Thean Yen Tan

Rapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR.

ABSTRACT Methicillin-resistant Staphylococcus aureussepticemia is associated with significant morbidity and mortality and requires treatment with intravenous glycopeptides. For blood cultures positive for gram-positive cocci, 24 to 48 h is required for the detection of S. aureus bacteremia and the provision of antibiotic susceptibility testing results. We describe a molecular biology-based assay that requires 2 h from the time of initial positivity of blood cultures. The assay correctly detected 96% of the S. aureus isolates including all methicillin-resistant S. aureus isolates. Clinical data collected during the study suggest that 28% of patients with S. aureus bacteremia do not receive early and appropriate treatment and that 10% of patients may initially be receiving inappropriate glycopeptide treatment.

Evaluation of Screening Methods To Detect Plasmid-Mediated AmpC in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis

ABSTRACT There are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampC genes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampC genes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiella spp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT- and DHA-like plasmid-borne ampC genes. The methods are simple enough for introduction into clinical microbiology laboratories.

Comparative in vitro activity of carbapenems against major Gram-negative pathogens: results of Asia-Pacific surveillance from the COMPACT II study.

Resistance rates amongst Gram-negative pathogens are increasing in the Asia-Pacific region. The Comparative Activity of Carbapenem Testing (COMPACT) II study surveyed the carbapenem susceptibility and minimum inhibitory concentrations (MICs) of doripenem, imipenem and meropenem against 1260 major Gram-negative pathogens isolated from hospitalised patients at 20 centres in five Asia-Pacific countries (New Zealand, the Philippines, Singapore, Thailand and Vietnam) during 2010. Pseudomonas aeruginosa (n=625), Enterobacteriaceae (n=500), and other Gram-negative pathogens including Acinetobacter baumannii (n=135) were collected from patients with bloodstream infection (32.2%), nosocomial pneumonia including ventilator-associated pneumonia (58.1%), and complicated intra-abdominal infection (9.7%), with 36.7% being isolated from patients in an Intensive Care Unit. As high as 29.8% of P. aeruginosa and 73.0% of A. baumannii isolates were not susceptible to at least a carbapenem, whereas the majority of Enterobacteriaceae (97.2%) were susceptible to all carbapenems. Respective MIC(50)/MIC(90) values (MICs for 50% and 90% of the organisms, respectively) of doripenem, imipenem and meropenem were: 0.38/8, 1.5/32 and 0.38/16 mg/L for P. aeruginosa; 0.023/0.094, 0.25/0.5 and 0.032/0.094 mg/L for Enterobacteriaceae; and 32/64, 32/128 and 32/64 mg/L for A. baumannii. Doripenem and meropenem had comparable activity against P. aeruginosa, both being more active than imipenem. All carbapenems were highly potent against Enterobacteriaceae, although imipenem demonstrated higher MIC values than doripenem and meropenem. The three carbapenems showed less activity against A. baumannii. The high prevalence of carbapenem resistance amongst important nosocomial pathogens (P. aeruginosa and A. baumannii) warrants rigorous infection control measures and appropriate antimicrobial use in the Asia-Pacific region.

Microbiological Characteristics, Presumptive Identification, and Antibiotic Susceptibilities of Staphylococcus lugdunensis

ABSTRACT This study validated abbreviated methods for the presumptive identification of Staphylococcus lugdunensis and studied the antibiotic susceptibilities of 106 isolates. The combination of positive responses to ornithine and pyrrolidonyl arylamidase identified all S. lugdunensis isolates. Resistance to penicillin and methicillin was detected in 27 and 5% of isolates, respectively.

