Salmonella enterotoxin (stn) gene is prevalent among strains of Salmonella enterica, but not among Salmonella bongori and other Enterobacteriaceae

Abstract All strains and serovars of Salmonella enterica such as serovar Typhimurium, Enteritidis, Dublin, Typhi, etc. were found to carry the Salmonella enterotoxin determinant stn as far as examined in PCR and hybridization studies. However, using MDCK cells for testing the toxicity of the strains under investigation, only a limited number of stn positive strains revealed phenotypically the Salmonella enterotoxin Stn. In contrast to S. enterica , other Enterobacteriaceae including Salmonella bongori were found neither genotypically nor phenotypically Stn toxin positive.     

Characterization and cloning of enterotoxin genes of Salmonella typhimurium

Five of fifty five strains of Salmonella typhimurium of human origin was hybridized with both the LT-A and LT-B gene of Escherichia coli. The remarkably erythromatous and indurated response on rabbit skin and significant elongation of Chinese Hamster Ovary (CHO) cells indicated the production of enterotoxin of these isolates. The Salmonella enterotoxin is heat-labile and is not a secretory product. The LT gene of E. coli was used to analyze the chromosome and plasmid DNA from Salmonella typhimurium strains for toxin gene sequences. Southern blot analysis demonstrated that the toxin gene was located on the plasmid but not on the chromosome. Restriction enzymes BamHI, EcoRI, HindIII and PstI were used to analyze the DNA isolated from salmonella strains Nos.22, 52, 55 and 59. Three DNA fragments with size of 5.2 Kb of strain 22, 5.0 Kb of strain 52 and 8.6 Kb of strain 59 were identified as containing the enterotoxin gene. Plasmid pUC19 was used as the vector to clone these DNA fragments in E. coli. The rabbit skin permeability test indicated that Salmonella enterotoxin could be synthesized at readily detectable levels in these transformed E. coli.     

Evaluation of ELISA and GM1-ELISA for detection of Salmonella enterotoxin.

The ELISA and GM1-ELISA, by using antiserum to purified Salmonella enterotoxin (SE), were standardized and carried out to screen salmonellae isolated from foods of animal origin for enterotoxigenicity. Of the 101 strains of Salmonella belonging to 15 different serogroups tested, 76 (75.24%) strains from 13 serogroups were found enterotoxigenic. ELISA correlated well with rabbit ligated ileal loop (RLIL) test for the detection of enterotoxin producing salmonellae with 24 test strains. ELISA also yielded positive reaction with 7 of 13 RLIL negative strains. GM1-ELISA could not be carried out as none of the 101 cell free culture supernatants (CFCS) were able to bind with GM1-ganglioside. ELISA and GM1-ELISA were also standardized with antiserum to cholera toxin for the detection of salmonellae producing cholera related enterotoxin. None of the 101 strains was found to produce cholera related enterotoxin. ELISA could detect as low as 15 ng/100 microliters of purified SE and 10 ng/100 microliters of cholera toxin when tested with their homologous antisera.     

Evaluation of Biken test for detection of Salmonella enterotoxin.

Biken test by using antiserum to purified Salmonella enterotoxin (SE) was standardized and carried out to screen salmonellae for their enterotoxigenicity. As many as 101 strains of Salmonella belonging to 15 different serotypes isolated from foods of animal origin were subjected to Biken test. Of these, 68 (67.32%) were found seropositive. The test correlated with the rabbit ligated ileal loop (RLIL) test completely for the detection of enterotoxin producing salmonellae with 24 test strains. However, 5 of the 13 strains which were negative in the RLIL test, yielded positive results with the Biken test.     

Induction of Salmonella enterotoxin (stn) gene expression by epithelial cells (IEC-6)

Prevalence of Salmonella enterotoxin (stn) gene among Salmonella enterica and S. bongori was investigated by polymerase chain reaction (PCR) and gene probe and its status of phenotypic expression was examined on chinese hamster ovary cells by cultivating the strains with conventional method for enterotoxin production and by cultivating the organisms in contact with intestinal epithelial cells of rats (IEC-6). All the 19 strains and serovars of S. enterica such as Typhimurium, Enteritidis, Newport, Weltevreden, Indiana, Gallinarum and Kentucky were found to carry stn gene as examined by PCR and gene probe but only a limited number of strains (13 out of 19) expressed phenotypically the enterotoxin when cultured by conventional method. Cultivation of organisms in contact with epithelial cells induced expression of stn gene phenotypically in all the 19 strains. In contrast to S. enterica, strains of S. bongori were found neither genotypically (stn) nor phenotypically (Stn) positive.     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

Influence of Food Microorganisms on Staphylococcal Growth and Enterotoxin Production in Meat

