The aim of this study was to investigate the prevalence and characterization of Listeria species and Listeria monocytogenes isolated from raw fish and open-air fish market environments. Eight hundred and sixty two samples including raw fish and fish market environments (samples from workers' hands, workers' knives, containers and work surface) were collected from the open-air fish markets in the Northern region of Iran.
A total of 103 group B streptococci (GBS) including 22 invasive, 21 non-invasive, and 60 colonizing isolates were collected in a Malaysian hospital (June 2010-October 2011). Isolates were characterized by conventional and molecular serotyping and analyzed for scpB, lmb, hylB, cylE, bac, bca and rib gene content. Antimicrobial susceptibility to penicillins, macrolides, lincosamides, quinolones and tetracyclines was determined using disk diffusion and the MICs for penicillin were determined by E-test. Molecular serotyping for all eight serotypes (Ia, Ib, II-VII) was in full accordance with conventional serotyping. Overall, taking CS and MS together, serotype VI was the most common capsular type (22.3 %) followed by VII (21.4 %), III (20.4 %), Ia (17.5 %), V (9.7 %), II (7.7 %) and IV (1 %). Susceptibility to beta-lactam antimicrobials was prevalent (100 %). Resistance rates for erythromycin, clindamycin and tetracycline were 23.3 %, 17.5 % and 71.8 %, respectively. PCR-virulence gene screening showed the presence of cylE, lmb, scpB and hylB in almost all the isolates while rib, bca, and bac genes were found in 29.1 %, 14.6 % and 9.7 % of the isolates. Certain genes were significantly associated with specific serotypes, namely, rib with serotypes Ia, II, III and VI; bca and bac with serotypes II and III. Furthermore, serotype Ia was significantly more common among patients with invasive infections (p
This study determined the risk factors and characteristics of vancomycin-resistant Enterococci (VRE) among individuals working with animals 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.
Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP2-149, VP1-145 and VP1-244. We investigate how these amino acids alter heparin-binding phenotype and shapes EV-A71 virulence in one-day old mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 149K/98E/145Q, termed KEQ) to generate KKQ, KKE, KEE, IEE and IEQ variants. We demonstrated that the weak heparin-binder IEE was highly lethal in mice. The initially strong heparin-binding IEQ variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased virus antigens in mice brain, with subsequent high virulence. IEE and IEQ-244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of IEQ attenuated the virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching between heparin-binding and weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.
Salmonella is an important food-borne pathogen causing disease in humans and animals worldwide. Salmonellosis may be caused by any one of over 2,500 serovars of Salmonella. Nonetheless, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Agona are the second most prevalent serovars isolated from humans and livestock products respectively. Limited knowledge is available about the virulence mechanisms responsible for diarrheal disease caused by them. To investigate the contribution of sopB, sopD and pipD as virulence factors in intracellular infections and the uniqueness of these bacteria becoming far more prevalent than other serovars, the infection model of Caenorhabditis elegans and phenotypic microarray were used to characterize their mutants. The strains containing the mutation in sopB, sopD and pipD genes were constructed by using latest site-specific group II intron mutagenesis approach to reveal the pathogenicity of the virulence factors. Overall, we observed that the mutations in sopB, sopD and pipD genes of both serovars did not exhibit significant decrease in virulence towards the nematode. This may indicate that these virulence effectors may not be universal virulence factors involved in conserved innate immunity. There are significant phenotypic differences amongst strains carrying sopB, sopD and pipD gene mutations via the analysis of biochemical profiles of the bacteria. Interestingly, mutant strains displayed different susceptibility to chemical stressors from several distinct pharmacological and structural classes when compared to its isogenic parental strains. These metabolic and chemosensitivity assays also revealed multiple roles of Salmonella virulence factors in nutrient metabolism and antibiotic resistance.
Mycobacterium iranicum is a newly reported mycobacterial species. We present the first comparative study of M. iranicum UM_TJL and other mycobacteria. We found M. iranicum to have a close genetic association with environmental mycobacteria infrequently associated with human infections. Nonetheless, UM_TJL is also equipped with many virulence genes (some of which appear to be the consequence of transduction-related gene transfer) that have been identified in established human pathogens. Taken all together, our data suggest that M. iranicum is an environmental bacterium adapted for pathogenicity in the human host. This comparative study provides important clues and forms the basis for future functional studies on this mycobacterium.
The role of Helicobacter pylori (HP) in non-ulcer dyspepsia is debatable. Eradicating HP will help a small group of non-ulcer dyspeptic patients. However, it is unclear which subgroup of patients will benefit from eradication therapy. The aim of the present study was to compare the cagA and cagE status, as well as vacA genotypes, of HP in non-ulcer dyspeptic patients who responded successfully to eradication therapy compared with those patients who did not.
To date, no genome of any of the species from the genus Spiroplasma has been completely sequenced. Long repetitive sequences similar to mobile units present a major obstacle for current genome sequencing technologies. Here, we report the assembly of the Spiroplasma melliferum KC3 genome into 4 contigs, followed by proteogenomic annotation and metabolic reconstruction based on the discovery of 521 expressed proteins and comprehensive metabolomic profiling. A systems approach allowed us to elucidate putative pathogenicity mechanisms and to discover major virulence factors, such as Chitinase utilization enzymes and toxins never before reported for insect pathogenic spiroplasmas.
Enterococcus, a Gram-positive facultative anaerobic cocci belonging to the lactic acid bacteria of the phylum Firmicutes, is known to be able to resist a wide range of hostile conditions such as different pH levels, high concentration of NaCl (6.5%), and the extended temperatures between 5(°)C and 65(°)C. Despite being the third most common nosocomial pathogen, our understanding on its virulence factors is still poorly understood. The current study was aimed to determine the prevalence of different virulence genes in Enterococcus faecalis and Enterococcus faecium. For this purpose, 79 clinical isolates of Malaysian enterococci were evaluated for the presence of virulence genes. pilB, fms8, efaAfm, and sgrA genes are prevalent in all clinical isolates. In conclusion, the pathogenicity of E. faecalis and E. faecium could be associated with different virulence factors and these genes are widely distributed among the enterococcal species.
Median enterococci counts of beach water samples gradually increased at statistically significant levels (χ2: 26.53, df: 4; p<0.0001) with increasing proximity to river influx. The difference in proportion of antibiotic resistant enterococci in beach water and river water samples was statistically significant (p<0.05) for the tested antibiotics with river isolates generally presenting higher resistance frequencies. Virulence genes cyl, esp, gelE and asa were detected at varying frequencies (7.32%, 21.95%, 100% and 63.41% respectively) among river isolates. On the other hand, the prevalence of these genes was lower (0%, 20%, 67.27% and 41.82% respectively) among beach water isolates. Multi-Locus-Sequence-Typing analysis of Enterococcus faecalis presented four sequence types (ST) one of which shared six out of seven tested loci with ST6, a member of the clonal complex of multi-drug resistant strains associated with hospital outbreaks.
Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.
The virulence of Vibrio parahaemolyticus is variable depending on its virulence determinants. A V. parahaemolyticus strain, in which the virulence is governed by the pirA and pirB genes, can cause acute hepatopancreatic necrosis disease (AHPND) in shrimps. Some V. parahaemolyticus that are non-AHPND strains also cause shrimp diseases and result in huge economic losses, while their pathogenicity and pathogenesis remain unclear. In this study, a non-AHPND V. parahaemolyticus, TJA114, was isolated from diseased Penaeus vannamei associated with a high mortality. To understand its virulence and adaptation to the external environment, whole-genome sequencing of this isolate was conducted, and its phenotypic profiles including pathogenicity, growth characteristics and nutritional requirements were investigated. Shrimps following artificial infection with this isolate presented similar clinical symptoms to the naturally diseased ones and generated obvious pathological lesions. The growth characteristics indicated that the isolate TJA114 could grow well under different salinity (10-55 p.p.t.), temperature (23-37 °C) and pH (6-10) conditions. Phenotype MicroArray results showed that this isolate could utilize a variety of carbon sources, amino acids and a range of substrates to help itself adapt to the high hyperosmotic and alkaline environments. Antimicrobial-susceptibility test showed that it was a multidrug-resistant bacterium. The whole-genomic analysis showed that this V. parahaemolyticus possessed many important functional genes associated with multidrug resistance, stress response, adhesions, haemolysis, putative secreted proteases, dedicated protein secretion systems and a variety of nutritional metabolic mechanisms. These annotated functional genes were confirmed by the phenotypic profiles. The results in this study indicated that this V. parahaemolyticus isolate possesses a high pathogenicity and strong environmental adaptability.