Antimicrobial susceptibility of bacterial pathogens associated with community-acquired respiratory tract infections in Asia: report from the Community-Acquired Respiratory Tract Infection Pathogen Surveillance (CARTIPS) study, 2009-2010

A multicentre resistance surveillance study [Community-Acquired Respiratory Tract Infection Pathogen Surveillance (CARTIPS)] investigating the susceptibilities of 2963 clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, meticillin-susceptible Staphylococcus aureus (MSSA) and Streptococcus spp. from Asia against 12 antimicrobial agents was undertaken from 2009 to 2010. Based on the breakpoints for oral penicillin V recommended by the Clinical and Laboratory Standards Institute, the prevalence of penicillin-non-susceptible S. pneumoniae (PNSSP) ranged from 46% to 100%. Azithromycin and clarithromycin exhibited variable resistance rates of 0-88% against S. pneumoniae, 0-57% against MSSA and 0-76.5% against Streptococcus spp. isolates. The prevalence of extended-spectrum β-lactamase-producing K. pneumoniae varied from 5.1% to 58.5%. β-Lactamase production rates amongst H. influenzae isolates ranged from 15% to 46.6% and amongst M. catarrhalis isolates from 90% to 100%. Amongst M. catarrhalis isolates, macrolide resistance and cefaclor resistance rates of 5.8% and 1.2%, respectively, were found, mainly in Mainland China. Levofloxacin resistance rates of 0-3.9% with a MIC(90) (minimum inhibitory concentration causing inhibition of 90% of isolates) of 1-2mg/L and moxifloxacin resistance rates of 0-1.7% with a MIC(90) of 0.125-0.5mg/L were found amongst PNSSP isolates. Moxifloxacin was very active against Streptococcus spp., H. influenzae and M. catarrhalis isolates, with MIC(90) values of 0.125-0.25, 0.032-0.5 and 0.064-0.125mg/L, respectively. These results from the CARTIPS study have confirmed some significant regional differences in the antimicrobial susceptibilities of S. pneumoniae, MSSA, K. pneumoniae, H. influenzae and Streptococcus spp. and emphasise the importance of antimicrobial surveillance programmes for guiding empirical therapy and for focusing interventional control of antimicrobial resistance in distinct geographic areas.

Detection of plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis.

Aims: This study investigated the prevalence of plasmid-mediated AmpC production in selected clinical isolates of Escherichia coli, Klebsiella species and Proteus mirabilis, and compared the results of boronic acid disc screening with conventional susceptibility testing for the detection of AmpC-positive isolates. Methods: E coli, Klebsiella species and P mirabilis with reduced susceptibility to amoxycillin-clavulanate, cefuroxime and cephalexin, but without phenotypic evidence of extended-spectrum β-lactamases were screened for AmpC activity using enzyme-extraction methods. The presence of plasmid-mediated ampC was determined by multiplex PCR. Antibiotic susceptibilities were determined using both disc and dilution-based methods. A disc-based screening method for detection of AmpC-producing strains was evaluated using boronic acid as an inhibitor of AmpC, and cefoxitin as the antibiotic substrate. Results: Plasmid-mediated ampC was present in 26% of study isolates, with CMY-like enzymes detected predominantly in E coli and DHA-like enzymes predominantly in Klebsiella pneumoniae. Current susceptibility methods failed to detect a significant proportion of plasmid-mediated AmpC-producing isolates, with 33% of such strains interpreted as susceptible to third-generation cephalosporins using current Clinical Laboratory Standards Institute breakpoints. The boronic acid disc method showed sensitivity and specificity of 90% and 98% respectively in detecting AmpC-positive isolates. Conclusion: The prevalence of plasmid-mediated ampC was high in the study population, and may be missed by conventional susceptibility testing methods. Inhibitor-based screening methods would improve detection of this emerging resistance phenotype.

Epidemiology and antimicrobial susceptibility profiles of pathogens causing urinary tract infections in the Asia-Pacific region: Results from the Study for Monitoring Antimicrobial Resistance Trends (SMART), 2010–2013

