The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231(T), Staphylococcus aurues ATCC 51650(T), methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44(T) and Pseudomonas aeruginosa ATCC 10145(T), respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.
DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.
Estimation of post-mortem interval (PMI) is crucial for time of death determination. The advent of DNA-based identification techniques forensic entomology saw the beginning of a proliferation of molecular studies into forensically important Calliphoridae (Diptera). The use of DNA to characterise morphologically indistinguishable immature calliphorids was recognised as a valuable molecular tool with enormous practical utility. The local entomofauna in most cases is important for the examination of entomological evidences. The survey of the local entomofauna has become a fundamental first step in forensic entomological studies, because different geographical distributions, seasonal and environmental factors may influence the decomposition process and the occurrence of different insect species on corpses. In this study, calliphorids were collected from 13 human corpses recovered from indoors, outdoors and aquatic conditions during the post-mortem examination by pathologists from the government hospitals in Malaysia. Only two species, Chrysomya megacephala and Chrysomya rufifacies were recovered from human corpses. DNA sequencing was performed to study the mitochondrial encoded COI gene and to evaluate the suitability of the 1300 base pairs of COI fragments for identification of blow fly species collected from real crime scene. The COI gene from blow fly specimens were sequenced and deposited in GenBank to expand local databases. The sequenced COI gene was useful in identifying calliphorids retrieved from human corpses.
Melioidosis is an infectious disease caused by Burkholderia pseudomallei and endemic in Southeast Asia. One hundred and forty six clinical isolates of B. pseudomallei from different states in Malaysia were obtained and molecular typing was carried out using pulsed-field gel electrophoresis (PFGE). Overall, nine clusters were successfully identified. Burkholderia pseudomallei isolates used in this study were found to be genetically diverse and there were differences in the clusters of isolates from peninsular and east Malaysia. BS9 cluster was the most common cluster and found in all the states while BS2 cluster only existed in a particular state. Based on the PFGE analysis, the distribution of different B. pseudomallei clinical isolates in Malaysia was mapped.
Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR) isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B).
We have analysed DNA fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) of 52 unrelated Burkholderia pseudomallei strains isolated from septicemic and localized infections from Malaysian subjects. A total of 38 PFGE types were observed among 36 septicemic and 16 localized strains with no predominant pattern. Type 25 was seen in 2 epidemiologically related strains, suggesting human to human transmission. Twelve PFGE types were shared among 26 strains (21 septicemic and 5 localized) showing close genetic relatedness with coefficient of similarity of 0.81 to 1.0. The other 26 strains (15 septicemic and 11 localized) were unrelated as shown by the similarity coefficient of < 0.8. This study showed that our B. pseudomallei strains in Malaysia were mainly heterogenous with no predominant type both in septicemic or localized strains.
Twenty-one Vibrio parahaemolyticus isolates representing 21 samples of coastal seawater from three beaches in peninsular Malaysia were found to be sensitive to streptomycin, norfloxacin and chloramphenicol. Resistance was observed to penicillin (100%), ampicillin (95.2%), carbenicilin (95.2%), erythromycin (95.2%), bacitracin (71.4%), cephalothin (28.6%), moxalactam (28.6%), kanamycin (19.1%), tetracycline (14.3%), nalidixic acid (9.5%) and gentamicin (9.5%). Plasmids of 2.6 to 35.8 mDa were detected among plasmid-containing isolates. All isolates carried the Vp-toxR gene specific to V. parahaemolyticus and were negative for the tdh gene, but only one isolate was positive for the trh gene. DNA fingerprinting of the isolates using ERIC-PCR and PFGE showed that the isolates belong to two major clonal groups, with several isolates from different locations in the same group, indicating the presence of similar strains in the different locations.
Seven isolates of Burkholderia pseudomallei from cases of melioidosis in human (2 isolates) and animal (2 isolates), cat (one isolate) and from soil samples (2 isolates) were examined for in vitro sensitivity to 14 antimicrobial agents and for presence of plasmid DNA. Randomly amplified polymorphic DNA (RAPD) analysis was used to type the isolates, using two arbitrary primers. All isolates were sensitive to chloramphenicol, kanamycin, carbenicillin, rifampicin, enrofloxacin, tetracycline and sulfamethoxazole-trimethoprim. No plasmid was detected in all the isolates tested. RADP fingerprinting demonstrated genomic relationship between isolates, which provides an effective method to study the epidemiology of the isolates examined.
