Type 1 pili enhance the invasion of Salmonella braenderup and Salmonella typhimurium to HeLa cells.

The relationship between type 1 pili-associated adhesion and invasion to HeLa cells by Salmonella braenderup and S. typhimurium was studied. When the clinical isolates of these strains were grown in L-broth, they showed both type 1 pili formation and mannose-sensitive adhesion to HeLa cells. On the other hand, the type 1 pili-defective mutants, which were obtained either by repeated subcultures on L-agar plates or by the transposon Tn1-insertion mutagenesis of the S. braenderup and S. typhimurium strains, concomitantly lost mannose-sensitive adhesion to HeLa cells. When the HeLa cells were incubated with Salmonella, the type 1 piliated strains invaded the HeLa cells with much higher infection rate than did the type 1 pili-defective strains. The invasion of type 1 piliated strains to HeLa cells was markedly inhibited in the presence of D-mannose. The infectivity of the strain, which lost type 1 pili but still had mannose-resistant adhesion, was slightly higher than that of the strains defective in both mannose-sensitive and mannose-resistant adhesion. These results suggested that type 1 pili have a role in enhancing the invasion of S. braenderup and S. typhimurium to HeLa cells.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

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.     

Serotypes,virulence factors,antibiotic sensitivity,beta-lactamase activity and plasmid analysis of Salmonella from children with diarrhea in Tripoli (Libya)

A total of 21 Salmonella strains isolated in Libya (16 from children with diarrhea and 5 from healthy controls) were serotyped and studied for their cell invasive ability, production of cytotoxin, antibiotic susceptibility, beta-lactamase activity and plasmid profiles. Eight different serotypes of Salmonella were identified: 6 S. saintpaul, 4 S. wien (1 from control), 2 S. newport, 2 S. muenchen (1 from control), 2 S. typhimurium (1 from control), 2 S. hadar (1 from control), 2 S. reading (1 from control), 1 S. kottbus. Twenty (95%) were positive in the invasiveness assay using HeLa cells, and all (100%) were negative for cytotoxin production in HT29 cells. More than 40% were resistant to ampicillin, cefalexin, cefamandole, cefoperazone, chloramphenicol, gentamicin, mezlocillin and trimethoprimsulphamethoxazole and 100% were susceptible to the new quinolones. Most (67%) of the strains harbored plasmids and 43% produced beta-lactamase. A strong association was observed between the presence of more than one plasmid, beta-lactamase activity, and multiple-resistance to antimicrobial agents and serotypes S. saintpaul and S. wien. Curing experiments with acridine orange showed that 2 plasmids (33 and 1.4 megadaltons) might be responsible for the resistance to chloramphenicol and gentamicin. The present study demonstrated that multiple-resistant salmonellae are widespread in Libya and the resistance is mainly plasmid mediated.     

Construction of isogenic gonococcal strains varying in the presence of a 4.2-kilobase cryptic plasmid.

A 4.2-kilobase (kb) cryptic plasmid is present in 96% of isolates of Neisseria gonorrhoeae. An inability to construct isogenic derivatives which vary in the presence of the 4.2-kb plasmid has prevented the study of its function. We report a method to deliver an intact 4.2-kb plasmid into plasmidless gonococcal strains. The method involved transformation with novel 15.7-kb hybrid penicillinase-producing (Pcr) plasmids, which were cointegrates containing two copies of the 4.2-kb plasmid arranged in tandem direct repeat plus one copy of the 7.2-kb Pcr plasmid pFA3. When the 15.7-kb hybrid Pcr plasmids were introduced into a gonococcal recipient lacking evident plasmids, they dissociated at a relatively high frequency into plasmids identical to their parents: the 4.2-kb cryptic plasmid and pFA10 (a stable 11.5-kb plasmid containing one copy of each of the 7.2-kb Pcr plasmid pFA3 and the 4.2-kb cryptic plasmid pFA1). Curing strains of their Pcr plasmids resulted in isogenic strains which varied only in the presence of the 4.2-kb plasmid. The presence of the autonomously replicating 4.2-kb plasmid did not affect a number of tested phenotypes, including auxotype, antibiotic sensitivity, and frequencies of variation of outer membrane protein II. The interpretation of the functional significance of the 4.2-kb plasmid was complicated, however, by the additional finding that each of three tested plasmid-free strains contained a chromosomal fragment of about 1.6 kb that hybridized under moderate stringency with a 1.65-kb HinfI fragment of the 4.2-kb plasmid.     

Genes on the 90-kilobase plasmid of Salmonella typhimurium confer low-affinity cobalamin transport: relationship to fimbria biosynthesis genes.

