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S Radu

Publications and source records attributed to S Radu.

At least 19 recordsLinked to original sources

DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14.

A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1,594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin (dso) and single strand origin (sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses.

Amino Acid Sequence↗

Prevalence of Staphylococcus aureus carriage by young Malaysian footballers during indoor training.

BACKGROUND: Research has shown that athletes are carriers of Staphylococcus aureus during physical activity. OBJECTIVE: To estimate the mean total plate count of S aureus carried by footballers before and after training at an indoor venue. METHODS: Forty Malay and 20 Indian students volunteered to participate. There was also a control group consisting of 40 Malay and 20 Indian students who were not active. The experimental group were active footballers who had played at school or club level. The subjects were healthy and free of skin infection. The experiment was divided into three sessions, with 20 subjects present at each. At each session, the subjects trained for one hour. Swabs were taken from the skin, nose, and ear before and after training. For the control group, swabs were taken only once from the skin, nose, and ear. The swabs were subjected to biochemical tests and then streaked and cultured aerobically in Baird Parker agar plates for 24 hours at 37 degrees C. Black colonies with a clear zone were presumed to be S aureus, and the mean total plate count of the colonies was estimated. Gram staining, catalase, coagulase slide, coagulase tube, acetoin production, o-nitrophenyl beta-D-galactopyranoside (ONPG), and mannitol fermentation tests were used to confirm the colonies as S aureus. A haemolysin test was conducted with human blood to confirm haemolytic activity. RESULTS: All subjects in the experimental group were carrying S aureus both before and after training. The estimated mean total counts of colonies from the skin, ear, and nose for the Malays before training were 33, 71, and 312 respectively. Counts after training were 21, 44, and 452 respectively. The results for the Indians were 72, 80, and 309 respectively before training and 55, 200, and 466 respectively after training. The positive results for Gram staining, catalase, coagulase slide, coagulase tube, acetoin production, ONPG, and mannitol fermentation tests were 100%, 96%, 95%, 95%, 93%, 93%, and 90% respectively. All subjects in the control group were also carrying S aureus. CONCLUSIONS: All of the players were carriers of S aureus during training. The decrease in total count from the skin for both races may be due to lysozyme activity lysing the bacterial cells. Contamination of the environment with these bacteria may have increased the estimated total plate count in the nose. The experimental group face a higher risk of infection because of lower immunity during training and higher rate of injuries compared with the control group.

Adolescent↗

Microbial analysis of Malaysian tempeh, and characterization of two bacteriocins produced by isolates of Enterococcus faecium.

AIMS: Isolation of bacteriocinogenic lactic acid bacteria (LAB) from the Malaysian mould-fermented product tempeh and characterization of the produced bacteriocin(s). METHODS AND RESULTS: LAB were present in high numbers in final products as well as during processing. Isolates, Enterococcus faecium B1 and E. faecium B2 (E. faecium LMG 19827 and E. faecium LMG 19828, respectively) inhibited Gram-positive indicators, including Listeria monocytogenes. Partially purified bacteriocins showed a proteinaceous nature. Activity was stable after heat-treatment except at alkaline pH values. Both strains displayed a bacteriostatic mode of action. Bacteriocin production was associated with late exponential/early stationary growth. Molecular mass, calculated by SDS-PAGE, was 3.4 kDa for B1 bacteriocin, and 3.4 kDa and 5.8 kDa for B2 bacteriocins. PCR screening of enterocin-coding genes revealed three amplified fragments in total genomic DNA that may correspond with PCR signals for enterocin P, enterocin L50A and enterocin L50B. Both B1 and B2 contained a 42-kb plasmid. No differences in bacteriocinogenic capacity were found between wild type strains and plasmid-cured strains. CONCLUSIONS: It was possible to isolate bacteriocinogenic E. faecium active against various Gram-positive bacteria from final products of tempeh. SIGNIFICANCE AND IMPACT OF THE STUDY: A first step in applying biopreservation to fermented South-east Asian foods is to obtain bacteriocinogenic LAB from this source. Such isolates may also be used for biopreservation of mould-fermented foods in general, including various types of mould-ripened cheese.

