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Genomic epidemiology of methicillin-resistant Staphylococcus sciuri carrying a SCCmec-mecC hybrid element
Infection, Genetics and Evolution 79 (2020) 104148
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Research paper
Genomic epidemiology of methicillin-resistant Staphylococcus sciuri carrying a SCCmec-mecC hybrid element
T
Gavin K. Paterson Easter Bush Pathology, Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK
A R T I C LE I N FO
A B S T R A C T
Keywords: mecA mecC SCCmec Staphylococci
The recognition in 2011 of the methicillin resistance determinate mecC among staphylococci has raised many questions over its evolution and epidemiology. While mecC has been best studied in Staphylococcus aureus it has also been described in at least nine other species of staphylococci. In most cases these studies are limited to single isolates. In the widespread animal commensal Staphylococcus sciuri mecC has been described in two isolates and is located within a distinct SCCmec-mecC composite element. In this study, a further 11 mecA/mecC S. sciuri isolated from dairy farms in England and Wales in 2015 and 2016 were genome sequenced and characterised. The results show that two variants of the SCCmec-mecC element are present in S. sciuri, differentiated by different ccr alleles and likely to have arisen by homologous recombination. A phylogeny of sixty genome-sequenced S. sciuri isolates was made using core genome multi-locus sequence typing and reveals a diverse population with the SCCmec-mecC element present in four distinct branches, indicative of four independent acquisitions by S. sciuri. Finally, the study identified the rapid clonal expansion of a mecA/mecC lineage of S. sciuri among dairy farms across a wide geographical area which may contribute to the future dissemination of this methicillin resistance cassette.
1. Introduction Methicillin resistance in staphylococci is typically encoded by mecA carried on the chromosomal mobile element, SCCmec (Peacock and Paterson, 2015). Although methicillin resistance conferred by plasmidencoded mecB and mutation of house-keeping genes have been reported in Staphylococcus aureus, these mechanisms currently appear to be rare (Ba et al., 2014; Ba et al., 2019; Becker et al., 2018). The mecA gene encodes an alternative penicillin-binding protein (PBP2a/PBP2’) which confers resistance to most β-lactam antimicrobials (Fuda et al., 2004; Hartman and Tomasz, 1984; Lim and Strynadka, 2002; Ubukata et al., 1989). While methicillin-resistant S. aureus (MRSA) encoding mecA is a prominent pathogen, mecA is also found in a range of other staphylococci isolated from carrier and diseased humans and animals (Becker et al., 2014b; McCarthy et al., 2015). In 2011 a variant of mecA was described in MRSA from humans (Garcia-Alvarez et al., 2011; Shore et al., 2011) and dairy cattle (Garcia-Alvarez et al., 2011) in Denmark, England and the Republic of Ireland. Sharing 68.7% nucleotide with the archetypal mecA of S. aureus N315, this variant was designated as mecC and is encoded in MRSA within a distinct SCCmec classified as type XI (Ito et al., 2012). mecC sequence divergence from the highly-conserved mecA meant that at the time of its description molecular methods to detect MRSA based on mecA PCR or PBP2a/PBP2’ slide agglutination
with monoclonal antibodies failed to reliably detect mecC MRSA. Subsequently mecC MRSA were found to be present in a wide range of host species and in many countries although at a much lower frequency than mecA MRSA (Becker et al., 2014a; Paterson et al., 2014a). Similarly to mecA, mecC has also been found in range of other staphylococci, namely; Staphylococcus xylosus (Loncaric et al., 2019; MacFadyen et al., 2018b), Staphylococcus pseudoxylosus (Harrison et al., 2013; MacFadyen et al., 2019b), Staphylocococus saprophyticus (Malyszko et al., 2014; Srednik et al., 2017), Staphylococcus caeli (MacFadyen et al., 2019a), Staphylococcus stepanovicii (Semmler et al., 2016), Staphylococcus warneri (Loncaric et al., 2019), Staphylococcus caprae (Loncaric et al., 2019), Staphylococcus edaphicus (Pantucek et al., 2018) and Staphylococcus sciuri (Harrison et al., 2014; Loncaric et al., 2019). In these species, where it has been investigated, mecC has been found either within a SCCmec type XI (Loncaric et al., 2019; MacFadyen et al., 2018b) or in a range of distinct SCCmec-like elements (Harrison et al., 2014; Harrison et al., 2013; Loncaric et al., 2013; MacFadyen et al., 2019a; Pantucek et al., 2018; Semmler et al., 2016). In the case of S. sciuri, mecC has been found encoded within a hybrid SCCmec element comprised of a mecA encoding SCCmec type VII and a mecC region consisting of the class E mec complex (mecI-mecR, mecC-blaZ) (Harrison et al., 2014). This element has been described in only two isolates of S. sciuri, originally in GVGS2 from a caesarean incision wound in a Belgian
E-mail address: [email protected]. https://doi.org/10.1016/j.meegid.2019.104148 Received 25 September 2019; Received in revised form 16 December 2019; Accepted 17 December 2019 Available online 17 December 2019 1567-1348/ © 2019 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 79 (2020) 104148 a Tested using Vitek2 against the following: cefoxitin screen, benylpenicillin, oxacillin, ceftiofur, cefquinome, amikacin, gentamicin, kanamycin, neomycin, enroflaxacin, inducible clindamycin, erythromycin, tilmicosin, tylosin, clindamycin, tetracycline, florfenicol, trimethoprim/sulfamethoxazole. Only resistance phenotypes are shown, susceptible unless stated. b Identified using ResFinder.
