Dietary exposure to acrylamide among the Malaysian adult population.

This study aimed to estimate the Malaysian adult population's current dietary exposure and margin of exposure (MOE) to the carcinogenic processing contaminant, acrylamide. A total of 448 samples from 11 types of processed foods were collected randomly throughout Malaysia in the year 2015 and 2016. Acrylamide was analysed in samples using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) with a limit of detection (LOD) of 10 µg/kg and a limit of quantification (LOQ) of 25 µg/kg. The highest average level of acrylamide (772 ± 752 µg/kg) was found in potato crisps, followed by French fries (415 ± 914 µg/kg) and biscuits (245 ± 195 µg/kg). The total acrylamide exposure for the adult Malaysian was 0.229 and 1.77 µg/kg body weight per day for average and high consumers, respectively. The MOE were 741 and 1875 for the average consumer based on cancer and non-cancer effects of acrylamide, respectively. Meanwhile, for high consumers, the MOE is 96 for cancer and 243 for non-cancer effects. These findings indicate potential carcinogenic risks from acrylamide exposure among Malaysian adults, especially in Malay and other Bumiputra groups compared to Chinese, Indian, and other ethnic groups, while non-cancer effects appeared less concerning.

Prevalence and Diversity of Antibiotic Resistant Escherichia coli From Anthropogenic-Impacted Larut River.

Aquatic environments, under frequent anthropogenic pressure, could serve as reservoirs that provide an ideal condition for the acquisition and dissemination of antibiotic resistance genetic determinants. We investigated the prevalence and diversity of antibiotic-resistant Escherichia coli by focusing on their genetic diversity, virulence, and resistance genes in anthropogenic-impacted Larut River. The abundance of E. coli ranged from (estimated count) Est 1 to 4.7 × 105 (colony-forming units per 100 ml) CFU 100 ml-1 to Est 1 to 4.1 × 105 CFU 100 ml-1 with phylogenetic group B1 (46.72%), and A (34.39%) being the most predominant. The prevalence of multiple antibiotic resistance phenotypes of E. coli, with the presence of tet and sul resistance genes, was higher in wastewater effluents than in the river waters. These findings suggested that E. coli could be an important carrier of the resistance genes in freshwater river environments. The phylogenetic composition of E. coli and resistance genes was associated with physicochemical properties and antibiotic residues. These findings indicated that the anthropogenic inputs exerted an effect on the E. coli phylogroup composition, diversification of multiple antibiotic resistance phenotypes, and the distribution of resistance genes in the Larut River.

Dynamics of fungal and bacterial microbiome associated with green-mould contaminated sawdust substrate of Pleurotus pulmonarius (grey oyster mushroom).

AIMS: Green-mould contamination is identified as one of the challenges faced by mushroom cultivation industry globally which believed to be caused by Trichoderma spp. METHODS AND RESULTS: To explore the dynamics of microbial population in mushroom substrate during commercial mushroom cultivation and how microbiota might play a role in green-mould contamination, we applied both culturing and targeted metagenomics approaches to identify microbiota in noncomposted sawdust substrates at different cultivation stages. The microbiological analysis showed that the green-mould contaminated substrates harboured higher total mesophilic bacteria count. The green-moulds isolated from the contaminated mushroom substrates were identified as Trichoderma pleurotum (n = 15; 93.8%) and Graphium penicillioides (n = 1; 6.3%). To our surprise, the targeted metagenomic analysis revealed that Graphium comprised 56.3% while Trichoderma consisted of only 36.1% of the total fungi population, suggesting that green-mould contamination might not be caused by Trichoderma alone, but also Graphium that grows very slowly in the laboratory. CONCLUSION: It is worthwhile to note that G. penicillioides was also isolated in the early stages of mushroom cultivation, but not T. pleurotum. The results indicated that the structure and composition of the bacterial population in the mushroom substrate varied and the bacterial population shifted along the cultivation process. SIGNIFICANCE AND IMPACT OF STUDY: This study revealed a possibility of G. penicillioides as an overlooked fungi causing green-mould contamination.

ß-Lactam Resistance in Upper Respiratory Tract Pathogens Isolated from a Tertiary Hospital in Malaysia.

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 ß-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 ß-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, ß-lactam antibiotics except penicillins remained largely effective against URTI pathogens in this region.

Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

Chemical and Antimicrobial Analyses of Juniperus chinensis and Juniperus seravschanica Essential Oils and Comparison with Their Methanolic Crude Extracts.

