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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 coliand Pseudomonas aeruginosa. Using MS2data 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. coliand 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 baumanniiisolated 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.

Lactococcus garvieaeis a well-known fish pathogen, and in recent years, a human pathogen of increasing clinical significance. However, not much is known about the variances in characteristics of strains isolated locally and overseas. This study aims at conducting comparative genomic analysis on local and overseas L. garvieaeisolates, to further understand the phylogenetic and virulence variances between the two groups. The genomic DNA of 11 local L. garvieaeisolates (fish 6, human 5) were sequenced, annotated and typed using multi-locus sequence typing (MLST). A total of five novel sequence types (STs) were found in the local isolates. Genotypic overlapping of the STs was observed between local fish and human isolates with overseas fish, food and human clinical isolates. Thereby, suggesting a possible transmission between fish or food and humans. Virulence genes (putative internalin and putative mucus adhesin) were found to be specific to genomic clusters (GC), GC2 and GC3. A higher incidence of resistance genes was also observed in local isolates (n=8, 72.72%) when compared to the overseas isolates (n=7, 41.18%). This study represents the first evidence of genetic variances amongst local and overseas isolates, and virulence characteristics specific to the phylogeny of L. garvieae.

Gastrointestinal carriage of Klebsiella pneumoniaeis a predisposing factor for liver abscess in several Asian countries. To determine whether hypervirulent K. pneumoniaein the gut may be transmitted through food, we screened a range of raw and ready-to-eat retail food by culture and recovered K. pneumoniaein 21% (147 of 698) of samples tested. Based on PCR, no K. pneumoniaeisolates carried the rmpAgene linked to community-acquired pyogenic liver abscess, providing no evidence of a link between food and liver disease. However, phenotypic resistance to multiple antibiotic classes was seen through disk diffusion tests, and carriage of genetic elements (wcaGand capsule types K1, K2, and K54) associated with increased virulence (8%, 11 of 147) was observed by PCR. Multidrug-resistant isolates were from raw vegetables, chicken or pork liver, and a ready-to-eat poultry dish; one multidrug-resistant K. pneumoniaeisolate from raw bean sprouts was resistant to a third-generation cephalosporin (ceftriaxone). Although K. pneumoniaemay be present in food without causing harm, we found isolates belonging to the K1 capsular serotype coexisting with the wcaGgene, one also conferring multidrug resistance. K. pneumoniaethat carry antibiotic resistance genes, regardless of pathogenicity, may increase the available genetic pool of resistance along the food chain. Hygienic food handling practices are necessary to lower risks of acquiring K. pneumoniaeand other opportunistic pathogens.

Streptococcus dysgalactiaecomprise two subspecies. Typically, S. dysgalactiaesubspecies equisimilis(SDSE) are large colony β-haemolytic Group C and Group G streptococci that cause pharyngitis, skin and soft tissue infections in humans. On the other hand, S. dysgalactiaesubspecies dysgalactiae(SDSD) are classically described as α-haemolytic Group C streptococci that are animal pathogens. We compared the genome sequences of five S. dysgalactiaeisolated from four cases of bacteraemia in women with breast cancer, and one from fish meat. One human isolate was SDSE, all other isolates were SDSD. Zoonotic SDSD infection may be under-recognised because of lack of patient and clinician awareness, and failure to distinguish SDSD from SDSE in the routine lab. The possibility of zoonotic SDSD should be suspected in patients with bacteraemia and ascending cellulitis of the upper limb with a history of handling raw fish and meat.

ABSTRACTWastewater 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 β-lactam antibiotics (blaNDM, blaKPC, blaSHV, blaCTX-M), amikacin [aac(6′)-Ib], co-trimoxazole (sul1, sul2, dfrA), ciprofloxacin (qnrA, qnrB), and the intI1gene. Among these resistant isolates, 80.7% were detected with intI1and 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 β-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 intI1genes.

ABSTRACTAgainst 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. cloacaeisolates with 5 log10CFU/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. cloacaeisolates 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. cloacaeisolates 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 vitrogrowth 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.

In the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus(MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe. We investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population. Our results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

ABSTRACTWe studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumanniiisolates, 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 lpxor pmrBgene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumanniiisolates that had developed polymyxin B resistance in vivocompared to isolates that had developed polymyxin B resistance in vitro.

ABSTRACTAmong 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-β-lactamase producers. Among older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.

ABSTRACTExtensively drug-resistant (XDR) Klebsiella pneumoniaeis 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. pneumoniaeisolates. Six NDM-1-producing and two OXA-181-producing K. pneumoniaestrains 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. pneumoniaeisolates over 240 h. The emergence of resistance against tigecycline was quantified using drug-free and selective (tigecycline at 3× the MIC) media. The in vitrogrowth 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 109CFU/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. pneumoniaeinfections, but its efficacy was strain specific.

