LipidA-IDER to Explore the Global Lipid A Repertoire of Drug-Resistant Gram-Negative Bacteria.

With the global emergence of drug-resistant bacteria causing difficult-to-treat infections, there is an urgent need for a tool to facilitate studies on key virulence and antimicrobial resistant factors. Mass spectrometry (MS) has contributed substantially to the elucidation of the structure-function relationships of lipid A, the endotoxic component of lipopolysaccharide which also serves as an important protective barrier against antimicrobials. Here, we present LipidA-IDER, an automated structure annotation tool for system-level scale identification of lipid A from high-resolution tandem mass spectrometry (MS2) data. LipidA-IDER was validated against previously reported structures of lipid A in the reference bacteria, Escherichia coli and Pseudomonas aeruginosa. Using MS2 data of variable quality, we demonstrated LipidA-IDER annotated lipid A with a performance of 71.2% specificity and 70.9% sensitivity, offering greater accuracy than existing lipidomics software. The organism-independent workflow was further applied to a panel of six bacterial species: E. coli and Gram-negative members of ESKAPE pathogens. A comprehensive atlas comprising 188 distinct lipid A species, including remodeling intermediates, was generated and can be integrated with software including MS-DIAL and Metabokit for identification and semiquantitation. Systematic comparison of a pair of polymyxin-sensitive and polymyxin-resistant Acinetobacter baumannii isolated from a human patient unraveled multiple key lipid A structural features of polymyxin resistance within a single analysis. Probing the lipid A landscape of bacteria using LipidA-IDER thus holds immense potential for advancing our understanding of the vast diversity and structural complexity of a key lipid virulence and antimicrobial-resistant factor. LipidA-IDER is freely available at https://github.com/Systems-Biology-Of-Lipid-Metabolism-Lab/LipidA-IDER.

In Vitro Pharmacodynamics of Fosfomycin against Carbapenem-Resistant Enterobacter cloacae and Klebsiella aerogenes.

The increase of carbapenem-resistant Enterobacterales (CRE) and lack of therapeutic options due to the scarcity of new antibiotics has sparked interest toward the use of intravenous fosfomycin against systemic CRE infections. We aimed to investigate the in vitro pharmacodynamics of fosfomycin against carbapenem-resistant Enterobacter cloacae and Klebsiella aerogenes Time-kill studies and population analysis profiles were performed with eight clinical CRE isolates, which were exposed to fosfomycin concentrations ranging from 0.25 to 2,048 mg/liter. The 24-h mean killing effect was characterized by an inhibitory sigmoid maximum effect (Emax) model. Whole-genome sequencing was performed to elucidate known fosfomycin resistance mechanisms. Fosfomycin MICs ranged from 0.5 to 64 mg/liter. The isolates harbored a variety of carbapenemase genes including blaIMP, blaKPC, and blaNDM Five out of eight isolates harbored the fosA gene, while none harbored the recently discovered fosL-like gene. Heteroresistant subpopulations were detected in all isolates, with two out of eight isolates harboring heteroresistant subpopulations at up to 2,048 mg/liter. In time-kill studies, fosfomycin exhibited bactericidal activity at 2 to 4 h at several fosfomycin concentrations (one isolate at ≥16 mg/liter, two at ≥32 mg/liter, two at ≥64 mg/liter, two at ≥128 mg/liter, and one at ≥512 mg/liter). At 24 h, bactericidal activity was only observed in two isolates (MICs, 0.5 and 4 mg/liter) at 2,048 mg/liter. From the Emax model, no significant bacterial killing was observed beyond 500 mg/liter. Our findings suggest that the use of fosfomycin monotherapy may be limited against CRE due to heteroresistance and rapid bacterial regrowth. Further optimization of intravenous fosfomycin dosing regimens is required to increase efficacy against such infections.

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.

Molecular mechanisms of azole resistance in Candida bloodstream isolates.

BACKGROUND: Antifungal resistance rates are increasing. We investigated the mechanisms of azole resistance of Candida spp. bloodstream isolates obtained from a surveillance study conducted between 2012 and 2015. METHODS: Twenty-six azole non-susceptible Candida spp. clinical isolates were investigated. Antifungal susceptibilities were determined using the Sensititre YeastOne® YO10 panel. The ERG11 gene was amplified and sequenced to identify amino acid polymorphisms, while real-time PCR was utilised to investigate the expression levels of ERG11, CDR1, CDR2 and MDR1. RESULTS: Azole cross-resistance was detected in all except two isolates. Amino acid substitutions (A114S, Y257H, E266D, and V488I) were observed in all four C. albicans tested. Of the 17 C. tropicalis isolates, eight (47%) had ERG11 substitutions, of which concurrent observation of Y132F and S154F was the most common. A novel substitution (I166S) was detected in two of the five C. glabrata isolates. Expression levels of the various genes differed between the species but CDR1 and CDR2 overexpression appeared to be more prominent in C. glabrata. CONCLUSIONS: There was interplay of various different mechanisms, including mechanisms which were not studied here, responsible for azole resistance in Candida spp in our study.