Strains of Salmonella typhi implicated in two separate cases of laboratory acquired infection from patients and the medical laboratory technologists who processed the patients' samples were analysed by pulsed-field gel electrophoresis. Although all four isolates were of bacteriophage type E1, PFGE was able to demonstrate that the strains responsible for the two laboratory acquired cases were not genetically related. The PFGE patterns of the isolates from the MLTs were found to be identical to those of the corresponding patients after digestion with restriction enzyme AvrII. This provided genetic as well as epidemiological evidence for the source of the laboratory acquired infections.
Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea among infants in developing countries. A total of 38 EPEC isolates, obtained from diarrhea patients of Hospital Miri, Sarawak, were investigated through plasmid profile, antibiotic resistance and randomly amplified polymorphic DNA (RAPD) analysis. From the 8 types of antibiotics used, all isolates were 100% resistant to furoxime, cephalothin and sulphamethoxazole and showed high multiple antibiotic resistant (MAR) indexes, ranging from 0.5 to 1.0. In plasmid profiling, 22 isolates (58%) showed the presence of one or more plasmids in the range 1.0 to 30.9 mDa. The dendrogram obtained from the results of the RAPD-PCR discriminated the isolates into 30 single isolates and 3 clusters at the level of 40% similarity. The EPEC isolates were highly diverse, as shown by their differing plasmid profiles, antibiotic resistance patterns and RAPD profiles.
Methicillin-resistant Staphylococcus aureus (MRSA) has been prevalent in our hospital over the last three years. Differentiation among MRSA strains by DNA typing in addition to antibiotic resistance pattern surveillance is crucial in order to implement infection control measures. The aim of this study was to characterize MRSA isolates from patients admitted to Hospital Universiti Kebangsaan Malaysia (HUKM) by phenotypic (analyses of antibiotic susceptibility pattern) and genotypic (PFGE) techniques to determine the genetic relatedness of the MRSA involved and to identify endemic clonal profiles of MRSA circulating in HUKM. Seventy one MRSA strains collected between January to March 2000 from patients from various wards in HUKM were tested for antimicrobial resistance and typed by pulsed-field gel electrophoresis (PFGE). Four major types of PFGE patterns were identified (A, B, C and D) among MRSA strains. Two predominant PFGE types were recognised, Type A (59.2%) and Type B (33.8%). Most of these strains were isolated from ICU, Surgical wards and Medical wards. MRSA strains with different PFGE patterns appeared to be widespread among wards. Strains with the same antibiotype could be of different PFGE types. Most of isolates were resistant to ciprofloxacin, erythromycin, gentamicin and penicillin. One isolate with a unique PFGE pattern Type D and susceptible to gentamicin was identified as a different clone. Some isolates obtained from the same patient showed different PFGE subtypes suggesting that these patients were infected/colonized with multiple MRSA strains. PFGE analysis suggests that MRSA strains with different PFGE types was propagated within our hospital. The relationship between antibiotic susceptibility and PFGE patterns was independent. The ability of PFGE technique in differentiating our MRSA strains make it a method of choice for investigating the source, transmission and spread of nosocomial MRSA infection, and thus an appropriate control programme can be implemented to prevent the spread of MRSA infection.
Short tandem repeat (STR) typing continues to be the primary workhorse in forensic DNA profiling. Therefore, the present review discusses the prominent role of STR marker in criminal justice system. All over the world, deoxyribonucleic acid (DNA) profiling provides evidence that may be used to convict criminals, as an irrefutable proof of wrongful convictions, invaluable links to the actual perpetrators of crimes, and could also deter some offenders from committing more serious offences. Clearly, DNA profiling tools have also aided forensic scientists to re-evaluate old cases that were considered closed as a result of inadequate evidence. In carrying out this review, a comprehensive electronic literature search using PubMed, ScienceDirect, Google Scholar and Google Search were used, and all works meeting the subject matter were considered, including reviews, retrospective studies, observational studies and original articles. Case reports presented here, further demonstrates the crucial role of forensic DNA profiling in mitigating and providing compelling evidence for the resolution of crimes. For case report 1, there was a 100% match between the DNA recovered from the items found in the crime scene, and the suspect's DNA sample collected via buccal swab following 15 STR loci examination. Case report 2 further highlights the indispensable contribution of DNA database in solving crime. Therefore, it has become very necessary for developing countries like Nigeria to develop a national DNA database and make policies and legislatures that will further expand and enable the practice of forensic genetics, particularly DNA profiling.
