Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance in Escherichia coli.

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.

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.

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.

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.

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.

Whole genome sequencing of multidrug resistant Enterobacterales identified in children and their household members within Siem Reap, Cambodia.

Objectives: To explore the association of recent hospitalization and asymptomatic carriage of multidrug-resistant Enterobacterales (MDRE) and determine the prevailing strains and antibiotic resistance genes in Siem Reap, Cambodia using WGS. Methods: In this cross-sectional study, faecal samples were collected from two arms: a hospital-associated arm consisted of recently hospitalized children (2-14 years), with their family members; and a community-associated arm comprising children in the matching age group and their family members with no recent hospitalization. Forty-two families in each study arm were recruited, with 376 enrolled participants (169 adults and 207 children) and 290 stool specimens collected from participants. The DNA of ESBL- and carbapenemase-producing Enterobacterales cultured from the faecal samples was subject to WGS on the Illumina NovaSeq platform. Results: Of the 290 stool specimens, 277 Escherichia coli isolates and 130 Klebsiella spp. were identified on CHROMagar ESBL and KPC plates. The DNA of 276 E. coli (one isolate failed quality control test), 89 Klebsiella pneumoniae, 40 Klebsiella quasipneumoniae and 1 Klebsiella variicola was sequenced. CTX-M-15 was the most common ESBL gene found in E. coli (n = 104, 38%), K. pneumoniae (n = 50, 56%) and K. quasipneumoniae (n = 16, 40%). The prevalence of bacterial lineages and ESBL genes was not associated with any specific arm. Conclusions: Our results demonstrate that MDRE is likely to be endemic within the Siem Reap community. ESBL genes, specifically blaCTX-M, can be found in almost all E. coli commensals, indicating that these genes are continuously propagated in the community through various unknown channels at present.

Comparative Activities of Novel Therapeutic Agents against Molecularly Characterized Clinical Carbapenem-Resistant Enterobacterales Isolates.

Limited treatment options exist for the treatment of carbapenem-resistant Enterobacterales (CRE) bacteria. Fortunately, there are several recently approved antibiotics indicated for CRE infections. Here, we examine the in vitro activity of various novel agents (eravacycline, plazomicin, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam) and comparators (tigecycline, amikacin, levofloxacin, fosfomycin, polymyxin B) against 365 well-characterized CRE clinical isolates with various genotypes. Nonduplicate isolates collected from the largest public health hospital in Singapore between 2007 and 2020 were subjected to antimicrobial susceptibility testing (broth microdilution or antibiotic gradient test strips). Susceptibilities were defined using Clinical and Laboratory Standards Institute (CLSI) or Food and Drug Administration (FDA) interpretative criteria. Sequence types and resistance mechanisms were characterized using short-read whole-genome sequencing. Overall, tigecycline and plazomicin exhibited the highest susceptibility rates (89.6% and 80.8%, respectively). However, the tigecycline susceptibility breakpoint utilized here may be outdated in view of prevailing pharmacokinetic-pharmacodynamic (PK/PD) data. Susceptibility varied by carbapenemase genotype; the ß-lactam/ß-lactamase inhibitor combinations were equally active (92.3 to 99.2% susceptible) against KPC producers, but only ceftazidime-avibactam retained high susceptibility (98.7%) against OXA-48-like producers. Against metallo-ß-lactamase producers, only plazomicin exhibited moderate activity (77.0% susceptible). Aminoglycoside activity was also influenced by carbapenemase genotypes. This work provides an insight into the comparative activity and presumptive utility of novel agents in this geographic region. IMPORTANCE This study determined the susceptibilities of carbapenem-resistant Enterobacterales isolates to various novel antimicrobial agents (ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, eravacycline, and plazomicin). Whole-genome sequencing was performed for all strains. Our study findings provide insights into the comparative activities of novel agents in this geographic region. Plazomicin and ceftazidime-avibactam exhibited the lowest nonsusceptibility rates and may be considered promising agents in the management of carbapenem-resistant Enterobacterales infections. We note also that antibiotic activity is influenced by genotypes and that understanding the geographic region's molecular epidemiology could aid in the definition of the presumptive utility of novel agents and contribute to antibiotic decision-making.

Genomic Surveillance of Carbapenem-Resistant Klebsiella pneumoniae from a Major Public Health Hospital in Singapore.

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 (n = 497, 86.4%), and KPC was the predominant carbapenemase (n = 235, 40.9%), followed by OXA-48-like (n = 128, 22.3%) and NDM (n = 93, 16.2%). Hypervirulence was detected in 59 (10.3%) isolates and was most common in the ST231 carbapenemase-producing isolates (21/59, 35.6%). Carbapenemase genes were associated with diverse plasmid replicons; however, there was an association of blaOXA-181/232 with ColKP3 plasmids. This study presents the complex and diverse epidemiology of the CRKP strains circulating in Singapore. Our study highlights the utility of WGS-based genomic surveillance in tracking the population dynamics of CRKP. IMPORTANCE In this study, we characterized carbapenem-resistant Klebsiella pneumoniae clinical isolates collected over a 12-year period in the largest public acute-care hospital in Singapore using whole-genome sequencing. The results of this study demonstrate significant genomic diversity with the presence of well-known epidemic, multidrug-resistant clones amid a diverse pool of nonepidemic lineages. Genomic surveillance involving comprehensive resistance, virulence, and plasmid gene content profiling provided critical information for antimicrobial resistance monitoring and highlighted future surveillance priorities, such as the emergence of ST231 K. pneumoniae strains bearing multidrug resistance, virulence elements, and the potential plasmid-mediated transmission of the blaOXA-48-like gene. The findings here also reinforce the necessity of unique infection control and prevention strategies that take the genomic diversity of local circulating strains into consideration.

Ceftolozane/Tazobactam Resistance and Mechanisms in Carbapenem-Nonsusceptible Pseudomonas aeruginosa.

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 ß-lactamases, especially metallo-ß-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 ß-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.IMPORTANCEPseudomonas 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 ß-lactam/ß-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 ß-lactams and ß-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.

In vitro Bactericidal Activities of Combination Antibiotic Therapies Against Carbapenem-Resistant Klebsiella pneumoniae With Different Carbapenemases and Sequence Types.

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 ß-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 ß-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.

Genomic characterization of carbapenem-non-susceptible Pseudomonas aeruginosa in Singapore.

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-ß-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.

Candida auris in Singapore: Genomic epidemiology, antifungal drug resistance, and identification using the updated 8.01 VITEKⓇ2 system.

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.

Do antimicrobial stewardship programme interventions reduce the rate of and protect against Clostridium difficile infection?

OBJECTIVES: 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. METHODS: 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. RESULTS: Whilst the median duration of antibiotic therapy was reduced by 2days (6days vs. 4 days; P<0.001), 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. CONCLUSION: 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.

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

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