In vitro Pharmacodynamics and PK/PD in Animals.

In the last decade, considerable advancements have been made to identify the pharmacokinetic/pharmacodynamic (PK/PD) index that defines the antimicrobial activity of polymyxins. Dose-fractionation studies performed in hollow-fiber models found that altering the dosing schedule had little impact on the killing or suppression of resistance emergence, alluding to AUC/MIC as the pharmacodynamic index that best describes polymyxin's activity. For in vivo efficacy, the PK/PD index that was the most predictive of the antibacterial effect of colistin against P. aeruginosa and A. baumannii was ƒAUC/MIC.

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.

Evaluating Polymyxin B-Based Combinations against Carbapenem-Resistant Escherichia coli in Time-Kill Studies and in a Hollow-Fiber Infection Model.

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 log10 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 log10 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.

In Vitro Activity of Polymyxin B in Combination with Various Antibiotics against Extensively Drug-Resistant Enterobacter cloacae with Decreased Susceptibility to Polymyxin B.

Against extensively drug-resistant (XDR) Enterobacter cloacae, combination antibiotic therapy may be the only option. We investigated the activity of various antibiotics in combination with polymyxin B using time-kill studies (TKS). TKS were conducted with four nonclonal XDR E. cloacae isolates with 5 log10 CFU/ml bacteria against maximum, clinically achievable concentrations of polymyxin B alone and in two-drug combinations with 10 different antibiotics. A hollow-fiber infection model (HFIM) simulating clinically relevant polymyxin B and tigecycline dosing regimens was conducted for two isolates over 240 h. Emergence of resistance was quantified using antibiotic-containing (3× MIC) media. Biofitness and stability of resistant phenotypes were determined. All XDR E. cloacae isolates were resistant to all antibiotics except for polymyxin B (polymyxin B MIC, 1 to 4 mg/liter). All isolates harbored metallo-ß-lactamases (two with NDM-1, two with IMP-1). In single TKS, all antibiotics alone demonstrated regrowth at 24 h, except amikacin against two strains and polymyxin B and meropenem against one strain each. In combination TKS, only polymyxin B plus tigecycline was bactericidal against all four XDR E. cloacae isolates at 24 h. In HFIM, tigecycline and polymyxin B alone did not exhibit any killing activity. Bactericidal kill was observed at 24 h for both isolates for polymyxin B plus tigecycline; killing was sustained for one isolate but regrowth was observed for the second. Phenotypically stable resistant mutants with reduced in vitro growth rates were observed. Polymyxin B plus tigecycline is a promising combination against XDR E. cloacae However, prolonged and indiscriminate use can result in resistance emergence.

Clinical Efficacy of Polymyxin Monotherapy versus Nonvalidated Polymyxin Combination Therapy versus Validated Polymyxin Combination Therapy in Extensively Drug-Resistant Gram-Negative Bacillus Infections.

Polymyxins have emerged as a last-resort treatment of extensively drug-resistant (XDR) Gram-negative Bacillus (GNB) infections, which present a growing threat. Individualized polymyxin-based antibiotic combinations selected on the basis of the results of in vitro combination testing may be required to optimize therapy. A retrospective cohort study of hospitalized patients receiving polymyxins for XDR GNB infections from 2009 to 2014 was conducted to compare the treatment outcomes between patients receiving polymyxin monotherapy (MT), nonvalidated polymyxin combination therapy (NVCT), and in vitro combination testing-validated polymyxin combination therapy (VCT). The primary and secondary outcomes were infection-related mortality and microbiological eradication, respectively. Adverse drug reactions (ADRs) between treatment groups were assessed. A total of 291 patients (patients receiving MT, n = 58; patients receiving NVCT, n = 203; patients receiving VCT, n = 30) were included. The overall infection-related mortality rate was 23.0% (67 patients). In the multivariable analysis, treatment of XDR GNB infections with MT (adjusted odds ratio [aOR], 8.49; 95% confidence interval [CI], 1.56 to 46.05) and NVCT (aOR, 5.75; 95% CI, 1.25 to 25.73) was associated with an increased risk of infection-related mortality compared to that with treatment with VCT. A higher Acute Physiological and Chronic Health Evaluation II (APACHE II) score (aOR, 1.14; 95% CI 1.07 to 1.21) and a higher Charlson comorbidity index (aOR, 1.28; 95% CI, 1.11 to 1.47) were also independently associated with an increased risk of infection-related mortality. No increase in the incidence of ADRs was observed in the VCT group. The use of an individualized antibiotic combination which was selected on the basis of the results of in vitro combination testing was associated with significantly lower rates of infection-related mortality in patients with XDR GNB infections. Future prospective randomized studies will be required to validate these findings.

