Complete Genome Sequence of Staphylococcus aureus Strain JKD6008, an ST239 Clone of Methicillin-Resistant Staphylococcus aureus with Intermediate-Level Vancomycin Resistance

We report here the complete 2.92-Mb genome sequence of a clinical isolate of methicillin-resistant Staphylococcus aureus subsp. aureus that demonstrates intermediate-level vancomycin resistance. The strain, named JKD6008, belongs to multilocus sequence type 239 and was isolated from the bloodstream of a patient in New Zealand in 2003.We have previously described the in vivo evolution of low-level vancomycin resistance in Staphylococcus aureus through comparative and functional genomic assessment of a pair of isogenic methicillin-resistant Staphylococcus aureus (MRSA) strains. The vancomycin-susceptible S. aureus (VSSA) strain JKD6009 was a patient wound isolate, whereas vancomycin-intermediate S. aureus (VISA) strain JKD6008 was recovered from the bloodstream of the same patient after 42 days of vancomycin treatment (5). Comparison of the partially assembled genomes of the two isolates revealed a single-point mutation in the sensor region of the two-component regulatory gene graS, which caused a significant reduction in the vancomycin susceptibility of JKD6008 (6). Here we report the fully assembled and annotated genome of S. aureus JKD6008.The genome sequence of S. aureus strain JKD6008 was determined by whole-genome shotgun sequencing using single-read 454 GS20 (Roche Diagnostics, Basel, Switzerland), Sanger (Applied Biosystems), and SOLiD (Applied Biosystems) sequencing technologies, producing approximately 20 times, 4 times, and 225 times coverage of the genome, respectively. GS20 reads were assembled using gsAssembler v2.0 software, resulting in 131 contigs (≥500 bp) totaling 2.83 Mbp (6, 10). Sanger paired-end reads (clone insert size, 3 to 5 kb) were combined with the GS20 contigs using Gap4 v4.11 software (3). Mate-pair SOLiD reads (3 to 5 kb) were aligned to the contigs using SHRiMP 1.3.2 software to identify and correct sequencing errors (11). Optical mapping produced a high-resolution XbaI chromosome restriction map, to which the contigs were aligned using MapSolver 2.1.1 (Opgen) to determine misassemblies. Gap closures were performed by PCR, followed by Sanger sequencing and primer walking of amplification products (3730S DNA Analyzer sequencer; Applied Biosystems). The assembly of the completed genome was confirmed to be correct by reference to the XbaI optical map.Protein-coding regions were predicted using GeneMarkS 4.6b software, tRNA genes using tRNAscan-SE 1.23, and rRNA genes using RNAmmer 1.2 (2, 8, 9). Gene products were assigned using HMMER 3.0 against the Pfam database (release 23) and BLAST 2.2.23 against RefSeq proteins (April 2010) and the Conserved Domain Database (v2.22) (1, 4). These automated analyses were followed by manual curation and comparisons with other completed S. aureus genomes.The genome of S. aureus strain JKD6008 consists of a circular 2,924,344-bp chromosome with a 34% G+C content and no extrachromosomal elements. A total of 2,766 coding DNA sequences, 82 tRNA genes, and 5 rRNA loci were detected. Over 70% of genes were assigned to specific Clusters of Orthologous Groups (COG) functional groups, and 42% were assigned an enzyme classification number (12).Initial analysis of the whole-genome sequence of JKD6008 confirmed it as a member of the ST239 complex, sharing 2,504 orthologous coding sequences (CDSs) with the recently described ST239 member TW20 (EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"FN433596.1","term_id":"269939526","term_text":"FN433596.1"}}FN433596.1). There are 17 copies of IS256 and a type III staphylococcal cassette chromosome mec element (SCCmec). Comparisons with 19 published S. aureus genomes revealed 20 CDS not present in any other S. aureus genome, although some of these 20 CDS have orthologs in other Staphylococcus species. JKD6008 also harbors a 28-kb integrated pSK1-like plasmid that is predicted to confer resistance to aminoglycosides and trimethoprim, as well as efflux-mediated antiseptic and disinfectant resistance (7).Nucleotide sequence accession number. The complete genome sequence has been deposited in NCBI GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"CP002120","term_id":"302749911","term_text":"CP002120"}}CP002120.     

