The 106-kilobase plasmid of Salmonella braenderup and the 100-kilobase plasmid of Salmonella typhimurium are not necessary for the pathogenicity in experimental models

Among 1.041 clinical isolates (77 serovars) of Salmonella which had been derived from cases with acute enterocolitis, 601 (58%) contained one or more plasmids. Large serovar-specific plasmids were seen in 95 of 307 isolates (31%) of Salmonella typhimurium, in 34 of 34 isolates (100%) of Salmonella enteritidis and in 36 of 38 isolates (94.7%) of Salmonella braenderup: the sizes of which were 100, 60 and 106 kilobases (kb), respectively. In order to determine the role of these plasmids in pathogenicity for enterocolitis, the plasmids were eliminated from some strains of S. braenderup and S. typhimurium and the pathogenicity of the plasmid-less strains was compared with that of the parent strains by invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, lesion of mucosal tissue and the Sereny test. The virulence of all the plasmid-less strains was as strong as that of the plasmid-bearing strains in these pathogenicity assay systems. We therefore concluded that the 106-kb plasmid of S. braenderup and the 100-kb plasmid of S. typhimurium are not necessary for their pathogenicity in the experimental models: invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, and Sereny test.     

Distribution of virulence plasmids within Salmonellae

The virulence region of the Salmonella dublin 50 MDa plasmid shared homology with 678 of 1021 salmonellae tested in colony hybridization experiments. The majority of S. dublin, S. typhimurium and S. enteritidis isolates tested hybridized with the region whereas, with the exception of S. hessarek, S. pullorum and S. gallinarum, other serotypes did not. Homologous virulence regions were plasmid encoded. In S. typhimurium a common 60 MDa plasmid was present in all phage types tested but not in DT4, DT37 and DT170. Smaller plasmids showing partial homology were found in DT12, DT18, DT193 and DT204C. In S. enteritidis a distinct plasmid profile for each of eight phage types was observed. Hybridizing plasmids were found in DT3, DT4, DT8, DT9 and DT11 whereas DT7, which was plasmid free, and DT10 and DT14, which harboured plasmids, did not hybridize. The extent of homology shared between S. dublin, S. typhimurium and S. enteritidis virulence plasmids was about 10 MDa and appeared conserved. Virulence plasmids from S. typhimurium and S. enteritidis did not show homology with a region of the S. dublin 50 MDa plasmid which was not associated with virulence functions whereas plasmids of about 24 MDa and 38 MDa in some S. typhimurium phage types did. The association of conserved virulence regions upon differing plasmids within salmonellae is discussed with reference to possible mechanisms of distribution and evolution of virulence genes.     

Characterization of a small cryptic plasmid from Salmonella enteritidis that affects the growth of Escherichia coli

Abstract We examined the plasmid content of 25 clinical isolates of Salmonella enteritidis , and detected the presence of small plasmids (3–5.3 kb) in 9 of them, alone, or in addition to the large, so-called virulence plasmid. A 5.3-kb plasmid isolated as unique extrachromosomal DNA from a strain responsible for a high-mortality outbreak was characterized by restriction mapping and cloning. The plasmid replicon was localized in a 1.7-kb fragment, that hybridized with three of the small plasmids detected in S. enteritidis , and with another small plasmid from Salmonella typhimurium . A strain of Escherichia coli carrying this plasmid, or a cloned 3.7-kb Pvu II restriction fragment, showed a slower growth rate, especially in minimal medium, as well as a noticeable increase in DNA methyltransferase activity.     

Prevalence of the virulence plasmids of nontyphoid Salmonella in the serovars isolated from humans and their association with bacteremia.

To determine if the virulence plasmid is one of the elements contributing to Salmonella bacteremia in humans, 436 clinical Salmonella isolates of different serovars were examined by a specific multiplex polymerase chain reaction assay for the presence of a virulence plasmid. These serovars showed differences in their ability to produce particular disease syndrome in humans. In the serovars usually causing bacteremia without concomitant gastroenteritis (primary bacteremia), i.e., S. choleraesuis, S. dublin, and S. enteritidis in this study, the rate of virulence plasmid carriage was 100%, while among those occasionally generating bacteremia following an episode of gastroenteritis (secondary bacteremia), the majority were plasmidless. Only a portion of S. typhimurium strains harbored a virulence plasmid; however, the rates of virulence plasmid carriage in S. typhimurium were not statistically different between non-fecal and fecal isolates (90% vs. 85%, 0.1 < P < 0.9). These results indicate that the virulence plasmids may be important for primary bacteremia, but not secondary bacteremia, to occur.     

