Prevalence and antibiotic resistance of Salmonella Enteritidis and Salmonella Typhimurium in raw chicken meat at retail markets in Malaysia.

Salmonellosis is one of the major food-borne diseases in many countries. This study was carried out to determine the occurrence of Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in raw chicken meat from wet markets and hypermarkets in Selangor, as well as to determine the antibiotic susceptibility profile of S. Enteritidis and S. Typhimurium. The most probable number (MPN) in combination with multiplex polymerase chain reaction (mPCR) method was used to quantify the Salmonella spp., S. Enteritidis, and S. Typhimurium in the samples. The occurrence of Salmonella spp., S. Enteritidis, and S. Typhimurium in 120 chicken meat samples were 20.80%, 6.70%, and 2.50%, respectively with estimated quantity varying from <3 to 15 MPN/g. The antibiogram testing revealed differential multi-drug resistance among S. Enteritidis and S. Typhimurium isolates. All the isolates were resistance to erythromycin, penicillin, and vancomycin whereas sensitivity was recorded for Amoxicillin/Clavulanic acid, Gentamicin, Tetracycline, and Trimethoprim. Our findings demonstrated that the retail chicken meat could be a source of multiple antimicrobial-resistance Salmonella and may constitute a public health concern in Malaysia.

Formation of biofilm by Listeria monocytogenes ATCC 19112 at different incubation temperatures and concentrations of sodium chloride.

Biofilm formation can lead to various consequences in the food processing line such as contamination and equipment breakdowns. Since formation of biofilm can occur in various conditions; this study was carried out using L. monocytogenes ATCC 19112 and its biofilm formation ability tested under various concentrations of sodium chloride and temperatures. Cultures of L. monocytogenes ATCC 19112 were placed in 96-well microtitre plate containing concentration of sodium chloride from 1-10% (w/v) and incubated at different temperature of 4 °C, 30 °C and 45 °C for up to 60 h. Absorbance reading of crystal violet staining showed the density of biofilm formed in the 96-well microtitre plates was significantly higher when incubated in 4 °C. The formation of biofilm also occurs at a faster rate at 4 °C and higher optical density (OD 570 nm) was observed at 45 °C. This shows that storage under formation of biofilm that may lead to a higher contamination along the processing line in the food industry. Formation of biofilm was found to be more dependent on temperature compared to sodium chloride stress.

Prevalence and quantification of Listeria monocytogenes in chicken offal at the retail level in Malaysia.

A total of 216 chicken offal samples (chicken liver = 72; chicken heart = 72; chicken gizzard = 72) from wet markets and hypermarkets in Selangor, Malaysia, were examined for the presence and density of Listeria monocytogenes by using a combination of the most probable number and PCR method. The prevalence of L. monocytogenes in 216 chicken offal samples examined was 26.39%, and among the positive samples, the chicken gizzard showed the highest percentage at 33.33% compared with chicken liver (25.00%) and chicken heart (20.83%). The microbial load of L. monocytogenes in chicken offal samples ranged from <3 to 93.0 most probable number per gram. The presence of L. monocytogenes in chicken offal samples may indicate that chicken offal can act as a possible vehicle for the occurrence of foodborne listeriosis. Hence, there is a need to investigate the biosafety level of chicken offal in Malaysia.

Formation of biofilm by Listeria monocytogenes ATCC 19112 at different incubation temperatures and concentrations of sodium chloride

Biofilm formation can lead to various consequences in the food processing line such as contamination and equipment breakdowns. Since formation of biofilm can occur in various conditions; this study was carried out using L. monocytogenes ATCC 19112 and its biofilm formation ability tested under various concentrations of sodium chloride and temperatures. Cultures of L. monocytogenes ATCC 19112 were placed in 96-well microtitre plate containing concentration of sodium chloride from 1-10% (w/v) and incubated at different temperature of 4 ºC, 30 ºC and 45 ºC for up to 60 h. Absorbance reading of crystal violet staining showed the density of biofilm formed in the 96-well microtitre plates was significantly higher when incubated in 4 ºC. The formation of biofilm also occurs at a faster rate at 4 ºC and higher optical density (OD 570 nm) was observed at 45 ºC. This shows that storage under formation of biofilm that may lead to a higher contamination along the processing line in the food industry. Formation of biofilm was found to be more dependent on temperature compared to sodium chloride stress.

Listeria monocytogenes in retailed raw chicken meat in Malaysia.

