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

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

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

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

Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) infection or HCV-related liver diseases are now shown to cause more than 350,000 deaths every year. Adaptability of HCV genome to vary its composition and the existence of multiple strains makes it more difficult to combat the emergence of drug-resistant HCV infections. Among the HCV polyprotein which has both the structural and non-structural regions, the non-structural protein NS5B RNA-dependent RNA polymerase (RdRP) mainly mediates the catalytic role of RNA replication in conjunction with its viral protein machinery as well as host chaperone proteins. Lack of such RNA-dependent RNA polymerase enzyme in host had made it an attractive and hotly pursued target for drug discovery efforts. Recent drug discovery efforts targeting HCV RdRP have seen success with FDA approval for sofosbuvir as a direct-acting antiviral against HCV infection. However, variations in drug-binding sites induce drug resistance, and therefore targeting allosteric sites could delay the emergence of drug resistance. In this study, we focussed on allosteric thumb site II of the non-structural protein NS5B RNA-dependent RNA polymerase and developed a five-feature pharmacophore hypothesis/model which estimated the experimental activity with a strong correlation of 0.971 & 0.944 for training and test sets, respectively. Further, the Güner-Henry score of 0.6 suggests that the model was able to discern the active and inactive compounds and enrich the true positives during a database search. In this study, database search and molecular docking results supported by experimental HCV viral replication inhibition assays suggested ligands with best fitness to the pharmacophore model dock to the key residues involved in thumbs site II, which inhibited the HCV 1b viral replication in sub-micro-molar range.

Beta-actin variant is necessary for Enterovirus 71 replication.

Enterovirus 71 (EV71) is one of the main etiological agents of the Hand, Foot and Mouth Disease (HFMD) and has been known to cause fatal neurological complications such as herpangina, aseptic meningitis, poliomyelitis-like paralysis and encephalitis. EV71 is endemic in the Asia-Pacific region and causes occasional epidemics. In order to better understand EV71 infection, we compared the proteome between EV71-susceptible and EV71-resistant human Rhabdomyosarcoma (RD) cell line. We found significant differences in the ß-actin variants between the EV71-susceptible RD cells and EV71-resistant RD cells, suggesting that ß-actin, in association with other proteins such as annexin 2 is required in vesicular transport of EV71. This finding further support our previous study that actin potentially plays a role in pathogenesis and the establishment of the disease in HFMD.

A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection.

Enterovirus 71 (EV71) is a neurotropic pathogen that has been consistently associated with the severe neurological forms of hand, foot, and mouth disease. The lack of a relevant animal model has hampered our understanding of EV71 pathogenesis, in particular the route and mode of viral dissemination. It has also hindered the development of effective prophylactic and therapeutic approaches, making EV71 one of the most pressing public health concerns in Southeast Asia. Here we report a novel mouse model of EV71 infection. We demonstrate that 2-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal (i.p.) and oral routes of inoculation. The infected mice displayed progressive limb paralysis prior to death. The dissemination of the virus was dependent on the route of inoculation but eventually resulted in virus accumulation in the central nervous systems of both animal groups, indicating a clear neurotropism of the virus. Histopathological examination revealed massive damage in the limb muscles, brainstem, and anterior horn areas. However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion. Together, our observations support that young AG129 mice display polio-like neuropathogenesis upon infection with a non-mouse-adapted EV71 strain, making this mouse model relevant for EV71 pathogenesis studies and an attractive platform for EV71 vaccine and drug testing.

Environmental colonization and onward clonal transmission of carbapenem-resistant Acinetobacter baumannii (CRAB) in a medical intensive care unit: the case for environmental hygiene.

Background: In May 2015, we noticed an increase in carbapenem-resistant Acinetobacter baumannii (CRAB) infections in the Medical Intensive Care Unit (MICU). To investigate this, we studied the extent of environmental contamination and subsequent onward clonal transmission of CRAB. Methods: We conducted a one-day point prevalence screening (PPS) of the patients and environment in the MICU. We screened patients using endotracheal tube aspirates and swabs from nares, axillae, groin, rectum, wounds, and exit sites of drains. We collected environmental samples from patients' rooms and environment outside the patients' rooms. CRAB isolates from the PPS and clinical samples over the subsequent one month were studied for genetic relatedness by whole genome sequencing (WGS). Results: We collected 34 samples from seven patients and 244 samples from the environment. On the day of PPS, we identified 8 CRAB carriers: 3 who screened positive and 5 previously known clinical infections. We detected environmental contamination in nearly two-thirds of the rooms housing patients with CRAB. WGS demonstrated genetic clustering of isolates within rooms but not across rooms. We analysed 4 CRAB isolates from clinical samples following the PPS. One genetically-related CRAB was identified in the respiratory sample of a patient with nosocomial pneumonia, who was admitted to the MICU five days after the PPS. Conclusion: The extensive environmental colonization of CRAB by patients highlights the importance of environmental hygiene. The transmission dynamics of CRAB needs further investigation.

Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

INTRODUCTION: Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. AIM: To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. MATERIALS AND METHODS: Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. RESULTS: Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. CONCLUSION: The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being from Singapore.

Comparative proteome analyses of host protein expression in response to Enterovirus 71 and Coxsackievirus A16 infections.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main etiological agents of Hand, Foot and Mouth Disease (HFMD), a common disease among children and had caused several outbreaks in the Asia-Pacific region. Although being genetically close to each other, EV71 infection can cause serious and fatal neurological complications like encephalitis, myocarditis, acute flaccid paralysis (AFP) and aseptic meningitis, but not in CA16 infections. In this study, the cellular response of host cells infected with EV71 and CA16 was characterized and compared by 2-dimensional proteome analyses. A total of 16 proteins were identified to be differentially expressed in EV71 and CA16-infected host cells. Desmin and HSP27, both indirectly regulate the contraction of muscle cells, were significantly downregulated as a result of EV71 infection, suggesting a link to acute flaccid paralysis. The ability of EV71 to evade host immune system may be due to the downregulation of MHC-I synthesis proteins like protein disulfide isomerase A3 and calreticulin. Proteins such as nucleophosmin, nuclear ribonucleoprotein C, and eukaryotic translation initiation factor 2 were all downregulated significantly, suggesting the rapid shutting down of host translation machinery by EV71. These findings provide insight into the nature of high virulent EV71 infection as compared to CA16.