High Heterogeneity within Methicillin-Resistant Staphylococcus aureus ST398 Isolates,Defined by Cfr9I Macrorestriction-Pulsed-Field Gel Electrophoresis Profiles and spa and SCCmec Types

During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.Methicillin-resistant Staphylococcus aureus (MRSA) strains are an important cause of hospital-acquired infections worldwide (8). However, MRSA strains are not confined to health care settings, and during the last 10 years community-acquired MRSA has increasingly been reported (8). In 2003, a clone of MRSA associated with pig farming and not related to the traditional hospital- and community-acquired MRSA emerged in the Netherlands (37), where it now amounts to >30% of human MRSA cases (16). This clone has also been detected in healthy and sick animals, in food of animal origin, and in humans from other European countries, Canada, the United States, the Dominican Republic, and China (5, 7, 31, 38, 39). This emerging MRSA clone belongs to the multilocus sequence type ST398, which includes different spa types (mainly t011, t034, and t108). The majority of the ST398 isolates reported are MRSA, although methicillin-susceptible (MSSA) strains have been described as well (15, 34). Resistance to methicillin and other β-lactam antibiotics is caused by the mecA gene, which is located on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec). The SCCmec cassette consists of the mec gene complex, the ccr gene complex, and the junkyard regions. Based on the variability and combinations of these genetic elements, several types of SCCmec and several variants of the types have been described (9). Three SCCmec types (III, IVa, and V) were identified in ST398 isolates (25). However, recent investigations have shown that some ST398 isolates typed as SCCmec type III using the method of Zhang et al. (40) proved to be type V after further sequencing (21, 35).For typing S. aureus, pulsed-field gel electrophoresis (PFGE) of the whole genome by macrorestriction with the SmaI endonuclease is still considered as the “gold standard” (26). However, the isolates of the ST398 clone are nontypeable (NT) by PFGE using SmaI (3, 4). Consequently, comparison between these isolates and the typeable ones from humans and animals is not possible. The nontypeability is due to the action of a novel C5-cytosine methyltransferase which modifies the consensus sequence C^mCNGG at the second cytosine (3, 4). Other enzymes with a different recognition sequence from SmaI have been used for PFGE typing of the ST398 clone, including EagI and ApaI (22, 28, 31, 38), but the patterns obtained cannot be compared to S. aureus patterns generated with SmaI. XmaI, a neoschizomer of SmaI that recognizes the same sequence cutting at a different position, only generates partial digestions (3, 4). Recently, the use of Cfr9I, another neoschizomer of SmaI whose activity is not reduced on ST398 methylated DNA, has been recommended. This enzyme had been successfully used for typing SmaI NT macrolide-resistant Streptococcus pyogenes isolates (6, 30), and now it is being applied for typing ST398 isolates, i.e., from human origin (5, 11, 36) and, to a lesser extent, from animals (3, 36). The aim of this study was to characterize a large collection of recent ST398 isolates by Cfr9I PFGE as well as other methods (spa typing, multilocus sequence typing [MLST], and SCCmec typing). Most of them were recovered in Germany from different sources, including animals and foods.     

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.     

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.     

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.     

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.     

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.     

Genome Sequence of a Recently Emerged,Highly Transmissible,Multi-Antibiotic- and Antiseptic-Resistant Variant of Methicillin-Resistant Staphylococcus aureus,Sequence Type 239 (TW)