Anisakis spp. (Nematoda: Anisakidae) parasitize a wide range of marine animals, mammals serving as the definitive host and different fish species as intermediate or paratenic hosts. In this study, 18 fish species were investigated for Anisakis infection. Katsuwonus pelamis, Euthynnus affinis, Caranx sp., and Auxis thazard were infected with high prevalence of Anisakis type I, while Cephalopholis cyanostigma and Rastrelliger kanagurta revealed low prevalence. The mean intensity of Anisakis larvae in K. pelamis and A. thazard was 49.7 and 5.6, respectively. A total of 73 Anisakis type I larvae collected from K. pelamis and A. thazard were all identified as Anisakis typica by PCR-RFLP analysis. Five specimens of Anisakis from K. pelamis and 15 specimens from A. thazard were sequenced using ITS1-5.8S-ITS2 region and 6 specimens from A. thazard and 4 specimens from K. pelamis were sequenced in mtDNA cox2 region. Alignments of the samples in the ITS region showed 2 patterns of nucleotides. The first pattern (genotype) of Anisakis from A. thazard had 100% similarity with adult A. typica from dolphins from USA, whereas the second genotype from A. thazard and K. pelamis had 4 base pairs different in ITS1 region with adult A. typica from USA. In the mtDNA cox2 regions, Anisakis type I specimens from A. thazard and K. pelamis showed similarity range from 94% to 99% with A. typica AB517571/DQ116427. The difference of 4 bp nucleotides in ITS1 regions and divergence into 2 subgroups in mtDNA cox2 indicating the existence of A. typica sibling species in the Makassar Strait.
The antibiotic susceptibility profiles and the repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR)-determined genotypes of 109 Acinetobacter strains collected from the University Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia, in 1987 (N=21) and 1996-1998 (N=88) were established. Twelve antibiotic susceptibility profiles of antibiotics used at the UMMC were obtained. In descending order of effectiveness, imipenem, amikacin and ciprofloxacin were the most effective against the Acinetobacter strains. Compared with 1987 isolates, the isolates obtained in 1996-1998 had decreased susceptibility to these antibiotics and were tolerant to the antibiotics up to an MIC90 of > or =256 mg/L. REP-PCR DNA fingerprints of all the isolates revealed the presence of four Acinetobacter spp. lineages; 92% of all the isolates belonged to two dominant lineages (genotypes 1 and 4). Genotype 4 isolates predominant in 1987 showed increased resistance and antibiotic tolerance to imipenem, amikacin and ciprofloxacin compared with the 1996-1998 isolates. In contrast, genotype 1 isolates from 1996-1998 were mainly sensitive to these antibiotics. These findings demonstrate the presence of at least two independent Acinetobacter spp. lineages in the same hospital, and suggest the possibility that genotype 4 Acinetobacter spp. acquired the resistance phenotype in situ, whereas most of the genotype 1 isolates were probably introduced to the hospital in recent years.
Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.
Short tandem repeat (STR) polymorphisms were investigated in 341 unrelated Malay individuals (218 males and 123 females) living in or around Kuala Lumpur by using a forensic analysts kit. The following STRs were targeted: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA. The purpose of this study was to elucidate population genetics in Malaysia and calculate statistical parameters for forensic and anthropological research. Data on these STRs in the target population were obtained and subjected to statistical analysis. Accordance with the Hardy-Weinberg equilibrium was proven for all the loci targeted. The combined power of discrimination was greater than 0.9999999999, indicating that this multiplex system is an excellent tool for forensic casework. The allele frequency in the data were weighed against that in four other local populations (Chinese, Iranian, Belgian, and African). The average coefficient of correlation was strongest in the order of Africa (0.092522), Belgium (0.264822), Iran (0.404363), and China (0.706661). These results are consistent with what is known about the anthropological history of and prehistoric human migration in the Malay region. We believe that these data offer a valuable anthropological resource, being applicable to the statistical evaluation of DNA evidence in human identification, as well as the determination of ethnicity in healthy populations.
