Unique features of heat-stable enterotoxin of Shigella flexneri.

Sephadex G-100 fractions of ultrasonic lysate of Shigella felxneri were compared to the fractions of Escherichia coli lysates of Ent- , LT+ ST+, LT+ and ST+ strains. The range of molecular weight of S. flexneri ST fractions was the same as that of E. coli LT fractions. Rapid PF activity was associated with the ST peak in the case of S. flexneri, and followed the LT activity in the E. coli (LT+ ST+) fractions, and appeared in the same molecular weight range in the case of Ent- E. coli lysate. Cross neutralization could be demonstrated between S. flexneri ST and E. coli LT. Antigenic relationship between shigella ST and choleragen seemed to be less expressed and rather unilateral.     

Complementation and hybridization evidence for additional families of type I DNA restriction and modification genes in Salmonella serotypes.

Of eight Salmonella, serB-linked hsd genes for the restriction and modification of DNA transferred to Escherichia coli/Salmonella hybrids, only two--those with SM and ST (S. muenchen and S. thompson, respectively) specificities--may have weakly complemented rSB- and none complemented rK-. An A-specific DNA probe failed to hybridize to HindIII-restricted fragments of each of the hybrids, but an SB (S. typhimurium)-specific probe hybridized to DNA from the hybrid with ST specificity. These results indicate that additional families of the type I hsd genes may exist.     

Salmonella in surface waters of central New York state.

Six tributary streams and southern Cayuga Lake in central New York state were sampled for the presence of Salmonella on swabs immersed for 4 days. Of a total of 322 swabs, 39% yielded salmonellae. Swabs were cultured in tetrathionate enrichment at 41.5 degrees C. Isolations were made from brilliant green agar. Salmonellae were isolated from many sites on the streams and from some lake sites. Twenty-five serotypes (11 somatic antigen groups) and a distinct biotype of S. typhimurium (H2S negative) were found. Most frequent isolates, in order of decreasing occurrence, were S. typhimurium, S. thompson, S. oranienburg, and S. enteritidis. Several uncommon isolates also appeared. When tested for mouse infectivity, the isolates generally showed little or no virulence. The incidence of clinical salmonellosis among humans was low in the area and the variety of serotypes had not been noted among cattle. The presence of Salmonella in waters ranging in classification from potable to agricultural and industrial indicated the existence of a low level and undefined reservoirs of the bacteria in the region.     

Thermal inactivation of eight Salmonella serotypes on dry corn flour.

Dry heat was used to inactivate Salmonella newington, Salmonella typhimurium, Salmonella anatum, Salmonella kentucky, Salmonella cubana, Salmonella seftenberg, Salmonella thompson, and Salmonella tennessee in corn flour at 10 and 15% moisture. The flour was spray inoculated at 10(5) Salmonella cells per g and then stored at 49 degrees C (120 degrees F); viable Salmonella cells were counted on Trypticase (BBL Microbiology Systems) soy agar plates every 30 min for the first 4 h and then at 4-h intervals for 20 additional h of storage. After 24 h, 99.9% of all Salmonella cells were killed. S. thompson and S. tennessee were more resistant to heat inactivation than the other serotypes. Naturally occurring contamination by Salmonella spp. in dry food products could be significantly reduced with this treatment.     

Widespread occurrence of specific restriction endonucleases in Salmonella infantis, Salmonella thompson, and Salmonella blockley isolated from humans in Japan

Specific restriction endonucleases were detected in three serotypes of Salmonella spp. isolated from humans in Japan from 1970 to 1987: an isoschizomer of AvaII endonuclease at a frequency of 0.91 in Salmonella infantis, an isoschizomer of KpnI at a frequency of 0.34 in Salmonella thompson, and an isoschizomer of StyI at a frequency of 0.30 in Salmonella blockley. Of interest is that restriction endonuclease-producing S. thompson was detected at high frequencies in the 1970s but at low frequencies in the 1980s.     

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.     

Widespread occurrence of specific restriction endonucleases in Salmonella infantis, Salmonella thompson, and Salmonella blockley isolated from humans in Japan.

Specific restriction endonucleases were detected in three serotypes of Salmonella spp. isolated from humans in Japan from 1970 to 1987: an isoschizomer of AvaII endonuclease at a frequency of 0.91 in Salmonella infantis, an isoschizomer of KpnI at a frequency of 0.34 in Salmonella thompson, and an isoschizomer of StyI at a frequency of 0.30 in Salmonella blockley. Of interest is that restriction endonuclease-producing S. thompson was detected at high frequencies in the 1970s but at low frequencies in the 1980s.     

Lipopolysaccharide-specific IgG in egg yolk from two chicken flocks infected with Salmonella enteritidis

An indirect enzyme-linked immunosorbent assay (ELISA) was used to determine levels of IgG reacting with lipopolysaccharide from Salmonella enteritidis in samples of egg yolk from broiler breeder and egg-laying flocks, some of which were known to be infected with S. enteritidis. There was positive correlation between infection with salmonella and a high reading in the ELISA.     

Comparative Survival of Indicator Bacteria and Enteric Pathogens in Well Water

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.     