The 3.1-Mb genome of an outbreak methicillin-resistant Staphylococcus aureus (MRSA) strain (TW20) contains evidence of recently acquired DNA, including two large regions (635 kb and 127 kb). The strain is resistant to a wide range of antibiotics, antiseptics, and heavy metals due to resistance genes encoded on mobile genetic elements and also mutations in housekeeping genes.A 2-year outbreak of a highly transmissible methicillin-resistant Staphylococcus aureus (MRSA) strain (designated TW) in an intensive care unit (ICU) in London was recently reported (12). Acquisition of TW MRSA was four times more likely to be associated with bacteremia than was acquisition of other commonly found MRSA strains [>95% epidemic (E)MRSA-15 or EMRSA-16]. TW MRSA was also significantly more frequently isolated from vascular access device cultures but less frequently from carriage sites (anterior nares, axilla, and perineum), suggesting that TW differs in its colonization capacity from other MRSA strains. TW was initially defined by its extended antibiotic resistance pattern, being resistant to penicillin, methicillin, erythromycin, ciprofloxacin, gentamicin, neomycin, trimethoprim, and tetracycline (12). TW also had elevated minimum bactericidal concentrations (MBCs) for chlorhexidine and was resistant to a chlorhexidine-based antiseptic protocol effective against other MRSA strains in the ICU (6). TW20 (strain 0582) was a representative bacteremic isolate cultured on 21 October 2003 (12).Multilocus sequence typing (MLST) identified TW20 as sequence type 239 (ST239), an international health care-associated (HA) MRSA lineage prevalent in Asia (19, 38), South America (2, 37), and Eastern Europe (5, 33), which includes EMRSA-1, -4, -7, and -11 and the Brazilian, Portuguese, Hungarian, and Viennese clones (24). To investigate the genetic basis for increased resistance and transmissibility, the TW20 genome was completely sequenced, assembled, and finished and annotated as described previously (16, 25). The final finished genome (10) assembly contained 64,087 capillary reads, giving an average coverage of 13.3. At 3,075,806 bp, the TW20 genome is the largest S. aureus genome sequenced thus far. It consists of a single chromosome of 3,043,210 bp in size (Fig. ​(Fig.1)1) and 2 plasmids (pTW20_1 and pTW20_2), of 29,585 bp and 3,011 bp.Open in a separate windowFIG. 1.Schematic circular diagram of the S. aureus TW20 chromosome. Key for the circular diagram (outer to inner): outer colored segments on the gray outer ring represent genomic islands and horizontally acquired DNA (see the key in the figure); scale (in Mb); annotated CDSs colored according to predicted function are shown on a pair of concentric circles, representing both coding strands; S. aureus reciprocal Fasta matches shared with the S. aureus strains: MRSA252, (accession number {"type":"entrez-nucleotide","attrs":{"text":"BX571856","term_id":"49240382","term_text":"BX571856"}}BX571856) (16), MSSA476 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BX571857","term_id":"49243355","term_text":"BX571857"}}BX571857) (16), MW2 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BA000033","term_id":"47118312","term_text":"BA000033"}}BA000033) (4), N315 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BA000018","term_id":"47118324","term_text":"BA000018"}}BA000018) (20), Mu50 (accession number {"type":"entrez-nucleotide","attrs":{"text":"BA000017","term_id":"47208328","term_text":"BA000017"}}BA000017) (20), Mu3 (accession number {"type":"entrez-nucleotide","attrs":{"text":"AP009324","term_id":"156720466","term_text":"AP009324"}}AP009324) (23), COL (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000046","term_id":"57284222","term_text":"CP000046"}}CP000046) (13), NCTC8325 (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000253","term_id":"87201381","term_text":"CP000253"}}CP000253) (14), USA3000 FPR3757 (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000255","term_id":"87125858","term_text":"CP000255"}}CP000255) (11), JH9 (accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000703","term_id":"147739516","term_text":"CP000703"}}CP000703) (22), Newman (accession number {"type":"entrez-nucleotide","attrs":{"text":"AP009351","term_id":"150373012","term_text":"AP009351"}}AP009351) (3), and RF122 (accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ938182","term_id":"82655308","term_text":"AJ938182"}}AJ938182) (15); regions of the chromosome derived from a CC8 ancestor (light green) or the CC30 ancestor (brown). Color coding for TW20 CDS functions: dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, surface associated; cyan, degradation of large molecules; magenta, degradation of small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage and IS elements; gray, miscellaneous.TW20 belongs to clonal complex 8 (CC8), which contains strains NCTC8325 (ST8) (14), Newman (ST8) (3), USA300 (ST8) (11), and COL (ST250) (13). Comparative genomic analysis with these strains by reciprocal Fasta analysis (36) revealed that between 83.7 and 82.7% of protein coding sequences (CDSs) in the TW20 chromosome have reciprocal matches with CC8 members. The highest numbers of matches in any sequenced S. aureus strain, however, was with MRSA 252 (85.9% of CDSs). MRSA 252 (ST36) belongs to CC30 and is a representative of EMRSA-16 that has been a dominant MRSA clone in United Kingdom hospitals for more than 10 years (16). In comparison to CC8, most of the additional matches to MRSA 252 are to CDSs in horizontally acquired mobile genetic elements (MGEs) rather than to orthologous CDSs. A significant component of the S. aureus genome is derived from MGEs that contribute to the accessory genome (21). In the TW20 genome, 16.2% of the CDSs (12.6% of the total genomic DNA) are found in MGEs (Fig. ​(Fig.1).1). Both TW20 and MRSA 252 are representatives of successful hospital-associated MRSA lineages and have large accessory genomes that contain many of the CDSs associated with drug resistance.Methicillin resistance is conferred by a mecA gene on a type III staphylococcal cassette chromosome (SCC) mec element (SCCmecIII). TW20 has a composite SCC region of two SCC elements, SCCmercury and SCCmecIII, identical in structure to the type III SCCmec region found by Ito et al. (18) in an isolate from New Zealand in 1985. The SCCmecIII region is present in a part of the chromosome hypothesized to have been transferred from CC30 into a CC8 background as part of a large block of DNA (26). The approximate boundaries of the recombination were identified from pairwise comparisons of the TW20 chromosome with MRSA 252 (CC30) and USA300 TCH1516 (CC8). A marked shift in DNA percent identity of approximately 1 percentage point was observed across the approximate recombination breakpoints (data not shown), demonstrating that 635 kb (∼20.6% of the TW20 chromosome; SATW20_26800 to SATW20_03960) may have been transferred from a CC30 donor. This transfer event also contributes to the high level of reciprocal Fasta matches between TW20 and MRSA252 (ST36).The origins of SCCmecIII in the TW20 genome are unclear, since SCCmecIII has not been found in the CC30 lineage. Each of the SCC elements contains further MGEs: SCCmercury contains Tn554, encoding a streptomycin 3′-adenylyltransferase and an erythromycin resistance protein, ErmA1, and SCCmec contains an integrated plasmid, pT181, and ΨTn554, containing cadmium resistance CDSs. In addition to Tn554 and ΨTn554 in the SCCmec region, the TW20 chromosome contains an additional Tn554 and a Tn552 transposon, encoding the β-lactamase BlaZ, within an integrative conjugative element (ICE) (31).Further resistance determinants are found on plasmid pTW20_1. Importantly, it carries a gene encoding an antiseptic resistance protein, QacA, that confers resistance to antiseptics such as cationic biocides, quaternary ammonium salts, and diamidines via an export-mediated mechanism (29). In addition, part of the plasmid is highly similar (98 to 100% DNA identity) to the mer operon of the SCCmercury region found on the chromosome (Fig. ​(Fig.2).2). pTW20_1 also contains a homologue of the gene encoding the cadmium-transporting ATPase CadA, found in ΨTn554 of SCCmec. This region in pTW20_1 is bordered by IS431 elements, as it is in the chromosomal copy of SCCmercury. Notably, upstream of the SCCmercury mer operon, there is a CDS that encodes a putative NADH-binding protein. A fragment homologous to the 3′ end of this CDS is also present on pTW20_1 upstream of the mer operon and is truncated by an IS431 element. The absence of the 5′ region of this CDS on pTW20_1 suggests that this region, including the mer operon, may have arisen on the plasmid by recombination between chromosomal and plasmid IS431 elements. It is therefore possible that IS431-mediated recombination plays a role in the evolution of the SCC region.Open in a separate windowFIG. 2.Comparative analysis of the TW20 plasmid pTW20_1 with the mer operon of the TW20 SCC region. Pairwise comparisons of the TW20 SCC region containing the mer operon from the TW20 chromosome (top) with the TW20 plasmid pTW20_1 (bottom) using the Artemis Comparison Tool (ACT) (9) are shown. The colored bars separating each sequence (red and blue) represent matches identified by BlastN (1); red lines link matches in the same orientation, and blue lines link matches in the reverse orientation. CDSs associated with metal and drug resistance are marked, as are IS431 elements. Colored bars at the top of the figure indicate parts of the sequence found in the SCCmercury (blue) and SCCmec (green) elements, including ΨTn554 (yellow), that make up this region.Two other drug resistance genes, encoding a tetracycline resistance protein, TetM, and a