Jean-Pierre Lafont

Emergence of aminoglycoside 3-N-acetyltransferase IV in Escherichia coli and Salmonella typhimurium isolated from animals in France.

We studied two outbreaks of calf salmonellosis caused by apramycin and gentamicin-resistant Salmonella typhimurium strains. In both cases, the responsible strains were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, tetracycline, and trimethoprim; one strain was also resistant to nalidixic acid in one outbreak. A systematic survey of the intestinal Escherichia coli strains of calves from the two affected flocks showed that 11 of 24 animals sampled were also colonized by apramycin- and gentamicin-resistant E. coli strains. These isolates belonged to four biotypes and were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, tetracycline, trimethoprim, and nalidixic acid. All of the strains were resistant to high levels of apramycin (MICs, 512 to 1,024 micrograms/ml) and to gentamicin (MICs, 8 to 32 micrograms/ml), and these resistances were always transferred en bloc. In S. typhimurium, this coresistance was borne by plasmids that were approximately 39 kilobases long (outbreak 1) or 90 kilobases long (outbreak 2), whereas in E. coli, the coresistance was due to plasmids that were approximately 110 kilobases long in both outbreaks. The two plasmids of Salmonella and four plasmids of E. coli encoded type IV aminoglycoside 3-N-acetyltransferases. The intensive use of curative and preventive treatments in calf production could be responsible for the emergence of enzymic resistance to apramycin and gentamicin. Images

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

Comparative infectivity for axenic and specific-pathogen-free chickens of O2 Escherichia coli strains with or without virulence factors.

Adhesion to epithelial respiratory cells, iron acquisition, and production of K1 polysaccharide capsules have been proposed as potential virulence factors of avian Escherichia coli. These factors were studied by inoculating groups of axenic or specific-pathogen-free (SPF) chickens intratracheally with O2 E. coli strains after previous challenge with a wild strain of infectious bronchitis virus (IBV). In all experiments, the association between IBV and an E. coli strain endowed with the three virulence factors previously mentioned resulted in the most severe pathological effects, as measured by mortality, weight gains, lesions, and reisolation of E. coli from internal organs. An E. coli strain devoid of virulence factors was able only to induce mild pathological effects restricted to the respiratory tract when combined with IBV. Both E. coli strains were more invasive in axenic chickens than in SPF chickens. These results confirm the probable involvement of the three factors studied in the pathogenic properties of avian E. coli. This model can be used to assess the role of virulence factors, by comparing pairs of positive and negative isogenic strains.

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.

Spontaneous Implantation of Antibiotic-Resistant Enterobacteriaceae in the Digestive Tract of Chickens in the Absence of Selective Pressure

In the absence of selective pressure by antibiotics, resistant enterobacteria implanted rapidly in the intestinal tract of chickens, where these organisms subsequently persisted in high numbers. Food could be an important source of this contamination: resistant Escherichia coli present in small numbers in the diet became rapidly and persistently established in the gut. The human caretaker played a passive role in the spread of antibiotic-resistant bacteria between separate groups of chickens. Resistant enteric organisms colonized the gut of animals, with different population sizes. Some strains were able to reach high numbers (107 to 109/g), and other strains established themselves at a lower level (103 to 105/g), whereas a third type seemed to be only transient inhabitants, unable to persist.

Serological conservation and location of the adhesin of avian Escherichia coli type 1 fimbriae.

By inoculation of mice with purified type 1-like fimbriae isolated from an avian Escherichia coli strain, a monoclonal antibody (mAb G5) was obtained. mAb G5 reacted in an enzyme-linked immunosorbent assay (ELISA) with type 1-like and type 1A fimbriae differing in the molecular masses of their major fimbrial subunit and isolated from several avian E. coli strains. The specificity of mAb G5 for type 1 fimbriae was assessed in a whole bacteria ELISA with 16 reference E. coli strains expressing different types of fimbriae. Immunoblotting experiments showed that mAb G5 recognized the 29 kDa minor component of reference type 1A fimbriae which has been identified as the adhesin. mAb G5 also recognized the 29 kDa component of type 1-like and type 1A fimbriae expressed by avian E. coli strains, suggesting that the adhesin is antigenically conserved among these fimbriae. Immunoelectron microscopic studies gave evidence that the adhesin could be located mainly at the tip or both at the tip and along the fimbriae, depending on the strain.

Presence of eaeA sequences in pathogenic and nonpathogenic Escherichia coli strains isolated from weaned rabbits

Seventy-one Escherichia coli strains isolated from diarrhoeic weaned rabbits from different areas of France were tested for the presence of DNA sequences specific for the EPEC, EHEC, DAEC and EAggEC strains and 16 of them were tested for pathogenicity in animal experiments. High pathogenicity was observed only with strains unable to ferment rhamnose. DNA from all 55 rhamnose-negative O103, O26 and rough strains hybridised with the eaeA probe. Similar hybridisation was obtained with six non-pathogenic rhamnose-positive strains belonging to serogroups O128 and O132. No hybridisation was observed with the other probes. This is the first report of the presence of eaeA sequences in genomic DNA of non-pathogenic strains.

Detection of a second mechanism of resistance to gentamicin in animal strains of Escherichia coli.

One mechanism of plasmid-mediated resistance to gentamicin in Escherichia coli strains isolated from animals is due to the synthesis of the aminoglycoside 3-N-acetyltransferase type IV. A second mechanism of plasmid-mediated resistance to gentamicin was detected in animal strains of E. coli in France and is due to the production of the aminoglycoside 3-N-acetyltransferase type II. The molecular relationships among plasmids encoding this enzyme were studied. Images

Parameters controlling interbacterial plasmid spreading in a gnotoxenic chicken gut system: influence of plasmid and bacterial mutations.

Conjugative transfer of R plasmids R64 and R64drd-11 has been compared in vitro and in vivo without selective pressure by antibiotics in a simplified experimental system; the ecosystem was the bowel of germfree chickens, with the host bacteria almost isogenic, and the plasmids differing only in their conjugative transfer frequency. The spread of repressed and derepressed (drd) R plasmids in recipient bacterial populations was very extensive. The repressed phenotype had only a transient effect during the first 4 h. The level of implantation of the donor bacterial population seems to be of minor importance. Only with a poor recipient (con strain) could the spread of R plasmids be reduced and a steady state with a predominantly sensitive bacterial population be established. It is suggested that this steady state results from an equilibrium between the frequencies of R plasmid transfer and loss.

Probable transmission between animals of a plasmid encoding aminoglycoside 3-N-acetyltransferase IV and dihydrofolate reductase I.

Ten aminoglycoside-resistant Escherichia coli isolated from the faeces of healthy or diarrhoeic animals reared in the same herd were studied. These strains were resistant to high levels of apramycin and low levels of gentamicin. They were also resistant to streptomycin, tetracycline, trimethoprim and some to ampicillin, chloramphenicol, kanamycin or nalidixic acid. Two strains, isolated from a calf and a lamb, respectively, belonged to the same biotype. All the transconjugants resistant to gentamicin-apramycin were also resistant to streptomycin, tetracycline and trimethoprim. In all cases, these resistances were encoded by plasmids of 100 kb. Analysis of these plasmids by agarose gel electrophoresis after digestion by EcoRI or BamHI revealed their similarity. Hybridization with a 500-bp HpaI insert of plasmid pFE872 was observed with DNA from field strains and their transconjugants, demonstrating the presence on the 100-kb plasmids of the gene coding for a dihydrofolate reductase I. A single plasmid, designated pIP1831, could be observed in identical or different strains isolated from calves or lambs, suggesting the transmission of strains and plasmids between animals of different species.