Staphylococcus belongs to the Gram-positive low G + C content group of the Firmicutes division of bacteria. Staphylococcus aureus is an important human and veterinary pathogen that causes a broad spectrum of diseases, and has developed important multidrug resistant forms such as methicillin-resistant S. aureus (MRSA). Staphylococcus simiae was isolated from South American squirrel monkeys in 2000, and is a coagulase-negative bacterium, closely related, and possibly the sister group, to S. aureus. Comparative genomic analyses of closely related bacteria with different phenotypes can provide information relevant to understanding adaptation to host environment and mechanisms of pathogenicity.

The viscoelastic properties of mono-microbial biofilms produced by ocular and reference staphylococcal strains were investigated. The microorganisms were characterized for their haemolytic activity and agr typing and the biofilms, grown on stainless steel surface under static conditions, were analysed by Confocal Laser Scanning Microscopy. Static and dynamic rheometric tests were carried out to determine the steady-flow viscosity and the elastic and viscous moduli. The analysed biofilms showed the typical time-dependent behaviour of viscoelastic materials with considerable elasticity and mechanical stability except for Staphylococcus aureus ATCC 29213 biofilm which showed a very fragile structure. In particular, S. aureus 6ME biofilm was more compact than other staphylococcal biofilms studied with a yield stress ranging between 2 and 3Pa. The data obtained in this work could represent a starting point for developing new therapeutic strategies against biofilm-associated infections, such as improving the drug effect by associating an antimicrobial agent with a biofilm viscoelasticity modifier.

The opportunistic pathogens Staphylococcus aureus and Staphylococcus epidermidis represent major causes of severe nosocomial infection, and are associated with high levels of mortality and morbidity worldwide. These species are both common commensals on the human skin and in the nasal pharynx, but are genetically distinct, differing at 24% average nucleotide divergence in 1,478 core genes. To better understand the genome dynamics of these ecologically similar staphylococcal species, we carried out a comparative analysis of 324 S. aureus and S. epidermidis genomes, including 83 novel S. epidermidis sequences. A reference pan-genome approach and whole genome multilocus-sequence typing revealed that around half of the genome was shared between the species. Based on a BratNextGen analysis, homologous recombination was found to have impacted on 40% of the core genes in S. epidermidis, but on only 24% of the core genes in S. aureus. Homologous recombination between the species is rare, with a maximum of nine gene alleles shared between any two S. epidermidis and S. aureus isolates. In contrast, there was considerable interspecies admixture of mobile elements, in particular genes associated with the SaPIn1 pathogenicity island, metal detoxification, and the methicillin-resistance island SCCmec. Our data and analysis provide a context for considering the nature of recombinational boundaries between S. aureus and S. epidermidis and, the selective forces that influence realized recombination between these species.

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.

Staphylococcus schleiferi is an opportunistic pathogen in humans and dogs. Recent taxonomic reassignment of its subspecies (S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans) into two separate species (S. schleiferi and S. coagulans) lacks supporting data for diagnostic implications and clinical relevance. We aimed to confirm the reclassification of S. schleiferi by using genomic and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data for a large set of isolates from humans and animals to investigate their molecular epidemiology and clinical relevance. Routine MALDI-TOF analysis and Illumina sequencing were performed on 165 S. schleiferi isolates from the Netherlands. With 33 publicly available genomes, the study included 198 genomes from 149 dogs, 34 humans, and 15 other sources. The Type Strain Genome Server was used to identify species in the genomes, and the MALDI-TOF MS database was extended to improve species differentiation. MALDI-TOF did not discriminate between S. schleiferi and S. coagulans. Genome phylogeny distinguished the two species in two monophyletic clusters. S. schleiferi isolates originated from humans, while S. coagulans isolates were found in animals and three human isolates clustering with the animal isolates. The sialidase B gene (nanB) was a unique marker gene for S. schleiferi, whereas the chrA gene was exclusive for S. coagulans. The mecA gene was exclusively detected in S. coagulans, as were the lnu(A), blaZ, erm(B/C), tet(O/M), and aac(6')-aph(2'') genes. The MALDI-TOF database extension did not improve differentiation between the two species. Even though our whole-genome sequencing-based approach showed clear differentiation between these two species, it remains critical to identify S. schleiferi and S. coagulans correctly in routine diagnostics. IMPORTANCE This study clearly shows that S. schleiferi is a concern in human hospital settings, whereas S. coagulans predominantly causes infections in animals. S. coagulans is more resistant to antibiotics and can sometimes transmit to humans via exposure to infected dogs. Even though genome-based methods can clearly differentiate the two species, current diagnostic methods used routinely in clinical microbiology laboratories cannot distinguish the two bacterial species.

