Staphylococcus epidermidis Phages Transduce Antimicrobial Resistance Plasmids and Mobilize Chromosomal Islands.

Staphylococcus epidermidis is a leading opportunistic pathogen causing nosocomial infections that is notable for its ability to form a biofilm and for its high rates of antibiotic resistance. It serves as a reservoir of multiple antimicrobial resistance genes that spread among the staphylococcal population by horizontal gene transfer such as transduction. While phage-mediated transduction is well studied in Staphylococcus aureus, S. epidermidis transducing phages have not been described in detail yet. Here, we report the characteristics of four phages, 27, 48, 456, and 459, previously used for S. epidermidis phage typing, and the newly isolated phage E72, from a clinical S. epidermidis strain. The phages, classified in the family Siphoviridae and genus Phietavirus, exhibited an S. epidermidis-specific host range, and together they infected 49% of the 35 strains tested. A whole-genome comparison revealed evolutionary relatedness to transducing S. aureus phietaviruses. In accordance with this, all the tested phages were capable of transduction with high frequencies up to 10-4 among S. epidermidis strains from different clonal complexes. Plasmids with sizes from 4 to 19 kb encoding resistance to streptomycin, tetracycline, and chloramphenicol were transferred. We provide here the first evidence of a phage-inducible chromosomal island transfer in S. epidermidis Similarly to S. aureus pathogenicity islands, the transfer was accompanied by phage capsid remodeling; however, the interfering protein encoded by the island was distinct. Our findings underline the role of S. epidermidis temperate phages in the evolution of S. epidermidis strains by horizontal gene transfer, which can also be utilized for S. epidermidis genetic studies.IMPORTANCE Multidrug-resistant strains of S. epidermidis emerge in both nosocomial and livestock environments as the most important pathogens among coagulase-negative staphylococcal species. The study of transduction by phages is essential to understanding how virulence and antimicrobial resistance genes spread in originally commensal bacterial populations. In this work, we provide a detailed description of transducing S. epidermidis phages. The high transduction frequencies of antimicrobial resistance plasmids and the first evidence of chromosomal island transfer emphasize the decisive role of S. epidermidis phages in attaining a higher pathogenic potential of host strains. To date, such importance has been attributed only to S. aureus phages, not to those of coagulase-negative staphylococci. This study also proved that the described transducing bacteriophages represent valuable genetic modification tools in S. epidermidis strains where other methods for gene transfer fail.

The Genome of Staphylococcus epidermidis O47.

The skin colonizing coagulase-negative Staphylococcus epidermidis causes nosocomial infections and is an important opportunistic and highly adaptable pathogen. To gain more insight into this species, we sequenced the genome of the biofilm positive, methicillin susceptible S. epidermidis O47 strain (hereafter O47). This strain belongs to the most frequently isolated sequence type 2. In comparison to the RP62A strain, O47 can be transformed, which makes it a preferred strain for molecular studies. S. epidermidis O47's genome has a single chromosome of about 2.5 million base pairs and no plasmid. Its oriC sequence has the same directionality as S. epidermidis RP62A, S. carnosus, S. haemolyticus, S. saprophyticus and is inverted in comparison to Staphylococcus aureus and S. epidermidis ATCC 12228. A phylogenetic analysis based on all S. epidermidis genomes currently available at GenBank revealed that O47 is closest related to DAR1907. The genome of O47 contains genes for the typical global regulatory systems known in staphylococci. In addition, it contains most of the genes encoding for the typical virulence factors for S. epidermidis but not for S. aureus with the exception of a putative hemolysin III. O47 has the typical S. epidermidis genetic islands and several mobile genetic elements, which include staphylococcal cassette chromosome (SCC) of about 54 kb length and two prophages φO47A and φO47B. However, its genome has no transposons and the smallest number of insertion sequence (IS) elements compared to the other known S. epidermidis genomes. By sequencing and analyzing the genome of O47, we provide the basis for its utilization in genetic and molecular studies of biofilm formation.

Staphylococcus carnosus: from starter culture to protein engineering platform.

Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and "food grade" species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus.

Secretome analysis revealed adaptive and non-adaptive responses of the Staphylococcus carnosus femB mutant.