Purification and characterization of fimbriae from Salmonella enteritidis.

A human isolate of Salmonella enteritidis which displayed strong pellicle formation during static broth culture and mannose-sensitive hemagglutination produced fimbriae which were morphologically indistinguishable from type 1 fimbriae of members of the family Enterobacteriaceae. Fimbrin was purified to homogeneity, and the apparent molecular weight (Mr, 14,400) was markedly lower than that reported for the type 1 fimbrin of Salmonella typhimurium (Mr, 22,100). This fimbrin contained 40% hydrophobic amino acids and lacked cysteine. The sequence of the N-terminal 64 amino acids was determined, and sequence alignment revealed that although the 18 N-terminal residues of the S. enteritidis molecule shared considerable homology with Escherichia coli and S. typhimurium type 1 fimbrins, the S. enteritidis fimbrin lacked a 6- to 9-residue terminal sequence present in the other type 1 fimbrins and, after residue 18, shared little homology with the E. coli sequence. Antibodies raised to the purified S. enteritidis fimbrin bound to surface-exposed conformational epitopes on the native fimbriae and displayed pronounced serospecificity. These antibodies were used in the isolation of a nonfimbriated Tn10 insertion mutant which was unable to hemagglutinate.     

辽宁食源性沙门菌血清型、 耐药谱及PFGE分型特征

目的分析辽宁食源性沙门菌血清型、耐药谱及脉冲场凝胶电泳(PFGE)型别,探讨辽宁沙门菌污染的同源性,为食源性疾病溯源和预警提供基础。方法对辽宁省2015年食品中、食源性疾病中分离的41株沙门菌进行血清学分型、耐药试验、PFGE分子分型,采用Bio Numerics version 6.6软件分析,比较同源性。结果 41株菌分为15个血清型,居前三位的是15株肠炎沙门菌、5株德尔卑沙门菌、5株姆班达卡沙门菌(辽宁省内少见血清型);对41株菌进行15种抗生素的耐药试验,对单一一种抗生素的耐药率为100.0%,其中红霉素97.6%,萘啶酸61.0%,氨苄西林53.7%;41株菌共分为18种PFGE带型,带型分布分散,只有两种优势,一种带型包含20株菌,有14株肠炎沙门菌,6株其他沙门菌,相似度为92.7%~100%;另一种包含5株菌,4株姆班达卡沙门菌,1株鼠伤寒沙门菌,相似度为96.6%~100.0%。结论辽宁省食源性沙门菌的血清型以肠炎沙门菌为主,生肉制品是其主要污染来源;血清型与PFGE图谱带型分布广泛,相同血清型沙门菌的PFGE带型聚集成簇、菌株具有高度同源性;相同PFGE型别的菌株耐药谱一致或相似;沙门菌的耐药情况较严重。     

Interference ofSalmonella enteritidis andLactobacillus spp. with IL-8 levels and transepithelial electrical resistance of enterocyte-like caco-2 cells

Caco-2 cells (exhibiting characteristics of mature villus enterocytes) were used to determine bacteria (Salmonella enteritidis causing human gastroenteritis)-intestinal cell interactions. The interference of bacteria with the transepithelial electrical resistance (TEER) of filter-grown Caco-2 cells and the production of IL-8 after exposure of the cells to S. enteritidis 857 and/or Lactobacillus strains (L. gasseri LF221 and L. rhamnosus BGT10) was evaluated. The strain 857 decreased TEER of filter-grown Caco-2 cells; in contrast, lactobacilli had a little or no effect. The effect of S. enteritidis on the TEER decreased if Caco-2 cells were pre-incubated with lactobacilli. This strain induced high levels of IL-8 (which can lead to cell damage). Compared to the IL-8 synthesis after exposure of Caco-2 cells to S. enteritidis 857, simultaneous exposure of Caco-2 cells to S. enteritidis and lactobacilli inhibited the IL-8 synthesis after short recovery periods.     

Thin, aggregative fimbriae mediate binding of Salmonella enteritidis to fibronectin.

The binding of human fibronectin and Congo red by an autoaggregative Salmonella enteritidis strain was found to be dependent on its ability to produce thin, aggregative fimbriae, named SEF 17 (for Salmonella enteritidis fimbriae with an apparent fimbrin molecular mass of 17 kDa). Two other fimbrial types produced by S. enteritidis, SEF 14 and SEF 21, were not responsible for the aggregative phenotype or for fibronectin binding. SEF 17-negative TnphoA mutants which retained the ability to produce SEF 14 and SEF 21 were unable to bind human fibronectin or Congo red and lost the ability to autoaggregate. Only purified SEF 17 but not purified SEF 14 or SEF 21 bound fibronectin in a solid-phase binding assay. Furthermore, only SEF 17 was able to inhibit fibronectin binding to S. enteritidis whole cells in a direct competition enzyme-linked immunosorbent assay. These results indicate that SEF 17 are the fimbriae responsible for binding fibronectin by this enteropathogen.     

Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo

The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm. enteritidis 857. Con A significantly increased numbers of Salm. typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone. Con A had much less effect on rats infected with Salm. enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study. GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm. typhimurium S986 and significantly improved rat growth. GNA had little effect on infection by Salm. enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.     