This study aimed to determine the prevalence Listeria monocytogenes in raw chicken meat samples at hypermarkets and wet markets. Chicken drumsticks, breasts, and thighs were randomly selected. The most probable number (MPN) PCR method was used to quantify the L. monocytogenes in the samples. Listeria monocytogenes was detected in 20% of the samples. Occurrence of L. monocytogenes was highest in breast (42.03%) followed by drumstick (11.27%) and thigh (7.14%). Samples from hypermarkets showed higher occurrence (25.71%) of L. monocytogenes compared with wet markets (14.29%). The density of L. monocytogenes found in samples ranged from <3.0 to 16 MPN•g(-1). The presence of L. monocytogenes in raw chicken meat is unwanted but unpreventable. Thus, further research on the processing method to reduce and eliminate this kind of bacteria in chicken meat before consumption is necessary. The presence of L. monocytogenes in chicken samples suggests the importance of this pathogen in chicken. Thus, more study is needed to find ways to eliminate this pathogen from poultry.

Transfer of Campylobacter jejuni from raw to cooked chicken via wood and plastic cutting boards.

AIMS: We quantified Campylobacter jejuni transferred from naturally contaminated raw chicken fillets and skins to similar cooked chicken parts via standard rubberwood (RW) and polyethylene cutting boards (PE). METHODS AND RESULTS: RW and PE cutting boards (2.5 × 2.5 cm(2)) were constructed. RW surfaces were smooth and even, whereas PE was uneven. Scoring with scalpel blades produced crevices on RW and flaked patches on the PE boards. Raw chicken breast fillets or skin pieces (10 g) naturally contaminated with Camp. jejuni were used to contaminate the cutting boards (6.25 cm(2)). These were then briefly covered with pieces of cooked chicken. Campylobacter jejuni on raw chicken, the boards, and cooked chicken pieces were counted using a combined most-probable-number (MPN)-PCR method. The type of cutting board (RW, PE; unscored and scored) and temperature of cooked chicken fillets and skins were examined. Unscored PE and RW boards were not significantly different in regards to the mean transfer of Camp. jejuni from raw samples to the boards. The mean transfer of Camp. jejuni from scored RW was significantly higher than from scored PE. When the chicken fillets were held at room temperature, the mean transfer of Camp. jejuni from scored RW and PE was found to be 44.9 and 40.3%, respectively. CONCLUSIONS: RW and PE cutting boards are potential vehicles for Camp. jejuni to contaminate cooked chicken. Although cooked chicken maintained at high temperatures reduced cross-contamination via contaminated boards, a risk was still present. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of cooked chicken by Camp. jejuni from raw chicken via a cutting board is influenced by features of the board (material, changes caused by scoring) and chicken (types of chicken parts and temperature of the cooked chicken).

Evaluation of a probiotics, Bifidobacterium breve BBG-01, for enhancement of immunogenicity of an oral inactivated cholera vaccine and safety: a randomized, double-blind, placebo-controlled trial in Bangladeshi children under 5 years of age.

To evaluate the probiotic, Bifidobacterium breve strain Yakult (BBG-01), for safety and enhancement of immunogenicity in an oral inactivated cholera vaccine, a randomized double-blind placebo-controlled study was performed. Bangladeshi children under 5-year-old received BBG-01 or placebo for 4 weeks with two doses of oral cholera vaccine. Serum/fecal antibodies and fecal bacterial flora in the study participants were monitored. All adverse events were mild and transient and had no significant difference between the two groups. Immunological responses were similar comparing the two groups. A negative correlation between Bifidobacterium and Enterobacteriaceae in the probiotic group suggests a possible involvement of BBG-01 in alteration of the enteric bacterial flora. In conclusion, BBG-01 is well tolerated by Bangladeshi children although the post vaccinal immunostimulatory effect of BBG-01 was not evident.

Occurrence of thermophilic Campylobacter spp. contamination on vegetable farms in Malaysia.

The aim of the present study was to examine the prevalence of thermophilic Campylobacter spp. (Campylobacter jejuni and Campylobacter coli) in soil, poultry manure, irrigation water, and freshly harvested vegetables from vegetable farms in Malaysia. C. jejuni was detected in 30.4% and 2.7% of the soil samples, 57.1% and 0% of the manure samples, and 18.8% and 3% of the vegetable samples from farm A and farm B, respectively, when using the MPNPCR method. Campylobacter spp. was not found in any of the irrigation water samples tested. Therefore, the present results indicate that the aged manure used by farm A was more contaminated than the composted manure used by farm B. Mostly, the leafy and root vegetables were contaminated. C. coli was not detected in any of the samples tested in the current study. Both farms tested in this study were found to be contaminated by campylobacters, thereby posing a potential risk for raw vegetable consumption in Malaysia. The present results also provide baseline data on Campylobacter contamination at the farm level.