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.     

Evolution of the virulence plasmids of non-typhoid Salmonella and its association with antimicrobial resistance

Among more than 2,500 serovars, eight contain a virulence plasmid, including medically important Salmonella enterica serovars Choleraesuis, Dublin, Enteritidis, and Typhimurium. These serovar-specific virulence plasmids vary in size, but all contain the spv operon, which plays a role in the expression of the virulence. Genetically, these virulence plasmids are likely derived from a common ancestral plasmid possessing virulence-related genes and loci. Based on the analysis of the available DNA sequences of the plasmids, the phylogenetic path may be split into two: pSPV (virulence plasmid of S. Gallinarum-Pullorum) acquires an incompatibility-related locus that differs from that of the others. At some point, pSCV (virulence plasmid of S. Choleraesuis) and pSDV (virulence plasmid of S. Dublin) lose oriT by recombination or simply by deletion, making the two unable to be mobilized. On the other hand, pSEV (virulence plasmid of S. Enteritidis) also loses some DNA by deletion but not as extensively as pSCV, and therefore pSEV is closest to pSTV (virulence plasmid of S. Typhimurium) both genetically and biologically. The pSTV shows the least alternation during the evolution. There are two types of pSDV. pSDVu recombines with non-virulence 36.6-kb plasmid to acquire additional incompatibility trait to form pSDVr. Recent reports indicated that S. Choleraesuis and S. Typhimurium could generate different types of hybrid plasmids, which consisted of the serovar-specific virulence plasmid and an array of resistance gene cassettes. The recombination gives Salmonella a survival advantage in an unfavorable drug environment. The integration of resistance genes and additional replicons into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and poses a serious threat to public health.     

The traT protein is able to normalize the phenotype of a plasmid-carried permeability mutation of Salmonella typhimurium

The isolation of different classes of antibiotic-supersensitive outer membrane permeability mutants of Salmonella typhimurium has been described previously (Sukupolvi et al., 1984, Journal of Bacteriology 159, 704-712). One of these, the SS-A mutation, sensitizes the bacteria to gentian violet and to hydrophobic antibiotics. The phenotype of the SS-A mutant was restored to normal when a cloned fragment of the F plasmid, or the R plasmid R6-5, carrying the genes traS, T and D was introduced on a multicopy plasmid. The introduction of a plasmid carrying only the traT gene showed that this gene was sufficient to restore the phenotype. Only clones with functioning traT (irrespective of copy number) restored the normal antibiotic-resistant phenotype in the SS-A mutant. An incompatibility test using a donor strain which carried transposon Tn10 in the 60 MDa plasmid of S. typhimurium and a recipient in which Tn5 was placed close to the SS-A mutation indicated that the SS-A mutation was located in the 60 MDa virulence plasmid (previously called the cryptic plasmid) of S. typhimurium. The introduction of the large virulence plasmid carrying the SS-A mutant allele into wild-type S. typhimurium or Escherichia coli resulted in strains with a phenotype identical to that of the original SS-A mutant.     

Genetic and molecular characterization of an epidemic plasmid coding for multidrug resistance in Salmonella typhimurium of human origin

All 201 multidrug resistant Salmonella typhimurium strains isolated from epidemics in India contained nonconjugative (157 strains) or conjugative (44 strains) Inc F1me multiresistance plasmids. Two small R-plasmids of 7 MDa which coded for resistance to either ampicillin or streptomycin and sulfamethoxazole were also detected along with other plasmids. The small plasmids were members of group 1 and group 2 incompatibility groups. Restriction endonuclease analysis of conjugative (96 MDa) and nonconjugative (88 MDa) Inc F1me plasmids showed considerable similarity except for the presence of unique fragments among both the groups and the loss of fragments corresponding to the smaller size of the nonconjugative plasmid. A single Inc F1me plasmid appears responsible for various outbreaks of multiresistant S. typhimurium in different parts of India.     

