Lisa Nonaka

Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.

Incidence of Antibiotic Resistance in Campylobacter jejuni Isolated in Alberta, Canada, from 1999 to 2002, with Special Reference to tet(O)-Mediated Tetracycline Resistance

ABSTRACT Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 μg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 μg/ml). In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 μg/ml) were higher than those previously reported in C. jejuni (64 to 128 μg/ml). In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene. Four isolates resistant to high levels of tetracycline (MIC = 512 μg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C. jejuni. The tetracycline MICs for transconjugants were comparable to those of the donors. Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 μg/ml for Escherichia coli DH5α. In contrast, transfer to a strain of C. jejuni by using mobilization conferred an MIC of 128 μg/ml. DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 μg/ml) were identical to one other, although the tet(O) genes encoding a 512-μg/ml MIC demonstrated several nucleotide substitutions. The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution. In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3′-aminoglycoside phosphotransferase. Resistance to erythromycin was not detected in 203 isolates. In general, resistance to most antibiotics in C. jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years.

New Mg2+-Dependent Oxytetracycline Resistance Determinant Tet 34 in Vibrio Isolates from Marine Fish Intestinal Contents

ABSTRACT A new oxytetracycline (OTC) resistance (Otcr) determinant, Tet 34, was cloned from chromosomal DNA of Vibrio sp. no. 6 isolated from intestinal contents of cultured yellowtail (Seriola quinqueradiata). The transformant, containing cloned Tet 34, could grow in broth containing 25 μg of drug per ml with 10 mM MgCl2. Tet 34 encoded an open reading frame (ORF) 154 amino acids long. The amino acid sequence of the ORF was homologous to sequences of several bacterial xanthine-guanine phosphoribosyltransferases (XPRTs), which act in purine nucleotide salvage synthesis. Mg2+ binding site residues and the active site were highly conserved in XPRT and the ORF of Tet 34. The results suggest that Tet 34 encodes a new Mg2+-dependent Otcr mechanism.

16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

Molecular evidence for the ancient origin of the ribosomal protection protein that mediates tetracycline resistance in bacteria

The ribosomal protection proteins (RPPs) mediate the resistance to tetracycline (TC) in Gram-positive and Gram-negative bacteria. The RPPs display sequence similarity to translation elongation factors, EF-G/EF-2 and EF-Tu/EF-1α. To determine the evolutionary origin of the RPPs, we constructed a composite phylogenetic tree of the RPPs, EF-G/EF-2 and EF-Tu/EF-1α. This tree includes two universal trees for the EF-G/EF-2 and EF-Tu/EF-1α, which form clusters corresponding to the respective two groups of proteins from three superkingdoms. The cluster of RPPs was placed at a point between the EF-G/EF-2 and EF-Tu/EF-1α clusters. The branch length (substitutions/site) between the node for the RPP cluster and the primary divergence of the RPPs was statistically shorter than that between the node for this cluster and the primary divergence in the EF-G/EF-2 cluster. This indicates that the RPPs derived through duplication and divergence of the ancient GTPase before the divergence of the three superkingdoms. Furthermore, this suggests the RPPs’ extant function occurred before the streptomycetes that include the TC-producing strains. Therefore, the RPPs evolved independent of the presence of TCs and serve a function other than antibiotic resistance. The RPPs may provide ribosomal protection against other chemical substances in the environment.

Novel macrolide-resistance genes, mef(C) and mph(G), carried by plasmids from Vibrio and Photobacterium isolated from sediment and seawater of a coastal aquaculture site.

