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Carbapenemase-producing Enterobacterales (CPE) infection control practices are based on the paradigm that detected carriers in the hospital transmit to other patients who stay in the same ward. The role of plasmid-mediated transmission at population level remains largely unknown. In this retrospective cohort study over 4.7 years involving all multi-disciplinary public hospitals in Singapore, we analysed 779 patients who acquired CPE (1215 CPE isolates) detected by clinical or surveillance cultures. 42.0% met putative clonal transmission criteria, 44.8% met putative plasmid-mediated transmission criteria and 13.2% were unlinked. Only putative clonal transmissions associated with direct ward contact decreased in the second half of the study. Both putative clonal and plasmid-mediated transmission associated with indirect (no temporal overlap in patients' admission period) ward and hospital contact did not decrease during the study period. Indirect ward and hospital contact were identified as independent risk factors associated with clonal transmission. In conclusion, undetected CPE reservoirs continue to evade hospital infection prevention measures. New measures are needed to address plasmid-mediated transmission, which accounted for 50% of CPE dissemination. Carbapenemase-producing Enterobacterales cause healthcare-associated infections but modes of transmission are not well understood. Here, the authors find evidence of transmission without direct patient contact, indicating presence of undetected environmental reservoirs, whilst half of the transmission events are likely due to plasmid-mediated transmission. [ABSTRACT FROM AUTHOR]

Objectives: The increasing incidence of carbapenem-nonsusceptible Enterobacterales as major pathogens in healthcare associated infections (HAIs) is of paramount concern. To implement effective prevention strategies against carbapenem-nonsusceptible Enterobacterales (CnSE) HAIs, it is crucial to identify modifiable factors associated with these infections. We identified risk factors for CnSE-HAIs, and compared clinical outcomes of CnSE-HAI and carbapenem-sensitive Enterobacterales (CSE)-HAI patients. Methods: We conducted a multi-centre parallel matched case-control study in two 1700-bedded Singapore acute-care hospitals from 2014–2016. Patients with CnSE-HAIs and CSE-HAIs were compared to a common control group without HAIs (1:1:3 ratio), matched by time-at-risk and patient ward. Carbapenem nonsusceptible was defined as non-susceptibility to either meropenem or imipenem. Presence of healthcare associated infections were defined by the criteria provided by the European Centre for Disease Prevention and Control. Outcomes of CnSE-HAI and CSE-HAI patients were compared using multivariable logistic and cox regression; the models were adjusted for infection and treatment characteristics. Results: Eighty CnSE-HAI and 80 CSE-HAI patients were matched to 240 patients without HAIs. All CRE-HAIs patients had prior antibiotic exposure, with 44 (55.0%) with prior carbapenem exposure. The most common CnSE-HAIs were intra-abdominal infections (28.8%) and pneumonia (23.8%). The most common CnSE species was Klebsiella spp. (63.8%). In the risk factor analysis, presence of drainage devices [adjusted odds ratio (aOR), 2.19; 95% CI, 1.29 – 3.70] and prior carbapenem exposure (aOR,17.09; 95% CI, 3.06 – 95.43) independently predicted CnSE-HAIs. In the crude outcomes analysis, CnSE-HAI patients had higher all-cause in-hospital mortality and longer time to discharge compared to CSE-HAI patients. After adjusting for differences in receipt of antibiotics with reported susceptibility to the Enterobacterales, there was no significant difference in all-cause in-hospital mortality between the two groups (aOR, 1.76; 95% CI, 0.86–3.58). Time to discharge remained significantly longer in patients with CnSE-HAI (adjusted hazard ratio, 0.71; 95% CI, 0.51 – 0.98) after adjusting for disease severity, receipt of antibiotics with reported susceptibility and receipt of appropriate source control. Conclusion: Appropriate management of deep-seated Enterobacterales infections and reducing exposure to carbapenems may reduce risk of CnSE-HAIs in Singapore. Efforts to improve antimicrobial therapy in CnSE-HAI patients may improve patient outcomes. [ABSTRACT FROM AUTHOR]

Gastrointestinal carriage of Klebsiella pneumoniae is a predisposing factor for liver abscess in several Asian countries. To determine whether hypervirulent K. pneumoniae in the gut may be transmitted through food, we screened a range of raw and ready-to-eat retail food by culture and recovered K. pneumoniae in 21% (147 of 698) of samples tested. Based on PCR, no K. pneumoniae isolates carried the rmpA gene 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 (wcaG and 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. pneumoniae isolate from raw bean sprouts was resistant to a third-generation cephalosporin (ceftriaxone). Although K. pneumoniae may be present in food without causing harm, we found isolates belonging to the K1 capsular serotype coexisting with the wcaG gene, one also conferring multidrug resistance. K. pneumoniae that 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. pneumoniae and other opportunistic pathogens. Multidrug-resistant and potentially virulent K. pneumoniae was found in food. Food samples did not carry hypervirulent K. pneumoniae linked to liver abscess. Foodborne K. pneumoniae can be a potential reservoir of antibiotic resistance genes. Good food and personal hygiene are needed to help curb antimicrobial resistance. [ABSTRACT FROM AUTHOR]

