Guy Gerbaud

In vivo transfer of genetic information between gram-positive and gram-negative bacteria.

A 1427‐bp DNA fragment containing the kanamycin resistance gene, aphA‐3, of plasmid pIP1433 from Campylobacter coli was inserted into a shuttle vector. Full expression of aphA‐3 was obtained in Bacillus subtilis and in Escherichia coli. This DNA fragment was sequenced in its entirety and the starting point for aphA‐3 transcription in B. subtilis, C. coli and E. coli was determined by S1 nuclease mapping. The sequence of the promoter consists of the hexanucleotides TTGACA and TATAAT, with a spacing of 17 bp. The nucleotide sequence of the aphA‐3 gene from C. coli and from the streptococcal plasmid pJH1 are identical whereas they differ by two substitutions and deletion of a codon from that cloned from the staphylococcal plasmid pSH2. These results indicate a recent extension of the resistant gene pool of Gram‐positive cocci to Gram‐negative bacilli. From an analysis of the DNA sequences surrounding the promoter region, we concluded that the DNA fragment containing the aphA‐3 gene in plasmid pJH1 has evolved by deletions from a sequence similar to that found in plasmid pIP1433.

High-Level Chloramphenicol Resistance in Neisseria meningitidis

BACKGROUND Neisseria meningitidis is nearly always susceptible to the penicillins, the cephalosporins, and chloramphenicol. Between 1987 and 1996, however, chloramphenicol-resistant strains were isolated from 11 patients in Vietnam and 1 in France. METHODS The minimal inhibitory concentration of chloramphenicol was determined for the 12 isolates. The isolates were analyzed by monoclonal-antibody-based serotyping and subtyping, pulsed-field gel electrophoresis, and multilocus enzyme electrophoresis. Bacterial DNA was analyzed by hybridization, the polymerase chain reaction, and sequencing to identify the resistance gene and determine the origin of the resistance. RESULTS The isolates were resistant to chloramphenicol (minimal inhibitory concentration, > or =64 mg per liter) and produced an active chloramphenicol acetyltransferase. All 12 strains belonged to serogroup B but had a high degree of diversity, and 10 could not be typed with the use of monoclonal antibodies. The nucleotide sequence of the resistance gene and the flanking regions was identical to that of an internal portion of transposon Tn4451 that carries the catP gene in Clostridium perfringens. Moreover, this gene was located in the same genomic site in the chloramphenicol-resistant isolates. CONCLUSIONS The high-level chloramphenicol resistance that we describe in N. meningitidis isolates is of great concern, since in developing countries, chloramphenicol given intramuscularly is the standard therapy for meningococcal meningitis. The resistance to chloramphenicol is due to the presence of the catP gene on a truncated transposon that has lost mobility because of internal deletions, and the transformation of genetic material between strains of N. meningitidis probably played an important part in the dissemination of the gene.

Susceptibility of Campylobacter pyloridis to 20 antimicrobial agents.

The susceptibility of 50 strains of Campylobacter pyloridis, isolated from human gastric biopsies, to 20 antimicrobial agents representing the different families of antibiotics was tested. The pattern of susceptibility was similar to that of the C. jejuni-C. coli group except for ampicillin, cephalothin, and rifampin. The relative resistance to cefsulodin was also noted.

Efflux Pump Lde Is Associated with Fluoroquinolone Resistance in Listeria monocytogenes

ABSTRACT Five Listeria monocytogenes isolates (CLIP 21369, CLIP 73298, CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistant to fluoroquinolones during the screening for antibiotic resistance of 488 L. monocytogenes isolates from human cases of listeriosis in France. On the basis of a fourfold or greater decrease in the ciprofloxacin MIC in the presence of reserpine, fluoroquinolone resistance was attributed to active efflux of the drugs. The lde gene (Listeria drug efflux; formerly lmo2741) encodes a 12-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of secondary transporters that displayed 44% identity with PmrA from Streptococcus pneumoniae. Insertional inactivation of the lde gene in CLIP 21369 indicated that the corresponding protein was responsible for fluoroquinolone resistance and was involved in the level of susceptibility to dyes such as ethidium bromide and acridine orange.

Spectinomycin resistance in Neisseria spp. due to mutations in 16S rRNA.

ABSTRACT Spectinomycin resistance in clinical isolates of Neisseria meningitidis and Neisseria gonorrhoeae was found to be due to mutations G1064C and C1192U (Escherichia colinumbering) in 16S rRNA genes, respectively.

Presence of the Listeria tetracycline resistance gene tet(S) in Enterococcus faecalis.