ERX3374896 ERX3374901 ERX3374902 ERX3374904 ERX3381835 ERX3381812 ERX3381831 ERX3381834 ERX3382826 ERX3407607 ERX3407627 mecA/mecA1/mecC/blaZ/sal(A) mecA/mecA1/mecC/blaZ sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ str/sal(A)/tet(K) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ sal(A) cefoxitin/benylpenicillin/oxacillin/ceftiofur cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome/clindamycin/ cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome/clindamycin/ cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome/clindamycin/tetracycline/ cefoxitin/benylpenicillin/oxacillin/ ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome Sep-15 Sep-15 Sep-15 Sep-15 Sep-15 Feb-16 Sep-15 Sep-15 Feb-16 Feb-16 Feb-16 5073-5-63 5078-2-20 5079-SM14 5081-3-55 5162-EF3a 5183-EF324a 5202–5-64a 5205-5-69 5454-CC103A 5790-EF260 5810-BC71A
Somerset, England Cornwall, England Devon, England Lancashire, England Lancashire, England Staffordshire, England Devon, England Devon, England Carmarthenshire, Wales Shropshire England Monmouthshire, Wales
Nucleotide accession Acquired resistance genesb Resistance phenotypea Location Date of isolation Isolate
Table 1 Characteristics of study isolates.
G.K. Paterson
Blue cow in England (Harrison et al., 2014) and LP600 from an alpaca in Austria (Loncaric et al., 2013). In addition the SCCmec-mecC element has been reported in a single S. aureus isolate from a racehorse wound (Sekizuka et al., 2019). In each case the SCCmec-mecC hybrid element was highly conserved with the archetypal version reported originally in GVGS2 (Harrison et al., 2014). To examine the epidemiology and genomics of mecC in S. sciuri this study has isolated and sequenced the genomes of a further 11 mecA/mecC-positive methicillin-resistant S. sciuri, from dairy bulk tank milk in England and Wales.
2. Materials and methods 2.1. Isolation and identification of mecA/C S. sciuri Bulk tank milk from dairy farms in England and Wales was sampled for methicillin-resistant staphylococci via salt broth enrichment and plating on MRSA Brilliance agar (Oxoid, Basingstoke, UK) as described previously (Paterson et al., 2014b).
2.2. Whole-genome sequencing and analysis Isolates of interest were genome sequenced by Microbes NG (University of Birmingham, UK) using Illumina HiSeq technology with 2 × 250 bp paired-end reads as described previously (MacFadyen et al., 2018a). In all cases the SCCmec-mecC hybrid was assembled on a single contig. Phylogenetic relationships among S. sciuri isolates were assessed with SeqSphere+ software version 4.1.9 (Ridom, Münster, Germany) (Jünemann et al., 2013) using a core genome multilocus sequence typing (cgMLST) scheme based on all 60 isolates in this study with the S. sciuri type strain NCTC12103T (NZ_LS483305.1) used as the reference genome. Reference genome filters were a minimum length > 50 bases, a start and stop codon at the beginning and end of the gene, a homologous gene filter (requiring no multiple copies of a gene with BLAST overlap > 100 bp, identity > 90.0%), a gene overlap filter (requiring no overlap with other genes of more than four bases) and an exclude sequences filter (requiring no BLAST hit with overlap > 100 bp, identity > 90.0% in excluded sequences). The query genome BLAST search required a BLAST hit with overlap = 100%, identity > 90.0% in every query genome using BLAST options: word size = 11, mismatch penalty = −1, match reward = 1, gap open costs = 5, gap extension costs = 2. A query genome stop codon percentage filter was applied requiring a single stop codon at end of gene in > 80%query genomes. Omitting missing values from isolates this resulted in a scheme for this study of 1278 gene targets. The resultant tree was annotated in the Interactive Tree of Life (iTOL) (Letunic and Bork, 2019). Comparator S. sciuri isolates were those with assembled genomes available from NBCI, details and nucleotide accession are shown in Supplementary Information. Acquired resistance genes were identified using ResFinder3.1 (Zankari et al., 2012) and schematic comparison of the SCCmecmecC element performed using EasyFig (Sullivan et al., 2011).
2.3. Antimicrobial susceptibility testing This was performed using Vitek2 (bioMéurieux, Basingstoke, UK) following the manufacturer's instructions and using the AST-GP79 Gram-positive livestock card. The antimicrobials tested were: cefoxitin screen, benylpenicillin, oxacillin, ceftiofur, cefquinome, amikacin, gentamicin, kanamycin, neomycin, enroflaxacin, inducible clindamycin, erythromycin, tilmicosin, tylosin, clindamycin, tetracycline, florfenicol, trimethoprim/sulfamethoxazole with interpretation performed using The Clinical and Laboratory Standards Institute criterion.
2
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3. Results
present only in the SCCmec elements of S. pseudintermedius KM241 (AM904731.1) and S. sciuri TXG-24 (KX774481.1) both of which are distinct to that in SM14 and which encode only mecA and not mecC.
