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Applied and Environmental Microbiology, January 2004, p. 599-602, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.599-602.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Analysis of the gyrA Gene of Clinical Yersinia ruckeri Isolates with Reduced Susceptibility to Quinolones

Alicia Gibello, M. Concepción Porrero, M. Mar Blanco, Ana I. Vela, Pilar Liébana, Miguel A. Moreno, José F. Fernández-Garayzábal,* and Lucas Domínguez

Departamento Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

Received 9 June 2003/ Accepted 2 October 2003


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ABSTRACT
 
Antimicrobial susceptibility of seven clinical strains of Yersinia ruckeri representative of those isolated between 1994 and 2002 from a fish farm with endemic enteric redmouth disease was studied. All isolates displayed indistinguishable pulsed-field gel electrophoresis restriction patterns, indicating that they represented a single strain. However, considering both inhibition zone diameters (IZD) and MICs, the isolates recovered in 2001-2002 formed a separate cluster with lower levels of susceptibility to all the quinolones tested, especially nalidixic acid (NA) and oxolinic acid (OA), compared with the isolates recovered between 1994 and 1998. Analysis of the PCR product of the quinolone resistance-determining region of the gyrA gene from clinical isolates of Y. ruckeri with reduced susceptibility to OA and NA revealed a single amino acid substitution, Ser-83 to Arg-83 (Escherichia coli numbering). Identical substitution was observed in induced OA-resistant mutant strains, which displayed IZD and MICs of quinolones similar to those of the clinical isolates of Y. ruckeri with reduced susceptibility to these antimicrobial agents. These data indicate in that for Y. ruckeri, the substitution of Ser by Arg at position 83 of the gyrA gene is associated with reduced susceptibility to quinolones.


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INTRODUCTION
 
Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) or yersiniosis, a serious infectious disease of fish that causes high economic losses in the rainbow trout farming industry in many countries (6). Y. ruckeri is able to persist and remain infective in the aquatic environment for prolonged periods of time (20) and to colonize fish farm tank surfaces by forming biofilms (5), which favor the existence of recurrent infections. Although ERM can be controlled by vaccination, antimicrobial agents, mainly quinolones, such as oxolinic acid or flumequine, are still frequently used for the treatment of ERM outbreaks (9, 13, 19, 21). Increased resistance in Y. ruckeri after persistent exposure to oxolinic acid has been reported under in vitro conditions (18). Nevertheless, the isolation of quinolone-resistant clinical strains of Y. ruckeri is unusual (17, 21). Quinolones are very useful antimicrobial agents that have proven to be helpful in different clinical contexts in both human and veterinary medicine. Quinolones act by interfering with bacterial DNA supercoiling, and in gram-negative bacteria, DNA gyrase is considered the primary target of these antimicrobials (1, 8, 26). DNA gyrase contains two protein subunits, A and B, coded by the gyrA and gyrB genes, respectively (10, 11). The majority of quinolone-resistant bacterial isolates contain substitutions between positions 67 and 106 of the GyrA protein, leading to the categorization of this section as the quinolone resistance-determining region (QRDR) (28). The mechanisms of quinolone resistance have been widely studied for many gram-negative bacteria (1, 8, 11, 26), and basically, quinolone resistance is usually associated with mutations at residues Ser-83 and Asp-87 of the gyrase A subunit (3). However, there are no data about the structure and mutations in the genes involved in quinolone resistance in Y. ruckeri. This work was therefore carried out to characterize the QRDR of the gyrA gene in strains of Y. ruckeri with a remarkably reduced susceptibility to OA as well as other quinolones.

