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.

CAT III chloramphenicol resistance in Pasteurella haemolytica and Pasteurella multocida isolated from calves.

Chloramphenicol, which had been used extensively for antimicrobial veterinary therapy, was prohibited in Europe in 1994. Soon after it became available, resistance to this drug was detected, generally conferred by plasmids encoding inactivating enzymes, the chloramphenicol acetyltransferases (CAT), in Gram-negative as well as in Gram-positive bacteria. In the last few years, resistance to antibiotics emerged in Pasteurella strains from breeding herds and this evolution was followed by a national surveillance network. Chloramphenicol-resistance was more recently detected in multiresistant strains. We studied 25 strains of Pasteurella, selected for their resistance to chloramphenicol. Production of a CAT was demonstrated in all these strains. PCR amplification indicated that the CAT produced was of type III for 23 of them. In these strains, chloramphenicol-resistance was mediated by plasmids of about 5.1 kb. Southern blots on restriction fragments suggested a high degree of homology between these 5.1 kb plasmids. In the two other strains, production of a CAT type I was demonstrated, and the corresponding genes were either shown on a plasmid of 17 or 5.5 kb.

Comparison of random amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus-PCR for epidemiological studies of Salmonella.

Discrimination of 70 Salmonella strains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis. RAPD results on the 56 S. typhimurium isolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprints only were obtained. For the 14 S. enteritidis strains, a helpful discrimination was obtained with RAPD analysis, while ERIC-PCR resulted in a single fingerprint.

Resistance to fluoroquinolones in Escherichia coli isolated from poultry.

Quinolone-resistant Escherichia coli strains were isolated from poultry clinical samples in Saudi Arabia. The poultry flocks had been treated with oxolinic acid or flumequine prophylaxis. The measure of the uptake of fluoroquinolones showed that none of the strains had a reduced accumulation of quinolones. The result of complementation with the wild-type E. coli gyrA gene, which restored fluoroquinolone susceptibility, and the isolation of DNA gyrase from six isolates indicated that the resistant strains had an altered DNA gyrase. The minimum effective dose of ciprofloxacin for inhibition of supercoiling catalyzed by the isolated gyrases varied from 0.085 microgram/ml for a susceptible isolate (MIC < 4 micrograms/ml) up to 96 micrograms/ml for the more resistant one (strain 215, MIC > 64 micrograms/ml). For the same two isolates, the minimum effective doses of sparfloxacin varied from 0.17 up to 380 micrograms/ml. The in vitro selection of spontaneous single-step fluoroquinolone-resistant mutants using ciprofloxacin suggested that the more resistant mutants are likely the result of several mutations. These results also show that, as in human medicine, cross-resistance between older quinolones and fluoroquinolones can exist in veterinary isolates and reiterate the need for the prudent use of these drugs.

[Evolution of antimicrobial susceptibility in salmonellas from bovine origin in France.]. / Evolution de la sensibilité aux antibiotiques des salmonelles d'origine bovine en France.

Animals are a wide source of salmonellas for humans and their environment. Domestic ruminants play an important role because of their high susceptibility to salmonellas infection giving an acute disease with massive salmonella excretion and mortality if lack of treatment. As in human medicine use of antibiotics at therapeutic doses in animals can lead to the selection of resistant strains may be pathogenic for humans by direct contact or through environment and food. Taking into account the importance of salmonellosis on calves in rearing units, CNEVA Lyon has set up since 1982 a national network for antimicrobial resistance monitoring of main bacterial pathogens in bovine. This network allowed to detect a recent evolution of antimicrobial susceptibility in salmonella from dairy cows related with new problem of salmonellosis in cattle.

Tetracycline resistance determinants, Tet B and Tet M, detected in Pasteurella haemolytica and Pasteurella multocida from bovine herds.

Resistance to antibiotics has recently emerged in Pasteurella haemolytica and Pasteurella multocida isolated from bovine herds. Forty-two clinical strains resistant to antibiotics and isolated through a French national network from different origins were analysed for their resistance to tetracycline. The MICs of tetracycline ranged from 32-256 mg/L. The resistance determinants Tet B and Tet M were detected in two strains, in which they are probably chromosomal.

Survey of antimicrobial resistance in bacterial isolates from diseased cattle in France.

