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Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a global public health threat. In this study, we employed whole-genome sequencing (WGS) to determine the genomic epidemiology of a longitudinal collection of clinical CRKP isolates recovered from a large public acute care hospital in Singapore. Phylogenetic analyses, a characterization of resistance and virulence determinants, and plasmid profiling were performed for 575 unique CRKP isolates collected between 2009 and 2020. The phylogenetic analyses identified the presence of global high-risk clones among the CRKP population (clonal group [CG] 14/15, CG17/20, CG147, CG258, and sequence type [ST] 231), and these clones constituted 50% of the isolates. Carbapenemase production was common (

Pseudomonas aeruginosa is a clinically important pathogen implicated in many hospital-acquired infections. Its propensity to acquire broad-spectrum resistance has earned the organism its status as a severe public health threat requiring urgent control measures. While whole-genome sequencing-based genomic surveillance provides a means to track antimicrobial resistance, its use in molecular epidemiological surveys of P. aeruginosa remains limited, especially in the Southeast Asian region. We sequenced the whole genomes of 222 carbapenem-non-susceptible P. aeruginosa (CNPA) isolates collected in 2006–2020 at the largest public acute care hospital in Singapore. Antimicrobial susceptibilities were determined using broth microdilution. Clonal relatedness, multi-locus sequence types, and antimicrobial resistance determinants (acquired and chromosomal) were determined. In this study, CNPA exhibited broad-spectrum resistance (87.8% multi-drug resistance), retaining susceptibility only to polymyxin B (95.0%) and amikacin (55.0%). Carbapenemases were detected in 51.4% of the isolates, where IMP and NDM metallo-β-lactamases were the most frequent. Carbapenem resistance was also likely associated with OprD alterations or efflux mechanisms (ArmZ/NalD mutations), which occurred in strains with or without carbapenemases. The population of CNPA in the hospital was diverse; the 222 isolates grouped into 68 sequence types (ST), which included various high-risk clones. We detected an emerging clone, the NDM-1-producing ST308, in addition to the global high-risk ST235 clone which was the predominant clone in our population. Our results thus provide a “snapshot” of the circulating lineages of CNPA locally and the prevailing genetic mechanisms contributing to carbapenem resistance. This database also serves as the baseline for future prospective surveillance studies.

Objectives Candida auris (C. auris) has globally emerged as a multidrug-resistant pathogen. While it is known that there are four geographic clades, little is known about its genomic epidemiology in the Southeast Asian region. Laboratory identification can be challenging but the VITEKⓇ2 system (version 8.01 software) has recently updated its database to include C. auris. This study aimed to investigate the genomic epidemiology of C. auris isolated in Singapore and the susceptibility profiles in relation to ERG11 and FKS1 mutations. Methods Seven C. auris isolates from 2012–2018 were analysed using whole-genome sequencing, and antifungal susceptibility testing was performed. The performance of the updated VITEKⓇ2 system in identifying C. auris was also evaluated using these C. auris strains together with five closely related Candida species. Results Three clades were identified: South Asian (71.4%), South American (14.3%) and East Asian (14.3%). Local transmission was unlikely as there was no obviously identified cluster and most cases were likely to be imported at different time points following overseas hospitalisation exposure. Three isolates (42.9%) were multidrug-resistant. All South Asian strains were resistant to fluconazole and harboured ERG11 mutations, which were clade-specific. No FKS1 mutation was detected. The VITEKⓇ2 system was able to correctly identify most of the South Asian C. auris strains but misidentified the East Asian strain and gave a low discrimination result for the South American clade. Conclusion This study showed that the introduction of C. auris into Singapore was possibly over multiple episodes and from different sources. The VITEKⓇ2 System version 8.01 software has limited abilities in identifying C .auris.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is becoming increasingly problematic due to the limited effectiveness of new antimicrobials or other factors such as treatment cost. Thus, combination therapy remains a suitable treatment option. We aimed to evaluate the in vitro bactericidal activity of various antibiotic combinations against CRKP with different carbapenemase genotypes and sequence types (STs). Thirty-seven CRKP with various STs and carbapenemases were exposed to 11 antibiotic combinations (polymyxin B or tigecycline in combination with β-lactams including aztreonam, cefepime, piperacillin/tazobactam, doripenem, meropenem, and polymyxin B with tigecycline) in static time-kill studies (TKS) using clinically achievable concentrations. Out of the 407 isolate-combination pairs, only 146 (35.8%) were bactericidal (≥3 log10CFU/mL decrease from initial inoculum). Polymyxin B in combination with doripenem, meropenem, or cefepime was the most active, each demonstrating bactericidal activity in 27, 24, and 24 out of 37 isolates, respectively. Tigecycline in combination with β-lactams was rarely bactericidal. Aside from the lower frequency of bactericidal activity in the dual-carbapenemase producers, there was no apparent difference in combination activity among the strains with other carbapenemase types. In addition, bactericidal combinations were varied even in strains with similar STs, carbapenemases, and other genomic characteristics. Our findings demonstrate that the bactericidal activity of antibiotic combinations is highly strain-specific likely owing to the complex interplay of carbapenem-resistance mechanisms, i.e., carbapenemase genotype alone cannot predict in vitro bactericidal activity. The availability of WGS information can help rationalize the activity of certain combinations. Further studies should explore the use of genomic markers with phenotypic information to predict combination activity.

