Characterization of Chlorhexidine Resistance Among Clinical Isolates of Coagulase-Negative Staphylococcus Species in a Tertiary Care Center, Chennai.

Background Coagulase-negative Staphylococci (CoNS) are potential pathogens and are often associated with healthcare-associated infections (HAIs). Chlorhexidine (CHX) is the most widely used antiseptic to reduce colonization and infection by all Staphylococci, including CoNS. Resistance to CHX among CoNS has been observed over the past few years, consequent to its widespread use. Phenotypic tolerance or reduced susceptibility to CHX is conferred by plasmid-mediated qac group of genes, mainly qacA/B and smr, which cause activation of efflux pumps over the bacterial cell wall. This study aims to characterize the phenotypic and genotypic resistance exhibited by CoNS species against CHX. Methods After ethical approval, 148 consecutive, non-repetitive isolates of clinically significant CoNS species of hospitalized patients, isolated from blood samples and exudative specimens, were included in the study. Speciation was performed by conventional biochemical identification and automated methods. Antimicrobial susceptibility testing was performed by disc diffusion technique and for vancomycin by minimum inhibitory concentration (MIC) determination, as per Clinical Laboratory Standards Institute (CLSI) M-100 2023 guidelines. Methicillin resistance was detected using a cefoxitin disc. MIC for CHX was performed by agar dilution method; reduced susceptibility was considered when MIC to CHX ≥4 µg/mL. The simplex polymerase chain reaction (PCR) was carried out with suitable controls to detect qacA/B and smr. Statistical analysis was conducted to determine the association of qacA/B and smr genes with MIC of CHX in the study isolates. Results Fifteen different species of CoNS were obtained from clinical samples. A high percentage of resistance was observed against various classes of antibiotics. Methicillin resistance was observed in 69.6% (103/148) of isolates. Of 148 CoNS, 52.7% (78/148) of isolates exhibited reduced susceptibility to CHX with an MIC ≥4 µg/mL. These isolates exhibited a higher percentage of methicillin resistance (75.6%, 59/78). By PCR, 34.5% (51/148) of isolates carried either or both genes. Gene qacA/B was solely detected in 27.02% (40/148) of isolates, of which 14 were CHX-tolerant and the remaining 26 were CHX-susceptible. Gene smr was solely detected in 4.1% (6/148) of isolates comprising three isolates each in CHX-tolerant and susceptible categories. There were 3.4% (5/148) of isolates that harbored both genes, of which only one isolate was CHX-susceptible, while the other four were CHX-tolerant. A proportion of isolates that were phenotypically tolerant to CHX did not carry either or both genes. A significant statistical association was found between reduced susceptibility to CHX and the presence of antiseptic resistance genes in the study isolates (p-value=0.033942). Conclusion To our knowledge, this is the first study from South India to investigate CHX resistance among CoNS using phenotypic and genotypic methods. The rise of antiseptic resistance among CoNS is an emerging threat to current infection control practices. The presence of qacA/B and smr genes, especially in CHX susceptible isolates, is concerning since these resistance genes are located on transferable plasmids, and the isolates can develop resistance eventually upon exposure to CHX.

Virulence, Susceptibility Profile, and Clinical Characteristics of Pathogenic Coagulase-Negative Staphylococci.

