Examination of base pair variants of the strA-strB streptomycin resistance genes from bacterial pathogens of humans, animals and plants.

Sir, Resistance to the antibiotic streptomycin is an interesting case study due to the length of time since the discovery and introduction of streptomycin into clinical medicine (c. 55 years), and the continuing varied usage of this antibiotic in clinical medicine, animal husbandry and on crop plants to control certain plant diseases. The linked strA–strB streptomycin resistance (Sm) genes are notable because these genes are distributed among commensal and pathogenic bacteria isolated from humans, animals and plants. Isolates from humans and animals typically carry strA–strB on small broad-host-range non-conjugative plasmids such as RSF1010 or pBP1, while isolates from plants typically harbour strA–strB on large conjugative plasmids encoded within the transposon Tn5393. Tn5393 is a 6.7 kb Tn3family transposon, which encodes the strA–strB genes downstream of the tnpR resolvase gene; closely related variants may contain IS1133 or IS6100, insertion elements that provide promoters to express strA–strB, depending upon the species of isolation. Recently, several discoveries have expanded the breadth of host range of the strA–strB genes associated with Tn5393 sequences among plant-associated bacteria and clinical pathogens. A report detailing the complete sequence of pTP10, a 51.4 kb multiple antibiotic resistance (Ab) plasmid from the Gram-positive opportunistic human pathogen Corynebacterium striatum, revealed the presence of an apparently complete Tn5393 sequence which was interrupted in two places by the insertion of another transposon, Tn5715, and the insertion element IS1250. The Tn5393 sequences were 100% identical to the previously determined Tn5393 sequence from the plant pathogen Erwinia amylovora. Sequence remnants of Tn5393 elements linked to other Ab genes have also recently been detected in the chromosome of clinical isolates of Campylobacter jejuni and Pseudomonas aeruginosa. We analysed the strA–strB sequences currently published to determine if any sequence polymorphisms existed that could be useful in epidemiological studies to track the dissemination of this determinant. While the known strB sequences are 100% identical, a total of 5 bp of 804 bp are polymorphic among the strA sequences (Table). Since the majority of the strA sequences currently published were from non-conjugative plasmids, we cloned and sequenced strA from Tn5393 of plant pathogenic isolates of Pseudomonas syringae (A2, 7B12, 8C32) from the USA and Xanthomonas campestris (BV5-4a) from Argentina. Nucleotide sequencing was carried out using the Big Dye kit (ABI, Foster City, CA, USA) following the instructions of the manufacturer; sequence reactions were run at the Genetic Technologies Center, Texas A&M University. The individual strA nucleotide sequences generated in this study have been deposited in GenBank (accession numbers M77502, U20588, AF273681 and AF273682). Analysis of the newly determined strA sequences from the plant pathogens showed that these sequences were invariant from strA from E. amylovora (Table). The plant pathogen strA sequences differed by only 1 bp from the P. aeruginosa and Pasteurella multocida strA sequences (Table). The RSF1010 strA sequence was the most divergent from that of Tn5393 (Table). Analysis of the translated strA sequences indicated that the polymorphisms at nucleotides 1, 204, 467 and 470 would result in alterations of the StrA amino acid sequence (data not shown). The strA–strB sequence on RSF1010 is linked to the right inverted repeat of Tn5393, suggesting that the ancestral plasmid from which RSF1010 evolved obtained strA–strB via the insertion of Tn5393. Following the evolution of RSF1010 and other non-conjugative Sm plasmids, the strA sequence has diverged from that harboured on intact Tn5393 elements currently distributed among plant pathogenic bacteria. However, the recent discovery of the Tn5393 sequence on a C. striatum plasmid, and sequence remnants in C. jejuni and P. aeruginosa, indicates that the magnitude of gene transfer events of Tn5393-like elements has encompassed bacteria inhabiting diverse ecological niches, and has also included Gram-positive organisms. Along these lines, we had previously shown that the IS6100 element present in Tn5393 from the plant pathogen Xanthomonas campestris is 100% identical to an element also found in Mycobacterium fortuitum. The experience with Tn5393 and strA–strB-mediated