Juniperus chinensis and Juniperus seravschanica are commonly used in the traditional folk medicine to treat microbial infection. In this study, the essential oils obtained from the leaves of J. chinensis growing in Malaysia and J. seravschanica growing in Oman were analysed by head space-solid phase microextraction-gas chromatography mass spectrometry (HS-SPME-GC-MS) and screened for antimicrobial activities against Escherichia coli (NCTC 10418), Pseudomonas aeruginosa (NCTC 10662), Bacillus subtilis ATCC6059, Micrococcus luteus (ATCC 9341), Staphylococcus aureus (NCTC 6571), and methicillin-resistant S. aureus (MRSA; ATCC 33591). To compare the antimicrobial activities of extracts using different extraction methods, methanol extraction was performed to obtain crude extracts from the leaves of J. chinensis and J. seravschanica for antimicrobial analysis. The HS-SPME-GS-MS analysis of the essential oils from the leaves of J. chinensis and J. seravschanica identified 37 and 36 components, respectively. Essential oils from these two species had distinctive chemical component profiles, with α-pinene (27.2%) as the major component of J. chinensis essential oil, while dl-limonene (45.2%) constitutes the major component of J. seravschanica essential oil. Essential oils of these two species shared only six similar terpenoids compounds: α-pinene, ß-pinene, γ-elemene, sabinene, elemol, and 3-cyclohexen-1-ol. Overall, the essential oils showed antimicrobial activities against all the six bacterial strains tested, with the highest antagonistic activity against M. luteus and B. cereus; while, methanolic crude extracts showed the highest activities against S. aureus and MRSA strains. The methanolic crude extracts demonstrated significantly higher antibacterial activity against the Gram-positive bacteria (p < 0.005); while, the essential oils of Juniperus did not show significant differences between Gram-positive and Gram-negative bacteria. Future studies are needed to investigate the active compounds present in the essential oils and methanolic crude extracts that confer the selectivity in the antimicrobial activity.

Anthropogenic impacts on sulfonamide residues and sulfonamide resistant bacteria and genes in Larut and Sangga Besar River, Perak.

The environmental reservoirs of sulfonamide (SA) resistome are still poorly understood. We investigated the potential sources and reservoir of SA resistance (SR) in Larut River and Sangga Besar River by measuring the SA residues, sulfamethoxazole resistant (SMXr) in bacteria and their resistance genes (SRGs). The SA residues measured ranged from lower than quantification limits (LOQ) to 33.13 ng L-1 with sulfadiazine (SDZ), sulfadimethoxine (SDM) and SMX as most detected. Hospital wastewater effluent was detected with the highest SA residues concentration followed by the slaughterhouse and zoo wastewater effluents. The wastewater effluents also harbored the highest abundance of SMXr-bacteria (107 CFU mL-1) and SRGs (10-1/16S copies mL-1). Pearson correlation showed only positive correlation between the PO4 and SMXr-bacteria. In conclusion, wastewater effluents from the zoo, hospital and slaughterhouse could serve as important sources of SA residues that could lead to the consequent emergence of SMXr-bacteria and SRGs in the river.

Elucidating the survival and response of carbapenem resistant Klebsiella pneumoniae after exposure to imipenem at sub-lethal concentrations.

The increasing prevalence of antibiotic resistant pathogens poses a serious threat to global health. However, less emphasis has been placed to co-relate the gene expression and metabolism of antibiotic resistant pathogens. This study aims to elucidate gene expression and variations in metabolism of multidrug resistant Klebsiella pneumoniae after exposure to antibiotics. Phenotypic responses of three genotypically distinct carbapenem resistant Klebsiella pneumoniae (CRKP) strains untreated and treated with sub-lethal concentrations of imipenem were investigated via phenotype microarrays (PM). The gene expression and metabolism of the strain harboring blaNDM-1 before and after exposure to sub-lethal concentration of imipenem were further investigated by RNA-sequencing (RNA-Seq) and 1H NMR spectroscopy respectively. Most genes related to cell division, central carbon metabolism and nucleotide metabolism were downregulated after imipenem treatment. Similarly, 1H NMR spectra obtained from treated CRKP showed decrease in levels of bacterial end products (acetate, pyruvate, succinate, formate) and metabolites involved in nucleotide metabolism (uracil, xanthine, hypoxanthine) but elevated levels of glycerophosphocholine. The presence of anserine was also observed for the treated CRKP while FAPγ-adenine and methyladenine were only present in untreated bacterial cells. As a conclusion, the studied CRKP strain exhibited decrease in central carbon metabolism, cell division and nucleotide metabolism after exposure to sub-lethal concentrations of imipenem. The understanding of the complex biological system of this multidrug resistant bacterium may help in the development of novel strategies and potential targets for the management of the infections.