trimethoprim-resistant dihydrofolate reductase, DfrG, are found in a 31.3-kb region (Tn5801-like), similar to transposons/ICE found in the genomes of S. aureus strains Mu50 (20) and Mu3 (23) and Streptococcus agalactiae strain COH1 (35). In comparison to the Tn5801 elements in Mu50 and Mu3, the TW20 element contains an additional four CDSs, including dfrG, in the central region of the element.There are three prophage within the TW20 genome, two of which are similar to those previously found in sequenced S. aureus genomes: φSa1(TW20) is 43.3 kb in size, is integrated within the 5′ region of a lipase gene, and does not carry CDSs with homology to known virulence factors; φSa3(TW20) is 44.7 kb in size, is integrated in the phospholipase C gene, and carries the staphylococcal complement inhibitor SCIN (28), staphylokinase (30), and enterotoxin A (7) genes associated with virulence. Genes for two other enterotoxins, enterotoxins K and Q, are carried on a Staphylococcus aureus pathogenicity island (SaPI), SaPI1.At 127.2 kb, the third prophage, φSPβ-like(TW20), is markedly larger than the other two and does not display similarity with other S. aureus prophage. φSPβ-like(TW20) exhibits extended similarity with the φSPß-like region in the Staphylococcus epidermidis RP62a genome (13) (Fig. ​(Fig.3).3). Comparison of the two sequences reveals a region of sequence divergence and rearrangement in the center of the prophage. In φSPβ-like(TW20), this region contains CDSs associated with aminoglycoside resistance and streptothricin resistance (Fig. ​(Fig.3).3). In addition, φSPβ-like(TW20) contains a CDS that may have a role in promoting persistence of TW20 in the hospital setting. S. aureus possesses many surface-anchored proteins with the LPxTG motif, which bind host molecules (27). SATW20_21850 encodes an LPxTG motif surface-anchored protein that does not have orthologs in any of the genomes of the other sequenced S. aureus strains currently available. A highly similar CDS (95.1% amino acid identity), sesI (8), is present in the S. epidermidis φSPβ-like region (Fig. ​(Fig.3).3). A recent study by Söderquist et al. found that sesI was absent from normal S. epidermidis flora of healthy individuals without any health care association but was found in approximately 50% of clinical isolates causing invasive infections, leading them to suggest that this gene was a potential marker of invasive capacity (32). The presence of an LPxTG motif surface-anchored protein on an MGE in TW20 suggests that this strain has augmented its array of this family of functionally important proteins through a recent acquisition event and therefore this LPxTG motif surface-anchored protein may not be widely distributed in related strains. Genome sequencing of a global collection of ST239 strains revealed only 7% (3/42) of isolates were positive for orthologs of this CDS (14a). Work is under way to survey the wider distribution of this gene in the S. aureus population and investigate the function of the encoded protein.Open in a separate windowFIG. 3.Comparative analysis of φSPβ-like(TW20) prophage with the S. epidermidis RP62a φSPß-like prophage. Pairwise BlastN comparison of the S. aureus TW20 prophage φSPβ-like(TW20) region from the TW20 chromosome (top) with the S. epidermidis RP62a φSPß-like prophage region from the RP62a chromosome (bottom) (13) displayed in ACT is shown. The extent of the φSPβ-like(TW20) prophage in the TW20 sequence, which extends from SATW20_20290 to SATW20_21850, is marked by the pink horizontal bar.Evidence of adaptation to survive in a health care environment is also found in the core genome. Several housekeeping genes have alleles associated with antibiotic resistance. The TW20 DNA gyrase subunit A (GyrA) contains a leucine residue at position 84. The more-widespread residue in S. aureus GyrA proteins is serine, suggesting this is the plesiomorphic amino acid at this position. In vitro studies have demonstrated that substitution of Ser84Leu generates resistance to quinolones in S. aureus (34). TW20 exhibits low-level resistance to mupirocin. The TW20 isoleucyl-tRNA synthetase contains a phenylalanine residue at position 588. The substitution of Val588Phe has been shown to confer chromosomal low-level mupirocin resistance in S. aureus without significantly affecting fitness (17).In conclusion, genomic analysis of TW20 provides evidence of its adaptation to survive in a health care setting through acquisition of drug and antiseptic resistance genes carried on MGEs, large chromosomal insertions, and point mutations in housekeeping genes. The large size of the TW20 genome reflects the ability of the ST239 lineage to undergo prolonged and continuing evolution to adapt to the hospital environment. Further studies are under way to elucidate the components of the genome that promote transmission and interaction with the host.     