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

Hepatitis E virus (HEV) causes 20 million infections worldwide yearly, of which only about 3.3 million are symptomatic. In developed Asian countries, HEV strains detected in human sera and in food sources were genetically similar, suggesting that indigenous HEV infections may be largely food‐borne. To assess the burden of hepatitis E in Singapore, we performed a seroepidemiologic study of the infection. Additionally, we carried out HEV genotyping on archived, residual HEV IgM‐positive serum samples collected between 2014 and 2016 (n = 449), and on pig liver samples (n = 36) purchased from wet markets and supermarkets. Our study shows a rise in hepatitis E incidence (IgM) from 1.7 to 4.1 cases per 100,000 resident population from 2012 to 2016 and an increase in hepatitis E IgG positivity rate among residents from 14% in 2007 to 35% in 2016. Other findings also suggest the epidemiology of hepatitis E in Singapore has shifted, from it being mainly a disease imported from the Indian subcontinent, to one that is now increasingly prevalent in our resident population. Genotypes obtained from 143 human samples identified the majority to be genotype 3 (n = 121), 21 to be genotype 1 and one to be genotype 4. Further phylogenetic analyses suggest genotype 3a to be the cause of indigenous infections in residents, which showed genetic similarity to the genotype 3a strains detected in pig livers. This link between the strains in the majority of human samples and those in pig livers consumed by the public suggests a possible food‐borne route of HEV infection in Singapore. [ABSTRACT FROM AUTHOR]

Background: In 2015, Singapore had the first and only reported foodborne outbreak of invasive disease caused by the group B Streptococcus (GBS; Streptococcus agalactiae). Disease, predominantly septic arthritis and meningitis, was associated with sequence type (ST)283, acquired from eating raw farmed freshwater fish. Although GBS sepsis is well-described in neonates and older adults with co-morbidities, this outbreak affected non-pregnant and younger adults with fewer co-morbidities, suggesting greater virulence. Before 2015 ST283 had only been reported from twenty humans in Hong Kong and two in France, and from one fish in Thailand. We hypothesised that ST283 was causing region-wide infection in Southeast Asia. Methodology/Principal findings: We performed a literature review, whole genome sequencing on 145 GBS isolates collected from six Southeast Asian countries, and phylogenetic analysis on 7,468 GBS sequences including 227 variants of ST283 from humans and animals. Although almost absent outside Asia, ST283 was found in all invasive Asian collections analysed, from 1995 to 2017. It accounted for 29/38 (76%) human isolates in Lao PDR, 102/139 (73%) in Thailand, 4/13 (31%) in Vietnam, and 167/739 (23%) in Singapore. ST283 and its variants were found in 62/62 (100%) tilapia from 14 outbreak sites in Malaysia and Vietnam, in seven fish species in Singapore markets, and a diseased frog in China. Conclusions: GBS ST283 is widespread in Southeast Asia, where it accounts for a large proportion of bacteraemic GBS, and causes disease and economic loss in aquaculture. If human ST283 is fishborne, as in the Singapore outbreak, then GBS sepsis in Thailand and Lao PDR is predominantly a foodborne disease. However, whether transmission is from aquaculture to humans, or vice versa, or involves an unidentified reservoir remains unknown. Creation of cross-border collaborations in human and animal health are needed to complete the epidemiological picture. [ABSTRACT FROM AUTHOR]

Objectives: Owing to gene transposition and plasmid conjugation, New Delhi metallo-β-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. [ABSTRACT FROM AUTHOR]

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

Background: Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations. Methods: 100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (105 CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (TRLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of TRLU values was determined. External validation was further done using 50 additional CR-GNB. Results: Predictive accuracies of TRLU were high for when all antibiotic combinations and species were collectively analyzed (TRLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (TRLU = 0.81, UA = 91%), and lowest for AB (TRLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively. Conclusion: We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations. [ABSTRACT FROM AUTHOR]

