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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is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.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.

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.

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.

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.

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.

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.