Virulence studies of Salmonella enteritidis phage types

Salmonella enteritidis phage types (PTs) 8, 13a and 24 could be distinguished by their virulence for BALB/c mice, their plasmid content and plasmid fingerprint. Virulent strains expressed long-chain lipopolysaccharide and carried a 38 MDa plasmid indistinguishable from that in Salm. enteritidis PT 4. Avirulent strains were smooth but did not carry the 38 MDa plasmid. Possession of antibiotic resistance factors by some strains of Salm. enteritidis PT 24 did not contribute to the virulence of their host strains.     

Nucleotide sequence of mkaD, a virulence-associated gene of Salmonella typhimurium containing variable and constant regions

We have identified the nucleotide (nt) sequence of mkaD, a virulence-associated gene of the Salmonella typhimurium virulence plasmid, pEX102. The gene shows 98% homology on nt sequence level to mkfA, a corresponding gene of the S. typhimurium virulence plasmid pIP1350. The few nt changes, however, caused more extensive changes on the amino-acid level. The differences between mkaD and mkfA were clustered in distinct variable regions rather than being randomly scattered along the sequence. A third salmonellar virulence plasmid, pLT2, contained an mkaD gene identical to that of pEX102. Our observation suggests that the conserved virulence determinant on the plasmids of Salmonellae may contain different alleles of the same gene.     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.     

Effect of conjugative R plasmids on the virulence of antibiotic-sensitive Salmonella strains and their streptomycin-resistant mutants

The pathogenicity level of antibiotic sensitive and streptomycin resistant strains of S. typhimurium, S. paratyphi B and S. kottbus changed under the effect of identical R plasmids more frequently in contrary directions. The conjugative plasmids of antibiotic resistance widened the ranges of the virulence changes in the Salmonella serovars for albino mice. It was found that 7 out of 8 plasmids studied significantly decreased and increased the virulence of the antibiotic sensitive Salmonella strains. As a rule, R plasmids of various origin decreased the virulence of all the tested streptomycin chromosome resistant causative agents of salmonellosis.     

A common virulence region on plasmids from eleven serotypes of Salmonella

Cured derivatives of Salmonella dublin and S. typhimurium showed reduced virulence following oral infection of mice (10(4)-10(5)-fold for S. dublin, 10(2)-fold for S. typhimurium). Large plasmids from S. dublin and S. typhimurium independently restored virulence to the cured S. dublin but truncated S. dublin plasmids with deletions in a previously identified virulence region did not. This common virulence region identified in plasmids from S. dublin and S. typhimurium was shown to be carried on plasmids from 11 other serotypes of Salmonella but was absent from 10 plasmid-containing serotypes. TnA and Tn10 were transduced from the virulence region of two TnA-insertion mutants of S. dublin and one Tn10-insertion mutant of S. typhimurium that showed diminished virulence to recipient wild-type strains of S. dublin, S. enteritidis and S. typhimurium. Each transductant showed a decrease in mouse virulence within the range 10(3)-10(5). It is therefore proposed that similar virulence determinants are expressed in different serotypes. It was also shown that integration that occurred during curing was Tn10 dependent.     

Identification of a multimer resolution system involved in stabilization of the Salmonella dublin virulence plasmid pSDL2.

The Salmonella dublin virulence plasmid pSDL2 is a low-copy-number plasmid that is highly conserved in its host. Deletion of the 8-kb EcoRI C fragment downstream of the virulence region leads to plasmid instability and formation of multimers. We identified a multimer resolution system in the EcoRI C fragment composed of a trans-acting resolvase gene and a cis-acting resolution site. The resolvase gene, rsd, maps within a 2-kb EcoRV fragment and appears to be part of a multicistronic unit together with at least two other genes of unknown function. The derived protein, 28.7-kDa in size, is almost identical to the D protein of miniF. The C-terminal region was shown to have substantial similarity to the conserved C-terminal domains of the site-specific recombinases of the integrase family. The cis-acting resolution site, crs, is located upstream of rsd within a 628-bp SmaI-HpaI fragment. It contains eight direct incomplete 17-bp repeats followed by a segment rich in indirect repeats, the latter being homologous to the oriV1 sequence of miniF. crs contains the crossover site for specific recombination and mediates bidirectional promoter activity. A replicative function in analogy to that of oriV1 of F could not be demonstrated. The multimer resolution system was shown to stabilize pACYC184 and is dependent on the recA-mediated formation of multimeric plasmids. Screening different Salmonella serovars with a pSDL2-specific recombination assay revealed that only strains harboring a virulence plasmid encode for resolvase activity. Our results suggest that site-specific recombination contributes to the stable inheritance of pSDL2 and other Salmonella virulence plasmids.     

