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Background: Hypermucoviscous carbapenem-resistant Klebsiella pneumoniae (hmCRKp) is emerging globally and approaching the worst-case scenario in health care system. Aims: The main objective in this study was to determine the hypermucoviscous characteristics among the carbapenem-resistant K. pneumoniae (CRKp) isolated from a teaching hospital in Malaysia. The association of hypermucoviscous phenotype with the virulence traits and clinical presentations were also investigated. Methods: A retrospective study was conducted in University Malaya Medical Centre (UMMC). The presence of hypermucoviscous K. pneumoniae was identified among a collection of CRKp clinical isolates (first isolate per patient) from 2014 to 2015 using string test. Correlation between clinical and microbial characteristics of the hmCRKp was investigated. Results: A total of nine (7.5%) hmCRKp were detected among 120 CRKp isolates. Majority of the isolates were hospital acquired or health care-associated infections. None of the patients had typical pyogenic liver abscess. All of the hmCRKp isolates harbored carbapenemase genes and were multidrug resistant. K1/K serotype, peg-344, allS, and magA were not identified among hmCRKp isolates, whereas aerobactin siderophore receptor gene (iutA), iroB, rmpA, and rmpA2 were detected. Only three hmCRKp isolates were resistant to serum bactericidal. Conclusions: All the isolates presented inconclusive evidence for the interpretation of hypervirulence. Therefore, more study should be performed in the future to have a better understanding of the virulence mechanisms in correlation with the clinical and microbial determinants.

Non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) confers carbapenem resistance through a combination of chromosomal mutations and acquired non-carbapenemase resistance mechanisms. In this study, we aimed to evaluate the clinical and molecular profiles of NC-CRKP isolated from patients in a tertiary teaching hospital in Malaysia from January 2013 to October 2019. During the study period, 54 NC-CRKP-infected/colonised patients’ isolates were obtained. Clinical parameters were assessed in 52 patients. The all-cause in-hospital mortality rate among NC-CRKP patients was 46.2% (24/52). Twenty-three (44.2%) patients were infected, while others were colonised. Based on the Charlson Comorbidity Index (CCI) score, 92.3% (48/52) of the infected/colonised patients had a score of ≥ 1. Resistance genes found among the 54 NC-CRKP isolates were blaTEM, blaSHV, blaCTX-M, blaOXA, and blaDHA. Porin loss was detected in 25/54 (46.3%) strains. None of the isolated strains conferred carbapenem resistance through the efflux pumps system. In conclusion, only 25/54 (46.3%) NC-CRKP conferred carbapenem resistance through a combination of porin loss and the acquisition of non-carbapenemase resistance mechanisms. The carbapenem resistance mechanisms for the remaining strains (53.7%) should be further investigated as rapid identification and distinction of the NC-CRKP mechanisms enable optimal treatment and infection control efforts.

Aims: The fluid of Nepenthes gracilis harbors diverse bacterial taxa that could serve as a gene pool for the discovery of the new genre of antimicrobial agents against multidrug-resistant Klebsiella pneumoniae. The aim of this study was to explore the presence of antibacterial genes in the fluids of N. gracilis growing in the wild. Methods: Using functional metagenomic approach, fosmid clones were isolated and screened for antibacterial activity against three strains of K. pneumoniae. A clone that exhibited the most potent antibacterial activity was sent for sequencing to identify the genes responsible for the observed activity. The secondary metabolites secreted by the selected clone was sequentially extracted using hexane, chloroform, and ethyl acetate. The chemical profiles of a clone (C6) hexane extract were determined by gas chromatography/mass spectrometry (GC-MS). Results: Fosmid clone C6 from the fluid of pitcher plant that exhibited antibacterial activity against three strains of K. pneumoniae was isolated using functional metagenome approach. A majority of the open reading frames detected from C6 were affiliated with the largely understudied Acidocella genus. Among them, the gene that encodes for coproporphyrinogen III oxidase in the heme biosynthesis pathway could be involved in the observed antibacterial activity. Based on the GC-MS analysis, the identities of the putative bioactive compounds were 2,5-di-tert-butylphenol and 1-ethyl-2-methyl cyclododecane. Conclusions: The gene that encodes for coproporphyrinogen III oxidase in the heme biosynthesis pathway as well as the secondary metabolites, namely 2,5-di-tert-butylphenol and 1-ethyl-2-methyl cyclododecane could be the potential antibacterial molecules responsible for the antibacterial activity of C6.