Multiple Genetic Mutations Associated with Polymyxin Resistance in Acinetobacter baumannii.

We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo. All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro.

In vitro pharmacodynamics of various antibiotics in combination against extensively drug-resistant Klebsiella pneumoniae.

Extensively drug-resistant (XDR) Klebsiella pneumoniae is an emerging pathogen in Singapore. With limited therapeutic options available, combination antibiotics may be the only viable option. In this study, we aimed to elucidate effective antibiotic combinations against XDR K. pneumoniae isolates. Six NDM-1-producing and two OXA-181-producing K. pneumoniae strains were exposed to 12 antibiotics alone and in combination via time-kill studies. A hollow-fiber infection model (HFIM) with pharmacokinetic validation was used to simulate clinically relevant tigecycline-plus-meropenem dosing regimens against 2 XDR K. pneumoniae isolates over 240 h. The emergence of resistance against tigecycline was quantified using drug-free and selective (tigecycline at 3× the MIC) media. The in vitro growth rates were determined and serial passages on drug-free and selective media were carried out on resistant isolates obtained at 240 h. Both the polymyxin B and tigecycline MICs ranged from 1 to 4 mg/liter. In single time-kill studies, all antibiotics alone demonstrated regrowth at 24 h, except for polymyxin B against 2 isolates. Tigecycline plus meropenem was found to be bactericidal in 50% of the isolates. For the isolates that produced OXA-181-like carbapenemases, none of the 55 tested antibiotic combinations was bactericidal. Against 2 isolates in the HFIM, tigecycline plus meropenem achieved a >90% reduction in bacterial burden for 96 h before regrowth was observed until 10(9) CFU/ml at 240 h. Phenotypically stable and resistant isolates, which were recovered from tigecycline-supplemented plates post-HFIM studies, had lower growth rates than those of their respective parent isolates, possibly implying a substantial biofitness deficit in this population. We found that tigecycline plus meropenem may be a potential antibiotic combination for XDR K. pneumoniae infections, but its efficacy was strain specific.

Electron transport chain capacity expands yellow fever vaccine immunogenicity.

Vaccination has successfully controlled several infectious diseases although better vaccines remain desirable. Host response to vaccination studies have identified correlates of vaccine immunogenicity that could be useful to guide development and selection of future vaccines. However, it remains unclear whether these findings represent mere statistical correlations or reflect functional associations with vaccine immunogenicity. Functional associations, rather than statistical correlates, would offer mechanistic insights into vaccine-induced adaptive immunity. Through a human experimental study to test the immunomodulatory properties of metformin, an anti-diabetic drug, we chanced upon a functional determinant of neutralizing antibodies. Although vaccine viremia is a known correlate of antibody response, we found that in healthy volunteers with no detectable or low yellow fever 17D viremia, metformin-treated volunteers elicited higher neutralizing antibody titers than placebo-treated volunteers. Transcriptional and metabolomic analyses collectively showed that a brief course of metformin, started 3 days prior to YF17D vaccination and stopped at 3 days after vaccination, expanded oxidative phosphorylation and protein translation capacities. These increased capacities directly correlated with YF17D neutralizing antibody titers, with reduced reactive oxygen species response compared to placebo-treated volunteers. Our findings thus demonstrate a functional association between cellular respiration and vaccine-induced humoral immunity and suggest potential approaches to enhancing vaccine immunogenicity.

Optimizing hollow-fiber-based pharmacokinetic assay via chemical stability study to account for inaccurate simulated drug clearance of rifampicin.

