Electroporation enhances the ability of lactobacilli to remove cholesterol.

The objective of the present study was to evaluate the effect of electroporation on the membrane properties of lactobacilli and their ability to remove cholesterol in vitro. The growth of lactobacilli cells treated at 7.5 kV/cm for 4 ms was increased by 0.89 to 1.96 log(10) cfu/mL upon fermentation at 37 °C for 20 h, the increase being attributed to the reversible and transient formation of pores and defragmentation of clumped cells. In addition, an increase of cholesterol assimilation as high as 127.2% was observed for most cells electroporated at a field strength of 7.5 kV/cm for 3.5 ms compared with a lower field strength of 2.5 kV/cm. Electroporation also increased the incorporation of cholesterol into the cellular membrane, as shown by an increased cholesterol:phospholipids ratio (50.0-59.6%) upon treatment at 7.5 kV/cm compared with treatment at 2.5 kV/cm. Saturation of cholesterol was observed in different regions of the membrane bilayer such as upper phospholipids, apolar tail, and polar heads, as indicated by fluorescence anisotropy using 3 fluorescent probes. Electroporation could be a useful technique to increase the ability of lactobacilli to remove cholesterol for possible use as cholesterol-lowering adjuncts in the future.

Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol.

Fifteen strains of Lactobacillus and Bifidobacterium were screened based on their ability to adhere to hydrocarbons via the determination of cellular hydrophobicity. Lactobacillus acidophilus ATCC 314, L. acidophilus FTCC 0291, Lactobacillus bulgaricus FTCC 0411, L. bulgaricus FTDC 1311, and L. casei ATCC 393 showed greater hydrophobicity and, thus, were selected for examination of cholesterol-removal properties. All selected strains showed changes in cellular fatty acid compositions, especially total fatty acids and saturated and unsaturated fatty acids in the presence of cholesterol compared with those grown in the absence of cholesterol. In addition, we found that cells grown in media containing cholesterol were more resistant to sonication and enzymatic lysis compared with those grown without cholesterol. We further evaluated the location of the incorporated cholesterol via the insertion of fluorescence probes into the cellular membrane. In general, enrichment of cholesterol was found in the regions of the phospholipid tails, upper phospholipids, and polar heads of the cellular membrane phospholipid bilayer. Our results also showed that lactobacilli were able to reduce cholesterol via conversion of cholesterol to coprostanol, aided by the ability of strains to produce cholesterol reductase. Our results provided experimental evidence to strengthen the hypothesis that probiotics could remove cholesterol via the incorporation of cholesterol into the cellular membrane and conversion of cholesterol to coprostanol. The strains studied may be potential health adjunct cultures in fermented dairy products with possible in vivo hypocholesterolemic effects.

Inhibitory effect of oxalic acid on bacterial spoilage of raw chilled chicken.

Oxalic acid was evaluated as a treatment for reducing populations of naturally occurring microorganisms on raw chicken. Raw chicken breasts were dipped in solutions of oxalic acid (0, 0.5, 1.0, 1.5, and 2.0%, wt/vol) for 10, 20, and 30 min, individually packed in oxygen-permeable polyethylene bags, and stored at 4 degrees C. Total plate counts of aerobic bacteria and populations of Pseudomonas spp. and Enterobacteriaceae on breasts were determined before treatment and after storage for 1, 3, 7, 10, and 14 days. The pH and Hunter L, a, and b values of the breast surface were measured. Total plate counts were ca. 1.5 and 4.0 log CFU/g higher on untreated chicken breasts after storage for 7 and 14 days, respectively, than on breasts treated with 0.5% oxalic acid, regardless of dip time. Differences in counts on chicken breasts treated with water and 1.0 to 2.0% of oxalic acid were greater. Populations of Pseudomonas spp. on chicken breasts treated with 0.5 to 2.0% oxalic acid and stored at 4 degrees C for 1 day were less than 2 log CFU/g (detection limit), compared with 5.14 log CFU/g on untreated breasts. Pseudomonas grew on chicken breasts treated with 0.5% oxalic acid to reach counts not exceeding 3.88 log CFU/g after storage for 14 days. Counts on untreated chicken exceeded 8.83 log CFU/g at 14 days. Treatment with oxalic acid caused similar reductions in Enterobacteriaceae counts. Kocuria rhizophila was the predominant bacterium isolated from treated chicken. Other common bacteria included Escherichia coli and Empedobacter brevis. Treatment with oxalic acid caused a slight darkening in color (decreased Hunter L value), retention of redness (increased Hunter a value), and increase in yellowness (increased Hunter b value). Oxalic acid has potential for use as a sanitizer to reduce populations of spoilage microorganisms naturally occurring on raw chicken, thereby extending chicken shelf life.

