The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant

To gain an understanding of the role of fimbriae and flagella in the adherence and colonisation of Salmonella enterica serotype Enteritidis in chickens, an in-vitro gut adherence assay was developed and used to assess the adherence of a wild-type Enteritidis strain and isogenic non-fimbriate and non-flagellate mutant strains. Enteritidis strain S1400/94, a clinical isolate virulent in chickens, was shown to possess genes which encoded type 1, SEF14, SEF17, plasmid-encoded and long polar fimbriae. Mutant strains unable to elaborate these fimbriae were created by allelic exchange. Each fimbrial operon was inactivated by the insertion of an antibiotic resistance gene cassette. In addition, fliC, motAB and cheA loci, which encode the major subunit of the flagellum, the energy-translation system for motility and one of the chemotaxis signalling proteins, respectively, were similarly inactivated. Non-flagellate mutant strains were significantly less adherent than the wild-type strain, whereas mutant strains defective for the elaboration of any of the types of fimbriae adhered as well as the wild-type strain. A flagellate but non-motile (paralysed) mutant strain and a smooth-swimming chemotaxis-deficient mutant strain were shown to be less adherent than the wild-type strain, but that observation depended on the assay conditions used.

Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis

Salmonella enteritidis expresses flagella and several finely regulated fimbriae, including SEF14, SEF17 and SEF21 (type 1). A panel of mutants was prepared in three strains of S. enteritidis to elucidate the role of these surface appendages in the association with and invasion of cultured epithelial cells. In all assays, the naturally occurring regulatory-defective strain 27655R associated with tissue culture cells significantly more than wild-type progenitor strains LA5 and S1400/94. Compared with wild-type strains, SEF14 mutants had no effect on association and invasion, whereas SEF17, SEF21 and aflagellate mutants showed significant reductions in both processes. Histological examination suggested a role for SEF17 in localized, aggregative adherence, which could be specifically blocked by anti-SEF17 sera and purified SEF17 fimbriae. SEF21-mediated association was neutralized by mannose and a specific monoclonal antibody, although to observe enhanced association it was necessary for the bacteria to be in fimbriate phase prior to infection. Additionally, aflagellate mutants associated and invaded less than motile bacteria. This study demonstrated the potential for multifactorial association and invasion of epithelial cells which involved SEF17 and SEF21 fimbriae, and flagella-mediated motility.

Effect of pH, temperature and surface contact on the elaboration of fimbriae and flagella by Salmonella serotype Enteritidis

Survival of enteric pathogens exposed to various environmental stresses depends upon a number of protective responses, some of which are associated with induction of virulence determinants. Flagella and fimbriae are putative virulence determinants of Salmonella spp, and ELISAs specific for the detection of flagella and SEF21, SEF14 and SEF17 fimbriae were used to assess the effect of temperature and pH upon their elaboration by isolates of Salmonella serotype Enteritidis in planktonic growth and on the surface of two-dimensional gradient agar plates, For three phage type 4 isolates of Enteritidis of comparative clinical provenance, similar phenotypes for the elaboration of these surface antigens were observed. SEF14 fimbriae were elaborated in planktonic growth at 37 degrees C, but not 20 degrees C, at pH 4.77 and above but not at pH 4.04; whereas on agar gradient plates SEF14 fimbriae were elaborated poorly but with best yields at pH 4.04, SEF17 fimbriae were elaborated in planktonic growth at 20 degrees C, but not at 37 degrees C, at pH 6.18 and above but not at pH 5.09 or below; whereas on agar gradient plates SEF17 fimbriae were elaborated well even at pH 4.65, SEF21 fimbriae were expressed very poorly under all conditions tested, Planktonic growth at 37 degrees C induced least flagella whereas growth at 20 degrees C, and particularly surface growth at lower pH values, induced a 'hyper-flagellate' phenotype, Single colonies allowed to form on gradient agar plates were shown to generate different colonial morphologies which were dependent on initial pH. These results demonstrate that the physicochemical environment is an important determinant of bacterial response, especially the induction of putative virulence factors.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

