Since its discovery in 1969, enterovirus 71 (EV71) has been recognised as a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) associated with severe neurological sequelae in a small proportion of cases. There has been a significant increase in EV71 epidemic activity throughout the Asia-Pacific region since 1997. Recent HFMD epidemics in this region have been associated with a severe form of brainstem encephalitis associated with pulmonary oedema and high case-fatality rates. The emergence of large-scale epidemic activity in the Asia-Pacific region has been associated with the circulation of three genetic lineages that appear to be undergoing rapid evolutionary change. Two of these lineages (B3 and B4) have not been described previously and appear to have arisen from an endemic focus in equatorial Asia, which has served as a source of virus for HFMD epidemics in Malaysia, Singapore and Australia. The third lineage (C2) has previously been identified [Brown, B.A. et al. (1999) J. Virol. 73, 9969-9975] and was primarily responsible for the large HFMD epidemic in Taiwan during 1998. As EV71 appears not to be susceptible to newly developed antiviral agents and a vaccine is not currently available, control of EV71 epidemics through high-level surveillance and public health intervention needs to be maintained and extended throughout the Asia-Pacific region. Future research should focus on (1) understanding the molecular genetics of EV71 virulence, (2) identification of the receptor(s) for EV71, (3) development of antiviral agents to ameliorate the severity of neurological disease and (4) vaccine development to control epidemics. Following the successful experience of the poliomyelitis control programme, it may be possible to control EV71 epidemics if an effective live-attenuated vaccine is developed.
Yersinia is a Gram-negative bacteria that includes serious pathogens such as the Yersinia pestis, which causes plague, Yersinia pseudotuberculosis, Yersinia enterocolitica. The remaining species are generally considered non-pathogenic to humans, although there is evidence that at least some of these species can cause occasional infections using distinct mechanisms from the more pathogenic species. With the advances in sequencing technologies, many genomes of Yersinia have been sequenced. However, there is currently no specialized platform to hold the rapidly-growing Yersinia genomic data and to provide analysis tools particularly for comparative analyses, which are required to provide improved insights into their biology, evolution and pathogenicity.
The severity of disease outcome in dyspepsia has been attributed to Helicobacter pylori virulence genes. The aim of this study was to determine the distribution of H. pylori virulence genes (cagA, babA2, and dupA) and to determine whether or not there arises a significant correlation with clinical dyspepsia outcomes.
The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.
Molecular analysis of Malaysian Vibrio cholerae was carried out using a multiple-locus variable-number tandem repeat analysis (MLVA) assay based on 7 loci of V. cholerae. The discriminatory ability of the assay was compared with pulsed-field gel electrophoresis (PFGE) using 43 Malaysian V. cholerae isolated from various sources. In addition, the virulotypes of the strains were determined. Based on MLVA, 38 allelic profiles were obtained (F = 0.63) while PFGE generated 35 pulsotypes (F = 0.71). Simpson's index of diversity for different VNTR loci ranged from 0.59 to 0.92. The combined loci increased the discriminatory index to 0.99 which was comparable with PFGE (D = 0.99). Most of the environmental non-O1/non-O139 strains harbored rtxA, rstR, toxR, and hlyA only, and the virulotype of this serogroup was significantly different (P < .01) from clinical/environmental O1 and environmental O139 strains. In conclusion, the MLVA assay developed in this study was a useful genotyping tool with comparable discriminatory power with PFGE. In addition, the combination of the two approaches can further distinguish the strains from different sources and geographical regions of isolation.
Legionella pneumophila are intracellular pathogens, associated with human disease, attributed to the presence and absence of certain virulent genes. In this study, virulent gene loci (lvh and rtxA regions) associated with human disease were determined. Thirty-three cooling tower water isolates, isolated between 2004 to 2006, were analyzed for the presence of these genes by PCR method. Results showed that 19 of 33 (57.5%) of the L. pneumophila serogroup 1 isolates have both the genes. Six (18.2%) of the isolates have only the lvh gene and 2 (6.1%) of the isolates have only the rtxA gene. However, both genes were absent in 6 (18.2%) of the L. pneumophila isolates. The result of our study provides some insight into the presence of the disease causing L. pneumophila serogroup 1 in the environment. Molecular epidemiological studies will provide better understanding of the prevalence of the disease in Malaysia.
The quorum sensing (QS) system has been used by many opportunistic pathogenic bacteria to coordinate their virulence determinants in relation to cell-population density. As antibiotic-resistant bacteria are on the rise, interference with QS has been regarded as a novel way to control bacterial infections. As such, many plant-based natural products have been widely explored for their therapeutic roles. These natural products may contain anti-QS compounds that could block QS signals generation or transmission to combat QS pathogens. In this study, we report the anti-QS activities of four different Chinese herbal plant extracts: Poria cum Radix pini, Angelica dahurica, Rhizoma cibotii and Schizonepeta tenuifolia, on Pseudomonas aeruginosa PAO1. All the plants extracted using hexane, chloroform and methanol were tested and found to impair swarming motility and pyocyanin production in P.aeruginosa PAO1, particularly by Poria cum Radix pini. In addition, all the plant extracts also inhibited violacein production in C.violaceum CV026 up to 50% while bioluminescence activities were reduced in lux-based E. coli biosensors, pSB401 and pSB1075, up to about 57%. These anti-QS properties of the four medicinal plants are the first documentation that demonstrates a potential approach to attenuate pathogens’ virulence determinants.
