Oral administration of a combination of select lactic acid bacteria strains to reduce the Salmonella invasion and inflammation of broiler chicks.

One-day-old chicks are susceptible to infection by strains of Salmonella enterica subspecies. Because multistrain probiotics are suggested to be more effective than monostrain probiotics due to the additive and synergistic effects, in this study, we prepared a multistrain formula A (MFA) consisting of 4 lactic acid bacteria (LAB) strains selected by enhancing the TNF-α production for mouse macrophage 264.7 cells. The antagonistic effect of this MFA against the cecal colonization, viscera invasiveness, as well as the inflammation of 1-d-old chicks challenged with Salmonella Typhimurium were then assayed. One-day-old chicks were fed with MFA from d 1 to d 3, and on d 4, chicks were challenged with Salmonella Typhimurium (200 µL, 10(6) cfu/mL). The livers, spleens, and cecal tonsils of chicks were then removed on d 3 and 6 postinfection. Compared with the multistrain formula B (MFB) which consisted of LAB strains selected at random, the efficacy of MFA to reduce the Salmonella counts recovered from the cecal tonsils, spleens, and livers of chicks were significantly higher. Moreover, when the levels of proinflammatory cytokines, such as IL-1ß, IL-6, interferon (IFN)-γ, and anti-inflmmatory cytokine, that is, IL-10, in cecal tonsils were measured by reverse-transcription real-time quantitative PCR; it was found that chicks fed with MFA for 3 d had lower levels of IL-1ß, IL-6, IFN-γ and a higher level of IL-10 in the cecal tonsils of chicks as compared with those of the chicks fed with MFB or without LAB. These results suggest that multistrain probiotics consisting of LAB strains selected by immunomodulatory activity and adherence are more effective than those consisting of strains selected at random in antagonistic effect against Salmonella colonization, invasion, and the induced inflammation.

Contamination of Salmonella Schwarzengrund cells in chicken meat from traditional marketplaces in Taiwan and comparison of their antibiograms with those of the human isolates.

Salmonella Schwarzengrund is one of the infective Salmonella serotypes for humans and food animals, such as poultry and swine. Because consumption of foods containing salmonellae due to cross contamination or inadequate cooking may lead to human salmonellosis, in this report, the prevalence of Salmonella Schwarzengrund contamination in chicken meat samples purchased from different traditional marketplaces in Taiwan between 2000 and 2006 was investigated. In addition, 228 Salmonella Schwarzengrund strains isolated from these chicken meat samples and 30 human isolates obtained between 2004 and 2006 were compared for their antimicrobial susceptibility. Results showed that the prevalence of Salmonella Schwarzengrund contamination in raw chicken meat samples was 30.5%. Of all of the Salmonella isolates from chicken meat, Salmonella Schwarzengrund accounted for 39.3%. On the other hand, of the total Salmonella strains isolates from humans between 2004 and 2006, Salmonella Schwarzengrund accounted for 2.8%. All these chicken meat isolates and human isolates were multidrug-resistant and demonstrated high resistance to ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, nalidixic acid, trimethoprim-sulfamethoxazole, and chloramphenicol. For gentamicin and kanamycin, however, the resistance gradually declined. The antibiogram study may indicate the abuse of some antibiotics for both humans and chickens. Also, transmission of Salmonella Schwarzengrund strains between humans and food of animal origin is possible.

Screening from pickled vegetables the potential probiotic strains of lactic acid bacteria able to inhibit the Salmonella invasion in mice.

AIMS: To screen from pickled vegetables the potential probiotic lactic acid bacteria (LAB) strains with antagonistic activity against Salmonella invasion in host. METHODS AND RESULTS: Probiotic properties including acid and bile tolerance as well as inhibition on pathogenic bacteria were used for screening of LAB strains from pickled vegetables. Two strains, i.e Pediococcus pentosaceus MP12 and Lactobacillus plantarum LAP6, were selected and further assayed for their activities against Salmonella invasion in mouse liver and spleen. For these two LAB strains, strain LAP6 was able to adhere to the mouse intestinal epithelium cells. CONCLUSIONS: In screening of the probiotic strains able to inhibit the Salmonella invasion in host, factors other than the adherence to host intestinal epithelium may contribute some roles. SIGNIFICANCE AND IMPACT OF THE STUDY: Probiotic LAB strains with activity against Salmonella invasion in host could be isolated from vegetable origins. These strains may be used for vegetable processing.

