Abstract
Forty-six Yersinia enterocolitica O:3 clinical isolates resistant to nalidixic acid were studied. The use of molecular typing techniques, other indicators of resistance patterns, the plasmid profile, and the presence of genes that encode aminoglycoside-modifying enzyme production suggested to us a clonal dissemination of the studied strains.
Yersinia enterocolitica produces common enteric diseases of humans and animals (2, 4) and shows susceptibility to most of the antimicrobial agents used clinically (17, 19). All clinical strains of Y. enterocolitica O:3 isolated in our environment from 1979 to 1993 were susceptible to nalidixic acid, ciprofloxacin, and norfloxacin. However, in the last few years, clinical isolates that are resistant to chloramphenicol (CHL) (16), streptomycin (STR) (5, 18), sulfonamides, trimethoprim-sulfamethoxazole (SXT) (23), and nalidixic acid have been identified.
The purpose of this study was to surveil resistance to nalidixic acid and fluoroquinolones in clinical Y. enterocolitica O:3 isolates. This epidemiological analysis of the Yersinia sp. isolates was performed by using genotypic and phenotypic markers to determine the possible clonal origin of the strains. From 1996 to 2002, 46 nalidixic acid-resistant strains of Y. enterocolitica O:3 were isolated from patients' feces. Five clinical strains that are susceptible to nalidixic acid were used as a reference for the typing experiments. Strains were isolated in cefsulodin-irgasan-novobiocin agar (22), identified with the API System (Biomerieux, Marcy l'Etoile, France), and serotyped with Y. enterocolitica antiserum (Bio-Rad, Marnes-la Coquette, France). Epidemiological analysis was performed by chromosomal DNA digestion with XbaI (Pharmacia Biotech, Piscataway, N.J.) (1, 8, 13), followed by pulsed-field gel electrophoresis (PFGE) with a gene navigator system (Pharmacia Biotech) through a 1.2% agarose gel with initial to final switch times ranging from 5 to 8 s at 200 V for 22 h at 14°C in 0.5× Tris-borate-EDTA buffer. Tenover's criteria were used to define the relationship among the studied strains (24). PFGE demonstrated three similar patterns, which differed in only one band. The most frequent subtype contains 46 strains. This study shows a close epidemiological relationship among strains of Y. enterocolitica O:3 (3, 10, 11).
Antimicrobial susceptibility was determined by two different methods. The disk diffusion method on Mueller-Hinton agar plates (Difco Laboratories, Detroit, Mich.) (15) for studies of nonquinonole agents was used. The antibiotics used were amikacin, apramycin, gentamicin, kanamycin, spectinomycin (SPT), STR, CHL, SXT, ampicillin, cefotaxime, and ceftriaxone. Quinolone susceptibility was determined by using the microdilution method according to NCCLS guidelines (14).
All Y. enterocolitica strains studied showed susceptibility to apramycin, gentamicin, kanamycin, cefotaxime, and ceftriaxone; all were resistant to ampicillin. We only found differences in resistance to STR, SPT, CHL, and SXT. With one exception, all isolates were resistant to STR. Resistance to CHL and SXT was widespread (46 and 42 resistant strains, respectively).
The MICs of nalidixic acid were 2,048 μg/ml for 24 (52%) strains, 1,024 μg/ml for 6 (13%) strains, 512 μg/ml for 3 (6.5%) strains, 256 μg/ml for 7 (15.2%) strains, and 128 μg/ml for 6 (13%) strains. The MICs of drugs tested against the susceptible strains ranged from 4 to 8 μg/ml (Table 1). All the studied isolates were susceptible to the tested fluoroquinolones, which had MICs ranging from 0.03 to 2 μg/ml. All quinolones showed similar intrinsic activity. The MIC at which 90% of strains were killed for ciprofloxacin, levofloxacin, and trovafloxacin was 1 μg/ml, and that for moxifloxacin and ofloxacin was 2 μg/ml.