Contribution of fimbriae and flagella of Salmonella enteritidis to colonization and invasion of chicks

Isogenic mutants of Salmonella enteritidis defective for the elaboration of fimbrial types SEF14, SEF17, SEF21 and flagella were used to study the contribution these organelles made to colonization, invasion and lateral transfer in young chicks. The caecum, liver and spleen were colonized within 24 h following oral inoculation of 1-day-old chicks with 10(5) wild-type S. enteritidis strain LA5. However, for some mutants, the numbers of organisms recovered from internal organs was reduced significantly, particularly at 24 h post-inoculum, which supported the hypothesis that the organelles contribute to invasion and dissemination to internal organs. Specifically, mutations affecting SEF17, SEF21 and flagella contributed to a delay in colonization of the spleen, and those affecting SEF21 and flagella delayed colonization of the liver. Lower numbers of bacteria were recovered from the caecum with mutants deficient in elaboration of SEF21. Sentinel birds were colonized by LA5 or EAV40 (14(-), 17(-), 21(-), fla(-)) directly from the environment within 2 days, although a consistent slight delay was observed with the multiple mutant. Overall, our data suggest a collective role for SEF17, SEF21 and flagella, but not SEF14, in the early stages of colonization and invasion of young chicks by S. enteritidis, but these surface appendages appear unnecessary for colonization of birds from their immediate environment.

Adhesion of Salmonella enterica var Enteritidis strains lacking fimbriae and flagella to rat ileal explants cultured at the air interface or submerged in tissue culture medium

Rat ileal air interface and submerged explant models were developed and used to compare the adhesion of Salmonella enterica var Enteritidis wild-type strains with that of their isogenic single and multiple deletion mutants. The modified strains studied were defective for fimbriae, flagella, motility or chemotaxis and binding was assessed on tissues with and without an intact mucus layer. A multiple afimbriate/aflagellate (fim(-)/fla(-)) strain, a fimbriate but aflagellate (fla(-)) strain and a fimbriate/flagellate but non-motile (mot(-)) strain bound significantly less extensively to the explants than the corresponding wild-type strains. With the submerged explant model this difference was evident in tissues with or without a mucus layer, whereas in the air interface model it was observed only in tissues,vith an intact mucus layer. A smooth swimming chemotaxis-defective (che(-)) strain and single or multiple afimbriate strains bound to explants as well as their corresponding wild-type strain. This suggests that under the present experimental conditions fimbriae were not essential for attachment of S. enterica var Enteritidis to rat ileal explants, However; the possession of active flagella did appear to be an important factor. in enabling salmonellae to penetrate the gastrointestinal mucus layer and attach specifically to epithelial cells.

The role of SEF14 and SEF17 fimbriae in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces

To gain an understanding of the role of fimbriae and flagella in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces, the extent of adherence of viable wild-type strains to a polystyrene microtitration plate was determined by a crystal violet staining assay, Elaboration of surface antigens by adherent bacteria was assayed by fimbriae- and flagella-specific ELISAs, Wild-type Enteritidis strains adhered well at 37 degrees C and 25 degrees C when grown in microtitration wells in Colonisation Factor Antigen broth, but not in other media tested, At 37 degrees C, adherent bacteria elaborated copious quantities of SEF14 fimbrial antigen, whereas at 25 degrees C adherent bacteria elaborated copious quantities of SEF17 fimbrial antigen. Non-fimbriate and non-flagellate knock-out mutant strains were also assessed in the adherence assay. Mutant strains unable to elaborate SEF14 and SEF17 fimbriae adhered poorly at 37 degrees C and 25 degrees C, respectively, but adherence was not abolished. Non-motile mutant strains showed reduced adherence whilst type-1, PEF and LPF fimbriae appeared not to contribute to adherence in this assay. These data indicate that SEF17 and SEF14 fimbriae mediate bacterial cell aggregation on inanimate surfaces under appropriate growth conditions.

