Sequence-Based Characterization of Tn5801-Like Genomic Islands in Tetracycline-Resistant Staphylococcus pseudintermedius and Other Gram-positive Bacteria from Humans and Animals

Abstract

Antibiotic resistance in pathogens is often associated with mobile genetic elements, such as genomic islands (GI) including integrative and conjugative elements (ICEs). These can transfer resistance genes within and between bacteria from humans and/or animals. The aim of this study was to investigate whether Tn5801-like GIs carrying the tetracycline resistance gene, tet(M), are common in Staphylococcus pseudintermedius from pets, and to do an overall sequences-based characterization of Tn5801-like GIs detected in Gram-positive bacteria from humans and animals. A total of 27 tetracycline-resistant S. pseudintermedius isolates from Danish pets (1998-2005) were screened for tet(M) by PCR. Selected isolates (13) were screened for GI- or ICE-specific genes (int Tn5801 or xis Tn916 ) and their tet(M) gene was sequenced (Sanger-method). Long-range PCR mappings and whole-genome-sequencing (Illumina) were performed for selected S. pseudintermedius-isolates (seven and three isolates, respectively) as well as for human S. aureus isolates (seven and one isolates, respectively) and one porcine Enterococcus faecium isolate known to carry Tn5801-like GIs. All 27 S. pseudintermedius were positive for tet(M). Out of 13 selected isolates, seven contained Tn5801-like GIs and six contained Tn916-like ICEs. Two different Tn5801-like GI types were detected among S. pseudintermedius (Tn5801 and GI6287) - both showed high similarity compared to GenBank sequences from human pathogens. Two distinct Tn5801-like GI types were detected among the porcine E. faecium and human S. aureus isolates (Tn6014 and GI6288). Tn5801-like GIs were detected in GenBank-sequences from Gram-positive bacteria of human, animal or food origin worldwide. Known Tn5801-like GIs were divided into seven types. The results showed that Tn5801-like GIs appear to be relatively common in tetracycline-resistant S. pseudintermedius in Denmark. Almost identical Tn5801-like GIs were identified in different Gram-positive species of pet and human origin, suggesting that horizontal transfer of these elements has occurred between S. pseudintermedius from pets and human pathogens, including S. aureus.

Keywords: GIs; ICEs; Tn916; horizontal gene transfer; integrative and conjugative elements; mobile genetic elements; tet(M); transmission.

FIGURE 1

Schematic illustration of Tn5801 and the strategy for PCR mapping of full-length Tn5801-like elements. (A) Illustration of the 25 kb putative integrative-conjugative element (ICE) integrated into the 3′end of a GMP synthetase gene guaA (black dotted arrow) in Staphylococcus aureus Mu50. Open reading frames (ORFs) are illustrated with arrows. Besides tet(M) (striped arrow), Tn5801 consists of three structural domains containing genes associated with conjugation (dark gray arrows), regulation (light gray arrows), and recombination (white dotted arrow) typically of elements belonging to the Tn916-family. ORFs whose functions are unknown (SAV393, SAV404, SAV406, SAV410, and SAV413-415) are illustrated with white arrows. SAV415 are predicted to encode a transposase. Direct repeats of 11 bp located in both ends of Tn5801 are chromosomal junctions and compose a putative core site for integration of the element (Ito et al., 2003). (B) Schematic illustration of primers (small arrows) designed based on the Tn5801 sequence (BA000017) and predicted PCR-products (rectangles) with expected product size listed below (see Supplementary Tables S2 and S3). (C) Schematic illustration of results of the PCR mapping of Tn5801-like genomic islands (GIs) for seven pet-associated S. pseudintermedius isolates, seven human S. aureus isolates and one porcine Enterococcus faecium isolate.

FIGURE 2

Illustration of Tn5801-like GIs from Gram-positive bacteria fully sequenced in this study. Gray arrows illustrate ORFs identical (sav393-sav397, and sav399-sav415) or similar (sav393-like-sav415-like) to corresponding ORFs in Tn5801, black arrows illustrate ORFs which were not annotated in Tn5801. Striped arrow: tet(M), white dotted arrow: Tn5801-like integrase gene (int), black dotted arrow: guaA, tnp: tranposase. Direct repeats of 11 bp located in both ends of Tn5801-like elements are chromosomal junctions and compose a putative core site for integration of the element (Ito et al., 2003). Underlined nucleotides differ from Tn5801. (A) Tn5801 from S. pseudintermedius 9841787-1 isolated from a dog and 9841998-1 isolated from a cat – sequenced GIs from the two isolates were 100% identical. GI6287 from S. pseudintermedius 200108299-1 isolated from a dog. (B) Tn6014 from human S. aureus 1680 (C) GI6288 sequenced from E. faecium CICYT205.

FIGURE 3

Comparison of full length Tn5801-like GIs from this study (5) and full-length Tn5801-like elements detected in sequences from GenBank (11). A multiple alignment of selected Tn5801-like sequences constructed and visualized with CLC (version 7.5.1) revealed overall seven Tn5801 GI types based on similar organization and DNA identity >99%. Element sizes are shown at the end of the sequences (to the right). EFA, E. faecalis; SAG, Streptococcus agalactiae; SA, Staphylococcus aureus; SP: LG, Lactococcus garvieae; SM, Streptococcus mitis. (EF_TX0133C/Bahaman: two strains TX0133C and TX0133B).