The aim of this study was to determine whether mef(C) and mph(G), originally found on the transferable multi‐drug plasmid pAQU1 from Photobacterium damselae subsp. damselae isolated from seawater of a fish farm, are responsible for conferring macrolide resistance. Since these genes are localized head‐to‐tail on pAQU1 and only four nucleotides exist between them, the single‐ and combination‐effect of these genes was examined. When mph(G) alone was introduced to Escherichia coli, the minimum inhibitory concentrations (MICs) against erythromycin, clarithromycin and azithromycin increased, whereas introduction of mef(C) alone did not influence macrolide susceptibility. Introduction of both mef(C) and mph(G) dramatically increased the MICs to the same three macrolides, i.e. >512 μg ml−1, >512 μg ml−1 and 128 μg ml−1 respectively. These results suggest that the macrolide phosphotransferase encoded by mph(G) is essential for macrolide resistance, while the efflux pump encoded by mef(C) is required for high‐level macrolide resistance. The tandem‐pair arrangements of the mef(C) and mph(G) genes were conserved on plasmids ranging in size from 240 to 350 kb of the 22 erythromycin‐resistant strains belonging to Vibrio and Photobacterium obtained from the fish farm. Sixteen of 22 plasmids ranged in size from 300 to 350 kb. This is the first report of novel macrolide resistance genes originating from a marine bacterium.

Various pAQU plasmids possibly contribute to disseminate tetracycline resistance gene tet(M) among marine bacterial community.

Emergence of antibiotic-resistant bacteria in the aquaculture environment is a significant problem for disease control of cultured fish as well as in human public health. Conjugative mobile genetic elements (MGEs) are involved in dissemination of antibiotic resistance genes (ARGs) among marine bacteria. In the present study, we first designed a PCR targeting traI gene encoding essential relaxase for conjugation. By this new PCR, we demonstrated that five of 83 strains isolated from a coastal aquaculture site had traI-positive MGEs. While one of the five strains that belonged to Shewanella sp. was shown to have an integrative conjugative element of the SXT/R391 family (ICEVchMex-like), the MGEs of the other four strains of Vibrio spp. were shown to have the backbone structure similar to that of previously described in pAQU1. The backbone structure shared by the pAQU1-like plasmids in the four strains corresponded to a ~100-kbp highly conserved region required for replication, partition and conjugative transfer, suggesting that these plasmids constituted “pAQU group.” The pAQU group plasmids were shown to be capable of conjugative transfer of tet(M) and other ARGs from the Vibrio strains to E. coli. The pAQU group plasmid in one of the examined strains was designated as pAQU2, and its complete nucleotide sequence was determined and compared with that of pAQU1. The results revealed that pAQU2 contained fewer ARGs than pAQU1 did, and most of the ARGs in both of these plasmids were located in the similar region where multiple transposases were found, suggesting that the ARGs were introduced by several events of DNA transposition into an ancestral plasmid followed by drug selection in the aquaculture site. The results of the present study indicate that the “pAQU group” plasmids may play an important role in dissemination of ARGs in the marine environment.

The novel mef(C)–mph(G) macrolide resistance genes are conveyed in the environment on various vectors

BACKGROUND The novel tandem genes mef(C) and mph(G) have been reported in marine bacteria in Japan. This paper aimed to characterise the extent of environmental distribution of mef(C) and mph(G) as well as their dissemination and persistence in aquatic bacterial communities. METHODS Erythromycin-resistant bacteria were isolated from Japan, Taiwan and Thailand aquaculture sites. The mef(C)-mph(G) genes were detected by PCR. The size of mobile genetic elements conveying mef(C) and mph(G) was examined by Southern blotting. The conjugation rate was assessed by filter mating. RESULTS The mef(C)-mph(G) tandem genes were distributed in erythromycin-resistant isolates from aquaculture seawater in Japan and northern Taiwan and in animal farm wastewater in Thailand. A total of 29 bacterial isolates were positive for mef(C)-mph(G). The genes were found on vectors of various sizes. Partial sequencing of the traI relaxase gene revealed homology with a pAQU1-like plasmid, an IncA/C-type plasmid and an SXT/R391 family integrative conjugative element (SRI) as vectors. Thirteen isolates (45%) were positive for traI(pAQU-IncA/C-SRI), whereas the others were negative. The traI(pAQU-IncA/C-SRI)-positive isolates exhibited a higher transfer frequency (10-4-10-5 transconjugants/donor) than traI(pAQU-IncA/C-SRI)-negative isolates (<10-9). CONCLUSIONS These results suggest that mef(C)-mph(G) are coded on various vectors and are distributed among marine and wastewater bacteria in Asian countries. Vectors with traI(pAQU-IncA/C-SRI) play a role in the spread of mef(C)-mph(G).

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a ‘non-conjugative’ integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.