Staphylococcus schleiferi and Staphylococcus coagulans are opportunistic pathogens of animals and humans. They were previously classified as Staphylococcus schleiferi subs. schleiferi and Staphylococcus schleiferi subs. coagulans, respectively, and recently reclassified as separate species. S. coagulans, is frequently associated with dogs, whereas S. schleiferi is more commonly isolated from humans. Coagulase activity status is a defining characteristic of the otherwise closely related species. However, the use of coagulase tests originally developed to distinguish S. aureus from non-coagulase-producing staphylococci, for this purpose is questionable and the basis for their host preference has not been elucidated. In the current study, a putative coa gene was identified and correlated with coagulase activity measured using a chromogenic assay with human and bovine prothrombin (closely related to canine prothrombin). The results of the tests performed with human prothrombin showed greater reactivity of S. coagulans isolates from humans than isolates obtained from dogs with the same substrate. Our data suggest that unlike S. coagulans isolates from humans, isolates from dogs have more coagulase activity with bovine prothrombin (similar to canine prothrombin) than human prothrombin. Differences in nuc and 16s rRNA genes suggest a divergence in S. coagulans and S. schleiferi. Phenotypic and genotypic variation based on the number of IgG binding domains, and the numbers of tandem repeats in C-terminal fibronectin binding motifs was also found in protein A, and fibronectin-binding protein B respectively. This study identified a coa gene and associated phenotypic activity that differentiates S. coagulans and S. schleiferi and identified key phylogenetic and phenotypic differences between the species.

Infections caused by antibiotic-resistant Staphylococcus are a global concern. This is true in the Middle East, where increasingly resistant Staphylococcus aureus and Staphylococcus haemolyticus strains have been detected. While extensive surveys have revealed the prevalence of infections caused by antibiotic-resistant staphylococci in Europe, Asia, and North America, the population structure of antibiotic-resistant staphylococci recovered from patients and clinical settings in Egypt remains uncharacterized. We performed whole-genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital; 46 of the S. aureus genomes and all 10 of the S. haemolyticus genomes carry mecA, which confers methicillin resistance. Supplemented with additional publicly available genomes from the other parts of the Middle East (34 S. aureus and 6 S. haemolyticus), we present the largest genomic study to date of staphylococcal isolates from the Middle East. These genomes include 20 S. aureus multilocus sequence types (MLST), including 3 new ones. They also include 9 S. haemolyticus MLSTs, including 1 new one. Phylogenomic analyses of each species' core genome largely mirrored those of the MLSTs, irrespective of geographical origin. The hospital-acquired spa t037/ST239-SCCmec III/MLST CC8 clone represented the largest clade, comprising 22% of the S. aureus isolates. Like S. aureus genome surveys of other regions, these isolates from the Middle East have an open pangenome, a strong indicator of gene exchange of virulence factors and antibiotic resistance genes with other reservoirs. Our genome analyses will inform antibiotic stewardship and infection control plans in the Middle East. IMPORTANCE Staphylococci are understudied despite their prevalence within the Middle East. Methicillin-resistant Staphylococcus aureus (MRSA) is endemic to hospitals in Egypt, as are other antibiotic-resistant strains of S. aureus and S. haemolyticus. To provide insight into the strains circulating in Egypt, we performed whole-genome sequencing of 56 S. aureus and 10 S. haemolyticus isolates from Alexandria Main University Hospital. Through analysis of these genomes, as well as all available S. aureus and S. haemolyticus genomes from the Middle East (n = 40), we were able to produce a picture of the diversity in this region more complete than those afforded by traditional molecular typing strategies. For example, we identified 4 new MLSTs. Most strains harbored genes associated with multidrug resistance, toxin production, biofilm formation, and immune evasion. These data provide invaluable insight for future antibiotic stewardship and infection control within the Middle East.

