A Transferable 20-Kilobase Multiple Drug Resistance-Conferring R Plasmid (pKL0018) from a Fish Pathogen ( Lactococcus garvieae ) Is Highly Homologous to a Conjugative Multiple Drug Resistance-Conferring Enterococcal Plasmid

Abstract

Lactococcus garvieae, the causative agent of lactococcosis, has evolved strains that are highly resistant to antibiotics. Here, the 20,034-bp sequence of L. garvieae conjugative plasmid pKL0018 was determined. It contained two ermB genes and one tetS gene and a backbone more than 96% identical to that of pRE25, an Enterococcus faecalis plasmid from dry sausage.Lactococcus garvieae, a gram-positive bacterium that causes lactococcosis, has caused serious economic damage to fish aquaculture worldwide (11). The appearance of L. garvieae strains resistant to macrolides and tetracycline following antibiotic treatment practices has compounded the problem. The newly acquired resistance was attributed partly to a transferable R plasmid(s) carried by the resistant strains, based on studies done as early as 1990 (1).Of 170 strains of L. garvieae isolated from cultured Seriola species (yellowtail, amberjack, and kingfish) from nine prefectures in Japan in 2002, most have been reported to have high frequencies of erythromycin (EM), lincomycin (LCM), and oxytetracycline resistance, and all isolates possessed ermB and tetS genes (6). Moreover, the yellowtail-derived L. garvieae isolates appeared to be homogenous and very different from isolates obtained from other fish, terrestrial animals, and food plants (4,5,6). Recently, of 146 L. garvieae strains isolated from 1999 to 2006 from yellowtail farms in three prefectures in Japan, 46 strains had high levels of resistance to EM, LCM, and tetracycline and were found to be carrying transferable R plasmids that carry ermB and tetS genes, as evidenced by conjugation, Southern blotting, and PCR methods (7).In this study, the complete nucleotide sequence of R plasmid pKL0018 from a multiple-drug-resistant, gram-positive L. garvieae strain isolated from yellowtail was determined and analyzed.L. garvieae strains were cultured in Todd-Hewitt broth (Difco) and 2% NaCl at 25°C. Enterococcus faecalis strain OG1SS was used as recipient cells of R plasmid. The cells were cultured in Todd-Hewitt broth (Difco) at 37°C. R plasmid pKL0018, isolated from EM-, LCM-, and tetracycline-resistant L. garvieae strains from Kagoshima, Japan, in 2000, was sequenced using cloning vector pBluescript (Stratagene).R plasmid was transferred in mixed culture with E. faecalis OG1SS for 18 h at 37°C and was selected by culture in medium containing 150 g/ml of streptomycin and 100 g/ml of EM. Purified plasmid DNA was randomly fragmented by sonication, cloned into the sequencing vector pBluescript (Stratagene), and transformed into Escherichia coli JM109 by electroporation. Clones were then sequenced using a Thermosequenase sequencing kit (Amersham-Biotech) with an LC4200 (Li-Cor) automated DNA sequencer. Data were assembled and analyzed using the ATSQ program in GENETYX v. 7.0 (SDC Software Development Co.), BLASTX of NCBI (http://www.ncbi.nlm.nih.gov), ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/), and BLAST 2 sequences (http://ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi).Sequence of R plasmid pKL0018. The R plas...

Lactococcus garvieae: an emerging bacterial pathogen of fish

“…Genes related to multiple drug resistance carried on pKL0018 were identified as the tetracycline resistance gene tet(S) and macrolide resistance genes encoding 23S rRNA methyltransferases (ermB1 and ermB2). This work constituted the first report of the sequence of a Gram-positive bacterium-derived plasmid from a fish patho gen (Maki et al 2009). The presence of tet(S) and another ribosomal protection protein gene, tet(M), was simultaneously detected in Japanese L. garvieae marine isolates (Kim et al 2004).…”

“…The remaining 74% of highly resistant isolates were shown to carry the same resistance genes present on the R plasmid, suggesting carriage of either an integrated R plasmid or transferable low frequency plasmids. Further characterisation of the R plasmid pKL0018, described by Maki et al (2008), revealed high sequence homology to pRE25, a plasmid found in E. faecalis isolated from dried sausage (Maki et al 2009). Genes related to multiple drug resistance carried on pKL0018 were identified as the tetracycline resistance gene tet(S) and macrolide resistance genes encoding 23S rRNA methyltransferases (ermB1 and ermB2).…”

The Plasmidome of Firmicutes: Impact on the Emergence and the Spread of Resistance to Antimicrobials

“…doi:10.1128/microbiolspec.PLAS-0039-2014.f7 ASMscience.org/MicrobiolSpectrum . These ISs also facilitate the co-integration with other RCR (e.g., pC221, which is cointegrated in pRE25) or theta replication plasmids as pheromone responsive (116,(119)(120)(121) or some pSK41-like elements. Figure 16 shows the diversity of Inc18 RIPs that can be identified by typing systems.…”

“…Analysis of the same AbR genes in different species (cfr, bac, lincosamide resistance genes) reflects the impact of recombination events between genes, MGEs, and different populations of Firmicutes (Staphylococcus, Clostridium, Lactobacillus, Lactococcus, and Enterococcus) and other Gram-negative organisms (200,204) in the gastrointestinal tract of animals and humans (120,144,205,206).…”

Different Genetic Supports for the tet (S) Gene in Enterococci

“…Genes involved in ribosomal protection, such as tet(S), tet(M), tet (O), tet(Q), and tet(W), were identified in conjugative transposons (CTns), phage-derived structures, and/or conjugative plasmids (3,4,5,11,12,16). The tet(S) gene has been found among Firmicutes and gammaproteobacteria from diverse ecological sources since the early 1950s (1, 2, 10, 11) (http://faculty.washington.edu/marilynr/tetweb2.pdf), and several genetic platforms carrying tet(S), such as CTn6000, CTn916S, pKL0018, and pK214, have been described (5,9,11). Among enterococci, the genetic context of tet(S) has been characterized in only a single Enterococcus casseliflavus isolate from animal origin (5,15).…”