Prediction of epidemic cholera due to Vibrio cholerae O1 in children younger than 10 years using climate data in Bangladesh.

To determine if a prediction of epidemic cholera using climate data can be made, we performed autoregression analysis using the data recorded in Dhaka City, Bangladesh over a 20-year period (1983-2002) comparing the number of children aged <10 years who were infected with Vibrio cholerae O1 to the maximum and minimum temperatures and rainfall. We formulated a simple autoregression model that predicts the monthly number of patients using earlier climate variables. The monthly number of patients predicted by this model agreed well with the actual monthly number of patients where the Pearson's correlation coefficient was 0.95. Arbitrarily defined, 39.4% of the predicted numbers during the study period were within 0.8-1.2 times the observed numbers. This prediction model uses the climate data recorded 2-4 months before. Therefore, our approach may be a good basis for establishing a practical early warning system for epidemic cholera.

Characterization of new O3:K6 strains and phylogenetically related strains of Vibrio parahaemolyticus isolated in Taiwan and other countries.

AIMS: We analysed the genetic divergence in the pandemic new O3:K6 and phylogenetically related (new O3:K6-like) strains and compare these two groups in terms of virulence and other biological traits. METHODS AND RESULTS: A total of 160 new O3:K6, new O3:K6-like and other strains of Vibrio parahaemolyticus isolated in Taiwan and other countries were collected and their clonal relationships analysed using SfiI-pulsed-field gel electrophoresis. All of the new O3:K6 and new O3:K6-like strains were grouped in cluster I with five new patterns identified. A O6:K18 strain was identified as a new member of the new O3:K6-like strains in addition to O4:K68, O1:KUT and O1:K25 strains. All of the lipopolysaccharide preparations of the selected strains exhibited closely spaced quadruplet banding patterns with similar mobility. The two groups of strains exhibited 100% sequence identity in the internal sequences of the toxR and laf genes, and also displayed similar virulence properties as determined with a suckling mouse model. CONCLUSIONS: The new O3:K6 and new O3:K6-like strains were highly similar in virulence and in several other phenotypical and genotypical traits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrated the spread and divergence of the pandemic and related clone of V. parahaemolyticus with similar virulence.

Prevalence and serodiversity of the pandemic clone among the clinical strains of Vibrio parahaemolyticus isolated in southern Thailand.

We collected diarrhoea specimens in two hospitals in southern Thailand in 1999 to examine whether infection by the Vibrio parahaemolyticus pandemic clone is prevalent. V. parahaemolyticus was isolated from 317 specimens. Seventy-six per cent of the isolated strains had the pandemic clone-specific characteristics (tdh+, trh-, and an unique toxRS sequence detectable by GS-PCR) and an associated characteristic (the ORF8 sequence of f237 phage). These strains belonged to the three pandemic servovars with the O3:K6 strains being dominant and three other serovars (O1:K25, O1:K41 and O4:K12). We also found O1:K25 and O1:K41 strains with the pandemic clone-specific characteristics among the strains isolated from the international travellers who left Thailand and three other Asian countries between 1998 and 1999, verifying pandemic potential of these strains. The results demonstrate prevalence of infection by the pandemic clone in southern Thailand and suggest emergence of various serovariants in this area and their implication in international spread.

Application of polymerase chain reaction for detection of Vibrio parahaemolyticus associated with tropical seafoods and coastal environment.

AIMS: To study the incidence of Vibrio parahaemolyticus in seafoods, water and sediment by molecular techniques vs conventional microbiological methods. METHODS AND RESULTS: Of 86 samples analysed, 28 recorded positive for V. parahaemolyticus by conventional microbiological method, while 53 were positive by the toxR-targeted PCR, performed directly on enrichment broth lysates. While one sample of molluscan shellfish was positive for tdh gene, trh gene was detected in three enrichment broths of molluscan shellfish. CONCLUSIONS: Direct application of PCR to enrichment broths will be useful for the rapid and sensitive detection of potentially pathogenic strains of V. parahemolyticus in seafoods. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus is an important human pathogen responsible for food-borne gastroenteritis world-wide. As, both pathogenic and non-pathogenic strains of V. parahaemolyticus exist in the seafood, application of PCR specific for the virulence genes (tdh & trh) will help in detection of pathogenic strains of V. parahaemolyticus and consequently reduce the risk of food-borne illness.

