C Le Bouguénec

High-level chromosomal gentamicin resistance in Streptococcus agalactiae (group B).

This is the first report of high-level gentamicin resistance in a group B streptococcus. Strain B128 of serotype II was isolated from an infected leg wound in 1987. B128 was resistant to high levels of gentamicin as well as of all other available aminoglycosides and was also resistant to tetracyclines. No bactericidal synergism was found between ampicillin or vancomycin and any of these aminoglycosides. Gentamicin, kanamycin, streptomycin, and tetracycline resistance determinants transferred by conjugation into a plasmid-free group B streptococcus recipient at a frequency of 10(-8) to 10(-9) transconjugants per donor cell. No transconjugants were detected when streptococci of groups A, C, and G, Streptococcus sanguis, or Enterococcus faecalis was used as a recipient. No plasmids were detected in B128 or in any of the four transconjugants tested. By DNA-DNA hybridization, homology was detected between gene aac6/aph2, of E. faecalis origin, and a 2.4-kilobase HindIII chromosomal fragment of B128; homology to the genes aph3 and aadE, of E. faecalis origin, was found with HindIII chromosomal fragments of the same size (3.0 kilobases). Strains like B128, which potentially can be responsible for severe neonatal infections, are of great clinical concern, since there are to date no antibiotic combinations exhibiting bactericidal synergism against them. Images

The afa-related gene cluster in necrotoxigenic and other Escherichia coli from animals belongs to the afa-8 variant

Six hundred and nine necrotoxigenic Escherichia coli type 1 and 2 (NTEC1 and NTEC2) and non-NTEC isolated in Western and Southern Europe, North Africa and Canada from diseased calves, pigs, humans, poultry, and 55 isolated from asymptomatic calves were studied for the identification of afa-related sequences to the recently described afa-7 and afa-8 gene cluster variants from two bovine Escherichia coli (Lalioui et al., 1999). Colony hybridization and PCR assays for the afaD-7, afaE-7, afaD-8 and afaE-8 identified the afa-related sequences to the afa-8 gene cluster in most (67/79; 85%) of the E. coli positive with the Afa-f family probe and in 14 additional strains negative with the Afa-f probe. No E. coli was positive for the afa-7 gene cluster. The existence of afa-8 positive strains was thus confirmed among bovine E. coli and for the first time among porcine, poultry and human E. coli. Sequencing of the afaE-8 amplicon of nine strains from the different host species showed a high degree of conservation (>95% at the DNA level; >92% at the amino-acid level). The afa-8 gene cluster was more frequent in E. coli from diseased calves (18%) than from piglets (12%), humans (6%) and poultry (5%). Bovine NTEC2 (26%) were more frequently positive than NTEC 1 (20%) and non-NTEC (11%). E. coli isolated from asymptomatic calves were rarely positive: one NTEC2 (3%) and no non-NTEC. The afa-8 gene cluster was located on the Vir plasmid in 11/23 NTEC2, but no plasmid localization was detected in NTEC1 or non-NTEC.

Genetic basis of antibiotic resistance in Aerococcus viridans.

Resistance to at least one of the following antibiotics was found in eight wild-type strains of Aerococcus viridans: erythromycin (six strains), tetracycline and minocycline (five strains), chloramphenicol (one strain), and high levels of streptomycin (one strain). None of the strains transferred any of their antibiotic resistance markers into streptococcal, enterococcal, or A. viridans recipients by conjugation. By DNA-DNA hybridization experiments, the ermB gene of transposon Tn917, of Enterococcus faecalis origin, was detected in five of the six strains resistant to erythromycin and was localized for one strain on the chromosome and for four strains on nonconjugative small (4.7- to 4.9-kilobase) plasmids. The tetM gene of the conjugative transposon Tn916, of E. faecalis origin, was localized on the chromosome of four of the five strains resistant to tetracycline and minocycline; in three of these strains a structure similar to that of Tn916 was found. Homology to the tetO gene of pUA466, of Campylobacter jejuni origin, was detected on the chromosome of the fifth strain. No sequence homology was detected in any strain with probes corresponding to the tetL gene of group B Streptococcus origin, to the ermA gene of the transposon Tn554 of Staphylococcus aureus origin, or to the cat genes of either pC194 or pC221 of S. aureus origin. Images

Location of antibiotic resistance markers in clinical isolates of Enterococcus faecalis with similar antibiotypes.

Eight wild-type strains of Enterococcus faecalis, resistant to chloramphenicol (Cmr), erythromycin (Emr), tetracycline (Tcr), and minocycline (Mnr), were examined for the genetic basis of their antibiotic resistance, Five of the strains transferred all of their antibiotic resistance markers by conjugation, while the other three strains transferred only Tcr and Mnr. Cmr and Emr determinants were localized by DNA-DNA hybridization experiments, in which the Cmr gene of plasmid pIP501, of group B Streptococcus origin, and the Emr gene of transposon Tn917, of E. faecalis origin, served as probes. A chromosomal location was found for the nonconjugative Cmr and Emr markers of one wild-type strain. In two strains these markers were carried by nonconjugative plasmids, and in the other strains they were carried by plasmids that transferred by conjugation. Plasmids isolated from three transconjugants resistant to tetracycline but susceptible to minocycline bore nucleotide sequences homologous to the tetL gene. Nucleotide sequences homologous to conjugative transposon Tn916, of E. faecalis origin, were detected by hybridization in the tetracycline-minocycline-resistant transconjugants. Three of these transconjugants were plasmid free, while four harbored conjugative cryptic plasmids. Sequences homologous to Tn916 were also found on two conjugative plasmids, one of which appeared to be a conjugative cryptic plasmid that had acquired chromosomal Tcr Mnr markers during transfer. Images

Conjugative R plasmids in Streptococcus faecium (group D).

Ten isolates of Streptococcus faecium were found to be resistant to penicillin, tetracycline, macrolides and related drugs, streptomycin, and kanamycin, and four strains were resistant to chloramphenicol. Six of these 10 strains transferred all their resistance markers (except penicillin) by conjugation at a low frequency (10(-7) to 10(-9)). Several plasmids of different molecular weights were found in each of the wild-type strains. In 5 of 11 transconjugant strains, R plasmids were detected which had molecular weights identical to those of the plasmids found in the corresponding donor strain. Each of the six other transconjugants harbored one plasmid with a size different from those found in the corresponding donor strain, suggesting the occurrence of molecular events during or after conjugative transfer. None of the five tetracycline-resistant transconjugants contained detectable satellite DNA, HindIII restriction enzyme fingerprints of S. faecium resistance plasmids were different from the HindIII patterns of macrolide, aminoglycoside, and tetracycline resistance plasmids from other strains of streptococci. Images

Dra/AfaE Adhesin of Uropathogenic Dr/Afa+ Escherichia coli Mediates Mortality in Pregnant Rats

ABSTRACT Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE+afaD and afaE afaD+ mutants. The clinical E. coli strain (afaE+afaD+) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE+afaD+ strain, followed by the group infected with the afaE+afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+afaD strain > afaE+afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+E. coli strains in vitro.