gyrA and parC Mutations and Associated Quinolone Resistance in Vibrio anguillarum Serotype O2b Strains Isolated from Farmed Atlantic Cod (Gadus morhua) in Norway

Abstract

MIC testing of Vibrio anguillarum isolates recovered from diseased farmed Atlantic cod revealed oxolinic acid MICs of ≤0.001, 0.06, and 16 μg ml⁻¹. Single gyrA Ser-Ile substitutions were identified at position 83 of the intermediate and resistant strains, while a parC Ser-Leu substitution at position 85 was found only in the resistant strain.

Recent advances in the field of larval production, regulated wild fisheries, and favorable market prices make farming of marine species, in particular cod, increasingly commercially attractive, and in recent years, cod farming has expanded considerably in Norway, Iceland, and Scotland. Farmed cod are susceptible to a range of bacterial diseases, the most significant of which are associated with Vibrio anguillarum infection (5), particularly with serotypes O2a and O2b.

Although commercial vaccines are available and appear to provide a significant degree of protection against some strains of V. anguillarum (L. C. Martinsen, H. Mikelsen, V. Lund, K. Gravningen, and M. B. Schrøder, presented at the 12th EAFP International Conference, Copenhagen, Denmark, 11 to 16 September 2005), there remain considerable problems regarding infection with V. anguillarum at all stages of the cod culture cycle. Outbreaks of bacterial disease necessitate antibiotic treatment, and oxolinic acid (OA), a 4-quinolone, is presently the antibiotic of choice. In the present study, six clinical strains isolated from clinically diseased, farmed Atlantic cod (Gadus morhua) and displaying three different degrees of susceptibility to quinolone antibiotics following disc diffusion analysis were investigated. All of the cod populations from which the bacteria were isolated had been previously treated with OA provided as an oral in-feed premix. The identities of all isolates were confirmed using a limited range of morphological characteristics, traditional biochemical tests, and agglutination testing with specific antisera.

MICs for enrofloxacin and nalidixic acid were established using a microplate broth dilution method, VetMic GN (National Veterinary Institute, Uppsala, Sweden), according to the manufacturer's instructions, with the exception of incubation, which was performed at 22°C for 5 days. OA MICs were established using a microplate broth dilution (256 through 0.0001 μg ml⁻¹) method as previously described (9) and were incubated at 22°C for 5 days. Disc diffusion assays were performed using OA (30 μg), flumequine (30 μg), florfenicol (30 μg), and oxytetracycline (80 μg) Neo-Sensitabs (Rosco, Taastrup, Denmark) on Müller-Hinton agar (Difco, MI). The results for disc diffusion are summarized in Table 1. The eight isolates displayed three different levels of sensitivity, which for the purposes of this study are termed sensitive (OA MIC, ≤0.0001 μg ml⁻¹), intermediate (OA MIC, 0.06 μg ml⁻¹), and resistant (OA MIC, 16 μg ml⁻¹). MICs for OA established in the presence of serial dilutions (256 through 0.0001 μg ml⁻¹) of the efflux pump inhibitor Phe-Arg β-napthylamide (EPI) (Sigma, St. Louis, MO) did not show any decrease.

TABLE 1.

Summary of MICs and disc diffusion zones for the studied isolates

Strain designation	Date of isolation	MIC (μg ml−1) for:			Disc diffusion zone (mm) for:
		Enrofloxacin	Nalidixic acid	Oxolinic acid	Oxolinic acid	Flumequine
04/09/363-5036	30 July 2004	≤0.03	≤1	≤0.0001	47	52
04/09/363-5034	30 July 2004	≤0.03	≤1	NDa	49	54
04/09/422-5063	10 September 2004	0.5	16	0.06	30	37
04/09/367-5042	12 August 2004	0.25	16	0.06	27	36
04/09/367-5043	12 August 2004	0.25	8	0.06	28	37
04/09/494-5106	11 October 2004	1	>128	16	0	17

^aND, not determined.

The quinolone resistance-determining regions (QRDR) of gyrA and parC were amplified using the degenerate primers gyrAF (5′-GAYGGNYTNAARCCNGTNCA-3′) and gyrAR (5′-GCCATNCCNACNGCDATNCC-3′) as previously described (10). This PCR, following electrophoresis and ethidium bromide staining, produced a single band of approximately 400 bp, subsequently identified (by DNA sequencing) as comprising both gyrA and parC products. The PCR products were cloned (TOPO TA; Invitrogen Groningen, The Netherlands) and transformed into Top10 (Invitrogen) chemically competent cells, following the manufacturer's instructions. Multiple clones of both gyrA and parC from each isolate were then sequenced using M13 primers, a DYEnamic ET dye terminator cycle sequencing kit, and a MEGABACE 1000 capillary sequencer (Amersham Biosciences, NJ). Contiguous sequences were assembled, aligned, and compared using the Sequencher program (Gene Codes Corp., Ann Arbor, MI). The identities of the obtained sequences were established using BLAST search analysis (1). Following trimming of the degenerate primer sequences, fragments of 382 and 385 bp were obtained. BLAST analysis (for sensitive strains) of the 382-bp fragment revealed DNA and amino acid identities of 100% with gyrA of V. anguillarum (AB201277), while the 385-bp fragment displayed DNA and amino acid identities of 99% and 100%, respectively, for V. anguillarum parC (BAF33487 and AB114417).