Antimicrobial stewardship programmes (ASPs) have often been recommended as a viable solution to minimise the incidence of Clostridium difficile infection (CDI), which can be life-threatening. This study aimed to evaluate whether ASP interventions have contributed to reducing CDI rates.A retrospective review of ASP interventions issued from January 2013 to April 2014 was performed using data from the ASP database of Singapore General Hospital, a 1600-bed tertiary-care hospital in Singapore. A total of 283 interventions satisfied the inclusion criteria, of which commonly audited antibiotics were piperacillin/tazobactam (41.3%) and carbapenems (54.8%). Comparisons were made at 30days post-intervention between those with accepted or rejected interventions. The primary outcome was CDI incidence; secondary outcomes included length of hospitalisation post-intervention, 30-day mortality and CDI recurrence rate.Whilst the median duration of antibiotic therapy was reduced by 2days (6days vs. 4 days; P0.001), acceptance of ASP interventions did not alter primary CDI incidence at 30days (P=0.644) post-intervention. However, reduced CDI recurrence rates were observed for patients positive for CDI in the accepted patient group compared with the rejected group (0% vs. 37.5%; P=0.03), with no difference in CDI 30-day mortality between the two groups.Intervention acceptance did not contribute to a significant reduction in CDI incidence but may be associated with lower recurrence rates, although further studies are required.

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.

Pseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel β-lactam/β-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections., This study established the in vitro activity of ceftolozane/tazobactam (C/T) and its genotypic resistance mechanisms by whole-genome sequencing (WGS) in 195 carbapenem-nonsusceptible Pseudomonas aeruginosa (CNSPA) clinical isolates recovered from Singapore between 2009 and 2020. C/T susceptibility rates were low, at 37.9%. Cross-resistance to ceftazidime/avibactam was observed, although susceptibility to the agent was slightly higher, at 41.0%. Whole-genome sequencing revealed that C/T resistance was largely mediated by the presence of horizontally acquired β-lactamases, especially metallo-β-lactamases. These were primarily disseminated in well-recognized high-risk clones belonging to sequence types (ST) 235, 308, and 179. C/T resistance was also observed in several non-carbapenemase-producing isolates, in which resistance was likely mediated by β-lactamases and, to a smaller extent, mutations in AmpC-related genes. There was no obvious mechanism of resistance observed in five isolates. The high C/T resistance highlights the limited utility of the agent as an empirical agent in our setting. Knowledge of local molecular epidemiology is crucial in determining the potential of therapy with novel agents. IMPORTANCE Pseudomonas aeruginosa infection is one of the most difficult health care-associated infections to treat due to the ability of the organism to acquire a multitude of resistance mechanisms and express the multidrug resistance phenotype. Ceftolozane/tazobactam (C/T), a novel β-lactam/β-lactamase inhibitor combination, addresses an unmet medical need in patients with these multidrug-resistant P. aeruginosa infections. Our findings demonstrate geographical variation in C/T susceptibility owing to the distinct local molecular epidemiology. This study adds on to the growing knowledge of C/T resistance, particularly mutational resistance, and will aid in the design of future β-lactams and β-lactamase inhibitors. WGS proved to be a useful tool to understand the P. aeruginosa resistome and its contribution to emerging resistance in novel antimicrobial agents.