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.

Characterization of Burkholderia pseudomallei isolated in Thailand and Malaysia.

A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis. All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested. Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand. RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates. The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate. The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B. pseudomallei in epidemiological investigation.

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.

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.

Prevalence of Bacillus cereus in selected foods and detection of enterotoxin using TECRA-VIA and BCET-RPLA.

Enterotoxigenic Bacillus cereus was detected in cooked foods (17), rice noodles (3), wet wheat noodles (2), dry wheat noodles (10), spices (8), grains (4), legumes (11) and legume products (3). One hundred ninety-four (42.3%), 70 (15.3%) and 23 (5.2%) of the 459 presumptive B. cereus colonies isolated from PEMBA agar were identified as B. cereus, Bacillus thuringiensis and B. mycoides, respectively. B. cereus isolates were examined for growth temperature, pH profile and enterotoxin production using both TECRA-VIA and BCET-RPLA kits. One hundred seventy-eight (91.8%) and 164 (84%) of the strains were enterotoxigenic as determined using TECRA-VIA and BCET-RPLA, respectively. Eighty-two (50%) of the enterotoxigenic strains were capable of growing at 5 degrees C, and 142 (86.6%) grew at 7 degrees C within 7 days of incubation. The enterotoxigenic strains did not grow at pH 4.0 but 69 (42.0%) of the strains were able to grow at pH 4.5 within 7 days at 37 degrees C. The isolates were resistant to ampicillin (98.8%), cloxallin (100%) and tetracycline (61.0%), and susceptible to chloroamphenicol (87%), erythromycin (77.4%), gentamycin (100%) and streptomycin (98.7%).

Identification of lactic acid bacteria constituting the predominating microflora in an acid-fermented condiment (tempoyak) popular in Malaysia.

Tempoyak is a traditional Malaysian fermented condiment made from the pulp of the durian fruit (Durio zibethinus). Salt is sometime added to proceed fermentation at ambient temperature. In various samples obtained from night markets, lactic acid bacteria (LAB) were the predominant microorganisms, ranging from log 8.4 to log 9.2 cfu g(-1). No other microorganisms were present to such a level. These samples contained reduced amount of saccharose, glucose and fructose but increased amount of D- and L-lactic acid and acetic acid compared with samples of non-fermented durian fruit. Sixty-four isolates of LAB were divided into five groups by use of a few phenotypic tests. A total of 38 strains of LAB were selected for comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their whole cell protein patterns with a SDS-PAGE database of LAB. These strains were also examined for their carbohydrate fermentation patterns by use of API 50 CH. Isolates belonging to the Lactobacillus plantarum group were shown to be the predominant members of the LAB flora. In addition, isolates belonging to the Lactobacillus brevis group, Leuconostoc mesenteroides, Lactobacillus mali, Lactobacilus fermentum and an unidentified Lactobacillus sp. were also observed. A high degree of diversity among isolates belonging to the Lb. plantarum group was demonstrated by analysis of their plasmid profiles.

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.

Prevalence of Salmonella in broilers at retail outlets, processing plants and farms in Malaysia.

A study was conducted to estimate the prevalence of Salmonella among broilers retailed at wet-markets and processing plants. Litter and feed samples obtained from both broiler and breeder farms were also examined for Salmonella. A total of 158 out of 445 (35.5%) and 52 out of 104 (50.0%) broiler carcasses obtained from wet-markets and processing plants were contaminated with Salmonella, respectively. Salmonella was isolated from 14 out of 98 (14.3%) samples of intestinal content. Litter samples from broiler and breeder farms were positive for Salmonella, 8/40 (20%) and 2/10 (20%), respectively. Salmonella isolates (230) belonging to 15 different serovars were isolated. Predominant serovars were S. enteritidis, S. muenchen, S. kentucky and S. blockley.

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.

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.