With increasing multidrug resistance coupled to a poor development pipeline, clinicians are exploring antimicrobial combinations to improve treatment outcomes. In vitro hollow-fiber infection model (HFIM) is employed to simulate human in vivo drug clearance and investigate pharmacodynamic synergism of antibiotics. Our overarching aim was to optimize the HFIM-based pharmacokinetic (PK) assay by using rifampicin and polymyxin B as probe drugs. An ultrapressure liquid chromatography tandem mass spectrometry method was validated for the quantification of rifampicin and polymyxin B components. In vitro profiling studies demonstrated that the experimental PK profiles of polymyxin B monotherapy were well correlated with the human population PK data while monotherapy with rifampicin failed to achieve the expected maximum plasma concentration. Chemical stability studies confirmed polymyxin B was stable in broth at 37 °C up to 12 h while rifampicin was unstable under the same conditions over 12 and 80 h. The calculated mean clearance of rifampicin due to chemical degradation was 0.098 ml/min accounting for 12.2 % of its clinical total clearance (CL = 0.8 ml/min) based on population PK data. Our novel finding reinforces the importance to optimize HFIM-based PK assay by performing chemical stability study so as to account for potential discrepancy between experimental and population PK profiles of antimicrobial agents.

In Vitro Susceptibility to Ceftazidime-Avibactam and Comparator Antimicrobial Agents of Carbapenem-Resistant Enterobacterales Isolates.

The emergence of carbapenem-resistant Enterobacterales (CRE) has been recognized as a significant concern globally. Ceftazidime/avibactam (CZA) is a novel ß-lactam/ß-lactamase inhibitor that has demonstrated activity against isolates producing class A, C, and D ß-lactamases. Here-in, we evaluated the in vitro activity of CZA and comparator antimicrobial agents against 858 CRE isolates, arising from the Southeast Asian region, collected from a large tertiary hospital in Singapore. These CRE isolates mainly comprised Klebsiella pneumoniae (50.5%), Escherichia coli (29.4%), and Enterobacter cloacae complex (17.1%). Susceptibility rates to levofloxacin, imipenem, meropenem, doripenem, aztreonam, piperacillin/tazobactam, cefepime, tigecycline, and polymyxin B were low. CZA was the most active ß-lactam agent against 68.9% of the studied isolates, while amikacin was the most active agent among all comparator antibiotics (80% susceptibility). More than half of the studied isolates (51.4%) identified were Klebsiella pneumoniae carbapenemase (KPC)-2 producers, 25.9% were New Delhi metallo-ß-lactamase (NDM) producers, and Oxacillinase (OXA)-48-like producers made up 10.7%. CZA was the most active ß-lactam agent against KPC-2, OXA-48-like, and Imipenemase (IMI) producers (99.3% susceptible; MIC50/90: ≤1/2 mg/L). CZA had excellent activity against the non-carbapenemase-producing CRE (91.4% susceptible; MIC50/90: ≤1/8 mg/L). Expectedly, CZA had no activity against the metallo-ß-lactamases (MBL)-producing CRE (NDM- and Imipenemase MBL (IMP) producers; 27.2% isolates), and the carbapenemase co-producing CRE (NDM + KPC, NDM + OXA-48-like, NDM + IMP; 3.0% isolates). CZA is a promising addition to our limited armamentarium against CRE infections, given the reasonably high susceptibility rates against these CRE isolates. Careful stewardship and rational dosing regimens are required to preserve CZA's utility against CRE infections.

Predictors and Outcomes of Healthcare-Associated Infections Caused by Carbapenem-Nonsusceptible Enterobacterales: A Parallel Matched Case-Control Study.

Objectives: The increasing incidence of carbapenem-nonsusceptible Enterobacterales as major pathogens in healthcare associated infections (HAIs) is of paramount concern. To implement effective prevention strategies against carbapenem-nonsusceptible Enterobacterales (CnSE) HAIs, it is crucial to identify modifiable factors associated with these infections. We identified risk factors for CnSE-HAIs, and compared clinical outcomes of CnSE-HAI and carbapenem-sensitive Enterobacterales (CSE)-HAI patients. Methods: We conducted a multi-centre parallel matched case-control study in two 1700-bedded Singapore acute-care hospitals from 2014-2016. Patients with CnSE-HAIs and CSE-HAIs were compared to a common control group without HAIs (1:1:3 ratio), matched by time-at-risk and patient ward. Carbapenem nonsusceptible was defined as non-susceptibility to either meropenem or imipenem. Presence of healthcare associated infections were defined by the criteria provided by the European Centre for Disease Prevention and Control. Outcomes of CnSE-HAI and CSE-HAI patients were compared using multivariable logistic and cox regression; the models were adjusted for infection and treatment characteristics. Results: Eighty CnSE-HAI and 80 CSE-HAI patients were matched to 240 patients without HAIs. All CRE-HAIs patients had prior antibiotic exposure, with 44 (55.0%) with prior carbapenem exposure. The most common CnSE-HAIs were intra-abdominal infections (28.8%) and pneumonia (23.8%). The most common CnSE species was Klebsiella spp. (63.8%). In the risk factor analysis, presence of drainage devices [adjusted odds ratio (aOR), 2.19; 95% CI, 1.29 - 3.70] and prior carbapenem exposure (aOR,17.09; 95% CI, 3.06 - 95.43) independently predicted CnSE-HAIs. In the crude outcomes analysis, CnSE-HAI patients had higher all-cause in-hospital mortality and longer time to discharge compared to CSE-HAI patients. After adjusting for differences in receipt of antibiotics with reported susceptibility to the Enterobacterales, there was no significant difference in all-cause in-hospital mortality between the two groups (aOR, 1.76; 95% CI, 0.86-3.58). Time to discharge remained significantly longer in patients with CnSE-HAI (adjusted hazard ratio, 0.71; 95% CI, 0.51 - 0.98) after adjusting for disease severity, receipt of antibiotics with reported susceptibility and receipt of appropriate source control. Conclusion: Appropriate management of deep-seated Enterobacterales infections and reducing exposure to carbapenems may reduce risk of CnSE-HAIs in Singapore. Efforts to improve antimicrobial therapy in CnSE-HAI patients may improve patient outcomes.