Background Knowledge on the epidemiology, genotypic and phenotypic features of antimicrobial-resistant (AMR) ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) and their association with hospital-acquired infections (HAIs) are limited in Malaysia. Therefore, we evaluated the AMR features and resistance mechanisms of the ESKAPEE pathogens collected in a tertiary hospital located in the capital of Malaysia. Methods A total of 378 AMR-ESKAPEE strains were obtained based on convenience sampling over a nine-month study period (2019–2020). All strains were subjected to disk diffusion and broth microdilution assays to determine the antimicrobial susceptibility profiles. Polymerase chain reaction (PCR) and DNA sequence analyses were performed to determine the AMR genes profiles of the non-susceptible strains. Chi-square test and logistic regression analyses were used to correlate the AMR profiles and clinical data to determine the risk factors associated with HAIs. Results High rates of multidrug resistance (MDR) were observed in A. baumannii, K. pneumoniae, E. coli, and S. aureus (69–89%). All organisms except E. coli were frequently associated with HAIs (61–94%). Non-susceptibility to the last-resort drugs vancomycin (in Enterococcus spp. and S. aureus), carbapenems (in A. baumannii, P. aeruginosa, and Enterobacteriaceae), and colistin (in Enterobacteriaceae) were observed. Both A. baumannii and K. pneumoniae harbored a wide array of extended-spectrum β-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA). Metallo-β-lactamase genes (blaVEB, blaVIM, blaNDM) were detected in carbapenem-resistant strains, at a higher frequency compared to other local reports. We detected two novel mutations in the quinolone-resistant determining region of the gyrA in fluoroquinolone-resistant E. coli (Leu-102-Ala; Gly-105-Val). Microbial resistance to ampicillin, methicillin, and cephalosporins was identified as important risk factors associated with HAIs in the hospital. Conclusion Overall, our findings may provide valuable insight into the microbial resistance pattern and the risk factors of ESKAPEE-associated HAIs in a tertiary hospital located in central Peninsular Malaysia. The data obtained in this study may contribute to informing better hospital infection control in this region.

The emergence of carbapenemase-producing Enterobacteriaceae has become a major global concern. OXA-48-like carbapenemase gene and its variants have been increasingly reported worldwide. This study reported the first OXA-181-producing Klebsiella quasipneumoniae isolate in Malaysia. This bacterium was isolated from blood specimen of a three-year-old boy with Alagille syndrome who had liver biopsy on October 2016. He had undergone liver transplant in India ten months previously. The genotypic and phenotypic characteristics of the strain were elucidated in this study. To our best knowledge, this is the first report of OXA-181-producing K. quasipneumoniae in Malaysia.

Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.

Staphylococcus aureus expresses diverse proteins at different stages of growth. The immunodominant staphylococcal antigen A (IsaA) is one of the proteins that is constitutively produced by S. aureus during colonisation and infection. SACOL2584 (or isaA) is the gene that encodes this protein. It has been suggested that IsaA can hydrolyse cell walls, and there is still need to study isaA gene disruption to analyse its impact on staphylococcal phenotypes and on alteration to its transcription and protein profiles. In the present study, the growth curve in RPMI medium (which mimics human plasma), autolytic activity, cell wall morphology, fibronectin and fibrinogen adhesion and biofilm formation of S. aureus SH1000 (wildtype) was compared to that of S. aureus MS001 (isaA mutant). RNA sequencing and liquid chromatography–tandem mass spectrometry were carried out on samples of both S. aureus strains taken during the exponential growth phase, followed by bioinformatics analysis. Disruption of isaA had no obvious effect on the growth curve and autolysis ability or thickness of cell walls, but this study revealed significant strength of fibronectin adherence in S. aureus MS001. In particular, the isaA mutant formed less biofilm than S. aureus SH1000. In addition, proteomics and transcriptomics showed that the adhesin/biofilm-related genes and hemolysin genes, such as sasF, sarX and hlgC, were consistently downregulated with isaA gene disruption. The majority of the upregulated genes or proteins in S. aureus MS001 were pur genes. Taken together, this study provides insight into how isaA disruption changes the expression of other genes and has implications regarding biofilm formation and biological processes.