Optical Mapping Reveals a Large Genetic Inversion between Two Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a highly versatile and evolving bacterium of great clinical importance. S. aureus can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of ∼500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS1181 element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.Staphylococcus aureus is a gram-positive bacterium of immense clinical importance. This opportunistic pathogen is capable of causing a wide range of diseases from skin and soft-tissue infections to life-threatening bacteremia, endocarditis, and osteomyelitis (14). Approximately 75% of the S. aureus genome is composed of a core genome that is common in all strains, and 25% of the genome is composed of variable regions which can differ between different strains (4, 16, 24-26). S. aureus evolves primarily by introducing single nucleotide polymorphisms in its core genome and by acquiring mobile genetic elements (MGEs) through horizontal gene transfer. These MGEs include pathogenicity/genomic islands, plasmids, transposons, and bacteriophages that become integrated in the chromosome (4, 11, 16, 31, 32). Despite being a heterogeneous organism, genetic recombination in S. aureus is proposed to be rather rare (20, 24, 29, 35). Its clones are more likely to evolve by point mutations than by recombination events (12). The MGEs contribute to the phenotypic and genotypic diversity seen with the S. aureus population. Acquisition of the staphylococcal cassette chromosome (SCCmec) elements through site-specific recombinases has led to the epidemic of methicillin-resistant S. aureus (MRSA) strains in hospitals and communities all over the world (6, 10, 15). In recent years, the integration of arginine catabolite mobile element in the USA300 lineage of MRSA has been proposed to give the pathogen its epidemiological advantage, including traits for surviving in low-pH conditions and oxygen tension environments (11). In addition, chromosomal replacements have been observed within lineages of sequence type 34 (ST34) and ST42 (34) and ST8 and ST30 (13).Genomic rearrangements, such as inversions, have been observed with genomes of enteric bacteria, such as Salmonella enterica, Shigella flexneri, Yersinia pestis KIM, Escherichia coli (K12 and O157:H7), and group A Streptococcus pyogenes (8, 9, 18, 27, 28, 30, 37). No genomic inversions in S. aureus have been reported to date. With the use of optical mapping, large genomic rearrangements, such as inversions, that would otherwise be missed with other comparative genotyping approaches, including microarray analysis, can be identified. Optical mapping uses high-resolution restriction maps (optical maps) of a bacterial genome to determine its genomic organization (5, 21, 23, 33, 36). These optical maps can be compared to an in silico (virtual) restriction map of a known genome sequence and can be used to identify gene rearrangements and their locations. Using optical mapping in conjunction with subsequent site-specific PCR and sequencing, we report the identification, approximate location, and partial characterization of an ∼500-kb DNA element in NRS387, a USA800 strain that was found to be inverted relative to USA300FPR3757. Identification of IS1181 elements and a novel 73-bp element at both ends of the ∼500-kb element in NRS387 could suggest the possibility of an inversion event in an ancestral strain of NRS387.     

Organoselenium Coating on Cellulose Inhibits the Formation of Biofilms by Pseudomonas aeruginosa and Staphylococcus aureus

Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria frequently develop biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the dressing. In contrast, the antimicrobial agent selenium can be covalently attached to the surfaces of a dressing, prolonging its effectiveness. We examined the effectiveness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation. Colony biofilm assays revealed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs confirmed these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate-buffered saline. These results demonstrate that 0.2% selenium in a coating on cellulose discs effectively inhibits bacterial attachment and biofilm formation and that, unlike other antimicrobial agents, longer periods of exposure to an aqueous environment do not compromise the effectiveness of the coating.Among the most difficult bacterial infections encountered in treating patients are wound infections, which may occur in burn victims (10), patients with traumatic wounds (33), people with diabetes (27), and patients with surgical wounds (29, 31). Two of the more common causative agents of wound infections are Staphylococcus aureus and Pseudomonas aeruginosa (10, 27, 29, 31, 33). Such infections often lead to fatality; the mortality rate among patients infected with P. aeruginosa ranges from 26% to 55% (9, 49), while mortality from S. aureus infection ranges from 19% to 38% (28, 46, 50). As opportunistic pathogens, S. aureus and P. aeruginosa cause few infections in healthy individuals but readily cause infection once host defenses are compromised, such as with the removal of skin from burns (10). S. aureus infection originates from the normal flora of either the patient or health care workers (48), while P. aeruginosa is acquired from the environment surrounding the patient (41). Once established on the skin, S. aureus and P. aeruginosa are then able to colonize the wound. Infection results if the organisms proliferate in the wound environment.Both P. aeruginosa and S. aureus often exist within burn wounds as biofilms (43, 47). A biofilm is presently defined as a sessile microbial community characterized by cells that are irreversibly attached either to a substratum or to each other (16). Biofilms, which can attain over 100 μm in thickness, are made up of multiple layers of bacteria in an exopolysaccharide matrix (12, 16, 42). Sauer et al. showed that P. aeruginosa biofilms form in distinct developmental stages: reversible attachment, irreversible attachment, two stages of maturation, and a dispersion phase (42). Clinically, biofilms present serious medical management problems through their association with different chronic infections (37). During vascular catheter-related infections and sepsis, biofilms serve as a reservoir of bacteria from which planktonic cells detach and spread throughout the tissue and/or enter the circulatory system, resulting in bacteremia or septicemia (15). Factors specific to the bacterium may influence the formation of bacterial biofilms at different infection sites or surfaces. For example, during the initial attachment stage the flagellum, lipopolysaccharide, and possibly outer membrane proteins play a major role in bringing P. aeruginosa into proximity with the surface as well as mediating the interaction with the substratum (12). Using the murine model of thermal injury, we recently showed that P. aeruginosa forms a biofilm within the thermally injured tissues (43). Clinically, the surgeons debride the infected or dead tissues; however, a few microorganisms may remain on the tissue surface and reinitiate biofilm formation.Antibiotics, silver, or chitosan, attached to or embedded in gauze, have been shown to be efficacious in preventing wound infection (21, 24, 26, 36). However, the resistance of P. aeruginosa and S. aureus to available antibiotics severely limits the choices for antibiotic treatment (13, 40). Additionally, silver compounds, such as silver nitrate and silver sulfadiazine, leaching from dressings are toxic to human fibroblasts even at low concentrations (20, 25). Thus, effective alternative antimicrobial agents that contact the thermally injured/infected tissues and prevent the development of bacterial biofilms are required. Previous studies have shown that selenium (Se) can be covalently bound to a solid matrix and retain its ability to catalyze the formation of superoxide radicals (O₂^·−) (30). These superoxide radicals inhibit bacterial attachment to the solid surface (30). In this study, we examined the ability of a newly synthesized organoselenium-methacrylate polymer (Se-MAP) to block biofilm formation by both S. aureus and P. aeruginosa. These bacteria were chosen since they cause a major share of wound infections and because drug-resistant forms of these bacteria have become a serious problem in the treatment and management of these wound infections (6, 13, 17, 18, 38). Results of the study show that 0.2% (wt/wt) Se in Se-MAP covalently attached to cellulose discs inhibited P. aeruginosa and S. aureus biofilm formation. This could lead to the development of a selenium-based antimicrobial coating for cotton materials that will prevent the bacterial attachment and colonization that can ultimately lead to bacterial biofilm formation during chronic infections.     

