Methicillin-Resistant Macrococcus bohemicus Encoding a Divergent SCCmecB Element

A methicillin-resistant Macrococcus isolate from canine otitis, H889678/16/1, was whole-genome sequenced using HiSeq technology to identify the species, antimicrobial resistance determinates and their genomic context. H889678/16/1 belonged to the newly described species Macrococcus bohemicus. It encoded mecB within a novel SCCmec element most similar to that of Macrococcus canis KM45013T. This SCCmecH889678/16/1 element also encoded blaZm and fusC, but no other resistance determinates were found in the H889678/16/1 genome. The ccrA and ccrB recombinase genes within SCCmecH889678/16/1 were distinct from those previously described in staphylococci and macrococci and therefore designated here as ccrAm3 and ccrBm3. Our study represents, to the best of our knowledge, the first description of mecB being encoded by M. bohemicus and of methicillin resistance in this species. Furthermore, the SCCmec described here is highly dissimilar to other such elements and encodes novel ccr genes. Our report demonstrates a wider distribution of mecB among Macrococcus species and expands the genomic context in which mecB may be found. The potential for dissemination of mec genes from Macrococcus to related but more pathogenic Staphylococcus species highlights the need to understand the epidemiology of these genes in macrococci.


Introduction
The genus Macrococcus is closely related to Staphylococcus and consists of eleven species typically found as commensals in a range of animal hosts [1]. However, there is a growing appreciation that some macrococci may also act as opportunistic pathogens in different animals. For instance, M. caseolyticus has been isolated from mastitis in dairy cattle [2,3], canine dermatitis [4], canine otitis [2], an outbreak high-mortality systemic infection in broiler chickens [5], ovine abscesses [6] and cases of embryo mortality in greater white-fronted geese (Anser albifrons) [7]. Macrococcus canis has also been isolated from a range of canine infections [4] and a small number of isolates of different Macrococcus species have come from human clinical samples, suggesting a potential role, albeit infrequently, in human infections as well [8].
mecB has been found to be encoded by various genetic elements in Macrococcus; different SCCmec elements [11,13], different plasmids [11,14,16] and different ΨSCC elements [8,14]. Importantly, Antibiotics 2020, 9, 590 2 of 8 there has been a single report of a human isolate of Staphylococcus aureus encoding mecB [17] on a plasmid almost identical to a M. canis plasmid also encoding mecB [14]. This raises the strong possibility of the exchange of methicillin resistance determinates between these two genera and highlights the need to better understand the epidemiology and genomics of mec genes among Macrococcus.
Macrococcus bohemicus was first described in 2018 on the basis of a single isolate cultured from a human traumatic knee wound sample collected in 2003 in the Czech Republic [8]. Subsequently, a second M. bohemicus isolate coming from bovine milk in the Republic of Ireland and collected in 2017 has been described [18]. Both isolates have been genome sequenced.
To the best of our knowledge, no mec gene or methicillin resistance has been described in M. bohemicus, and herein we describe the first example of such, a canine otitis isolate H889678/16/1 encoding mecB within a distant SCCmec element and carrying novel ccrA and ccrB alleles, designated here as ccrAm3 and ccrBm3.

Results and Discussion
2.1. Isolation and Whole-Genome Sequencing of Methicillin-Resistant M. bohemicus H889678/16/1 H889678/16/1 was isolated from mixed growth cultured from a canine otitis sample collected from a cocker spaniel in Scotland in 2016. Also isolated were an Enterobacter sp., yeast (likely Malassezia pachydermatis) and Aerococcus viridans. H889678/16/1 was identified phenotypically as a presumed Macrococcus sp. and considered to be methicillin resistant on the basis of resistance to oxacillin when tested by Vitek2. H889678/16/1 was also resistant to benzylpenicillin and fusidic acid but susceptible to all the other antimicrobials tested. H889678/16/1 was whole-genome sequenced using HiSeq technology to resolve its identity to the species level and to determine the genetic basis for methicillin resistance. The resultant assembled draft genome consisted of 49 contigs totalling 2,497,285 bp in length, with a GC% content of 33.89%. The average genome coverage was 42.9-fold. H889678/16/1 was identified as belonging to M. bohemicus using the Type Strain Genome Server [19] and showed a dDDH value of 83.3 against M. bohemicus type strain CCM 7100.