A cloned fragment of Salmonella typhimurium DNA complemented the defect in cobalamin uptake of Escherichia coli or S. typhimurium btuB mutants, which lack the outer membrane high-affinity transport protein. This DNA fragment did not carry btuB and was derived from the 90-kb plasmid resident in S. typhimurium strains. The cobalamin transport activity engendered by this plasmid had substantially lower affinity and activity than that conferred by btuB. Complementation behavior and maxicell analyses of transposon insertions showed that the cloned fragment encoded five polypeptides, at least two of which were required for complementation activity. The nucleotide sequence of the coding region for one of these polypeptides, an outer membrane protein of about 84,000 Da, was determined. The deduced polypeptide had properties typical of outer membrane proteins, with an N-terminal signal sequence and a predicted preponderance of beta structure. This outer membrane protein had extensive amino acid sequence homology with PapC and FaeD, two E. coli outer membrane proteins involved in the export and assembly of pilus and fimbria subunits on the cell surface. This homology raises the likelihood that the observed cobalamin transport did not result from the production of an authentic transport system but that overexpression of one or more outer membrane proteins allowed leakage of cobalamins through the perturbed outer membrane. These results also suggest that the 90-kb plasmid carries genes encoding an adherence mechanism.     

重组质粒在减毒沙门氏菌中的稳定性及其对细菌侵袭力的影响

将含有外源基因的重组真核表达质粒pcDNA3-F和pCI-F转化减毒鼠伤寒门氏菌,探讨质粒类型和插入片段对重组质粒在细菌内的稳定性和细菌侵袭力的影响。结果表明,外源质粒可降低减毒沙门氏菌在体外的增殖能力和侵袭力,也影响细菌在鸡体内的存活力;就质粒类型而言,pCI的影响大于pcDNA3,而以携带外源基因的重组质粒影响较为显著;外源基因插入也影响质粒在宿主菌内的稳定性。提示利用减毒鼠伤寒沙门氏菌为载体传递DNA疫苗研究时,要考虑质粒类型与其在宿主菌内稳定性的关系、携带外源基因重组质粒对载体菌侵袭力和存活力的影响等问题。     

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.     

Biological properties of plasmid-free strains of Salmonella typhimurium and S. dublin

The study of Salmonella virulent strains has revealed that the characteristic feature of such strains is the presence of plasmids with a molecular weight of 90.2-91.5 kb for S. typhimurium and 77.2-78.5 kb for S. dublin. From Salmonella strains harboring only a single plasmid, variants with no plasmid at all have been obtained. These variants possess lower virulence for mice infected through enteral and intraperitoneal routes; besides, they lose their capacity for penetration into epithelial cells of HeLa line. S. typhimurium and S. dublin have shown decreased multiplication rate in vivo in comparison with the parent strains, while the multiplication rates in vitro were similar. These results suggest that the products of plasmid genes are either responsible for the virulent properties of salmonellae, or they have regulatory functions, thus controlling the work of chromosomal genes.     

Salmonella typhimurium LT7 and LT2 strains carrying the imp operon on colIa.

The imp operon is carried on a transmissible plasmid, ColIa, in original isolates of Salmonella typhimurium LT7. LT2 strain recipients of F' factors from LT7 strains harboring ColIa can acquire ColIa and imp under nonselective conditions. Thus, S. typhimurium LT2 strains that have received plasmids by conjugal transfer from LT7 strains might be inadvertently harboring ColI factors.     

Shigella dysenteriae type 1 carrying LPS biosynthesis genes of Salmonella typhimurium affects both Invasive Plasmid AntigenH (IpaH) secretion and invasion

A Shigella dysenteriae 1 strain isolated from an epidemic in West Bengal, India. The strain contained six plasmids including a large virulence plasmid. A plasmid, pPR1347 carrying both the rfb gene cluster and the rfc gene of Salmonella typhimurium has been transferred to this invasive Shigella dysenteriae 1 strain by triparental cross with a very low frequency. Only five stable (100%) clones were isolated after examining several thousand colonies. All five transconjugants were Sereny negative and were unable to invade the HeLa cells. Transconjugants exhibited strong cross reactivity with S. dysenteriae 1 antisera but they showed weak reaction with Salmonella typhimurium antisera. Plasmid profiles of the transconjugants were unaltered as compared with the wild type strain except for the presence of pPR 1347. The transconjugants regained their invasive property after elimination (curing) of pPR 1347. However, Shigella-infected convalescent phase serum was able to detect IpaABCD proteins from whole cell lysate and culture supernatant of transconjugants and cured (pPR 1347) transconjugants. A 60kDa IpaH protein was not secreted into the culture supernatant by the transconjugants. Synthesis of lipopolysaccharides (LPS) of the hybrid strains was increased within the region of 43 to 67kDa in comparison with the wild type S. dysenteriae 1 strains.     

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.     

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.     

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Molecular analysis confirms food source and simultaneous involvement of two distinct but related subgroups of Salmonella typhimurium bacteriophage type 10 in major interprovincial Salmonella outbreak.

