Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona.

This study describes the characterization of the recently described Salmonella genomic island 1 (SGI1) (D. A. Boyd, G. A. Peters, L.-K. Ng, and M. R. Mulvey, FEMS Microbiol. Lett. 189:285-291, 2000), which harbors the genes associated with the ACSSuT phenotype in a Canadian isolate of Salmonella enterica serovar Typhimurium DT104. A 43-kb region has been completely sequenced and found to contain 44 predicted open reading frames (ORFs) which comprised approximately 87% of the total sequence. Fifteen ORFs did not show any significant homology to known gene sequences. A number of ORFs show significant homology to plasmid-related genes, suggesting, at least in part, a plasmid origin for the SGI1, although some with homology to phage-related genes were identified. The SGI1 was identified in a number of multidrug-resistant DT120 and S. enterica serovar Agona strains with similar antibiotic-resistant phenotypes. The G+C content suggests a potential mosaic structure for the SGI1. Emergence of the SGI1 in serovar Agona strains is discussed.

Use of antimicrobials in veterinary medicine and mechanisms of resistance.

This review deals with the application of antimicrobial agents in veterinary medicine and food animal production and the possible consequences arising from the widespread and multipurpose use of antimicrobials. The various mechanisms that bacteria have developed to escape the inhibitory effects of the antimicrobials most frequently used in the veterinary field are reported in detail. Resistance of bacteria to tetracyclines, macrolide-lincosamide-streptogramin antibiotics, beta-lactam antibiotics, aminoglycosides, sulfonamides, trimethoprim, fluoroquinolones and chloramphenicol/florfenicol is described with regard to enzymatic inactivation, decreased intracellular drug accumulation and modification/protection/replacement of the target sites. In addition, basic information is given about mobile genetic elements which carry the respective resistance genes, such as plasmids, transposons, and gene cassettes/integrons, and their ways of spreading via conjugation, mobilisation, transduction, and transformation.

Antimicrobial resistance in pasteurella and mannheimia: epidemiology and genetic basis.

Isolates of the genera Pasteurella and Mannheimia cause a wide variety of diseases of great economic importance in poultry, pigs, cattle and rabbits. Antimicrobial agents represent the most powerful tools to control such infections. However, increasing rates of antimicrobial resistance may dramatically reduce the efficacy of the antimicrobial agents used to control Pasteurella and Mannheimia infections. This review presents a short summary of the infections caused by Pasteurella and Mannheimia isolates in food-producing animals and the possibilities of preventing and controlling primary and secondary pasteurellosis. Particular reference is given to antimicrobial chemotherapy and the resistance properties of Pasterurella and Mannheimia isolates. The genetic basis of the most predominant resistance properties such as resistance to beta-lactam antibiotics, tetracyclines, aminoglycosides, sulfonamides, and chloramphenicol is discussed. This is depicted with reference to the role of plasmids and transposons in the spread of the resistance genes among Pasteurellaceae and members of other bacterial families and genera.

Mechanisms of quinolone resistance in Salmonella.

As in other Gram-negative bacteria, mechanisms of resistance to quinolones in Salmonella include target gene mutations, active efflux, and decreased outer membrane permeability. However, the exact contribution of these individual mechanisms to resistance, which may nevertheless interplay to reach high-level resistance, has not yet clearly been defined as in other bacteria such as Escherichia coli. This paper reviews the current state of knowledge of quinolone resistance mechanisms in Salmonella by comparison with that of E. coli and future directions of research with particular attention to the recent development of efflux pump inhibitors as possible means of avoiding the emergence and spread of fluoroquinolone resistance.

Nonenzymatic chloramphenicol resistance mediated by IncC plasmid R55 is encoded by a floR gene variant.

The IncC plasmid R55, initially described in the 1970s and isolated from Klebsiella pneumoniae, confers nonenzymatic chloramphenicol resistance. The gene coding for this resistance was cloned and sequenced and shows 95 to 97% nucleotide identity with the recently reported floR gene from Salmonella enterica serovar Typhimurium DT104 and from Escherichia coli animal isolates, respectively, conferring cross-resistance to florfenicol.

Characterization of high-level fluoroquinolone resistance in Escherichia coli O78:K80 isolated from turkeys.

Fluoroquinolone resistance was characterized in Escherichia coli O78:K80 isolated from diseased turkeys. The level of resistance to fluoroquinolones of the isolates appeared closely correlated with substitutions in GyrA and ParC, but not with the production of the AcrAB efflux pump. Among isolates highly resistant to ciprofloxacin (MIC 8 mg/L) and harbouring identical substitutions (two in GyrA and one in ParC), two close but distinguishable ribotypes were identified. This indicated that at least two independent selection events may have occurred.

Plasmid-mediated florfenicol resistance encoded by the floR gene in Escherichia coli isolated from cattle.