Since 1982, a national veterinary network has been involved in the monitoring of resistance to antimicrobial agents in the main pathogenic bacteria isolated from diseased cattle in France. It is based on 40 regional veterinary diagnostic laboratories and managed by a central reference laboratory (CNEVA Lyon). Highly standardized methods are used in the diagnostic laboratories. This network collects up-to-date information on antimicrobial resistance in veterinary isolates and gathers strains for specific studies on fastidious bacteria and for the analysis of mechanisms of resistance to antibiotics. Such a permanent survey is essential to establish a rational veterinary antibiotic policy. It could be connected to other compatible systems developed in other fields such as human medicine, food, and environment, to evaluate the importance of resistance and R-factors spread for public health. The limits and perspectives of this surveillance system are discussed.

Results of passive and active immunization directed against ferric aerobactin in experimental enterobacterial infections in mice and chickens.

Production of aerobactin has been reported to be a virulence factor in members of the family Enterobacteriaceae. To investigate the protection afforded by humoral immunity directed towards aerobactin in infectious diseases caused by aerobactin-producing strains, we tested the efficacy of mAbAERO1, a murine monoclonal antibody directed to ferric aerobactin, which, in vitro, was found to impair the growth of aerobactin-dependent strains of Enterobacteriaceae under iron-limited conditions. The mortality of mice experimentally infected with the aerobactin-producing strains Escherichia coli V2019 (LD50 = 3.5 x 10(5) CFU/mice) or Klebsiella pneumoniae Caroli (LD50 = 1.3 CFU/mice) was not reduced when 1 mg of mAbAERO1 was injected intravenously 1 h before or 1 h after bacterial challenge. Nor was mortality reduced after challenge with either E. coli V2019 or K. pneumoniae Caroli, even though the active immunization of mice with purified FeAero (ferric aerobactin) conjugated with thyroglobulin as followed by a rise in systemic anti-FeAero antibodies. Lastly, chicks born of hens immunized with FeAero showed evidence of antibody transmission towards FeAero, but were not protected when challenged with E. coli MT78, an aerobactin-producing strain highly virulent for chickens. Therefore, under the experimental conditions tested, humoral immunity against aerobactin appeared to play only a minor role in protection against infections caused by aerobactin-producing members of the family Enterobacteriaceae. However, other experimental models should be tested to confirm these observations.

Clonal diffusion of EPEC-like Escherichia coli from rabbits as detected by ribotyping and random amplified polymorphic DNA assays.

The genetic diversity and clonal relationships among 77 Escherichia coli strains isolated in France from diarrhoeic rabbits and that belonged to seven O serogroups including the predominant O103 serogroup, were estimated by ribotyping and random amplified polymorphic DNA (RAPD) assays. Fifteen ribotypes were defined. Most of the highly pathogenic O103 strains could be assigned to two major groups. Non-pathogenic strains were clearly distinguished. RAPD assays generally matched ribotyping, or gave more precision for subdividing strains from the two main O103 groups. The results on strains isolated from different areas and over a 9-year period showed the relevance of the association of these two methods for the survey of the spread of strains in breeding flocks and illustrated clonal diffusion in rabbit production structures.

Value of plasmid profiling, ribotyping, and detection of IS200 for tracing avian isolates of Salmonella typhimurium and S. enteritidis.

Seventy selected strains of Salmonella typhimurium and S. enteritidis isolated from related poultry flocks in three independent geographical areas were characterized by phenotypic and genotypic methods to compare the usefulness of the methods in epidemiological studies. The 56 S. typhimurium isolates were poorly discriminated by their biotypes, resistance patterns, and plasmid profiles. Nine different ribotypes were obtained after DNA digestion by BglII, PvuII, and SmaI. Seven IS200 types, characterized by six to nine copies of IS200 on the chromosome, were detected after digestion of genomic DNA by PstI. These studies resulted in the definition of 15 clonal lineages distributed in three clusters. The 14 S. enteritidis strains were not discriminated either by ribotyping or by detection of IS200 (IS200 typing), but were separated on the basis of antibiotic resistance and plasmid profiling. The stability of the insertion sequence type was confirmed by inoculation of an S. typhimurium strain to axenic chickens reared for 15 weeks in sterile isolators.

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.

Conjugative transfer and in vitro/in vivo stability of the broad-host-range IncP R751 plasmid in Brucella spp.

Escherichia coli strain K12 BM14, carrying plasmid R751 (51.4 kb; Tpr, Tra+, IncP), was used as donor in matings with reference strains of the six Brucella nomenspecies, not known so far to harbor naturally occurring plasmids. R751 was easily transferred to and between Brucella spp., at frequencies ranging between 10(-1) and 10(-4). All Brucella transconjugants stably maintained plasmid R751 both in vitro and in vivo in our experimental conditions. No genetic modification of the plasmid during and after the conjugal transfer process could be deduced from the comparative restriction analysis (BamHI, HindIII, and EcoRI) in each Brucella transconjugant and in the E. coli donor.

