Staphylococcus sciuri C2865 from a distinct subspecies cluster as reservoir of the novel transferable trimethoprim resistance gene, dfrE, and adaptation driving mobile elements

Four methicillin-resistant Staphylococcus sciuri (MRSS) strains isolated from stranded dogs showed trimethoprim (TMP) resistance, while all staphylococcal TMP resistant dihydrofolate reductase genes (dfr) were negative. An in-depth whole-genome-sequencing approach on strain C2865 was followed for resistome and mobilome profiling, and for comparative genomics with S. sciuri group available genomes. Lack of species host tropism was observed, with MRSS C2865 placed at a separate sub-branch within S. sciuri species, close to the average nucleotide identity to be considered a different species (95-96%). S. sciuri proved a pronounced accessory genome (73% of genes), while MRSS C2865 distinctively harboured the highest total gene number and highest number of unique genes, with 75% associated to the recognised mobilome. A novel multidrug resistance mosaic plasmid (pUR2865-34) with several adaptive, mobilization (oriT mimic) and segregational stability (Type Ib par system) traits and two small single resistance plasmids were identified. Plasmid pUR2865-34 enclosed a novel staphylococcal TMP resistance gene, named dfrE, which shared highest identity with dfr of soil-related Paenibacillus anaericanus (68%). DfrE conferred high-level TMP resistance in S. aureus and Escherichia coli. Database searches revealed that dfrE was formerly denoted (dfr_like) in an Exiguobacterium spp. from a fish-farm sediment and that was present but unnoticed in several staphylococcal and onemacrococcal genomes with different epidemiological backgrounds. Novel chromosomal site-specific mobile islands with resourceful traits were identified, including a multidrug-resistant SCCmec cassette lacking cassette chromosome recombinase (Ccr) genes, a staphylococcal pathogenicity island of the SaPI4 family, and three unrelated siphoviridae prophages, two of which enclosed recombinases with the conserved Ccr-motif. We reveal a novel staphylococcal TMP resistance dfrE gene already present in diverse bacterial backgrounds. We confirm the ubiquity, high genome plasticity and low host tropism of S. sciuri highlighting its role as a resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability. Author summary Staphylococcus spp. are ubiquitous bacteria present in diverse ecological niches, including humans, animals and the environment. They are clinically relevant opportunistic pathogens and are notorious for their ability to acquire antimicrobial resistance (AMR) and virulence properties, resulting in a significant impact for Public Health. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information across bacterial species. Staphylococcus sciuri represents one of the most ancestral species in the genus and has been suggested a reservoir for AMR genes. Here, following a refined whole genome sequencing approach we determined the entire genome of an animal and environment-associated multidrug resistant (MDR) S. sciuri strain uncovering a novel acquired staphylococcal TMP resistance gene already spread among different bacterial species from different epidemiological backgrounds. We also reveal several additional MGEs, including a novel MDR mobilizable plasmid that encloses several adaptive and stabilization features, and novel mobilizable chromosomal islands with resourceful traits, including three unrelated prophages. Together with comparative genomics, we confirm the ubiquity, high intraspecies heterogenicity, genome plasticity and low host tropism of this species, highlighting its role as resourceful reservoir for evolutionary novel features contributing to its extraordinary versatility and adaptability.

Author summary 49 Staphylococcus spp. are ubiquitous bacteria present in diverse ecological niches, including 50 humans, animals and the environment. They are clinically relevant opportunistic pathogens and 51 are notorious for their ability to acquire antimicrobial resistance (AMR) and virulence properties, 52 resulting in a significant impact for Public Health. Mobile genetic elements (MGEs) play a central 53 role in this adaptation process and are a means to transfer genetic information across bacterial 54 species. Staphylococcus sciuri represents one of the most ancestral species in the genus and has 55 been suggested a reservoir for AMR genes. Here, following a refined whole genome sequencing 56 approach we determined the entire genome of an animal and environment-associated multidrug 57 resistant (MDR) S. sciuri strain uncovering a novel acquired staphylococcal TMP resistance gene 58 already spread among different bacterial species from different epidemiological backgrounds. We 59 also reveal several additional MGEs, including a novel MDR mobilizable plasmid that encloses 60 several adaptive and stabilization features, and novel mobilizable chromosomal islands with 61 resourceful traits, including three unrelated prophages. Together with comparative genomics, we 62 confirm the ubiquity, high intraspecies heterogenicity, genome plasticity and low host tropism of 63 this species, highlighting its role as resourceful reservoir for evolutionary novel features 64 contributing to its extraordinary versatility and adaptability.    Potential genes or chromosomal elements involved in conjugation, such as integrative or 186 conjugative elements or conjugative transposons, which could mediate intercellular transfer of 187 identified MGEs, were not detected. The genome location of the methicillin-susceptible mecA1 gene, two intrinsic dhfr genes (dhfr1 and dhfr2, the former next to a thy gene), a bacterial 189 chromosomal hicA-antitoxin hicB (Type II TA system), as well as the most relevant adaptive genes   190 from the novel chromosomally-located MGEs are displayed ( Fig 1B) encompassing the accessory genome (73%) (Fig 2A). The pan genome curve revealed not 197 saturated while the core genome was limited to less than 1,600 genes ( Fig 2B). This indicates an 198 open pan genome and high genetic diversity in S. sciuri. This is further supported by a high 199 prevalence of "cloud genes" (genes found in up to 15% of the strains), which corresponds to 200 approximately 43.2% of the pan genome (Fig 2A)   The dfrE-carrying region suggested a transposon-like structure encompassing an IS1216E copy, 296 the res/rec2 and res/rec3 resolvase genes, as well as the dfrE and thy genes (4,345 bp) ( Fig 3B). imperfect IRs downstream of thy was present ( Fig 3B). Here, the pMC2-carrying Tn3-like tnpA 300 gene, including the 38-bp upstream flanking IR, has been replaced by res/rec2. This truncated 301 region (IS1216E-res/rec2-res/rec3-dfrE-thy) was conserved in all additional dfrE-enclosing 302 strains (Table 3). Of note, two 6-bp identical DRs (5'-AAAAGC-3') and two 11-bp imperfect DRs     345 Based on the CRAmod assay, all four ica-locus variant carrying S. sciuri strains were biofilm 346 formers, while they were considered weak biofilm formers by the CV assay (S4 Fig). By this later 347 method, both positive controls, strains SA113 and DSM1104, confirmed strong biofilm formers.