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.

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.

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.

Bacteriocin-producing lactic Acid bacteria isolated from traditional fermented food.

Lactic Acid Bacteria (LAB) isolated from several traditional fermented foods such as "tempeh", "tempoyak" and "tapai" were screened for the production of bacteriocin. One strain isolated from "tempeh" gives an inhibitory activity against several LAB. The strain was later identified as Lactobacillus plantarum BS2. Study shows that the inhibitory activity was not caused by hydrogen peroxide, organic acids or bacteriophage. The bacteriocin production was maximum after 10 hours of incubation with an activity of 200 AU/ml. The bacteriocin was found to be sensitive towards trypsin, α-chymotrypsin, ß-chymotrypsin, α-amylase and lysozyme.

Random amplified polymorphic DNA analysis to differentiate strains of Vibrio vulnificus isolated from cockles and shrimps.

A random amplified polymorphic DNA (RAPD) fingerprinting method has been developed to differentiate Vibrio vulnificus strains isolated. Twenty-nine strains isolated from cockles and twenty-one strains isolated from shrimps were analyzed. A total of 10 primers were screened with Vibrio vulnificus strains to identify those capable of generating DNA polymorphisms and two primers were selected. Primer GEN 1-50-01 and GEN 1-50-08 produced polymorphisms in most strains tested, with the band sizes ranging from 10.0 to 0.25 kb pair. Dendrogram analysis showed that primer GEN 1-50-01 produced 10 clusters and 24 single strains at a 40% similarity, whereas primer GEN 1-50-08 produced 11 clusters and 20 single strains at a 40% similarity. This study revealed the potential use of PCR fingerprinting in epidemiological studies.

Detection of Vancomycin-Resistant Enterococcus Spp. (VRE) from Poultry.

Twenty-eight isolates of E. faecalis and 5 isolates of E. hirae were isolated from chicken samples obtained from markets in Sri Serdang, Selangor. They were tested for susceptibility to vancomycin and other antimicrobial agents. All of the isolates showed multiple resistance to the antibiotic tested. All Enterococcus spp. were resistant (100%) to ceftaxidime, cephalothin, erythromycin, gentamicin, kanamycin, nalidixic acid and streptomycin. Resistance was also observed to norfloxacin (97%), tetracycline (91%), penicillin (85%), bacitracin (82%), chloramphenicol (61%) and the least resistance was to ampicillin (27%). High prevalence to vancomycin resistance was detected among the E. faecalis (27of 28) and E. hirae (4 of 5) isolates. The multiple antibiotic resistance index ranging between 0.64 to 1.0 showed that all strains tested originated from high-risk contamination. Plasmid profile analysis of Enterococcus spp. revealed plasmid DNA bands ranging in size from 1.3 to 35.8 megadalton but some isolates were plasmidless. No correlation could be made between plasmid patterns and antibiotic resistance.

Ultrasound enhanced growth and cholesterol removal of Lactobacillus fermentum FTDC 1311 in the parent cells but not the subsequent passages.

The aim of this study was to evaluate the effect of ultrasound on the intestinal adherence ability, cell growth, and cholesterol removal ability of parent cells and subsequent passages of Lactobacillus fermentum FTDC 1311. Ultrasound significantly decreased the intestinal adherence ability of treated parent cells compared to that of the control by 11.32% (P<0.05), which may be due to the protein denaturation upon local heating. Growth of treated parent cells also decreased by 4.45% (P<0.05) immediately upon ultrasound (0-4h) and showed an increase (P<0.05) in the viability by 2.18-2.34% during the later stage of fermentation (12-20 h) compared to that of the control. In addition, an increase (P<0.05) in assimilation of cholesterol (>9.74%) was also observed for treated parent cells compared to that of the control, accompanied by increased (P<0.05) incorporation of cholesterol into the cellular membrane. This was supported by the increased ratio of membrane cholesterol:phospholipids (C:P), saturation of cholesterol in the apolar regions, upper phospholipids regions, and polar regions of membrane phospholipids of parent cells compared to that of the control (P<0.05). However, such traits were not inherited by the subsequent passages of treated cells (first, second, and third passages). Our data suggested that ultrasound treatment could be used to improve cholesterol removal ability of parent cells without inducing permanent damage/defects on treated cells of subsequent passages.