3.1. Isolation of methicillin-resistant mecA/C S. sciuri A survey of 363 dairy farms in England and Wales in September 2015 and February 2016 led to the isolation of methicillin-resistant staphylococci from the bulk tank milk of 18 of these (4.96%) (unpublished data). Three of these 18 isolates were methicillin-resistant S. sciuri encoding both mecA and mecC and further convenience sampling of bulk tank milk led to the isolation of an additional 8 such isolates all from different dairy farms (Table 1).
3.3. Phylogenetic analysis of mecA/C S. sciuri To assess the phylogenetic relationships among S. sciuri isolates encoding mecA/mecC and with other S. sciuri isolates a cgMLST scheme with 1278 gene targets was generated. This comprised the 11 S. sciuri mecA/C isolates described here, the two previously described S. sciuri mecA/C isolates GVGS2 and LP600 along with the 47 further isolates whose assembled genomes were available on public databases (Supplementary Information Table 1). The host source of these 60 isolates are: cattle (42 isolates), Asian Rice (7), Human (4) and one isolate each from Pig, African Social Spider, Mouse, Alpaca, Grey Squirrel, Virginia Opossum and source unknown. Isolates came from 11 countries: Canada (28 isolates), United Kingdom (13), India (7), USA (3), China (2), Czech Republic (2) and singles from Australia, Austria, Chile, Denmark, and Sweden. All of the isolates encoded the mecA1 allotype found ubiquitously in S. sciuri but none of the isolates from the public database encoded both mecA and mecC. Two human isolates encoded mecA without mecC (Fig. 2). In each case these isolates are distantly related to those with mecA/mecC and encode mecA within a SCCmec with no resemblance to the SCCmec-mecC element (data not shown). The phylogeny shows a diverse population of S. sciuri and that the SCCmec-mecC element is present in four distinct branches each likely indicating an independent acquisition event (Fig. 2). Of particular note are the cluster of eight highly-related isolates which correspond to the eight referred to above which encode identical SCCmec-mecC elements. The mean pairwise difference among these 8 isolates is only 6.4 gene targets, range 4–11. The farms from which these isolates came were located in six different counties with a mean pairwise distance between farms of 95 miles, range 9–233 miles. The closest distance being any two positive farms is 9 miles and the furthest is 96 miles.
3.2. Analysis of SCCmec in mecA/C S. sciuri Genome sequencing of these 11 mecA/C S. sciuri revealed that they all carried a SCCmec-mecC hybrid element in the orfX/rlmH region which was highly conserved between the isolates and with similarity to that previously reported in GVGS2 (Harrison et al., 2014). For comparative purposes this region was defined from the start codon of orfX/ rlmH to the inverted repeat of the attL2 site. In each of the 11 bulk tank milk isolates this region was 43,723 bp in length and was 100% identical in 8 of the isolates. Two isolates (CC103a and 3–55) shared identical sequences to each other in this region which differed from those aforementioned 8 by 15 SNPs, the final isolate (EF3a) shared those 15 SNPs plus two further unique SNPs. Taking SM14 as a representative of these bulk tank milk isolates the SCCmec-mecC hybrid element was compared with that region previously reported in S. sciuri GVGS2 which was isolated from Belgium Blue cattle in England (Harrison et al., 2014). This revealed the elements are closely-related sharing a high degree of synteny and nucleotide conservation, Fig. 1. However, there were notable differences around the ccr genes. No mobile genetic elements are apparent in this region suggesting this rearrangement has occurred through homologous recombination. Consistent with this suggestion is the finding that the 5′ portion of a hypothetical protein gene in this region is conserved between the two isolates but has a highly divergent 3′ sequence and may therefore have been a site for a recombination event within the SCCmec-mecC element. The SCCmec-mecC element of GVGS2 carries the recombinase ccrA1B5 genes whereas SM14 and the other 10 bulk tank milk isolates possess ccrA3B3. Furthermore an ORF downstream of the ccr genes in GVGS2 is absent in SM14 with an ORF present upstream of the ccr genes in SM14 absent in GVGS2. The GVGS2 gene encodes a 244 amino acid protein belonging to the short-chain dehydrogenases/reductases superfamily (Conserved Domain Database accession: cl25409) and a standard nucleotide BLAST search shows it to be present in a small number of staphylococcal isolates belonging to S. sciuri, S. pseudintermedius, S. simulans, S. aureus and S. haemolyticus (data not shown). The SM14 gene in question encodes a 96 amino acid protein belonging to the NeuB Nacetylneuraminate synthase superfamily (Conserved Domain Database accession: cl30262). A standard nucleotide BLAST search shows it to be
3.4. Phenotypic resistance and other antimicrobial resistance determinates Consistent with the presence of mecA, mecC and blaZ, all 11 isolates were resistant to the β-lactms penicillin, cefoxitin, ceftiofur and oxacillin with all but 2 was resistant to cefquinome (Table 1). All isolates encoded sal(A) which confer moderate resistance to lincosamides and high-level resistance to streptogramin A in S. sciuri (Hot et al., 2014). However, only three were resistant to clindamycin as assessed by Vitek2 (MIC ≥4). The specintomycin resistance gene str being present in 6 isolates and a single tetracycline resistant isolate encoded tet(K). No other phenotypic resistance was observed among the antimicrobials tested and no other antimicrobial resistance determinates were noted.
