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

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

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.

Oral vs Intravenous Antibiotics for Patients With Klebsiella pneumoniae Liver Abscess: A Randomized, Controlled Noninferiority Study.

BACKGROUND: Klebsiella pneumoniae liver abscess (KLA) is emerging worldwide due to hypermucoviscous strains with a propensity for metastatic infection. Treatment includes drainage and prolonged intravenous antibiotics. We aimed to determine whether oral antibiotics were noninferior to continued intravenous antibiotics for KLA. METHODS: This noninferiority, parallel group, randomized, clinical trial recruited hospitalized adults with liver abscess and K. pneumoniae isolated from blood or abscess fluid who had received ≤7 days of effective antibiotics at 3 sites in Singapore. Patients were randomized 1:1 to oral (ciprofloxacin) or intravenous (ceftriaxone) antibiotics for 28 days. If day 28 clinical response criteria were not met, further oral antibiotics were prescribed until clinical response was met. The primary endpoint was clinical cure assessed at week 12 and included a composite of absence of fever in the preceding week, C-reactive protein <20 mg/L, and reduction in abscess size. A noninferiority margin of 12% was used. RESULTS: Between November 2013 and October 2017, 152 patients (mean age, 58.7 years; 25.7% women) were recruited, following a median 5 days of effective intravenous antibiotics. A total of 106 (69.7%) underwent abscess drainage; 71/74 (95.9%) randomized to oral antibiotics met the primary endpoint compared with 72/78 (92.3%) randomized to intravenous antibiotics (risk difference, 3.6%; 2-sided 95% confidence interval, -4.9% to 12.8%). Effects were consistent in the per-protocol population. Nonfatal serious adverse events occurred in 12/72 (16.7%) in the oral group and 13/77 (16.9%) in the intravenous group. CONCLUSIONS: Oral antibiotics were noninferior to intravenous antibiotics for the early treatment of KLA. CLINICAL TRIALS REGISTRATION: NCT01723150.

Acquisition of Plasmid with Carbapenem-Resistance Gene blaKPC2 in Hypervirulent Klebsiella pneumoniae, Singapore.

The convergence of carbapenem-resistance and hypervirulence genes in Klebsiella pneumoniae has led to the emergence of highly drug-resistant superbugs capable of causing invasive disease. We analyzed 556 carbapenem-resistant K. pneumoniae isolates from patients in Singapore hospitals during 2010-2015 and discovered 18 isolates from 7 patients also harbored hypervirulence features. All isolates contained a closely related plasmid (pKPC2) harboring blaKPC-2, a K. pneumoniae carbapenemase gene, and had a hypervirulent background of capsular serotypes K1, K2, and K20. In total, 5 of 7 first patient isolates were hypermucoviscous, and 6 were virulent in mice. The pKPC2 was highly transmissible and remarkably stable, maintained in bacteria within a patient with few changes for months in the absence of antimicrobial drug selection pressure. Intrapatient isolates were also able to acquire additional antimicrobial drug resistance genes when inside human bodies. Our results highlight the potential spread of carbapenem-resistant hypervirulent K. pneumoniae in Singapore.

Genomic Study of blaIMI-Positive Enterobacter cloacae Complex in Singapore over a Five-Year Study Period.

The blaIMI gene is rarely detected outside the Enterobacter genus. Genomic characterization of 87 blaIMI-positive Enterobacter cloacae complex members revealed that the largest phylogenomic clade was made up of E. cloacae subsp. cloacae (71.3%), followed by the newly described species E. bugandensis (13.8%), E. sichuanensis (10.3%), and E. roggenkampii (4.6%). IMI-1 was the predominant carbapenemase variant (86/87, 98.9%). All the blaIMI genes were associated with chromosomally integrated Xer-dependent integrative mobile elements (IMEXs), with two new variants detected.

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.

Antecedent Carbapenem Exposure as a Risk Factor for Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae and Carbapenemase-Producing Enterobacteriaceae.

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

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

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

2015 Epidemic of Severe Streptococcus agalactiae Sequence Type 283 Infections in Singapore Associated With the Consumption of Raw Freshwater Fish: A Detailed Analysis of Clinical, Epidemiological, and Bacterial Sequencing Data.