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 (E(max)) 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 bla(IMP), bla(KPC), and bla(NDM). 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 E(max) 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.

Population pharmacokinetic studies have suggested that high polymyxin B (PMB) doses (&ge, 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 &ge, 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&ndash, 34,884 IU/kg/day) and 14 days (IQR, 7&ndash, 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&ndash, 13 days). Higher daily PMB dose (aOR,1.01, 95% CI, 1.00&ndash, 1.02) and higher number of concurrent nephrotoxins (aOR, 2.14, 95% CI, 1.03&ndash, 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.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli., Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans that recognize bacterial metabolites. This study shows that MAIT cells exert potent antimicrobial activity against both cell-associated and extracellular forms of Escherichia coli, including strains that are resistant to the last resort antibiotics carbapenems.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans, and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against E. coli clinical strains in a manner dependent on the activity of cytolytic proteins, but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to TCR-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living E. coli. Furthermore, MAIT cell-mediated bacterial control extend to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity, and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli.One Sentence SummaryPotent antimicrobial activity of human MAIT cells overcomes carbapenem-resistance in control of Escherichia coli

Tze-Peng Lim,1,2 Reyna Wang,1 Gang Quan Poh,3 Tse-Hsien Koh,4 Thean-Yen Tan,5 Winnie Lee,1 Jocelyn Qi-Min Teo,1 Yiying Cai,1 Thuan-Tong Tan,6 Pui Lai Rachel Ee,3 Andrea L Kwa1,3,7 1Department of Pharmacy, Singapore General Hospital, Singapore, Singapore; 2SingHealth Duke-NUS Medicine Academic Clinical Programme, Singapore, Singapore; 3Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore, Singapore; 4Department of Microbiology, Singapore General Hospital, Singapore, Singapore; 5Department of Laboratory Medicine, Changi General Hospital, Singapore, Singapore; 6Department of Infectious Diseases, Singapore General Hospital, Singapore, Singapore; 7Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore Objectives: Treatment for nosocomial bloodstream infections (BSI) caused by multidrug-resistant (MDR) Gram-negative bacteria (GNB) is challenging. Rising antimicrobial resistance, especially in extended spectrum beta-lactamase production, inadvertently increases empiric carbapenem consumption. Three antipseudomonal carbapenems (imipenem, meropenem [MER], and doripenem [DOR]) are available commercially against MDR GNB in Singapore. The study aims to determine the most optimal empiric carbapenem dosing regimens (CDR) and evaluate their cost-effectiveness for GNB-BSI in the face of increasing MDR GNB. Methods: Carbapenem minimum inhibitory concentrations (MICs) were generated for non-repeat GNB-BSI obtained in 2013–2014 from two hospitals. Monte Carlo simulations were used to assess the cumulative fraction of response (CFR) of various CDRs using the percentage of time above MIC for 40% (%T>MIC of 40%) as the pharmacokinetic (PK)–pharmacodynamic (PD) parameter for efficacy. Carbapenem costs were based on patient antibiotic costs. Antibiotic cost-effectiveness was calculated as total daily drug cost/CFR. Results: A total of 1,140 bloodstream isolates were collected. They comprised 116 Acinetobacter baumannii, 237 Pseudomonas aeruginosa, and 787 Enterobacteriaceae. All CDRs achieved ~40, ~80, and ≥90% CFRs against A. baumannii, P. aeruginosa, and Enterobacteriaceae, respectively. Against P. aeruginosa, MER 2g every 8h infused over 3h and DOR 1g every 8h infused over 4h achieved CFRs 84 and 81%, respectively. Against Enterobacteriaceae, the cost of MER 2g every 8h infused over 3h was the lowest among the three carbapenems at $0.40/percentage of CFR. Conclusion: This study demonstrates the utility of PK–PD modeling to formulate the optimal selection of a cost-effective empiric CDR in antibiotics guidelines and formulary inclusion. The findings support the selection of high MER doses of prolonged infusions as empiric coverage for GNB-BSI in our institutions. Keywords: empiric carbapenem regimens, multidrug resistant, Gram-negative bacteria, bloodstream infections