Forty-four microorganisms were studied for their influence on staphylococcal growth and enterotoxin production. Inhibition was found to be more common than stimulation. Two types of inhibition were observed: inhibition of staphylococcal growth, and inhibition of enterotoxin formation with no apparent effect on growth. By use of a plate test, 12 of the 44 food microorganisms were found to inhibit staphylococcal growth at 35 C. Of the 12, 3 also inhibited growth at 25 C. No significant differences in inhibition were observed with the 15 strains of enterotoxigenic staphylococci. In meat slurries, inhibition of staphylococcal growth was found to be greater at 25 C than at 35 C. Results on inhibition obtained from the plate test could not be correlated with the effect of the organisms in slurries. Environmental conditions were found to affect markedly the influence of food microorganisms on staphylococci. Of the 44 food microorganisms studied, only Bacillus cereus was observed to stimulate significantly staphylococcal growth and enterotoxin formation. Stimulation was more pronounced with Staphylococcus aureus 196E than with other strains of enterotoxigenic staphylococci. Bacillus megaterium and Brevibacterium linens were inhibited by staphylococci. These organisms were completely inhibited when inoculated in mixed cultures with staphylococci. In pure cultures, good staphylococcal growth was found to be accompanied by enterotoxin production; however, in the presence of food microorganisms, good staphylococcal growth occurred without the formation of detectable levels of enterotoxin A.     

Antibiotic sensitivity and pathogenetic factors of hospital strains of Pseudomonas aeruginosa isolated from patients treated in a resuscitation unit]

Strains of Pseudomonas aeruginosa were isolated from patients treated in the Centre of Thermal Affections in 1985-1989. It was shown that 72.9, 59.3, 33.8 and 54.2 per cent of the isolates were sensitive to cefotaxime, tobramycin, gentamicin and polymyxin, respectively. The study of pathogenicity factors of the isolates revealed that 83 per cent of the strains produced thermolabile enterotoxin, 79.6 per cent of the strains had adhesive activity and 71.1 per cent of the strains produced hemolysin. The study detected combinations of various pathogenicity factors. 42.3 per cent of the isolates had both adhesive and enterotoxigenic properties. Adhesiveness and hemolytic activity were shown by 13.5 per cent of the strains. 16.9 per cent of the strains produced both enterotoxin and hemolysin. Adhesive activity, enterotoxigenicity and hemolysin production were observed in 6.7 per cent of the strains. It was noted that the strains of P. aeruginosa resistant to polymyxin mainly produced enterotoxin (18.6 per cent) and those resistant to cefotaxime had adhesive activity (34.0 per cent).     

Enterotoxin and cytotoxin production by Salmonella enteritidis strains isolated from gastroenteritis outbreaks

Seventy-six Salmonella enteritidis , three Salmonella virchow and one Salmonella braedenrup strains were screened for enterotoxigenicity by using the Chinese hamster ovary (CHO), Y1 adrenal, Vero and HeLa cell tests. All the strains gave positive reactions for enterotoxin production, except one, and the relative sensitivity to the toxin exhibited by the different cell lines was evaluated. An enterotoxic activity has been identified in sonicated extracts of Salm. enteritidis: This enterotoxin was purified on Agarose A-5m (Bio-Rad) and Superose 12 HR 10/30 column. The enterotoxic activity was eluted from the Superose column in the first peak. Like Vibrio cholerae toxin CT and Escherichia coli enterotoxin LT, it was blocked by GM₁ ganglioside, but at a higher concentration. In addition, a cytotoxic factor has been partially identified. The procedure for isolating the cytotoxin included ammonium sulphate precipitation, size-exclusion chromatography and anion exchange chromatography. This cytotoxin factor caused inhibition of protein synthesis in cultured cells, as determined by flow cytometry and [³H]-leucine incorporation. Flow cytometry analysis also showed an activation of CHO cells when exposed to this cytotoxic factor resulting in a state of active growth. Cytotoxic activity was not blocked by gangliosides.     

Virulence factors from Aeromonas species

Isolates (116) of Aeromonas were obtained from various sources and subjected to tests to establish their virulence factors. A high number of the isolates (69.8%) were found to be enterotoxigenic. The isolates from snails had more enterotoxigenic strains (73.3%), while those from cattle faeces had the lowest (33.3%). Haemolysin production was found to be high (60.3%) amongst the isolates, and human isolates gave the highest number of haemolysin producing strains (70.6%), while the least number (33.3%) was obtained from cattle strains. About 50% of the strains produced both enterotoxin and haemolysin. The enzyme profile of the isolates included amylase, lecithinase, lipase and protease. There was no definite pattern in the elaboration of these enzymes and the production of haemolysin and enterotoxin, thus inferring that the production of these factors is not specific to the source. Two isolates were seen to produce none of these enzymes, and one was positive for enterotoxin and haemolysin production, leaving only one isolate which yielded none of these factors. The work adds more support to the pathogenicity of Aeromonas species, and indicates the existence of non-pathogenic strains.     

