By means of the gentamicin HEp-2 cell invasion assay, it was demonstrated that 82% of the Campylobacters tested were cell-invasive, including 83% of isolates from bloody diarrhoea and 80% of isolates from watery diarrhoea. The large number of invasive strains from watery diarrhoea suggests the possible role of invasiveness in the production of watery diarrhoea. Whether this stage can progress further to more severe symptoms such as bloody diarrhoea remains to be elucidated. Whether this progression to bloody diarrhoea occurs as a result of toxin production is still debatable. In Vero cells, invasion was less efficient and intracellular multiplication was not observed.
Limited information is available on tropical ticks and tick-borne bacteria affecting the health of humans and animals in the Southeast Asia region. Francisella tularensis is a tick-borne bacterium which causes a potentially life-threatening disease known as tularemia. This study was conducted to determine the occurrence of Francisella spp. in questing ticks collected from Malaysian forest reserve areas. A total of 106 ticks (mainly Dermacentor and Haemaphysalis spp.) were examined for Francisella DNA using a Polymerase chain reaction (PCR) assay targeting the bacterial 16S rDNA. Francisella DNA was detected from 12 Dermacentor ticks. Sequence analysis of the amplified 16S rDNA sequences (1035 bp) show >99% identity with that of Francisella endosymbiont reported in a tick from Thailand. A dendrogram constructed based on the bacterial 16S rDNA shows that the Francisella spp. were distantly related to the pathogenic strains of F. tularensis. Three Francisella-positive ticks were identified as Dermacentor atrosignatus, based on sequence analysis of the tick mitochondrial 16S rRNA gene. Further screening of cattle and sheep ticks (Haemaphysalis bispinosa and Rhipicephalus microplus) and animal samples (cattle, sheep, and goats) did not yield any positive findings. Our findings provide the first molecular data on the occurrence of a Francisella strain with unknown pathogenicity in Dermacentor questing ticks in Malaysia.
Anaplasma spp. are Gram-negative obligate intracellular, tick-borne bacteria which are of medical and veterinary importance. Little information is available on Anaplasma infection affecting domestic and wildlife animals in Malaysia. This study investigated the presence of Anaplasma spp. in the blood samples of domestic and wildlife animals in Peninsular Malaysia, using polymerase chain reaction (EHR-PCR) assays targeting the 16S rRNA gene of Anaplasmataceae. High detection rates (60.7% and 59.0%, respectively) of Anaplasma DNA were noted in 224 cattle (Bos taurus) and 78 deer (77 Rusa timorensis and one Rusa unicolor) investigated in this study. Of the 60 amplified fragments obtained for sequence analysis, Anaplasma marginale was exclusively detected in cattle while Anaplasma platys/Anaplasma phagocytophilum was predominantly detected in the deer. Based on sequence analyses of the longer fragment of the 16S rRNA gene (approximately 1000 bp), the occurrence of A. marginale, Anaplasma capra and Candidatus Anaplasma camelii in cattle, Candidatus A. camelii in deer and Anaplasma bovis in a goat was identified in this study. To assess whether animals were infected with more than one species of Anaplasma, nested amplification of A. phagocytophilum, A. bovis and Ehrlichia chaffeensis DNA was performed for 33 animal samples initially screened positive for Anaplasmataceae. No amplification of E. chaffeensis DNA was obtained from animals investigated. BLAST analyses of the 16S rDNA sequences from three deer (R. timorensis), a buffalo (Bubalus bubalis) and a cow (B. taurus) reveal similarity with that of Candidatus Anaplasma boleense strain (GenBank accession no.: KX987335). Sequence analyses of the partial gene fragments of major surface protein (msp4) gene from two deer (R. timorensis) and a monitor lizard (Varanus salvator) show the detection of a strain highly similar (99%) to that of A. phagocytophilum strain ZJ-China (EU008082). The findings in this study show the occurrence of various Anaplasma species including those newly reported species in Malaysian domestic and wildlife animals. The role of these animals as reservoirs/maintenance hosts for Anaplasma infection are yet to be determined.
This study reports the molecular detection of Theileria spp. from six cattle farms, a sheep farm and a goat farm located at different states in Peninsular Malaysia. Animal blood samples were screened for the presence of Theileria DNA using a conventional polymerase chain reaction (PCR) assay. A total of 155 (69.2%) of 224 cattle investigated were PCR-positive for Theileria DNA. The occurrences of Theileria spp. ranged from 17.5% to 100.0% across six cattle farms. Theileria DNA was detected from 90.0% of 40 sheep but none of 40 goats examined in this study. Sequence analyses of amplified 18S rRNA partial fragments (335-338bp) confirmed the identification of Theileria buffeli, Theileria sergenti, and Theileria sinensis in representative samples of cattle and ticks. T. luwenshuni was identified in the infected sheep. The high occurrences of Theileria spp. in our farm animals highlight the needs for appropriate control and preventive measures for theileriosis.