A florfenicol resistance gene almost identical to floR of Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coli isolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104.

The French antibiotic resistance monitoring programs.

Surveillance of antimicrobial resistance in bacteria from animal origin in France is organised by the French Agency for Food Safety (Agence Française de Sécurité Sanitaire des Aliments, AFSSA) through two types of networks. The first collects non-human zoonotic Salmonella strains in one centre (AFSSA, Paris) where they are tested for their antimicrobial susceptibility. The others, managed by AFSSA Lyon, deal with bovine pathogenic strains and are multicentric, that is they collecting antibiotic sensitivity and other data from the local public veterinary diagnostic laboratories. This requires standardisation of the methods used in each partner laboratory. Statistical analysis of any change in French resistance patterns can be monitored by these three networks either as a function of strain pathogenicity and/or of the ecological origin of the isolate. The system also encourages efficient collaboration between veterinarians and the laboratory. Such collaboration improves both the quality of routine antibiotic testing and understanding of the molecular mechanisms underlying resistance.

Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar typhimurium.

The occurrence of active efflux and cell wall modifications were studied in Salmonella enterica serovar Typhimurium mutants that were selected with enrofloxacin and whose phenotypes of resistance to fluoroquinolones could not be explained only by mutations in the genes coding for gyrase or topoisomerase IV. Mutant BN18/21 exhibited a decreased susceptibility to ciprofloxacin (MIC = 0.125 microg/ml) but did not have a mutation in the gyrA gene. Mutants BN18/41 and BN18/71 had the same substitution, Gly81Cys in GyrA, but exhibited different levels of resistance to ciprofloxacin (MICs = 2 and 8 microg/ml, respectively). None of the mutants had mutations in the parC gene. Evidence for active efflux was provided by a classical fluorimetric method, which revealed a three- to fourfold decrease in ciprofloxacin accumulation in the three mutants compared to that in the parent strain, which was annulled by addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In mutant BN18/71, a second fluorimetric method also showed a 50% reduction in the level of accumulation of ethidium bromide, a known efflux pump substrate. Immunoblotting and enzyme-linked immunosorbent assay experiments with an anti-AcrA antibody revealed that the resistance phenotype was strongly correlated with the expression level of the AcrAB efflux pump and suggested that decreased susceptibility to ciprofloxacin due to active efflux probably related to overproduction of this pump could occur before that due to gyrA mutations. Alterations were also found in the outer membrane protein and lipopolysaccharide profiles of the mutants, and these alterations were possibly responsible for the decrease in the permeability of the outer membrane that was observed in the mutants and that could act synergistically with active efflux to decrease the level of ciprofloxacin accumulation.

Occurrence of a Salmonella enterica serovar typhimurium DT104-like antibiotic resistance gene cluster including the floR gene in S. enterica serovar agona.

Recently a chromosomal locus possibly specific for Salmonella enterica serovar Typhimurium DT104 has been reported that contains a multiple antibiotic resistance gene cluster. Evidence is provided that Salmonella enterica serovar Agona strains isolated from poultry harbor a similar gene cluster including the newly described floR gene, conferring cross-resistance to chloramphenicol and florfenicol.

Comparative studies of mutations in animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp. suggest a counterselection of highly fluoroquinolone-resistant strains in the field.

The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 microg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 microg/ml, versus 16 microg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 microg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure.

A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella typhimurium DT104.

A new chloramphenicol resistance gene from Salmonella typhimurium DT104, designated floR, also conferring resistance to florfenicol, was characterized. Sequence analysis of the deduced FloR protein suggested that it belongs to the 12-TMS (transmembrane segments) multidrug efflux pumps family. The floR gene, and the downstream sequenced tetR and tetA tetracycline resistance genes, were surrounded by two class 1 integrons. The first one contained the resistance gene aadA2 and a deleted sulI resistance gene. The second one contained the beta-lactamase gene pse1 and a complete sulI gene. Thus, the floR gene is included in a multiresistance locus of at least 12.5 kb. Its particular organization and chromosomal location could be involved in the antibioresistance pattern stability of the DT104 Salmonella typhimurium strains.

Genetic diversity of Ornithobacterium rhinotracheale strains isolated from poultry in France.

Twenty-three strains of Ornithobacterium rhinotracheale from various origins, associated with respiratory pathology of birds, were compared using plasmid profiles, ribotyping and Random Amplified Polymorphic DNA (RAPD) in order to achieve a precise strain characterization as well as to highlight the relationships between these strains. No plasmid could be detected. These strains were poorly discriminated by ribotyping although different enzymes were used. The RAPD analysis has given reproducible DNA fingerprints and a good level of discrimination. This method can be used with only one or two primers to differentiate the O. rhinotracheale strains and could be used in epidemiological studies.

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.

[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.