Trends in carbapenem-resistant Enterobacteriaceae (CRE) collected from hospitals nationwide in Singapore over 3 years are presented. Hospital isolates with imipenem or meropenem minimum inhibitory concentrations (MICs) of 1mg/L were sent to the National Public Health Laboratory for further investigation. A total of 400 CRE were submitted, 227 (56.8%) of which carried a carbapenemase gene. blaNDMwas the most common (130/400; 32.5%), followed by blaOXA-48-like(blaOXA-48, -181, -232) (55/400; 13.8%). Interestingly, four isolates bearing dual carbapenemase genes were also detected. KPC- and OXA-48-like-producing Klebsiella pneumoniaewere fingerprinted by DiversiLab®rep-PCR. Locally, KPC producers do not appear to have clonal dissemination. In contrast, OXA-48-like producers were found to have a greater degree of clustering than KPC producers.

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

Carbapenem resistance due to metallo-β-lactamases (MBLs) in Enterobacteriaceae has gained much prominence in recent months. The emergence and subsequent spread of New Delhi metallo-β-lactamase-1 (NDM-1) constitutes a potential threat in terms of the management of affected patients and institutional infection control efforts. We evaluated an in-house prepared meropenem impregnated Mac-Conkey agar versus CHROMagar KPC.

Klebsiella pneumoniaeis the major cause of liver abscesses in several Asian countries. Differences in the type of circulating Klebsiellastrains and/or the genetic make up of the host seem to be plausible explanations for this.

Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of <102CFU were from ST23, were resistant to phagocytosis and serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.

ABSTRACTA surveillance study was performed in four Singapore public hospitals from 2006 to 2008 to determine the correlation between antibiotic prescription and Gram-negative bacterial antimicrobial resistance. Targeted organisms included ceftriaxone- and ciprofloxacin-resistant Escherichia coliand Klebsiella pneumoniae, as well as imipenem-resistant Pseudomonas aeruginosaand Acinetobacterspp. Antibiotic prescription data were collated in the WHO anatomical therapeutic chemical (ATC)/defined daily dose (DDD) format, while antibiotic resistance was expressed as incidence density adjusted for total inpatient-days every quarter. Individual trends were determined by linear regression, while possible associations between antibiotic prescription and resistance were evaluated via cross-correlation analysis. Results over 3 years indicated significantly rising incidence densities of ceftriaxone- and ciprofloxacin-resistant E. coliand imipenem-resistant Acinetobacterspp. (blood isolates only). Antimicrobial-resistant Klebsiella pneumoniaerates declined. The prescription rates of piperacillin-tazobactam, ertapenem, meropenem, ciprofloxacin, and levofloxacin increased significantly, while imipenem and moxifloxacin prescription decreased. Cross-correlation analysis demonstrated possible associations between prescription of fluoroquinolones and ciprofloxacin-resistant E. coli(R2= 0.46), fluoroquinolones and ceftriaxone-resistant E. coli(R2= 0.47), and carbapenems and imipenem-resistant Acinetobacterspp. (R2= 0.48), all at zero time lag. Changes in meropenem prescription were associated with a similar trend in imipenem-resistant Acinetobacterblood isolates after a 3-month time lag. No correlation was found between cephalosporin use and resistance. In conclusion, our data demonstrated correlation between prescription of and Gram-negative bacterial resistance to several, but not all, key antimicrobial agents in Singapore hospitals. In areas where Gram-negative bacterial resistance is endemic and prescription of broad-spectrum antimicrobial agents is high, factors other than antimicrobial usage may be equally important in maintaining high resistance rates.

ABSTRACTThere are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiellaspp., and Proteusspp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampCgenes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampCgenes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiellaspp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT- and DHA-like plasmid-borne ampCgenes. The methods are simple enough for introduction into clinical microbiology laboratories.

ABSTRACTCapsular serotypes, magA, and rmpAhave been documented in high prevalence for Klebsiella pneumoniaeliver abscess. To investigate the regional difference and the correlation of capsular serotype, magA, and rmpAwith virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of magAand rmpAwas determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of magAand rmpA.The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%). magAwas restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried rmpA. Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did rmpA-positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of rmpA-positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than rmpAcontributed to virulence. The characteristics of K. pneumoniaeliver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than magAand rmpAin determining virulence in K. pneumoniaeliver abscess.