Virulent strains of Salmonella enteritidis disrupt the epithelial barrier of Caco-2 and HEp-2 cells

To confirm the existence in nature of Salmonella enteritidis strains of different degrees of virulence and to elucidate the mechanisms underlying the effects of such strains on the epithelial barrier function, the consequences of infection of Caco-2 cells and HEp-2 cells with 15 S. enteritidis strains in a chicken infection model were examined. The more virulent strains of S. enteritidis, which are biofilm producers in adherence test medium, were able to disrupt HEp-2 and Caco-2 monolayers, as shown by transmonolayer electrical resistance and lactate dehydrogenase activity. In contrast, the low-virulence strains of S. enteritidis, which do not produce biofilms in adherence test medium, had no effect on the same cells. An avirulent rough mutant of Salmonella minnesota exhibited a pattern of behaviour similar to that of the low virulence strains of S. enteritidis, whilst a clinical Salmonella typhi strain caused rapid injury to the monolayers. The effect of supernatants of Salmonella cultures in adherence test medium on the integrity of Caco-2 cell monolayers indicated that the high-virulence S. enteritidis strains, but not the low-virulence strains, release a soluble factor when incubated under optimum biofilm-forming conditions, which enables the disruption of the integrity of Caco-2 monolayers.     

Molecular phylogenetic typing of pandemic isolates of Salmonella enteritidis

Salmonella enteritidis is now the most common Salmonella serovar in many countries. We have used cloned DNA probes to analyze genome interrelationships between strains chosen to represent the current S. enteritidis pandemic, and included designated type strains of the seven subspecies of Salmonella in order to compare the levels of discrimination of probes. DNA sequence divergence and rearrangements were analyzed in and around the rfa, fim and umuDC loci, and around insertion sites of the Salmonella-specific DNA insertion element, IS200. The S. enteritidis isolates showed a high degree of genome homogeneity. Chromosomal genetic loci exhibited characteristic DNA sequence divergence between subspecies of Salmonella, but no intraserovar divergence or difference with the subspecies I type strain was observed for S. enteritidis. The locus umuDC was not found in S. enteritidis. S. enteritidis contains a conserved and a variable site of insertion of insertion sequence IS200 and the analysis of DNA rearrangements around the second of these sites showed that three distinct evolutionary lines or races exist within pandemic isolates associated with human gasteroenteritis. IS200 profiles of a range of U.K. isolates of the epidemic phage type PT4 showed that all belonged to a single clonal line.     

Type 1 fimbriae of Salmonella enteritidis.

Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21.     

Rapid identification of Salmonella typhimurium, S. enteritidis and S. virchow isolates by polymerase chain reaction based fingerprinting methods.

In this study we used and evaluated three rapid molecular typing methods for the identification of three frequent, clinically significant Salmonella serovars on the basis of the ease, simplicity and reproducibility of the chosen methods. We determined the genetic diversity among several isolates of Salmonella enteritidis, S. typhimiurium and S. virchow, and compared them with other enterobacteria by using the repetitive extragenic palindromic (REP) sequences, the enterobacterial repetitive intergenic consensus (ERIC) sequences, and the 16S-23S rDNA intergenic spacer region (ITS 1). The objective was to evaluate their potential application to discriminate among members of the species Salmonella enterica subspecies enterica using the genetic diversity of the group found by genomic fingerprinting. The three different serovars of Salmonella studied gave reproducible and distinguishable profiles using whichever of the above mentioned polymerase chain reaction (PCR) methods assayed. The conserved patterns in each serovar allowed for easy differentiation from other serovars of Salmonella.     

Mutation of flgM attenuates virulence of Salmonella typhimurium, and mutation of fliA represses the attenuated phenotype.

Salmonella typhimurium ST39 exhibits reduced virulence in mice and decreased survival in mouse macrophages compared with the parent strain SL3201. Strain ST39 is nonmotile, carries an indeterminate deletion in and near the flgB operon, and is defective in the mviS (mouse virulence Salmonella) locus. In flagellum-defective strains, the flgM gene product of S. typhimurium negatively regulates flagellar genes by inhibiting the activity of FliA, the flagellin-specific sigma factor. In this study, flgM of wild-type S. typhimurium LT2 was found to complement the mviS defect in ST39 for virulence in mice and for enhanced survival in macrophages. Transduction of flgM::Tn10dCm into the parent strain SL3201 resulted in attenuation of mouse virulence and decreased survival in macrophages. However, a flgM-fliA double mutant was fully virulent in mice and survived in macrophages at wild-type levels. Thus, the absolute level of FliA activity appears to affect the virulence of S. typhimurium SL3201 in mice. DNA hybridization studies showed that flgM-related sequences were present in species other than Salmonella typhimurium and that sequences related to that of fliA were common among members of the family Enterobacteriaceae. Our results demonstrate that flgM and fliA, two genes previously shown to regulate flagellar operons, are also involved in the regulation of expression of virulence of S. typhimurium and that this system may not be unique to the genus Salmonella.