FemABX peptidyl transferases are involved in non-ribosomal pentaglycine interpeptide bridge biosynthesis. Here, we characterized the phenotype of a Staphylococcus carnosus femB deletion mutant, which was affected in growth and showed pleiotropic effects such as enhanced methicillin sensitivity, lysostaphin resistance, cell clustering, and decreased peptidoglycan cross-linking. However, comparative secretome analysis revealed a most striking difference in the massive secretion or release of proteins into the culture supernatant in the femB mutant than the wild type. The secreted proteins can be categorized into typical cytosolic proteins and various murein hydrolases. As the transcription of the murein hydrolase genes was up-regulated in the mutant, they most likely represent an adaption response to the life threatening mutation. Even though the transcription of the cytosolic protein genes was unaltered, their high abundance in the supernatant of the mutant is most likely due to membrane leakage triggered by the weakened murein sacculus and enhanced autolysins.

Characterization of a mazEF toxin-antitoxin homologue from Staphylococcus equorum.

Toxin-antitoxin (TA) systems encoded in prokaryotic genomes fall into five types, typically composed of two distinct small molecules, an endotoxic protein and a cis-encoded antitoxin of ribonucleic or proteinaceous nature. In silico analysis revealed seven putative type I and three putative type II TA systems in the genome of the nonpathogenic species strain Staphylococcus equorum SE3. Among these, a MazEF system orthologue termed MazEF(seq) was further characterized. 5' rapid amplification of cDNA ends (RACE) revealed the expression and the transcriptional start site of mazE(seq), indicating an immediately upstream promoter. Heterologous expression of the putative toxin-encoding mazF(seq) gene imposed growth cessation but not cell death on Escherichia coli. In vivo and in vitro, MazF(seq) was shown to cleave at UACAU motifs, which are remarkably abundant in a number of putative metabolic and regulatory S. equorum gene transcripts. Specific interaction between MazF(seq) and the putative cognate antitoxin MazE(seq) was demonstrated by bacterial two-hybrid analyses. These data strongly suggest that MazEF(seq) represents the first characterized TA system in a nonpathogenic Staphylococcus species and indicate that MazEF modules in staphylococci may also control processes beyond pathogenicity.

What distinguishes highly pathogenic staphylococci from medium- and non-pathogenic?

Members of the genus Staphylococcus are widespread as commensals of humans and animals where they colonize the skin or mucous membranes. While this coexistence remains mostly untroubled, especially for the healthy host, the bacteria may pose a serious threat for the human or animal host when they get access to inner layers of the body through breaches in skin or membranes. Among the members of the genus a wide span exists in the ability to cope with the hostile conditions encountered in the bloodstream of the living host as a scarce supply of certain nutrients, attacks of the immune system, or anti-infective measures undertaken in the clinical field. In this respect, Staphylococcus aureus is by far the most versatile species of the genus. Its equipment with a huge repertoire of different virulence factors and additional supportive gene products that increase the capability to survive within the living host makes S. aureus the leading pathogen not only within the genus but also one of the most threatening microorganisms regarding hospitally and community-acquired infections. Compared with S. aureus, the other virulent species of the genus like S. epidermidis, S. lugdunensis, S. saprophyticus, and S. haemolyticus have a more limited arsenal of virulence factors resulting in a specialized spectrum of diseases and a generally lower degree of pathogenicity. Besides the highly and medium-pathogenic staphylococci, the genus comprises also species like S. carnosus, S. xylosus, and S. equorum that are generally inconspicuous regarding clinical occurrences. Some strains of this group are used in the food industry and can be graded as non-pathogenic. This review aims to work out the differences between the pathogenic properties of highly and medium-pathogenic staphylococcal species and to draw a comparison between the pathogenic species and the food-grade S. carnosus TM300.

Phylogeny of the staphylococcal major autolysin and its use in genus and species typing.

The major staphylococcal autolysin Atl is an important player in cell separation and daughter cell formation. In this study, we investigated the amino acid sequences of Atl proteins derived from 15 staphylococcal and 1 macrococcal species representatives. The overall organization of the bifunctional precursor protein consisting of the signal peptide, a propeptide (PP), the amidase (AM), six repeat sequences (R(1) to R(6)), and the glucosaminidase (GL) was highly conserved in all of the species. The most-conserved domains were the enzyme domains AM and GL; the least-conserved regions were the PP and R regions. An Atl-based phylogenetic tree for the various species representatives correlated well with the corresponding 16S rRNA-based tree and also perfectly matched the phylogenetic trees based on core genome analysis. The phylogenetic distance analysis of 18 AtlA proteins of various Staphylococcus aureus strains and 15 AtlE proteins of S. epidermidis revealed that both species representatives formed a relatively homogeneous cluster. Two S. epidermidis strains, M23864:W1 and VCU116, were identified by Atl typing that clustered far more distantly and belonged to either S. caprae and S. capitis or a new subspecies. Here we show that Atl typing is a useful tool for staphylococcal genus and species typing by using either the highly conserved AM domain or the less-conserved PP domain.