Objectives In patients with a history of carbapenemase-producing, carbapenem-resistant Enterobacteriaceae (CP-CRE), the need for CP-CRE targeted treatment in subsequent sepsis episodes is unclear. This study aimed to characterise the incidence of subsequent CP-CRE infective episodes in individuals with prior CP-CRE colonisation and/or infection, and identify predictors for these subsequent CP-CRE infections. Methods All adult inpatients with CP-CRE detected from any site between June 2012 and May 2014 at a tertiary-care hospital were prospectively followed for two years to assess for any subsequent CP-CRE infections. Potential factors to which patients were exposed during the follow-up period were collected from medical records and analysed. Results A total of 171 patients were enrolled. Of 151 patients who entered the follow-up period, 16 (10.6%) developed a subsequent CP-CRE infection. The median time to a subsequent infective episode was 24.5 days (12–105 days). The type of carbapenemase was highly conserved within index and subsequent paired episodes (16 of 17 pairs). Patients with first CP-CRE isolated from intra-abdominal or respiratory sources were ≥7 times more likely to develop a subsequent infection, while most rectal carriers remain colonised. For carriers (n = 133), Klebsiella spp. (OR 4.7) and OXA carbapenemase (OR 9.4) were significant predictors of subsequent infection. In patients with initial infection (n = 18), end-stage renal failure requiring dialysis (OR 22.0) was the only predisposing factor. Conclusion The incidence of subsequent infections in patients with prior colonisation was low. Consideration for CP-CRE targeted therapy is recommended in patients on dialysis and previous CP-CRE infections involving the bloodstream and/or respiratory tract.

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 log(10) 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.

Candida auris (C. auris) has globally emerged as a multidrug-resistant pathogen. While it is known that there are four geographic clades, little is known about its genomic epidemiology in the Southeast Asian region. Laboratory identification can be challenging but the VITEKSeven C. auris isolates from 2012-2018 were analysed using whole-genome sequencing, and antifungal susceptibility testing was performed. The performance of the updated VITEKThree clades were identified: South Asian (71.4%), South American (14.3%) and East Asian (14.3%). Local transmission was unlikely as there was no obviously identified cluster and most cases were likely to be imported at different time points following overseas hospitalisation exposure. Three isolates (42.9%) were multidrug-resistant. All South Asian strains were resistant to fluconazole and harboured ERG11 mutations, which were clade-specific. No FKS1 mutation was detected. The VITEKThis study showed that the introduction of C. auris into Singapore was possibly over multiple episodes and from different sources. The VITEK

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.

The original article [1] contains an error whereby the author, Pui Lai Rachel Ee’s name is incorrectly presented.

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 &deg, C, correlation to viable counts was significantly higher compared to untreated samples (p &lt, 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p &lt, 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.

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 log(10) 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 (T(RLU)) 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 T(RLU) 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.

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.

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.

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.

Background In patients with history of carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CPCRE), the need for CPCRE targeted treatment in subsequent sepsis episodes is unclear. We determine the likelihood of CPCRE infection (CI) in patients previously colonized (PC) or infected (PI) with CPCRE and relatedness of both episodes. Methods Adult inpatients with CPCRE isolated from any site in June 2012–May 2014 at a tertiary-care hospital were prospectively followed for 2 years to assess for subsequent CI. Bacteria isolates from paired episodes were subjected to Illumina HiSeq2500 and multilocus sequence typing. Results Six of 25 (24%) PI and 11 of 152 (7%) PC patients had subsequent CI—overall incidence was 9.6%. KP was most commonly implicated. While bacteria species differed in four cases, the carbapenemase type was conserved in all but one. Those with initial bacteremia, intra-abdominal (IA) or lung infection (n = 6) were five times more likely to develop CI. Only 33% of PI vs. 62% of PC patients had subsequent infections of the same clonal group. For PC, KP (OR 9.3) and OXA carbapenemase (OR 12.8) significantly predicted for subsequent CI. In PI, chronic renal failure requiring dialysis (OR 70.2) and KPC enzyme (OR 14) were predisposing factors. In-hospital mortality was observed in six cases. Initial Subsequent Time (Days) Site Bacteria Gene Site Bacteria Gene U KP OXA-48 B KP OXA-48 26 IA EC KPC-2 L KP OXA-1; KPC-2 17 B KP KPC-2, ;OXA-1 B KP KPC-2; OXA-1 8 S KP OXA-48 S KP OXA-48 146 L KP KPC-2 B KP KPC-2; OXA-1 91 U KP KPC-2 S KP KPC-2 45 ECO KPC-2 R KP OXA-1 IA KP OXA-1 6 L KP KPC-2 B KP KPC-2 82 L KP OXA-1; KPC-2 U KP KPC-2 23 R ECO KPC-2 IA ECO KPC 16 R KP NDM-1; OXA-1; OXA-181; S KP NDM-1; OXA-181; 13 S EC OXA-1; OXA-48 S ECO OXA-1; OXA-181 93 IA KP KPC-2 B ECO OXA-181 311 R KP KPC-2 U KP KPC-2; OXA-181 250 R KP KPC-2; OXA-1; OXA-9 L KP KPC 6 R KP OXA-1; OXA-181 B ECO OXA-181 5 R KP KPC-2; OXA-1 S KP KPC 367 B, blood; IA, intra-abdominal; L, lung; R, rectal; S, skin soft tissue; U, urine; EC, Enterobacter cloacae; ECO, Escherichia coli; KP, Klebsiella pneumoniae Conclusion Incidence of CI in carriers is low. Patients with IA and respiratory CI in the preceding 93 days are candidates for CPCRE treatment; empiric therapy should be active against the carbapenemase identified in the index episode. Disclosures All authors No reported disclosures.

