Using cultured mouse fibroblast L929 cells, this study demonstrated the hemolytic and cytotoxic activities and induction of apoptosis in cells infected with Orientia tsutsugamushi. Low levels of hemolytic activity were detected using heavily infected cells. No hemolysin or cytotoxin were detected in the infected culture fluid regardless of the pathogenicity of the O. tsutsugamushi strains in mice. Using propidium iodide uptake assay, acridine orange/ethidium bromide staining and terminal deoxynucleotide transferase-mediated dUTP-digoxigenin nick-end labeling assay, apoptosis was observed in L929 cells infected with Karp and Gilliam strains.
In Malaysia, Shigella spp. was reported to be the third commonest bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "gold standard". This study aimed to detect the prevalence of four Shigella virulence genes present concurrently, in randomly selected Malaysian strains via a rapid multiplex PCR (mPCR) assay.
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.
The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70% of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of DNA 0.8 pg microl(-1). This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies.
Vibrio cholerae infection is mainly caused acute diarrhoea disease. Bacteraemia due to non-O1 V. cholerae is rare and mainly reported in liver cirrhotic patients. We report one case of non-O1 V. cholerae bacteraemia in splenectomised thalassaemic patient who presented with septic shock secondary to abdominal sepsis. She had undergone emergency laporatomy and was managed in the intensive care unit for nine days. She was treated with meropenem and doxycyline and discharged well after fourteen days of admission. The V. cholerae was identified by API 20NE, serotype and polymerase chain reaction showed as non-O1, non-O139 strain. Besides known cholera-like toxin and El Tor hemolysin, with increasing reported cases of V. cholerae bacteraemia, there is possibility of other virulence factors that allow this organism to invade the bloodstream.
Methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398) has frequently been detected in pigs and pig handlers. However, in Malaysia, sampling 360 pigs and 90 pig handlers from 30 farms identified novel ST9-spa type t4358-staphylococcal cassette chromosome mec type V MRSA strains that were found to transiently colonize more than 1% of pigs and 5.5% of pig handlers.
The β-1,6-glucanases are ubiquitous enzymes which appear to be implicated in the morphogenesis and have the ability to become virulence factor in plant-fungal symbiotic interaction. To our knowledge, no report on ß-1,6-glucanases purification from Trichoderma longibrachiatum has been made, although it has been proven to have a significant effect as a biocontrol agent for several diseases. Therefore, the aim of this study was to purify β-1,6- glucanase from T. longibrachiatum T28, with an assessment on the physicochemical properties and substrate specificity. β-1,3-glucanase enzyme, from the culture filtrate of T. longibrachiatum T28, was successively purified through precipitation with 80% acetone, followed by anionexchange chromatography on Neobar AQ and chromatofocusing on a Mono P HR 5/20 column. (One β-1,6-glucanase) band at 42kDa in size was purified, as shown by the SDS-PAGE. The physicochemical evaluation showed an optimum pH of 5 and optimum temperature of 50°C for enzyme activity with an ability to maintain 100% enzyme stability. Enzyme activity was slightly reduced by 10-20% in the presence of 20 mM of Zn2+, Ca2+, Co2+, Mg2+, Cu2+, Mn2+ and Fe2+. The highest β-1,6-glucanase hydrolysis activity was obtained on pustulan due to the similarity of β-glucosidic bonds followed by laminarin, glucan and cellulose. Therefore, it can be concluded that the characterization of ß-1,6-glucanase secreted by T. longibrachiatum in term of molecular weight, responsed to selected physicochemical factors and the substrate specificity are approximately identical to other Trichoderma sp.
Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.
We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.
Helicobacter pylori-related disease is at least partially attributable to the genotype of the infecting strain, particularly the presence of specific virulence factors. We investigated the prevalence of a novel combination of H. pylori virulence factors, including the cag pathogenicity island (PAI), and their association with severe disease in isolates from the three major ethnicities in Malaysia and Singapore, and evaluated whether the cag PAI was intact and functional in vitro. Polymerase chain reaction (PCR) was used to detect dupA, cagA, cagE, cagT, cagL and babA, and to type vacA, the EPIYA motifs, HP0521 alleles and oipA ON status in 159 H. pylori clinical isolates. Twenty-two strains were investigated for IL-8 induction and CagA translocation in vitro. The prevalence of cagA, cagE, cagL, cagT, babA, oipA ON and vacA s1 and i1 was >85%, irrespective of the disease state or ethnicity. The prevalence of dupA and the predominant HP0521 allele and EPIYA motif varied significantly with ethnicity (p < 0.05). A high prevalence of an intact cag PAI was found in all ethnic groups; however, no association was observed between any virulence factor and disease state. The novel association between the HP0521 alleles, EPIYA motifs and host ethnicity indicates that further studies to determine the function of this gene are important.
