and to characterize the staphylococcal cassette chromosome mec (SCCmec) types among MRSA in a tertiary care center. Materials and Methods A total of 133 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion method. Methicillin resistance was screened using cefoxitin disc; mecA and mecC genes were detected using polymerase chain reaction (PCR). PCR was done to detect 12 virulence factors such as hla, hlb, fnbA, fnbB, sea, seb, sec, icaA, clfA, tst, pvl, and eta. SCCmec typing wa...
Background Staphylococcus haemolyticus has emerged as an important multidrug-resistant nosocomial... more Background Staphylococcus haemolyticus has emerged as an important multidrug-resistant nosocomial pathogen. Linezolid is useful in the treatment of severe infections caused by methicillin-resistant Staphylococci. Resistance to linezolid in Staphylococci is due to one or more of the following mechanisms: acquisition of the cfr (chloramphenicol florfenicol resistance) gene, mutation in the central loop of domain V of the 23S rRNA, and mutation in the rplC and rplD genes. This study was carried out to detect and characterize resistance to linezolid among the clinical isolates of Staphylococcus haemolyticus. Materials and Methods The study included 84 clinical isolates of Staphylococcus haemolyticus. Susceptibility to various antibiotics was determined by disc diffusion method. Minimum inhibitory concentration (MIC) was determined by agar dilution method for linezolid. Methicillin resistance was screened using oxacillin and cefoxitin disc. Polymerase chain reaction was done to detect me...
Introduction Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings;... more Introduction Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings; woefully their usage has been reduced by the emergence and distribution of resistance mainly due to aminoglycoside modifying enzymes (AME).Purpose This study was performed to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of gram-negative bacteria. This study also aimed to detect the presence of AMEs that are prevalent in gram-positive bacteria, among gram negatives.Materials and Methods A total number of 386 clinical isolates were included in this study. Polymerase chain reaction revealed the prevalence rate of AMEs screened [aac(6′)-lb, aac(3′)-I, aac(3′)-II, aac(3′)-VI, ant(2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, aph(2′)-Id, aac (6′)-Ie- aph(2′)-Ia, and aph(3′)-IIIa]. Conjugation experiment was performed for the clinical isolates which harbored any one of the AME which was prevalent in gram-positive bacter...
BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resi... more BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resistant Klebsiella pneumoniae. Resistance to carbapenems in K. pneumoniae is mainly due to metallo-beta-lactamases (NDM, IMP, and VIM) and class D oxacillinase (OXA-48-like). AIM AND OBJECTIVE: This study was undertaken to detect the genes encoding for carbapenemase in K. pneumoniae and to determine the clonal relatedness of selected isolates of K. pneumoniae producing NDM and OXA-48 by pulsed-field gel electrophoresis method (PFGE). MATERIALS AND METHODS: The isolates were collected over a period of 1 year. A total of 370 clinically significant, nonduplicate isolates of K. pneumoniae were included in this study. Phenotypic tests for the detection of carbapenemases were performed for all the isolates. Polymerase chain reaction (PCR) was carried out for the detection of carbapenemase genes such as blaKPC, blaIMP, blaVIM,blaNDM, and blaOXA-48. PFGE was performed, and the PFGE profiles were a...
Pulmonary infections are the most common clinical manifestations of Nocardia species. There is an... more Pulmonary infections are the most common clinical manifestations of Nocardia species. There is an increase in cases of nocardial infections occurring worldwide attributable to the increase in the immunosuppressed population. The availability of molecular methods has aided the detection of more number of cases as well as unusual species. Still, it remains one of the most underdiagnosed pathogens. Recognition of drug resistance in this organism has now mandated early and precise identification with speciation for effective treatment and management. Nocardial species identity can predict antimicrobial susceptibility and guide clinical management. Here, we report two cases of pulmonary nocardiosis caused by unusual species of Nocardia, namely, N. cyriacigeorgica and N. beijingensis identified by 16S rRNA gene-based sequencing. These cases are being reported for their rarity.
BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hyp... more BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hypervirulent K. pneumoniae (hvKP), new variant of K. pneumoniae, can cause invasive infections in young healthy individuals as well as in the immunocompromised population. Hypervirulent strains frequently belong to capsular serotypes K1 or K2. Emergence of antimicrobial resistance in hvKP is a cause for concern. AIM AND OBJECTIVE: The present study was done to detect the K1 and K2 serotypes among clinical isolates of K. pneumoniae, spectrum of infections caused by them and presence of common beta-lactamases encoding genes in them. MATERIALS AND METHODS: A total of 370 isolates of K. pneumoniae, isolated from various clinical samples over a period of 1 year was included in this study. Antibiotic susceptibility testing to various classes of antimicrobials was done as per Clinical and Laboratory Standard Institute guidelines. The presence of K2A (specific to serotype K2), magA (specific to se...
Journal of clinical and diagnostic research : JCDR, 2013
Detection of carbapenem hydrolyzing class D beta lactamase OXA-181, (a variant of OXA-48) in Ente... more Detection of carbapenem hydrolyzing class D beta lactamase OXA-181, (a variant of OXA-48) in Enterobacteriaceae, is important, to institute appropriate therapy and to initiate preventive measures. This study was done to determine the presence of OXA 48 and its derivative OXA-181 in Enterobacteriaceae of pathogenic significance. One hundred and eleven non-repetitive Enterobacteriaceae isolates which were resistant to any of the cephalosporin subclasses III and which exhibited reduced susceptibility to carbapenems were included in the study. Minimum inhibitory concentrations (MICs) to imipenem and meropenem was determined by broth microdilution. Production of carbapenamase was screened by Modified Hodge test (MHT). Polymerase Chain Reaction (PCR) was done to detect the presence of bla OXA-181 and bla OXA-48 .Coexistence of other carbapenemase encoding genes, namely, NDM-1, VIM, IMP and KPC were also looked for, by PCR. Of all the isolates which were tested, only 2 (1.8%) revealed the ...
Purpose: Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spect... more Purpose: Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR). Materials and Methods: Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25) and K. pneumoniae (n = 52) were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL). Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test). Results: Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origen -Citrobacter freundii), DHA (Dhahran Hospital, Saudi Arabia), ACC (Ambler class C), EBC (Amp C origen -Enterobacter cloacae) groups. ESBL was co-produced in 54 isolates. Conclusions: Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.
The metallo-β-lactamase gene bla VIM-2 was identified in a strain of Pseudomonas aeruginosa isola... more The metallo-β-lactamase gene bla VIM-2 was identified in a strain of Pseudomonas aeruginosa isolated in India. The integron encoding bla VIM-2 was virtually identical to those recently found in the United States and Russia. These unusual structures are likely to have arisen from an ancestral integron predating the formation of the 3′ conserved sequence.
BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the em... more BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the emergence of multidrug resistance in these organisms limits the treatment options for serious infections caused by them. K. pneumoniae carbapenemase (KPC) is one of the clinically significant Class A beta-lactamases. AIM AND OBJECTIVE: This study was aimed to detect the KPC and its coexistence with other beta-lactamases in K. pneumoniae. MATERIALS AND METHODS: A total of 370 isolates, collected over a period of 1 year, were included in this study. The source of these isolates were urine (n = 170), exudative specimens (n = 132), respiratory secretions such as bronchial wash, endotracheal aspirate, and pleural fluid (n = 38), and blood (n = 30). For all the isolates, antibiotic susceptibility tests by disc diffusion, modified Hodge test, and KPC screening test were done. Polymerase chain reaction (PCR) was performed for the detection of KPC and the copresence of other beta-lactamases genes. ...
