Dispersal in Campylobacter spp. of aphA-3, a kanamycin resistance determinant from gram-positive cocci.

DNA annealing studies indicated that kanamycin resistance in Campylobacter strains from various geographical areas is encoded by a gene structurally related to aphA-3 of gram-positive cocci. This finding confirms the transfer of genetic material between gram-positive and gram-negative bacteria under natural conditions. Images

We recently reported that high-level resistance to kanamycin in C. coli BM2509 was due to the presence of a plasmid-kanamycin (MIC, >2,000 pLg/ml). Six strains that were found to be resistant to kanamycin were also resistant to tetracycline (MIC, .64 [Lg/ml) ( Table 1). All the strains, except BM2635, were resistant to streptomycin (MIC, >1,024 pug/ml), and two strains ( Table 1) were resistant to ampicillin (MIC, >128 ,ug/ml). Resistance to kanamycin and tetracycline was transferable by conjugation in all strains except BM2638. Resistance to ampicillin and streptomycin never transferred, and we did not succeed in transferring erythromycin resistance (MIC, -1,024 VLg/ml) from strains 981 and BM2509.
The six strains studied were resistant to kanamycin but susceptible to tobramycin (3' deoxykanamycin B), indicating that the 3'-hydroxyl group is the site of modification. The 945 Vol. 32, No. 6  (20) " Genetic symbols are according to Novick et al. (14) and Davies and Smith (8).
distribution of nucleotide sequences related to aphA-J, aphA-2, and aphA-3 genes ( Table 2) in total DNA of kanamycin-resistant Campylobacter strains was studied by dot blot hybridization under high-stringency conditions (19). We detected homology with a probe specific for aphA-3 ( Fig. 1) but not with DNA fragments internal to aphA-1 and aphA-2 (data not shown). In similar experiments, we did not find homology between total DNA of strains BM2509 and 981 resistant to erythromycin and probes specific for the genes encoding erythromycin esterases (ereA and ereB) and ribosomal methylases (ermA, ermB, ermC, ermD, and ermF) (2). Plasmid DNA from transconjugants and strain BM2638 was purified (4), digested with HincII ( Fig. 2A) or BgIII (Fig.  3A), and analyzed by agarose gel electrophoresis. The kanamycin resistance plasmids had distinct restriction patterns. Strains BM2633 and BM2634, isolated from different patients in Thailand, harbored similar plasmids which appeared different from the plasmid present in strain BM2635, also isolated in Thailand but from an animal source. The plasmid in strain BM2638, which did not transfer kanamycin and tetracycline resistance to other Campylobacter strains, had a distinct restriction endonuclease profile and a smaller molecular weight ( Fig. 2A, Table 1). The plasmid DNA digested by HincIl was transferred to a nitrocellulose filter (12) and hybridized to the 32P-labeled M13mp8laphA-3 probe (Fig. 2B). The kanamycin resistance gene was located on the largest HincII-DNA fragment of all plasmids, except that of strain BM2638, which did not hybridize (Fig. 2B). Since total DNA of BM2638 hybridized with the same probe ( Fig. 1), the kanamycin resistance gene was tentatively assigned to a chromosomal location.
We previously suggested that kanamycin resistance in Campylobacter spp. results from the acquisition of a gene from a gram-positive bacterium (11,23). Plasmid DNA digested by BgIII was transferred to a nitrocellulose filter 1 2 3 4 5 6 7 8 (12) and hybridized to pMAK175 DNA 32p labeled in vitro by nick translation (Fig. 3B). This plasmid is representative of the tetracycline resistance plasmids of Campylobacter spp. (20). Although they had different restriction endonuclease patterns (Fig. 3A), plasmids mediating kanamycin resistance shared extensive sequence homology with pMAK175 (Fig. 3B). The plasmid in strain BM2638 which does not encode kanamycin or tetracycline resistance did not hybridize to the pMAK175 probe. This observation and the stability of the plasmids in their original hosts and also in C. fetus subsp. fetuis BM2560 support the notion that kanamycin resistance in Campylobacter spp. results from the acquisition of a gene rather than of a replicon en bloc. We recently established that resistance to kanamycin in C. coli BM2509 was due to a plasmid-encoded APH(3') of type III, an enzyme not detected previously in gram-negative bacteria (11,23). In this report, we extend this notion to Campylobacter strains independently isolated in various countries. The fact that the corresponding aphA-3 genes are . Plasmid DNA was digested with Bg/ll, fractionated by electrophoresis in a 0.8% agarose gel, transferred to a nitrocellulose filter, and hybridized with a 32P-Iabeled pMAK175 probe. DNAs of pMAK175 and strain BM2560 were used as positive and negative controls, respectively. Bacteriophage DNA digested with Pstl was used as the internal size standard. located on different plasmids or in the chromosome is compatible with the presence of this resistance determinant on a transposable element. However, direct evidence for a transposon in Campylobacter species is still lacking. Although emergence of kanamycin resistance in Campylobacter spp. constitutes the first example of transfer of genetic material between gram-positive and gram-negative bacteria under natural conditions (23), the gene flux between these two groups of microorganisms is not limited to this resistance gene or to this bacterial genus. Tetracycline resistance in Campylobacter spp. is due to the presence of the determinant tetO (19). This gen'e exhibits 76% sequence identity with tetM from Streptococcus species, and the substitutions are scattered throughout, suggesting that the two genes diverged from a common ancestor (19). Analysis of the optimal codon usage and of the transcription and translation signals of tetO and the finding of this gene in Enterococcus' and Streptococcus species of various groups indicate that tetO, as well as tetM, originates in grampositive cocci (19; R. Zilhao et al., submitted for publication). It also appears that streptomycin resistance by adenylylation of the antibiotic in Campylobacter spp. is due to the presence of a 6-aminoglycoside nucleotidyltransferase, an enzyme confined so far to gram-positive cocci (H. Pinto-Alphandary et al., manuscript in preparation). In all the cases stuidied to date, antibiotic resistance in Campylobacter spp. appears to originate in gram-positive cocci. The fact that a fundamental difference in gene expression seems to exist between members of the family Enteroba'cteriaceae and Campylobacter species is consistent with this observation (9). The bla gene and the replication machinery of pBR322 (9) 'and genes aphA-2 and aadA (A. Labigne, personal communication) from members of the Enterobac-teriac-eae are not expressed in Campylobacter spp. In contrast, aphA-1 and aadA encoding kanamycin and streptomycin resistance, respectively, in gram-negative bacteria have been found in a Campylobacter-like organism (16; Pinto-Alphandary et al., in preparation).
Transfer of genetic material from gram-positive bacteria to members of the family Enterobacteriaceaqe (1, 6) and to Neisseria gonorrhoeae (13) has also been documented. Since this gene flux involves microorganisms that are extremely distantly related, we postulated that transfer under natural conditions could have occurred only by transformation or conjugation followed by illegitimate recombination (23). In view of this hypothesis, it i's all the more interesting that transfer of plasmid DNA by conjugation from gramnegative to gram-positive bacteria (21) and vice versa (P. Trieu-Cuot et al., submitted for publication) ha's recently been obtained under laboratory conditions. We thank F. M6graud, M. J. Rivera, and S. Supavez for the gift of bacterial strains. B.P. was the recipient of a fellowship from the Centre International des Etudiants et Stagiaires.