The Y. ruckeri strains used in this study, except the collection ones, are representative of those clinical strains isolated from different diseased rainbow trout during the ERM outbreaks diagnosed in the same fish farm between 1994 and 2002 (Table 1). Oxolinic acid was usually used for the treatment of the clinical ERM outbreaks as medicated feed at a level of 30 mg of oxolinic acid/kg of body weight/day for 10 days. Collection strains CECT 955 and CECT 956 of Y. ruckeri, isolated from ERM diseased rainbow trout and chinook salmon, respectively, were supplied from the Spanish Type Culture Collection. Clinical isolates of Y. ruckeri were biochemically characterized by using the API 20E system (bioMérieux España, S.A., Madrid), and identification was further confirmed by PCR (7). Clinical Y. ruckeri isolates were also molecularly characterized by pulsed-field gel electrophoresis (PFGE) with the enzyme XbaI (Promega Co), according to the specifications of Blanco et al. (4). The restriction enzyme XbaI was used according to the manufacturer's recommendations. Antimicrobial susceptibilities to tetracycline (30 µg), sulfisoxazole (300 µg), florfenicol (30 µg), and the quinolones nalidixic acid (NA) (30 µg), oxolinic acid (OA) (2 µg), flumequine (30 µg), and enrofloxacin (5 µg) were tested by the disk diffusion method according to the National Committee for Clinical Laboratory Standards (15). Inocula were prepared from an overnight Columbia blood agar plate by suspending three colonies in 5 ml of Mueller-Hinton broth (Oxoid, United Kingdom), adjusted to 0.5 McFarland turbidity and further diluted 1/100 in sterile distilled water (23). MICs to NA and OA were determined by the microdilution method, with U-bottom 96-well microtiter plates (Lab-Center, Mostoles, Madrid, Spain) (23). Serial twofold dilutions of the antimicrobial agents (final concentrations ranging from 0.5 to 128 µg ml-1 for NA and from 0.125 to 256 µg ml-1 for OA) were prepared in the microtiter plates. The inoculum was adjusted as described above, and 50 µl was dispensed into each well of the microplate. Mueller-Hinton and microdilution plates were all read after 24 h of incubation at 30°C.


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  		TABLE 1. Susceptibilities to quinolonesa of the Y. ruckeri strains used in this study

The DNA fragment containing the putative QRDR of the gyrA gene from all Y. ruckeri strains used in this work was generated by PCR amplification. Bacterial chromosomal DNA was extracted by the method of Lawson et al. (12). The PCR amplifications were performed in 1x reaction buffer containing 50 ng of chromosomal bacterial DNA, 0.2 mM (each) deoxynucleotide triphosphates, 2.5 U of polymerase, and 1 µM (each) primers. For the gyrA gene, a 467-bp DNA fragment was amplified with primers YER-AF (5'-ATGTGGCGGAATATTGGTTGC-3') and YER-AR (5'-TCGGACGTGCGTTACCAGATGT-3), designed upon the DNA sequence data of the homologous gene in Y. pestis (GenBank accession number AF282314). The amplifications were performed in a PT-100 thermal cycler (MJ Research), and PCR was carried out with AmpliTaq Gold polymerase (Applied Biosystems) by using an initial activation of the enzyme and denaturation of template DNA at 95°C for 5 min, followed by 30 cycles at 95°C for 1 min, annealing at 56°C for 1 min 30 s, and extension for 1 min at 72°C, followed by a final extension step of 72°C for 5 min. PCR products were purified for automated DNA sequencing with a QIAquick PCR purification kit (QIAGEN). DNA sequencing was performed by the Centro de Investigaciones Biológicas sequencing facilities (Madrid, Spain), with the Dye Deoxy (dRhodamine) terminator cycle sequencing Kit (Applied Biosystems) in an automatic ABI Prism 373 DNA sequencer with software provided by the manufacturer.

In order to examine the role of gyrase A in Y. ruckeri with reduced susceptibility to quinolones, OA-resistant mutants were obtained from the susceptible strain 1692 by a stepwise selection procedure. Approximately 106 CFU were inoculated in Mueller-Hinton (MH) broth containing OA at 1/2 MIC (62.5 ng ml-1), as recommended by Rodgers (18). After 48 h of incubation at 30°C, 100 µl of culture was surface plated onto MH plates without and with OA (1.5 µg of OA ml-1). Serial passages were repeated until a resistant mutant was obtained. The resistant population was compared with the total population for calculation of the mutation rate. One mutant strain (1692OA-RM) was selected for antimicrobial susceptibility, gyrA sequence analysis, and PFGE molecular characterization studies as described above.