Immune effect of heat-killed multistrain of Lactobacillus acidophilus against Salmonella typhimurium invasion to mice.

AIMS: This study attempted to determine whether lactic acid bacteria (LAB) could have a better probiotic function when used as a multistrain mixture, i.e. Mix-LAB, than when used as a monostrain. To this end, three strains of Lactobacillus acidophilus, specifically strain LAP5, LAF1 and LAH7, were heat-killed and mixed. This heat-killed Mix-LAB was used to evaluate the effectiveness of multistrain in inhibiting Salmonella invasion into cultured cells and into organs (spleen and liver) of live mice. METHODS AND RESULTS: BALB/c mice were orally administered with heat-killed Mix-LAB or sterile normal saline (control) for seven consecutive days and then challenged with orally administered Salmonella typhimurium on day 8. Results showed that, at day 6 after the challenge, the mice which had received Mix-LAB exhibited lower rates (P < 0.05) of Salmonella invasion into liver and spleen than did the control mice. Also, before the Salmonella challenge, the serum tumour necrosis factor-alpha (TNF-alpha) levels were not significantly different (P > 0.05) between these two groups of mice. After the challenge, however, the serum TNF-alpha level was significantly elevated (P < 0.05) in the control group, but not significantly changed in the Mix-LAB fed mice. To investigate possible factors involved in heat-killed Mix-LABs antagonistic effect on Salmonella invasion of mouse organs, heat-killed single strain and Mix-LAB were evaluated for ability to inhibit Salmonella invasion into cultured human intestinal Int-407 and Caco-2 cells. Results showed that none of the heat-killed strains were able to protect these cultured cells from Salmonella invasion, even though strains of LAP5 and Mix-LAB were adherent to them. However, study of the activation of murine macrophage Raw 264.7 cells showed that heat-killed Mix-LAB stimulated TNF-alpha production, nitric oxide release, and increased phagocytic activity in macrophages. CONCLUSIONS: Our findings suggest that heat-killed Mix-LAB can inhibit Salmonella invasion of mouse organs through the immunomodulating role of activated macrophage. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of heat-killed Mix-LAB to prevent bacterial infection in mice was found to be more significant than that of viable monostrain. This effect may be due to the activation of the immune system rather than to the adherence of LAB to the intestine epithelium.

Pulsed-field gel electrophoresis, plasmid profiles and phage types for the human isolates of Salmonella enterica serovar Enteritidis obtained over 13 years in Taiwan.

AIMS: Plasmid profile, phage typing, and pulsed-field gel electrophoresis (PFGE) patterns of 124 Salmonella Enteritidis strains isolated in 1998-2002 in Taiwan were analysed and the results were compared with those of the 63 strains obtained in 1991-1997, so that molecular subtypes and epidemic strains for Salmonella Enteritidis over a 13-year period (1991-2002) could be elucidated. METHODS AND RESULTS: A total of 124 strains of Salmonella Enteritidis isolated from human in Taiwan between 1998 and 2002 were analysed by PFGE, plasmid analysis and phage typing. The results obtained were compared with those of the 63 strains obtained in 1991-1997, so that the clonal relationships for a total of 187 strains obtained over 13 years could be elucidated. For PFGE, restriction enzymes XbaI, SpeI and NotI were used for chromosomal DNA digestion. Results showed 28 PFGE pattern combinations for the 187 Salmonella strains. Of them, pattern X3S3N3 was the major subtype as 130 strains isolated from different locations during 1991-2002 showed this PFGE pattern. For all these 187 strains, the genetic similarity was higher than 80%. Plasmid analysis showed 17 distinct types, which consist of one to four plasmids and the predominant phage type of those strains was PT4 (71.6%) and PT6a (13.4%). The three methods identified different degrees of polymorphism in the following order: plasmid profile (18 types, D = 0.659) > PFGE (28 types, D = 0.512) > phage typing (13 types, D = 0.438). As PFGE patterns, phage type and plasmid profile were combined for subtyping, the 187 strains could be grouped into 46 subtypes and the discriminatory index was raised to 0.795. For these 46 subtypes, the predominant one was X3S3N3/P1/PT4, which contained 77 (41%) isolates. CONCLUSIONS: Most of the Salmonella Enteritidis strains from sporadic cases were with pattern X3S3N3. They were the prevalent and may be the epidemic strains found in Taiwan during 1991-2002. The present study suggested that the several variants were derived from a single clonal line and the genome for strains of Salmonella Enteritidis are highly conserved over a 13-year period (1991-2002). SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here are useful for epidemiolgical study of salmonellosis caused by Salmonella Enteritidis in Taiwan. Comparing the data of the present study with those obtained for strains from other countries, the major subtypes for Salmonella Enteritidis infection in the world can be elucidated.

Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites.

AIMS: To evaluate the possibility for discrimination of Bacillus cereus and B. thuringiensis using 16S rRNA and gyrB gene based PCR methods, and to obtain the sequences of the primer annealing sites so that the PCR results may be explained. METHODS AND RESULTS: Based on the sequence difference in the variable region (V1) of 16S rRNA and in the gyrB gene between B. cereus and B. thuringiensis, PCR primers specific to these Bacillus spp. were designed. When these primers were used to discriminate B. cereus and B. thuringiensis, six of 82 B. cereus strains were identified as B. thuringiensis while 67 of 73 B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing sites showed that there is no clear-cut difference in the V1 region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. CONCLUSIONS: Although 16S rDNA based probes and gyrB gene based PCR primers have been suggested for the discrimination of B. cereus and B. thuringiensis strains, when a large number of Bacillus strains was tested, results showed that discrimination between B. cereus and B. thuringiensis is difficult. Therefore, to distinguish B. thuringiensis from B. cereus, a single feature, such as the presence of a parasporal crystal protein or cry gene, may sometimes be reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: Discrimination between B. cereus and B. thuringiensis is a challenging debate to which this paper makes a contribution.

Combination of immunomagnetic separation and polymerase chain reaction for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples.

A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i.e., the IMS-mPCR method) was developed for simultaneous detection of Listreria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs-e.g., n x 10(7) to n x 10(4) or n x 10(6) to n x 10(3)--the organism presenting the lower numbers might go undetected. Following the enrichment step with universal preenrichment (UP) broth, if an IMS method using equal quantities of anti-Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.

Analysis of Salmonella enteritidis strains isolated from food-poisoning cases in Taiwan by pulsed field gel electrophoresis, plasmid profile and phage typing.

AIMS: To establish the molecular typing data for Salmonella enteritidis due to its increasing role in Salmonella infections in Taiwan. METHODS AND RESULTS: Sixty-three Salm. enteritidis strains isolated from related and unrelated patients suffering from food-borne poisoning during 1991-97 were collected and subjected to pulsed field gel electrophoresis (PFGE), plasmid analysis and phage typing. For PFGE, XbaI, SpeI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 63 Salmonella strains, 10 PFGE pattern combinations were found. Of these, pattern X3 S3 N3 was the major subtype, since 46 strains isolated from different locations at different times during 1991-97 showed this PFGE pattern. Plasmid analysis showed only three plasmid profiles and phage typing showed that most of the Salmonella strains were of the phage type PT4. CONCLUSION: Most of the Salm. enteritidis strains circulating in Taiwan are of very similar genetic types or are highly related and that strains of PFGE pattern X3 S3 N3 are the prevalent and recirculating strains of Salm. enteritidis which caused food-poisoning cases in Taiwan in 1991-97. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information that in Salmonella infection, certain subtypes of Salm. enteritidis should be scrutinized.

PCR primers designed from malic acid dehydrogenase gene and their use for detection of Escherichia coli in water and milk samples.

Escherichia coli has been the appropriate focus for monitoring of potential enteric pathogens in water and foods. Although several methods have been used for the detection or enumeration of E. coli cells in water and foods, the time and accuracy limitations of these methods suggest the need of a rapid and specific method. By comparison of the gene sequences coding for malic acid dehydrogenase (mdh) of E. coli and non-E. coli strains, two oligonucleotides were designed and their possible use as E. coli-specific PCR primers was tested. All of the 110 E. coli strains tested, including non-pathogenic and various pathogenic strains, generated the expected PCR products with Mw equal to 392 bp. On the other hand, only 97 of these 110 E. coli strains were detectable using the BAM gas production method. With the exception of Shigella strains, non-E. coli strains, including strains of the family of Enterobacteriaceae, did not generate any false positive PCR results. When this PCR system was used for the monitoring of E. coli cells inoculated into water and milk samples, as low as 10(0) cfu per 100 ml of water or per ml of milk sample could be detected if an 8 h preculture step was performed prior to the PCR. Including the preculture step, the whole PCR detection process may be completed within 12 h.