M. bohemicus H889678/16/1 Encodes mecB within a Novel SCCmec Element
ResFinder analysis of the H889678/16/1 genome showed that it encoded mecB and fusC. Further analysis showed that mecB was within a mec gene complex with blaZm, but no other antimicrobial resistance determinates were apparent in the genome. All three genes, mecB, blaZ m and fusc, were located on a single contig 265 kbp in size (JACEGF000000003) and encoded within a SCCmec element in the orfX/rlmH region. The insertion of the element into the orfX/rlmH region has to date been a reliable indication of a chromosomal location for SCCmec elements. Additional evidence for this being the case in H889678/16/1 are the large size of the SCCmec-containing contig, the absence of any plasmid features in this contig as detected by PlasmidFinder and the presence of numerous housekeeping genes on this contig, including those likely to be essential for viability, such as gyrA and gyrB. This SCCmec element, designated as SCCmec H889678/16/1 , was most similar to, but distinct from, the mecB-encoding SCCmec of M. canis KM45013 T (Figure 1) [13]. SCCmec H889678/16/1 is also distinct from the ΨSCC element found in the M. bohemicus type strain CCM7100 that lacks a mec gene complex and ccr genes ( Figure 1) [8]. The only other described M. bohemicus isolate, DPC 7215 [18], also lacks any mec gene and possesses an orfX/rlmH region distinct from that of H889678/16/1 (data not shown). SCCmec H889678/16/1 is 57,612 bp in size, as defined by the length from the two outermost direct repeats inclusive ( Figure 1). The mec gene complex of mecI, mecR1, mecB and blaZ m in SCCmec H889678/16/1 was highly conserved with those of SCCmec KM45013 , with each gene pair sharing 98-99% nucleotide identity. The other large region conserved between these two elements is a series of eight genes located near the ccr genes which encode the DNA repair protein RadC, a helix-turn-helix domain protein and six hypothetical proteins. This region of similarity extends into portions of the two flanking genes and, in the apparent absence of adjacent mobile genetic elements, may indicate their acquisition by homologous recombination. SCCmec H889678/16/1 also contained blocks of similarity with the ΨSCC element found in the M. bohemicus CCM7100 T , indicating a mosaic structure likely arising through multiple horizontal genetic transfer events.

SCCmec H889678/16/1 Encodes Novel Recombinase Genes ccrA3m and ccrB3m
A notable feature of the SCCmec H889678/16/1 is the relative lack of similarity shown by the ccr genes with those of SCCmec KM45013 (Figure 1), with the ccr genes being responsible for the excision and integration of SCCmec elements in and out of the genome. Indeed, SCCmec H889678/16/1 ccr genes share limited nucleotide identity with known Macrococcus and Staphylococcus ccr genes, 43.5-63.0% in the case of ccrB and 41.5-54.4% in the case of ccrA. Following precedent [11,13], we propose the designation of the SCCmec H889678/16/1 ccr genes as ccrAm3 and ccrBm3. A phylogenetic analysis of ccr genes highlights the distinctness of ccrAm3 and ccrBm3 (Figure 2). Whilst ccrAm3 and ccrBm3 belong to their respective Macrococcus ccr gene branches, they are distant to these counterparts, with ccrAm3 and ccrBm3 closest to the ancestral forms of the macrococcal ccr genes.
In conclusion, we report the first example, to the best of our knowledge, of methicillin resistance and mecB in the newly described species M. bohemicus. This is only the third description of a M. bohemicus isolate and appears to be the first isolation from a dog. The mecB gene in this isolate is encoded within a distinct SCCmec element containing novel ccr alleles. This expands our knowledge on the distribution and genomic context of methicillin resistance determinates among Macrococcus which themselves are opportunistic pathogens, but which may also act as a genetic reservoir for the more pathogenic and related Staphylococcus.  limited nucleotide identity with known Macrococcus and Staphylococcus ccr genes, 43.5-63.0% in the case of ccrB and 41.5-54.4% in the case of ccrA. Following precedent [11,13], we propose the designation of the SCCmecH889678/16/1 ccr genes as ccrAm3 and ccrBm3. A phylogenetic analysis of ccr genes highlights the distinctness of ccrAm3 and ccrBm3 (Figure 2). Whilst ccrAm3 and ccrBm3 belong to their respective Macrococcus ccr gene branches, they are distant to these counterparts, with ccrAm3 and ccrBm3 closest to the ancestral forms of the macrococcal ccr genes.

Whole-Genome Sequencing
Whole-genome sequencing was performed by Microbes NG (University of Birmingham, Birmingham UK) using Illumina technology with 2 × 250 bp paired-end reads. Genomic DNA was purified with solid-phase reversible immobilization beads, and libraries were prepared using Nextera XT Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's protocol with the following modifications: two nanograms of DNA instead of one were used as input, and PCR elongation time was increased to 1 min from 30 seconds. Reads were trimmed using Trimmomatic version 0.30 [20], using a sliding window quality cut-off of 15. Genome assembly was done de novo using SPAdes, version 3.7, with default parameters for 250 bp Illumina reads [21] and annotated by the NCBI Prokaryotic Genome Annotation Pipeline [22].

Genome Analysis
Genome-based identification was done using the Type Strain Genome Server [17] (https://tygs. dsmz.de/). Acquired resistance genes were identified using ResFinder-3.1 [23] employing the thresholds of 60% for percentage identity and minimum length of 60%. Visual inspection and formatting of the genome for Figure 1 was performed using Artemis 17.0.1 [24]. Schematic comparison of mecB regions was performed using EasyFig 2.2.5 [25]. Plasmid features were searched for using PlasmidFinder 2.0 [26] with the thresholds of 60% for percentage identity and minimum length of 60%. The phylogenetic relationship of ccr genes was assessed in MEGA X [27] using a single representative of each type (nucleotide accession numbers provided in Figure 2), aligning the nucleotide sequences with MUSCLE and producing a maximum-likelihood tree using a general time-reversible (GTR) model. The final tree was produced using the Interactive Tree of Life (iTOL) [28].

Nucleotide Accession Numbers
The whole-genome sequencing reads and annotated assembly of M. bohemicus H889678/16/1 are available under the GenBank accession numbers SRR12266687 and JACEGF000000000, respectively.