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 log 10 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-β-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.

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

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.

Singapore is an international travel and medical hub and faces a genuine threat for import and dissemination of bacteria with broad-spectrum resistance. In this review, we described the current landscape and management of carbapenem resistance in Gram-negative bacteria (GNB) in Singapore. Notably, the number of carbapenem-resistant Enterobacteriaceae has exponentially increased in the past two years. Resistance is largely mediated by a variety of mechanisms. Polymyxin resistance has also emerged. Interestingly, two Escherichia coli isolates with plasmid-mediated mcr-1 genes have been detected. Evidently, surveillance and infection control becomes critical in the local setting where resistance is commonly related to plasmid-mediated mechanisms, such as carbapenemases. Combination antibiotic therapy has been proposed as a last-resort strategy in the treatment of extensively drug-resistant (XDR) GNB infections, and is widely adopted in Singapore. The diversity of carbapenemases encountered, however, presents complexities in both carbapenemase detection and the selection of optimal antibiotic combinations. One unique strategy introduced in Singapore is a prospective in vitro combination testing service, which aids physicians in the selection of individualized combinations. The outcome of this treatment strategy has been promising. Unlike countries with a predominant carbapenemase type, Singapore has to adopt management strategies which accounts for diversity in resistance mechanisms.

Whilst studies have shown that antimicrobial stewardship programmes (ASPs) can effectively reduce antibiotic utilisation, cost of care and even antimicrobial resistance rates, ASPs should avoid the perception that the goal is primarily to reduce antibiotic purchases and costs, instead of focusing on improving the quality of care. In addition, to address the concern of primary physicians who deemed that ASPs' choices of antibiotics were often inadequate, the impact of ASPs on patient safety should be monitored and evaluated. The aim of this study was to analyse the impact of ASP interventions on patient safety in Singapore General Hospital (SGH), a 1559-bed, large, acute, tertiary-care hospital in Singapore. A retrospective database review of data on ASP interventions issued between October 2008 and September 2010 was performed. The database maintained by the ASP team detailed patients' demographic data as well as outcomes of issued interventions. The ASP recommended 1256 interventions in a total of 1249 admissions in six departments. Shorter average length of stay (mean ± standard deviation 19.4 ± 19.9 days vs. 24.2 ± 24.2 days) was observed among patients of physicians who accepted ASP suggestions compared with patients of physicians who rejected ASP interventions (P

As data on procalcitonin utility in antibiotics discontinuation [under an antimicrobial stewardship program (ASP)] in patients with malignancies are lacking, we aimed to evaluate the utility of procalcitonin in an ASP in patients with malignancies. We conducted a retrospective review of the ASP database of all patients with malignancies in whom at least one procalcitonin level was taken and our ASP had recommended changes in carbapenem regimen, from January to December 2011. We compared clinical outcomes between two groups of patients: patients whose physicians accepted and those whose physicians rejected ASP interventions. There were 749 carbapenem cases reviewed. Ninety-nine were suggested to either de-escalate, discontinue antibiotics, or narrow the spectrum of empiric treatment, based on procalcitonin trends. While there was no statistical difference in the mortality within 30 days post-ASP intervention (accepted: 8/65 patients vs. rejected: 9/34 patients; p = 0.076), the median duration of carbapenem therapy was significantly shorter (5 vs. 7 days; p = 0.002). Procalcitonin use safely facilitates decisions on antibiotics discontinuation and de-escalation in patients with malignancies in the ASP.