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.Cysteine thiols in proteins fulfill an important and diverse set of cellular functions. In particular, they participate in enzymatic catalysis; in metal coordination, such as in the generation of Fe-S-clusters; and in determining the spatial structure of proteins via disulfide bond formation (3, 22, 23, 38). Cysteines are strong nucleophiles amenable to posttranslational modifications by reactive oxygen species (ROS) and reactive nitrogen species, leading to disulfides; to sulfenic, sulfinic, or sulfonic acids; mixed disulfides with low-molecular-weight (LMW) thiols (S thiolations); and S nitrosylations (7, 16, 17, 27).The redox status of the cytoplasm is under physiological conditions in a reduced state. Thus, most cysteines are present as free thiols (6). Because aerobic organisms have to cope with oxidative stress caused by ROS, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals, they need to employ effective mechanisms that maintain the reduced state. In gram-negative bacteria, the thiol-disulfide balance is accomplished by the glutathione (GSH) system, a thiol-based redox buffer. The GSH system consists of glutaredoxin (Grx), GSH (γ-glutamylcysteinyl glycine), GSH reductase, and GSH peroxidase (34). Reduction of disulfides occurs via sequential electron transfer from glutaredoxin and reduced GSH; oxidized GSH (GSSG) is reduced by the NADPH-dependent GSH reductase. GSH peroxidase enables the direct detoxification of ROS by GSH oxidation.However, many gram-positive bacteria lack genes for GSH biosynthesis. Actinomycetes instead use a thiol redox buffer based on mycothiol (50). Bacillus subtilis, Staphylococcus aureus, and other gram-positive bacteria rely on different thiol redox buffers based on cysteine, the novel 398-Da bacillithiol (BSH), or coenzyme A (CoA) (15, 52). To maintain the reduced state of the cytoplasm, most bacteria use enzymatic systems for disulfide bond reduction such as the thioredoxin (Trx) system, which is highly conserved in gram-negative bacteria (3, 10). The Trx system consists of thioredoxin (TrxA) and the NADPH-dependent thioredoxin reductase (TrxB).Any imbalance in the cellular redox state caused by ROS elicits expression of a repertoire of different proteins, commonly under the control of a redox-sensing regulator: for example, OxyR in Escherichia coli and PerR, OhrR, SarZ, and Spx in B. subtilis and S. aureus, respectively (11, 12, 41, 55, 58, 64-66). The subsequently induced proteins detoxify ROS and restore and protect the normal physiological redox state in the cell.Besides ROS and reactive nitrogen species, so-called “reactive electrophilic species” (RES) affect the thiol redox balance. RES include different chemical compounds such as aldehydes, quinones, and the azo compound diamide (2, 43, 45, 46, 53, 66). Quinones and aldehydes have electron-deficient centers that result in thiol-(S) alkylation of cysteine. Exposure of cells to diamide induces the oxidative as well as the electrophile stress response in B. subtilis (43, 45, 53). The toxicity of diamide is based on disulfide bond formation (40), which was recently visualized in B. subtilis and S. aureus by the fluorescence alkylation of oxidized thiols (FALKO) assay (32, 64). It was thought that the formation of nonnative inter- and intramolecular disulfide bonds results in damage of proteins.However, more recent findings demonstrate that diamide stress leads also to S thiolations: formation of disulfide bonds between proteins and LMW thiols (8, 13, 33). S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35, 47) and signal transduction (39, 42). In B. subtilis, we have identified a few cytoplasmic proteins that are S cysteinylated (33). In addition, the organic peroxide sensor OhrR was inactivated by an S bacillithiolation in B. subtilis (42).Cysteine, BSH, and CoA are also dominant LMW thiols in S. aureus (52). In this study, we have investigated in more detail the extents of S thiolations and inter- and intramolecular disulfide bond formation of B. subtilis and S. aureus in response to disulfide stress. The results showed that exposure to diamide leads to S thiolations in S. aureus. Using a nonreducing/reducing sodium dodecyl sulfate (SDS) diagonal electrophoresis approach, proteins with intermolecular disulfide bonds could be distinguished from proteins with intramolecular disulfide bonds (57). The results support that the majority of reversible thiol oxidations are based on S thiolations rather than disulfide bonds between proteins. Depletion of the free cysteine pool in B. subtilis after exposure to diamide supports this finding. To assess if GSH may have a bearing on the thiol redox buffer of B. subtilis, the gshF gene of Listeria monocytogenes (gshF_Lm) was expressed in B. subtilis, enabling GSH biosynthesis (29). Although GSH production does not enhance the resistance to oxidative stress in B. subtilis, it participates in the formation of S thiolations.     