Isolation of the replication and partitioning regions of the Salmonella typhimurium virulence plasmid and stabilization of heterologous replicons.

Although the virulence plasmid of Salmonella typhimurium has a copy number of one to two per chromosome, plasmid-free segregants are produced at a rate less than 10(-7) per cell per generation. Three regions appear to be involved in the maintenance of this virulence plasmid. The first two, repB and repC, are functional replicons hybridizing with IncFII and IncFI plasmids, respectively, neither exhibiting the segregational stability of the parent virulence plasmid. The third region, par, cloned as a 3.9-kilobase Sau3A fragment, is not a functional replicon but exhibits incompatibility with the virulence plasmid. Subsequent tests revealed the ability of this 3.9-kilobase par insert to increase the stability of pACYC184 in S. typhimurium from less than 34% to 99% plasmid-containing cells after 50 generations. In addition, the par region increased the stability of oriC, R388, and repC replicons in both S. typhimurium and Escherichia coli hosts. The par region encodes 44,000- and 40,000-molecular-weight proteins essential for the Par+ phenotype but not for the Inc+ phenotype. Although actual sequestering of plasmids within the cell was not demonstrated, all results indicate that the par region described is an actual partitioning locus, similar in organization to those described for plasmids F, P1, and NR1.     

Preliminary characterization of a food-borne multiple-antibiotic-resistant Salmonella typhimurium strain

Plasmid characterization studies were conducted on a Salmonella typhimurium strain isolated from pasteurized milk and from a symptomatic patient during the 1985 Illinois salmonellosis outbreak. This strain (Hf) was reported to possess an unusual plasmid profile which distinguished it from all Salmonella strains isolated in the United States prior to 1984. Antibiotic susceptibility testing revealed that the strain was resistant to tetracycline, erythromycin, clindamycin, sulfisoxazole, sulfadiazene, triple sulfa, cefoperazone, streptomycin, mezlocillin, piperacillin, carbenicillin, penicillin, ampicillin, and kanamycin. Plasmid analysis revealed that the strain possessed four plasmids with sizes of approximately 158, 98, 10.2, and 6.0 kilobase pairs (kb). Successive transfer at 43 degrees C led to increased antibiotic sensitivity in 75.5% of the isolates screened. Electroporation and calcium chloride treatment were each used to transform plasmid-free Escherichia coli strains with the plasmid pool from S. typhimurium Hf. Plasmids introduced by transformation ranged in size from 4.4 to 23.2 kb and correlated with resistance to penicillin G, ampicillin, carbenicillin, cephalothin, cefoperazone, cefamandole, mezlocillin, piperacillin, and in some cases, tetracycline and kanamycin. DNA-DNA hybridization experiments localized these resistance genes to a highly duplicated 6.3-kb fragment of the total EcoRI restriction digest of the S. typhimurium Hf plasmid pool.     

Preliminary characterization of a food-borne multiple-antibiotic-resistant Salmonella typhimurium strain.