Fig. 1. Schematic comparison of the SCCmec-mecC elements in S. sciuri isolates GVGS2 (accession HG515014) and SM14 (representative of the dairy bulk tank milk isolates described in this study). Selected key genes are highlighted. Regions of homology are represented by bands connecting the two sequences, with the percentage identity key shown on the bottom right. Red denotes normal sequence alignment and blue denotes inverted sequence alignment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3
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Fig. 2. Phylogenetic relationships among an international collection of whole-genome sequenced S. sciuri isolates. Neighbour-joining phylogeny generated using a core genome multilocus sequence typing scheme produced from all 60 isolates in this study and consisting of 1278 gene targets present in all isolates. Isolates positive for mecA and mecC are highlighted in yellow, those positive for mecA are highlighted in blue and the S. sciuri type strain is highlighted in orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion
present in the second isolate LP600 (Loncaric et al., 2019). To further knowledge of this SCCmec-mecC element, this study has genome sequenced 11 dairy cattle isolates of mecA/mecC S. sciuri. Genomic analysis reveals that two variants of this SCCmec-mecC element are present among S. sciuri isolates. The prototype described in GVGS2 (Harrison et al., 2014) and a second among the dairy isolates in this study. While highly conserved with the GVGS2 element this second variant is distinguished most prominently by the carriage of different ccr alleles to those in GVGS2. Phylogenetic analysis of a global collection of 60 S.
The recognition of the mecC methicillin resistant determinate in staphylococci has raised many questions regarding its evolution and epidemiology. In the widespread animal commensal S. sciuri it is encoded within a composite SCCmec-mecC element, but this has only been described in two S. sciuri isolates to date. It has been fully characterised in one isolate, the assembled element in GVGS2 (Harrison et al., 2014) (Fig. 1) with read mapping indicating a highly similar element is 4
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References
sciuri sequenced isolates indicates the independent acquisition of this SCCmec-mecC element on four occasions by this population of S. sciuri. Of particular note is the apparent clonal expansion of 8 highly-related isolates encoding an identical SCCmec-mecC element. These were each present on different farms up to 233 miles apart and their close phylogenetic relationship would strongly suggest transmission events between the farms or from a common source. The very limited genetic diversity among these isolates may further indicate a relatively recent expansion and it will be of interest to monitor the epidemiology of this clone in future. Further spread may be problematic through the dissemination of methicillin resistance and/or should this clone has a propensity for causing infections. While the isolates in this study were isolated from bulk tank milk, no link to bovine mastitis can be apportioned here. However, S. sciuri including methicillin-resistant isolates can cause bovine mastitis (Khazandi et al., 2018) and so veterinarians and veterinary microbiology laboratories need to be conscious of the possible presence of mecA/mecC isolates from bovine mastitis and other infections. Indeed, the original mecA/mecC isolate GVGS2 came from a Caesarean incision wound infection (Harrison et al., 2014). Other resistance genes were uncommon among the 11 dairy mecA/ mecC isolates. Consistent with the report of Hot et al. (2014) all isolates carried sal(A).
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5. Conclusions This work expands our understanding of the epidemiology of mecC among staphylococci. Specially, a novel variant of the S. sciuri SCCmecmecC composite element is described and core genome phylogenetic analysis indicates that the mecA/mecC cassette has been acquired on at least four occasions by this staphylococcal species. The clonal expansion of a lineage of mecA/mecC S. sciuri among several dairy farms across a wide geographical area may be of future concern through the spread of resistance or if this is a virulent clone. Acknowledgements Genome sequencing was provided by MicrobesNG (http://www. microbesng.uk), which is supported by the BBSRC (grant number BB/ L024209/1). We gratefully thank National Milk Laboratories Ltd. for their assistance in providing bulk tank milk samples. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author contributions Gavin K. Paterson: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2019.104148. 5
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(In press) 2019 Nov 6. pii: dkz459. [Epub ahead of print]. Semmler, T., Harrison, E.M., Lübke-Becker, A., Ulrich, R.G., Wieler, L.H., Guenther, S., Stamm, I., Hanssen, A.-M., Holmes, M.A., Vincze, S., Walther, B., 2016. A look into the melting pot: the mecC-Harboring region is a recombination Hot spot in Staphylococcus stepanovicii. PLoS One 11, e0147150. Shore, A.C., Deasy, E.C., Slickers, P., Brennan, G., O’Connell, B., Monecke, S., Ehricht, R., Coleman, D.C., 2011. Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA, mecI, mecR1, blaZ, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 55, 3765–3773. Srednik, M.E., Archambault, M., Jacques, M., Gentilini, E.R., 2017. Detection of a mecCpositive Staphylococcus saprophyticus from bovine mastitis in Argentina. J. Glob. Antimicrob. Resist. 10, 261–263. Sullivan, M.J., Petty, N.K., Beatson, S.A., 2011. Easyfig: a genome comparison visualizer. Bioinformatics (Oxford, England) 27, 1009–1010. Ubukata, K., Nonoguchi, R., Matsuhashi, M., Konno, M., 1989. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillinresistant S. aureus specific penicillin-binding protein. J. Bacteriol. 171, 2882–2885. Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O., Aarestrup, F.M., Larsen, M.V., 2012. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67, 2640–2644.