Development and use of PCR primers for the investigation of C1, C2 and C3 enterotoxin types of Staphylococcus aureus strains isolated from food-borne outbreaks.

Staphylococcus aureus is a major food-borne pathogen in many countries. Enterotoxins produced by S. aureus strains include staphylococcal enterotoxins (SEs) A, B, C, D, E and G, H, I, etc. For SEC, in addition to the three major SEC subtypes, i.e., SEC1, C2 and C3, other molecular variants may exist. Although the detection methods and the distribution of SEA, B, C, D, E types of S. aureus in staphylococcal infections or food-borne outbreaks have been well documented, the differentiation method and the distribution of SEC subtypes in staphylococcal infections are rarely reported. In this study, four polymerase chain reaction (PCR) primers used in pairs (ENTC1/ENTCR, ENTC2/ENTCR and ENTC3/ENTCR) for the specific detection of SEC1, C2 and C3 genes of S. aureus strains were developed. When 39 SEC S. aureus strains isolated from fecal samples of randomly selected diarrheal patients associated with food-borne outbreaks in central Taiwan in 6 years (1995-2000) were analyzed, it was found that the major SEC subtypes for these S. aureus strains were SEC2 and C3.

Bacillus cereus group strains, their hemolysin BL activity, and their detection in foods using a 16S RNA and hemolysin BL gene-targeted multiplex polymerase chain reaction system.

Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group strains. It is also a target enterotoxin for the most commonly used B. cereus detection kit, i.e., the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (BCET-RPLA) test kit. A survey of the HBL activities and the cytotoxicities to the Chinese hamster ovary (CHO) cells for the B. cereus group strains, however, showed that although only part of the B. cereus group strains are HBL active, all strains show cytotoxicity to the CHO cells. Thus, methods that allow the detection of not only the HBL but also of the B. cereus group strains are important. In this study, by comparison of the gene sequences of the 16S rRNA for B. cereus group and other bacteria strains, we designed primers B16S1 and B16S2 specific to all the B. cereus group strains. In addition, because HBL is a major enterotoxin, we also designed HBL gene-specific polymerase chain reaction (PCR) primers, i.e., Hm1 and Hm2, that generated the same results as those of the hemolysis and BCET-RPLA assays. Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B. cereus group cells and the possible presence of their HBL enterotoxins. Also, all these PCR systems allowed the detection of n x 10(0) CFU B. cereus cells per g of food sample if an 8-h enrichment step was performed prior to the PCR.

Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks.

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.

Development and use of polymerase chain reaction for the specific detection of Salmonella Typhimurium in stool and food samples.

Salmonella Typhimurium is one of the most important Salmonella serovars that may cause foodborne disease and human salmonellosis infection. Detection of this organism in the clinical samples of persons with gastroenteritis and the food samples associated with such persons may allow us to trace the cause of disease. Because malic acid dehydrogenase, an enzyme of the citric acid cycle, is common to organisms, the gene (mdh) coding for this enzyme was selected for the design of Salmonella Typhimurium-specific polymerase chain reaction (PCR) primers. By comparison of the mdh gene sequences of Salmonella Typhimurium and other Salmonella serotypes and of some isolates of other genera, two oligonucleotides were designed and used as PCR primers for the specific detection of Salmonella Typhimurium. The molecular weight of the PCR product was 261 bp as expected. Salmonella serovars other than Salmonella Typhimurium and isolates of other genera in the Enterobacteriaceae that is closely related to Salmonella did not generate any false-positive results. When this primer pair was used for the detection of Salmonella Typhimurium cells artificially inoculated into human stool specimens and food samples, such as milk and raw chicken meat, levels as low as 10(0) CFU per 0.1 g of stool specimen or per ml of milk or food homogenate could be detected if an 8- to 12-h preculture step using combined lactose-tetrathionate broth was performed prior to the PCR.

Use of pulsed field gel electrophoresis as an epidemiological tool for analysis of sporadic associated strains of Salmonella typhi isolated in Taiwan.