Antibiosis↗

Genetic diversity of clinical and environmental strains of Salmonella enterica serotype Weltevreden isolated in Malaysia.

The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.

DNA, Bacterial↗

Detection of Escherichia coli O157:H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses.

Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.

Animals↗

Occurrence of the vanA and vanC2/C3 genes in Enterococcus species isolated from poultry sources in Malaysia.

Enterococcus species isolated from poultry sources were characterized for their resistance to antibiotics, plasmid content, presence of van genes and their diversity by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The results showed that all isolates were multi-resistance to the antibiotics tested. Ampicillin (15/70) followed by chloramphenicol (37/70) were the most active antibiotics tested against the Enterococcus spp. isolates, while the overall resistant rates against the other antibiotics were between 64.3% to 100%. All vancomycin-resistant E. faecalis, E. durans, E. hirae and E. faecium isolates tested by the disk diffusion assay were positive in PCR detection for presence of vanA gene. All E. casseliflavus isolates were positive for vanC2/C3 gene. However, none of the Enterococcus spp. isolates were positive for vanB and vanC1 genes. Plasmids ranging in sizes between 1.1 to ca. 35.8 MDa were detected in 38/70 of the Enterococcus isolates. When the genetic relationship among all isolates of the individual species were tested by RAPD-PCR, genetic differences detected suggested a high genetic polymorphisms of isolates in each individual species. Our results indicates that further epidemiological studies are necessary to elucidate the role of food animals as reservoir of VRE and the public health significance of infections caused by Enterococcus spp.

Animals↗

Molecular characterization of Salmonella weltevreden isolated from poultry: evidence of conjugal transfer of plasmid and antibiotic resistance.

Ten strains of Salmonella weltevreden isolated from poultry sources were examined and found to contain plasmid DNA ranging in size from 1.8 to 68.5 MD. All isolates were susceptible to carbenicillin, cephalothin, ceftriazone, gentamicin, kanamycin and nalidixic acid, but resistance to bacitracin (100%), penicillin G (100%), rifampicin (100%), sulphamethoxazole (100%), cefuroxime (80%) and tetracycline (60%) was recorded. The 55 MD plasmid of strain SW5 determined resistance to penicillin G and tetracycline, which was transmissible to the E. coli K12 recipient at a frequency of 3.52 x 10(-5) transconjugants per input donor cell. The results of arbitrarily primed polymerase chain reaction (AP-PCR), using two 10-mer oligonucleotides and PCR-ribotyping to differentiate between the ten strains of S. weltevreden were compared. The strains were separated into ten different genome types by AP-PCR but were indistinguishable by PCR-ribotyping. These results suggest that poultry may constitute a reservoir for disseminating antibiotic resistance and that AP-PCR may be a valuable tool for epidemiological studies.

Animals↗

Detection and molecular characterization of the zot gene in Vibrio cholerae and V. alginolyticus isolates.

A total of 11 Vibrio cholerae isolates from 1996-1998 outbreaks in Malaysia and 4 V. alginolyticus were analyzed. Isolates were characterized by polymerase chain reaction (PCR) and Southern hybridization for the presence of the gene encoding zonula occludens toxin (zot). Screening of zot gene by PCR revealed the presence of this gene in V. cholerae and V. alginolyticus. The zot gene from one V. cholerae Ogawa isolate that was cloned in a pCR 2.1 TOPO vector was sequenced. The sequences obtained were 99% homologous to the zot gene sequence from the Gene Bank.

Base Sequence↗

Prevalence of Listeria spp and Listeria monocytogenes in meat and fermented fish in Malaysia.