2003–2005. McCarthy, A.J., Harrison, E.M., Stanczak-Mrozek, K., Leggett, B., Waller, A., Holmes, M.A., Lloyd, D.H., Lindsay, J.A., Loeffler, A., 2015. Genomic insights into the rapid emergence and evolution of MDR in Staphylococcus pseudintermedius. J. Antimicrob. Chemother. 70, 997–1007. Pantucek, R., Sedlacek, I., Indrakova, A., Vrbovska, V., Maslanova, I., Kovarovic, V., Svec, P., Kralova, S., Kristofova, L., Keklakova, J., Petras, P., Doskar, J., 2018. Staphylococcus edaphicus sp. nov., isolated in Antarctica, Harbors the mecC gene and Genomic Islands with a suspected role in adaptation to extreme environments. Appl. Environ. Microbiol. 84. Paterson, G.K., Harrison, E.M., Holmes, M.A., 2014a. The emergence of mecC methicillinresistant Staphylococcus aureus. Trends Microbiol. 22, 42–47. Paterson, G.K., Morgan, F.J.E., Harrison, E.M., Peacock, S.J., Parkhill, J., Zadoks, R.N., Holmes, M.A., 2014b. Prevalence and properties of mecC methicillin-resistant Staphylococcus aureus (MRSA) in bovine bulk tank milk in Great Britain. J. Antimicrob. Chemother. 69, 598–602. Peacock, S.J., Paterson, G.K., 2015. Mechanisms of methicillin resistance in Staphylococcus aureus. Annu. Rev. Biochem. 84, 577–601. Sekizuka, T., Niwa, H., Kinoshita, Y., Uchida-Fujii, E., Inamine, Y., Hashino, M., Kuroda, M., 2019. Identification of a mecA/mecC-positive MRSA ST1-t127 isolate from a racehorse in Japan. J. Antimicrob. Chemother. https://doi.org/10.1093/jac/dkz459.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Research paper
Genomic epidemiology of methicillin-resistant Staphylococcus sciuri carrying a SCCmec-mecC hybrid element
T
Gavin K. Paterson Easter Bush Pathology, Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, UK
A R T I C LE I N FO
A B S T R A C T
Keywords: mecA mecC SCCmec Staphylococci
The recognition in 2011 of the methicillin resistance determinate mecC among staphylococci has raised many questions over its evolution and epidemiology. While mecC has been best studied in Staphylococcus aureus it has also been described in at least nine other species of staphylococci. In most cases these studies are limited to single isolates. In the widespread animal commensal Staphylococcus sciuri mecC has been described in two isolates and is located within a distinct SCCmec-mecC composite element. In this study, a further 11 mecA/mecC S. sciuri isolated from dairy farms in England and Wales in 2015 and 2016 were genome sequenced and characterised. The results show that two variants of the SCCmec-mecC element are present in S. sciuri, differentiated by different ccr alleles and likely to have arisen by homologous recombination. A phylogeny of sixty genome-sequenced S. sciuri isolates was made using core genome multi-locus sequence typing and reveals a diverse population with the SCCmec-mecC element present in four distinct branches, indicative of four independent acquisitions by S. sciuri. Finally, the study identified the rapid clonal expansion of a mecA/mecC lineage of S. sciuri among dairy farms across a wide geographical area which may contribute to the future dissemination of this methicillin resistance cassette.
1. Introduction Methicillin resistance in staphylococci is typically encoded by mecA carried on the chromosomal mobile element, SCCmec (Peacock and Paterson, 2015). Although methicillin resistance conferred by plasmidencoded mecB and mutation of house-keeping genes have been reported in Staphylococcus aureus, these mechanisms currently appear to be rare (Ba et al., 2014; Ba et al., 2019; Becker et al., 2018). The mecA gene encodes an alternative penicillin-binding protein (PBP2a/PBP2’) which confers resistance to most β-lactam antimicrobials (Fuda et al., 2004; Hartman and Tomasz, 1984; Lim and Strynadka, 2002; Ubukata et al., 1989). While methicillin-resistant S. aureus (MRSA) encoding mecA is a prominent pathogen, mecA is also found in a range of other staphylococci isolated from carrier and diseased humans and animals (Becker et al., 2014b; McCarthy et al., 2015). In 2011 a variant of mecA was described in MRSA from humans (Garcia-Alvarez et al., 2011; Shore et al., 2011) and dairy cattle (Garcia-Alvarez et al., 2011) in Denmark, England and the Republic of Ireland. Sharing 68.7% nucleotide with the archetypal mecA of S. aureus N315, this variant was designated as mecC and is encoded in MRSA within a distinct SCCmec classified as type XI (Ito et al., 2012). mecC sequence divergence from the highly-conserved mecA meant that at the time of its description molecular methods to detect MRSA based on mecA PCR or PBP2a/PBP2’ slide agglutination
with monoclonal antibodies failed to reliably detect mecC MRSA. Subsequently mecC MRSA were found to be present in a wide range of host species and in many countries although at a much lower frequency than mecA MRSA (Becker et al., 2014a; Paterson et al., 2014a). Similarly to mecA, mecC has also been found in range of other staphylococci, namely; Staphylococcus xylosus (Loncaric et al., 2019; MacFadyen et al., 2018b), Staphylococcus pseudoxylosus (Harrison et al., 2013; MacFadyen et al., 2019b), Staphylocococus saprophyticus (Malyszko et al., 2014; Srednik et al., 2017), Staphylococcus caeli (MacFadyen et al., 2019a), Staphylococcus stepanovicii (Semmler et al., 2016), Staphylococcus warneri (Loncaric et al., 2019), Staphylococcus caprae (Loncaric et al., 2019), Staphylococcus edaphicus (Pantucek et al., 2018) and Staphylococcus sciuri (Harrison et al., 2014; Loncaric et al., 2019). In these species, where it has been investigated, mecC has been found either within a SCCmec type XI (Loncaric et al., 2019; MacFadyen et al., 2018b) or in a range of distinct SCCmec-like elements (Harrison et al., 2014; Harrison et al., 2013; Loncaric et al., 2013; MacFadyen et al., 2019a; Pantucek et al., 2018; Semmler et al., 2016). In the case of S. sciuri, mecC has been found encoded within a hybrid SCCmec element comprised of a mecA encoding SCCmec type VII and a mecC region consisting of the class E mec complex (mecI-mecR, mecC-blaZ) (Harrison et al., 2014). This element has been described in only two isolates of S. sciuri, originally in GVGS2 from a caesarean incision wound in a Belgian