DNA microarray based detection of genes involved in safety and technologically relevant properties of food associated coagulase-negative staphylococci.

Aim of the work was to design a polynucleotide based DNA microarray as screening tool to detect genes in food associated coagulase-negative staphylococci (CNS). A focus was laid on genes with potential health concern and technological relevance. The microarray contained 220 probes for genes encoding antibiotic resistances, hemolysins, toxins, amino acid decarboxylases (e.g. biogenic amine formation), binding proteins to extracellular matrix (ECM), lipases, proteases, stress response factors, or nitrate dissimilation. Hybridization of genomic DNA isolated from 32 phenotypically characterized CNS permitted to detect numerous genes, corresponding with the phenotype. However, numerous hybridization signals were obtained for genes without any detectable phenotype. The antibiotic resistance genes blaZ, lnuA, and tetK involved in ß-lactam, lincomycin and tetracycline resistance, respectively, were rarely identified in CNS, however, all species contained some strains with such resistance genes. Decarboxylase genes involved in biogenic amine formation were detected regularly in Staphylococcus carnosus, S. condimenti, S. piscifermentans and S. equorum, but was rarely correlated with the phenotype. The same applied for the fibrinogen (clf) and fibronectin (fbp) binding protein genes, whose phenotype (binding assay) was only correlated in S. equorum and Staphylococcus succinus. Although some CNS showed hemolytic activity and enterotoxin production (Immunoblot) the corresponding genes could not be verified. Technological relevant genes such as proteases or lipases revealed good hybridization signals. In addition, genes involved in nitrate dissimilation (nre, nar, nir), catalase (kat), or superoxide dismutase (sod) were well detected. Interestingly, genes involved in dissimilatory nitrate reduction were more prevalent in strains of S. carnosus, S. condimenti and S. piscifermentans than of S. equorum, S. succinus and S. xylosus.

Genomic differences between the food-grade Staphylococcus carnosus and pathogenic staphylococcal species.

By comparative analyses based on the newly sequenced genome of the meat starter bacterium Staphylococcus carnosus TM300, we observed remarkable differences in the content of mobile genetic elements between non-pathogenic and pathogenic staphylococci. While the latter reveal highly flexible genomes with various mobile elements indicating frequent exchange and rearrangement of genomic material, S. carnosus shows a conspicuous lack of those elements carrying only remnants of a prophage and a genomic island in its genome. Furthermore, the S. carnosus genome is significantly poor in repetitive sequences. Despite being known as completely avirulent, S. carnosus reveals also various gene products with similarity to proteins annotated as virulence factors in S. aureus. In addition, the genome carries a number of mutationally inactivated genes including those of the global regulatory systems agr and sae. Our data indicate that S. carnosus has adapted to the constant environmental conditions encountered as part of a starter culture population by a reductive evolution leading to gene loss and inactivation.

Role of the twin-arginine translocation pathway in Staphylococcus.

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (DeltatatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of DeltatatAC and Deltatat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.

Genome analysis of the meat starter culture bacterium Staphylococcus carnosus TM300.

The Staphylococcus carnosus genome has the highest GC content of all sequenced staphylococcal genomes, with 34.6%, and therefore represents a species that is set apart from S. aureus, S. epidermidis, S. saprophyticus, and S. haemolyticus. With only 2.56 Mbp, the genome belongs to a family of smaller staphylococcal genomes, and the ori and ter regions are asymmetrically arranged with the replichores I (1.05 Mbp) and II (1.5 Mbp). The events leading up to this asymmetry probably occurred not that long ago in evolution, as there was not enough time to approach the natural tendency of a physical balance. Unlike the genomes of pathogenic species, the TM300 genome does not contain mobile elements such as plasmids, insertion sequences, transposons, or STAR elements; also, the number of repeat sequences is markedly decreased, suggesting a comparatively high stability of the genome. While most S. aureus genomes contain several prophages and genomic islands, the TM300 genome contains only one prophage, PhiTM300, and one genomic island, nuSCA1, which is characterized by a mosaic structure mainly composed of species-specific genes. Most of the metabolic core pathways are present in the genome. Some open reading frames are truncated, which reflects the nutrient-rich environment of the meat starter culture, making some functions dispensable. The genome is well equipped with all functions necessary for the starter culture, such as nitrate/nitrite reduction, various sugar degradation pathways, two catalases, and nine osmoprotection systems. The genome lacks most of the toxins typical of S. aureus as well as genes involved in biofilm formation, underscoring the nonpathogenic status.