Serratia marcescens is a well-known cause of nosocomial infections and outbreaks, particularly in immunocompromised patients with severe underlying disease. An outbreak due to S. marcescens infection was detected from 13 to 22 February 2001 at the intensive care unit (ICU) of our institution. We used pulsed-field gel electrophoresis (PFGE) typing to analyse the outbreak strains involved.
To determine and compare the pulsed-field gel electrophoresis (PFGE) patterns of endemic MRSA strains in 2 major Malaysian hospitals and to compare the PFGE patterns with antibiotypes of the strains studied.
Short repetitive regions in autosomal and Y chromosomes known as short tandem repeats (STRs) are currently used for DNA profiling in crime investigations. However, DNA profiling requires a sufficient quality and quantity of DNA template, which is often not obtained from trace evidence or degraded biological samples collected at the scene of a crime. Here, we assessed autosomal and male DNA components extracted from crime scene and mock casework samples using the Casework Direct Kit, Custom and compared the results against those obtained by extraction of matching samples using well-established Maxwell 16 System DNA IQ Casework Pro Kit. The quantity and quality of extracted DNA obtained using both Casework Direct Kit, Custom and Maxwell 16 System DNA IQ Casework Pro Kit were analyzed using PowerQuant Systems followed by autosomal and Y-chromosome STR profiling using GlobalFiler Express PCR Amplification Kit and PowerPlex Y23 System, respectively. Our results showed that the Casework Direct Kit and Maxwell 16 DNA IQ Casework Pro Kit have more or less equal capacity to extract inhibitor free DNA, but that the latter produces slightly better quality and more DNA template and subsequently higher numbers of STR allele calls for autosomal and Y-STR analyses. Nonetheless, the Casework Direct Kit, Custom is the quicker and cheaper option for extraction of good, clean DNA from high content material and might best be used for extraction of reference samples. Such reference DNA samples typically come from buccal swabs or freshly drawn blood. So, in general, they can confidently be expected to have a high nucleic acid content and to be inhibitor-free.
As a new crop in Malaysia, forty-four Bambara groundnut (Vigna subterranea L. verdc.) genotypes were sampled from eleven distinct populations of different origins to explore the genetic structure, genetic inconsistency, and fixation index. The Bambara groundnut, an African underutilized legume, has the capacity to boost food and nutrition security while simultaneously addressing environmental sustainability, food availability, and economic inequalities. A set of 32 ISSRs were screened out of 96 primers based on very sharp, clear, and reproducible bands which detected a total of 510 loci with an average of 97.64% polymorphism. The average calculated value of PIC = 0.243, RP = 5.30, H = 0.285, and MI = 0.675 representing the efficiency of primer set for genetic differentiation among the genotypes. The ISSR primers revealed the number of alleles (Na = 1.97), the effective number of alleles (Ne = 1.38), Nei's genetic diversity (h = 0.248), and a moderate level of gene flow (Nm = 2.26) across the genotypes studied. The estimated Shannon's information index (I = 0.395) indicates a high level of genetic variation exists among the accessions. Based on Nei's genetic dissimilarity a UPMGA phylogenetic tree was constructed and grouped the entire genotypes into 3 major clusters and 6 subclusters. PCA analysis revealed that first principal component extracted maximum variation (PC1 = 13.92%) than second principal component (PC2 = 12.59%). Bayesian model-based STRUCTURE analysis assembled the genotypes into 3 (best ΔK = 3) genetic groups. The fixation-index (Fst) analysis narrated a very great genetic diversity (Fst = 0.19 to 0.40) exists within the accessions of these 3 clusters. This investigation specifies the effectiveness of the ISSR primers system for the molecular portrayal of V. subterranea genotypes that could be used for genetic diversity valuation, detection, and tagging of potential genotypes with quick, precise, and authentic measures for this crop improvement through effective breeding schemes.