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.

High genetic homology between plasmids of human and animal origins conferring resistance to the aminoglycosides gentamicin and apramycin.

Escherichia coli and Salmonella strains resistant to gentamicin and apramycin were isolated from cattle in France and Belgium and from patients in hospitals. Homology between plasmids of both human and animal origins encoding aminoglycoside 3-N-acetyltransferase was revealed by digestion with several restriction endonucleases and confirmed by hybridization with different replicon-specific probes.

Surface antigens from Escherichia coli O2 and O78 strains of avian origin.

Fimbriae from O2 and O78 virulent strains of avian Escherichia coli were compared with type 1A fimbriae with regard to the apparent molecular weights of their subunits and their antigenic relationships. Under static broth culture conditions, most O78 strains expressed fimbriae closely related to those of type 1A. Under the same culture conditions, another type of fimbriae, sharing some common properties with type 1A fimbriae, was observed only on O2 strains; however, these fimbriae differed from type 1A fimbriae in the apparent molecular weights of their subunits and in the expression of specific epitopes. They were called type 1-like fimbriae. Homologies in lipopolysaccharide and outer membrane protein profiles were also demonstrated among the strains expressing type 1-like fimbriae, which suggests the existence of a clonal relationship among O2:K1 avian E. coli strains. The O78 strains studied did not appear to be clonally related.

Implantation and in vivo antagonistic effects of antibiotic-susceptible Escherichia coli strains administered to premature newborns.

Two antibiotic-susceptible and non-pathogenic Escherichia coli strains were administered to hospitalized premature infants in order to protect them from intestinal colonization by hospital-acquired antibiotic-resistant enteric organisms (EOs). Three groups of 16 premature newborns received respectively strain ECA, strain EMO and both strains simultaneously. A fourth group was used as a control. Resistant EOs became spontaneously established in the digestive tract of a majority of the unadministered children. Both ECA and EMO were able to colonize the digestive tract of a majority of the subjects, and reached high population numbers (greater than 10(7)/g) in the faeces. Both strains appeared as able to reduce significantly the establishment of antibiotic-resistant EOs. This effect was more prominent with EMO, which also impaired the implantation of ECA when both strains had been administered simultaneously. The use of such innocuous microorganisms could thus constitute an additional means of preventing nosocomial infections of intestinal origin.

Virulent Escherichia coli strains for chicks bind fibronectin and type II collagen.

125I-fibronectin and 125I-collagen (type II) binding was detected in Escherichia coli strains isolated from chickens and poults. High fibronectin binding-strains also bind the 29 kD aminoterminal fragment of fibronectin. Binding properties in strain CK28 were partially characterized. The highest binding of 125I-fibronectin and 125I-collagen for strain CK28 was obtained with bacteria grown at 33 degrees C. Binding of 125I-fibronectin, its 125I-29 kD fragment, and 125I-collagen, was very rapid, reaching a maximum in 5 min. Binding of 125I-fibronectin and 125I-collagen was considerably inhibited by preincubation of bacteria with unlabelled fibronectin and unlabelled type I collagen respectively, but not inhibited with human immunoglobulin G or bovine serum albumin. Inhibition experiments showed that the reversibility of 125I-fibronectin binding was estimated at approximately 50%, while reversibility for 125I-collagen binding was higher than 90%. Receptors for fibronectin, its 29 kD fragment, and collagen were released from the bacterial surface by treatment at different temperatures, and surface material released at 100 degrees C inhibited binding. There was cross-inhibition for both fibronectin and collagen binding when unlabelled fibronectin and unlabelled collagen were used as inhibitors, suggesting that binding receptors for both proteins may be closely located.

Detection of apramycin resistant Enterobacteriaceae in hospital isolates.

Apramycin is a recently developed aminoglycoside restricted to veterinary therapy. Production of a 3-aminoglycoside acetyltransferase type IV (AAC(3)-IV) conferring cross-resistance to this drug and to gentamicin was detected in 1984 in France in bacteria of bovine origin. This mechanism of resistance was apparently confined to animals. We have studied 17 strains resistant to apramycin and gentamicin isolated in 5 hospitals in Belgium. Conjugative plasmids encoding an AAC(3)IV were present in 14 isolates. Comparison of the restriction fingerprints revealed 6 different plasmid patterns: 8 plasmids belonged to 2 groups sharing extensive intragroup homology and 4 were not related to the other replicons. These results indicate dissemination of plasmids within and between hospitals, but also of the gene encoding an AAC(3)IV.

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.