Characterization of toxin production of coagulase-negative staphylococci isolated from food and starter cultures.

In this study a comprehensive analysis of toxin production of food associated coagulase-negative staphylococci (CNS) was investigated. The strains belong to the following staphylococcal species, Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus equorum, Staphylococcus piscifermentans, Staphylococcus succinus, and Staphylococcus xylosus, which were isolated from fermented food and starter cultures. A collection of 330 strains were analyzed with respect to their hemolytic activity. 59% of the strains exhibited weak to moderate hemolytic activity with human blood and 34% with sheep blood after 48 h incubation. A selection of 35 strains were tested by immunoblot analysis for their ability to produce toxins, such as the most common staphylococcal enterotoxins (SEs), the toxic shock syndrome toxin 1 (TSST-1), and the exfoliative toxin A (ETA). 18 of the 35 strains produced at least one of the toxins with the SED and SEH being the most common. These indicate that the use of CNS in food production demands a safety evaluation.

Understanding the physiology and adaptation of staphylococci: a post-genomic approach.

Staphylococcus aureus as well as coagulase-negative staphylococci are medically highly important pathogens characterized by an increasing resistance rate toward many antibiotics. Although normally being skin and mucosa commensals, some staphylococcal species and strains have the capacity to cause a wide range of infectious diseases. Many of these infections affect immunocompromised patients in hospitals. However, community-acquired staphylococcal infections due to resistant strains are also currently on the rise. In the light of this development, there is an urgent need for novel anti-staphylococcal therapeutic and prevention strategies for which a better understanding of the physiology of these bacteria is an essential prerequisite. Within the past years, staphylococci have been in the focus of genomic research, resulting in the determination and publication of a range of full-genome sequences of different staphylococcal species and strains which provided the basis for the design and application of DNA microarrays and other genomic tools. Here we summarize the results of the project group 'Staphylococci' within the research network 'Pathogenomics' giving new insights into the genome structure, molecular epidemiology, physiology, and genetic adaptation of both S. aureus and coagulase-negative staphylococci.

Microevolution of cytochrome bd oxidase in Staphylococci and its implication in resistance to respiratory toxins released by Pseudomonas.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and frequently coinfect the lungs of cystic fibrosis patients. P. aeruginosa secretes an arsenal of small respiratory inhibitors, like pyocyanin, hydrogen cyanide, or quinoline N-oxides, that may act against the commensal flora as well as host cells. Here, we show that with respect to their susceptibility to these respiratory inhibitors, staphylococcal species can be divided into two groups: the sensitive group, comprised of pathogenic species such as S. aureus and S. epidermidis, and the resistant group, represented by nonpathogenic species such as S. carnosus, S. piscifermentans, and S. gallinarum. The resistance in the latter group of species was due to cydAB genes that encode a pyocyanin- and cyanide-insensitive cytochrome bd quinol oxidase. By exchanging cydB in S. aureus with the S. carnosus-specific cydB, we could demonstrate that CydB determines resistance. The resistant or sensitive phenotype was based on structural alterations in CydB, which is part of CydAB, the cytochrome bd quinol oxidase. CydB represents a prime example of both microevolution and the asymmetric pattern of evolutionary change.

Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions.

It is well known that biofilm formation by pathogenic staphylococci on implanted medical devices leads to "chronic polymer-associated infections." Bacteria in these biofilms are more resistant to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which genes are up-regulated in Staphylococcus aureus biofilm cells, we carried out a comparative transcriptome analysis. Biofilm growth was simulated on dialysis membranes laid on agar plates. Staphylococci were cultivated planktonically in Erlenmeyer flasks with shaking. mRNA was isolated at five time points from cells grown under both conditions and used for hybridization with DNA microarrays. The gene expression patterns of several gene groups differed under the two growth conditions. In biofilm cells, the cell envelope appeared to be a very active compartment since genes encoding binding proteins, proteins involved in the synthesis of murein and glucosaminoglycan polysaccharide intercellular adhesin, and other enzymes involved in cell envelope synthesis and function were significantly up-regulated. In addition, evidence was obtained that formate fermentation, urease activity, the response to oxidative stress, and, as a consequence thereof, acid and ammonium production are up-regulated in a biofilm. These factors might contribute to survival, persistence, and growth in a biofilm environment. Interestingly, toxins and proteases were up-regulated under planktonic growth conditions. Physiological and biochemical tests for the up-regulation of urease, formate dehydrogenase, proteases, and the synthesis of staphyloxanthin confirmed the microarray data.