Using the sequences obtained from sensitive clones as a basis for comparison, we identified a single serine-isoleucine substitution at position 83 (related to V. anguillarum AB201277) in the gyrA genes of both the intermediate and the resistant strains and a single serine-leucine substitution at position 85 (relating to V. anguillarum BAF33487) in parC of the resistant strain.

Partial gyrB gene sequences approximately 1,100 bp in length and covering the gyrB QRDR (6) were amplified in one sensitive isolate, two intermediate isolates, and the resistant isolate by using primers V. anguillarum gyrBF (5′-GTC TGC ATG GTG TCG GTG T-3′) and V. anguillarum gyrBR (5′-CAC AGC CTA ATG CGG TGA TA-3′) and sequenced using internal primers gyrB1 (5′-CAT TTA CTG CTT TAC CAA-3′), gyrB2 (5′-GTT GAG TCA GCA ATG GGT-3′), gyrB4 (5′-CTA GAG AAC TTA GGA TCC-3′), and gyrB5 (5′-TAA TCC CAT CGT CAC GCT-3′) (all produced in this study). Identical gyrB sequences (submitted to GenBank under accession numbers EF534721 through EF534724) were derived from all investigated strains, indicating the noninvolvement of this locus in the observed quinolone resistance.

Although specific virulence-related traits (2, 11, 15) have been extensively studied, gyrA and parC have only recently been described for V. anguillarum (12). Although some quinolone resistance in gram-negative bacteria has been attributed to plasmid-borne genetic elements (8) and transmembrane efflux pumps (17), it is in the main associated with mutations in particular areas (QRDR) of the gyrA and parC genes. Such mutations have been identified in quinolone-resistant strains of a number of fish-pathogenic bacteria, including Vibrio anguillarum in Japan (12), Aeromonas salmonicida (4, 13), Yersinia ruckeri (3), and Flavobacterium psychrophilum (7).

Although not functionally tested, the two amino acid substitutions identified correlate directly with incremental increases in quinolone resistance. It is therefore concluded, given the evidence from similar studies with other bacteria, that these mutations are probably responsible for the increased quinolone resistance in the studied isolates, although additional mutations in other genes, such as parE, or the presence of plasmid-bound qnr-like elements (14) cannot be discounted. As MICs showed no decrease when determined in the presence of EPI, efflux pumps are not considered to constitute a resistance mechanism in the present case.

MIC (OA) values for both sensitive isolates and those harboring gyrA position 83 Ser-Ile substitutions in the present study (0.06 μg ml⁻¹) appear to be somewhat lower than those for equivalent strains studied by Okuda et al. (0.39 μg ml⁻¹) (12). However, the Ser-Leu mutation at position 85 in parC identified in the resistant isolate in the present study appears to award a significantly higher MIC (16 μg ml⁻¹) than the Glu-Gly substitution at position 90 (V. anguillarum BAF33487) of the Japanese field isolates described by Okuda et al. (6.25 μg ml⁻¹) (12). That Okuda et al. (12) found higher MICs (25 μg ml⁻¹) in laboratory-induced strains with the same gyrA and parC sequences as field strains suggests that mutations outside the investigated areas can also be involved in quinolone resistance.

Although only a few intermediate strains and a single resistant strain have been identified to date, the discovery of quinolone resistance among bacteria pathogenic for Atlantic cod at this early stage in the development of the Norwegian cod farming industry is disturbing but not surprising. Some of the fish from which the current isolates were recovered had been treated eight times with OA during the two preceding years. OA has generally been considered to be an effective compound for treatment of bacterial infections in cod. This may be due to the higher bioavailability of OA in cod than in salmon (16). Currently, only four (effectively) antibiotics are licensed for use in aquaculture in Norway, and two of these are quinolones (OA and flumequine). It is therefore important that resistance levels are kept to a minimum in fish-pathogenic bacteria. The development of quinolone resistance in one of the major pathogens of cod at this early stage of the industry's development indicates a requirement for both a conservative approach to antibiotic use and further development/use of effective vaccines.

Nucleotide sequence accession numbers.

The established sequences have been submitted to GenBank under accession numbers DQ234973 through DQ234978 (gyrA), EF534721 through EF534724 (gyrB), and EF469663 through EF469668 (parC).