The rise of antimicrobial resistance (AMR) among clinically important bacteria, including respiratory pathogens, is a growing concern for public health worldwide. Common causative bacteria for upper respiratory tract infections (URTIs) include Streptococcus pneumoniae and Haemophilus influenzae, and sometimes Staphylococcus aureus. We assessed the β-lactam resistant trends and mechanisms of 150 URTI strains isolated in a tertiary care hospital in Kuala Lumpur Malaysia. High rates of non-susceptibility to penicillin G (38%), amoxicillin-clavulanate (48%), imipenem (60%), and meropenem (56%) were observed in S. pneumoniae. Frequent mutations at STMK and SRNVP motifs in PBP1a (41%), SSNT motif in PBP2b (32%), and STMK and LKSG motifs in PBP2x (41%) were observed in S. pneumoniae. H. influenzae remained highly susceptible to most β-lactams, except for ampicillin. Approximately half of the ampicillin non-susceptible H. influenzae harboured PBP3 mutations (56%) and only blaTEM was detected in the ampicillin-resistant strains (47%). Methicillin-susceptible S. aureus (MSSA) strains were mostly resistant to penicillin G (92%), with at least two-fold higher median minimum inhibitory concentrations (MIC) for all penicillin antibiotics (except ticarcillin) compared to S. pneumoniae and H. influenzae. Almost all URTI strains (88–100%) were susceptible to cefcapene and flomoxef. Overall, β-lactam antibiotics except penicillins remained largely effective against URTI pathogens in this region.

Background: Klebsiella pneumoniae is a major opportunistic pathogen frequently associated with nosocomial infections, and often poses a major threat to immunocompromised patients. In our previous s...

Cholera is an acute diarrheal illness caused by the Gram-negative bacterium Vibrio cholerae. The pathogen is known for its ability to form biofilm that confers protection against harsh environmental condition and as part of the colonisation process during infection. Coaggregation is a process that facilitates the formation of biofilm. In a preliminary in vitro study, high coaggregation index and biofilm production were found between V. cholerae with human commensals namely Escherichia coli and Enterobacter cloacae. Building upon these results, the effects of coaggregation were further evaluated using adult BALB/c mouse model. The animal study showed no significant differences in mortality and fluid accumulation ratio between treatment groups infected with V. cholerae alone and those infected with coaggregation partnership (V. cholerae with E. coli or V. cholerae with E. cloacae). However, mild inflammation was detected in both partnering pairs. Higher density of V. cholerae was recovered from faecal samples of mice co-infected with E. coli and V. cholerae in comparison with other groups at 24 h post-infection. This partnership also elicited slightly higher levels of interleukin-5 (IL-5) and interleukin-10 (IL-10). Nonetheless, the involvement of autoinducer-2 (AI-2) as the signalling molecules in quorum sensing system is not evident in this study. Since E. coli is one of the common commensals, our result may suggest the involvement of commensals in cholera development.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a great concern, as carbapenems are the last-line therapy for multidrug-resistant Gram-negative bacteria infections. This study aims to report the epidemiology of CRKP in a teaching hospital in Malaysia based on the molecular genotypic and clinical characteristics of the isolates. Sixty-three CRKP strains were isolated from a tertiary teaching hospital from January 2016 until August 2017. Carbapenemase genes were detected in 55 isolates, with blaOXA-48 (63.5%) as the predominant carbapenemase gene, followed by blaNDM (36.5%). At least one porin loss was detected in nine isolates. Overall, 63 isolates were divided into 30 clusters at similarity of 80% with PFGE analysis. Statistical analysis showed that in-hospital mortality was significantly associated with the usage of central venous catheter, infection or colonization by CRKP, particularly NDM-producers. In comparison, survival analysis using Cox proportional hazards regression identified a higher hazard ratio for patients with a stoma and patients treated with imipenem but a lower hazard ratio for patients with NDM-producing CRKP. OXA-48 carbapenemase gene was the predominant carbapenemase gene in this study. As CRKP infection could lead to a high rate of in-hospital mortality, early detection of the isolates was important to reduce their dissemination.

OXA-48-like carbapenemase gene remains a hidden threat, as different OXA-48 variants have varying presentations of susceptibility to antibiotics that might affect the treatment decisions. Rapid detection and differentiation of OXA-48-like carbapenemase genes are critical for targeted treatment and infection control. In this study, we aimed to develop high-resolution melting (HRM) analysis for the differentiation of OXA-48 variants. HRM analysis is a post-polymerase chain reaction (post-PCR) method for identification of small variations in nucleic acid sequences based on the PCR dissociation curve. A total of 82 bacterial strains, which consisted of Enterobacteriaceae and non-Enterobacteriaceae, were collected from a tertiary teaching hospital. The sensitivity and specificity of the assay were determined, and the developed assay was evaluated using the collected isolates against conventional-sequencing method. Overall, the developed assay was able to detect isolates that harboured OXA-48 and OXA232/OXA-181 by showing two distinct peaks at 81.1 &plusmn, 0.2 &deg, C and 82.1 &plusmn, C, respectively. The detection limit of the assay was 1.6 x 10-6 ng/&micro, l for OXA-48 and 1.8 x 10-7 ng/&micro, l for OXA-232/OXA-181. This assay showed 100% specificity when evaluated on a panel of 37 isolates comprised of different species of bacteria and yeasts. When the assay with isolates collected in the year 2016 was first evaluated, the assay showed comparable results with conventional PCR-sequencing method where 34 OXA-48 and OXA-232/OXA-181 were detected. By using HRM analysis, the presence of OXA-48-like variants could be easily identified within 3 hours from the pure culture.