Prevalence of the pandemic genotype of Vibrio parahaemolyticus in Dhaka, Bangladesh, and significance of its distribution across different serotypes.

Sixty-six strains of Vibrio parahaemolyticus belonging to 14 serotypes were isolated from hospitalized patients in Dhaka, Bangladesh, from January 1998 to December 2000. Among these, 48 strains belonging to four serotypes had the pandemic genotype and possessed the tdh gene. A marker (open reading frame ORF8) for a filamentous phage previously thought to correspond to the pandemic genotype was found to have a poor correlation with the pandemic genotype.

Automated ribotyping differentiates vibrio parahaemolyticus O3:K6 strains associated with a Texas outbreak from other clinical strains.

Automated ribotyping with a Qualicon Riboprinter was used to determine whether clinical isolates of Vibrio parahaemolyticus O3:K6 recovered during two U.S. outbreaks of oyster-associated gastroenteritis in 1998 were related to each other and to a previously identified highly virulent Asian clone of this serotype. The patterns produced using the restriction enzymes Eco RI and Pst I suggest that the outbreak in the Northeastern United States was caused by a single strain closely related to the Asian clone. In contrast, it appears that multiple strains were involved in the Texas outbreak and that the predominant type was genetically distinct from the Northeastern and Asian clone.

The toxR gene of Vibrio (Listonella) anguillarum controls expression of the major outer membrane proteins but not virulence in a natural host model.

To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two beta-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a virulence gene(s).

Molecular epidemiologic analysis of Vibrio cholerae O1 isolated during the 1997-8 cholera epidemic in southern Thailand.

An unusually high incidence of Vibrio cholerae O1 infection was observed in southern Thailand between late December 1997 and March 1998. Fifty-seven V. cholerae O1 strains were isolated in five provinces during this epidemic and were examined. They were El Tor Ogawa strains exhibiting similar antibiograms. All strains were resistant to tetracycline, which had not been reported in Thailand since 1993. The ribotypes. hybridization patterns with ctx and zot gene probes, arbitrarily primed PCR profiles, and pulsed-field gel electrophoresis profiles of the representative strains were compared with the clinical strains isolated from patients in India and Bangladesh in 1997 and 1998 and from international travellers originating from various Asian countries during the 1992-8 period. All southern Thailand strains and the 1998 international traveller strain of Thai origin showed indistinguishable genetic fingerprinting patterns that were distinct from those of other test strains. The results suggest that a tetracycline-resistant clone newly emerged in late December 1997 caused the large epidemic in southern Thailand and that the variants with a slightly different antibiogram appeared during the course of the spreading epidemic.

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.

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.

Molecular evidence of clonal Vibrio parahaemolyticus pandemic strains.

The upsurge in worldwide incidence of Vibrio parahaemolyticus infection in the last 5 years has been attributed to the recent appearance of three serotypes with pandemic potential: O3:K6, O4:K68, and O1:K untypeable (KUT). Thirty-five strains of these serotypes, isolated from different countries over 4 years, were characterized by ribotyping and pulsed-field gel electrophoresis to determine their origin. The ribotypes of the strains of these serotypes were indistinguishable, except for a Japanese tdh- negative O3:K6 strain and a U.S. clinical O3:K6 isolate, which had slightly different profiles. The migration patterns of the NotI-digest of the total DNA of the strains were similar, and only slight variations were observed between the serotypes. By contrast, the O3:K6 and O1:KUT strains isolated before 1995 and strains of other serotypes had markedly different profiles. The O4:K68 and O1:KUT strains most likely originated from the pandemic O3:K6 clone.

A unique and common restriction fragment pattern of the nucleotide sequences homologous to the genome of vf33, a filamentous bacteriophage, in pandemic strains of Vibrio parahaemolyticus O3:K6 O4:K68, and O1:K untypeable.

Vf33 is a filamentous bacteriophage isolated from Vibrio parahaemolyticus. We performed Southern blot hybridization analysis to examine the distribution of Vf33-related genetic elements in the pandemic strains (O3:K6 strains isolated between 1995 and 1997, O4:K68 and O1:K untypeable strains isolated between 1997 and 1999) of V. parahaemolyticus. Nucleotide sequences homologous to the Vf33 DNA were detected in all 57 test strains including pandemic and non-pandemic strains. However, the profiles of hybridization, including the restriction fragment length polymorphism, with nine Vf33-derived DNA probes exhibited by the pandemic strains were identical and were different from those by the non-pandemic strains. The results support the hypothesis that the pandemic strains are clonal, and suggest a possibility that they have acquired (a) new gene(s) via a Vf33-like filamentous phage.