Roles of CcrA and CcrB in Excision and Integration of Staphylococcal Cassette Chromosome mec,a Staphylococcus aureus Genomic Island

The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci, mecA, is carried on a genomic island, SCCmec (for staphylococcal cassette chromosome mec). The chromosomal excision and integration of types I to IV SCCmec are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCCmec. Purified CcrB but not CcrA was shown to mediate the gel shift of chromosomal target integration sequences (attB) in electrophoretic mobility shift assays. However, preincubation of CcrB-DNA complexes with increasing concentrations of CcrA blocked gel shift. The interaction of CcrB and CcrA was confirmed by Escherichia coli two-hybrid analysis. SCCmec excision mediated by plasmid-encoded and inducible ccrA, ccrB, or both genes was assessed by PCR in Staphylococcus aureus. CcrB alone could mediate excision but excision was at an alternate att site (attR2) within the right extremity of SCCmec. In contrast, both CcrB and CcrA were required to mediate excision at the chromosomal attB site (called attR when SCCmec is integrated). Insertion of a plasmid containing the SCCmec att site (attS) into the chromosome required both CcrA and CcrB, but CcrA overexpression lowered integration frequency. Thus, while CcrB binds DNA, interaction between CcrA and CcrB, in a precise ratio, is required for attB site-specific excision and SCCmec chromosomal insertion.Staphylococcus aureus is one of the most common causes of serious human bacterial infections, both in the hospital and the community (33). Therapy of these infections is made more difficult by the development of resistance to drugs with antistaphylococcal activity such as the beta-lactam antibiotics. Resistance to beta-lactam antibiotics in staphylococci is mediated by a beta-lactamase and by a beta-lactam-resistant target transpeptidase, penicillin-binding protein 2a (PBP2a) (4, 5, 8). However, while the beta-lactamase has a narrow substrate specificity, limited to penicillins, PBP2a resists inactivation by all beta-lactam antibiotics and can cross-link peptidoglycan when all other target PBPs are rendered nonfunctional by beta-lactams. The latter is called methicillin resistance and is the most important clinical resistance phenotype among staphylococci (8) The gene for PBP2a, mecA, is located on a genomic island called SCCmec (for staphylococcal cassette chromosome mec) that is integrated into the staphylococcal chromosome at a specific site. In addition to mecA, all SCCmec elements carry intact or mutant mecA regulators (mecR1/mecI) and genes that mediate the site-specific integration and excision of SCCmec (ccr genes) (14). SCCmec elements have been typed according to the sequences of the ccr and mec complexes with five cores (types I to V) being prevalent but with considerable variation in the genetic organization within each element (14-17, 22).SCCmec is presumed to be a mobile genetic element, which can integrate into and excise from the chromosome by site-specific recombination between a site on SCCmec (attS) and one on the chromosome (attB). attB comprises the last 15 bp of a highly conserved gene called orfX that is located near the S. aureus origin of replication (15, 19). When SCCmec is inserted, the attB sequence is duplicated at the other end of the element with the site in orfX now called attR and the one abutting the non-orfX end of SCCmec designated attL. When SCCmec excises, the attB site is reconstituted in the chromosome and the two ends of the element come together to form attS within a nonreplicating circular version of SCCmec.The site-specific recombination of SCCmec is catalyzed by its encoded ccr recombinases, CcrA and CcrB for types I to IV and CcrC for type V. CcrA and CcrB belong to a family of large serine invertase and resolvases which consist of resolvases, invertases, phage integrases, and transposases (6, 10, 29, 31). All of them contain a conserved catalytic motif and some contain DNA-binding domains at either the N or the C terminus. The catalytic domains can either function as both integrases and excisases or as only integrases that require additional proteins to mediate excision (6, 29, 30, 31).The ccrA and ccrB genes are part of two-gene operons of 1,350 and 1646 bp in S. aureus strain N315 encoding proteins of 52.6 and 62.7 kDa, respectively. Although there is considerable variation at the amino acid level among the CcrA and CcrB proteins found in types I to IV SCCmec, plasmid-encoded CcrA and CcrB recombinases from each type can excise SCCmec from any of the others (23). However, CcrC can only excise type V SCCmec (16). There has been little examination of the role of each of these proteins in recombination or in DNA binding. In the present study we sought to define the precise roles of CcrA and CcrB in DNA binding and in the excision and integration of SCCmec in S. aureus. This is the first step in understanding the host range of SCCmec and how it may move among staphylococcal isolates in nature.     