To characterise the cag pathogenicity island in Helicobacter pylori (H. pylori) isolates by analysing the strains' vacA alleles and metronidazole susceptibilities in light of patient ethnicity and clinical outcome.
Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.
Chronic bacterial infections occur as a result of the infecting pathogen's ability to live within a biofilm, hence escaping the detrimental effects of antibiotics and the immune defense system. Burkholderia pseudomallei, a gram-negative facultative pathogen, is distinctive in its ability to survive within phagocytic and non-phagocytic cells, to persist in vivo for many years and subsequently leading to relapse as well as the development of chronic disease. The capacity to persist has been attributed to the pathogen's ability to form biofilm. However, the underlying biology of B. pseudomallei biofilm development remains unresolved.
A pair of primers targeting the hlyA gene for Vibrio cholerae which could distinguish the classical from El Tor biotypes was designed and combined with other specific primers for ompW, rfb complex, and virulence genes such as ctxA, toxR, and tcpI in a multiplex PCR (m-PCR) assay. This m-PCR correctly identified 39 V. cholerae from clinical, water and seafood samples. The efficiency of this multiplex PCR (m-PCR) was compared with conventional biochemical and serogrouping methods. One O139 and 25 O1 V. cholerae strains including 10 environmental strains harbored all virulence-associated genes except 1 clinical strain which only had toxR and hlyA genes. Thirteen environmental strains were classified as non-O1/non-O139 and had the toxR and hlyA genes only. The detection limit of m-PCR was 7 x 10(4) cfu/ml. The m-PCR test was reliable and rapid and reduced the identification time to 4 h.
Colony morphology variation is a characteristic of Burkholderia pseudomallei primary clinical isolates, associated with variations in expression of virulence factors. Here, we performed comparative investigations on adhesion, invasion, plaque-forming abilities and protein profiles of B. pseudomallei wild-type (WT) and a small colony variant (SCV). The percentage of SCV adherence to A549 cells was significantly higher (2.73%) than WT (1.91%). In contrast, WT was significantly more efficient (0.63%) than SCV (0.31%) in invasiveness and in inducing cellular damage. Using 2-DE and MALDI TOF/TOF, 263 and 258 protein spots were detected in WT and SCV, respectively. Comparatively, 49 proteins were differentially expressed in SCV when compared with WT. Of these, 31 proteins were up-regulated, namely, nucleoside diphosphate kinase (Ndk), phosphoglycerate kinase (Pgk), thioredoxin (TrxA), putative ferritin DPS-family DNA-binding protein (DPS) and oxidoreductase (AhpC) that are known to be involved in adhesion, intracellular survival and persistence. However, among the 18 down-regulated proteins, enolase (Eno), elongation factor (EF-Tu) and universal stress-related proteins were associated with invasion and virulence. Differences observed in these protein profiles provide ample clues to their association with the morphotypic and phenotypic characteristics of colony variants, providing additional insights into the potential association of B. pseudomallei colony morphotypes with disease pathogenesis.
Cladophialophora bantiana is a dematiaceous fungus with a predilection for causing central nervous system (CNS) infection manifesting as brain abscess in both immunocompetent and immunocompromised patients. In this paper, we report comprehensive genomic analyses of C. bantiana isolated from the brain abscess of an immunocompetent man, the first reported case in Malaysia and Southeast Asia. The identity of the fungus was determined using combined morphological analysis and multilocus phylogeny. The draft genome sequence of a neurotrophic fungus, C. bantiana UM 956 was generated using Illumina sequencing technology to dissect its genetic fundamental and basic biology. The assembled 37.1 Mb genome encodes 12,155 putative coding genes, of which, 1.01% are predicted transposable elements. Its genomic features support its saprophytic lifestyle, renowned for its versatility in decomposing hemicellulose and pectin components. The C. bantiana UM 956 was also found to carry some important putative genes that engaged in pathogenicity, iron uptake and homeostasis as well as adaptation to various stresses to enable the organism to survive in hostile microenvironment. This wealth of resource will further catalyse more downstream functional studies to provide better understanding on how this fungus can be a successful and persistent pathogen in human.
