Methicillin resistance in Staphylococcus pseudintermedius encoded within novel staphylococcal cassette chromosome mec (SCCmec) variants

Abstract Background Staphylococcus pseudintermedius is a common opportunistic pathogen of companion dogs and an occasional human pathogen. Treatment is hampered by antimicrobial resistance including methicillin resistance encoded by mecA within the mobile genetic element SCCmec. Objectives SCCmec elements are diverse, especially in non-Staphyloccocus aureus staphylococci, and novel variants are likely to be present in S. pseudintermedius. The aim was to characterize the SCCmec elements found in four canine clinical isolates of S. pseudintermedius. Material and methods Isolates were whole-genome sequenced and SCCmec elements were assembled, annotated and compared to known SCCmec types. Results and discussion Two novel SSCmec are present in these isolates. SCCmec7017-61515 is characterized by a novel combination of a Class A mec gene complex and a type 5 ccr previously only described in composite SCCmec elements. The other three isolates share a novel composite SCCmec with features of SCCmec types IV and VI. Conclusions S. pseudintermedius is a reservoir of novel SSCmec elements that has implications for understanding antimicrobial resistant in veterinary and human medicine.


Introduction
Staphylococcus pseudintermedius a coagulase-positive staphylococci, previously confused with Staphylococcus intermedius, was first recognized as a separate species in 2005 1 following the re-classification of S. intermedius into two species; S. intermedius and S. pseudintermedius.Together with Staphylcococus delphini 2 and Staphylococcus cornubiensis 3 -both separately describedthey form the S. intermedius group.A further proposed species, 'Staphylococcus ursi' (not validly published at the time writing) has been suggested as additional member of this group. 4. pseudintermedius is a common commensal of healthy companion dogs residing on the cutaneous and mucosal membranes, with the mouth, nose, groin and perineum-rectum the most commonly colonized regions. 56][7][8][9] Such is the prevalence of S. pseudintermedius canine pyoderma that it is a leading cause of antimicrobial prescriptions in small animal practice. 10][13] Methicillin resistance in S. pseudintermedius, as in other staphylococci, 14 is conferred by mecA, 15 encoding an alternative penicillin-binding protein PBP2a. 16mecA being carried by a variable mobile genetic element known as staphylococcal cassette chromosome mec (SCCmec). 17SCCmec diversity is best studied in S. aureus where 15 types, I-XV, are currently recognized with nomenclature being governed by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) (https://www.sccmec.org/0][21] SCCmec include joining (J) regions, defined by the areas between the orfX, ccr and mec genes.Often the components within the J regions are non-essential, however, they can contain determinants of antimicrobial resistance, thereby providing the SCCmec the ability to encode for additional resistance mechanisms beyond methicillin.SCCmec elements are found widely among the Staphylococcus genus as well as in the related Gram-stain positive genera Mammaliicoccus and Macrococcus. 23While some SCCmec elements found in S. aureus are also found in other species, the diversity of SCCmec elements is even greater among non-S.aureus organisms such that the 'IWG-SCC has decided not to annotate new SCCmec subtypes in other species than S. aureus, due to the high complexity of the elements found in isolates other than S. aureus'. 21,24Instead an alternative nomenclature is 'SCCmec[NAME OF THE STRAIN]', has been adopted for non-aureus species. 21,24In the case of methicillin-resistant S. pseudintermedius SCCmec type IV and V predominant. 12,25n addition to the challenge S. pseudintermedius and methicillinresistant S. pseudintermedius presents in veterinary medicine, it is increasingly being recognized as a zoonotic human pathogen responsible for a range of infections, most commonly skin and soft tissue infections. 26ere we highlight the diversity of SCCmec elements encoded within staphylococci with the description of two novel SCCmec elements in S. pseudintermedius.

Ethics
Samples were collected through routine diagnostic procedures with the written informed consent of the owner and approved by the institutional Veterinary Ethical Review Committee (reference 28.21).