Occurrence and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus pseudintermedius in an Academic Veterinary Hospital

Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in human hospitals. The prevalence of hospital-acquired MRSA (HA-MRSA) infection among inpatients in intensive care units (ICUs) continues to increase steadily in Japan. Recently, cases of community-acquired MRSA (CA-MRSA) have been documented in persons without an established risk factor for HA-MRSA infection (14, 32, 36, 49).There has also been an increase in the number of reports of the isolation of MRSA from veterinarians and companion animals (5, 21, 23-26, 28, 31, 34, 38, 44, 50, 51, 53). Values reported for the prevalence of MRSA among veterinary staff include 17.9% in the United Kingdom (21), 10% in Japan (38), 3.9% in Scotland (13), and 3.0% in Denmark (28). Loeffler et al. reported that the prevalence of MRSA among dog patients and healthy dogs owned by veterinary staff members was 8.9% (21). In Japan, an MRSA isolate was detected in only one inpatient dog (3.8%) and could not be detected in any of 31 outpatient dogs (38). In the United States, MRSA isolates were detected in both dog (0.1%) and cat (0.1%) patients (31). The prevalence of MRSA among healthy dogs has been reported to be 0.7% (5). Hanselman et al. suggested that MRSA colonization may be an occupational risk for large-animal veterinarians (12). Recently, Burstiner et al. reported that the frequency of MRSA colonization among companion-animal veterinary personnel was equal to the frequency among large-animal veterinary personnel (6).In addition, other methicillin-resistant coagulase-positive staphylococci (MRCPS), such as methicillin-resistant Staphylococcus pseudintermedius (MRSP) and methicillin-resistant Staphylococcus schleiferi (MRSS), isolated from dogs, cats, and a veterinarian have been reported (11, 31, 38, 40, 52). MRSP isolates have also been detected among inpatient dogs (46.2%) and outpatient dogs (19.4%) in a Japanese veterinary teaching hospital (38). In Canada, however, MRSP and MRSS isolates were detected in only 2.1% and 0.5% of dog patients, respectively (11).Methicillin-resistant staphylococci produce penicillin-binding protein 2′, which reduces their affinity for β-lactam antibiotics. This protein is encoded by the mecA gene (48), which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is a mobile genetic element characterized by the combination of the mec and ccr complexes (16), and it is classified into subtypes according to differences in the junkyard regions (43). SCCmec typing can be used as a molecular tool (22, 27, 30, 33, 36, 55) for examining the molecular epidemiology of methicillin-resistant staphylococci.In this study, we investigated the occurrence and characteristics of MRCPS isolates in a veterinary hospital in order to establish the transmission route of MRCPS in a veterinary hospital and with a view to preventing the spread of MRCPS infection. In addition, we evaluated the factors associated with MRCPS. Further, as Heller et al. have reported the distribution of MRSA within veterinary hospital environments and suggested the necessity to review cleaning protocols of hospital environments (13), we also attempted to isolate MRCPS from environmental samples collected in a veterinary hospital for an evaluation of MRSA transmission cycle though environmental surfaces in the veterinary hospital.     