All clinical isolates of Y. ruckeri displayed identical biochemical profiles, numerical code 5105100, which is one of the typical profiles displayed by this species with the API 20E system (6). All the strains of Y. ruckeri gave also a unique amplification product of 575 bp, which is specific for this species (7), therefore corroborating the biochemical identification results. By PFGE only one indistinguishable restriction pattern was obtained from all clinical isolates (data not shown), demonstrating that a single strain was responsible for all the yersiniosis outbreaks diagnosed between 1994 and 2002 and therefore confirming the endemic nature of ERM in this fish farm.

By the disk diffusion method, all clinical isolates showed the same antimicrobial susceptibility pattern against tetracycline, sulfisoxazole, and florfenicol, with inhibition zone diameter (IZD) values of 28, 34, and 30 mm, respectively. The clinical isolates recovered between 2001 and 2002 exhibited lower IZD values against all the quinolones tested than the isolates recovered between 1994 and 1998 (Table 1), indicating a reduced susceptibility to these antimicrobials. The decrease in the IZD was especially remarkable with NA and OA. By the microdilution method, Y. ruckeri isolates 1692, 1821, 1857, and 1983 exhibited MICs of <=0.5 µg/ml for NA and <=0.125 µg/ml for OA. Similar MICs for OA have been reported previously (18). Strains 5563, 5591, and 5991 exhibited seven- and fivefold increases in MICs for NA and OA, respectively (Table 1). The 2001-2002 isolates formed a separate cluster, considering both IZD and MICs, with respect to the 1994-1998 strains, confirming the reduced susceptibility of these strains to NA and OA.

DNA sequence analysis of the PCR products containing the QRDR of the gyrA from the collection strains and clinical isolates of Y. ruckeri revealed a high homology with the gyrA gene of other enterobacterial species. The greatest degree of similarity was found with Y. pestis (89.5% identity in 467-nucleotide overlap), while Escherichia coli K-12, Enterobacter cloacae, and Salmonella enterica serovar Typhi showed a lower degree of similarity (80.7, 79.8, and 79.2%, respectively). The alignment of deduced amino acid sequences of the gyrA QRDR revealed a higher degree of similarity to Y. pestis (99.2% identity) and a lower degree of similarity to E. coli (91.79% identity in 133-residue overlap). Amino acid residues from positions 67 to 106 (E. coli numbering), which are known to cause quinolone resistance (26), were perfectly conserved in Y. ruckeri (Fig. 1). gyrA gene sequences of the strains CECT 956, 1692, 1821, 1857, and 1983 displayed 100% similarity. One silent nucleotide change was observed in the third nucleotide (T->C) of Ala-84 (E. coli numbering) in the strain CECT 955. Analysis of the Y. ruckeri gyrA sequence from the isolates with reduced susceptibility to quinolones (5563, 5591, and 5991) exhibited, with respect to the 1994-1998 isolates, one nucleotide substitution, such as the transversion of cytosine for adenine, leading to an amino acid substitution at position 83 (according to E. coli numbering of protein sequence), which resulted in a Ser-to-Arg change (Fig. 1). This substitution of Ser-83 with Arg-83 has been detected in quinolone-resistant clinical isolates of different bacterial species (10), including several bacterial fish pathogens (8, 16). The Ser-83-to-Arg substitution might itself lead to a high-level quinolone resistance by introducing a bulky amino acid residue into the GyrA protein and also by decreasing the hydrogen-binding capacity between amino acid residues (10). These single mutations of DNA gyrase A generally result in MICs four- to eightfold higher than those for the susceptible strain (3, 27), similar to those observed for the isolates recovered in 2001 and 2002. Additional factors, such as the drug efflux system or decreased permeability of the outer membrane, also have been described (2, 22), but they result in very low-level MIC increases as well as a coresistance to other antimicrobials, like tetracyclines or phenicols (25). In our study, Y. ruckeri isolates with and without reduced susceptibility to quinolones displayed identical IZD values with tetracycline and florfenicol, suggesting no changes in the susceptibility to these antimicrobials. Therefore, these data indicate that the mutation in the gyrA gene would be sufficient to explain the reduced susceptibility to quinolones of the clinical isolates of Y. ruckeri.