348
Instead, remarkable lack of correlation was observed with the non-biofilm former S. aureus 349 RN4220 and both RN4220 transformants carrying the pUR2865-34 plasmid (S319 and S320) In addition to S. sciuri C2865, the three additional TMP resistant S. sciuri strains carried the novel 357 dfrE gene and the ica-locus variant cluster, based on the PCR assays. Linearized-plasmid analysis 358 by gel electrophoresis revealed that these three strains also harbored a similar-sized plasmid as 359 pUR2865-34. Strains C2853, C2854 and C2855 were also resistant to tetracyclines, macrolides-360 lincosamides and aminoglycosides and harbored the corresponding tet(K), erm(B) and aacA-aphD 361 resistance genes, which were also harboured in mosaic plasmid pUR2865-34 (Table 4).

493
The LSR integrase gene, named int/recC2865-pp1, was detected at the left extremity of the phage. (attR, carried in the excised circularized element: attP) allocated at each phage boundary (Fig 7).

517
Comparative analysis of Int/RecC2865-pp2 revealed that eight S. sciuri genomes shared related 518 integrases (Fig 7B). In all but one case we could determine that the integrated elements were 519 incorporated truncating the same chromosomal gene as in MRSS C2865; however, no conservation 520 of the DRs was observed in MRSS C2865 (Fig 7).  (Fig 8). The leftmost 60.5-kb leftwards-transcribed arm carries genes whose predicted morphogenesis (tape measure) and cell lysis (holin, endolysin) (Fig 8).

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The leftmost located integrase gene, named int1/recC2865-pp3, belonged to the LSR subfamily and      (Fig 9A). None of the genes involved in interference of the 628 helper phage reproduction in favor of SscPIC2865 packaging were identified. This may be due to 629 lack of sequence conservation, typical of SaPI-containing genes. However, this 3' region carried 630 a putative transcriptional regulator, a RNA polymerase sigma factor and a putative helicase, which 631 might be involved in the replication of the element (Fig 9A). Interestingly, a putative mazF toxin 632 gene, coding for an endoribonuclease (mRNA interferase) of a Type II toxin-antitoxin system 633 (MazF/MazE) was identified in the accessory region of SscPIc2865 by HHpred (9.3e-20 E-value).

634
Twelve integrases sharing >80% amino acid identity to SscPIc2865 integrase were identified by 635 sequence comparison, all truncating the 3'-end of the ribosomal gene rpsR, (Fig 9B). Of these, 636 nine belong to S. sciuri and the rest to S. schleiferi, S. lentus and S. fleurettii. Remarkably, their 637 integrase-surrounding regions share the characteristic structure of SaPIs (data not shown).

638
Among the six groups of site-specific integrases detected so far among elements of the SaPI family, 639 SaPIs of integrase group I, with SaPI4 as prototype, also integrate at the 3' region of the ribosomal protein S18 (rpsR gene) [62]. However, the amino acid identity of SaPI4 (S. aureus MRSA252) 641 and SscPIC2865 integrases was considerably low (74%) (Fig 9B). To address whether SscPIC2865 642 integrase and relatives belong the SaPI4 family, the integration region (attL, attR) of these 643 elements was analyzed in further detail (Fig 9C). The SscPIC2865 perfect DR 5'-644 AAAGAAGAACAATAA-3' (15 bp) was detected at both extremities of the islands in all cases, 645 including in S. aureus MRSA252, except for S. sciuri strain BMSA1 (Fig 9C). Hence, we propose  Comparative analysis of SscPIC2865 TerS, mostly conserved in SaPI-like elements, identified 650 nine terminases sharing >80% identity to TerS of SscPIC2865 (Fig 9B). These corresponded to 651 strains harboring the SscPIC2865-conserved integrase in all cases but one (S. sciuri SNUC70).  Hence, the ribosomal protein S18 (rpsR gene) appears as a hub for integration of mobile islands 792 and should be consider when addressing staphylococcal chromosomal-integrated elements.  prophages to mediate such events. We reassure and stress the importance of refined genome-wide 857 ecological studies to understand and eventually refrain the recombination and mobilization 858 landscape of this critical opportunistic pathogen. We also highlight the need to profit from the 859 richness of genomic data so far available in combination with functional analyses to unveil 860 adaptive traits that otherwise will pass unnoticed among the overwhelming sequencing scenery.    chromosomal region after excision were tested by specific inverse and conventional PCR, 951 respectively (S1 Data, S1 Table). ATCC 25923 were used as positive controls for their strong biofilm-forming potential, while S. 992 aureus RN4220 (DSM 26309) was selected as negative control for biofilm production.