In order to characterize the subtypes of Salmonella typhi which cause sporadic disease in Taiwan, 55 isolates of Salm. typhi obtained from unrelated patients of sporadic cases during 1992-96 were subjected to chromosomal DNA digestion and pulsed field gel electrophoresis (PFGE). When DNAs of these 55 Salm. typhi strains were digested with XbaI, 41 PFGE patterns were observed. Strains sharing the same XbaI digestion pattern could not be further discriminated by PFGE analysis using SpeI and NotI as digestion enzymes. Thus, considerable genetic diversity exists among the Salm. typhi isolates. Although strains of the same patterns were mainly isolated during the same time, recirculation of certain infectious strains could be possible. When 12 antibiotics, i.e. ampicillin, trimethoprim/sulfamethoxazole, erythromycin, norfloxacin, tetracycline, sulphonamide, streptomycin, neomycin, chloramphenicol, kanamycin, cefoperazone and gentamycin were used to test the antibiotic susceptibility for these Salmonella isolates, only three antibiogram patterns were obtained and 49 of the 55 Salm. typhi isolates were found to belong to one pattern. Phage typing and plasmid profiles were also poor in discriminating these strains. Thus, PFGE alone may be used as a powerful tool for analysis of sporadic associated Salm. typhi strains.

Development and use of 16S rRNA gene targeted PCR primers for the identification of Escherichia coli cells in water.

The primary sequences of the V3 and V6 regions of the 16S rRNA gene of pathogenic and non-pathogenic strains of Escherichia coli were determined and compared with those obtained for a number of reference strains which belong to the family Enterobacteriaceae. Three oligonucleotide primers 16E1, 16E2 and 16E3 were designed and used in the polymerase chain reaction to identify specifically all E. coli isolates. When 16E1, 16E2 and 16E3 were used as primers for the identification of E. coli cells present in tap, underground and pond waters, as low as 1 cfu 100 ml-1 of water could be detected if an 8 h pre-culture step was performed prior to the PCR reaction.

Use of a multiplex PCR system for the simultaneous detection of heat labile toxin I and heat stable toxin II genes of enterotoxigenic Escherichia coil in skim milk and porcine stool.

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT 1-and ST 11-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 10(0) cells per ml of the sample could be detected. Without the preculture step, 10(4) CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR system can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water.

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non-E. coli bacteria. Its detection limit was 10(2)-10(3) cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10(0) cells 100 ml-1 of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.

Comparison of type A enterotoxigenic Staphylococcus aureus strains isolated from geographically far distant locations by pulsed field gel electrophoresis.

Staphylococcal enterotoxin A (SEA) is one of the major staphylococcal enterotoxins which may cause food-borne outbreaks. In order to investigate the difference in genomic types and to elucidate the most disseminated strains for enterotoxin A-producing strains of Staphylococcus aureus, a total of 60 SEA Staph. aureus strains isolated from food and clinical samples in Taiwan and 30 strains of the same enterotoxigenic type of strains obtained from geographically far distant locations were compared for their pulsed field gel electrophoresis (PFGE) patterns. The rare cutting endonuclease SmaI generated 10 distinct genome patterns for the 60 local SEA isolates and 15 and eight genome patterns, respectively, for the 20 and 10 SEA strains originally isolated from the USA and other countries. The local isolates are less diverse in genome patterns as compared to the US isolates. Of all these PFGE patterns, a certain pattern, such as pattern 3, is shared by the food and clinical isolates and the local and foreign isolates. Thus, although SEA Staph. aureus strains from geographically far distant locations showed considerable genetic diversity, PFGE pattern 3 strain might be one of the most disseminated strains.

Use of two 16S DNA targeted oligonucleotides as PCR primers for the specific detection of Salmonella in foods.

A 16S DNA targeted polymerase chain reaction (PCR) method specific for the detection of Salmonella isolates with various serotypes was developed. The primers used for such a PCR method were 16SF1 and 16SIII. 16SF1 is the reverse and complementary strand of 16SI which has been shown to be able to hybridize with Salmonella and Citrobacter spp. 16III on the other hand, is able to hybridize with Klebsiella and Serratia spp. in addition to Salmonella. Although 16SF1 and 16SIII were not specific to Salmonella only, when they were used as PCR primers, only the Salmonella isolates could be specifically detected. The interference from Citrobacter, Klebsiella and Serratia spp. could be prevented. None of the other non-Salmonella isolates including strains of the family of Enterobacteriaceae closely related to Salmonella would generate the false-positive reaction. When this PCR system was used for the detection of Salmonella cells artificially contaminated in food samples, results obtained were satisfactory. A detection limit of N x 10(0) cells per assay could be obtained.

Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods.

DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non-Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non-Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37 degrees C and/or 43 degrees C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella.