Virulence factors of salmonella ser. enteritidis strains isolated in Italy from food-borne outbreaks.

Twelve Salmonella ser. Enteritidis strains phage type 4 isolated in Italy from different food-borne outbreaks were characterized for the expression of different virulence traits, for antibiotic resistance, and for plasmid DNA profile. All the twelve S. Enteritidis strains were able to invade and multiply within HeLa cell monolayers, even if at a lower efficiency if compared to an invasive Shigella flexneri strain. The strains were not hemolytic and produced only a moderate-level cytotoxic effect on HeLa cell monolayers. Moreover, all the strains examined produced mannose-sensitive hemagglutination with chicken erythrocytes but were not able to adhere to tissue culture cells. The strains did not produce the hydroxamate-type siderophore aerobactin or the specific ferric-aerobactin receptor. The S. Enteritidis strains were resistant only to spectinomycin, and eleven strains harbored a 38 MDa non-conjugative plasmid, while one strain harbored a 64 MDa conjugative plasmid which carried a colicinogenic activity-encoding locus. The uniformity of antibiotic resistance pattern, of the plasmid DNA content, and of the virulence factors produced indicated that the S. Enteritidis clinical isolates examined are clonally-related.

Characterization of endemic Shigella flexneri strains in Somalia: antimicrobial resistance, plasmid profiles, and serotype correlation.

One hundred twelve Shigella flexneri strain isolated from children with diarrheal disease in Somalia in 1983, 1984, 1988, and 1989 were analyzed for serotype, plasmid profile, and genetic location of antimicrobial resistance determinants. The prevalent serotypes were 4 (46% of the isolates), 1b (16%), 2a (16%), 3a (12%), and 6 (8%). Each serotype was associated with a characteristic predominant plasmid profile, whereas no specific correlation between antimicrobial resistance patterns and single serotypes was found. All but three of the strains were resistant at least to ampicillin, chloramphenicol, spectinomycin, and tetracycline. Of these resistant strains, 41 were resistant to sulfonamide and streptomycin and 14 were resistant to trimethoprim or trimethoprim and kanamycin. The genes for resistance to ampicillin, chloramphenicol, spectinomycin, and tetracycline formed a linkage group located on the chromosome of the strains of all serotypes. The genes for resistance to sulfonamide and streptomycin were located on a 6.3-kb plasmid in strains of serotypes 1b, 2a, and 4. Conjugative trimethoprim or trimethoprim and kanamycin resistance plasmids with lengths of 80 to 110 kb were present in strains of serotypes 1b, 2a, 3a, and 4. The systematic presence of a chromosomal component in this uncommon genetic plasmid-chromosome configuration may play a role in the emergence of increased genetic stability of resistance patterns in S. flexneri.

Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose-negative Escherichia coli strains of serotype O4 isolated from children with diarrhea.

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin.

v-jun oncogene prevents terminal differentiation and suppresses muscle-specific gene expression in ASV-17-infected muscle cells.

Infection of replicating quail myoblasts with avian sarcoma virus 17 (ASV-17) results in the inhibition of terminal differentiation into multinucleated myotubes and in the acquisition of anchorage-independent proliferation. Expression of v-jun, the ASV-17 oncogene, concomitantly leads to the accumulation of the gag-jun polyprotein P65 in the nucleus and to the lack of expression of typical differentiation-specific genes such as myosin heavy chain (MHC) and alpha-actinin. Surprisingly, expression of desmin, the muscle-specific subunit of intermediate filaments, is conserved in ASV-17-transformed myoblasts. Analysis of clonal strains of transformed myoblasts suggests that (i) suppression of morphological and biochemical differentiation depends on the absence of muscle-specific gene transcripts; (ii) inhibition of muscle differentiation by v-jun does not depend on the transcriptional silencing of MyoD, a muscle-specific regulatory gene; (iii) expression of desmin is compatible with proliferation of ASV-17-transformed cells and is independent of v-jun and MyoD levels of expression. The present data suggest that nuclear localization of v-jun prevents terminal differentiation in myoblasts and selectively down-regulates muscle-specific genes in terminally differentiated myotubes. In this respect, the behaviour of v-jun is quite different from that of v-myc, thus suggesting that these two oncogenes, although both encoding nuclear proteins, may have different mechanisms of action.

Virulence plasmids of enteroinvasive Escherichia coli and Shigella flexneri integrate into a specific site on the host chromosome: integration greatly reduces expression of plasmid-carried virulence genes.

The ability of enteroinvasive Escherichia coli and Shigella flexneri to cause disease depends on the presence of a large virulence plasmid (pINV). In this report we show that pHN280, the pINV of the O135:K-:H- enteroivasive strain E. coli HN280, and pWR100, the pINV of S. flexneri serotype 5 strain M90T, are able to integrate into a specific site on the host chromosome. pINV-integrated HN280 and M90T strains required methionine (Met-) to grow in minimal medium, were noninvasive, did not produce contact-mediated hemolysin, and had lost the ability to bind Congo red (Crb-) at 37 degrees C. Immunoblots of whole bacterial extracts from pHN280-integrated HN280 derivatives revealed that integration severely reduced the expression of ipa and virG (icsA) plasmid genes. Met- HN280 and M90T derivative strains spontaneously generated Met+ revertants that either contained excised forms of pINV or had lost pINV. Restriction analysis of excised pINVs showed that they either were virtually identical to parental pINVs (precise excision) or had suffered some deletion (imprecise excision). Precisely excised pINVs expressed the full pattern of virulence, whereas imprecisely excised pINVs were always Crb- and noninvasive. The revertion to Met+ was shown to be recA dependent, indicating that homologous plasmid and chromosomal DNA sequences are involved in the integration-excision process. The maintainance of pINV through integration and downregulation of its virulence genes may represent an advantageous mechanism for enteroinvasive bacteria, particularly when they are outside host cells and/or have to face adverse environmental conditions.

Virulence factors of lactose-negative Escherichia coli strains isolated from children with diarrhea in Somalia.

Lactose-negative Escherichia coli strains were isolated at high frequency from children with diarrhea in Somalia during a 2-year study on diarrheal diseases. Sixty-four of these strains, considered to be a representative sample, were characterized for virulence factors, plasmid profiles, and antibiotic resistance. Of these strains, 5 were recognized as enteroinvasive E. coli (they were serotyped as O135:K-:H-), 6 belonged to classical enteropathogenic E. coli serotypes, 9 were able to adhere to tissue culture cells (of these, 4 showed a pattern of localized adherence and 1 was an enteropathogenic strain), 18 were both adherent and hemolytic, and 8 were simply hemolytic. None hybridized with 32P-labeled heat-labile or heat-stable (a and b) enterotoxin gene probes or produced moderate or high-level cytotoxic effects on HeLa cells. Of the 64 strains examined, 24 produced mannose-resistant hemagglutination with human, chicken, and monkey erythrocytes. One of these was serotyped as O4:K-:H8, and a rabbit O antiserum raised against this strain allowed us to establish that 23 strains had the same O antigen. The 23 O4 strains were hemolytic and were not enterotoxic for rabbit ileal loops, and intact bacteria were able to destroy tissue culture cell monolayers very rapidly. The uniformity of the antibiotic resistance pattern and of the plasmid DNA content, together with the fact that they were isolated in different years and in different children, suggests that the O4 strains must be epidemiologically relevant in Somalia. A possible diarrheagenic role for the adherent-hemolytic E. coli strains is also discussed.