Inappropriate antibiotic use contributes to antimicrobial resistance. Multi-faceted antimicrobial stewardship programmes (ASPs) are recommended for sustainable changes in prescribing practices. A multi-disciplinary ASP was established in October 2008 and piloted in the Departments of General Surgery, Renal Medicine and Endocrinology sequentially. To improve the quality of patient care via optimising the (1) choice, (2) dose, (3) route and (4) duration of antibiotics, a "whole-system" approach incorporating prospective review with immediate concurrent feedback (ICF), prescriber education (public or individualised), de-escalation of therapy, dose optimisation and parenteral-to-oral conversion, while recognising the autonomy of primary prescribers, was adopted. The audited department received a quarterly outcomes report and any common unaccepted practices would be addressed. Outcomes were analysed for 12 months post-ASP implementation. A total of 1,535 antibiotic prescriptions were reviewed. Antimicrobial use in 376 (24.5%) prescriptions was inappropriate. Of 596 interventions made, 70.2% were accepted. A reduction in audited antibiotics consumption resulted in acquisition cost savings of S$198,575 for the hospital. Patients' cost-savings attributable to ASP-initiated interventions were $91,194. The overall all-cause mortality rate and median monthly inpatient-days pre- and post-intervention remained stable. A "whole-system" ASP was effective in optimising antibiotic use in our hospital, without compromising clinical outcomes.

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 .

Limited knowledge of the local molecular epidemiology and the paucity of new effective antibiotics has resulted in an immense challenge in the control and treatment of extensively drug-resistant (XDR) Acinetobacter baumannii infections in Thailand. Antimicrobial combination regimens may be the only feasible treatment option in such cases. We sought to characterize the local molecular epidemiology and assess the bactericidal activity of various antibiotics individually and in combination against XDR A. baumannii in a Thai hospital. All XDR A. baumannii isolates from Thammasat University Hospital were collected between October 2010 and May 2011. Susceptibility testing was conducted according to reference broth dilution methods. Pulse-field gel electrophoresis was used to genotype the isolates. Carbapenemase genes were detected using polymerase chain reaction. In vitro testing of clinically-relevant concentrations of imipenem, meropenem, doripenem, rifampicin and tigecycline alone and in combination with polymyxin B was conducted using multiple combination bactericidal testing. Forty-nine polymyxin B-susceptible XDR A. baumannii isolates were identified. bla OXA-23 and bla OXA-51 genes were detected in all isolates. Eight clonally related clusters were identified, resulting in the initiation of several infection control measures. Imipenem, meropenem, doripenem, rifampicin, and tigecycline in combination with PB respectively, exhibited bactericidal killing in 100%, 100%, 98.0%, 100% and 87.8% isolates respectively at 24 hours. Molecular epidemiologic analysis can aid the early detection of infection outbreak within the institution, resulting in the rapid containment of the outbreak. Imipenem/meropenem/rifampicin in combination with polymyxin B demonstrated consistent bactericidal effect against 49 bla OXA-23-harbouring XDR A. baumannii clinical isolates, suggesting a role of combination therapy in the treatment of these infections.

The clinical advantages of prolonged (extended/continuous) infusion remain controversial. Previous studies and reviews have failed to show consistent clinical benefits of extending the infusion time. This meta-analysis sought to determine whether prolonged β-lactam infusions were associated with a reduction in mortality and improvement in clinical success. A search of PubMed, EMBASE and The Cochrane Library for randomised controlled trials (RCTs) and observational studies comparing prolonged infusion with intermittent bolus administration of the same antibiotic in hospitalised adult patients was conducted. Primary outcomes evaluated were mortality and clinical success. A total of 29 studies with 2206 patients (18 RCTs and 11 observational studies) were included in the meta-analysis. Compared with intermittent boluses, use of prolonged infusion appeared to be associated with a significant reduction in mortality [pooled relative risk (RR) = 0.66, 95% confidence interval (CI) 0.53-0.83] and improvement in clinical success (RR = 1.12, 95% CI 1.03-1.21). Statistically significant benefit was supported by non-randomised studies (mortality, RR = 0.57, 95% CI 0.43-0.76; clinical success, RR = 1.34, 95% CI 1.02-1.76) but not by RCTs (mortality, RR = 0.83, 95% CI 0.57-1.21; clinical success, RR = 1.05, 95% CI 0.99-1.12). The positive results from observational studies, especially in the face of increasing antibiotic resistance, serve to justify the imperative need to conduct a large-scale, well-designed, multicentre RCT involving critically ill patients infected with high minimum inhibitory concentration pathogens to clearly substantiate this benefit.

