Hau-Yang Tsen

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.

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 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.

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.

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 102–103 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 100 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.

Fiber entrapment of naringinase from Penicillium sp. and application to fruit juice debittering

Abstract Naringinase from Penicillium sp. was immobilized on cellulose triacetate by the fiber entrapment method. Although the optimum pH (3.7) and optimum temperature (55°C) of the fiber-entrapped enzyme were similar to those of the native form, the immobilized enzyme had better heat stability. Kinetic studies showed that the immobilized enzyme had higher K m values than its native form. When this immobilized naringinase was successively used in a column reactor for the hydrolysis of ρ-nitrophenyl-α- l -rhamnoside or naringin in a simulated fruit juice system or grapefruit juice, the enzyme column could be operated with satisfactory stability. In addition, when the natural grapefruit juice was recycled through the column reactor, no column blocking or filtering action of the catalyst bed was observed.

Possible Use of a Polymerase Chain Reaction Method for Specific Detection of Salmonella in Beef

Abstract A rapid and sensitive polymerase chain reaction (PCR) method for the detection of Salmonella isolates with different serotypes is described. Based on the DNA sequence of a cloned 1.8 kb Hin dIII DNA fragment which could hybridize with all the Salmonella isolates tested but not with any of the non- Salmonella isolates including Enterobacteriaceae closely related to Salmonella , oligonucleotide fragments ranging from 18- to 26-mer were synthesized and tested for their possible use as the PCR primers. Results showed that three oligonucleotides, called TS11, TS4 and TS5 could be used in pairs of TS11/TS4 and TS11/TS5 for the PCR detection of salmonellae with various serotypes. Under the conditions described, non- Salmonella isolates did not generate any false positive results. For primers TS11/TS4, the molecular weight of the PCR product amplified with Salmonella DNA was 1179 bp while for primers TS11/TS5, the molecular weight amplified was 375 bp. Primer TS5 could also be used as a checking probe to identify the PCR product amplified from primers TS11/TS4. Study of the detection sensitivity showed that DNA from N × 10 0 or N × 10 1 cells of Salmonella could be detected unambiguously either with primers TS11/TS5 or with primers TS11/TS4. When these PCR primers were used for the detection of salmonellae in beef, N × 10 0 ∼N × 10 1 cells per gram of beef could be detected and the endogenously contaminated microflora in the food sample did not interfere with the detection.

Possible use of a 1.8 kb DNA fragment for the specific detection of Salmonella in foods

Abstract The aim of this study was to find a specific DNA probe for the detection of Salmonella in foods. A 1.8 kb Hin dIII DNA fragment cloned from S. typhimurium was tested for its specificity by hybridization to Salmonella of different serotypes as well as to non- Salmonella bacteria. The 35 S-labeled Hin dIII DNA fragment hybridized with all of the common and uncommon Salmonella isolates but did not hybridize with the other bacteria tested. Hybridization efficiencies of this 1.8 kb DNA probe to Salmonella strains of different serotypes were nearly the same. When this probe was applied to the detection of Salmonella exogenously added to a variety of foods, bacterial flora naturally occurring in foods did not interfere with the hybridization assay. Thus, the hybridization specificity of this 1.8 kb DNA probe was established.

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.

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.