The annual prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysia has been estimated to be 30 % to 40 % of all S. aureus infections. Nevertheless, data on the antimicrobial resistance and genetic diversity of Malaysian MRSAs remain few. In 2009, we collected 318 MRSA strains from various wards of our teaching hospital located in Kuala Lumpur, the capital city of Malaysia, and performed antimicrobial susceptibility testing on these strains. The strains were then molecularly characterized via staphylococcal cassette chromosome (SCC) mec and virulence gene (cna, sea, seb, sec, sed, see, seg, seh, sei, eta, etb, Panton-Valentine leukocidin and toxic shock syndrome toxin-1) typing; a subset of 49 strains isolated from the intensive care unit was also typed using PFGE. Most strains were found to be resistant to ciprofloxacin (92.5 %), erythromycin (93.4 %) and gentamicin (86.8 %). The majority (72.0 %) of strains were found to harbour SCCmec type III-SCCmercury with the presence of ccrC, and carried the sea+cna gene combination (49.3 %), with cna as the most prevalent virulence gene (94.0 %) detected. We identified four PFGE clusters, with pulsotype C (n=19) as the dominant example in the intensive care unit, where this pulsotype was found to be associated with carriage of SCCmec type III and the sea gene (P=0.05 and P=0.02, respectively). In summary, the dominant MRSA circulating in our hospital in 2009 was a clone that was ciprofloxacin, erythromycin and gentamicin resistant, carried SCCmec type III-SCCmercury with ccrC and also harboured the sea+cna virulence genes. This clone also appears to be the dominant MRSA circulating in major hospitals in Kuala Lumpur.
Brown spot disease, caused by Cochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice (Oryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C. miyabeanus as a virulence factor during brown spot disease and the identification of the non-ribosomal protein synthetase (NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C. miyabeanus strains differing in virulence ability using ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution Orbitrap mass spectrometry (HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem-quadrupole mass spectrometry (MS/MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild-type and Cmnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin-insensitive plant species, our data demonstrate that tentoxin production by C. miyabeanus affects symptom development.
Polymorphisms of Helicobacter pylori cagA and vacA genes do exist and may contribute to differences in H. pylori infection and gastroduodenal diseases among races in the Malaysian population. This study was conducted to characterize the polymorphisms in H. pylori cagA and vacA in Malaysian population.
Nosocomial infections have become alarming with the increase of multidrug-resistant bacterial strains of Acinetobacter baumannii. Being the causative agent in ~80% of the cases, these pathogenic gram-negative species could be deadly for hospitalized patients, especially in intensive care units utilizing ventilators, urinary catheters, and nasogastric tubes. Primarily infecting an immuno-compromised system, they are resistant to most antibiotics and are the root cause of various types of opportunistic infections including but not limited to septicemia, endocarditis, meningitis, pneumonia, skin, and wound sepsis and even urinary tract infections. Conventional experimental methods including typing, computational methods encompassing comparative genomics, and combined methods of reverse vaccinology and proteomics had been proposed to differentiate and develop vaccines and/or drugs for several outbreak strains. However, identifying proteins suitable enough to be posed as drug targets and/or molecular vaccines against the multidrug-resistant pathogenic bacterial strains has probably remained an open issue to address. In these cases of novel protein identification, the targets either are uncharacterized or have been unable to confer the most coveted protection either in the form of molecular vaccine candidates or as drug targets. Here, we report a strategic approach with the 3,766 proteins from the whole genome of A. baumannii ATCC19606 (AB) to rationally identify plausible candidates and propose them as future molecular vaccine candidates and/or drug targets. Essentially, we started with mapping the vaccine candidates (VaC) and virulence factors (ViF) of A. baumannii strain AYE onto strain ATCC19606 to identify them in the latter. We move on to build small networks of VaC and ViF to conceptualize their position in the network space of the whole genomic protein interactome (GPIN) and rationalize their candidature for drugs and/or molecular vaccines. To this end, we propose new sets of known proteins unearthed from interactome built using key factors, KeF, potent enough to compete with VaC and ViF. Our method is the first of its kind to propose, albeit theoretically, a rational approach to identify crucial proteins and pose them for candidates of vaccines and/or drugs effective enough to combat the deadly pathogenic threats of A. baumannii.
Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free.
Shigella is an intracellular bacterial pathogen that causes bacterial dysentery called shigellosis. The assessment of pro- and anti-inflammatory mediators produced by immune cells against this bacteria are vital in identifying the effectiveness of the immune reaction in protecting the host. In Malaysia, Shigella is ranked as the third most common bacteria causing diarrheal disease among children below 5 years old. In the present study, we aim to examine the differential cytokine gene expressions of macrophages in response to two types of clinical strains of Shigella flexneri 2a (S. flexneri 2a) isolated from patients admitted in Hospital Universiti Sains Malaysia, Kelantan, Malaysia. THP-1-derived macrophages, as the model of human macrophages, were infected separately with S. flexneri 2a mild (SH062) and virulence (SH057) strains for 6, 12, and 24 h, respectively. The gene expression level of inflammatory mediators was identified using real-time quantitative polymerase chain reaction (RT-qPCR). The production of nitric oxide (NO) by the macrophages was measured by using a commercialized NO assay kit. The ability of macrophages to kill the intracellular bacteria was assessed by intracellular killing assay. Induction of tumor necrosis factor-alpha (TNFα), interleukin (IL)-1β, IL-6, IL-12, inducible NO synthase (iNOS), and NO, confirmed the pro-inflammatory reaction of the THP-1-derived macrophages in response to S. flexneri 2a, especially against the SH507 strain. The SH057 also induced a marked increase in the expression levels of the anti-inflammatory cytokine mRNAs at 12 h and 24 h post-infection. In the intracellular killing assay, both strains showed less viable, indicating the generation of pro-inflammatory cytokines in the presence of iNOS and NO was crucial in the stimulation of macrophages for the host defense against shigellosis. Transcription analysis of THP-1-derived macrophages in this study identifies differentially expressed cytokine genes that correlated with the virulence factor of S. flexneri 2a.
INTRODUCTION: Uropathogenic virulence factors have been identified by comparing the prevalence of these among urinary tract isolates and environmental strains. The uropathogenic-specific protein (USP) gene is present on the pathogenicity island (PAI) of uropathogenic Escherichia coli (UPEC) and, depending on its two diverse gene types and the sequential patterns of three open reading frame units (orfUs) following it, there is a method to characterize UPEC epidemiologically called PAIusp subtyping.
METHODOLOGY: A total of 162 UPEC isolates from Sabah, Malaysia, were tested for the presence of the usp gene and the sequential patterns of three orfUs following it using polymerase chain reaction (PCR). In addition, by means of triplex PCR, the prevalence of the usp gene was compared with other two VFs of UPEC, namely alpha hemolysin (α-hly) and cytotoxic necrotizing factor (cnf-1) genes encoding two toxins.
RESULTS: The results showed that the usp gene was found in 78.40% of UPEC isolates, indicating that its prevalence was comparable to that found in a previous study in Japan. The two or three orfUs were also associated with the usp gene in this study. All the PAIusp subtypes observed in Japan were present in this study, while subtype IIa was the most common in both studies. The usp gene was observed in a higher percentage of isolates when compared with α-hly and cnf-1 genes.
CONCLUSIONS: The findings in Japan and Sabah, East Malaysia, were similar, indicating that PAIusp subtyping is applicable to the characterization of UPEC strains epidemiologically elsewhere in the world.