Isolation, antimicrobial susceptibly testing and whole-genome sequencing
Canine S. pseudintermedius clinical isolates 6110-24416, 6127-64107, 7017-61515 and 10916-77753 were collected during routine veterinary diagnostic work at Easter Bush Pathology within the Royal (Dick) School of Veterinary Studies, University of Edinburgh, UK 7017-61515 came from a wound in 2016 and 10916-77753 from an infected digit in 2017.6110-24416 and 6127-64107 we isolated from canine pyoderma in 2013 and 2016 respectfully.Identification and antimicrobial susceptibly testing was performed by Vitek ® 2 (bioMérieux, Basingstoke, UK) using the AST-GP80 card and applying Clinical and Laboratory Standards Institute (CLSI) Veterinary Interpretation Guidelines, 27 antibiograms shown in Table S1 (available as Supplementary data at JAC Online), the oxacillin breakpoint being S ≤ 2 R ≥ 4 mg/L.Oxacillin disc diffusion was also performed using CLSI guidelines, the zone diameter breakpoint being S ≥ 18 R ≤ 17 mm. 27,28All four isolates were whole-genome-sequenced at MicrobesNG (Birmingham, UK).

Genome assembly and SCCmec identification
Illumina HiSeq technology with 2 × 250 bp paired-end reads and read trimming was performed by MicrobesNG.To generate a fully assembled SCCmec additional long read sequencing with Oxford Nanopore technology was carried out on isolates 6127-64107 and 10916-77753, also performed by MicrobesNG.In either case, read trimming was achieved using Trimmomatic v.0.30 29sing a sliding window quality cut-off of 15.De novo genome assembly was done using Unicycler v.0.4.8 30 using default parameters.All assemblies were annotated using the NCBI Prokaryotic Genome Annotation Pipeline. 31ontiguous sequences (contigs) containing the orfX and mecA genes were identified using BLAST, using the respective genes from S. aureus N315 Type II SCCmec (accession number D86934) as query sequences.For 6110-2416 and 7017-61515, contig JASSUT010000010 and JASSUS010000005 were identified, respectively, as containing the orfX and mecA genes.Initial analysis of 6127-64107 and 10916-77753 identified orfX and mecA located on different contigs.To resolve the SCCmec sequence, subsequent long read sequencing was carried out.This resulted in complete genome sequences with orfX and mecA located at 32 319..32 795 and 64 798..62 789 bp, respectively, within the genome of 6127-64107 and for 10916-77753, orfX and mecA, were located at 3 316..32792 and 64 370..62 361 bp, respectively.

Multi-locus sequence typing
Multi-locus sequence types (ST) were derived from the assembled genomes using the MLST tool available from the Center for Genomic Epidemiology (www.genomicepidemiology. org). 32

Results and discussion
The SCCmec of S. pseudintermedius 7017-61515 contains a novel meccomplex and ccr type combination S. pseudintermedius 7017-61515 was isolated from a canine wound with genome sequence-derived multi-locus sequence typing revealed this isolate to have a novel ST type, assigned ST1200.BLASTn analysis, with mecA from S. aureus N315, SCCmec Type II (accession D86934) as the query sequence, revealed carriage of mecA within the genome of 7017-61515 on MacFadyen and Paterson JASSUS010000005.To identify whether isolate 7017-61515 carried any SCCmec elements, manual examination of the region surrounding the mecA gene of 7017-61515 was undertaken.This showed the presence of mecR1 encoded upstream of mecA and on the opposite strand.Immediately downstream of mecR1 was mecI.Downstream of mecA was a hypervariable region followed by the insertion sequence IS431, which contained a frame-shift mutation.These features, taken together, indicate that the mecA gene of 7017-61515 is encoded within a Class A mec gene complex. 33Indeed, the mecA complex of 7017-61515 shared 99.30% nucleotide identity with the Class A mec gene complex from the Type II SCCmec of S. aureus N315.The mecA region was located 951 bp downstream of orfX, a 23S rRNA methyltransferase gene, indicating it was encoded within a SCCmec element. 33o characterize the SCCmec element, a search for the ccr genes was carried out.A single ccr gene was identified 6847 bp downstream from the mecA complex and shares 98% nucleotide identity with ccrC1 from the Type VII SCCmec of S. aureus PM1 (accession number AB462393).This constitutes a type 5 ccr gene complex. 33BLASTn analysis using the Class A mec gene complex as query, against the non-redundant nucleotide database, identified 500 staphylococci strains with this mec class and out of those only 118 had ccrC1 associated.Of the typed SCCmec elements, Type III (accession number AB037671) and Type XIV (accession number LC440647.1)are the only elements that contain both a Class A mec gene complex and a type 5 ccr gene complex.However, both the Type III and Type XIV SCCmec elements have the type 5 ccr as part of a composite element with other ccr types; as yet, there has been no type classification of a SCCmec that contains both Class A mec gene complex with a type 5 ccr.To the best of our knowledge, this is the first description of this combination of mec class and ccr type from a non-composite element.
Integration of SCCmec elements into the chromosome of staphylococci is achieved via an attB site, located at the 3′ end of the gene orfX. 34This integration results in the generation of direct repeat regions, attR and attL, which flank the element. 19Therefore, to establish the boundaries of the SCCmec element, the orfX region was searched to identify these repeat regions.The following search sequences, generated from previously identified attR and attL sequences, respectively, were used; gc [