Introduction Combination therapy is increasingly utilized against extensively-drug resistant (XDR) Gram negative bacteria (GNB). However, choosing a combination can be problematic as effective combinations are often strain-specific. An in vitro antibiotic combination testing (iACT) service, aimed to guide the selection of individualized and rationally optimized combination regimens within 48 hours, was developed. We described the role and feasibility of the iACT service in guiding individualized antibiotic combination selection in patients with XDR-GNB infections. Methods A retrospective case review was performed in two Singapore hospitals from April 2009–June 2014. All patients with XDR-GNB and antibiotic regimen guided by iACT for clinical management were included. The feasibility and role of the prospective iACT service was evaluated. The following patient outcomes were described: (i) 30-day in-hospital all-cause and infection-related mortality, (ii) clinical response, and (iii) microbiological eradication in patients with bloodstream infections. Results From 2009–2014, the iACT service was requested by Infectious Disease physicians for 39 cases (20 P. aeruginosa, 13 A. baumannii and 6 K. pneumoniae). Bloodstream infection was the predominant infection (36%), followed by pneumonia (31%). All iACT recommendations were provided within 48h from request for the service. Prior to iACT-guided therapy, most cases were prescribed combination antibiotics empirically (90%). Changes in the empiric antibiotic regimens were recommended in 21 (54%) cases; in 14 (36%) cases, changes were recommended as the empiric regimens were found to be non-bactericidal in vitro. In 7 (18%) cases, the number of antibiotics used in combination empirically was reduced by the iACT service. Overall, low 30-day infection-related mortality (15%) and high clinical response (82%) were observed. Microbiological eradication was observed in 79% of all bloodstream infections. Conclusions The iACT service can be feasibly employed to guide the timely selection of rationally optimized combination regimens, and played a role in reducing indiscreet antibiotic use.

BackgroundIncreasing 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.MethodsA 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.ResultsTwenty-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.ConclusionPrior 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.

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.

Objective Extreme drug-resistant Pseudomonas aeruginosa (XDR-PA) with decreased susceptibility to polymyxin B (PB) has emerged in Singapore, causing infections in immunocompromised hosts. Combination therapy may be the only viable therapeutic option until new antibiotics become available. The objective of this study is to assess the in vitro activity of various antibiotics against local XDR-PA isolates. Methods PA isolates from all public hospitals in Singapore were systematically collected between 2006 and 2007. MICs were determined according to CLSI guidelines. All XDR-PA isolates identified were genotyped using a PCR-based method. Time-kill studies (TKS) were performed with approximately 105 CFU/ml at baseline using clinically achievable unbound concentrations of amikacin (A), levofloxacin (L), meropenem (M), rifampicin (R) and PB alone and in combination. Bactericidal activity (primary endpoint) was defined as a ≥3 log10 CFU/ml decrease in the colony count from the initial inoculum at 24 hours. Results 22 clinical XDR-PA isolates with PB MIC 2–16 µg/ml were collected. From clonal typing, 5 clonal groups were identified and nine isolates exhibited clonal diversity. In TKS, meropenem plus PB, amikacin plus meropenem, amikacin plus rifampicin, amikacin plus PB exhibited bactericidal activity in 8/22, 3/22, 1/22 and 6/22 isolates at 24 hours respectively. Against the remaining ten isolates where none of the dual-drug combination achieved bactericidal activity against, only the triple-antibiotic combinations of ARP and AMP achieved bactericidal activity against 7/10 and 6/10 isolates respectively. Conclusion Bactericidal activity with sustained killing effect of ≥99.9% is critical for eradicating XDR-PA infections, especially in immunocompromised hosts. These findings underscore the difficulty of developing combination therapeutic options against XDR-PA, demonstrating that at least 3 antibiotics are required in combination and that efficacy is strain dependant.