The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.

Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources, stone fragments and sandy soil in James Ross Island, Antarctica. Here we describe properties of novel species of the genus that has near identical 16S rRNA gene sequence to However, compared to and the next closest relatives, the new species demonstrates considerable phylogenetic distance at whole genome level, average nucleotide identity<85 %, and inferred DNA-DNA hybridization <30%. It forms a separate branch in phylogenetic clade confirmed by multilocus sequence analysis of six housekeeping genes, ,, ,, and MALDI-TOF MS and key biochemical characteristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbours. In contrast to subsp. , strains were pyrrolidonyl arylamidase- and β-glucuronidase-positive, β -galactosidase-negative, nitrate was reduced and acid produced aerobically from d-mannose. Whole genome sequencing of 2.75 Mb large chromosome revealed the presence of a number of mobile genetic elements including the 27-kb-long pseudo staphylococcus cassette chromosome (ψSCC) harboring gene, two composite phage-inducible chromosomal islands probably essential to adaptation to extreme environment, and one complete and one defective prophage. Both strains are resistant to penicillin G, ampicillin, ceftazidin, methicillin, cefoxitin, and fosfomycin. We hypothesize that antibiotic resistance might represent an evolutionary advantage against beta-lactams producers, which are common in a polar environment. Based on these results, a novel species of the genus is described and named sp. nov. The type strain is P5085 (=CCM 8730 = DSM 104441). The description of sp. nov. enables the comparison of multidrug-resistant staphylococci from human and veterinary sources evolved in the globalized world to their geographically distant relative from the extreme Antarctic environment. Although this new species was not exposed to the pressure of antibiotic treatment in human or veterinary practice, mobile genetic elements carrying antimicrobial resistance genes were found in the genome. The presented genomic characteristics elucidate the evolutionary relationships in the genus with a special focus on antimicrobial resistance, pathogenicity and survival traits. Genes encoded on mobile genetic elements were arranged in unique combinations but retained conserved locations for the integration of mobile genetic elements. These findings point to enormous plasticity of the staphylococcal pangenome shaped by horizontal gene transfer. Thus can act not only as a reservoir of antibiotic resistance in a natural environment, but also as a mediator for the spread and evolution of resistance genes.

The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus , zebrafish Danio reiro , and the fruit fly Drosophila melanogaster . However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus . We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G 0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.