The Lancefield group C alpha-hemolytic Streptococcus dysgalactiae ssp. dysgalactiae (GCSD) causes systemic granulomatous inflammatory disease and high mortality rates in infected fish. Superantigen and streptolysin S genes are the most important virulence factors contributing to an invasive streptococcal infection. PCR amplification revealed that all strains isolated from moribund fish harbored the streptolysin S structural gene (sagA). GCSD fish isolates were PCR negative for emm, speA, speB, speC, speM, smeZ, and ssa. However, the size of the streptococcal pyrogenic exotoxin G (spegg) locus, a superantigen, in positive S. dysgalactiae fish and pig strains was variable. The ORF of the spegg locus of 26 GCSD fish strains and one GCSD pig strain was inserted with IS981SC. Interestingly, the ORF of the spegg locus of two fish strains of GCSD collected in Malaysia was inserted with an IS981SC-IS1161 hybrid IS element. The hybrid IS element was found in all of the GCSD fish isolates and one GCSD pig through PCR screening. Although no insertion sequence (IS) was detected in the spegg locus of S. dysgalactiae ssp. equisimilis (GCSE) strains, a five-nucleotide deletion mutation was detected in the ORF of the spegg locus of one GCSE strain at the supposed site of IS981SC insertion, resulting in a frameshift mutation.
A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.
Despite the association of methicillin-susceptible S. aureus (MSSA) with several life-threatening diseases, relatively little is known about their clinical epidemiology in Malaysia. We characterized MSSA isolates (n=252) obtained from clinical and community (carriage) sources based on spa sequencing and multilocus sequence typing (MLST). The prevalence of several important virulence genes was determined to further define the molecular characteristics of MSSA clones circulating in Malaysia. Among the 142 clinical and 110 community-acquired MSSA isolates, 98 different spa types were identified, corresponding to 8 different spa clonal clusters (spa-CCs). In addition, MLST analysis revealed 22 sequence types (STs) with 5 singletons corresponding to 12 MLST-CCs. Interestingly, spa-CC084/085 (MLST-CC15) (p=0.038), spa-non-founder 2 (MLST-ST188) (p=0.002), and spa-CC127 (MLST-CC1) (p=0.049) were identified significantly more often among clinical isolates. spa-CC3204 (MLST-CC121) (p=0.02) and spa-CC015 (MLST-CC45) (p=0.0002) were more common among community isolates. Five dominant MLST-CCs (CC8, CC121, CC1, CC45, and CC5) having clear counterparts among the major MRSA clones were also identified in this study. While the MSSA strains are usually genetically heterogeneous, a relatively high frequency (19/7.5%) of ST188 (t189) strains was found, with 57.8% of these strains carrying the Panton-Valentine leukocidin (PVL). Analysis of additional virulence genes showed a frequency of 36.5% and 36.9% for seg and sei and 0.8% and 6.3% for etb and tst genes, respectively. Arginine catabolic mobile element (ACME) was detected in 4 community isolates only. These represent the first isolates harbouring this gene in an Asian region. In conclusion, MSSA from the Malaysian community and their clinical counterparts are genetically diverse, but certain clones occur more often among clinical isolates than among carriage isolates and vice versa.
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.