TABLE 1.
Groups of Y. enterocolitica isolates obtained, listed by pulsotype and resistance profile
| PFGE subtype | Strain no. | Year obtained | Resistance patterna | Plasmid profile (kb) | MICc (μg/ml) | ANT(3")(9)b | APH(3")b |
|---|---|---|---|---|---|---|---|
| I | 1 | 1999 | Str Spt Chl | 512 | + | − | |
| 2 | 2000 | Str Spt Chli Sxt | 44, 37 | 4 | + | − | |
| II | 3 | 1998 | Str Spt Chl Sxt | 44 | 2,048 | + | − |
| 4 | 1999 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 5 | 2001 | Str Spt Chl Sxt | 44 | 128 | + | − | |
| III | 6 | 1997 | Str Spt Chl | 44 | 128 | + | − |
| 7 | 1997 | Str Spt Chl | 44 | 512 | + | − | |
| 8 | 2001 | Str Spt Chl | 44 | 256 | + | − | |
| 9 | 2001 | Str Spt Chl | 2,048 | − | − | ||
| 10 | 2001 | Spt Chli | 44 | 2,048 | − | − | |
| 11 | 2002 | Str Spt Chli Sxt | 44, 37 | 128 | + | − | |
| 12 | 2002 | Str Spt Chli Sxt | 8 | + | − | ||
| 13 | 2001 | Str Spt Chli | 44 | 1,024 | − | − | |
| 14 | 1996 | Str Spt Chl Sxti | 2,048 | + | − | ||
| 15 | 1998 | Str Spt Chl Sxti | 2,048 | + | − | ||
| 16 | 1997 | Str Spt Chl Sxt | 2,048 | + | − | ||
| 17 | 1997 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 18 | 1997 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 19 | 1998 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 20 | 1998 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 21 | 1998 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 22 | 1998 | Str Spt Chl Sxt | 57 | 2,048 | + | − | |
| 23 | 1998 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 24 | 1999 | Str Spt Chl Sxt | 2,048 | + | − | ||
| 25 | 2000 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 26 | 2001 | Str Spt Chl Sxt | 2,048 | + | − | ||
| 27 | 2001 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 28 | 2001 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 29 | 2001 | Str Spt Chl Sxt | 44, 37 | 2,048 | + | − | |
| 30 | 2001 | Str Spt Chl Sxt | 57 | 2,048 | + | − | |
| 31 | 2001 | Str Spt Chl Sxt | 44, 8 | 2,048 | + | − | |
| 32 | 2001 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 33 | 2001 | Str Spt Chl Sxt | 44 | 2,048 | + | − | |
| 34 | 1997 | Str Spt Chl Sxt | 44 | 1,024 | + | − | |
| 35 | 1998 | Str Spt Chl Sxt | 44 | 1,024 | + | − | |
| 36 | 1998 | Str Spt Chl Sxt | 1,024 | + | − | ||
| 37 | 2000 | Str Spt Chl Sxt | 44 | 1,024 | + | − | |
| 38 | 2001 | Str Spt Chl Sxt | 44 | 1,024 | + | − | |
| 39 | 1996 | Str Spt Chl Sxt | 57 | 512 | + | − | |
| 40 | 1999 | Str Spt Chl Sxt | 256 | + | − | ||
| 41 | 2000 | Str Spt Chl Sxt | 44 | 256 | + | − | |
| 42 | 2001 | Str Spt Chl Sxt | 44 | 256 | + | − | |
| 43 | 2001 | Str Spt Chl Sxt | 44 | 256 | + | − | |
| 44 | 2001 | Str Spt Chl Sxt | 44 | 256 | + | − | |
| 45 | 2001 | Str Spt Chl Sxt | 44 | 256 | + | − | |
| 46 | 1998 | Str Spt Chl Sxt | 44 | 128 | + | − | |
| 47 | 2001 | Str Spt Chl Sxt | 44 | 128 | + | − | |
| 48 | 2002 | Str Spt Chl Sxt | 37 | 128 | + | − | |
| 49 | 1997 | Str Spt Chl Sxt | 44 | 8 | + | − | |
| 50 | 2002 | Str Spt Chl Sxt | 44 | 8 | + | − | |
| 51 | 2002 | Str Spt Chl Sxt | 44 | 8 | + | − |
i, intermediate resistance.
