Macrococci are usually found as commensals on the skin and mucosa of animals and have been isolated from mammal-derived fermented foods; however, they can also act as opportunistic pathogens. Here, we used whole-genome sequencing, comparative genomics, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize Macrococcus sp. strains isolated from livestock and human-related specimens. Based on the results of polyphasic taxonomy, we propose the species Macrococcus psychrotolerans sp. nov. (type strain NRL/St 95/376T = CCM 8659T = DSM 111350T) belonging to the Macrococcus caseolyticus phylogenetic clade. It grows at 4°C, and the core genome of the isolates contains suspected genes contributing to low-temperature tolerance. Variable genetic elements include prophages, chromosomal islands, a composite staphylococcal cassette chromosome island, and many plasmids that affect the overall genome expansion and adaptation to specific ecological settings of the studied isolates. Large plasmids carrying the methicillin resistance gene mecB were identified in M. psychrotolerans sp. nov. strains and confirmed as self-transmissible to Staphylococcus aureus in vitro. In addition to plasmids with circular topology, a 150-kb-long linear plasmid with 14.1-kb-long inverted terminal repeats, harboring many IS elements and putative genes for a type IV secretion system was revealed. The described strains were isolated from human clinical material, food-producing animals, meat, and a wooden cheese board and have the potential to proliferate at refrigerator temperatures. Their presence in the food chain and human infections indicates that attention needs to be paid to this potential novel opportunistic pathogen.IMPORTANCEThe study offers insights into the phenotypic and genomic features of a novel species of the genus Macrococcus that occurs in livestock, food, and humans. The large number of diverse mobile genetic elements contributes to the adaptation of macrococci to various environments. The ability of the described microorganisms to grow at refrigerator temperatures, enabled by genes that are predicted to contribute to low-temperature tolerance, raises food safety concerns. Confirmed in vitro conjugative transfer of plasmid-borne mecB gene to S. aureus poses a significant risk of spread of broad β-lactam resistance. In addition, the intergeneric plasmid transfer to S. aureus is indicative of horizontal gene transfer events that may be more frequent than generally accepted. Determining a complete sequence and gene content of linear megaplasmid with exceptional topology for the Staphylococcaceae family suggests its possible role in shuttling adaptive traits through an exchange of genetic information.

• Keywords
• Gram-positive cocci, cephalosporin resistance, cold temperature tolerance, conjugation, food safety, linear plasmid, methicillin resistance,
• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Bacterial Proteins * genetics   metabolism   MeSH
• beta-Lactam Resistance * genetics   MeSH
• Livestock microbiology   MeSH
• Enterococcaceae * genetics   isolation & purification   drug effects   MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Humans MeSH
• Plasmids MeSH
• Gene Transfer, Horizontal MeSH
• Whole Genome Sequencing MeSH
• Staphylococcal Infections microbiology   MeSH
• Staphylococcus aureus * genetics   drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents * MeSH
• Bacterial Proteins * MeSH

Antibiotic-resistant strains of Staphylococcus aureus pose a significant threat in healthcare, demanding urgent therapeutic solutions. Combining bacteriophages with conventional antibiotics, an innovative approach termed phage-antibiotic synergy, presents a promising treatment avenue. However, to enable new treatment strategies, there is a pressing need for methods to assess their efficacy reliably and rapidly. Here, we introduce a novel approach for real-time monitoring of pathogen lysis dynamics employing the piezoelectric quartz crystal microbalance (QCM) with dissipation (QCM-D) technique. The sensor, a QCM chip modified with the bacterium S. aureus RN4220 ΔtarM, was utilized to monitor the activity of the enzyme lysostaphin and the phage P68 as model lytic agents. Unlike conventional QCM solely measuring resonance frequency changes, our study demonstrates that dissipation monitoring enables differentiation of bacterial growth and lysis caused by cell-attached lytic agents. Compared to reference turbidimetry measurements, our results reveal distinct alterations in the growth curve of the bacteria adhered to the sensor, characterized by a delayed lag phase. Furthermore, the dissipation signal analysis facilitated the precise real-time monitoring of phage-mediated lysis. Finally, the QCM-D biosensor was employed to evaluate the synergistic effect of subinhibitory concentrations of the antibiotic amoxicillin with the bacteriophage P68, enabling monitoring of the lysis of P68-resistant wild-type strain S. aureus RN4220. Our findings suggest that this synergy also impedes the formation of bacterial aggregates, the precursors of biofilm formation. Overall, this method brings new insights into phage-antibiotic synergy, underpinning it as a promising strategy against antibiotic-resistant bacterial strains with broad implications for treatment and prevention.