BACKGROUND: Streptococcus agalactiae (group B Streptococcus [GBS]) has not been described as a foodborne pathogen. However, in 2015, a large outbreak of severe invasive sequence type (ST) 283 GBS infections in adults epidemiologically linked to the consumption of raw freshwater fish occurred in Singapore. We attempted to determine the scale of the outbreak, define the clinical spectrum of disease, and link the outbreak to contaminated fish. METHODS: Time-series analysis was performed on microbiology laboratory data. Food handlers and fishmongers were screened for enteric carriage of GBS. A retrospective cohort study was conducted to assess differences in demographic and clinical characteristics of patients with invasive ST283 and non-ST283 infections. Whole-genome sequencing was performed on human and fish ST283 isolates from Singapore, Thailand, and Hong Kong. RESULTS: The outbreak was estimated to have started in late January 2015. Within the study cohort of 408 patients, ST283 accounted for 35.8% of cases. Patients with ST283 infection were younger and had fewer comorbidities but were more likely to develop meningoencephalitis, septic arthritis, and spinal infection. Of 82 food handlers and fishmongers screened, none carried ST283. Culture of 43 fish samples yielded 13 ST283-positive samples. Phylogenomic analysis of 161 ST283 isolates from humans and fish revealed they formed a tight clade distinguished by 93 single-nucleotide polymorphisms. CONCLUSIONS: ST283 is a zoonotic GBS clone associated with farmed freshwater fish, capable of causing severe disease in humans. It caused a large foodborne outbreak in Singapore and poses both a regional and potentially more widespread threat.

Clinical and Molecular Epidemiology of Carbapenem-Resistant Enterobacteriaceae Among Adult Inpatients in Singapore.

BACKGROUND: Since 2010, the incidence of carbapenem-resistant Enterobacteriaceae (CRE) has been increasing in Singapore. We analyzed the clinical and molecular epidemiology of CRE among adult inpatients in Singapore. METHODS: Quarterly incidence of unique subjects (per 100000 patient-days) with positive clinical and surveillance cultures for CRE were estimated based on mandatory data submitted to the National Public Health Laboratory by public hospitals between 2010 and 2015. CRE-positive adult inpatients were prospectively recruited from 6 public sector hospitals between December 2013 and April 2015. Subjects answered a standardized epidemiologic questionnaire and provided samples for this study. Further clinical information was extracted from subjects' electronic medical records. Whole-genome sequencing was performed on study isolates to determine transmission clusters. RESULTS: Incidence of CRE clinical cultures among adult inpatients plateaued from 2013 (range: 7.73 to 10.32 per 100000 patient-days) following an initial increase between 2010 and end-2012. We prospectively recruited 249 subjects. Their median age was 65 years, 108 (43%) were female, and 161 (64.7%) had carbapenemase-producing Enterobacteriaceae (CPE). On multivariate analysis, prior carbapenem exposure (OR: 3.23; 95% CI: 1.67-6.25) and hematological malignancies (OR: 2.85; 95% CI: 1.10-7.41) were associated with non-carbapenemase-producing CRE (NCPE) (n = 88) compared with CPE (n = 161) subjects. Among 430 CRE isolates from the 249 subjects, 307(71.3%) were CPE, of which 154(50.2%) were blaKPC-positive, 97(31.6%) blaNDM-positive, and 42 (13.7%) blaOXA-positive. Klebsiella pneumoniae (n = 180, 41.9%), Escherichia coli (n = 129, 30.0%) and Enterobacter cloacae (n = 62, 14.4%) were the main Enterobacteriaceae species. WGS (n = 206) revealed diverse bacterial strain type (STs). The predominant blaKPC-positive plasmid was pHS102707 (n = 62, 55.4%) and the predominant blaNDM-positive plasmid was pNDM-ECS01 (n = 46, 48.9%). Five transmission clusters involving 13 subjects were detected. CONCLUSIONS: Clinical CRE trend among adult inpatients showed stabilization following a rapid rise since introduction in 2010 potentially due to infection prevention measures and antimicrobial stewardship. More work is needed on understanding CPE transmission dynamics.

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.

Occurrences and Characterization of Antibiotic-Resistant Bacteria and Genetic Determinants of Hospital Wastewater in a Tropical Country.

Wastewater discharged from clinical isolation and general wards at two hospitals in Singapore was examined to determine the emerging trends of antibiotic resistance (AR). We quantified the concentrations of 12 antibiotic compounds by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibiotic-resistant bacteria (ARB), the class 1 integrase gene (intI1), and 16 antibiotic resistance genes (ARGs) that confer resistance to 10 different clinically relevant antibiotics. A subset of 119 antibiotic-resistant isolates were phylogenetically classified and tested for the presence of ARGs encoding resistance to ß-lactam antibiotics (blaNDM, blaKPC, blaSHV, blaCTX-M), amikacin [aac(6')-Ib], co-trimoxazole (sul1, sul2, dfrA), ciprofloxacin (qnrA, qnrB), and the intI1 gene. Among these resistant isolates, 80.7% were detected with intI1 and 66.4% were found to carry at least 1 of the tested ARGs. Among 3 sampled locations, the clinical isolation ward had the highest concentrations of ARB and the highest levels of ARGs linked to resistance to ß-lactam (blaKPC), co-trimoxazole (sul1, sul2, dfrA), amikacin [aac(6')-Ib], ciprofloxacin (qnrA), and intI1 We found strong positive correlations (P < 0.05) between concentrations of bacteria resistant to meropenem, ceftazidime, amikacin, co-trimoxazole, and ciprofloxacin and abundances of blaKPC, aac(6')-Ib, sul1, sul2, dfrA, qnrA, and intI1 genes.