Staphylococcus aureus NrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of deoxyribonucleotides during the cell cycle (29). Three main classes of RNRs are known. Class I RNRs are oxygen-dependent enzymes, class II RNRs are oxygen-independent enzymes, and class III RNRs are oxygen-sensitive enzymes. Class I RNRs are divided into two subclasses, subclasses Ia and Ib.Staphylococcus aureus is a Gram-positive facultative aerobe and a major human pathogen (24). S. aureus contains class Ib and class III RNRs, which are essential for aerobic and anaerobic growth, respectively (26). The class Ib NrdEF RNR is encoded by the nrdE and nrdF genes: NrdE contains the substrate binding and allosteric binding sites, and NrdF contains the catalytic site for ribonucleotide reduction. The S. aureus nrdEF genes form an operon containing a third gene, nrdI, the product of which, NrdI, is a flavodoxin (5, 33). Many other bacteria such as Escherichia coli (16), Lactobacillus lactis (17), and Mycobacterium and Corynebacterium spp. possess class Ib RNR operons that contain a fourth gene, nrdH (30, 44, 50), whose product, NrdH, is a thiol-disulfide redoxin (16, 17, 40, 43, 49). More-complex situations are found for some bacteria, where the class Ib RNR operon may be duplicated and one or more of the nrdI and nrdH genes may be missing or located in another part of the chromosome (29).NrdH proteins are glutaredoxin-like protein disulfide oxidoreductases that alter the redox state of target proteins via the reversible oxidation of their active-site dithiol proteins. NrdH proteins function with high specificity as electron donors for class I RNRs (9, 16-18). They are widespread in bacteria, particularly in those bacteria that lack glutathione (GSH), where they function as a hydrogen donor for the class Ib RNR (12, 16, 17). In E. coli, which possesses class Ia and class Ib RNRs, NrdH functions in vivo as the primary electron donor for the class Ib RNR, whereas thioredoxin or glutaredoxin is used by the class Ia NrdAB RNR (12, 17). NrdH redoxins contain a conserved CXXC motif, have a low redox potential, and can reduce insulin disulfides. NrdH proteins possess an amino acid sequence similar to that of glutaredoxins but behave functionally more like thioredoxins. NrdH proteins are reduced by thioredoxin reductase but not by GSH and lack those residues in glutaredoxin that bind GSH and the GSH binding cleft (39, 40). The structures of the E. coli and Corynebacterium ammoniagenes NrdH redoxins reveal the presence of a wide hydrophobic pocket at the surface, like that in thioredoxin, that is presumed to be involved in binding to thioredoxin reductase (39, 40). NrdI proteins are flavodoxin proteins that function as electron donors for class Ib RNRs and are involved in the maintenance of the NrdF diferric tyrosyl radical (5, 33). In Streptococcus pyogenes, NrdI is essential for the activity of the NrdHEF system in a heterologous E. coli in vivo complementation assay (33). Class Ib RNRs are proposed to depend on two specific electron donors, NrdH, which provides reducing power to the NrdE subunit, and NrdI, which supplies electrons to the NrdF subunit (33).In this report we identify an open reading frame (ORF) in S. aureus encoding an NrdH-like protein with partial sequence relatedness to the E. coli, Salmonella enterica serovar Typhimurium, L. lactis, and C. ammoniagenes NrdH proteins. In contrast to these bacteria, the S. aureus nrdH gene does not form part of the class Ib RNR operon. The S. aureus NrdH protein differs in its structure from the canonical NrdH in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that in vitro, S. aureus NrdH reduces protein disulfides and is an electron donor for the homologous class Ib NrdEF ribonucleotide reductase.     

Diverse Enterotoxin Gene Profiles among Clonal Complexes of Staphylococcus aureus Isolates from the Bronx,New York

Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillin-resistant and methicillin-sensitive S. aureus isolates, as well as wound- and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.As a commensal, Staphylococcus aureus colonizes the nasal mucosa of 20 to 40% of humans (54), and as a pathogen it causes pyogenic diseases and toxin-mediated diseases (38). S. aureus produces many different virulence factors, including enterotoxins (SEs), which can cause defined toxic shock syndromes (4). The characterization of some of these toxins led to the discovery of superantigens (41), which bind to major histocompatibility complex class II molecules and Vβ chains of T-cell receptors, resulting in the activation of large numbers of T cells (20 to 30%) and massive cytokine production (10, 18). These superantigen-induced “cytokine storms” are responsible for the toxic effects seen in staphylococcal entertoxin B (SEB)- and toxic shock syndrome toxin (TSST)-associated shock syndromes in S. aureus infections (13, 40, 47). To date, 19 SEs have been identified based on sequence homologies, and studies have reported enterotoxin genes in up to 80% of all S. aureus strains (4, 21). Although many new enterotoxins have been identified, i.e., seg ser and seu (33, 37, 44, 49), their precise functions have not been characterized yet. The majority of experimental work with SEs is still done with SEB, toxic shock syndrome toxin 1, and SEA (27, 31), because these toxins are commercially available. Most SEs are located on mobile elements in bacterial genomes such as plasmids or pathogenicity islands and can thus be easily transferred horizontally between strains (5, 34, 35). Certain SE genes are grouped together. For instance seg, sei, sem, sen, and seo are commonly found in a gene cluster (egc) on genomic island νSAβ (34), and sel and sek are often found together with seb or sec on S. aureus pathogenicity islands. Other staphylococcal superantigen genes are encoded on plasmids (sed, sej, and ser) or are linked to the antibiotic resistance cassette SCCmec (seh) (44, 55). Phage φ3 carries either sea (strain Mu50), sep (N315), or sea sek seq (MW2) (1, 29).Although a few clinical studies have attempted to correlate shock and outcome with the presence of certain SEs in patients with S. aureus infections (17, 28), the contribution of these toxins to outcome is still unclear. Recent papers have proposed the SEs are immunomodulators and that colonization with S. aureus strains that produce SEB may contribute to the pathogenesis of asthma, chronic rhinitis, and dermatitis (2, 36, 46, 48, 56). The superantigen function of SEs in supernatants of S. aureus cultures can be neutralized by serum of colonized patients (21, 23). With new data emerging implicating SEs in the pathogenesis of chronic allergic syndromes, production of monoclonal antibodies and or vaccine strategies targeting SEs may be considered (6, 24, 26, 30) in the future. It is therefore important to characterize the prevalence of SE genes in clinical S. aureus strains.In this study, we analyzed SE content in both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains that were cultured from wounds (including USA300) and bloodstream infections of patients from a defined geographical area. In addition, SEB production was quantified by enzyme-linked immunosorbent assay (ELISA) in S. aureus strains carrying the seb gene, and spa typing confirmed clonal diversity among S. aureus isolates from different patients, as well as clonal stability in serial isolates, and multilocus sequence typing (MLST) done on a subset of less common spa types. We conclude that SE genes are abundant in S. aureus strains, albeit less abundant in USA300. SE content and combination are highly diverse and therefore more discriminatory than pulsed-field gel electrophoresis (PFGE) and MLST typing, albeit stable in serial isolates. Quantification of SEB production demonstrates that enterotoxin secretion can vary greatly among strains, even if they belong to the same S. aureus lineage. Given the complexities of SE prevalence, regulation, and possible function, we propose that the association of these toxins with chronic allergic diseases or outcome may be oversimplified at present. Precise characterizations of SE function and secretion patterns in individual S. aureus clones are warranted.     

Integrative and Sequence Characteristics of a Novel Genetic Element,ICE6013, in Staphylococcus aureus

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant Staphylococcus aureus (MRSA) revealed a novel genetic element, ICE6013. The element is 13,354 bp in length, excluding a 6,551-bp Tn552 insertion. ICE6013 is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced S. aureus strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE6013 were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE6013 is predicted to encode 15 open reading frames, including an IS30-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE6013 is more closely related to ICEBs1 from Bacillus subtilis than to the only other potential integrative conjugative element known from S. aureus, Tn5801. Evidence of recombination between ICE6013 elements is also presented. In summary, ICE6013 is the first member of a new family of active, integrative genetic elements that are widely dispersed within S. aureus strains.ST239 is a globally distributed clonal group of methicillin-resistant Staphylococcus aureus (MRSA). Currently, ST239 is a major cause of MRSA infections in Asian hospitals (5, 18, 25, 37, 45, 64, 74). Pulsed-field gel electrophoresis has detected extensive chromosomal variation in local ST239 populations (3, 24, 52, 72). As ST239 has geographically spread and diversified, its variants have been given more than a dozen different names (20, 22, 24, 25, 49, 52, 61, 67, 68, 73), which reflects their clinical significance in various locales. The molecular basis for the ecological success of ST239 is unclear, but virulence-associated traits such as enhanced biofilm development and epidemiological characteristics such as a propensity to cause device-associated bacteremia and pulmonary infections have been highlighted (3, 19, 27, 54).Multilocus genetic investigations of the ST239 chromosome revealed that it is a hybrid with estimated parental contributions of approximately 20% and 80% from distantly related ST30- and ST8-like parents, respectively (58). Unusual for naturally isolated bacteria was the finding that these parental contributions were large chromosomal replacements rather than a patchwork of localized recombinations. It was postulated that conjugation might be responsible for the natural transfer of hundreds of kilobases of contiguous chromosomal DNA that resulted in ST239 (58). Recent genomic investigations have presented evidence that large chromosomal replacements also occur within Streptococcus agalactiae strains and that they can be mimicked with laboratory conjugation experiments (12). Importantly, conjugative transfer frequencies in S. agalactiae were found to be highest near three genomic islands (12), two of which were identified as being integrative conjugative elements (ICEs) (13).ICEs and conjugative transposons are synonyms and refer to genetic elements that are maintained by integration into a replicon and are transmitted by self-encoded conjugation functions (56). ICEs abound in the genomes of S. agalactiae (11), but only one potential ICE has been identified in staphylococci to date: Tn5801 was discovered through the genomic sequencing of S. aureus strain Mu50 (46). Tn5801 is most similar to a truncated genetic element, CW459tet(M), from Clostridium perfringens (57). Both Tn5801 and CW459tet(M) have Tyr recombinases, regulatory genes, and tetM modules that are similar to those of the prototypical gram-positive conjugative transposon, Tn916. Moreover, both Tn5801 and CW459tet(M) integrate into the same locus, guaA, at a nearly identical 11-bp sequence. Although the conjugative transfer module of CW459tet(M) is deleted (57), the conjugative transfer module of Tn5801 is similar to that of Tn916.We suspected that ST239 strains might carry novel accessory genes that contribute to their chromosomal variation and ecological success. To explore this possibility, we conducted a survey of chromosomal variation in ST239 using a PCR scanning approach. We report the discovery and partial characterization of a novel genetic element, ICE6013, that resulted from the survey.     

Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.Staphylococcus aureus is a gram-positive bacterium that lives as part of the normal microflora on the skin and mucous membranes of humans and animals. If S. aureus passes through the epithelial barrier and reaches internal organs, it can cause a variety of diseases, ranging from minor skin infections, such as furuncles or boils, to severe infections, such as bacteremia, pneumonia, osteomyelitis, or endocarditis. Despite the progress with antibiotics in the treatment of bacterial infections over the last 2 decades, the number of infections due to S. aureus has increased (11, 30). The infection rate has been correlated with an increase in the use of prosthetic and indwelling devices in modern medical practices (24, 26). S. aureus, as well as other coagulase-negative staphylococci, displays a strong capacity to irreversibly attach to the surface of implanted medical devices and forms multilayered communities of bacteria, known as biofilms, that grow embedded in a self-produced extracellular matrix (23). The biofilm formation process occurs in two steps: first, bacterial cells irreversibly attach to a surface, and second, they interact with each other and accumulate in multilayered cell clusters embedded in a self-produced extracellular matrix. Primary attachment is mediated by physico-chemical cell surface properties as well as specific factors that mediate the attachment to the host-derived extracellular matrix components that rapidly coat the biomaterial following insertion into the patient. Numerous proteins from the MSCRAMMs family (microbial surface components recognizing adhesive matrix molecules) are involved in the first step of S. aureus biofilm formation, such as clumping factors ClfA (37) and ClfB (41) and fibrinogen and fibronectin binding proteins (FnBPA and FnBPB) (25, 31). Once bacteria accumulate in multilayered cell clusters, most have no direct contact with the surface, and thus cell-to-cell interactions become essential for biofilm development and maintenance. An extracellular polysaccharide intercellular adhesin (PIA, or PNAG), produced by icaADBC operon-encoded enzymes, is currently the best-characterized element mediating intercellular interactions in vitro (8, 23, 34, 35, 38). Alternatively, a number of surface proteins can replace PIA/PNAG exopolysaccharide in promoting intercellular adhesion and biofilm development, including the surface protein Bap (9). All the tested staphylococcal isolates harboring the bap gene were shown to be strong biofilm producers, and inactivation of the icaADBC operon in bap-positive strains had no effect on in vitro biofilm formation (57). Remarkably, proteins homologous to Bap are involved in the biofilm formation process in diverse bacterial species (33). A second surface protein, SasG, as well as its homologous protein in Staphylococcus epidermidis, Aap, also mediates intercellular interactions and biofilm development in the absence of the ica operon (7, 51). More recently, two independent laboratories have shown that fibronectin binding proteins A and B (FnBPA and FnBPB) induce biofilm development of clinical isolates of S. aureus (45, 55). Finally, there is growing evidence that extracellular DNA, despite not being sufficient to replace PIA/PNAG exopolysaccharide, is an important S. aureus biofilm matrix component (50).During the course of a systematic mutagenesis study of the 17 two-component systems of S. aureus that aimed to identify biofilm-negative regulators, we found that S. aureus agr arlRS double mutants developed an alternative, ica-independent biofilm in a chemically defined medium, Hussain-Hastings-White (HHW) medium (56). This study focused on the identification of the proteinaceous compound responsible for the biofilm developed by S. aureus agr arlRS mutants. Here, we show that S. aureus protein A is responsible for the aggregative phenotype and capacity for biofilm formation displayed by this strain. Furthermore, overproduction of protein A in wild-type S. aureus strains or addition of soluble protein A to bacterial growth medium induced aggregation and biofilm development, suggesting that protein A does not need to be covalently linked to the cell wall to promote multicellular behavior. Moreover, deletion of the spa gene significantly decreased the capacity of S. aureus to colonize subcutaneously implanted catheters. Our findings support a novel role for protein A in promoting multicellular behavior and suggest that protein A-mediated biofilm development may have a critical function during the infection process of S. aureus.     