+, presence of indicated AGME; −, lack of indicated AGME.
MICs of nalidixic acid are given.
Plasmid DNA was obtained by the procedure of alkaline lysis (9, 12). Plasmid profile analysis was carried out for all strains, and five plasmid profiles could be identified. Four were as follows: A, those strains that only harbored one plasmid of 44 kb (34 strains); B, those with a plasmid of 57 kb (three strains); C, three strains, each one with two plasmids of 44 and 37 kb; and D, only one strain with two plasmids of 44 and 8 kb. The last profile, E, included a strain that carried a 37-kb plasmid.
Amplification by PCR was used to detect aadA and aphE, the genes that encoded the ANT(3") (9) and APH(3") aminoglycoside-modifying enzymes (AGMEs).
Two oligonucleotide primers, 5′-TGATTTGCTGGTTACGGTGAC-3′ and 5′-CGCTATGTTCTCTTGCTTTTG-3′, were used to amplify a 284-bp fragment of the aadA gene, a procedure we performed by following the methods described previously by Clark et al. (6).
Oligonucleotides 5′-CAGGAGGAACAGGAGGGTG-3′ and 5′-GGTAAGAAGTCGGGATTGA-3′ were used to amplify a 230-bp fragment of the aphE gene. Half a colony was resuspended in 25 μl of sterile distilled water and boiled for 10 min. Twenty-five microliters of a reaction mixture containing 20 mM Tris-HCl (pH 8.8), 100 mM potassium chloride, 30 mM magnesium chloride, gelatin (0.1%, wt/vol), 400 μM deoxynucleoside triphosphates, and a 1 μM concentration of each primer was added together with 2.5 U of Taq polymerase (Promega, Madison, Wis.). The reaction was amplified at the following temperature profiles: 30 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C, with a final extension at 72°C for 10 min.
The AGME study was also carried out for all strains. The aadA gene was found in 48 strains. Among the other three, which did not carry the aadA gene, two were resistant to STR and SPT, and the last one was susceptible to STR but was resistant to SPT. We did not find any strain carrying the aphE gene.
We observed an increase of nalidixic acid resistance in Y. enterocolitica, although the new quinolones are still highly active in vitro. These results agree with those described by Sánchez-Céspedes et al. (21). These authors showed the clonal spread of one strain that became resistant to nalidixic acid by acquiring mutations in the gyrA gene. Future studies of the mutations which are present in the region that determines the resistance to quinolones will allow us to relate the acquired nalidixic acid resistance levels to the presence of mutations. Plasmids of around 40 to 50 kb could be associated with virulence plasmids. It may be possible that in strains without plasmids, the virulence plasmid has been lost by storage. On the other hand, the low variability of the plasmid and AGME profile supports the possible clonal dissemination of one strain.
Our results suggest the clonal dissemination of a nalidixic acid-susceptible Y. enterocolitica strain that developed resistance to nalidixic acid, probably as a result of selective pressure exerted by the use of fluoroquinolones in humans and especially in animal hosts (7, 20, 25). Further experiments with additional, more discriminatory markers are in progress to confirm our results.
Acknowledgments
This study was supported by Ministerio de Sanidad y Consumo (project no. FIS 01/0210) and by Departamento de Educación y Cultura del Gobierno Autónomo de Aragón (project no. DGA PO 56/2001). P. Goñi was the recipient of Diputación General de Aragón, Department of Public Health, fellowship P49/97.
We thank R. Lozano Mantecón for reviewing the manuscript.
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