This paper describes the development and application of multilocus sequencing typing (MLST) and multi-virulence locus sequencing typing (MVLST) methods in determining the genetic variation and relatedness of 43 Vibrio cholerae strains of different serogroups isolated from various sources in Malaysia. The MLST assay used six housekeeping genes (dnaE, lap, recA, gyrB, cat and gmd), while the MVLST assay incorporated three virulence genes (ctxAB, tcpA and tcpI) and three virulence-associated genes (hlyA, toxR and rtxA). Our data showed that the dnaE and rtxA genes were the most conserved genes in V. cholerae O1 strains. Among the 12 studied genes, transitional substitutions that led to silent mutations were observed in all, except for gmd and hlyA, while non-synonymous substitutions occurred more frequently in virulence and virulence-associated genes. Five V. cholerae O1 strains were found to be the El Tor variant O1 strains because they harboured the classical ctxB gene. In addition, the classical ctxB gene was also observed in O139 V. cholerae. A total of 29 MLST types were observed, and this assay could differentiate V. cholerae within the non-O1/non-O139 serogroups. A total of 27 MVLST types were obtained. MVLST appeared to be more discriminatory than MLST because it could differentiate V. cholerae strains from two different outbreaks and could separate the toxigenic from the non-toxigenic subtypes. Although the O1 V. cholerae strains were closely related, the combined MLST and MVLST analyses differentiated the strains isolated from different localities. In conclusion, sequence-based analysis in this study provided a better understanding of mutation points and the type of mutations in V. cholerae. The MVLST assay is useful to characterise O1 V. cholerae strains, while combined analysis may improve the discriminatory power and is suitable for the local epidemiological study of V. cholerae.
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.
The ubiquitous Candida spp. is an opportunistic fungal pathogen which, despite treatment with antifungal drugs, can cause fatal bloodstream infections (BSIs) in immunocompromised and immunodeficient persons. Thus far, several major C. albicans virulence factors have been relatively well studied, including morphology switching and secreted degradative enzymes. However, the exact mechanism of Candida pathogenesis and the host response to invasion are still not well elucidated. The relatively recent discovery of the quorum-sensing molecule farnesol and the existence of quorum sensing as a basic regulatory phenomenon of the C. albicans population behavior has revolutionized Candida research. Through population density regulation, the quorum-sensing mechanism also controls the cellular morphology of a C. albicans population in response to environmental factors, thereby, effectively placing morphology switching downstream of quorum sensing. Thus, the quorum-sensing phenomenon has been hailed as the 'missing piece' of the pathogenicity puzzle. Here, we review what is known about Candida spp. as the etiological agents of invasive candidiasis and address our current understanding of the quorum-sensing phenomenon in relation to virulence in the host.
Staphylococcus aureus is a persistent human pathogen responsible for a variety of infections ranging from soft-tissue infections to bacteremia. It produces a variety of virulence factors which are responsible for specific acute staphylococcal toxaemia syndromes. The objective of this study was to determine the prevalence of a repertoire of toxin genes among Malaysian MRSA strains and their genetic diversity by PCR-RFLP of coa gene. One hundred eighty-eight strains (2003, 2004, 2007 and 2008) of methicillin-resistant S. aureus (MRSA) were screened for 20 genes encoding for extracellular virulence determinant (sea, seb, sec, sed, see, seg, seh, sei, sej, tst, eta, etb, etd) and adhesins (cna, etb, fnbA, fnbB, hlg, ica, sdrE). The genetic relatedness of these strains was determined by PCR-RFLP of coa gene and agr grouping. Majority of the strains were tested positive for efb and fnbA (96% each), ica (78%) and hlg (59%) genes. A total of 101 strains were positive for at least one type of staphylococcal enterotoxin genes with sea being the predominant. Genes for seb, sed, see, seh, sej, eta and etb were not detected in any of the MRSA strains. The prevalence of sea, sec and ica among strains isolated in 2008 was increased significantly (p< 0.05) compared to 2003. Most of the strains were of agr type I (97.5%) followed by agr type II (1.2%) and agr type III (0.6%). All sea, sei and tst gene-positive strains were of agr type I. The only etd positive strain was agr type III. PCR-RFLP of coa produced 47 different patterns. The number of strains with virulence factors (sea, sec and ica) had increased over the years. No direct correlation between PCR-RFLP- coa profiles and virulotypes was observed.
To study risk factors, contributing factors of bacterial and fungal endophthalmitis in Upper Egypt, test the isolated species sensitive to some therapeutic agents, and to investigate the air-borne bacteria and fungi in opthalmology operating rooms.
The genome of Mycobacterium massiliense M172, isolated from a human sputum sample, was sequenced using Illumina GA IIX technology and found to contain 5,204,460 bp, including putative genes for virulence and antibiotic resistance as well as a 92-kb genomic region most likely to correspond to a mycobacteriophage.