Isolation and Characterization of Aquatic-Borne Klebsiella pneumoniae from Tropical Estuaries in Malaysia.

Klebsiella pneumoniae is an opportunistic pathogen that is responsible for causing nosocomial and community-acquired infections. Despite its common presence in soil and aquatic environments, the virulence potential of K. pneumoniae isolates of environmental origin is largely unknown. Hence, in this study, K. pneumoniae isolated from the estuarine waters and sediments of the Matang mangrove estuary were screened for potential virulence characteristics: antibiotic susceptibility, morphotype on Congo red agar, biofilm formation, presence of exopolysaccharide and capsule, possession of virulence genes (fimH, magA, ugE, wabG and rmpA) and their genomic fingerprints. A total of 55 strains of K. pneumoniae were isolated from both human-distributed sites (located along Sangga Besar River) and control sites (located along Selinsing River) where less human activity was observed, indicated that K. pneumoniae is ubiquitous in the environment. However, the detection of potentially virulent strains at the downstream of Kuala Sepetang village has suggested an anthropogenic contamination source. In conclusion, the findings from this study indicate that the Matang mangrove estuary could harbor potentially pathogenic K. pneumoniae with risk to public health. More studies are required to compare the environmental K. pneumoniae strains with the community-acquired K. pneumoniae strains.

Quinolone Resistance Mechanisms Among Salmonella enterica in Malaysia.

The prevalence of quinolone-resistant Salmonella enterica is on the rise worldwide. Salmonella enterica is one of the major foodborne pathogens in Malaysia. Therefore, we aim to investigate the occurrence and mechanisms of quinolone resistance among Salmonella strains isolated in Malaysia. A total of 283 Salmonella strains isolated from food, humans, and animals were studied. The disk diffusion method was used to examine the quinolone susceptibility of the strains, and the minimum inhibitory concentration (MIC) values of nalidixic acid and ciprofloxacin were also determined. DNA sequencing of the quinolone resistance-determining regions (QRDRs) of gyrase and topoisomerase IV genes and the plasmid-borne qnr genes was performed. The transfer of the qnr gene was examined through transconjugation experiment. A total of 101 nalidixic acid-resistant Salmonella strains were identified. In general, all strains were highly resistant to nalidixic acid (average MICNAL, 170 µg/ml). Resistance to ciprofloxacin was observed in 30.7% of the strains (1 ≤ MICCIP ≤ 2 µg/ml). Majority of the strains contained missense mutations in the QRDR of gyrA (69.3%). Silent mutations were frequently detected in gyrB (75.2%), parC (27.7%), and parE (51.5%) within and beyond the QRDRs. Novel mutations were detected in parC and parE. The plasmid-borne qnrS1 variant was found in 36.6% of the strains, and two strains were found to be able to transfer the qnrS1 gene. Overall, mutations in gyrA and the presence of qnrS1 genes might have contributed to the high level of quinolone resistance among the strains. Our study provided a better understanding on the status of quinolone resistance among Salmonella strains circulating in Malaysia.

Diverse and abundant multi-drug resistant E. coli in Matang mangrove estuaries, Malaysia.

E.coli, an important vector distributing antimicrobial resistance in the environment, was found to be multi-drug resistant, abundant, and genetically diverse in the Matang mangrove estuaries, Malaysia. One-third (34%) of the estuarine E. coli was multi-drug resistant. The highest antibiotic resistance prevalence was observed for aminoglycosides (83%) and beta-lactams (37%). Phylogenetic groups A and B1, being the most predominant E. coli, demonstrated the highest antibiotic resistant level and prevalence of integrons (integron I, 21%; integron II, 3%). Detection of phylogenetic group B23 downstream of fishing villages indicates human fecal contamination as a source of E. coli pollution. Enteroaggregative E. coli (1%) were also detected immediately downstream of the fishing village. The results indicated multi-drug resistance among E. coli circulating in Matang estuaries, which could be reflective of anthropogenic activities and aggravated by bacterial and antibiotic discharges from village lack of a sewerage system, aquaculture farms and upstream animal husbandry.