Two hundred thirty-eight tetracycline- and minocycline-resistant clinical isolates of Enterococcus and Streptococcus spp. were investigated by dot blot hybridization for the presence of nucleotide sequences related to tet(S) (first detected in Listeria monocytogenes BM4210), tet(K), tet(L), tet(M), tet(O), tet(P), and tet(Q) genes. The tet(S) determinant was found in 22 strains of Enterococcus faecalis, associated with tet(M) in 9 of these isolates and further associated with tet(L) in 3 of these strains. tet(M) was detected in all strains of Streptococcus spp. and in all but 10 isolates of Enterococcus spp.; tet(L) was found in 93 enterococci and tet(O) was found in single isolates of E. faecalis and Streptococcus milleri. No hybridization with the tet(K), tet(P), and tet(Q) probes was observed. Transfer of tet(S) by conjugation to E. faecalis or to E. faecalis and L. monocytogenes was obtained from 8 of the 10 E. faecalis strains harboring only this tet gene. Hybridization experiments with DNAs of four donors and of the corresponding transconjugants suggested that tet(S) was located in the chromosome. These results indicate that the genetic support of tet(S) in E. faecalis is different from that in L. monocytogenes, where it is carried by self-transferable plasmids, and confirm the notion of exchange of genetic information between Enterococcus and Listeria spp. in nature. Images

Translocation of sequences encoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez

SummarySalmonella ordonez strain BM2000 carries kanamycin (Km), ampicillin (Ap), spectinomycin (Sp), chloramphenicol (Cm), tetracyline (Tc), and sulfonamide (Su) resistance and production of colicin Ib (Cib). The Km and Cib characters were carried by a 97kb IncI1 plasmid (pIP565). In addition to the Km and Cib traits, all or part of the other antibiotic resistance (R) determinants could be transferred by conjugation from S. ordonez to Escherichia coli where all the acquired characters are borne by an IncI1 plasmid, designated complete or partial composite plasmid respectively. DNA from pIP565 and composite plasmids and total DNA from strain BM2000 were studied by agarose and polyacrylamide gel electrophoresis following digestion with restriction endonucleases, and by Southern hybridization. These comparative analyses enabled us a) to show that acquisition by pIP565 of resistance to all or some of the antibiotics was due to the insertion of a single DNA fragment into the receptor plasmid; b) to detect two types of composite plasmids with regard to the specificity of insertion into pIP565 and the mapping of the inserts; c) to demonstrate that the ApCmSpSuTc resistance determinants were integrated into S. ordonez BM2000 chromosomal DNA; d) to map the restriction fragments of the translocatable sequence integrated into strain BM2000 chromosome or into pIP565.The results obtained suggest that two distinct mechanisms for the translocation of the R determinants coexist in S. ordonez BM2000. Recombination between two of the four directly repeated copies of the IS-like sequence (IS1522) present in S. ordonez chromosome leads to the circularisation of all or part of the AmCmSpSuTc R determinants and is followed by either 1) a second recombination with the copy of IS1522 in pIP565 (Type I composite plasmids), or 2) transposition of precise groups of characters in various sites of pIP565 (Type II composite plasmids).

Persistence of an antibiotic resistance plasmid in intestinal Escherichia coli of chickens in the absence of selective pressure.

We studied eight strains of Escherichia coli resistant to high levels of trimethoprim that were isolated over a 6-week period in a commercial breeding flock of broilers. The strains originated from fecal samples and from a carcass immediately after slaughter. Seven of eight strains belonged to the same infrequent biotype. They were also resistant to ampicillin and streptomycin, and some were resistant to tetracycline and potassium tellurite. All the strains transferred trimethoprim and ampicillin resistance to E. coli. Analysis of the donors and of the transconjugants by agarose gel electrophoresis after digestion by restriction endonucleases and by nucleic acid hybridization indicated that resistance to trimethoprim (dfrI) and to ampicillin (bla TEM-1) was mediated by a 65-kilobase plasmid, pIP1531. Persistence of resistance to trimethoprim and ampicillin in this flock was therefore due to two cumulative factors, both occurring in the absence of selective pressure, namely the dissemination of a particular plasmid between strains and the ability of an atypical E. coli strain to stably colonize many animals. Images

rpoB Mutation Conferring Rifampin Resistance in Streptococcus pyogenes

ABSTRACT Streptococcus pyogenes BM4478 and Staphylococcus aureus BM4479 were isolated from a patient undergoing rifampin therapy. High-level resistance to rifampin was due to the following mutations in the rpoB gene: Ser522Leu in strain BM4478 and His526Asn and Ser574Leu in strain BM4479.

Aminoglycoside Resistance Gene ant(4′)-IIb of Pseudomonas aeruginosa BM4492, a Clinical Isolate from Bulgaria

ABSTRACT The ant(4′)-IIb gene of Pseudomonas aeruginosa BM4492, which encodes an aminoglycoside 4′-O-adenylyltransferase, was identified as a coding sequence of 756 bp corresponding to a protein with a calculated mass of 27,219 Da. Analysis of the deduced sequence indicated that the protein was related to aminoglycoside 4′-O-adenylyltransferases IIa and Ia found in P. aeruginosa and gram-positive bacteria, respectively. The enzyme conferred resistance to amikacin and tobramycin but not to dibekacin, gentamicin, or netilmicin. The ant(4′)-IIb gene had a chromosomal location in five of six clinical isolates of P. aeruginosa tested and was plasmid borne in the remaining strain. The ant(4′)-IIb gene was detected by PCR in some clinical strains of P. aeruginosa from the same hospital but not in members of other bacterial genera.