A multilocus sequence analysis using mitochondria-encoded cytochrome c oxidase subunit I (COI), cytochrome B (CytB), NADH dehydrogenase subunit 5 (ND5); nuclear encoded 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) genes was performed to determine the levels of genetic variation between the closely related species Haematobia irritans Linnaeus and Haematobia exigua de Meijere. Among these five genes, ND5 and CytB genes were found to be more variable and informative in resolving the interspecific relationships of both species. In contrast, the COI gene was more valuable in inferring the intraspecific relationships. The ribosomal 18S and 28S sequences of H. irritans and H. exigua were highly conserved with limited intra- and inter-specific variation. Molecular evidence presented in this study demonstrated that both flies are genetically distinct and could be differentiated based on sequence analysis of mitochondrial genes.
Anaplasma spp. infects a wide variety of wildlife and domestic animals. This study describes the identification of a novel species of Anaplasma (Candidatus Anaplasma pangolinii) from pangolins (Manis javanica) and Anaplasma bovis from wild boars (Sus scrofa) in Malaysia. Based on 16S rRNA gene sequences, Candidatus Anaplasma pangolinii is identified in a distinct branch within the family Anaplasmataceae, exhibiting the closest sequence similarity with the type strains of Anaplasma bovis (97.7%) and Anaplasma phagocytophilum (97.6%). The sequence also aligned closely (99.9%) with that of an Anaplasma spp. (strain AnAj360) detected from Amblyomma javanense ticks. The nearly full length sequence of the 16S rRNA gene derived from two wild boars in this study demonstrated the highest sequence similarity (99.7%) to the A. bovis type strain. Partial 16S rRNA gene fragments of A. bovis were also detected from a small population of Haemaphysalis bispinosa cattle ticks in this study. Our finding suggests a possible spread of two Anaplasma species in the Malaysian wildlife and ticks. The zoonotic potential of the Anaplasma species identified in this study is yet to be determined.
Rickettsioses of the typhus group (TG) and spotted fever group (SFG) are emerging bacterial infections worldwide, especially in the tropics. Only a few studies on these pathogens and their respective clinical diseases have been conducted in Malaysia. Here, we performed a seroprevalence study among 544 healthy, afebrile indigenous people (Orang Asli) from peninsular Malaysia for TG and SFG rickettsioses in nine rural and peri-urban settlements. The study population encompassed children, adolescents, and adults. The overall seroprevalence of rickettsiosis in the Orang Asli was 48.5%, with 27.9% seroprevalence against TG rickettsiae and 20.6% seroprevalence against SFG rickettsiae. In 7.9% of the study participants, antibodies against both rickettsial groups were found. The highest seropositivity rates against TG and SRG rickettsiae were detected in young children and adults. Overall, there were no gender differences. Seroprevalences were similar among inhabitants of different settlements, except for two localities. More studies are needed to shed more light on the ecology and risk factors for TG and SFG rickettsioses in Malaysia.
This study was conducted to determine the occurrence of Anaplasma spp. in the blood samples of cattle, goats, deer and ticks in a Malaysian farm. Using polymerase chain reaction (PCR) and sequencing approach, Anaplasma spp. was detected from 81(84.4%) of 96 cattle blood samples. All blood samples from 23 goats and 22 deer tested were negative. Based on the analysis of the Anaplasma partial 16S ribosomal RNA gene, four sequence types (genotypes 1 to 4) were identified in this study. Genotypes 1-3 showed high sequence similarity to those of Anaplasma platys/ Anaplasma phagocytophilum, whilst genotype 4 was identical to those of Anaplasma marginale/ Anaplasma centrale/ Anaplasma ovis. Anaplasma DNA was detected from six (5.5%) of 109 ticks which were identified as Rhipicephalus (formely known as Boophilus) microplus ticks collected from the cattle. This study reported for the first time the detection of four Anaplasma sequence types circulating in the cattle population in a farm in Malaysia. The detection of Anaplasma DNA in R. microplus ticks in this study provides evidence that the ticks are one of the potential vectors for transmission of anaplasmosis in the cattle.
Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
This study reports for the first time molecular detection of Anaplasma platys infection in 4 (13.3%) of 30 Malaysian dogs investigated. A low occurrence (3.3%) of Babesia gibsoni was also noted, being detected in one of the 30 dogs. Rickettsia, Bartonella, Orientia tsutsugamushi, and Ehrlichia DNA were not detected in the dog blood samples. The role of A. platys as an agent of canine anaplasmosis and its transmission through Rhipicephalus sanguineus ticks merits further investigation.
Burkholderia pseudomallei the causative agent of melioidosis, is being increasingly recognized as an important cause of morbidity and mortality in South East Asia. Biofilm formation of B. pseudomallei may be responsible for dormancy, latency and relapse of melioidosis. Based on the colonial morphology of the bacteria on B. pseudomallei selective agar medium, seven distinct morphotypes were identified. This study was conducted to assess the in vitro biofilm produced by B. pseudomallei and to investigate possible correlation between B. pseudomallei morphotypes with biofilm forming abilities of the isolates. Using a standard biofilm crystal violet staining assay, comparison was made between the biofilm forming ability of 76 isolates of B. pseudomallei and Burkholderia thailandensis ATCC 700388. Amongst the blood isolates, 30.2% were considered as high biofilm producers and 27.9% were low producers, 33.3% of the pus isolates were considered as high and 16% low biofilm producers. Most of the isolates were identified as morphotype group 1 which displayed a rough centre with irregular circumference on the agar medium. However, we did not find any correlation of B. pseudomallei morphotypes with biofilm forming abilities (p > 0.05). Additional studies are needed to identify internal and external factors which contribute to the high and low biofilm formation of B. pseudomallei.
Melioidosis has been recognized as an important cause of sepsis in the tropics. The disease caused by an environmental saprophyte Burkholderia pseudomallei, affects mostly adults with underlying immunocompromised conditions. In this study, the enzymatic profiles of 91 clinical and 9 environmental isolates of B. pseudomallei were evaluated using the APIZYM system, in addition to assessment of protease, phospholipase C and sialidase activities using agar plate methods and other assays. The activity of 10 enzymes - alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase were detected in >75% of the clinical isolates. The majority of B. pseudomallei isolates in this study exhibited protease and phospholipase activities. No sialidase activity was detected. Five Burkholderia thailandensis isolates had similar APIZYM profiles as B. pseudomallei clinical isolates except for the lower detection rate for N-acetyl-β-glucosaminidase. The subtle differences in the number of enzymes secreted and the levels of enzymatic activities of phenotypically identical clinical and environmental strains of B. pseudomallei give weight to the fact that the causative agent of melioidodis originates from the environment.
The biodiversity and the killer activity of yeasts isolated from various types of fermented food in Malaysia were investigated in this study. Of 252 yeasts isolated from 48 fermented food samples in this study, 19 yeast species were identified based on sequence analysis of the ITS1-5.8S-ITS2 partial fragments of the yeasts. A total of 29 (11.5%) of the yeast isolates demonstrated killer activity to at least one Candida species tested in this study; including 22 isolates of Trichosporon asahii, 4 isolates of Pichia anomala, and one isolate each of Pichia norvegensis, Pichia fermentans and Issatchenkia orientalis, respectively. The presence of killer yeasts reflects antagonism that occurs during microbial interaction in the fermented food, whereby certain yeasts produce killer toxins and possibly other toxic substances in competition for limited nutrients and space. The anti-Candida activity demonstrated by killer yeasts in this study should be further explored for development of alternative therapy against candidiasis.
Ciprofloxacin, a quinolone with good intracellular penetration may possibly be used for treatment of melioidosis caused by Burkholderia pseudomallei, but problems with resistance may be encountered. Amino acid substitutions in gyrA/gyrB have given rise to fluoroquinolone resistance in various microorganisms. Using published primers for gyrA and gyrB, PCR was performed on 11 isolates of B. pseudomallei with varying degrees of sensitivity to ciprofloxacin, followed by DNA sequencing to detect possible mutations. Results showed an absence of any point mutation in either gene. Local isolates have yet to develop full resistance to ciprofloxacin and probably other mechanisms of resistance may have been involved in the decreased sensitivity to ciprofloxacin.
The increased frequency of antibiotic resistance is known to be associated with the dissemination of integrons in the Enterobacteriaceae. This study determined the prevalence and type of integrons amongst 160 extended-spectrum beta-lactamase producing enterobacterial isolates kept in our culture collection. Integrons were detected in 98(61.3%) isolates, including 28(62.2%) Escherichia coli, 34(64.2%) Klebsiella spp., 27(61.4%), Enterobacter spp. and 9(50.0%) Citrobacter spp. investigated in this study. Restriction analysis of the integron gene fragments revealed that class I integron was the principal integron detected in 92(57.5%) of our isolates. Class II integron was detected in 6(3.8%) of our isolates, while no class III integron was detected in this study. The high rates of integron prevalence particularly of the class I integron in the E. coli and Klebsiella spp. concur with previous studies in other geographical regions. The higher (≥50%) integron prevalence of Citrobacter and Enterobacter isolates comparing to previous studies suggests the potential of these isolates as sources for dissemination of resistance determinants. The finding in this study serves as a basis for further study on the antibiotic resistance mechanisms of enterobacterial species in this teaching hospital.