E-mail address: [email protected]. https://doi.org/10.1016/j.meegid.2019.104148 Received 25 September 2019; Received in revised form 16 December 2019; Accepted 17 December 2019 Available online 17 December 2019 1567-1348/ © 2019 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 79 (2020) 104148 a Tested using Vitek2 against the following: cefoxitin screen, benylpenicillin, oxacillin, ceftiofur, cefquinome, amikacin, gentamicin, kanamycin, neomycin, enroflaxacin, inducible clindamycin, erythromycin, tilmicosin, tylosin, clindamycin, tetracycline, florfenicol, trimethoprim/sulfamethoxazole. Only resistance phenotypes are shown, susceptible unless stated. b Identified using ResFinder.
ERX3374896 ERX3374901 ERX3374902 ERX3374904 ERX3381835 ERX3381812 ERX3381831 ERX3381834 ERX3382826 ERX3407607 ERX3407627 mecA/mecA1/mecC/blaZ/sal(A) mecA/mecA1/mecC/blaZ sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ str/sal(A)/tet(K) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ str/sal(A) mecA/mecA1/mecC/blaZ sal(A) cefoxitin/benylpenicillin/oxacillin/ceftiofur cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome/clindamycin/ cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome/clindamycin/ cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome/clindamycin/tetracycline/ cefoxitin/benylpenicillin/oxacillin/ ceftiofur/cefquinome cefoxitin/benylpenicillin/oxacillin/ceftiofur cefoxitin/benylpenicillin/oxacillin/ceftiofur/cefquinome Sep-15 Sep-15 Sep-15 Sep-15 Sep-15 Feb-16 Sep-15 Sep-15 Feb-16 Feb-16 Feb-16 5073-5-63 5078-2-20 5079-SM14 5081-3-55 5162-EF3a 5183-EF324a 5202–5-64a 5205-5-69 5454-CC103A 5790-EF260 5810-BC71A
Somerset, England Cornwall, England Devon, England Lancashire, England Lancashire, England Staffordshire, England Devon, England Devon, England Carmarthenshire, Wales Shropshire England Monmouthshire, Wales
Nucleotide accession Acquired resistance genesb Resistance phenotypea Location Date of isolation Isolate
Table 1 Characteristics of study isolates.
G.K. Paterson
Blue cow in England (Harrison et al., 2014) and LP600 from an alpaca in Austria (Loncaric et al., 2013). In addition the SCCmec-mecC element has been reported in a single S. aureus isolate from a racehorse wound (Sekizuka et al., 2019). In each case the SCCmec-mecC hybrid element was highly conserved with the archetypal version reported originally in GVGS2 (Harrison et al., 2014). To examine the epidemiology and genomics of mecC in S. sciuri this study has isolated and sequenced the genomes of a further 11 mecA/mecC-positive methicillin-resistant S. sciuri, from dairy bulk tank milk in England and Wales.
2. Materials and methods 2.1. Isolation and identification of mecA/C S. sciuri Bulk tank milk from dairy farms in England and Wales was sampled for methicillin-resistant staphylococci via salt broth enrichment and plating on MRSA Brilliance agar (Oxoid, Basingstoke, UK) as described previously (Paterson et al., 2014b).
2.2. Whole-genome sequencing and analysis Isolates of interest were genome sequenced by Microbes NG (University of Birmingham, UK) using Illumina HiSeq technology with 2 × 250 bp paired-end reads as described previously (MacFadyen et al., 2018a). In all cases the SCCmec-mecC hybrid was assembled on a single contig. Phylogenetic relationships among S. sciuri isolates were assessed with SeqSphere+ software version 4.1.9 (Ridom, Münster, Germany) (Jünemann et al., 2013) using a core genome multilocus sequence typing (cgMLST) scheme based on all 60 isolates in this study with the S. sciuri type strain NCTC12103T (NZ_LS483305.1) used as the reference genome. Reference genome filters were a minimum length > 50 bases, a start and stop codon at the beginning and end of the gene, a homologous gene filter (requiring no multiple copies of a gene with BLAST overlap > 100 bp, identity > 90.0%), a gene overlap filter (requiring no overlap with other genes of more than four bases) and an exclude sequences filter (requiring no BLAST hit with overlap > 100 bp, identity > 90.0% in excluded sequences). The query genome BLAST search required a BLAST hit with overlap = 100%, identity > 90.0% in every query genome using BLAST options: word size = 11, mismatch penalty = −1, match reward = 1, gap open costs = 5, gap extension costs = 2. A query genome stop codon percentage filter was applied requiring a single stop codon at end of gene in > 80%query genomes. Omitting missing values from isolates this resulted in a scheme for this study of 1278 gene targets. The resultant tree was annotated in the Interactive Tree of Life (iTOL) (Letunic and Bork, 2019). Comparator S. sciuri isolates were those with assembled genomes available from NBCI, details and nucleotide accession are shown in Supplementary Information. Acquired resistance genes were identified using ResFinder3.1 (Zankari et al., 2012) and schematic comparison of the SCCmecmecC element performed using EasyFig (Sullivan et al., 2011).