Artificial Cells, Nanomedicine, and Biotechnology, 2017
Pseudomonas aeruginosa is a problematic human pathogen resistant to almost all available antibiot... more Pseudomonas aeruginosa is a problematic human pathogen resistant to almost all available antibiotics. The important prerequisite for these drugs to target this bacterium is an efficient delivery system. Siderophore-mediated drug delivery system is a promising approach to carry out antibiotics to the cells. Pyochelin, a siderophore of P. aeruginosa, was successfully synthesized in a five-step procedure. PEGylated liposomal pyochelin-antibiotic (L-Pch-Ab) carrier was fabricated by thin-film hydration method. L-Pch-Ab had an average size of 90.31 ± 0.11 nm holding a negative zeta potential at À54.12 ± 0.03 mV (PDI <2). The MIC determined by broth dilution method against three clinical strains isolated from burn wounds showed that L-Pch-Ab significantly reduced (16 mg/ml) the MIC values than those of free antibiotics. In the time kill assay, L-Pch-Ab was bactericidal against all strains at most time intervals at 2 Â and 4 Â MIC up to 24 h. TEM observations revealed that L-Pch-Ab was actively taken up by P. aeruginosa and exhibited membrane deformation within 2 h. Developed L-Pch-Ab fused intimately with the outer membrane of MDRPa and exhibited effective antibacterial activity than free Ab. Furthermore, L-Pch-Ab kills MDRPa within infected HaCaT keratinocytes without any cytotoxic effects at 4Â MIC concentrations after 72 h. Thus, the specific targeting of L-Pch-Ab with its higher efficacy to deliver drug by limiting the toxicity will be a novel approach to fight infections caused by P. aeruginosa.
Artificial cells, nanomedicine, and biotechnology, Jan 31, 2017
In this study, we examined the efficacy of liposomal oleic acid-based antibiotic formulations on ... more In this study, we examined the efficacy of liposomal oleic acid-based antibiotic formulations on 32 strains of multidrug-resistant Pseudomonas aeruginosa (MDRPa). The average size of liposomes were 93.12 ± 2.3 nm holding a negative zeta potential at -57.3 ± 0.89. Liposomal antibiotic formulations were tested against 32 MDRPa strains isolated from burn wounds and urine samples, which exhibited an MIC of ≤8 μg/mL, whereas MIC of free antibiotics ranged from 32 to >1024 μg/mL. The results clearly indicate that the liposomes composed of naturally occurring oleic acid, could be used therapeutically either alone or in combination with antibiotics to effectively treat P. aeruginosa infections.
BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resi... more BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resistant Klebsiella pneumoniae. Resistance to carbapenems in K. pneumoniae is mainly due to metallo-beta-lactamases (NDM, IMP, and VIM) and class D oxacillinase (OXA-48-like). This study was undertaken to detect the genes encoding for carbapenemase in K. pneumoniae and to determine the clonal relatedness of selected isolates of K. pneumoniae producing NDM and OXA-48 by pulsed-field gel electrophoresis method (PFGE). The isolates were collected over a period of 1 year. A total of 370 clinically significant, nonduplicate isolates of K. pneumoniae were included in this study. Phenotypic tests for the detection of carbapenemases were performed for all the isolates. Polymerase chain reaction (PCR) was carried out for the detection of carbapenemase genes such as bla KPC , bla IMP , bla VIM , bla NDM , and bla OXA-48 . PFGE was performed, and the PFGE profiles were analyzed and compared using BioNumerics version 7.6. Of the 370 isolates of K. pneumoniae, carbapenemase genes were detected in 13.78% (51/370). bla OXA-48 was the prevalent gene detected followed by bla NDM and bla KPC . Thirty strains of K. pneumoniae selected by PFGE analysis were divided into five clusters (A, B, C, D, and E). Cluster C was the major type detected carrying bla NDM and bla OXA-48 genes. CONCLUSION: bla OXA-48 was the most prevalent gene detected in this study. PCR is useful in detecting carbapenemase genes, especially bla NDM , which may show false susceptibility to carbapenems. There was no direct correlation detected between PFGE profiles and antibiotic susceptibility pattern. PFGE has revealed the genomic diversity among isolates, thereby suggesting heterogeneity in strain circulation within intensive care unit and wards of the hospital. Monitoring and molecular typing is essential to curtail the spread of multidrug-resistant strains and control the outbreaks of infection.
Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminogl... more Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminoglycoside by producing aminoglycoside modifying enzymes (AMEs). In enterococci, high level resistance to aminoglycosides is mediated by acquisition of plasmid mediated genes encoding for aminoglycoside modifying enzymes (AMEs). High level gentamicin resistance (MIC ! 500μg /mL) is predominantly mediated by aac(6′)-Ie-aph(2″)-Ia, encoding the bifunctional aminoglycoside modifying enzyme AAC(6′)-APH(2″). This enzyme eliminates the synergistic activity of gentamicin when combined with a cell wall active agent. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id and ant(4′)-1a have also been detected in enterococci. Objective This study was carried out to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of Enterococci. Materials and Methods A total number of 150 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion. Minimum Inhibitory Concentration (MIC) was ascertained by agar dilution method. Polymerase chain reaction was done to screen the following AMEs (aac(6′)-Ie-aph(2″)-Ia; aph(2″)-Ib; aph(2″)-Ic; aph(2″)-Id and aph(3′)-IIIa genes). Results 51.3% of the study isolates exhibited high level gentamicin resistance. Polymerase chain reaction revealed that aph(3′)-111a is the most prevalent AME, followed by aac(6′)-1e-aph(2″)-1a. The combination of both the genes were detected in 44.1% of the study isolates. The rest of the AMEs and their combinations were not encountered in this study. 8.6% of the study isolates did not harbour any AME genes screened for, but was phenotypically resistant to gentamicin. In contrast 31.3% anchored the AME genes but phenotypically appeared susceptible to gentamicin. Conclusion This study indicates the high-level aminoglycoside resistance disseminated among Enterococci in our geographical region. It also emphasizes the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbour one or more AMEs genes leading to therapeutic failure.
BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the em... more BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the emergence of multidrug resistance in these organisms limits the treatment options for serious infections caused by them. K. pneumoniae carbapenemase (KPC) is one of the clinically significant Class A beta-lactamases. This study was aimed to detect the KPC and its coexistence with other beta-lactamases in K. pneumoniae. A total of 370 isolates, collected over a period of 1 year, were included in this study. The source of these isolates were urine (n = 170), exudative specimens (n = 132), respiratory secretions such as bronchial wash, endotracheal aspirate, and pleural fluid (n = 38), and blood (n = 30). For all the isolates, antibiotic susceptibility tests by disc diffusion, modified Hodge test, and KPC screening test were done. Polymerase chain reaction (PCR) was performed for the detection of KPC and the copresence of other beta-lactamases genes. RESULTS: Among the 370 isolates, 41 were resistant to the carbapenem by disc diffusion and minimum inhibitory concentration tests. Screen test using ertapenem and the boronic acid disk was positive in 14 isolates. Only one isolate harbored KPC gene by PCR, and it was co-produced with SHV-12 and CTX-M-15. CONCLUSION: PCR remains the gold standard for detection of KPC compared with any other phenotypic methods. Early detection of these genes helps in initiating proper antibiotic treatment.
Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens in health... more Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens in health care-associated infections. Fluoroquinolone resistance has emerged in these pathogens. In this study, we aimed to determine the occurrence of plasmid-mediated quinolone resistance (PMQR) determinants (qnrA, qnrB, qnrS, aac(6′)-Ib-cr, oqxAB, and qepA) by polymerase chain reaction (PCR) and the transmissibility of plasmid-borne resistance determinants in clinical isolates of P. aeruginosa and A. baumannii. The study included P. aeruginosa (85) and A. baumannii (45) which were nonduplicate, clinically significant, and ciprofloxacin resistant. Antibiotic susceptibility testing was done by disk diffusion method for other antimicrobial agents, namely amikacin, ceftazidime, piperacillin/tazobactam, ofloxacin, levofloxacin, and imipenem. Minimum inhibitory concentration of ciprofloxacin was determined. Efflux pump activity was evaluated using carbonyl-cyanide m-chlorophenylhydrazone (CCCP). The presence of PMQR genes was screened by PCR amplification. Transferability of PMQR genes was determined by conjugation experiment, and plasmid-based replicon typing was performed. Results Resistance to other classes of antimicrobial agents was as follows: ceftazidime (86.9%), piperacillin/tazobactam (73.8%), imipenem (69.2%), and amikacin (63.8%). The minimal inhibitory concentration (MIC)50 and MIC90 for ciprofloxacin were 64 and greater than or equal to 256 µg/mL, respectively. There was a reduction in MIC for 37 (28.4%) isolates with CCCP. In P. aeruginosa, 12 (14.1%) isolates harbored qnrB, 12 (14.1%) qnrS, 9 (10.5%) both qnrB and qnrS, 66 (77.6%) aac(6′)-Ib-cr, and 3 (3.5%) oqxAB gene. In A. baumannii, qnrB was detected in 2 (4.4%), 1 (2.2%) harbored both the qnrA and qnrS, 1 isolate harbored qnrB and qnrS, 21 (46.6%) aac(6′)-Ib-cr, and 1 (2.2%) isolate harbored oqxAB gene. Notably, qepA gene was not detected in any of the study isolates. Conjugation experiments revealed that 12 (9.2%) were transferable. Of the transconjugants, seven (58.3%) belonged to IncFII type plasmid replicon, followed by four (33.3%) IncA/C and one (8.3%) IncFIC type. Conclusion The plasmid-mediated resistance aac(6′)-Ib-cr gene is primarily responsible for mediating fluoroquinolone resistance in clinical isolates of P. aeruginosa and A. baumannii. The predominant plasmid type is IncFII.
Introduction Fluoroquinolones are widely used broad-spectrum antibiotics. Recently, increased rat... more Introduction Fluoroquinolones are widely used broad-spectrum antibiotics. Recently, increased rate of resistance to this antibiotic has been observed in Klebsiella pneumoniae. The aim of the present study was to determine the presence of quinolone resistance determining regions (QRDR) mutation genes and plasmid-mediated quinolone resistance (PMQR) determinants in clinical isolates of ciprofloxacin-resistant K. pneumoniae. The study included 110 nonduplicate ciprofloxacin-resistant K. pneumoniae clinical isolates. Antibiotic susceptibility testing by disk diffusion method and minimum inhibitory concentration (MIC) by agar dilution methods for ciprofloxacin was performed according to the recommendations of Clinical Laboratory Standards Institute. The presence of QRDR genes and PMQR genes was screened by polymerase chain reaction (PCR) amplification. Result All 110 isolates were resistance to ciprofloxacin, levofloxacin, and ofloxacin. As much as 88% of the isolates exhibited high-level of MIC to ciprofloxacin. Among the 110 isolates, 94(85%) harbored gyrA and 85 (77%) gyrB. The parC and parE genes were detected in 88 (80%) and 64 (58%) isolates. qnrB was detected in 13 (12%) isolates and qnrS in 5 (4.5%) isolates. Two (1.8%) isolates carried both qnrB and qnrS genes. The acc (6')-Ib-cr gene was found in 98 (89%) isolates and oqxAB was detected in 7 (6.3%) isolates. One (0.9%) isolate carried qnrB, acc(6')-Ib-cr and oqxAB genes. Conclusion The prevalence of acc (6')-Ib-cr gene is high among PMQR determinants, followed by qnrB, oqxAB and qnrS.
BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hyp... more BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hypervirulent K. pneumoniae (hvKP), new variant of K. pneumoniae, can cause invasive infections in young healthy individuals as well as in the immunocompromised population. Hypervirulent strains frequently belong to capsular serotypes K1 or K2. Emergence of antimicrobial resistance in hvKP is a cause for concern. The present study was done to detect the K1 and K2 serotypes among clinical isolates of K. pneumoniae, spectrum of infections caused by them and presence of common beta-lactamases encoding genes in them. A total of 370 isolates of K. pneumoniae, isolated from various clinical samples over a period of 1 year was included in this study. Antibiotic susceptibility testing to various classes of antimicrobials was done as per Clinical and Laboratory Standard Institute guidelines. The presence of K2A (specific to serotype K2), magA (specific to serotype K1), and rmpA genes was detected by multiplex polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (TEM, SHV, and CTX-M), plasmid-mediated AmpCs (MOX, CIT, DHA, ACC, EBC, and FOX), and carbapenemase genes (IMP, VIM, NDM, KPC, and OXA-48) were also determined by PCR. RESULTS: Among the 370 isolates, 8 harbored K2A gene and one harbored magA. rmpA gene was detected in three isolates along with K1 or K2 serotypes. Seven K2A-positive isolates were resistant to one or more classes of antimicrobials. The studied ESBL genes were present in four isolates. Two isolates harbored carbapenemase genes (NDM-1, OXA-48) along with ESBLs. CONCLUSION: K2 serotype is more prevalent among hvKP isolates. They can harbor ESBLs and Carbapenemase genes. K1 serotype is rather uncommon in K. pneumoniae. Acquisition of multidrug-resistant genes by these strains adds to their virulence and limits the treatment options.
Background Methicillin-resistant Staphylococcus aureus (MRSA) is a widely recognized multidrug-re... more Background Methicillin-resistant Staphylococcus aureus (MRSA) is a widely recognized multidrug-resistant bacteria presenting a major therapeutic challenge to clinicians. Staphylococcus aureus possesses a number of pathogenicity factors that attribute to the severity of infections. This study was undertaken to investigate the common virulence genes in clinical isolates of Staphylococcus aureus, determine their antimicrobial susceptibility profile, and to characterize the staphylococcal cassette chromosome mec (SCCmec) types among MRSA in a tertiary care center. Materials and Methods A total of 133 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion method. Methicillin resistance was screened using cefoxitin disc; mecA and mecC genes were detected using polymerase chain reaction (PCR). PCR was done to detect 12 virulence factors such as hla, hlb, fnbA, fnbB, sea, seb, sec, icaA, clfA, tst, pvl, and eta. SCCmec typing wa...
Background Staphylococcus haemolyticus has emerged as an important multidrug-resistant nosocomial... more Background Staphylococcus haemolyticus has emerged as an important multidrug-resistant nosocomial pathogen. Linezolid is useful in the treatment of severe infections caused by methicillin-resistant Staphylococci. Resistance to linezolid in Staphylococci is due to one or more of the following mechanisms: acquisition of the cfr (chloramphenicol florfenicol resistance) gene, mutation in the central loop of domain V of the 23S rRNA, and mutation in the rplC and rplD genes. This study was carried out to detect and characterize resistance to linezolid among the clinical isolates of Staphylococcus haemolyticus. Materials and Methods The study included 84 clinical isolates of Staphylococcus haemolyticus. Susceptibility to various antibiotics was determined by disc diffusion method. Minimum inhibitory concentration (MIC) was determined by agar dilution method for linezolid. Methicillin resistance was screened using oxacillin and cefoxitin disc. Polymerase chain reaction was done to detect me...
Introduction Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings;... more Introduction Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings; woefully their usage has been reduced by the emergence and distribution of resistance mainly due to aminoglycoside modifying enzymes (AME).Purpose This study was performed to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of gram-negative bacteria. This study also aimed to detect the presence of AMEs that are prevalent in gram-positive bacteria, among gram negatives.Materials and Methods A total number of 386 clinical isolates were included in this study. Polymerase chain reaction revealed the prevalence rate of AMEs screened [aac(6′)-lb, aac(3′)-I, aac(3′)-II, aac(3′)-VI, ant(2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, aph(2′)-Id, aac (6′)-Ie- aph(2′)-Ia, and aph(3′)-IIIa]. Conjugation experiment was performed for the clinical isolates which harbored any one of the AME which was prevalent in gram-positive bacter...
BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resi... more BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resistant Klebsiella pneumoniae. Resistance to carbapenems in K. pneumoniae is mainly due to metallo-beta-lactamases (NDM, IMP, and VIM) and class D oxacillinase (OXA-48-like). AIM AND OBJECTIVE: This study was undertaken to detect the genes encoding for carbapenemase in K. pneumoniae and to determine the clonal relatedness of selected isolates of K. pneumoniae producing NDM and OXA-48 by pulsed-field gel electrophoresis method (PFGE). MATERIALS AND METHODS: The isolates were collected over a period of 1 year. A total of 370 clinically significant, nonduplicate isolates of K. pneumoniae were included in this study. Phenotypic tests for the detection of carbapenemases were performed for all the isolates. Polymerase chain reaction (PCR) was carried out for the detection of carbapenemase genes such as blaKPC, blaIMP, blaVIM,blaNDM, and blaOXA-48. PFGE was performed, and the PFGE profiles were a...
Pulmonary infections are the most common clinical manifestations of Nocardia species. There is an... more Pulmonary infections are the most common clinical manifestations of Nocardia species. There is an increase in cases of nocardial infections occurring worldwide attributable to the increase in the immunosuppressed population. The availability of molecular methods has aided the detection of more number of cases as well as unusual species. Still, it remains one of the most underdiagnosed pathogens. Recognition of drug resistance in this organism has now mandated early and precise identification with speciation for effective treatment and management. Nocardial species identity can predict antimicrobial susceptibility and guide clinical management. Here, we report two cases of pulmonary nocardiosis caused by unusual species of Nocardia, namely, N. cyriacigeorgica and N. beijingensis identified by 16S rRNA gene-based sequencing. These cases are being reported for their rarity.
BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hyp... more BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hypervirulent K. pneumoniae (hvKP), new variant of K. pneumoniae, can cause invasive infections in young healthy individuals as well as in the immunocompromised population. Hypervirulent strains frequently belong to capsular serotypes K1 or K2. Emergence of antimicrobial resistance in hvKP is a cause for concern. AIM AND OBJECTIVE: The present study was done to detect the K1 and K2 serotypes among clinical isolates of K. pneumoniae, spectrum of infections caused by them and presence of common beta-lactamases encoding genes in them. MATERIALS AND METHODS: A total of 370 isolates of K. pneumoniae, isolated from various clinical samples over a period of 1 year was included in this study. Antibiotic susceptibility testing to various classes of antimicrobials was done as per Clinical and Laboratory Standard Institute guidelines. The presence of K2A (specific to serotype K2), magA (specific to se...
Journal of clinical and diagnostic research : JCDR, 2013
Detection of carbapenem hydrolyzing class D beta lactamase OXA-181, (a variant of OXA-48) in Ente... more Detection of carbapenem hydrolyzing class D beta lactamase OXA-181, (a variant of OXA-48) in Enterobacteriaceae, is important, to institute appropriate therapy and to initiate preventive measures. This study was done to determine the presence of OXA 48 and its derivative OXA-181 in Enterobacteriaceae of pathogenic significance. One hundred and eleven non-repetitive Enterobacteriaceae isolates which were resistant to any of the cephalosporin subclasses III and which exhibited reduced susceptibility to carbapenems were included in the study. Minimum inhibitory concentrations (MICs) to imipenem and meropenem was determined by broth microdilution. Production of carbapenamase was screened by Modified Hodge test (MHT). Polymerase Chain Reaction (PCR) was done to detect the presence of bla OXA-181 and bla OXA-48 .Coexistence of other carbapenemase encoding genes, namely, NDM-1, VIM, IMP and KPC were also looked for, by PCR. Of all the isolates which were tested, only 2 (1.8%) revealed the ...
Purpose: Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spect... more Purpose: Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR). Materials and Methods: Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25) and K. pneumoniae (n = 52) were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL). Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test). Results: Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origen -Citrobacter freundii), DHA (Dhahran Hospital, Saudi Arabia), ACC (Ambler class C), EBC (Amp C origen -Enterobacter cloacae) groups. ESBL was co-produced in 54 isolates. Conclusions: Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.
The metallo-β-lactamase gene bla VIM-2 was identified in a strain of Pseudomonas aeruginosa isola... more The metallo-β-lactamase gene bla VIM-2 was identified in a strain of Pseudomonas aeruginosa isolated in India. The integron encoding bla VIM-2 was virtually identical to those recently found in the United States and Russia. These unusual structures are likely to have arisen from an ancestral integron predating the formation of the 3′ conserved sequence.
BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the em... more BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the emergence of multidrug resistance in these organisms limits the treatment options for serious infections caused by them. K. pneumoniae carbapenemase (KPC) is one of the clinically significant Class A beta-lactamases. AIM AND OBJECTIVE: This study was aimed to detect the KPC and its coexistence with other beta-lactamases in K. pneumoniae. MATERIALS AND METHODS: A total of 370 isolates, collected over a period of 1 year, were included in this study. The source of these isolates were urine (n = 170), exudative specimens (n = 132), respiratory secretions such as bronchial wash, endotracheal aspirate, and pleural fluid (n = 38), and blood (n = 30). For all the isolates, antibiotic susceptibility tests by disc diffusion, modified Hodge test, and KPC screening test were done. Polymerase chain reaction (PCR) was performed for the detection of KPC and the copresence of other beta-lactamases genes. ...
Artificial Cells, Nanomedicine, and Biotechnology, 2017
Pseudomonas aeruginosa is a problematic human pathogen resistant to almost all available antibiot... more Pseudomonas aeruginosa is a problematic human pathogen resistant to almost all available antibiotics. The important prerequisite for these drugs to target this bacterium is an efficient delivery system. Siderophore-mediated drug delivery system is a promising approach to carry out antibiotics to the cells. Pyochelin, a siderophore of P. aeruginosa, was successfully synthesized in a five-step procedure. PEGylated liposomal pyochelin-antibiotic (L-Pch-Ab) carrier was fabricated by thin-film hydration method. L-Pch-Ab had an average size of 90.31 ± 0.11 nm holding a negative zeta potential at À54.12 ± 0.03 mV (PDI <2). The MIC determined by broth dilution method against three clinical strains isolated from burn wounds showed that L-Pch-Ab significantly reduced (16 mg/ml) the MIC values than those of free antibiotics. In the time kill assay, L-Pch-Ab was bactericidal against all strains at most time intervals at 2 Â and 4 Â MIC up to 24 h. TEM observations revealed that L-Pch-Ab was actively taken up by P. aeruginosa and exhibited membrane deformation within 2 h. Developed L-Pch-Ab fused intimately with the outer membrane of MDRPa and exhibited effective antibacterial activity than free Ab. Furthermore, L-Pch-Ab kills MDRPa within infected HaCaT keratinocytes without any cytotoxic effects at 4Â MIC concentrations after 72 h. Thus, the specific targeting of L-Pch-Ab with its higher efficacy to deliver drug by limiting the toxicity will be a novel approach to fight infections caused by P. aeruginosa.
Artificial cells, nanomedicine, and biotechnology, Jan 31, 2017
In this study, we examined the efficacy of liposomal oleic acid-based antibiotic formulations on ... more In this study, we examined the efficacy of liposomal oleic acid-based antibiotic formulations on 32 strains of multidrug-resistant Pseudomonas aeruginosa (MDRPa). The average size of liposomes were 93.12 ± 2.3 nm holding a negative zeta potential at -57.3 ± 0.89. Liposomal antibiotic formulations were tested against 32 MDRPa strains isolated from burn wounds and urine samples, which exhibited an MIC of ≤8 μg/mL, whereas MIC of free antibiotics ranged from 32 to >1024 μg/mL. The results clearly indicate that the liposomes composed of naturally occurring oleic acid, could be used therapeutically either alone or in combination with antibiotics to effectively treat P. aeruginosa infections.
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Papers by Uma Sekar