The relative efficacy of different strain combinations of lactic acid bacteria in the reduction of populations of Salmonella enterica Typhimurium in the livers and spleens of mice

Multispecies probiotics have been reported to be more effective than monostrain probiotics in health promoting for the host. In this study, 12 lactic acid bacteria (LAB) strains were selected based on the level of induction of tumor necrosis factor (TNF)-alpha in RAW 264.7 macrophage cells. Their adherence to Caco-2 cells and inhibitory effects on Salmonella invasion of Caco-2 cells were compared. Strains with different probiotic properties were then combined and BALB/c mice were fed with LAB strains for 63 days; then the mice were challenged with Salmonella on day 64. For Salmonella-unchallenged mice that received a multistrain combination of LAB strains that have greater TNF-alpha production in macrophages, greater adherence and inhibit Salmonella invasion of Caco-2 cells to a greater extent, their peritoneal macrophages had greater phagocytic activity. For Salmonella-challenged mice, a significant reduction of Salmonella cells in the livers and spleens of the mice was observed 8 days post challenge. The addition of 12% skim milk powder together with LAB strain combinations significantly enhanced the reduction of Salmonella cells in the mice livers and spleens. In conclusion, we have shown that LAB strain combinations with particular probiotic properties when fed to mice can inhibit Salmonella invasion of the liver and spleen

A comparison of antibiograms for the Salmonella typhimurium isolates from humans and domestic or other animals in Taiwan

The antibiograms of 45 human isolates and 87 animal isolates of Salmonella typhimurium collected from 1990 to 1996 in Taiwan were investigated. The antibiotics used were tetracycline (Te), sulfisoxazole (G), ampicillin (Am), chloramphenicol (C), streptomycin (S), trimethoprim-sulfamethoxazole (Sxt), kanamycin (K), gentamicin (Gm), norfloxacin (Nor) and cefoperazone (Cfp). Resistance to antibiotics for these Salmonella isolates was studied, and the results obtained for human and animal isolates were compared. it was found that the antibiograms for human and animal isolates are quite similar The major resistant type for these Salmonella strains is TeGAmSC. Both the human and animal isolates are highly resistant to first-line antibiotics, such as tetracycline, sulfisoxazole, ampicillin, streptomycin of chloramphenicol; but are sensitive to fluoroquinolone antibiotics, such as norfloxacin and the third generation antibiotic of cephalosporin, such as cefoperazone and gentamicin. Between 93% and 100% of the local strains is inhibited by these antibiotics. Also, a significant fraction of these S. typhimurium isolates are multidrug resistant strains. For example, 58.6% of the animal isolates and 68.9% of the human isolates are multidrug-resistant. In conclusion, the antibiograms for human and animal isolates of S. typhimurium are similar. These results may be owing to the fact that Taiwan is geographically a small island and S. typhimurium strains are the common infective strains for human and domestic animals. Also, a high fraction of these strains was found to be drug resistant, which may be attributed to the fact that antibiotics are not strictly restricted for use in Taiwan

Molecular typing of Salmonella enterica serovars Typhimurium, Typhi, and Enteritidis isolated in Taiwan

Salmonella enterica serovars Typhimurium, Typhi, and Enteritidis are serious food pathogens which may cause human disease and/or animal infections. In an attempt to elucidate the clonal relationship in each of these species, to find the most disseminated and recirculating strains in food poisoning cases, and to discern the possible transmission of these strains from different origins and areas, we have used phage typing, antibiograms and molecular typing methods, such as plasmid profiles, pulsed field gel electrophoresis (PFGE), and random amplified polymorphic DNA (RAPD) to identify subtypes of these Salmonella strains. The results showed that in Salmonella Typhimurium and Typhi strains, considerable genetic diversity were found while in S. Enteritidis, high genetic similarity was observed. Also possibly, the most disseminated and recirculating strains of S. Typhimurium and S. Enteritidis were identified. Strains of these common subtypes might be the most prevalent strains and transmission of strains between different areas and origins might be possible

Pulsed field gel electrophoresis for animal Salmonella enterica serovar Typhimurium isolates in Taiwan

Salmonella enterica subspecies enterica serovar Typhimurium is a common pathogen for humans and animals. In order to trace the clonal relationship and to find the circulating strains between human and animal isolates, chromosomal DNAs from 87 serovar Typhimurium strains isolated from animals (pigs were the majority) were subjected to XbaI and SpeI digestion and pulsed field gel electrophoresis (PFGE). For the 87 animal isolates, 38 PFGE pattern combinations were obtained. As the subtyping results from animal isolates were compared with those from the 45 human isolates, it was found that 14 of the animal isolates and 13 of the human isolates shared a common PFGE pattern combination, i.e., pattern XgSf (or called X5S4). When these human and animal isolates were subjected to antibiotic susceptibility test using I I antibiotics, it was found that strains of pattern XgSf (X5S4) belong to a common antibiogram pattern which is tetracycline, gentamicin, ampicillin, streptomycin and chloramphenicol resistant. Since most of the animal and human strains in pattern XgSf were originally isolated from various areas over different years, strains of this PFGE pattern may be the most epidemic strains which circulating between human and animal sources. (C) 2002 Elsevier Science B.V. All rights reserved

Comparison of the partial 16S rRNA gene sequences and development of oligonucleotide probes for the detection of Escherichia coli cells in water and milk

The time and accuracy limitations of the commonly used methods for Escherichia coli detection in food and water suggest the need for rapid and specific methods. DNA hybridization is one of these methods. To develop E. coli-specific DNA probes, the primary sequences of the V3 and V6 regions of the 16S rRNA genes of non-pathogenic and pathogenic E. coli strains were determined and compared with those of the non-E. coli strains, including strains of the family of Enterobacteriaceae. Two oligonucleotides named as;16El and EV6 were then designed from the sequence unique to the E. coli cells. They were P-32-labelled and tested for their colony-hybridization specificities to the non-pathogenic and pathogenic E. coli strains. Results showed that ail the E. coli strains tested generated positive hybridization signals, while none of the non-E. coli strains tested gave a false positive result (except for Shigella spp.) When these probes were used for the detection of E. coli cells in contaminated tap-water and milk samples it was found that as low as 10(0)cfu 100 ml(-1) of tap-water or ml(-1) milk could be detected if an 8-h preculture step was performed prior to the hybridization assay. (C) 1999 Academic Press

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 mi 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

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

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 fi om 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 anal sis showed only three plasmid profiles and phage typing show-ed that most of the Salmonella strains were of the phage type PT4. Conclusions: Most of the Salm. enteritidis strains circulating in Taiwan are of ver 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 ill 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 M-w equal to 392 bp. On the other hand, only 97 of these 110 E. roll 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 degrees 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. (C) 2001 Elsevier Science B.V. All rights reserved