2.3. Antimicrobial susceptibility testing This was performed using Vitek2 (bioMéurieux, Basingstoke, UK) following the manufacturer's instructions and using the AST-GP79 Gram-positive livestock card. The antimicrobials tested were: cefoxitin screen, benylpenicillin, oxacillin, ceftiofur, cefquinome, amikacin, gentamicin, kanamycin, neomycin, enroflaxacin, inducible clindamycin, erythromycin, tilmicosin, tylosin, clindamycin, tetracycline, florfenicol, trimethoprim/sulfamethoxazole with interpretation performed using The Clinical and Laboratory Standards Institute criterion.
2
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3. Results
present only in the SCCmec elements of S. pseudintermedius KM241 (AM904731.1) and S. sciuri TXG-24 (KX774481.1) both of which are distinct to that in SM14 and which encode only mecA and not mecC.
3.1. Isolation of methicillin-resistant mecA/C S. sciuri A survey of 363 dairy farms in England and Wales in September 2015 and February 2016 led to the isolation of methicillin-resistant staphylococci from the bulk tank milk of 18 of these (4.96%) (unpublished data). Three of these 18 isolates were methicillin-resistant S. sciuri encoding both mecA and mecC and further convenience sampling of bulk tank milk led to the isolation of an additional 8 such isolates all from different dairy farms (Table 1).
3.3. Phylogenetic analysis of mecA/C S. sciuri To assess the phylogenetic relationships among S. sciuri isolates encoding mecA/mecC and with other S. sciuri isolates a cgMLST scheme with 1278 gene targets was generated. This comprised the 11 S. sciuri mecA/C isolates described here, the two previously described S. sciuri mecA/C isolates GVGS2 and LP600 along with the 47 further isolates whose assembled genomes were available on public databases (Supplementary Information Table 1). The host source of these 60 isolates are: cattle (42 isolates), Asian Rice (7), Human (4) and one isolate each from Pig, African Social Spider, Mouse, Alpaca, Grey Squirrel, Virginia Opossum and source unknown. Isolates came from 11 countries: Canada (28 isolates), United Kingdom (13), India (7), USA (3), China (2), Czech Republic (2) and singles from Australia, Austria, Chile, Denmark, and Sweden. All of the isolates encoded the mecA1 allotype found ubiquitously in S. sciuri but none of the isolates from the public database encoded both mecA and mecC. Two human isolates encoded mecA without mecC (Fig. 2). In each case these isolates are distantly related to those with mecA/mecC and encode mecA within a SCCmec with no resemblance to the SCCmec-mecC element (data not shown). The phylogeny shows a diverse population of S. sciuri and that the SCCmec-mecC element is present in four distinct branches each likely indicating an independent acquisition event (Fig. 2). Of particular note are the cluster of eight highly-related isolates which correspond to the eight referred to above which encode identical SCCmec-mecC elements. The mean pairwise difference among these 8 isolates is only 6.4 gene targets, range 4–11. The farms from which these isolates came were located in six different counties with a mean pairwise distance between farms of 95 miles, range 9–233 miles. The closest distance being any two positive farms is 9 miles and the furthest is 96 miles.
3.2. Analysis of SCCmec in mecA/C S. sciuri Genome sequencing of these 11 mecA/C S. sciuri revealed that they all carried a SCCmec-mecC hybrid element in the orfX/rlmH region which was highly conserved between the isolates and with similarity to that previously reported in GVGS2 (Harrison et al., 2014). For comparative purposes this region was defined from the start codon of orfX/ rlmH to the inverted repeat of the attL2 site. In each of the 11 bulk tank milk isolates this region was 43,723 bp in length and was 100% identical in 8 of the isolates. Two isolates (CC103a and 3–55) shared identical sequences to each other in this region which differed from those aforementioned 8 by 15 SNPs, the final isolate (EF3a) shared those 15 SNPs plus two further unique SNPs. Taking SM14 as a representative of these bulk tank milk isolates the SCCmec-mecC hybrid element was compared with that region previously reported in S. sciuri GVGS2 which was isolated from Belgium Blue cattle in England (Harrison et al., 2014). This revealed the elements are closely-related sharing a high degree of synteny and nucleotide conservation, Fig. 1. However, there were notable differences around the ccr genes. No mobile genetic elements are apparent in this region suggesting this rearrangement has occurred through homologous recombination. Consistent with this suggestion is the finding that the 5′ portion of a hypothetical protein gene in this region is conserved between the two isolates but has a highly divergent 3′ sequence and may therefore have been a site for a recombination event within the SCCmec-mecC element. The SCCmec-mecC element of GVGS2 carries the recombinase ccrA1B5 genes whereas SM14 and the other 10 bulk tank milk isolates possess ccrA3B3. Furthermore an ORF downstream of the ccr genes in GVGS2 is absent in SM14 with an ORF present upstream of the ccr genes in SM14 absent in GVGS2. The GVGS2 gene encodes a 244 amino acid protein belonging to the short-chain dehydrogenases/reductases superfamily (Conserved Domain Database accession: cl25409) and a standard nucleotide BLAST search shows it to be present in a small number of staphylococcal isolates belonging to S. sciuri, S. pseudintermedius, S. simulans, S. aureus and S. haemolyticus (data not shown). The SM14 gene in question encodes a 96 amino acid protein belonging to the NeuB Nacetylneuraminate synthase superfamily (Conserved Domain Database accession: cl30262). A standard nucleotide BLAST search shows it to be
3.4. Phenotypic resistance and other antimicrobial resistance determinates Consistent with the presence of mecA, mecC and blaZ, all 11 isolates were resistant to the β-lactms penicillin, cefoxitin, ceftiofur and oxacillin with all but 2 was resistant to cefquinome (Table 1). All isolates encoded sal(A) which confer moderate resistance to lincosamides and high-level resistance to streptogramin A in S. sciuri (Hot et al., 2014). However, only three were resistant to clindamycin as assessed by Vitek2 (MIC ≥4). The specintomycin resistance gene str being present in 6 isolates and a single tetracycline resistant isolate encoded tet(K). No other phenotypic resistance was observed among the antimicrobials tested and no other antimicrobial resistance determinates were noted.