Enterococcus faecium is an opportunistic pathogen with a remarkable ability to acquire resistance toward multiple antibiotics, including those of last-resort drugs such as vancomycin and daptomycin. The occurrence of vancomycin-resistant E. faecium is on the rise and there is a need to understand the virulence of this organism. One of the factors that contributes to the virulence is the ability to form biofilms. Since bacteria in biofilm state are more resistant to antibiotics and host immune response, understanding the molecular mechanism of biofilm development is important to control biofilm-related diseases. The aim of this study was to determine the global gene expression profiles of an E. faecium strain, VREr5, during the early event of sessile growth compared with its planktonic phase through RNA-sequencing approach. The results clearly illustrated distinct expression profiles of the planktonic and biofilm cells. A total of 177 genes were overexpressed in the biofilm cells. Most of them encode for proteins involved in adherence, such as the ebpABCfm locus. Genes associated with plasmid replication, gene exchange, and protein synthesis were also upregulated during the early event of biofilm development. Furthermore, the transcriptome analysis also identified genes such as fsrB, luxS, and spx that might suppress biofilm formation in VREr5. The putative biofilm-related bee locus was found to be downregulated. These new findings could provide caveats for future studies on the regulation and maintenance of biofilm and development of biomarkers for biofilm-related diseases.
Postweaning diarrhea caused by pathogenic Escherichia coli, in particular verotoxigenic E. coli (VTEC), has caused significant economic losses in the pig farming industry worldwide. However, there is limited information on VTEC in Malaysia. The objective of this study was to characterize pathogenic E. coli isolated from post-weaning piglets and growers with respect to their antibiograms, carriage of extended-spectrum beta-lactamases, pathotypes, production of hemolysins and fimbrial adhesins, serotypes, and genotypes.
There is a geographic variation in Helicobacter pylori (HP) genotypes and virulence factors. Cytotoxin associated genes A (cagA) and E (cagE), and certain vacuolating cytotoxin (vacA) genotypes are associated with peptic ulcer disease (PUD). There is also a different prevalence of PUD among different ethnic groups in Malaysia. The present study compared the distribution of vacA alleles and cagA and cagE status in three ethnic groups residing in Kuala Lumpur, Malaysia, and their association with clinical outcome.
Avian influenza viruses are highly infectious micro-organisms that primarily affect birds. Nevertheless, they have also been isolated from a number of mammals, including humans. Avian influenza virus can cause large economic losses to the poultry industry because of its high mortality. Although there are pathogenic variants with a low virulence and which generally cause only mild, if any, clinical symptoms, the subtypes H5 and H7 can mutate from a low to a highly virulent (pathogenic) virus and should be taken into consideration in eradication strategies. The primary source of infection for commercial poultry is direct and indirect contact with wild birds, with waterfowl forming a natural reservoir of the virus. Live-poultry markets, exotic birds, and ostriches also play a significant role in the epidemiology of avian influenza. The secondary transmission (i.e., between poultry farms) of avian influenza virus is attributed primarily to fomites and people. Airborne transmission is also important, and the virus can be spread by aerosol in humans. Diagnostic tests detect viral proteins and genes. Virus-specific antibodies can be traced by serological tests, with virus isolation and identification being complementary procedures. The number of outbreaks of avian influenza seems to be increasing - over the last 5 years outbreaks have been reported in Italy, Hong Kong, Chile, the Netherlands, South Korea, Vietnam, Japan, Thailand, Cambodia, Indonesia, Laos, China, Pakistan, United States of America, Canada, South Africa, and Malaysia. Moreover, a growing number of human cases of avian influenza, in some cases fatal, have paralleled the outbreaks in commercial poultry. There is great concern about the possibility that a new virus subtype with pandemic potential could emerge from these outbreaks. From the perspective of human health, it is essential to eradicate the virus from poultry; however, the large number of small-holdings with poultry, the lack of control experience and resources, and the international scale of transmission and infection make rapid control and long-term prevention of recurrence extremely difficult. In the Western world, the renewed interest in free-range housing carries a threat for future outbreaks. The growing ethical objections to the largescale culling of birds require a different approach to the eradication of avian influenza.
Previously we have shown that very virulent infectious bursal disease viruses (vvIBDV) that are SspI, TaqI and StyI positive (92/04, 97/61 and 94/B551) but not SspI and TaqI positive and StyI negative (94/273) cause high mortality, up to 80% in specific-pathogen-free chickens with significant damage of the bursal as well as nonbursal tissues. In this study, we sequenced the VP2 gene (1351 bp) of the 92/04, 94/273 and 94/B551 and compared them with other IBDV strains. All the isolates have the unique amino acid residues at positions 222A, 256I, 294I and 299S found in other vvIBDV strains. The deduced VP2 amino acids encoded by 92/04 is identical to the vvIBDV strains from Israel (IBDVKS), Japan (OKYM) and Europe (UK661), whereas the 94/273 and 94/B551 isolates have one to three amino acid substitutions. The 94/273 has two amino acid substitutions at positions 254 G to S and at 270 A to E that have not been reported before from vvIBDV strains. The 94/B551 also has one amino acid substitution at position 300 E to S, which is uncommon among other vvIBDV strains. However, phylogenetic analysis suggested that the isolates are very close to each other and all of them may have derived from the same origin as vvIBDV strains isolated from China, Japan and Europe. Even though antigenic index analysis of the 94/273 and 94/B551 indicated that the isolates are unique compared to other IBDV strains, their antigenic variation remain to be determined by monoclonal antibody study.