Background Coagulase-negative staphylococci (CoNS) are emerging as clinically significant pathogens. A high proportion of methicillin resistance along with intense biofilm-producing ability render CoNS-related infections challenging to treat. This study was undertaken to investigate the mechanisms of methicillin resistance, identify genes encoding for virulence, and their association with clinical outcomes among clinical isolates of Staphylococci in a tertiary care center. Methods A total of 203 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by the disc diffusion method. Methicillin resistance was screened using cefoxitin disc, mecA and mecC genes were detected using polymerase chain reaction (PCR). PCR was performed to detect five virulence genes: atlE, aap, fbe, embp, and icaAB. Staphylococcal cassette chromosome mec (SCCmec) types were identified by multiplex PCR. Statistical analysis was performed using SPSS software (IBM Inc., Armonk, New York). The Chi-squared test was used to compare the distribution of virulence genes among methicillin-susceptible resistant CoNS. A p-value of less than 0.5 was considered significant. Results In the current study, 60% (122/203) of CoNS were methicillin-resistant, and SCCmec type I was the most common. Among the 203 CoNS, 24.6% (50/203) isolates harbored one or more virulence genes in them.  Conclusion CoNS have relatively low virulence as only 24.6% of isolates carried the virulence genes. Nevertheless, the variety of diseases linked to these species indicates the necessity for accurate identification and precise reporting of antimicrobial susceptibility to avoid adverse outcomes.

Spectrum of Virulence Factors in Clinical Isolates of Staphylococcus aureus and Prevalence of SCCmec Types in Methicillin-Resistant Staphylococcus aureus in a Tertiary Care Center.

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; m ecA 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 was done by multiplex PCR. Results Of the 133 clinical isolates, 54 (40.6%) were MRSA. The most common virulence gene detected was hlb (61.6%), hla (39%), and fnbA (37%). SCCmec type I was the most predominant. Mortality rate of 6.7% was observed among patients with staphylococcal infections. Univariate analysis of mortality associated virulence genes did not reveal any significant association between virulence genes and mortality. Conclusion The distribution of virulence genes is similar in both MRSA and methicillin-sensitive Staphylococcus aureus . MRSA belongs to the SCCmec types I to IV. Possession of multiple virulence factors and multidrug resistance profile makes Staphylococcus aureus a formidable pathogen in clinical settings.

Detection of a Novel G2603T Mutation in cfr Harboring Linezolid-Resistant Staphylococcus haemolyticus : First Report from India.

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 mecA, cfr and mutations in the V domain of the 23S rRNA gene. Results Resistance to linezolid was exhibited by 3 of the 84 study isolates with MIC more than 128 µg/mL. The cfr gene was detected in all the three isolates. The G2603T mutation was observed in the domain V of the 23S rRNA among two isolates, whereas one isolate lacked any mutation. Conclusion The emergence and spread of linezolid-resistant Staphylococcus haemolyticus isolates carrying G2603T mutation in the domain V of the 23S rRNA and harboring the cfr gene pose a threat in clinical practice.

Plasmid-Mediated Fluoroquinolone Resistance in Pseudomonas aeruginosa and Acinetobacter baumannii.

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 . Materials and Methods 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.

Molecular Characterization of High-Level Aminoglycoside Resistance among Enterococcus Species.

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.

Fluoroquinolone Resistance in Clinical Isolates of Klebsiella Pneumonia e.

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. Material and Methods 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.

Spectrum of Aminoglycoside Modifying Enzymes in Gram-Negative Bacteria Causing Human Infections.

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 bacteria [aph(3')-IIIa, aac(6')-Ie-aph(2')-Ia]. Results aac(6')-lb is the most prevalent AME, followed by aac(3')-I, aph(3')-VI, aac(3')-VI, and aac(3')-II. The AMEs such as ant (2')-I, ant(4')-IIb, aac(3')-III, aac(3')-IV, aph(2')-Ib, aph(2')-Ic, and aph(2')-Id were not established in our study isolates. The rate of prevalence of aph(3')-IIIa, aac(6')-Ie-aph(2')-Ia-the AMEs encountered in gram-positive and their co-existence was 19.68% and the conjugation experiment revealed their transfer via plasmids. Conclusion This is the first report from India revealing the presence and prevalence of AMEs which are often encountered among gram-positive bacteria in gram negatives and their presence on conjugative plasmids.