Fig. 1. Schematic comparison of the SCCmec-mecC elements in S. sciuri isolates GVGS2 (accession HG515014) and SM14 (representative of the dairy bulk tank milk isolates described in this study). Selected key genes are highlighted. Regions of homology are represented by bands connecting the two sequences, with the percentage identity key shown on the bottom right. Red denotes normal sequence alignment and blue denotes inverted sequence alignment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3
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Fig. 2. Phylogenetic relationships among an international collection of whole-genome sequenced S. sciuri isolates. Neighbour-joining phylogeny generated using a core genome multilocus sequence typing scheme produced from all 60 isolates in this study and consisting of 1278 gene targets present in all isolates. Isolates positive for mecA and mecC are highlighted in yellow, those positive for mecA are highlighted in blue and the S. sciuri type strain is highlighted in orange. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion
present in the second isolate LP600 (Loncaric et al., 2019). To further knowledge of this SCCmec-mecC element, this study has genome sequenced 11 dairy cattle isolates of mecA/mecC S. sciuri. Genomic analysis reveals that two variants of this SCCmec-mecC element are present among S. sciuri isolates. The prototype described in GVGS2 (Harrison et al., 2014) and a second among the dairy isolates in this study. While highly conserved with the GVGS2 element this second variant is distinguished most prominently by the carriage of different ccr alleles to those in GVGS2. Phylogenetic analysis of a global collection of 60 S.
The recognition of the mecC methicillin resistant determinate in staphylococci has raised many questions regarding its evolution and epidemiology. In the widespread animal commensal S. sciuri it is encoded within a composite SCCmec-mecC element, but this has only been described in two S. sciuri isolates to date. It has been fully characterised in one isolate, the assembled element in GVGS2 (Harrison et al., 2014) (Fig. 1) with read mapping indicating a highly similar element is 4
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References
sciuri sequenced isolates indicates the independent acquisition of this SCCmec-mecC element on four occasions by this population of S. sciuri. Of particular note is the apparent clonal expansion of 8 highly-related isolates encoding an identical SCCmec-mecC element. These were each present on different farms up to 233 miles apart and their close phylogenetic relationship would strongly suggest transmission events between the farms or from a common source. The very limited genetic diversity among these isolates may further indicate a relatively recent expansion and it will be of interest to monitor the epidemiology of this clone in future. Further spread may be problematic through the dissemination of methicillin resistance and/or should this clone has a propensity for causing infections. While the isolates in this study were isolated from bulk tank milk, no link to bovine mastitis can be apportioned here. However, S. sciuri including methicillin-resistant isolates can cause bovine mastitis (Khazandi et al., 2018) and so veterinarians and veterinary microbiology laboratories need to be conscious of the possible presence of mecA/mecC isolates from bovine mastitis and other infections. Indeed, the original mecA/mecC isolate GVGS2 came from a Caesarean incision wound infection (Harrison et al., 2014). Other resistance genes were uncommon among the 11 dairy mecA/ mecC isolates. Consistent with the report of Hot et al. (2014) all isolates carried sal(A).
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5. Conclusions This work expands our understanding of the epidemiology of mecC among staphylococci. Specially, a novel variant of the S. sciuri SCCmecmecC composite element is described and core genome phylogenetic analysis indicates that the mecA/mecC cassette has been acquired on at least four occasions by this staphylococcal species. The clonal expansion of a lineage of mecA/mecC S. sciuri among several dairy farms across a wide geographical area may be of future concern through the spread of resistance or if this is a virulent clone. Acknowledgements Genome sequencing was provided by MicrobesNG (http://www. microbesng.uk), which is supported by the BBSRC (grant number BB/ L024209/1). We gratefully thank National Milk Laboratories Ltd. for their assistance in providing bulk tank milk samples. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author contributions Gavin K. Paterson: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Writing - Review & Editing, Visualization, Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2019.104148. 5
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