Growth of Salmonella enteritidis in artificially contaminated eggs: the effects of inoculum size and suspending media

Growth profiles of two isolates of Salmonella enteritidis phage type (PT) 4 inoculated into either the albumen of whole shell eggs or into separated albumen were found to be markedly affected by the size of the inoculum and the composition of the medium used to suspend the cells prior to inoculation. Using our model with an inoculum of two cells, multiplication of the Salmonella was not seen in 93% of eggs held at 20 degreesC for 8 days. In approximately 7% of eggs, however, growth occurred during the 8 days of storage. If the inoculum equaled or exceeded 25 cells per egg when eggs were subsequently stored at 20 degreesC, or 250 cells per egg when eggs were stored at 30 degreesC, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum was two cells. High levels of growth were also seen more frequently if the inoculum was suspended in buffered peptone water or maximal recovery diluent rather than in phosphate buffered saline. Growth of Salmonella in separated albumen occurred very infrequently (1.1% of samples) at low inoculum levels and did not become significant until the inoculum was 250 cells or greater. Growth in the albumen was unaffected by the composition of the suspending medium. Provided that the inoculum was approximately 2 cells per egg and the bacteria were suspended in PBS, observed growth profiles of S. enteritidis inoculated into the albumen of whole eggs resembled those in naturally contaminated eggs. (C) 2001 Elsevier Science B.V. All rights reserved.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef-pef region, Ranked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI-orf9 region although polymorphism was evident. Crown Copyright (C) 2001 Published by Elsevier Science B.V. All rights reserved.

Virulence in the chick model and stress tolerance of Salmonella enterica serovar Orion var. 15+

Three Salmonella enterica serovar Orion var. 15+ isolates of distinct provenance were tested for survival in various stress assays. All were less able to survive desiccation than a virulent S. Enreritidis strain, with levels of survival similar to a rpoS mutant of the S. Enteritidis strain, whereas one isolate (F3720) was significantly more acid tolerant. The S. Orion var. 15+ isolates were motile by flagellae and elaborated type-1 and curli-like fimbriae; surface organelles that are considered virulence determinants in Salmonella pathogenesis. Each adhered and invaded HEp-2 tissue culture cells with similar proficiency to the S. Enteritidis control but were significantly less virulent than S. En teritidis in the one-day-old and seven-day-old chick model. Given an oral dose of 1 x 10(3) cfu to one-day-old chicken, S. Orion var. 15+ isolates colonised 25% of liver and spleens examined at 24 h whereas S. Enteritidis colonised 100% of organs by the same with the same dose. Given an oral dose of 1 x 10(7) cfu at seven-day old, S. Orion var. 15+ failed to colonise livers and spleens in any bird examined at 24 h whereas S. Enteritidis colonised 50% of organs by the same with the same dose. Based on the number of internal organs colonised, one of the three S. Orion var. 15+ isolates tested (strain F3720) was significantly more invasive than the other two (B1 and B7). Also, strain F3720 was shed less than either B1 or B7 supporting the concept that there may be an inverse relationship between the ability to colonise deep tissues and to persist in the gut. These data are discussed in the light that S. Orion var. 15+ is associated with sporadic outbreaks of human infection rather than epidemics.

Diversity of strains of Salmonella enterica serotype Enteritidis from English poultry farms assessed by multiple genetic fingerprinting

Reliable and sufficiently discriminative methods are needed for differentiating individual strains of Salmonella enterica serotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism was PstI-SphII ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of XhaI-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins of Salmonella serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.

Molecular typing of Salmonella serotypes prevalent in animals in England: assessment of methodology