The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) has now become a global sentinel event. CRAB infections often instigate severe clinical complications and are potentially fatal, especially for debilitated patients. The present study aimed to conduct molecular characterization on CRAB isolated from patients in the intensive care unit from 2015 to 2016 and determine the risk factors associated with patients’ mortality. One hundred CRAB isolates were retrospectively selected and included in this study. Antimicrobial susceptibility testing showed that all isolates remained susceptible to colistin, even though 62% of them conferred resistance to all other classes of antibiotics tested. OXA carbapenemase gene was found to be the predominant carbapenemase gene, with 99% of the isolates coharbouring blaOXA-23-like and blaOXA-51-like carbapenemase genes. All isolates were carrying intact CarO genes, with the presence of various degree of nucleotide insertion, deletion and substitution. Overall, PFGE subtyped the isolates into 13 distinct pulsotypes, with the presence of 2 predominant pulsotypes. Univariate analysis implied that age, infection/colonization by CRAB, ethnicity, comorbidity and CRAB specimen source were significantly associated with in-hospital mortality. Multivariate analysis identified a higher risk of mortality for patients who are of Chinese ethnicity with diabetes as an underlying disease. As CRAB infection could lead to high rate of mortality, comprehensive infection control measures are needed to minimize the spread of this pathogen.

Carbapenem resistant Enterobacteriaceae is a growing concern worldwide including Malaysia. The emergence of this pathogen is worrying because carbapenem is one of the 'last-line' antibiotics. The main objective of this study was to determine the prevalence of genetic mechanisms and clinical risk factors of carbapenem resistant Klebsiella pneumoniae (K. pneumoniae) in Malaysia. In this study, seventeen carbapenem resistant K. pneumoniae strains isolated from a tertiary teaching hospital in 2013 were studied. Minimal inhibitory concentration (MIC) of the bacterial strains was determined and genes associated with carbapenemases and extended-spectrum-beta-lactamases (ESBLs) were sequenced and compared with the closest representatives published in public domains. All strains were also sub-typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Statistical analyses were performed to determine the correlation between risk factors for acquiring carbapenem resistant K. pneumoniae and in-hospital mortality. The predominant carbapenemase was bla OXA-48, detected in 12 strains (70.59%). Other carbapenemases detected in this study were bla KPC-2, bla IMP-8, bla NMC-A and bla NDM-1. Nine different pulsotypes were identified and nine strains which were affiliated with ST101, the predominant sequence type had similar PFGE patterns (similarity index of 85%). Based on univariate statistical analysis, resistance to imipenem and usage of mechanical ventilation showed a statistically significant effect separately to in-hospital mortality. The diverse genetic mechanisms harbored by these carbapenem resistant K. pneumoniae facilitates its spread and complicates its detection. Thus, correlation between microbiological trends with host characteristics and clinical factors will provide a better insight of rational treatment strategies and pathogen control.

The increasing prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has greatly affected the clinical efficacy of β-lactam antibiotics in the management of urinary tract infections (UTIs). The limited treatment options have resulted in the increased use of carbapenem. However, flomoxef could be a potential carbapenem-sparing strategy for UTIs caused by ESBL-producers. Here, we compared the in vitro susceptibility of UTI-associated ESBL-producers to flomoxef and established β-lactam antibiotics. Fifty Escherichia coli and Klebsiella pneumoniae strains isolated from urine samples were subjected to broth microdilution assay, and the presence of ESBL genes was detected by polymerase chain reactions. High rates of resistance to amoxicillin-clavulanate (76–80%), ticarcillin-clavulanate (58–76%), and piperacillin-tazobactam (48–50%) were observed, indicated by high minimum inhibitory concentration (MIC) values (32 µg/mL to 128 µg/mL) for both species. The ESBL genes blaCTX-M and blaTEM were detected in both E. coli (58% and 54%, respectively) and K. pneumoniae (88% and 74%, respectively), whereas blaSHV was found only in K. pneumoniae (94%). Carbapenems remained as the most effective antibiotics against ESBL-producing E. coli and K. pneumoniae associated with UTIs, followed by flomoxef and cephamycins. In conclusion, flomoxef may be a potential alternative to carbapenem for UTIs caused by ESBL-producers in Malaysia.