Carbapenemase-producing Enterobacteriaceae: Risk factors for infection and impact of resistance on outcomes.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) have increased in recent years leading to limitations of treatment options. The present study was undertaken to detect CPE, risk factors for acquiring them and their impact on clinical outcomes. METHODS: This retrospective observational study included 111 clinically significant Enterobacteriaceae resistant to cephalosporins subclass III and exhibiting a positive modified Hodge test. Screening for carbapenemase production was done by phenotypic methods, and polymerase chain reaction was performed to detect genes encoding them. Retrospectively, the medical records of the patients were perused to assess risk factors for infections with CPE and their impact. The data collected were duration of hospital stay, Intensive Care Unit (ICU) stay, use of invasive devices, mechanical ventilation, the presence of comorbidities, and antimicrobial therapy. The outcome was followed up. Univariate and multivariate analysis of the data were performed using SPSS software. RESULTS: Carbapenemase-encoding genes were detected in 67 isolates. The genes detected were New Delhi metallo-ß-lactamase, Verona integron-encoded metallo-ß-lactamase, and oxacillinase-181.Although univariate analysis identified risk factors associated with acquiring CPE infections as ICU stay (P = 0.021), mechanical ventilation (P = 0.013), indwelling device (P = 0.011), diabetes mellitus (P = 0.036), usage of multiple antimicrobial agents (P = 0.007), administration of carbapenems (P = 0.042), presence of focal infection or sepsis (P = 0.013), and surgical interventions (P = 0.016), multivariate analysis revealed that all these factors were insignificant. Mortality rate was 56.7% in patients with CPE infections. By both univariate and multivariate analysis of impact of the variables on mortality in these patients, the significant factors were mechanical ventilation (odds ratio [OR]: 0.141, 95% confidence interval [CI]: 0.024-0.812) and presence of indwelling invasive device (OR: 8.034; 95% CI: 2.060-31.335). CONCLUSION: In this study, no specific factor was identified as an independent risk for acquisition of CPE infection. However, as it is evident by multivariate analysis, there is an increased risk of mortality in patients with CPE infections when they are ventilated and are supported by indwelling devices.

Detection of Vancomycin Resistance among Enterococcus faecalis and Staphylococcus aureus.

INTRODUCTION: Vancomycin remains the drug of choice for resistant gram positive infections caused by Enterococcus spp and Methicillin resistant Staphylococcus aureus (MRSA). Increased use of vancomycin has led to frank resistance and increase in MIC (MIC creep). Vancomycin intermediate Staphylococcus aureus (VISA), Vancomycin resistant Staphylococcus aureus (VRSA) & Vancomycin resistant Enterococci (VRE) are important emerging nosocomial pathogens resulting in treatment failures. AIM: This study was undertaken to detect vancomycin resistance among clinical isolates of Staphylococcus aureus and Enterococcus faecalis by phenotypic and genotypic methods. MATERIALS AND METHODS: The study was conducted in a 1850 bedded university teaching hospital from November 2013 to April 2014. Non-repetitive, consecutive clinically significant Staphylococcus aureus (109) and Enterococcus faecalis (124) were included in this study. They were identified up to species level by conventional and automated methods. Susceptibility to various antibiotics was tested by disc diffusion method. MIC of vancomycin was determined by agar dilution method. Inducible resistance to clindamycin was detected by the D test. Methicillin resistance in Staphylococcus aureus (MRSA) was screened using cefoxitin disc. All isolates were subjected to polymerase chain reaction (PCR) to detect van A and van B genes. RESULTS: Out of 109 Staphylococcus aureus isolates, 54 were MRSA. By MIC there was no resistance observed to vancomycin.MIC50 was 1µg/ml. None of the isolates harboured van A and van B. Among Enterococcus faecalis, sixteen isolates (12.9%) and four isolates (3.2%) exhibited resistance to vancomycin and teicoplanin by disc diffusion respectively. All isolates were susceptible to linezolid. Van A was detected in 2, van B in 7 and one had both van A and van B. CONCLUSION: PCR remains the gold standard for diagnosis of vancomycin resistance. There was no resistance observed to vancomycin among Staphylococci though the MIC creep detected is a cause for concern. Eight percent of Enterococci were vancomycin resistant.