A total of 9599 isolates of Gram-negative bacteria (GNB) causing urinary tract infections (UTIs) were collected from 60 centres in 13 countries in the Asia-Pacific region from 2010-2013. These isolates comprised Enterobacteriaceae species (mainly Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Klebsiella oxytoca, Enterobacter cloacae and Morganella morganii) and non-fermentative GNB species (predominantly Pseudomonas aeruginosa and Acinetobacter baumannii). In vitro susceptibilities were determined by the agar dilution method and susceptibility profiles were determined using the minimum inhibitory concentration (MIC) interpretive breakpoints recommended by the Clinical and Laboratory Standards Institute in 2015. Production of extended-spectrum β-lactamases (ESBLs) amongst E. coli, K. pneumoniae, P. mirabilis and K. oxytoca isolates was determined by the double-disk synergy test. China, Vietnam, India, Thailand and the Philippines had the highest rates of GNB species producing ESBLs and the highest rates of cephalosporin resistance. ESBL production and hospital-acquired infection (isolates obtained ≥48 h after admission) significantly compromised the susceptibility of isolates of E. coli and K. pneumoniae to ciprofloxacin, levofloxacin and most β-lactams, with the exception of imipenem and ertapenem. However, >87% of ESBL-producing E. coli strains were susceptible to amikacin and piperacillin/tazobactam, indicating that these antibiotics might be appropriate alternatives for treating UTIs due to ESBL-producing E. coli. Fluoroquinolones were shown to be inappropriate as empirical therapy for UTIs. Antibiotic resistance is a serious problem in the Asia-Pacific region. Therefore, continuous monitoring of evolutionary trends in the susceptibility profiles of GNB causing UTIs in Asia is crucial.

In Vitro Antibiotic Synergy in Extensively Drug-Resistant Acinetobacter baumannii: the Effect of Testing by Time-Kill, Checkerboard, and Etest Methods

ABSTRACT This study examined the in vitro effects of polymyxin B, tigecycline, and rifampin combinations on 16 isolates of extensively drug-resistant Acinetobacter baumannii, including four polymyxin-resistant strains. In vitro synergy was demonstrated in 19 (40%) of a possible 48 isolate-antibiotic combinations by time-kill methods, 8 (17%) by checkerboard methods, and only 1 (2%) by Etest methods. There was only slight agreement between Etest and checkerboard methods and no agreement between results obtained by other methods.

Clinical Significance of Coagulase-Negative Staphylococci Recovered from Nonsterile Sites

ABSTRACT Laboratory criteria were used to select coagulase-negative staphylococci isolated from nonsterile body sites for further investigation. Fifty-seven percent of the study isolates were clinically significant, predominantly causing community-acquired soft tissue infections. There were species-related differences in virulence, with 91% of Staphylococcus lugdunensis isolates clinically significant compared with 11% of S. haemolyticus isolates.

Risk Factors, Molecular Epidemiology and Outcomes of Ertapenem-Resistant, Carbapenem-Susceptible Enterobacteriaceae: A Case-Case-Control Study

Background Increasing prevalence of ertapenem-resistant, carbapenem-susceptible Enterobacteriaceae (ERE) in Singapore presents a major therapeutic problem. Our objective was to determine risk factors associated with the acquisition of ERE in hospitalized patients; to assess associated patient outcomes; and to describe the molecular characteristics of ERE. Methods A retrospective case-case-control study was conducted in 2009 at a tertiary care hospital. Hospitalized patients with ERE and those with ertapenem-sensitive Enterobacteriaceae (ESE) were compared with a common control group consisting of patients with no prior gram-negative infections. Risk factors analyzed included demographics; co-morbidities; instrumentation and antibiotic exposures. Two parallel multivariate logistic regression models were performed to identify independent variables associated with ERE and ESE acquisition respectively. Clinical outcomes were compared between ERE and ESE patients. Results Twenty-nine ERE cases, 29 ESE cases and 87 controls were analyzed. Multivariate logistic regression showed that previous hospitalization (Odds ratio [OR], 10.40; 95% confidence interval [CI], 2.19–49.20) and duration of fluoroquinolones exposure (OR, 1.18 per day increase; 95% CI, 1.05–1.34) were unique independent predictors for acquiring ERE. Duration of 4th-generation cephalosporin exposure was found to predict for ESE acquisition (OR, 1.63 per day increase; 95% CI, 1.05–2.54). In-hospital mortality rates and clinical response rates were significantly different between ERE and ESE groups, however ERE infection was not a predictor of mortality. ERE isolates were clonally distinct. Ertapenem resistance was likely to be mediated by the presence of extended-spectrum β-lactamases or plasmid-borne AmpC in combination with impermeability due to porin loss and/or efflux pumps. Conclusion Prior hospitalization and duration of fluoroquinolone treatment were predictors of ERE acquisition. ERE infections were associated with higher mortality rates and poorer clinical response rates when compared to ESE infections.