ABSTRACTThe number of infections attributable to community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) in Singapore is progressively increasing. Most cases in the past 2 years were caused by Panton-Valentine leukocidin-positive isolates belonging to sequence type 30, according to multilocus sequence typing. This has clearly become the predominant sequence type among CA-MRSA isolates in Singapore.

The number of infections attributable to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Singapore is progressively increasing. Most cases in the past 2 years were caused by Panton-Valentine leukocidin-positive isolates belonging to sequence type 30, according to multilocus sequence typing. This has clearly become the predominant sequence type among CA-MRSA isolates in Singapore.

A prospective cross-sectional study was performed to determine the continuing shift in the molecular epidemiology of meticillin-resistant Staphylococcus aureus(MRSA) in Singapore. In total, 666 MRSA isolates from screening cultures performed between 7 and 20 January 2013 were obtained from all seven public sector hospitals in Singapore and were subjected to molecular typing using multilocus variable-number tandem-repeat fingerprinting with confirmatory multilocus sequencing typing for clustered isolates. Isolates were also tested for the presence of the orfX–ACME insert and the high-level mupirocin resistance gene ileS-2. The major circulating clones in Singaporean hospitals were ST22 (63.2%), ST45 (18.9%) and ST239 (10.7%). The orfX–ACME insert was only found in ST239 isolates (31/71, 43.7%), but ileS-2 was found in 207 (31.1%) of the MRSA isolates, varying between 10.0% and 47.8% among the hospitals. In conclusion, the molecular epidemiology of MRSA in Singaporean hospitals has continued to change, with ST45 now replacing ST239 in addition to the ongoing replacement of the latter by ST22. Although a greater proportion of ST239 isolates carry the orfX–ACME insert, the actual clinical impact may be marginal as ST239 MRSA continues to decline. Finally, high-level mupirocin resistance rates are remarkably high in local healthcare-associated MRSA, with implications for MRSA decolonisation and infection prevention. Further surveillance is required to monitor the changing epidemiological trends.

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 vitropharmacodynamics of fosfomycin against carbapenem-resistant Enterobacter cloacaeand 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.

The blaIMIgene is rarely detected outside the Enterobactergenus. Genomic characterization of 87 blaIMI-positive Enterobacter cloacaecomplex members revealed that the largest phylogenomic clade was made up of E. cloacaesubsp. cloacae(71.3%), followed by the newly described species E. bugandensis(13.8%), E. sichuanensis(10.

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. baumanniito polymyxin B-based antibiotic combinations using flow cytometry.

We report the first outbreak of vancomycin-resistant Enterococcus faeciumcolonization and infection among inpatients in the hematology ward of an acute tertiary care public hospital in Singapore. Two cases of bacteremia and 4 cases of gastrointestinal carriage were uncovered before implementation of strict infection control measures resulted in control of the outbreak.

ABSTRACTAnalysis of hospital-acquired methicillin-resistant Staphylococcus aureusstrains isolated from a tertiary public hospital in Singapore revealed that multisusceptible strains had gradually started to replace the endemic multiresistant strain (ST239-MRSA-III) since 2002. Molecular typing showed that this was a predominantly clonal outbreak of a UK-EMRSA-15 strain (ST22-MRSA-IV).

ABSTRACTThirty-six isolates of carbapenem-resistant Pseudomonas aeruginosawere studied. Pulsed-field gel electrophoresis revealed the presence of two clones. One clone carried a blaIMP-1gene identical to that first described in Japan. The other clone carried a blaIMP-1variant containing four silent mutations. One isolate with a unique pulsed-field gel electrophoresis pattern contained blaIMP-7.

ABSTRACTFour carbapenem-resistant Pseudomonasspp. were isolated from patients in Singapore. One Pseudomonas putidaisolate contained a blaIMP-1identical to that first described in Japan. The sequence of a variant blaIMP-1in Pseudomonas fluorescenscontained four silent mutations compared with the original sequence. The remaining P. putidaisolates contained blaVIM-6, a novel VIM gene variant.

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).

To guide the timely selection of antibiotic combinations against carbapenem-resistant Gram-negative bacteria (CR-GNB), an in vitrotest with a short turnaround time is essential. We developed an in vitroATP bioluminescence assay to determine effective antibiotic combinations against CR-GNB within 6 h.

ABSTRACTPolymyxin 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 log10CFU/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 log10CFU/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.

ABSTRACTNine imipenem-resistant Pseudomonas aeruginosaisolates were found to contain a variety of metallo-ß-lactamase genes, including blaIMP-1, blaIMP-7, blaVIM-2, blaVIM-6, and the novel blaIMP-26. Multilocus sequence typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the epidemic clones from 2000 have not become established.