Salmonella enterica serotypes Derby, Mbandaka, Montevideo, Livingstone, and Senftenberg were among the 10 most prevalent serotypes isolated from farm animals in England and Wales in 1999. These serotypes are of potential zoonotic relevance; however, there is currently no "gold standard" fingerprinting method for them. A collection of isolates representing the former serotypes and serotype Gold Coast were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), and ribotyping. The success of the molecular methods in identifying DNA polymorphisms was different for each serotype. Plasmid profiling was particularly useful for serotype Derby isolates, and it also provided a good level of discrimination for serotype Senftenberg. For most serotypes, we observed a number of nontypeable plasmid-free strains, which represents a limitation of this technique. Fingerprinting of genomic DNA by ribotyping and PFGE produced a significant variation in results, depending on the serotype of the strain. Both PstI/SphI ribotyping and XbaI-PFGE provided a similar degree of strain differentiation for serotype Derby and serotype Senftenberg, only marginally lower than that achieved by plasmid profiling. Ribotyping was less sensitive than PFGE when applied to serotype Mbandaka or serotype Montevideo. Serotype Gold Coast isolates were found to be nontypeable by XbaI-PFGE, and a significant proportion of them were found to be plasmid free. A similar situation applies to a number of serotype Livingstone isolates which were nontypeable by plasmid profiling and/or PFGE. In summary, the serotype of the isolates has a considerable influence in deciding the best typing strategy; a single method cannot be relied upon for discriminating between strains, and a combination of typing methods allows further discrimination.

Expression of type 1 fimbriae (SEF 21) of Salmonella enterica serotype Enteritidis in the early colonisation of the rat intestine

The involvement of type 1 fimbriae in colonisation of the rat gastrointestinal tract in vivo was investigated with Salmonella enterica serotype Enteritidis LA5 and a mutant of LA5 denoted EAV3 unable to elaborate type 1 fimbriae (SEF 21), Rats were given a single dose of LA5 or EAV3 or a 1:1 mixture of both, LA5 was found in higher numbers in the stomach and small intestine than EAV3 at 6 h after infection with a single strain, but not after 6 days, LA5 did not out-compete EAV3 when the strains were administered together. Indeed, after 6 and 21 days, EAV3 was found in the distal small intestine and large intestine in far higher numbers than LA5. These findings suggest that SEF 21 have an important role(s) in the early stages of infection in vivo, However, SEF 21 expression may disadvantage the pathogen in the longer term as indicated by EAV3 out-competing LA5 in the gut at 21 days.

Association between cyclohexane resistance in Salmonella of different serovars and increased, resistance to multiple antibiotics, disinfectants and dyes

A panel of 388 salmonellas of animal and human origin, comprising 35 serotypes, was tested for resistance to cyclohexane and to a range of antibiotics, disinfectants and dyes. Cyclohexane resistance was detected in 41 isolates (10.6%): these comprised members of the serovars Binza (1 of 15), Dublin (1 of 24), Enteritidis (1 of 61), Fischerkietz (4 of 5), Livingstone (9 of 11), Montevideo (1 of 32), Newport (4 of 23), Saint-paul (1 of 3), Senftenberg (10 of 24) and Typhimurium (9 of 93). Most (39 of 41) of the cyclohexane-resistant isolates were from poultry. Statistical analysis showed that the cyclohexane-resistant strains were significantly more resistant than the cyclohexane-susceptible strains to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid, tetracycline, trimethoprim, cetrimide and triclosan. The multiresistance patterns seen,were typical of those caused by efflux pumps, such as AcrAB. The emergence of such resistance may play an important role in the overall antibiotic resistance picture of Salmonella, with particular effect on ciprofloxacin.

Role of the mar locus in virulence of Salmonella enterica serovar Typhimurium DT104 in chickens

The virulence of a Salmonella enterica serovar Typhimurium DT014 strain in which marA was insertionally inactivated was compared to its isogenic parent in vitro and in vivo. In vitro, the numbers of the marA mutant phagocytosed by porcine lung macrophages were significantly increased, while survival at 24 h inside macrophages and adherence to human gut cells were significantly reduced in comparison with the parent strain. In vivo, the marA inactivated strain, in competition with its parent strain, persisted for a shorter period in chickens, was present in the caeca at significantly lower levels and invaded the deeper organs to a significantly lesser extent. Therapeutic antibiotic treatment of one group of chickens with oxytetracycline favoured the persistence of both the parent strain and, to a lesser extent, the marA inactivated strain; but interestingly, increased tetracycline resistance of Salmonella isolates after treatment of birds with antibiotic was seen only for the parent strain. Further work is needed to elucidate how mar is involved in virulence and if its inactivation can minimise the ability of bacteria to become antibiotic-resistant in vivo.