This study describes the killer phenotypes of tropical environmental yeasts and the inhibition effects of the culture filtrates on the biofilm of Candida albicans. A total of 26 (10.5%) of 258 yeast isolates obtained from an environmental sampling study demonstrated killer activity to Candida species. The killer yeasts were identified as species belonging to the genus Aureobasidium, Pseudozyma, Ustilago and Candida based on sequence analysis of the ITS1-5.8S-ITS2 region of the yeasts. Pseudozyma showed the broadest killing effects against sensitive strains of Candida. New species of Ustilago and Pseudozyma demonstrating killer phenotypes were identified in this study. Interestingly, more than 50% reduction in the metabolic activity of Candida albicans biofilm was noted after exposure to the culture filtrates of the nine killer yeasts. Purification and characterization of toxin and metabolites are essential for understanding the yeast killing effects.
Candida glabrata has been reported as the second or third most common yeast species isolated from patients with vaginitis and invasive candidiasis. This study was aimed to determine the genetic diversity, antifungal susceptibility and enzymatic profiles of C. glabrata isolated from vaginal and blood samples in the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre. A random amplified polymorphic DNA (RAPD) analysis method, using M13 and (GTG)5 primers, was used for strain differentiation of C. glabrata isolates. Antifungal susceptibility testing of C. glabrata isolates was determined using E-test against amphotericin B, caspofungin, fluconazole and voriconazole and microbroth dilution method against clotrimazole. The enzymic profiles of C. glabrata were determined using APIZYM semi-quantitation kit and egg-yolk agar method. A total of 14 RAPD patterns were identified amongst C. glabrata isolates investigated this study. Susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole was noted. Approximately one third of the isolates demonstrated resistance to clotrimazole (MIC>=1 µg/ml). A single isolate of C. glabrata was resistant to caspofungin (MIC:1.5 µg/ml). Enzymatic activities of acid and alkaline phosphatase, aminopeptidases, esterase and lipase and phospholipase were detected in the C. glabrata isolates. The genetic diversity and antifungal susceptibility profiles of C. glabrata isolates were presented in this study. Continued surveillance and monitoring of the incidence and antifungal resistance in C. glabrata isolates is necessary.
Routine use of selective media improves diagnosis of Burkholderia pseudomallei, but resources may be limited in endemic developing countries. To maximise yield in the relatively low-prevalence setting of Kuala Lumpur, Malaysia, B. pseudomallei selective agar and broth were compared with routine media for 154 respiratory specimens from patients with community-acquired disease. Selective media detected three additional culture-positive specimens and one additional melioidosis patient, at a consumables cost of US$75. Burkholderia pseudomallei was not isolated from 74 diabetic foot ulcer samples. Following careful local evaluation, focused use of selective media may be cost-effective.
A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.
Carbapenems are the primary choice of treatment for severe Pseudomonas aeruginosa infection. However, the emergence of carbapenem resistance due to the production of metallo-β-lactamases (MBLs) is of global concern. In this study, 90 imipenem- (IPM- or IP-) resistant P. aeruginosa (IRPA) isolates, including 32 previously tested positive and genotyped for MBL genes by PCR, were subjected to double-disk synergy test (DDST), combined disk test (CDT), and imipenem/imipenem-inhibitor (IP/IPI) E-test to evaluate their MBLs detection capability. All three methods were shown to have a sensitivity of 100%. However, DDST was the most specific of the three (96.6%), followed by IP/IPI E-test interpreted based on the single criteria of IP/IPI ≥8 as positive (62.1%), and CDT was the least specific (43.1%). Based on the data from this evaluation, we propose that only IRPA with IP MIC >16 μg/mL and IP/IPI ≥8 by IP/IPI E-test should be taken as positive for MBL activity. With the new dual interpretation criteria, the MBL IP/IPI E-test was shown to achieve 100% sensitivity as well as specificity for the IRPA in this study. Therefore, the IP/IPI E-test is a viable alternative phenotypic assay to detect MBL production in IRPA in our population in circumstances where PCR detection is not a feasible option.