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  		FIG. 1. Comparison of deduced amino acid sequences of GyrA QRDRs of Yersinia ruckeri and several type strains of Enterobacteriaceae species. Amino acid positions (E. coli numbering) are given above the sequences. Dots indicate identity among all the proteins. Variations in amino acid residues with respect to E. coli sequence are in boldface. The Yersinia ruckeri sequence represents strains CECT 956, 1692, 1821, 1857, and 1983. The OA-RM Y. ruckeri sequence represents strains 5563, 5591, 5991, and 1692OA-RM.

The selection of OA-resistant mutant strains was obtained after six serial passages on MH agar containing OA at 62.5 ng ml-1, with a mutation rate of 10-8, which is considered a normal frequency of mutation (14). The PFGE restriction patterns of the 1692OA-RM mutant and the parent strain were also undistinguishable (data not shown). IZD values and MICs for 1692OA-RM were similar to those for the clinical strains of Y. ruckeri with reduced susceptibility to quinolones (Table 1). Similarly, sequence analysis of the PCR product of the gyrA QRDR from this mutant carried the same amino acid substitution, Ser-83 to Arg-83, observed in clinical isolates 5563, 5591, and 5991. These results corroborate those obtained with the clinical isolates and provide evidence that in Y. ruckeri the substitution of Ser with Arg at position 83 of the gyrA gene is associated with reduced susceptibility to quinolones.

Selection of in vitro-resistant strains of Y. ruckeri by repeated exposures of a susceptible strain to OA, suggest that acquired resistance may occur in vivo during the quinolone therapy, as has been observed for several bacteria (24). No quinolone-resistant clinical strains of Y. ruckeri have been reported until now, despite the fact that these antimicrobials have been widely used under field conditions for the treatment of different bacterial infections in aquaculture, including ERM. Nevertheless, the isolation of strains of Y. ruckeri for which MICs were elevated, similar to those found in this study, from diseased and healthy carrier fishes (21) would make advisable the establishment of surveillance systems in order to have timely information about antimicrobial resistance trends that can be used for selecting the best therapeutic options.

Nucleotide sequence accession numbers. The partial nucleotide sequences corresponding to the gyrA gene of Y. ruckeri CECT 956 and the isolate 1692 have been assigned the accession number AJ426042 in the GenBank/EMBL database. The partial nucleotide sequence corresponding to the gyrA gene of Y. ruckeri CECT 955 has been assigned the accession number AJ426043 in the GenBank/EMBL database.


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ACKNOWLEDGMENTS
 
This work was supported by project ACU00-004-C2-2 of the Spanish Ministry of Sciences and Technology and by Dibaq-Diproteg S.A.


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FOOTNOTES
 
* Corresponding author. Mailing address: Dpto. Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain. Phone: 34 91 3943716. Fax: 34 91 3943908. E-mail: garayzab{at}vet.ucm.es. Back


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Applied and Environmental Microbiology, January 2004, p. 599-602, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.599-602.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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  • Colquhoun, D. J., Aarflot, L., Melvold, C. F. (2007). gyrA and parC Mutations and Associated Quinolone Resistance in Vibrio anguillarum Serotype O2b Strains Isolated from Farmed Atlantic Cod (Gadus morhua) in Norway. Antimicrob. Agents Chemother. 51: 2597-2599 [Abstract] [Full Text]  
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