Detection of Salmonella enterotoxin using rabbit ileal loops.

The presence of an enterotoxin produced by Salmonella in broth culture has been demonstrated by using the rabbit ileal loop model. Response by the animal to enterotoxin in sterile culture supernatant fluids is enhanced when the intestinal lumen is washed with a mucolytic agent prior to the administration of toxin. Fluid secretion is untreated intestinal loops was also observed if enterotoxin was administered with a live, invasive Salmonella strain which did not evoke a secretory response. A limited survey of Salmonella isolated for clinical and food sources indicated the common occurrence of enterotoxin production, and stock cultures maintained the ability to produce the toxin. The host-adapted species which were tested varied in their ability to produce enterotoxin.     

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.     

Seasonal distribution and age structure of those ill with salmonellosis due to causative agents with different drug resistances

The medicinal resistance of Salmonella strains isolated on the territory of the Moldavian SSR in 1985 was studied. The greatest number of multiresistant microorganisms was shown to occur among S. java, S. virchow and S. infantis, i.e. the serovars dominating in the etiological structure of Salmonella infections on this territory. The seasonal distribution of cases of Salmonella infections and the age structure of patients with these infections induced by multiresistant strains and antibiotic-sensitive strains were found to have essential differences.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Immune reactions of Ornithodoros papillipes ticks (Argasidae) to the administration of different microorganisms

It has been shown that ticks possess cellular factors of organism protection which fulfil functions of seizure and digestion of different microorganisms in a way similar to phagocytosis. In response to the introduction of bacteria lysozyme quantity increases in tick's haemolymph that exerts a bactericide effect on the introduced strains Micrococcus lysodeikticus 2665, Staphilococcus aureus 209 and Salmonella typhimurium IT-2. When administering phage FX-174 into tick's haemolymph a persistence of phage particles during 1-2 months is observed. Specific antibodies developing in response to introduced phage corpuscles were not found.     

Use of microbial antagonism to reduce pathogen levels on produce and meat products: a review

Lactic acid bacteria (LAB) are often utilized to control food-borne pathogens on produce and on cooked, fermented, or refrigerated meats. Most research to date has focused on the inhibition of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella, Clostridium botulinum, and spoilage microorganisms. LAB are excellent candidates for reducing pathogen levels on foods because they inhibit the growth of these microorganisms through various mechanisms without causing unacceptable sensory changes. This review provides an up-to-date look at research directed at maximizing the use of LAB by selecting the most appropriate strains, by learning how to apply them to foods most effectively, and by gaining an understanding of the mechanism by which they inhibit pathogens.     

Shigella dysenteriae 1-like cytotoxic enterotoxins produced by Salmonella strains.

A Salmonella enteritidis strain produced a cytotoxin in addition to heat-labile (LT) and heat-stable (ST) enterotoxins. Two strains of serotypes Salmonella kapemba and Salmonella thompson were LT and ST negative, but exhibited a cytotoxic effect. After Sephadex G-100 fractionation of the crude S. enteritidis material, some high and low molecular fractions had both cytotonic and cytotoxic activities. Of the two other salmonellae, only some high molecular fractions contained the cytotoxic substance. Neutralization experiments revealed an antigenic relationship between the cytotoxins studied and Shigella dysenteriae 1 enterotoxin. On the basis of cross neutralization and other data, it seems that cytotoxic and LT-like characters are carried by the same molecule. In S. thompson and S. kapemba the LT fails to exert a biological effect, although it is antigenically related to the LT of Escherichia coli.     

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.     

Extrachromosomal Control of Methicillin Resistance and Toxin Production in Staphylococcus aureus

All of 41 naturally occurring coagulase-positive methicillin-resistant strains of Staphylococcus aureus isolated in various laboratories were resistant to several antibiotics and were lipase-negative. Most strains produced hemolysins, and 38 strains produced enterotoxin B. Acriflavine treatment of four strains resulted in elimination of resistance to methicillin and mercury; in one strain, resistance to cadmium was also lost. Production of enterotoxin B and beta-hemolysin was eliminated in all four strains and penicillinase production was eliminated in one strain. In transduction experiments, methicillin resistance and enterotoxin B production were transferred together at a frequency of 0.2 x 10(-8) to 1.1 x 10(-8) by use of ultraviolet-induced phage lysates from naturally lysogenic methicillin-resistant strains. Cotransductions of resistance to mercury and cadmium, as well as production of penicillinase and beta-hemolysin, were obtained to some extent. The extrachromosomal character of these determinants and their possible genetic association are discussed.