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Carbapenemase-producing Enterobacteriaceae (CPE) are a group of drug-resistant Gram-negative pathogens that are classified as a critical threat by the World Health Organization (WHO). Conventional methods of detecting antibiotic-resistant pathogens do not assess the resistance mechanism and are often time-consuming and laborious. We have developed a magnetic digital microfluidic (MDM) platform, known as MDM Carba, for the identification of CPE by measuring their ability to hydrolyze carbapenem antibiotics. MDM Carba offers the ability to rapidly test CPE and reduce the amount of reagents used compared with conventional phenotypic testing. On the MDM Carba platform, tests are performed in droplets that function as reaction chambers, and fluidic operations are accomplished by manipulating these droplets with magnetic force. The simple droplet-based magnetic fluidic operation allows easy system automation and simplified hands-on operation. Because of the unique “power-free” operation of MDM technology, the MDM Carba platform can also be operated manually, showing great potential for point-of-care testing in resource-limited settings. We tested 27 bacterial isolates on the MDM Carba platform, and the results showed sensitivity and specificity that were comparable to those of the widely used Carba NP test. MDM Carba may shorten the overall turnaround time for CPE identification, thereby enabling more timely clinical decisions for better clinical outcomes. MDM Carba is a technological platform that can be further developed to improve diagnostics for other types of antibiotic resistance with minor modifications. Ministry of Education (MOE) Nanyang Technological University Published version The authors would like to thank Scott Cunningham and Robin Patel from the Mayo Clinic, Rochester, MN, for sharing the reference isolates used in this study and Lew Wen Siang and Gerard Joseph Lim at Nanyang Technological University, Singapore, for helping with vibrating-sample magnetometer measurements. Yi Zhang and Shawn Vasoo are grateful for the funding support from NTUitive Gap Fund NGF-2020-08-002. Yi Zhang is thankful for funding support from the Singapore Ministry of Education Grant Tier 1 RG39/19 and the Nanyang Technological University Startup Grant.

Movement of patients in a health care network poses challenges for the control of carbapenemase-producing Enterobacteriaceae (CPE). We aimed to identify intra- and interfacility transmission events and facility type-specific risk factors of CPE in an acute-care hospital (ACH) and its intermediate-term and long-term-care facilities (ILTCFs). Serial cross-sectional studies were conducted in June and July of 2014 to 2016 to screen for CPE. Whole-genome sequencing was done to identify strain relatedness and CPE genes (blaIMI, blaIMP-1, blaKPC-2, blaNDM-1, and blaOXA-48). Multivariable logistic regression models, stratified by facility type, were used to determine independent risk factors. Of 5,357 patients, half (55%) were from the ACH. CPE prevalence was 1.3% in the ACH and 0.7% in ILTCFs (P = 0.029). After adjusting for sociodemographics, screening year, and facility type, the odds of CPE colonization increased significantly with a hospital stay of ≥3 weeks (adjusted odds ratio [aOR], 2.67; 95% confidence interval [CI], 1.17 to 6.05), penicillin use (aOR, 3.00; 95% CI, 1.05 to 8.56), proton pump inhibitor use (aOR, 3.20; 95% CI, 1.05 to 9.80), dementia (aOR, 3.42; 95% CI, 1.38 to 8.49), connective tissue disease (aOR, 5.10; 95% CI, 1.19 to 21.81), and prior carbapenem-resistant Enterobacteriaceae (CRE) carriage (aOR, 109.02; 95% CI, 28.47 to 417.44) in the ACH. For ILTCFs, presence of wounds (aOR, 5.30; 95% CI, 1.01 to 27.72), respiratory procedures (aOR, 4.97; 95% CI, 1.09 to 22.71), vancomycin-resistant enterococcus carriage (aOR, 16.42; 95% CI, 1.52 to 177.48), and CRE carriage (aOR, 758.30; 95% CI, 33.86 to 16,982.52) showed significant association. Genomic analysis revealed only possible intra-ACH transmission and no evidence for ACH-to-ILTCF transmission. Although CPE colonization was predominantly in the ACH, risk factors varied between facilities. Targeted screening and precautionary measures are warranted. Agency for Science, Technology and Research (A*STAR) Ministry of Health (MOH) Published version This study was funded by a Singapore Ministry of Health’s Communicable Diseases Public Health Research Grant (CDPHRG/0008/2014) and an Agency for Science, Technology, and Research (A*STAR) grant (IAF311018). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We studied the performance of an algorithm combining multiplex polymerase chain reaction with phenotypic detection of extended-spectrum β-lactamases and carbapenemases directly from positive blood culture bottles in patients with gram-negative bacteremia and found good concordance with routine cultures. Such an algorithm may be a tool to improve time to optimal therapy in patients with gram-negative bacteremia.