Staphylococci can cause a wide array of infections that can be life threatening. These infections become more deadly when the isolates are antibiotic resistant and thus harder to treat. Many resistance determinants are plasmid-mediated; however, staphylococcal plasmids have not yet been fully characterized. In particular, plasmids and their contributions to antibiotic resistance have not been investigated within the Arab states, where antibiotic use is not universally regulated. Here, we characterized the putative plasmid content among 56 Staphylococcus aureus and 10 Staphylococcus haemolyticus clinical isolates from Alexandria, Egypt. Putative plasmid sequences were detected in over half of our collection. In total, we identified 72 putative plasmid sequences in 27 S. aureus and 1 S. haemolyticus isolates. While these isolates typically carried one or two plasmids, we identified one isolate-S. aureus AA53-with 11 putative plasmids. The plasmid sequences most frequently encoded a Rep_1, RepL, or PriCT_1 type replication protein. As expected, antibiotic resistance genes were widespread among the identified plasmid sequences. Related plasmids were identified amongst our clinical isolates; homologous plasmids present in multiple isolates clustered into 11 groups based upon sequence similarity. Plasmids from the same cluster often shared antibiotic resistance genes, including blaZ, which is associated with β-lactam resistance. Our analyses suggest that plasmids are a key factor in the pathology and epidemiology of S. aureus in Egypt. A better characterization of plasmids and the role they contribute to the success of Staphylococci as pathogens will guide the design of effective control strategies to limit their spread.

Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used "survival" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.

Staphylococcus spp. colonize commensally on the human skin. Some commensal coagulase-negative staphylococci and Staphylococcus aureus are also involved in nosocomial infections. Bacteria were collected from skin healed from pressure injury (PI). After the collection time points, some patients suffered from recurrent PI (RPI). This study analyzed the characteristics of Staphylococcus spp. on healed skin before recurrence between healed skin that suffered from RPI within 6 weeks (RPI group) and healed skin that did not suffer within the duration (non-RPI group) by Staphylococcus spp.-specific sequencing. Of the seven patients in the RPI group, two were dominated by S. aureus and four by Staphylococcus caprae, coagulase-negative human commensal staphylococci in the RPI group. Using mouse models, both S. caprae and S. aureus, but not Staphylococcus epidermidis, colonized on skin healed from injury at significantly higher rates than normal skin. Although subcutaneous injection of S. caprae did not induce lesion formation, the bacterium exhibited high hemolytic activity on human red blood cells. Lesion formation by subcutaneous injection of S. aureus was significantly suppressed in the presence of S. caprae. The hemolytic activity of rabbit blood cells of S. aureus was suppressed by S. caprae, whereas the hemolytic activity of S. caprae was dramatically suppressed by S. aureus. Data indicated that each of the two Staphylococcus spp. suppresses the pathogenicity of the other and that the imbalance between the two is associated with RPI.

Staphylococcus agnetis is an emerging pathogen in chickens but has been most commonly isolated from sub-clinical mastitis in bovines. Previous whole-genome analyses for known virulence genes failed to identify determinants for the switch from mild ductal infections in cattle to severe infections in poultry. We now report identification of a family of 15 kbp, 17-19 gene mobile genetic elements (MGEs) specific to chicken osteomyelitis and dermatitis isolates of S. agnetis. These MGEs can be present in multiple copies per genome. The MGE has been vectored on a Staphylococcus phage that separately lysogenized two S. agnetis osteomyelitis strains. The S. agnetis genome from a broiler breeder case of ulcerative dermatitis contains 2 orthologs of this MGE, not associated with a prophage. BLASTn and phylogenetic analyses show that there are closely related intact MGEs found in genomes of S. aureus. The genome from a 1980s isolate from chickens in Ireland contains 3 copies of this MGE. More recent chicken isolates descended from that genome (Poland 2009, Oklahoma 2010, and Arkansas 2018) contain 2 to 4 related copies. Many of the genes of this MGE can be identified in disparate regions of the genomes of other chicken isolates of S. aureus. BLAST searches of the NCBI databases detect no similar MGEs outside of S. aureus and S. agnetis. These MGEs encode no proteins related to those produced by Staphylococcus aureus Pathogenicity Islands, which have been associated with the transition of S. aureus from human to chicken hosts. Other than mobilization functions, most of the genes in these new MGEs annotate as hypothetical proteins. The MGEs we describe appear to represent a new family of Chromosomal Islands (CIs) shared amongst S. agnetis and S. aureus. Further work is needed to understand the role of these CIs/MGEs in pathogenesis. Analysis of horizontal transfer of genetic elements between isolates and species of Staphylococci provides clues to evolution of host-pathogen interactions as well as revealing critical determinants for animal welfare and human diseases.

Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia.

Staphylococcus species are emerging opportunistic pathogens that cause outbreaks of hospital and community-acquired infections. Some of these bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat due to their resistance to multiple antibiotics. We carried out a comparative study on the lipidome adaptations in response to starvation in the two most common coagulase-negative Staphylococcus species: a S. epidermidis strain sensitive to ampicillin and erythromycin and a S. haemolyticus strain resistant to both. The predominant fatty acid composition in glycerolipids was (17:0-15:0) in both bacteria. During the exponential phase, the two bacterial lipidomes were similar. Both were dominated by diacylglycerol (DAG), phosphatidylglycerol (PG), lysyl-phosphatidylglycerol (Lysyl-PG) and Diglucosyl-diacylglycerol (DGDG). Alanyl-PG was detected in small amounts in both bacterial lipids. N-succinyl-lysyl-PG was detected only in S. haemolyticus, while lysyl-DAG only in S. epidermidis. As the two bacteria entered stationary phase, both lipidomes became essentially nitrogen-free. Both bacteria accumulated large amounts of free fatty acids. Strikingly, the lipidome of S. epidermidis became dominated by cardiolipin (CL), while that of S. haemolyticus was simplified to DGDG and PG. The S. epidermidis strain also produced acyl-phosphatidylglycerol (APG) in the stationary phase.

Staphylococcus aureus and Staphylococcus epidermidis are two major skin associated bacteria, and Substance P (SP) is a major skin neuropeptide. Since bacteria are known to sense and response to many human hormones, we investigated the effects of SP on Staphylococci virulence in reconstructed human epidermis model and HaCaT keratinocytes. We show that SP is stimulating the virulence of S. aureus and S. epidermidis in a reconstructed human epidermis model. qRT-PCR array analysis of 64 genes expressed by keratinocytes in the response to bacterial infection revealed a potential link between the action of SP on Staphylococci and skin physiopathology. qRT-PCR and direct assay of cathelicidin and human β-defensin 2 secretion also provided that demonstration that the action of SP on bacteria is independent of antimicrobial peptide expression by keratinocytes. Considering an effect of SP on S. aureus and S. epidermidis, we observed that SP increases the adhesion potential of both bacteria on keratinocytes. However, SP modulates the virulence of S. aureus and S. epidermidis through different mechanisms. The response of S. aureus is associated with an increase in Staphylococcal Enterotoxin C2 (SEC2) production and a reduction of exolipase processing whereas in S. epidermidis the effect of SP appears mediated by a rise in biofilm formation activity. The Thermo unstable ribosomal Elongation factor Ef-Tu was identified as the SP-interacting protein in S. aureus and S. epidermidis. SP appears as an inter-kingdom communication factor involved in the regulation of bacterial virulence and essential for skin microflora homeostasis.

Ozenoxacin (OZN) belongs to a new generation of non-fluorinated quinolones for the topical treatment of skin infections which has shown to be effective in the treatment of susceptible and resistant Gram-positive cocci. The mutant prevention concentration (MPC) of ozenoxacin, levofloxacin and ciprofloxacin was determined in quinolone-susceptible and -resistant strains including methicillin-susceptible S. aureus, methicillin-resistant S. aureus, methicillin-susceptible S. epidermidis and methicillin-resistant S. epidermidis with different profile of mutation in the quinolone resistance determining regions (QRDR). The MPC value of OZN for the methicillin-susceptible S. aureus strain susceptible to quinolones, without mutations in QRDR, was 0.05 mg/L, being 280-fold lower than that observed with ciprofloxacin and levofloxacin. In methicillin-susceptible and-resistant S. aureus strains with mutations in the gyrA or/and grlA genes the MPC of OZN went from 0.1 to 6 mg/L, whereas the MPC of levofloxacin and ciprofloxacin was > 50 mg/L for the same strains. For methicillin-susceptible and-resistant S. epidermidis the results were similar to those abovementioned for S. aureus. According to our results, the MPC of OZN was far below the quantity of ozenoxacin achieved in the epidermal layer, suggesting that the in vivo selection of mutants, if it occurs, will take place at low frequency. Ozenoxacin is an excellent candidate for the treatment of bacterial infections caused by susceptible and quinolone-resistant staphylococci isolated usually from skin infections.