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS.

Fosfomycin-based combination therapy has emerged as an attractive option in our armamentarium due to its synergistic activity against carbapenem-resistant Gram-negative bacteria (CRGNB). The ability to simultaneously measure fosfomycin and other antibiotic drug levels will support in vitro and clinical investigations to develop rational antibiotic combination dosing regimens against CRGNB infections. We developed an analytical assay to measure fosfomycin with nine important antibiotics in human plasma and cation-adjusted Mueller-Hinton II broth (CAMHB). We employed a liquid-chromatography tandem mass spectrometry method and validated the method based on accuracy, precision, matrix effect, limit-of-detection, limit-of-quantification, specificity, carryover, and short-term and long-term stability on U.S. Food & Drug Administration (FDA) guidelines. Assay feasibility was assessed in a pilot clinical study in four patients on antibiotic combination therapy. Simultaneous quantification of fosfomycin, levofloxacin, meropenem, doripenem, aztreonam, piperacillin/tazobactam, ceftolozane/tazobactam, ceftazidime/avibactam, cefepime, and tigecycline in plasma and CAMHB were achieved within 4.5 min. Precision, accuracy, specificity, and carryover were within FDA guidelines. Fosfomycin combined with any of the nine antibiotics were stable in plasma and CAMHB up to 4 weeks at -80 °C. The assay identified and quantified the respective antibiotics administered in the four subjects. Our assay can be a valuable tool for in vitro and clinical applications.

Therapeutic drug monitoring of meropenem and piperacillin-tazobactam in the Singapore critically ill population - A prospective, multi-center, observational study (BLAST 1).

PURPOSE: To determine percentage of patients with sub-therapeutic beta-lactam exposure in our intensive care units (ICU) and to correlate target attainment with clinical outcomes. MATERIALS AND METHODS: Multi-centre, prospective, observational study was conducted in ICUs from three hospitals in Singapore from July 2016 to May 2018. Adult patients (≥21 years) receiving meropenem or piperacillin-tazobactam were included. Four blood samples were obtained during a dosing interval to measure and determine attainment of therapeutic targets: unbound beta-lactam concentration above (i) minimum inhibitory concentration (MIC) at 40% (meropenem) or 50% (piperacillin) of dosing interval (40-50%fT > MIC) and (ii) 5 × MIC at 100% of dosing interval (100%fT > 5 × MIC). Correlation to clinical outcomes was evaluated using Cox regression. RESULTS: Beta-lactam levels were highly variable among 61 patients, with trough meropenem and piperacillin levels at 21.5 ± 16.8 mg/L and 101.6 ± 81.1 mg/L respectively. Among 85 sets of blood samples, current dosing practices were able to achieve 94% success for 40-50%fT > MIC and 44% for 100%fT > 5 × MIC. Failure to achieve 40-50%fT > MIC within 48 h was significantly associated with all-cause mortality (HR: 9.0, 95% CI: 1.8-45.0), after adjustment for APACHE II score. Achievement of 100%fT > 5 × MIC within 48 h was significantly associated with shorter length of hospital stay. CONCLUSION: Current dosing practices may be suboptimal for ICU patients. Beta-lactam TDM may be useful.

In vitro antibiotic synergy in extensively drug-resistant Acinetobacter baumannii: the effect of testing by time-kill, checkerboard, and Etest methods.

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.