Elimination of Extracellular Adenosine Triphosphate for the Rapid Prediction of Quantitative Plate Counts in 24 h Time-Kill Studies against Carbapenem-Resistant Gram-Negative Bacteria.

Traditional in vitro time-kill studies (TKSs) require viable plating, which is tedious and time-consuming. We used ATP bioluminescence, with the removal of extracellular ATP (EC-ATP), as a surrogate for viable plating in TKSs against carbapenem-resistant Gram-negative bacteria (CR-GNB). Twenty-four-hour TKSs were conducted using eight clinical CR-GNB (two Escherichia coli, two Klebsiella spp., two Acinetobacter baumannii, two Pseudomonas aeruginosa) with multiple single and two-antibiotic combinations. ATP bioluminescence and viable counts were determined at each timepoint (0, 2, 4, 8, 24 h), with and without apyrase treatment. Correlation between ATP bioluminescence and viable counts was determined for apyrase-treated and non-apyrase-treated samples. Receiver operator characteristic curves were plotted to determine the optimal luminescence threshold to discriminate between inhibitory/non-inhibitory and bactericidal/non-bactericidal combinations, compared to viable counts. After treatment of bacteria with 2 U/mL apyrase for 15 min at 37 °C, correlation to viable counts was significantly higher compared to untreated samples (p < 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p < 0.01) and bactericidal (p = 0.03) combinations against CR-GNB compared to untreated samples, when all species were collectively analyzed. We found that ATP bioluminescence can potentially replace viable plating in TKS. Our assay also has applications in in vitro and in vivo infection models.

Clinical Experience with High-Dose Polymyxin B against Carbapenem-Resistant Gram-Negative Bacterial Infections-A Cohort Study.

Population pharmacokinetic studies have suggested that high polymyxin B (PMB) doses (≥30,000 IU/kg/day) can improve bacterial kill in carbapenem-resistant Gram-negative bacteria (CR-GNB). We aim to describe the efficacy and nephrotoxicity of patients with CR-GNB infections prescribed high-dose PMB. A single-centre cohort study was conducted from 2013 to 2016 on septic patients with CR-GNB infection and prescribed high-dose PMB (~30,000 IU/kg/day) for ≥72 h. Study outcomes included 30-day mortality and acute kidney injury (AKI) development. Factors associated with AKI were identified using multivariable regression. Forty-three patients with 58 CR-GNB received high-dose PMB; 57/58 (98.3%) CR-GNB were susceptible to PMB. The median daily dose and duration of high-dose PMB were 32,051 IU/kg/day (IQR, 29,340-34,884 IU/kg/day) and 14 days (IQR, 7-28 days), respectively. Thirty-day mortality was observed in 7 (16.3%) patients. AKI was observed in 25 (58.1%) patients with a median onset of 8 days (IQR, 6-13 days). Higher daily PMB dose (aOR,1.01; 95% CI, 1.00-1.02) and higher number of concurrent nephrotoxins (aOR, 2.14; 95% CI; 1.03-4.45) were independently associated with AKI. We observed that a sizable proportion developed AKI in CR-GNB patients described high-dose PMB; hence, the potential benefits must be weighed against increased AKI risk. Concurrent nephrotoxins should be avoided to reduce nephrotoxicity.

Determining the Development of Persisters in Extensively Drug-Resistant Acinetobacter baumannii upon Exposure to Polymyxin B-Based Antibiotic Combinations Using Flow Cytometry.

Polymyxin B-based combinations are increasingly prescribed as a last-line option against extensively drug-resistant (XDR) Acinetobacter baumannii It is unknown if such combinations can result in the development of nondividing persister cells in XDR A. baumannii We investigated persister development upon exposure of XDR A. baumannii to polymyxin B-based antibiotic combinations using flow cytometry. Time-kill studies (TKSs) were conducted in three nonclonal XDR A. baumannii strains with 5 log10 CFU/ml bacteria against polymyxin B alone and polymyxin B-based two-drug combinations over 24 h. At different time points, samples were obtained and enumerated by viable plating and flow cytometry. Propidium iodide and carboxyfluorescein succinimidyl ester dyes were used to differentiate between live and dead cells and between dividing and nondividing cells, respectively, at the single-cell level, and nondividing live cells were resuscitated and characterized phenotypically. Our results from viable plating showed that polymyxin B plus meropenem and polymyxin B plus rifampin were each bactericidal (>99.9% kill compared to the initial inoculum) against 2/3 XDR A. baumannii strains at 24 h. By flow cytometry, however, none of the combinations were bactericidal against XDR A. baumannii at 24 h. Further analysis using cellular dyes in flow cytometry revealed that upon exposure to polymyxin B-based combinations, XDR A. baumannii entered a viable but nondividing persister state. These bacterial cells reinitiated division upon the removal of antibiotic pressure and did not have a growth deficit compared to the parent strain. We conclude that persister cells develop in XDR A. baumannii upon exposure to polymyxin B-based combinations and that nonplating methods appear to complement viable-plating methods in describing the killing activity of polymyxin B-based combinations against XDR A. baumannii.

Risk factors and outcomes associated with the isolation of polymyxin B and carbapenem-resistant Enterobacteriaceae spp.: A case-control study.

