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Carbapenem-resistant Enterobacteriaceae (CRE) can be mechanistically classified into carbapenemase-producing Enterobacteriaceae (CPE) and non-carbapenemase-producing carbapenem nonsusceptible Enterobacteriaceae (NCPCRE). We sought to investigate the effect of antecedent carbapenem exposure as a risk factor for NCPCRE versus CPE. Among all patients with CRE colonization and infection, we conducted a case-control study comparing patients with NCPCRE (cases) and patients with CPE (controls). The presence of carbapenemases was investigated with phenotypic tests followed by PCR for predominant carbapenemase genes. We included 843 unique patients with first-episode CRE, including 387 (45.9%) NCPCRE and 456 (54.1%) CPE. The resistance genes detected in CPEs were bla NDM (42.8%), bla KPC (38.4%), and bla OXA-48-like (12.1%). After adjusting for confounders and clustering at the institutional level, the odds of prior 30-day carbapenem exposure was three times higher among NCPCRE than CPE patients (adjusted odds ratio [aOR], 3.48; 95% confidence interval [CI], 2.39 to 5.09; P  < 0.001). The odds of prior carbapenem exposure and NCPCRE detection persisted in stratified analyses by Enterobacteriaceae species ( Klebsiella pneumoniae and Escherichia coli ) and carbapenemase gene ( bla NDM and bla KPC ). CPE was associated with male gender (aOR, 1.45; 95% CI, 1.07 to 1.97; P  = 0.02), intensive care unit stay (aOR, 1.84; 95% CI, 1.24 to 2.74; P  = 0.003), and hospitalization in the preceding 1 year (aOR, 1.42; 95% CI, 1.01 to 2.02; P  = 0.05). In a large nationwide study, antecedent carbapenem exposure was a significant risk factor for NCPCRE versus CPE, suggesting a differential effect of antibiotic selection pressure., (Copyright © 2019 American Society for Microbiology.)

Polymyxin B-based combinations have emerged as a mainstay treatment against carbapenem-resistant Escherichia coli (CREC). We investigated the activity of polymyxin B-based two-antibiotic combinations against CREC using time-kill studies (TKS) and validated the findings in a hollow-fiber infection model (HFIM). TKS were conducted using 5 clinical CREC strains at 5 log 10 CFU/ml against 10 polymyxin B-based two-antibiotic combinations at maximum clinically achievable concentrations. HFIMs simulating dosing regimens with polymyxin B (30,000U/kg/day) and tigecycline (100 mg every 12 h) alone and in combination were conducted against two CREC strains at 5 log 10 CFU/ml over 120 h. Emergence of resistance was quantified using antibiotic-containing media. Phenotypic characterization (growth rate and stability of resistant phenotypes) of the resistant isolates was performed. All five CREC strains harbored carbapenemases. Polymyxin B and tigecycline MICs ranged from 0.5 mg/liter to 2 mg/liter and from 0.25 mg/liter to 8 mg/liter, respectively. All antibiotics alone did not have bactericidal activity at 24 h in the TKS, except for polymyxin B against two strains. In combination TKS, only polymyxin B plus tigecycline demonstrated both bactericidal activity and synergy in two out of five strains. In the HFIM, polymyxin B alone was bactericidal against both CREC strains before regrowth was observed at 8 h. Phenotypically stable polymyxin B-resistant mutants were observed for both strains, with a reduced growth rate observed in one strain. Tigecycline alone resulted in a slow reduction in bacterial counts. Polymyxin B plus tigecycline resulted in rapid and sustained bactericidal killing up to 120 h. Polymyxin B plus tigecycline is a promising combination against CREC. The clinical relevance of our results warrants further investigations., (Copyright © 2016 American Society for Microbiology.)

Background: Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations., Methods: 100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (105 CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (TRLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of TRLU values was determined. External validation was further done using 50 additional CR-GNB., Results: Predictive accuracies of TRLU were high for when all antibiotic combinations and species were collectively analyzed (TRLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (TRLU = 0.81, UA = 91%), and lowest for AB (TRLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively., Conclusion: We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.

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., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

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 4(th)-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.

Objective: Carbapenem-resistant Acinetobacter baumannii (CR-AB) is an emerging cause of nosocomial infections worldwide. Combination therapy may be the only viable option until new antibiotics become available. The objective of this study is to identify potential antimicrobial combinations against CR-AB isolated from our local hospitals., Methods: AB isolates from all public hospitals in Singapore were systematically collected between 2006 and 2007. MICs were determined according to CLSI guidelines. All CR-AB isolates were genotyped using a PCR-based method. Clonal relationship was elucidated. Time-kill studies (TKS) were conducted with polymyxin B, rifampicin and tigecycline alone and in combination using clinically relevant (achievable) unbound concentrations., Results: 31 CR AB isolates were identified. They are multidrug-resistant, but are susceptible to polymyxin B. From clonal typing, 8 clonal groups were identified and 11 isolates exhibited clonal diversity. In single TKS, polymyxin B, rifampicin and tigecycline alone did not exhibit bactericidal activity at 24 hours. In combination TKS, polymyxin plus rifampicin, polymyxin B plus tigecycline and tigecycline plus rifampicin exhibited bactericidal killing in 13/31, 9/31 and 7/31 isolates respectively at 24 hours. Within a clonal group, there may be no consensus with the types of antibiotics combinations that could still kill effectively., Conclusion: Monotherapy with polymyxin B may not be adequate against polymyxin B susceptible AB isolates. These findings demonstrate that in-vitro synergy of antibiotic combinations in CR AB may be strain dependant. It may guide us in choosing a pre-emptive therapy for CR AB infections and warrants further investigations.

Outbreaks of carbapenem-resistant Acinetobacter species have emerged, especially in Singapore. Combination therapy may be the only viable option until new antibiotics are available. The objective of this study was to identify potential antimicrobial combinations against carbapenem-resistant Acinetobacter baumannii and Acinetobacter species in Singapore. From an ongoing surveillance program, two isolates of A. baumannii and an isolate of Acinetobacter species that were multidrug resistant were selected on the basis of their unique resistance mechanisms. The two A. baumannii isolates carried the carbapenemase bla(OXA-23)-like gene and the Acinetobacter species carried a metallo-beta-lactamase IMP-4 gene. Time-kill studies were conducted with approximately 10(5) CFU ml(-1) at baseline with 0.5 times minimum inhibitory concentrations (MICs) of polymyxin B and tigecycline, and at a maximally achievable clinical concentration of meropenem(64 microg ml(-1)) and rifampicin(2 microg ml(-1)), alone and in combinations. The MICs (microg ml(-1)) of Acinetobacter species A105, A. baumannii AB112 and A. baumannii AB8879 to polymyxin B/tigecycline/rifampicin/meropenem were found to be 1/0.5/4/64, 1/4/4/32 and 2/2/2/64, respectively. In time-kill studies, enhanced combined killing effects were observed in the tigecycline-rifampicin combination; the tigecycline-rifampicin and rifampicin-polymyxin B combination; and the rifampicin-polymyxin B combination for Acinetobacter species A105, A. baumannii AB112 and A. baumannii AB8879, respectively, with >5 log kill at 24 h suggesting synergism, with no regrowth observed at 72 h. These findings demonstrate that in vitro synergy of antibiotic combinations in carbapenem-resistant Acinetobacter species may be strain dependent. It may guide us in choosing a preemptive therapy for carbapenem-resistant Acinetobacter species infections and warrants further investigations.