Fermented fish and meat samples were purchased from supermarket and wet market for microbiological analysis of Listeria species and Listeria monocytogenes contamination. Listeria species were isolated from 17 (73.9%) of 23 samples of imported frozen beef, 10 (43.5%) of the 23 samples of local beef and 14 (56%) of the 25 samples of fermented fish from wet market. Listeria monocytogenes occurred in 15 (75%) of the frozen beef samples, 6 (30.4%) of the 23 samples of local meat and 3 (12%) of the 25 samples from fermented fish. Listeria species was not isolated from any of the 23 samples of imported frozen beef from supermarket and from the 5 samples of buffalo meat examined. This highlights the possibility of Listeria spp or L. monocytogenes to persist in meat and fermented fish in wet market and raises the problem of illness due to the handling and consumption of Listeria-contaminated meat or fermented fish are likely as evidence by the high contamination rates of samples sold at the wet market.

Fish Products↗

Improvement in raw sago starch degrading enzyme production from Acremonium sp. endophytic fungus using carbon and nitrogen sources.

Attempts were made to improve the growth of endophytic fungus Acremonium sp. and its raw sago starch degrading enzyme (RSSDE) production using different nitrogen and carbon sources at varying pH values and temperatures. It was observed that growth and enzyme activity levels were highest with peptone and sodium nitrate as the nitrogen sources and raw sago starch as the carbon source of which the optimum concentrations were 0.5 g/l, 3 g/l, and 20 g/l, respectively. Cell growth and RSSDE production reached their optimum at pH 5.0 and incubation temperature of 30 degrees C. Under these conditions, the enzyme production was significantly increased by 19- to 22-folds compared to the activity obtained in the original basal medium.

Journal Article↗

Rapid isolation and detection of Escherichia coli O157:H7 by use of rainbow agar O157 and PCR assay.

This study has evaluated the use of a commercially available Rainbow agar O157 and polymerase chain reaction (PCR) assays for the detection of Shiga-like toxin producing Escherichia coli and to serotype E. coli O157:H7 from raw meat. The Rainbow agar O157 was found to be selective and sensitive for the screening of the E. coli O157 from artificially and naturally contaminated meat samples. Shiga-like toxin producing E. coli were identified with two primer pairs that amplified fragments of the SLT-I (384 bp) and SLT-II (584 bp). E. coli O157:H7 was serotyped with a primer pair specified for the H7 flagellar gene, which amplify specific DNA fragments (625 bp) from all E. coli O157:H7 strains. The use of Rainbow agar O157 described allows for the presumptive isolation of E. coli O157 in 24 hours. Identification and confirmation of the presumptive isolates as E. coli O157:H7 by PCR assays require additional 6-8 hours. The above-mentioned screening and identification procedures should prove to be a very useful method since it allows for the specific detection of E. coli O157:H7.

Animals↗

Detection and molecular characterization of Vibrio vulnificus from coastal waters of Malaysia.

A total of 57 Vibrio vulnificus isolates from coastal water were characterized for their antimicrobial resistance, plasmid profiles and were typed by the PCR-based techniques: a random amplification of polymorphic DNA (RAPD) method and the enterobacterial repetitive intergenic consensus sequence (ERIC) method. All isolates were susceptible to chloramphenicol, nalidixic acid, tetracycline and trimethoprim-sulfamethoxazole. Fifty-one isolates were resistant to one or more of the other antibiotics tested. Plasmid analysis indicated that only 18 isolates carried small plasmids of 1.6 to 16 megadaltons. Analysis of the RAPD and ERIC DNA fingerprints of the V. vulnificus isolates with Gel Compare and cluster analysis software revealed significant genetic heterogeneity among these isolates. The combination of RAPD and ERIC analysis allowed us to distinguish all isolates. Thus, the combination of the two techniques is recommended for epidemiological investigation.

Base Sequence↗

Molecular characterization of Vibrio cholerae O1 and non-O1 from human and environmental sources in Malaysia.