Functional Genomic Analysis of Two Staphylococcus aureus Phages Isolated from the Dairy Environment

The genomes of the two lytic mutant Staphylococcus aureus bacteriophages, vB_SauS-phiIPLA35 (phiIPLA35) and vB_SauS-phiIPLA88 (phiIPLA88), isolated from milk have been analyzed. Their genomes are 45,344 bp and 42,526 bp long, respectively, and contain 62 and 61 open reading frames (ORFS). Enzymatic analyses and sequencing revealed that the phiIPLA35 DNA molecule has 3′-protruding cohesive ends (cos) 10 bp long, whereas phiIPLA88 DNA is 4.5% terminally redundant and most likely is packaged by a headful mechanism. N-terminal amino acid sequencing, mass spectrometry, bioinformatic analyses, and functional analyses enabled the assignment of putative functions to 58 gene products, including DNA packaging proteins, morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, modification, and replication. Point mutations in their lysogeny control-associated genes explain their strictly lytic behavior. Muralytic activity associated with other structural components has been detected in virions of both phages. Comparative analysis of phiIPLA35 and phiIPLA88 genome structures shows that they resemble those of φ12 and φ11, respectively, both representatives of large genomic groupings within the S. aureus-infecting phages.Staphylococcus aureus is an important etiologic agent of food-borne diseases due to its ability to produce heat-resistant staphylococcal enterotoxins (SEs) when it grows in foods. In fact some S. aureus strains may produce up to 20 serologically distinct SEs, which could be responsible for food poisoning (30). SEs have been divided initially into serological types SEA through SEE, and recently the existence of new types of SEs has also been reported (5).S. aureus strains harboring enterotoxin genes have been isolated from a variety of foods (38) including dairy products (9, 46, 56). Mastitis caused by this pathogen and poor hygienic processing conditions are the most important sources of dairy product contamination. Growth of enterotoxigenic S. aureus in both raw milk and dairy products poses a potential health hazard to consumers. In this context, new biocontrol strategies to prevent growth of S. aureus, suitable to be applied in the food industry, are being explored.Currently, there is a renewed interest in exploiting the antimicrobial potential of bacterial viruses for bacterial-control applications in agriculture, aquaculture, and the food industry (11, 18, 23, 49). In fact, the use of phages for the treatment of infectious diseases (or phage therapy) has a long successful history in the countries of Eastern Europe (or former Soviet Union) (50). Specifically, S. aureus bacteriophages have been assayed in the treatment of venous leg ulcers and eye infections (22, 42).Prior to any phage application, genome analysis is a prerequisite to examine the safety of the phages, specifically, traits which might enhance the virulence of the infected bacterium. In addition, genome analysis might uncover novel antibacterial targets or agents (33) with promising biotechnological applications (6). For example, various lytic phage proteins (endolysins) have shown great potential in veterinary and human medicine for the treatment and prophylaxis of infections (12) and have been applied as biocontrol agents in dairy products (36). Several technologies employing phages and endolysins for pathogen detection and decontamination have also been patented (7).To date, genomes of over 47 S. aureus phages are available in public databases. The number of known, strictly lytic phages is limited to the close-knit Myoviridae genus of the SPO1-like viruses, containing phages K, Twort, and G1. Apart from this group, a large number of genomes from unclassified Siphoviridae in lysogenic S. aureus strains are available (26, 37). Some temperate bacteriophages may play an important role in the pathogenicity of S. aureus by carrying virulence factors, mediating lateral gene transfer, and even facilitating the adaptation of the pathogen during infection (1, 21, 52).In previous work, we have characterized phiIPLA35 and phiIPLA88 S. aureus phages (17). These two lytic phages, previously named φ35 and φ88, were selected as mutants of the temperate phages φA72 and φH5, respectively, isolated from raw bovine milk. They belong to the Siphoviridae family of double-stranded DNA bacterial viruses in the order Caudovirales. Remarkably, these phages infect S. aureus of bovine and dairy origin while clinical isolates appear to be resistant. Both phiIPLA35 and phiIPLA88 are very well adapted to the dairy environment and effectively inhibit S. aureus growth in milk and curd-manufacturing processes (17, 20).In this study, we have sequenced and annotated the genomes of both bacteriophages, elucidated their physical genome structures, and identified peptidoglycan hydrolytic activities. Comparative genome analysis also allowed us to put phiIPLA35 and phiIPLA88 into a phylogenetic context.