Plasmid characterization studies were conducted on a Salmonella typhimurium strain isolated from pasteurized milk and from a symptomatic patient during the 1985 Illinois salmonellosis outbreak. This strain (Hf) was reported to possess an unusual plasmid profile which distinguished it from all Salmonella strains isolated in the United States prior to 1984. Antibiotic susceptibility testing revealed that the strain was resistant to tetracycline, erythromycin, clindamycin, sulfisoxazole, sulfadiazene, triple sulfa, cefoperazone, streptomycin, mezlocillin, piperacillin, carbenicillin, penicillin, ampicillin, and kanamycin. Plasmid analysis revealed that the strain possessed four plasmids with sizes of approximately 158, 98, 10.2, and 6.0 kilobase pairs (kb). Successive transfer at 43 degrees C led to increased antibiotic sensitivity in 75.5% of the isolates screened. Electroporation and calcium chloride treatment were each used to transform plasmid-free Escherichia coli strains with the plasmid pool from S. typhimurium Hf. Plasmids introduced by transformation ranged in size from 4.4 to 23.2 kb and correlated with resistance to penicillin G, ampicillin, carbenicillin, cephalothin, cefoperazone, cefamandole, mezlocillin, piperacillin, and in some cases, tetracycline and kanamycin. DNA-DNA hybridization experiments localized these resistance genes to a highly duplicated 6.3-kb fragment of the total EcoRI restriction digest of the S. typhimurium Hf plasmid pool.     

Salmonella typhimurium DT 193: differentiation of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence (spv) gene probe

M.D. HAMPTON, E.J. THRELFALL, J.A. FROST, L.R. WARD AND B. ROWE. 1995. Of over 2000 isolates of Salmonella typhimurium DT 193 from humans examined in the 2 year period 1991–92, 93% were antibiotic-resistant with the most common R-types being ASSuT (38%) and T (29%). Fourteen plasmid profiles were identified in DT 193 R-type ASSuT with the majority of isolates being characterized by a single plasmid of 80 MDa (pDEP 34) which in addition to coding for ASSuT, also hybridized with a spv gene probe prepared from the 50 MDa Salm. dublin serovar-specific plasmid. On the basis of restriction fragment length polymorphisms, two variant lines of pDEP 34-like plasmids were identified and a third line which had lost the genes coding for resistance to ampicillin, streptomycin and sulphonamides, was recognized. Although 18 plasmid profile types were identified in DT 193 R-type T, all isolates carried a high mol. wt plasmid which coded for tetracycline resistance only. Further discrimination was achieved on the basis of hybridization of tetracycline resistance plasmids with the spv gene probe and restriction enzyme fingerprinting. These results demonstrate that Salm. typhimurium DT 193 can be rapidly subdivided by antibiogram and that further subdivision can be achieved on the basis of plasmid profile, plasmid fingerprint and hybridization with a spv gene probe.     

Cloning of genes related to exo-beta-glucanase production in Saccharomyces cerevisiae: characterization of an exo-beta-glucanase structural gene

The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain (exg1-2) with highly reduced extracellular exo-beta-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb HindIII-XhoI fragment. Exg+ strains and Exg- mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg+ wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg+ wt strains. The EXG1 gene transformed Schizosaccharomyces pombe, yielding extracellular EXG activity which showed cross-reactivity with anti-S. cervisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg+ strain. Genetic and Southern analysis of several stable Exg- transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exg1-2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related in some way to EXG production in S. cerevisiae.     

Antibodies to lipopolysaccharide and outer membrane proteins of Salmonella enteritidis PT4 are not involved in protection from experimental infection

BALB/c and Schofield mice were inoculated with formalin-killed bacteria prepared from strains of Salmonella enteritidis belonging to phage type (PT) 4 and carrying a 38 MDa plasmid and expressing long-chain lipopolysaccharide, or strains without a 38 MDa plasmid or lacking the ability to express lipopolysaccharide. Vaccinated mice were challenged with viable bacteria belonging to a virulent strain of S. enteritidis (PT4). Mice surviving this viable challenge were examined for a humoral antibody response to membrane antigens of S. enteritidis (PT4) that might relate to the possession of a given virulence property. BALB/c mice immunized with any of the test antigens were found to be immune to S. enteritidis (PT4), and this immunity was protective. Serum antibodies, of the IgG class, were detected to OmpA and a minor outer membrane protein (OMP) of 31 kDa. Schofield mice also raised IgG antibodies to these outer membrane proteins; however, non-immunized mice of this strain were resistant to infection. The virulence of S. enteritidis (PT4) was also tested using mice belonging to strains B10D2 (new), Biozzi (high), Biozzi (low), C3HeJ, B10ITYR and C57/L.