Infective endocarditis is a rare condition in humans and is associated with high illness and death rates. We describe a case of infective endocarditis caused by Staphylococcus succinus bacteria in France. We used several techniques for susceptibility testing for this case to determine the oxacillin profile.

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing public and occupational health concern. As transmission of MRSA can occur via contact with fomites, it is crucial to have sensitive methods for sampling of bacteria. The overall aim of this study was to obtain knowledge about methods and strategies for quantitative sampling Staphylococcus species on surfaces.

In bacteria, gene regulation is one of the fundamental characteristics of survival, colonization and pathogenesis. Operons play a key role in regulating expression of diverse genes involved in metabolism and virulence. However, operon structures in pathogenic bacteria have been determined only by in silico approaches that are dependent on factors such as intergenic distances and terminator/promoter sequences. Knowledge of operon structures is crucial to fully understand the pathophysiology of infections. Presently, transcriptome data obtained from growth curves in a defined medium were used to predict operons in Staphylococcus aureus. This unbiased approach and the use of five highly reproducible biological replicates resulted in 93.5% significantly regulated genes. These data, combined with Pearson's correlation coefficients of the transcriptional profiles, enabled us to accurately compile 93% of the genome in operon structures. A total of 1640 genes of different functional classes were identified in operons. Interestingly, we found several operons containing virulence genes and showed synergistic effects for two complement convertase inhibitors transcribed in one operon. This is the first experimental approach to fully identify operon structures in S. aureus. It forms the basis for further in vitro regulation studies that will profoundly advance the understanding of bacterial pathophysiology in vivo.

Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches.

The approach of sequencing or genotyping to characterize the pathogenic potential of staphylococci from orthopedic device-related infection (ODRI) has been applied in recent studies. These studies described the genomic carriage of virulence in clinical strains and compared it with those in commensal strains. Only a few studies have directly correlated genomic profiles to patient outcome and phenotypic virulence properties in periprosthetic joint infections (PJIs). We investigated the association between genomic variations and virulence-associated phenotypes (biofilm-forming ability and antimicrobial resistance) in 111 staphylococcal strains isolated from patients with PJI and the infection outcome (resolved/unresolved). The presence of a strong biofilm phenotype in Staphylococcus aureus and an antibiotic-resistant phenotype in Staphylococcus epidermidis were both associated with treatment failure of PJI. In S. epidermidis, multidrug resistance (MDR) and resistance to rifampicin were associated with unresolved infection. Sequence type 45 (ST45) and ST2 were particularly enriched in S. aureus and S. epidermidis, respectively. S. epidermidis ST2 caused the majority of relapses and was associated with MDR and strong biofilm production, whereas ST215 correlated with MDR and non/weak biofilm production. S. aureus agr II correlated with resolved infection, while S. epidermidis agr I was associated with strong biofilm production and agr III with non/weak production. Collectively, our results highlight the importance of careful genomic and phenotypic characterization to anticipate the probability of the strain causing treatment failure in PJI. Due to the high rate of resistant S. epidermidis strains identified, this study provides evidence that the current recommended treatment of rifampicin and a fluoroquinolone should not be administered without knowledge of the resistance pattern. IMPORTANCE This study addresses the presence and frequency of particular genetic variants and virulence factors found in staphylococcal bacteria causing periprosthetic joint infection (PJI) of the hip and knee to ascertain their clinical relevance as predictors of treatment failure. We characterized the genetic virulence traits of a large collection of clinical staphylococci isolated from patients with PJI and evaluated their association with the patient's infection outcome. The results showed that S. aureus strains that produced strong biofilms and S. epidermidis strains with resistance to several antibiotics associated significantly with unresolved infection. Some particular genetic variants associated with biofilm formation and multidrug resistance. These traits should be considered important risk factors for the diagnosis and treatment guidance in PJI.