ag]tatca[tc]aaatgatgcggttt and aacc[tg]catca[tc][tc][at][ac]c[tc]gataag[ct]
. The attL search sequence did not reveal any corresponding match, however, the attR search sequence gave two matches starting 23.7 kbp from the 3′-end of orfX.These two matches appear to be direct repeats reminiscent of known attL sites 35 separated by 32 bp, and were assigned attL1-1 and attL1-2 [Figure 1(a and b)].As the attR search sequence did not reveal an attR site within orfX, manual inspection of the 3′ end of the gene was carried out.From this inspection an attR sequence could be identified.This attR sequence differed from the attR sites used to generate the search sequence pattern, which were primarily associated with ccrAB carriage, with the attR of 7017-61515 being homologous to the attR sequences of SCCmec carrying only ccrC [Figure 1(b)].Our previous work revealed that the attR sites differ between SCCmec that carry either ccrAB and ccrC, which would explain why the initial attR search sequence did not find a match within the 3′ end of the orfX gene. 35Further to this, the central 8 bp of the attR sequence of 7017-61515 did not contain the traditional TATCATAA conserved bases but TACCACAA.Although this is different from the previously identified central 8 bp sequences, the central cytosine, thought to be essential for attB and attSCC recombination, is still present. 34Therefore, the identified attR site from 7017-61515 was considered accurate, resulting in a SCCmec element of 24 779 bp.
SCCmec 7017-61 515 contains three J regions, defined as the areas between the orfX, mec and ccr genes.The whole element shares 92% identity with the Type V SCCmec element from S. aureus WIS (accession number AB121219), although only 59% coverage [Figure 1(a)].One region of similarity corresponds to the mec region, though this region varies between the two SCCmec, as the Type V SCCmec contains a Class C2 mec gene complex, rather than Class A. The other region of similarity is the J2 region containing ccrC1, as well as putative proteins.The J3 region shares the least amount of similarity, with the J3 region of 7017-61515 lacking the type 1 restriction-modification genes, with the exception of a truncated hsdM gene, present within the Type V SCCmec [Figure 1(a)].

Isolates 6110-24416, 6127-64107 and 10916-77753 contain a novel composite SCCmec
S. pseudintermedius isolates 6110-24416, 6127-64107 and 10916-77753 were found to be methicillin resistant, with MLST revealing them to be ST561, ST41, ST668, respectively.Initial sequence analysis, as described before, revealed that all three isolates carried mecA.Manual inspection of the region surrounding mecA identified the presence of a truncated mecR1, with insertion sequence (IS) elements IS431 and IS1272, upstream and downstream of mecA, respectively.Therefore, the mec complex encoded by all three isolates is Class B. 33 All three isolates' complexes shared 99% nucleotide identity with the Class B mec of S. aureus HDE288 (accession number AF411935), with 98% coverage for both 6110-24416 and 10916-77753, and 83% coverage for 6127-6107.The lower coverage for 6127-6107 was found to be due to the presence of an additional IS element, located immediately downstream of mecA, putatively associated with the ISL3 family (Figure 2).
To determine which SCCmec type these isolates carried, identification of the ccr genes was undertaken.Two copies of both ccrA and ccrB were found, located 16 779 bp upstream and 1929 bp downstream of the mec complex (Figure 2).nBLAST analysis against previously identified ccrAB genes, revealed the ccr complex upstream was type 4, sharing 99% identity with ccrAB4, whereas the ccr complex downstream was type 2, sharing 97% identity with ccrAB2, 33 suggesting the identified SCCmec may be a composite of Type IVa and VI (Figure 3).Supporting this hypothesis, is the presence of a fusC gene within the region containing the ccrAB4 genes, which could be attributed to type VI SCCmec (Figure 3).To establish the boundaries of the SCCmec, att sites were determined.Using the search sequence mentioned previously, an attR site was found within the 3′ end of the orfX, with an attL site discovered 60 391, 48 755 and 48 275 bp from attR, for isolates 6110-24416, 6127-6107 and 10916-77753, respectively (Figure 2).Resulting in SCCmec elements of 61 259 bp for isolate 6110-24416, with isolates 6127-6107 and 10916-77753 having elements of 49 294 bp and 48 809 bp in size, respectively.