Background: Acinetobacter baumannii is an important nosocomial pathogen that has become increasingly resistant to multiple antibiotics. Genetic manipulation of MDR A. baumannii is useful especially for defining the contribution of each active efflux mechanism in multidrug resistance. Existing methods rely on the use of an antibiotic selection marker and are not suited for multiple gene deletions. Results: A tellurite-resistant (sacB+, xylE+) suicide vector, pMo130-TelR, was created for deleting the adeFGH and adeIJK operons in two clinical MDR A. baumannii, DB and R2 from Singapore. Using a two-step selection, plasmid insertion recombinants (first-crossover) were selected for tellurite resistance and the deletion mutants (secondcrossover) were then selected for loss of sacB. The DNA deletions were verified by PCR while loss of gene expression in the ΔadeFGH, ΔadeIJK and ΔadeFGHΔadeIJK deletion mutants was confirmed using qRT-PCR. The contribution of AdeFGH and AdeIJK pumps to MDR was defined by comparing antimicrobial susceptibilities of the isogenic mutants and the parental strains. The deletion of adeIJK produced no more than eight-fold increase in susceptibility to nalidixic acid, tetracycline, minocycline, tigecycline, clindamycin, trimethoprim and chloramphenicol, while the deletion of adeL-adeFGH operon alone had no impact on antimicrobial susceptibility. Dye accumulation assays using H33342 revealed increased dye retention in all deletion mutants, except for the R2ΔadeFGH mutant, where a decrease was observed. Increased accumulation of ethidium bromide was observed in the parental strains and all pump deletion mutants in the presence of efflux inhibitors. The efflux pump deletion mutants in this study revealed that only the AdeIJK, but not the AdeFGH RND pump, contributes to antimicrobial resistance and dye accumulation in MDR A. baumannii DB and R2. Conclusions: The marker-less gene deletion method using pMo130-TelR is applicable for creating single and multiple gene deletions in MDR A. baumannii. The adeFGH and adeIJK operons were successfully deleted separately and together using this method and the impact of each efflux pump on antimicrobial resistance could be defined clearly. [ABSTRACT FROM AUTHOR]

Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance-conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system. [ABSTRACT FROM AUTHOR]

A new national antimicrobial resistance surveillance program in Singapore public hospitals that uses WHONET detected high levels of methicillin resistance among Staphylococcus aureus (35.3%), carbapenem resistance among Acinetobacter spp. (49.6%), and third-generation cephalosporin resistance among Klebsiella pneumoniae (35.9%) hospital isolates in 2006. Antimicrobial drug resistance is a major problem in Singapore. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Traditional in vitro time-kill studies (TKSs) require viable plating, which is tedious and time-consuming. We used ATP bioluminescence, with the removal of extracellular ATP (EC-ATP), as a surrogate for viable plating in TKSs against carbapenem-resistant Gram-negative bacteria (CR-GNB). Twenty-four-hour TKSs were conducted using eight clinical CR-GNB (two Escherichia coli, two Klebsiella spp., two Acinetobacter baumannii, two Pseudomonas aeruginosa) with multiple single and two-antibiotic combinations. ATP bioluminescence and viable counts were determined at each timepoint (0, 2, 4, 8, 24 h), with and without apyrase treatment. Correlation between ATP bioluminescence and viable counts was determined for apyrase-treated and non-apyrase-treated samples. Receiver operator characteristic curves were plotted to determine the optimal luminescence threshold to discriminate between inhibitory/non-inhibitory and bactericidal/non-bactericidal combinations, compared to viable counts. After treatment of bacteria with 2 U/mL apyrase for 15 min at 37 °C, correlation to viable counts was significantly higher compared to untreated samples (p < 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p < 0.01) and bactericidal (p = 0.03) combinations against CR-GNB compared to untreated samples, when all species were collectively analyzed. We found that ATP bioluminescence can potentially replace viable plating in TKS. Our assay also has applications in in vitro and in vivo infection models. [ABSTRACT FROM AUTHOR]

The rapid emergence of novel multidrug-resistant organisms coupled with the slow development of new antibiotics is of great concern. With the discovery of New Delhi metallo-beta-lactamase 1 (NDM-1)-producing Klebsiella pneumoniae, clinicians are faced with problems in the treatment of infections caused by this multidrug-resistant organism. Therapeutic experiences are limited. We share our experience of the successful treatment of a febrile neutropenic patient with NDM-1-producing K. pneumoniae bacteraemia. [ABSTRACT FROM AUTHOR]

The ST131 multilocus sequence type (MLST) of Escherichia coli is a globally successful pathogen whose dissemination is increasing rates of antibiotic resistance. Numerous global surveys have demonstrated the pervasiveness of this clone; in some regions ST131 accounts for up to 30% of all E. coli isolates. However, many regions are underrepresented in these published surveys, including Africa, South America, and Asia. We collected consecutive bloodstream E. coli isolates from three countries in Southeast Asia; ST131 was the most common MLST type. As in other studies, the C2/H30Rx clade accounted for the majority of ST131 strains. Clinical risk factors were similar to other reported studies. However, we found that nearly all of the C2 strains in this study were closely related, forming what we denote the SEA-C2 clone. The SEA-C2 clone is enriched for strains from Asia, particularly Southeast Asia and Singapore. The SEA-C2 clone accounts for all of the excess resistance and virulence of ST131 relative to non-ST131 E. coli. The SEA-C2 strains appear to be locally circulating and dominant in Southeast Asia, despite the intuition that high international connectivity and travel would enable frequent opportunities for other strains to establish themselves. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the emergence of zoonotic streptococcus canis infections in Singapore.

Although Streptococcus bovis infections in adults are associated with endocarditis and bowel neoplasms, S. bovis-associated meningitis is rare in neonates. We describe the case of a neonate with Ivemark syndrome, which possibly predisposed her to infection with this bacterium. [ABSTRACT FROM AUTHOR]