Increasing resistance to polymyxin, a last-line antibiotic, is a growing public health concern worldwide. The primary objective of this study was to identify predictors for the isolation of polymyxin-resistant (PR) carbapenem-resistant Enterobacteriaceae (CRE) among hospitalized patients. The secondary objective was to describe the clinical outcomes of patients with PR-CRE infections. A retrospective case-control study including patients admitted to Singapore General Hospital between June 2012 and June 2016 was conducted. Cases were defined as patients who had clinical cultures from which a PR-CRE was isolated. Controls were randomly selected from patients with polymyxin-susceptible (PS) CRE admitted during the same period, and frequency-matched to site of isolation. We included 37 PR cases and 111 PS controls. Polymyxin resistance was detected predominantly in Enterobacter spp. (54.1%) and Klebsiella pneumoniae (43.2%). Multilocus sequence typing showed little clonal relatedness among the isolates. mcr-1 was detected in two PR-CRE isolates. Multivariable analyses showed that PR-CRE isolation was associated with prior polymyxins (adjusted odds ratio (OR), 21.31; 95% confidence interval (CI), 3.04-150.96) and carbapenem exposures (OR 3.74; CI 1.13-12.44), when adjusted for time at risk and bacteria species. In PR-CRE patients with infections, the 30-day all-cause in-hospital mortality was 50.0% as compared to 38.1% in patients with PS-CRE (P = 0.346). Prior polymyxin and carbapenem exposures were independent risk factors for isolation of PR-CRE. Outcomes of PR-CRE and PS-CRE infections were similar in this study.

Correction to: Candidemia in a major regional tertiary referral hospital - epidemiology, practice patterns and outcomes.

[This corrects the article DOI: 10.1186/s13756-017-0184-1.].

Rapid Antibiotic Combination Testing for Carbapenem-Resistant Gram-Negative Bacteria within Six Hours Using ATP Bioluminescence.

To guide the timely selection of antibiotic combinations against carbapenem-resistant Gram-negative bacteria (CR-GNB), an in vitro test with a short turnaround time is essential. We developed an in vitro ATP bioluminescence assay to determine effective antibiotic combinations against CR-GNB within 6 h. We tested 42 clinical CR-GNB strains (14 Acinetobacter baumannii, 14 Pseudomonas aeruginosa, and 14 Klebsiella pneumoniae strains) against 74 single antibiotics and two-antibiotic combinations. Bacteria (approximately 5 log10 CFU/ml) were incubated with an antibiotic(s) at 35°C; ATP bioluminescence was measured at 6 h and 24 h; and the measurements were compared to viable counts at 24 h. Receiver operating characteristic (ROC) curves were used to determine the optimal luminescence thresholds (TRLU) for distinguishing between inhibitory and noninhibitory combinations. The areas under the 6-h and 24-h ROC curves were compared using the DeLong method. Prospective validation of the established thresholds was conducted using 18 additional CR-GNB. The predictive accuracy of TRLU for the 6-h ATP bioluminescence assay was 77.5% when all species were analyzed collectively. Predictive accuracies ranged from 73.7% to 82.7% when each species was analyzed individually. Upon comparison of the areas under the 6-h and 24-h ROC curves, the 6-h assay performed significantly better than the 24-h assay (P < 0.01). Predictive accuracy remained high upon prospective validation of the 6-h ATP assay (predictive accuracy, 79.8%; 95% confidence interval [CI], 77.6 to 81.9%), confirming the external validity of the assay. Our findings indicate that our 6-h ATP bioluminescence assay can provide guidance for prospective selection of antibiotic combinations against CR-GNB in a timely manner and may be useful in the management of CR-GNB infections.

Candidemia in a major regional tertiary referral hospital - epidemiology, practice patterns and outcomes.

BACKGROUND: Candidemia is a common cause of nosocomial bloodstream infections, resulting in high morbidity and mortality. This study was conducted to describe the epidemiology, species distribution, antifungal susceptibility patterns and outcomes of candidemia in a large regional tertiary referral hospital. METHODS: A retrospective surveillance study of patients with candidemia was conducted at Singapore General Hospital between July 2012 and December 2015. In addition, incidence densities and species distribution of candidemia episodes were analysed from 2008 to 2015. RESULTS: In the period of 2012 to 2015, 261 candidemia episodes were identified. The overall incidence was 0.14/1000 inpatient-days. C. glabrata (31.4%), C. tropicalis (29.9%), and C. albicans (23.8%) were most commonly isolated. The incidence of C. glabrata significantly increased from 2008 to 2015 (Coefficient 0.004, confidence interval 0-0.007, p = 0.04). Fluconazole resistance was detected primarily in C. tropicalis (16.7%) and C. glabrata (7.2%). fks mutations were identified in one C. albicans and one C. tropicalis. Candidemia episodes caused by C. tropicalis were more commonly encountered in patients with haematological malignancies (p = 0.01), neutropenia (p < 0.001) and higher SAPS II scores (p = 0.02), while prior exposure to echinocandins was associated with isolation of C. parapsilosis (p = 0.001). Echinocandins (73.3%) were most commonly prescribed as initial treatment. The median (range) time to initial treatment was 1 (0-9) days. The 30-day in-hospital mortality rate was 49.8%. High SAPS II score (Odds ratio, OR 1.08; 95% confidence interval, CI 1.05-1.11) and renal replacement therapy (OR 5.54; CI 2.80-10.97) were independent predictors of mortality, while drain placement (OR 0.44; CI 0.19-0.99) was protective. CONCLUSIONS: Decreasing azole susceptibilities to C. tropicalis and the emergence of echinocandin resistance suggest that susceptibility patterns may no longer be sufficiently predicted by speciation in our institution. Candidemia is associated with poor outcomes. Strategies optimising antifungal therapy, especially in the critically-ill population, should be explored.

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours.

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.