• Keywords
• Staphylococcus aureus, Antimicrobial treatment, Multidrug-resistant bacteria, Phage therapy, Phage-antibiotic synergy, Piezoelectric biosensor,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacteriophages physiology   MeSH
• Bacteriolysis * drug effects   MeSH
• Biosensing Techniques * methods   instrumentation   MeSH
• Lysostaphin pharmacology   MeSH
• Quartz Crystal Microbalance Techniques * methods   MeSH
• Staphylococcus aureus * drug effects   virology   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Lysostaphin MeSH

Novel species of coagulase-negative staphylococci, which could serve as reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens from the genus Staphylococcus, are recognized in human and animal specimens due to advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St 16/872T = CCM 9024T = LMG 31872T = DSM 111349T). The genomic analysis revealed numerous variable genomic elements, including staphylococcal cassette chromosome (SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbCIccrDE integrated into the ribosomal protein L7 serine acetyltransferase gene rimL. SbCIccrDE has a cassette chromosome recombinase (ccr) gene complex with a typical structure found in SCCs. Based on nucleotide and amino acid identity to other known ccr genes and the distinct integration site that differs from the canonical methyltransferase gene rlmH exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative genomic analysis of SbCIccrDE with related islands shows that they can accumulate virulence and antimicrobial resistance factors creating novel resistance elements, which reflects the evolution of SCC. The spread of these resistance islands into established pathogens such as Staphylococcus aureus would pose a great threat to the healthcare system. IMPORTANCE The coagulase-negative staphylococci are important opportunistic human pathogens, which cause bloodstream and foreign body infections, mainly in immunocompromised patients. The mobile elements, primarily the staphylococcal cassette chromosome mec, which confers resistance to methicillin, are the key to the successful dissemination of staphylococci into healthcare and community settings. Here, we present a novel species of the Staphylococcus genus isolated from human clinical material. The detailed analysis of its genome revealed a previously undescribed genomic island, which is closely related to the staphylococcal cassette chromosome and has the potential to accumulate and spread virulence and resistance determinants. The island harbors a set of conserved genes required for its mobilization, which we recognized as novel cassette chromosome recombinase genes ccrDE. Similar islands were revealed not only in the genomes of coagulase-negative staphylococci but also in S. aureus. The comparative genomic study contributes substantially to the understanding of the evolution and pathogenesis of staphylococci.

• Keywords
• cassette chromosome recombinase, coagulase-negative staphylococci, comparative genomics, genomic islands, gram-positive pathogens, mobile genetic elements, phylogenetic analysis, polyphasic taxonomy,
• Publication type
• Journal Article MeSH

Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

• Keywords
• Kayvirus, Staphylococcus aureus, abortive infection, bacteriophage evolution, bacteriophage therapy, bacteriophages, cell death, lysogeny, phage resistance, phage therapy,
• MeSH
• Humans MeSH
• Lysogeny MeSH
• Membrane Proteins genetics   MeSH
• Prophages * genetics   MeSH
• Staphylococcus Phages genetics   MeSH
• Staphylococcal Infections * microbiology   MeSH
• Staphylococcus aureus genetics   MeSH
• Staphylococcus MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH

Staphylococci from the Staphylococcus intermedius-Staphylococcus hyicus species group include numerous animal pathogens and are an important reservoir of virulence and antimicrobial resistance determinants. Due to their pathogenic potential, they are possible causative agents of zoonoses in humans; therefore, it is important to address the properties of these strains. Here we used a polyphasic taxonomic approach to characterize the coagulase-negative staphylococcal strain NRL/St 03/464T, isolated from the nostrils of a healthy laboratory rat during a microbiological screening of laboratory animals. The 16S rRNA sequence, MALDI-TOF mass spectrometry and positive urea hydrolysis and beta-glucuronidase tests clearly distinguished it from closely related Staphylococcus spp. All analyses have consistently shown that the closest relative is Staphylococcus chromogenes; however, values of digital DNA-DNA hybridization <35.3% and an average nucleotide identity <81.4% confirmed that the analyzed strain is a distinct Staphylococcus species. Whole-genome sequencing and expert annotation of the genome revealed the presence of novel variable genetic elements, including two plasmids named pSR9025A and pSR9025B, prophages, genomic islands and a composite transposon that may confer selective advantages to other bacteria and enhance their survival. Based on phenotypic, phylogenetic and genomic data obtained in this study, the strain NRL/St 03/464T (= CCM 9025T = LMG 31873T = DSM 111348T) represents a novel species with the suggested name Staphylococcus ratti sp. nov.

• Keywords
• Hyicus-Intermedius species group, Staphylococcus, genomic island, laboratory rat, taxonomy, variable genetic element, whole genome sequencing,
• Publication type
• Journal Article MeSH