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

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

Tracking inter-institutional spread of NDM and identification of a novel NDM-positive plasmid, pSg1-NDM, using next-generation sequencing approaches.

OBJECTIVES: Owing to gene transposition and plasmid conjugation, New Delhi metallo-ß-lactamase (NDM) is typically identified among varied Enterobacteriaceae species and STs. We used WGS to characterize the chromosomal and plasmid molecular epidemiology of NDM transmission involving four institutions in Singapore. METHODS: Thirty-three Enterobacteriaceae isolates (collection years 2010-14) were sequenced using short-read sequencing-by-synthesis and analysed. Long-read single molecule, real-time sequencing (SMRTS) was used to characterize genetically a novel plasmid pSg1-NDM carried on Klebsiella pneumoniae ST147. RESULTS: In 20 (61%) isolates, blaNDM was located on the pNDM-ECS01 plasmid in the background of multiple bacterial STs, including eight K. pneumoniae STs and five Escherichia coli STs. In six (18%) isolates, a novel blaNDM-positive plasmid, pSg1-NDM, was found only in K. pneumoniae ST147. The pSg1-NDM-K. pneumoniae ST147 clone (Sg1-NDM) was fully sequenced using SMRTS. pSg1-NDM, a 90 103 bp IncR plasmid, carried genes responsible for resistance to six classes of antimicrobials. A large portion of pSg1-NDM had no significant homology to any known plasmids in GenBank. pSg1-NDM had no conjugative transfer region. Combined chromosomal-plasmid phylogenetic analysis revealed five clusters of clonal bacterial NDM-positive plasmid transmission, of which two were inter-institution clusters. The largest inter-institution cluster involved six K. pneumoniae ST147-pSg1-NDM isolates. Fifteen patients were involved in transmission clusters, of which four had ward contact, six had hospital contact and five had an unknown transmission link. CONCLUSIONS: A combined sequencing-by-synthesis and SMRTS approach can determine effectively the transmission clusters of blaNDM and genetically characterize novel plasmids. Plasmid molecular epidemiology is important to understanding NDM spread as blaNDM-positive plasmids can conjugate extensively across species and STs.

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 Activities of Ceftazidime-Avibactam, Aztreonam-Avibactam, and a Panel of Older and Contemporary Antimicrobial Agents against Carbapenemase-Producing Gram-Negative Bacilli.

Among 177 carbapenemase-producing Gram-negative bacilli (108 KPC, 32 NDM, 11 IMP, 8 OXA-48, 4 OXA-181, 2 OXA-232, 5 IMI, 4 VIM, and 3 SME producers), aztreonam-avibactam was active against all isolates except two NDM producers with elevated MICs of 8/4 and 16/4 mg/liter; ceftazidime-avibactam was active against all KPC-, IMI-, SME-, and most OXA-48 group-producing isolates (93%) but not metallo-ß-lactamase producers. Among older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.

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.

The Nod1, Nod2, and Rip2 axis contributes to host immune defense against intracellular Acinetobacter baumannii infection.

Acinetobacter baumannii is a major extensively drug-resistant lethal human nosocomial bacterium. However, the host innate immune mechanisms controlling A. baumannii are not well understood. Although viewed as an extracellular pathogen, A. baumannii can also invade and survive intracellularly. However, whether host innate immune pathways sensing intracellular bacteria contribute to immunity against A. baumannii is not known. Here, we provide evidence for the first time that intracellular antibacterial innate immune receptors Nod1 and Nod2, and their adaptor Rip2, play critical roles in the sensing and clearance of A. baumannii by human airway epithelial cells in vitro. A. baumannii infection upregulated Rip2 expression. Silencing of Nod1, Nod2, and Rip2 expression profoundly increased intracellular invasion and prolonged the multiplication and survival of A. baumannii in lung epithelial cells. Notably, the Nod1/2-Rip2 axis did not contribute to the control of A. baumannii infection of human macrophages, indicating that they play cell type-specific roles. The Nod1/2-Rip2 axis was needed for A. baumannii infection-induced activation of NF-κB but not mitogen-activated protein kinases. Moreover, the Nod1/2-Rip2 axis was critical to induce optimal cytokine and chemokine responses to A. baumannii infection. Mechanistic studies showed that the Nod1/2 pathway contributed to the innate control of A. baumannii infection through the production of ß-defensin 2 by airway epithelial cells. This study revealed new insights into the immune control of A. baumannii and may contribute to the development of effective immune therapeutics and vaccines against A. baumannii.