A total of 31 strains of Vibrio cholerae O1 (10 from outbreak cases and 7 from surface water) and non-O1 (4 from clinical and 10 from surface water sources) isolated between 1993 and 1997 were examined with respect to presence of cholera enterotoxin (CT) gene by PCR-based assays, resistance to antibiotics, plasmid profiles and random amplified polymorphic DNA (RAPD) analysis. All were resistant to 9 or more of the 17 antibiotics tested. Identical antibiotic resistance patterns of the isolates may indicate that they share a common mode of developing antibiotic resistance. Furthermore, the multiple antibiotic resistance indexing showed that all strains tested originated from high risk contamination. Plasmid profile analysis by agarose gel electrophoresis showed the presence of small plasmids in 12 (7 non-O1 and 5 O1 serotypes) with sizes ranging 1.3-4.6 MDa. The CT gene was detected in all clinical isolates but was present in only 14 (6 O1 serotype and 8 non-O1 serotype) isolates from environmental waters. The genetic relatedness of the clinical and environmental Vibrio cholerae O1 and non-O1 strains was investigated by RAPD fingerprinting with four primers. The four primers generated polymorphisms in all 31 strains of Vibrio cholerae tested, producing bands ranging from < 250 to 4500 bp. The RAPD profiles revealed a wide variability and no correlation with the source of isolation. This study provides evidence that Vibrio cholerae O1 and non-O1 have significant public health implications.

Cholera↗

Genotypic and phenotypic relationship in Burkholderia pseudomallei indicates colonization with closely related isolates.

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.

Animals↗

Characterization of Vibrio vulnificus isolated from cockles (Anadara granosa): antimicrobial resistance, plasmid profiles and random amplification of polymorphic DNA analysis.

Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.

Animals↗

Survival of Vibrio spp. including inoculated V. cholerae 0139 during heat-treatment of cockles (Anadara granosa).

The effect of heat-treatment on the internal temperature of raw cockles (Anadara granosa) and survival of their intrinsic flora of Vibrio spp. as well as of inoculated V. cholerae 0139 was examined. The cockles were purchased from markets in Malaysia and had an average weight including shells of 8.90+/-2.45 g. In one experiment heatpenetration of individual cockles was examined. Cockles weighing < 8 g (including shell) exhibited maximum internal temperatures of between 50 and 75 degrees C when heated in water at 99 degrees C for 10 s and 71-93 degrees C when heated for 30 s. Cockles weighing > 12 g exhibited maximum internal temperatures between 42 and 58 degrees C when heated in water at 99 degrees C for 10 s and 56-69 degrees C when heated for 30 s. In another experiment, heat-treatment of 10 cockles treated as a group at 99 degrees C for 10 or 30 s resulted in reduction of levels of intrinsic Vibrio spp. (enumerated directly on thiosulphate-citrate-bile salt sucrose agar; TCBS) from 5.73 to 3.15 log cfu g(-1) or below 1 log cfu g(-1), respectively. The levels of Vibrio spp. after heat-treatment decreased with an increase in numbers of cockles grouped together during treatment. In a third experiment V. cholerae 0139 was inoculated into cockles and subjected to heat-treatment at 99 degrees C for 0, 10, 15, 20, 25 or 30 s. The levels of Vibrio spp. in uninoculated, non-heat-treated cockles was 4.89 log cfu g(-1) on TCBS, and the predominant species were V. parahaemolyticus and V. alginolyticus. V. cholerae 0139 inoculated into cockles with an average weight of 13.5+/-1.90 g (including shell) decreased for samples examined immediately after heat-treatment from 6 log cfu g(-1) initially to 3.5 log cfu g(-1) after 25 s and < 1 log cfu g(-1) (TCBS) after 30 s of heat-treatment. The most probable number method by enrichment in alkaline peptone water gave in general within 1 log unit higher counts than TCBS direct enumeration. TCBS direct enumeration and MPN counts were up to 2.38 or 1.30 log units higher, respectively, for samples heat-treated for 20 s or longer and stored for 6 h at 30 degrees C before examination, than for samples heat-treated for same periods of time and examined immediately. This study shows that a mild heat-treatment of cockles for up to 25 s is inadequate to ensure a large reduction in numbers of Vibrio spp., including V. cholerae 0139.

Animals↗

Molecular analysis of Dichelobacter nodosus isolated from footrot in sheep in Malaysia.

Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5'GGGCCC3'), SfiI (5'GGCCNNNNNGGCC3') and SmaI ('5CCCGGG3') enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.

Animals↗