Funding: Bioinformatics and Computational Biology analyses were supported by the University of St Andrews Bioinformatics Unit that is funded by a Wellcome Trust ISSF award (grant 097831/Z/11/Z). M.T.G.H is supported by the Scottish Infection Research Network and Chief Scientist Office through the Scottish Healthcare Associated Infection Prevention Institute consortium funding (CSO Reference: SIRN10). Objectives Methicillin-resistant Staphylococcus aureus(MRSA) has spread across countries and healthcare settings, with different ecological niches for different clones. It is crucial to understand the comparative epidemiology of MRSA clones between healthcare settings, and independent factors associated with colonization of specific clones. Methods We conducted annual cross-sectional surveillance studies in a network comprising an acute-care hospital and six closely-affiliated intermediate- and long-term care facilities in Singapore, in June-July, 2014-2016. 5,394 patients contributed 16,045 nasal, axillary and groin samples for culture and MRSA isolates for whole genome sequencing. Multivariable multilevel multinomial regression models were constructed to assess for independent factors associated with MRSA colonization. Results MRSA clonal complex(CC) 22 was more prevalent in the acute-care hospital(n=256/493; 51.9%) and intermediate-care(n=348/634; 54.9%) than long-term care(n=88/351; 25.1%) facilities, with clones besides CC22 and CC45 being more prevalent in long-term care facilities(n=144/351; 41.0%) (P

Background: The risk factors for methicillin-resistant Staphylococcus aureus (MRSA) colonization can differ between acute-care, subacute-care, and long-term care facilities, but comparative information is lacking. We compared risk factors for MRSA colonization contemporaneously between an acute-care hospital (ACH) and its affiliated intermediate- and long-term care facilities (ILTCFs). Methods: Serial cross-sectional studies were conducted in a 1,600-bed tertiary-care ACH and its 6 affiliated ILTCFs in Singapore, in June–July 2014–2016. Separate nasal, axillary, and groin swabs were taken and cultured for MRSA. MRSA isolates were subject to whole-genome sequencing. Clinical and epidemiological data were obtained from medical records. To account for clustering, multivariable 2-level multinomial logistic regression models were constructed to assess factors associated with colonization of specific MRSA clones, in the ACH and ILTCFs, respectively. Results: In total, 8,873 samples from 2,985 patients in the ACH and 7,172 samples from 2,409 patients and residents in ILTCFs were included in the study. Patients and residents in the ILTCFs (29.7%) were more likely to be colonized with MRSA than patients in the ACH (12.6%) (P < .0001). The predominant MRSA clones were clonal complexes (CC)22 (n = 692, 46.7%) and CC45 (n = 494, 33.4%), contributing to 80% of MRSA isolates. For ACH patients, after adjusting for age, gender, comorbidities, prior exposures to antibiotics and percutaneous devices, presence of wounds, and screening year, prior MRSA carriage in the preceding 12 months was the strongest predictor of colonization with all MRSA clones: CC22 (aOR, 14.71; 95% CI, 6.17–34.48); CC45 (aOR, 7.75; 95% CI, 2.70–22.22); and others (aOR, 22.22; 95% CI, 3.83–125.00). Hospital stay >14 days was also positively associated with colonization with MRSA CC22 (aOR, 2.67; 95% CI, 1.22–5.88), but not the other clones. For ILTCF patients and residents, after adjusting for age, comorbidities, prior exposure to antibiotics, presence of wounds, and screening year, prior MRSA carriage was a significant predictor of colonization with MRSA CC22 (aOR, 2.72; 95% CI, 1.35–5.46), and CC45 (aOR, 2.36; 95 % CI, 1.06–5.24), but not with other clones. Additionally, prior exposure to a percutaneous device and being male were respectively positively associated with colonization by MRSA CC22 (aOR, 2.70; 95% CI, 1.19–6.17) and CC45 (aOR, 2.17; 95% CI, 1.11–4.26). Conclusions: Prior MRSA carriage was a common risk factor for colonization with the predominant MRSA clones in both the ACH and ILTCFs. Hospital stay >14 days and exposure to percutaneous devices were additional risk factors for CC22 colonization in the ACH and ILTCFs, respectively. Pre-emptive contact precautions for prior MRSA-carriers on admission and active screening for long-stayers in the ACH could prevent intra- and interinstitutional MRSA transmission.Funding: NoneDisclosures: None

Funding: This research was supported by the Small Innovative Grant (SIG/15033) and Communicable Diseases – Public Health Research Grant (CDPHRG/0008/2014) awarded by the National Healthcare Group and Ministry of Health Singapore respectively. Objectives With the widespread use of antiseptics in healthcare facilities for the prevention of methicillin-resistant Staphylococcus aureus (MRSA) transmission, there are concerns for antiseptic tolerance and resistance. We sought to understand the use of chlorhexidine and octenidine, qac genes carriage and reduced antiseptic susceptibilities. Methods A serial cross-sectional study was conducted in an acute care hospital and three extended-care facilities of a healthcare network in June-July, 2014-2016. Two of the extended-care facilities were exposed to intranasal octenidine and universal daily chlorhexidine/octenidine bathing. The minimum inhibitory concentration (MIC) levels and qac genes were determined by broth microdilution tests and whole genome sequencing respectively. Multivariable logistic regression was used to assess for the independent associations between antiseptic exposures, qac genes and reduced antiseptic susceptibilities. Results A total of 878 MRSA isolates were obtained. There were associations between qacA/B carriage and chlorhexidine (adjusted odds ratio [aOR]: 7.80; 95% confidence interval [CI]: 3.25-18.71) and octenidine (aOR: 11.79; 95%CI: 5.14-27.04) exposures. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility (MIC≥4mg/L) (aOR: 3.15; 95%CI: 1.14-8.74). Carriage of qacA/B (aOR: 10.65: 95%CI: 4.14-27.40) or qacC (aOR: 2.55; 95%CI: 1.22-5.32) had an association with reduced chlorhexidine susceptibility; while MRSA sequence type modified the association. However, we found no direct association between (i) antiseptics use and qacC carriage, (ii) octenidine exposure and reduced susceptibility and (iii) reduced octenidine susceptibility and qacA/B or qacC carriage. Conclusions Antiseptic exposures were associated with qac genes carriage. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility, requiring continued surveillance for the emergence of resistance. Postprint

Intranasal octenidine, an antiseptic alternative to mupirocin, can be used for methicillin-resistant Staphylococcus aureus (MRSA) decolonisation in the prevention of nosocomial transmission. A controlled before–after study was conducted in three extended-care hospitals in Singapore. All inpatients with >48 h stay were screened for MRSA colonisation in mid-2015(pre-intervention) and mid-2016(post-intervention). Hospital A: universal daily chlorhexidine bathing throughout 2015 and 2016, with intranasal octenidine for MRSA-colonisers in 2016. Hospital B: universal daily octenidine bathing and intranasal octenidine for MRSA-colonisers in 2016. Hospital C: no intervention. In 2015, MRSA prevalence was similar among the hospitals (Hospital A: 38.5%, Hospital B: 48.1%, Hospital C: 43.4%, P = 0.288). From 2015 to 2016, MRSA prevalence reduced by 58% in Hospital A (Adj OR 0.42, 95% CI 0.20–0.89) and 43% in Hospital B (Adj OR 0.57, 95% CI 0.39–0.84), but remained similar in Hospital C (Adj OR 1.19, 95% CI 0.60–2.33), after adjusting for age, gender, comorbidities, prior MRSA carriage, prior antibiotics exposure and length of hospital stay. Compared with the change in MRSA prevalence from 2015 to 2016 in Hospital C, MRSA prevalence declined substantially in Hospital A (Adj OR 0.35, 95% CI 0.13–0.97) and Hospital B (Adj OR 0.48, 95% CI 0.22–1.03). Topical intranasal octenidine, coupled with universal daily antiseptic bathing, can reduce MRSA colonisation in extended-care facilities.

Vancomycin-resistant enterococci (VRE) are an important cause of nosocomial infections in acute-care hospitals (ACHs), intermediate-care facilities (ITCFs), and long-term care facilities (LTCFs). This study contemporaneously compared the epidemiology and risk factors for VRE colonization in different care settings in a health care network. We conducted a serial cross-sectional study in a 1,700-bed ACH and its six closely affiliated ITCFs and LTCFs in June and July of 2014 to 2016. Rectal swab or stool specimens were cultured for VRE. Multivariable logistic regression was used to assess for independent risk factors associated with VRE colonization. Of 5,357 participants, 523 (9.8%) were VRE colonized. VRE prevalence was higher in ACHs (14.2%) than in ITCFs (7.6%) and LTCFs (0.8%). Common risk factors between ACHs and ITCFs included prior VRE carriage, a longer duration of antibiotic therapy, surgery in the preceding 90 days, and the presence of a skin ulcer. Independent risk factors specific to ACH-admitted patients were prior methicillin-resistant Staphylococcus aureus carriage, a higher number of beds per room, prior proton pump inhibitor use, and a length of stay of >14 days. For ITCFs, a length of stay of >14 days was inversely associated with VRE colonization. Similarities and differences in risk factors for VRE colonization were observed between health care settings. VRE prevention efforts should target the respective high-risk patients. MOH (Min. of Health, S’pore) Published version

This work was supported by the Ministry of Health, Singapore (Communicable Diseases–Public Health Research Grant), the Wellcome Trust (Institutional Strategic Support Fund award [grant 097831/Z/11/Z] to the University of St Andrews Bioinformatics Unit), and the Scottish Infection Research Network and Chief Scientist Office (Scottish Healthcare Associated Infection Prevention Institute consortium funding [Chief Scientist Office reference SIRN10] to K. P. and M. T. G. H.). Background: Methicillin-resistant Staphylococcus aureus (MRSA) is the most common healthcare-associated multidrug-resistant organism. Despite the interconnectedness between acute care hospitals (ACHs) and intermediate- and long-term care facilities (ILTCFs), the transmission dynamics of MRSA between healthcare settings is not well understood. Methods: We conducted a cross-sectional study in a network comprising an ACH and 5 closely affiliated ILTCFs in Singapore. A total of 1700 inpatients were screened for MRSA over a 6-week period in 2014. MRSA isolates underwent whole-genome sequencing, with a pairwise single-nucleotide polymorphism (Hamming distance) cutoff of 60 core genome single-nucleotide polymorphisms used to define recent transmission clusters (clades) for the 3 major clones. Results: MRSA prevalence was significantly higher in intermediate-term (29.9%) and long-term (20.4%) care facilities than in the ACH (11.8%) (P < .001). The predominant clones were sequence type [ST] 22 (n = 183; 47.8%), ST45 (n = 129; 33.7%), and ST239 (n = 26; 6.8%), with greater diversity of STs in ILTCFs relative to the ACH. A large proportion of the clades in ST22 (14 of 21 clades; 67%) and ST45 (7 of 13; 54%) included inpatients from the ACH and ILTCFs. The most frequent source of the interfacility transmissions was the ACH (n = 28 transmission events; 36.4%). Conclusions: MRSA transmission dynamics between the ACH and ILTCFs were complex. The greater diversity of STs in ILTCFs suggests that the ecosystem in such settings might be more conducive for intrafacility transmission events. ST22 and ST45 have successfully established themselves in ILTCFs. The importance of interconnected infection prevention and control measures and strategies cannot be overemphasized. Postprint

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.

We report what we believe to be the first six cases of daptomycin-non-susceptible Staphylococcus aureus infections from Singapore. These strains were rapidly isolated after bacteraemic patients were switched to daptomycin following initial prolonged unsuccessful therapy with vancomycin, despite confirmation of daptomycin susceptibility just prior to initiating daptomycin therapy. The majority of post-vancomycin therapy strains exhibited marked thickening of their cell walls on electron microscopic examination. In patients with persistent S. aureus bacteraemia, therapeutic failure with daptomycin may occur if used as salvage therapy following vancomycin failure, notwithstanding initial susceptibility testing results.

Background We studied the multiplex PCR panel (BioFire Blood Culture ID panel, “BCID”) with phenotypic testing using the Rosco Diagnostica Rapid ESBL Screen kit 98022 (RE) and the Neo-Rapid CARB kit 98024 (RC) for extended-spectrum β-lactamase (ESBL)/carbapenemase producing Gram negative bacilli (CPGNB) detection directly from blood culture bottles, in patients with Gram negative bacteremia. Methods The RE and RC kits were evaluated in a verification phase with 98 blood cultures, comprising 43 spiked with GNB: 23 Escherichia coli, 9 Klebsiella pneumoniae, 7 Enterobacter cloacae, 2 Serratia marcescens, one Pseudomonas aeruginosa, one Acinetobacter baumanii complex with varying resistance genotypes (11 CTX-M-15, 5 CTX-M9, one SHV-18, one SHV-3, one TEM-10, 3 IMI, 4 IMP, 4 KPC, 2 NDM, one OXA-23+OXA-51-like, 3 OXA-232, one OXA-48, one SME-1, 2 VIM-1, 2 AmpC from reference and clinical isolate banks, and ATCC 25922), and 54 clinical blood cultures with GNB (5 phenotypic ESBL-positive, one KPC, 48 no known β-lactamase). In a prospective phase, a further 123 clinical blood cultures positive for GNB were tested simultaneously with the BCID, RE and RC kits. Results In the verification phase, the RE kit detected 24/25 of ESBL-positive samples (sensitivity 96%, specificity 99%). The RE kit did not detect the 2 AmpC-producers, and was positive for a K. oxytoca isolate, which are known to produce chromosomally encoded β-lactamases. The RC kit detected 11/22 of CPGNB (sensitivity 50%, specificity 100%). It missed IMI, OXA-23+OXA-51-like, OXA-232, OXA-48, SME-1 and VIM CPGNB (weak carbapenemases), but detected NDM, KPC, IMP. In the prospective phase, the RE kit detected 20/20 ESBL-positive blood culture samples (sensitivity 100%). The single OXA-48 positive sample was detected by both the RE and RC kits. The 123 blood cultures had a total of 125 panel-represented targets detectable by BCID. The BCID detected 124 /125 (missed one K. pneumoniae in a polymicrobial bacteremia), and there were 2 Proteus false-positives (sensitivity 99%, specificity 98%). No KPC-positive samples were detected by BCID. Conclusion An algorithm comprising the BCID and the RE/RC kits applied to positive blood cultures allows both rapid and accurate pathogen identification and detection of ESBLs and some carbapenemases (e.g., KPC, NDM, IMP). This may allow the institution of timelier, directed therapy. Disclosures S. Vasoo, bioMerieux: Grant Investigator, Research support. Rosco Diagnostica: In-kind support, Research support. R. Banerjee, Accelerate Diagnostics, Biomerieux, BioFire: Grant Investigator, Research grant and Research support. R. Patel, CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Research grant - monies paid to Mayo Clinic. Curetis, Specific Technologies, Selux Dx, GenMark Diagnostics, PathoQuest and Genentech: Consultant and Scientific Advisor, Consulting fee - monies paid to Mayo Clinic. ASM and IDSA: Travel reimbursement and editor’s stipends, Travel reimbursement and editor’s stipends. NBME, Up-to-Date and the Infectious Diseases Board Review Course: Varies, Honoraria. Mayo Clinic: Employee, Salary.

Limited knowledge of the local molecular epidemiology and the paucity of new effective antibiotics has resulted in an immense challenge in the control and treatment of extensively drug-resistant (XDR) Acinetobacter baumannii infections in Thailand. Antimicrobial combination regimens may be the only feasible treatment option in such cases. We sought to characterize the local molecular epidemiology and assess the bactericidal activity of various antibiotics individually and in combination against XDR A. baumannii in a Thai hospital. All XDR A. baumannii isolates from Thammasat University Hospital were collected between October 2010 and May 2011. Susceptibility testing was conducted according to reference broth dilution methods. Pulse-field gel electrophoresis was used to genotype the isolates. Carbapenemase genes were detected using polymerase chain reaction. In vitro testing of clinically-relevant concentrations of imipenem, meropenem, doripenem, rifampicin and tigecycline alone and in combination with polymyxin B was conducted using multiple combination bactericidal testing. Forty-nine polymyxin B-susceptible XDR A. baumannii isolates were identified. bla OXA-23 and bla OXA-51 genes were detected in all isolates. Eight clonally related clusters were identified, resulting in the initiation of several infection control measures. Imipenem, meropenem, doripenem, rifampicin, and tigecycline in combination with PB respectively, exhibited bactericidal killing in 100%, 100%, 98.0%, 100% and 87.8% isolates respectively at 24 hours. Molecular epidemiologic analysis can aid the early detection of infection outbreak within the institution, resulting in the rapid containment of the outbreak. Imipenem/meropenem/rifampicin in combination with polymyxin B demonstrated consistent bactericidal effect against 49 bla OXA-23-harbouring XDR A. baumannii clinical isolates, suggesting a role of combination therapy in the treatment of these infections.