More than 2,000 confirmed cases of food poisoning occurred in the four Atlantic provinces of Canada and in Ontario during the second and third quarters of 1984. Salmonella typhimurium phage type 10 was identified as the etiologic agent, and cheddar cheese was implicated as the source of infection. Strains isolated from infected humans and from cheese were indistinguishable by biotyping, antibiotic resistance typing, and phage typing. Plasmid analysis confirmed cheese as the source of infection and revealed the presence of two molecular subgroups of bacteriophage type 10. Group I strains carried 57-, 22.3-, and 3.4-kilobase (kb) plasmids; group II strains carried 57-, 4.6-, and 3.4-kb plasmids. Digestion with endonucleases HaeIII, HpaII, and AvaIII indicated that the 3.4-kb plasmids were identical. This outbreak was, therefore, caused by a mixed infection with two distinct but related bacteria. Group I strains are fairly common among Canadian S. typhimurium phage type 10 isolates, whereas group II strains appeared to be unique to this outbreak.     

Bacteriophage P22 H5 transfection and infection of plasmid Salmonella strains

The results of the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5 to constructed Salmonella typhimurium F'- and R+-strains LT2 WT-R and SA118 demonstrated that in these salmonellae the effectiveness of transfection depended on the specificity of the interrelation of plasmids with host strains. Plasmids RA1, R538-1 and RP1 stimulated the transfection of S. typhimurium strain LT2 WT-R, but suppressed the transfection ability of S. typhimurium strain SA118. At the same time the expression of the function of plasmids R446b and R64-11 did not depend on the host strain, as the former did not affect and the latter suppressed the release of transfectants in both Salmonella strains. The presence of plasmids R124, RA1, R64-11 and R724 in strain SA118, heat-sensitive in respect to the synthesis of cell-wall lipopolysaccharide, not only led to a decrease in the effectiveness of transfection; the effectiveness of the inoculation of bacteriophage P22 H5 was also suppressed 10(4) times in the presence of plasmid R124 and at least 10(10) times in the presence of 3 other plasmids. The development of resistance to S-specific bacteriophage P22 H5 was not linked with disturbances in the adsorption of this bacteriophage. Besides, the addition of CaCl2 into the medium completely removed the limitation of infection with bacteriophage P22 H5, determined by plasmid R124.     

Novel virulence properties of the Salmonella typhimurium virulence-associated plasmid: immune suppression and stimulation of splenomegaly

Mice infected subcutaneously with wild-type Salmonella typhimurium, SR11, developed a significant splenomegaly when compared with mice infected with an equal number of a plasmid-cured strain. Further, the bacterial load in the spleen at 14 days after infection, measured as colony-forming units per gram tissue, was significantly higher in mice infected with the parent strain than in mice infected with the plasmid-cured strain. These data confirm the previously reported plasmid-associated ability of Salmonella to multiply within the spleen. In addition, lymph node cells (LNC) from mice infected with the parent strain had a significantly reduced ability to proliferate in response to concanavalin A, a T-cell mitogen, and to heat-killed S. typhimurium cells when compared with LNC isolated from mice infected with the plasmid-cured strain. Finally, reintroduction of a functional Tn5-tagged 90-kb plasmid into a plasmid-free strain restored its capacity to cause a marked splenomegaly and to suppress lymph node cell proliferation in BALB/c mice. These data demonstrate that the 90-kb plasmid of highly virulent S. typhimurium strains mediates several novel pathogenic properties in infected mice: (1) enhancement of the ability of Salmonella to multiply within the spleen; (2) stimulation of a splenic inflammatory response as displayed by marked splenomegaly; and (3) a general suppression of lymphocyte responsiveness to both T-cell mitogens and specific Salmonella antigens.     

Evolution of genes on the Salmonella Virulence plasmid phylogeny revealed from sequencing of the virulence plasmids of S. enterica serotype Dublin and comparative analysis

Salmonella enterica serotype Dublin harbors an approximately 80-kb virulence plasmid (pSDV), which mediates systemic infection in cattle. There are two types of pSDV: one is pSDVu (pOU1113) in strain OU7025 and the other pSDVr (pOU1115) in OU7409 (SD Lane) and many clinical isolates. Sequence analysis showed that pSDVr was a recombinant plasmid (co-integrate) of pSDVu and a plasmid similar to a 35-kb indigenous plasmid (pOU1114) of S. Dublin. Most of the F-transfer region in pSDVu was replaced by a DNA segment from the pOU1114-like plasmid containing an extra replicon and a pilX operon encoding for a type IV secretion system to form pSDVr. We reconstructed the particular evolutionary history of the seven virulence plasmids of Salmonella by comparative sequence analysis. The whole evolutionary process might begin with two different F-like plasmids (IncFI and IncFII), which then incorporated the spv operon and fimbriae operon from the chromosome to form the primitive virulence plasmids. Subsequently, these plasmids descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. Our results suggest that the phylogeny of virulence plasmids as a result of frequent recombination provides the opportunity for rapid evolution of Salmonella in response to the environmental cues.     

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.