Variable Responses to Carbon Utilization between Planktonic and Biofilm Cells of a Human Carrier Strain of Salmonella enterica Serovar Typhi.

Salmonella enterica serovar Typhi (S. Typhi) is a foodborne pathogen that causes typhoid fever and infects only humans. The ability of S. Typhi to survive outside the human host remains unclear, particularly in human carrier strains. In this study, we have investigated the catabolic activity of a human carrier S. Typhi strain in both planktonic and biofilm cells using the high-throughput Biolog Phenotype MicroArray, Minimum Biofilm Eradication Concentration (MBEC) biofilm inoculator (96-well peg lid) and whole genome sequence data. Additional strains of S. Typhi were tested to further validate the variation of catabolism in selected carbon substrates in the different bacterial growth phases. The analyzes of the carbon utilization data indicated that planktonic cells of the carrier strain, S. Typhi CR0044 could utilize a broader range of carbon substrates compared to biofilm cells. Pyruvic acid and succinic acid which are related to energy metabolism were actively catabolised in the planktonic stage compared to biofilm stage. On the other hand, glycerol, L-fucose, L-rhamnose (carbohydrates) and D-threonine (amino acid) were more actively catabolised by biofilm cells compared to planktonic cells. Notably, dextrin and pectin could induce strong biofilm formation in the human carrier strain of S. Typhi. However, pectin could not induce formation of biofilm in the other S. Typhi strains. Phenome data showed the utilization of certain carbon substrates which was supported by the presence of the catabolism-associated genes in S. Typhi CR0044. In conclusion, the findings showed the differential carbon utilization between planktonic and biofilm cells of a S. Typhi human carrier strain. The differences found in the carbon utilization profiles suggested that S. Typhi uses substrates mainly found in the human biliary mucus glycoprotein, gallbladder, liver and cortex of the kidney of the human host. The observed diversity in the carbon catabolism profiles among different S. Typhi strains has suggested the possible involvement of various metabolic pathways that might be related to the virulence and pathogenesis of this host-restricted human pathogen. The data serve as a caveat for future in-vivo studies to investigate the carbon metabolic activity to the pathogenesis of S. Typhi.

Comparative genomics of closely related Salmonella enterica serovar Typhi strains reveals genome dynamics and the acquisition of novel pathogenic elements.

BACKGROUND: Typhoid fever is an infectious disease of global importance that is caused by Salmonella enterica subsp. enterica serovar Typhi (S. Typhi). This disease causes an estimated 200,000 deaths per year and remains a serious global health threat. S. Typhi is strictly a human pathogen, and some recovered individuals become long-term carriers who continue to shed the bacteria in their faeces, thus becoming main reservoirs of infection. RESULTS: A comparative genomics analysis combined with a phylogenomic analysis revealed that the strains from the outbreak and carrier were closely related with microvariations and possibly derived from a common ancestor. Additionally, the comparative genomics analysis with all of the other completely sequenced S. Typhi genomes revealed that strains BL196 and CR0044 exhibit unusual genomic variations despite S. Typhi being generally regarded as highly clonal. The two genomes shared distinct chromosomal architectures and uncommon genome features; notably, the presence of a ~10 kb novel genomic island containing uncharacterised virulence-related genes, and zot in particular. Variations were also detected in the T6SS system and genes that were related to SPI-10, insertion sequences, CRISPRs and nsSNPs among the studied genomes. Interestingly, the carrier strain CR0044 harboured far more genetic polymorphisms (83% mutant nsSNPs) compared with the closely related BL196 outbreak strain. Notably, the two highly related virulence-determinant genes, rpoS and tviE, were mutated in strains BL196 and CR0044, respectively, which revealed that the mutation in rpoS is stabilising, while that in tviE is destabilising. These microvariations provide novel insight into the optimisation of genes by the pathogens. However, the sporadic strain was found to be far more conserved compared with the others. CONCLUSIONS: The uncommon genomic variations in the two closely related BL196 and CR0044 strains suggests that S. Typhi is more diverse than previously thought. Our study has demonstrated that the pathogen is continually acquiring new genes through horizontal gene transfer in the process of host adaptation, providing novel insight into its unusual genomic dynamics. The understanding of these strains and virulence factors, and particularly the strain that is associated with the large outbreak and the less studied asymptomatic Typhi carrier in the population, will have important impact on disease control.

Detection of multiple potentially pathogenic bacteria in Matang mangrove estuaries, Malaysia.

The deltaic estuarine system of the Matang Mangrove Forest Reserve of Malaysia is a site where several human settlements and brackish water aquaculture have been established. Here, we evaluated the level of fecal indicator bacteria (FIB) and the presence of potentially pathogenic bacteria in the surface water and sediments. Higher levels of FIB were detected at downstream sampling sites from the fishing village, indicating it as a possible source of anthropogenic pollution to the estuary. Enterococci levels in the estuarine sediments were higher than in the surface water, while total coliforms and E. coli in the estuarine sediments were not detected in all samples. Also, various types of potentially pathogenic bacteria, including Klebsiella pneumoniae, Serratia marcescens and Enterobacter cloacae were isolated. The results indicate that the Matang estuarine system is contaminated with various types of potential human bacterial pathogens which might pose a health risk to the public.

Variations in motility and biofilm formation of Salmonella enterica serovar Typhi.

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) exhibits unique characteristics as an intracellular human pathogen. It causes both acute and chronic infection with various disease manifestations in the human host only. The principal factors underlying the unique lifestyle of motility and biofilm forming ability of S. Typhi remain largely unknown. The main objective of this study was to explore and investigate the motility and biofilm forming behaviour among S. Typhi strains of diverse background. RESULTS: Swim and swarm motility tests were performed with 0.25% and 0.5% agar concentration, respectively; while biofilm formation was determined by growing the bacterial cultures for 48 hrs in 96-well microtitre plate. While all S. Typhi strains demonstrated swarming motility with smooth featureless morphology, 58 out of 60 strains demonstrated swimming motility with featureless or bull's eye morphology. Interestingly, S. Typhi strains of blood-borne origin exhibited significantly higher swimming motility (P < 0.05) than stool-borne strains suggesting that swimming motility may play a role in the systemic invasion of S. Typhi in the human host. Also, stool-borne S. Typhi displayed a negative relationship between motility and biofilm forming behaviour, which was not observed in the blood-borne strains. CONCLUSION: In summary, both swimming and swarming motility are conserved among S. Typhi strains but there was variation for biofilm forming ability. There was no difference observed in this phenotype for S. Typhi strains from diverse background. These findings serve as caveats for future studies to understand the lifestyle and transmission of this pathogen.

Genome sequence and comparative pathogenomics analysis of a Salmonella enterica Serovar Typhi strain associated with a typhoid carrier in Malaysia.

Salmonella enterica serovar Typhi is a human pathogen that causes typhoid fever predominantly in developing countries. In this article, we describe the whole genome sequence of the S. Typhi strain CR0044 isolated from a typhoid fever carrier in Kelantan, Malaysia. These data will further enhance the understanding of its host persistence and adaptive mechanism.

Insights from the genome sequence of a Salmonella enterica serovar Typhi strain associated with a sporadic case of typhoid fever in Malaysia.

Salmonella enterica serovar Typhi is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths globally. Herein, we describe the whole-genome sequence of the Salmonella Typhi strain ST0208, isolated from a sporadic case of typhoid fever in Kuala Lumpur, Malaysia. The whole-genome sequence and comparative genomics allow an in-depth understanding of the genetic diversity, and its link to pathogenicity and evolutionary dynamics, of this highly clonal pathogen that is endemic to Malaysia.

Whole-genome sequences and comparative genomics of Salmonella enterica serovar Typhi isolates from patients with fatal and nonfatal typhoid fever in Papua New Guinea.

Many of the developing countries of the Southeast Asian region are significantly affected by endemic typhoid fever, possibly as a result of marginal living standards. It is an important public health problem in countries such as Papua New Guinea, which is geographically close to some of the foci of endemicity in Asia. The severity of the disease varies in different regions, and this may be attributable to genetic diversity among the native strains. Genome sequence data on strains from different countries are needed to clearly understand their genetic makeup and virulence potential. We describe the genomes of two Salmonella Typhi isolates from patients with fatal and nonfatal cases of typhoid fever in Papua New Guinea. We discuss in brief the underlying sequencing methodology, assembly, genome statistics, and important features of the two draft genomes, which form an essential step in our functional molecular infection epidemiology program centering on typhoid fever. The comparative genomics of these and other isolates would enable us to identify genetic rearrangements and mechanisms responsible for endemicity and the differential severity of pathogenic salmonellae in Papua New Guinea and elsewhere.