Novel SCCmec element in Staphylococcus pseudintermedius
The SCCmec within isolates 6110-24416, 6127-6107 and 10916-77753 share a significant amount of homology, however, due to the size discrepancies of the elements, further examination of the J regions was carried out.The J region between orfX and the ccrBA4 genes contained type 1 restrictionmodification genes, hsdRMS.However, only isolate 6127-6107 had an intact set of these genes, as isolate 10916-77753 had an IS element, IS1252, inserted into the hsdS gene, with isolate 6110-24416 having an integrative conjugative element (ICE) within its copy of hsdS (Figure 2).To further characterize the ICE of 6110-24416, comparative analyses were carried out with known ICE of other staphylococci.This analysis revealed that the ICE of 6110-24416 belongs to the ICE6013 family (subfamily 3), as it shared 94% nucleotide coverage and 97% nucleotide identity with the ICE6013 of S. intermedius NCTC 11048 (accession number UHDP00000000). 36Work carried out by Sansevere et al. demonstrated that ICE6013, of subfamily 1, can conjugatively transfer between different S. aureus backgrounds but did not detect any mobilization of chromosomal DNA. 36This suggests that the ICE6013 within the SCCmec of 6110-24416 is unlikely to be involved in the mobilization of the SCCmec, it may still have an impact on the SCCmec's ability to excise from the chromosome.However, it should be noted that ICE6013 are diverse, with seven subfamilies described, 36 allowing for the possibility that ICE6013 from different subfamilies may mobilize chromosomal DNA on excision.

Conclusion
Here, we present the discovery of two novel SCCmec elements within four clinical isolates of S. pseudintermedius from companion dogs.This finding expands our knowledge of the diverse range of this important staphylococcal antimicrobial resistance determinant, with relevance for both human and veterinary medicine.However, to gain a comprehensive understanding of atypical SCCmec elements, further research is warranted to elucidate their diversity, epidemiology and implications for understanding antimicrobial resistance effectively.

Figure 1 .
Figure 1.Overview of S. pseudintermedius 7017-61515 SCCmec and associated att sites.(a) S. aureus WIS corresponds to Type V SCCmec, accession number AB121219, with S. pseudintermedius 7017-61515 corresponding to this isolates SCCmec region.Bands connecting the two sequences represent regions of homology, with the percentage identity key shown on the right.Inverted sequence alignment is shown in red, normal sequence alignment is shown in blue.Key features associated with SCCmec elements are labelled.att sites are highlighted by filled circles and labelled above/below.(b) Comparison of attR/L sites of only ccrC-containing SCCmec.Conserved nucleotide bases are represented by an asterisk, for the core 8 bp region, which is represented in black, bold font.The central cytosine, indicated by a black triangle, thought to be essential for recombination between attB and attSCC. 34Inverted repeats are marked by the underlined bases.Sequences of known attR and attL sites associated with ccrC [from S. aureus WIS (WIS), S. aureus JCSC6082 (JCSC6082), AB373032; S. aureus 55-99-44 (55-99-44), MG674089] were aligned and compared with those identified in S. pseudintermedius 7017-61515.This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2 .
Figure 2. Overview of SCCmec elements in isolates 6110-24416, 6127-64107 and 10916-77753.The SCCmec region from each isolate is labelled with the corresponding name.Regions of homology are highlighted by the bands connecting the sequences, with the percentage identity key shown on the right.Inverted sequence alignment is shown in red, normal sequence alignment is shown in blue.Key features associated with SCCmec elements are labelled.att sites are highlighted by filled circles and labelled above/below.The ICE6013 of 6110-24416 has been labelled and lack of connecting bands indicates this region is missing from the SCCmec of 6127-64107 and 10916-77753.This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.