Characterization of the Structure and Biological Functions of a Capsular Polysaccharide Produced by Staphylococcus saprophyticus

Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections in women. S. saprophyticus strain ATCC 15305 carries two staphylococcal cassette chromosome genetic elements, SCC_15305RM and SCC_15305cap. The SCC_15305cap element carries 13 open reading frames (ORFs) involved in capsular polysaccharide (CP) biosynthesis, and its G+C content (26.7%) is lower than the average G+C content (33.2%) for the whole genome. S. saprophyticus strain ATCC 15305 capD, capL, and capK (capD_Ssp, capL_Ssp, and capK_Ssp) are homologous to genes encoding UDP-FucNAc biosynthesis, and gtaB and capI_Ssp show homology to genes involved in UDP-glucuronic acid synthesis. S. saprophyticus ATCC 15305 CP, visualized by immunoelectron microscopy, was extracted and purified using anionic-exchange and size exclusion chromatography. Analysis of the purified CP by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy and gas-liquid chromatography revealed two types of branched tetrasaccharide repeating units composed of the following: Sug represents two stereoisomers of 2-acetamido-2,6-dideoxy-hexos-4-ulose residues, one of which has an arabino configuration. The encapsulated ATCC 15305 strain was resistant to complement-mediated opsonophagocytic killing by human neutrophils, whereas the acapsular mutant C1 was susceptible. None of 14 clinical isolates reacted with antibodies to the ATCC 15305 CP. However, 11 of the 14 S. saprophyticus isolates were phenotypically encapsulated based on their resistance to complement-mediated opsonophagocytic killing and their failure to hemagglutinate when cultivated aerobically. Ten of the 14 clinical strains carried homologues of the conserved staphylococcal capD gene or the S. saprophyticus gtaB gene, or both. Our results suggest that some strains of S. saprophyticus are encapsulated and that more than one capsular serotype exists.Approximately 13 million women develop urinary tract infections (UTIs) annually in the United States, with a recurrence rate between 25% and 44% (45). Staphylococcus saprophyticus is second only to Escherichia coli as a cause of uncomplicated UTI in young women (45, 46). A novobiocin-resistant member of the coagulase-negative staphylococci (60), S. saprophyticus has rarely exhibited resistance to other antibiotics (25). However, a recent report (19) indicated that methicillin-resistant S. saprophyticus isolates have emerged in Japan. The gastrointestinal tract and the vagina are the major reservoirs of S. saprophyticus (18, 30) and the likely sources of recurrent infection (20, 37, 49). Approximately 40% of patients with S. saprophyticus UTI present with acute pyelonephritis (22, 30). These patients experience symptoms more severe than those of patients infected by E. coli (24), and they are more likely to develop recurrent infections (21).A number of potential virulence factors have been identified in S. saprophyticus. Gatermann et al. showed that in a rodent model of ascending UTI, the production of urease contributes to S. saprophyticus growth and pathogenicity in the bladder (10, 12). Other putative virulence factors of S. saprophyticus include a surface-associated lipase (11, 51, 53), the collagen binding protein SdrI (52), and a cell wall-anchored hemagglutinin protein that mediates the binding of S. saprophyticus to sheep erythrocytes, fibronectin, and human uroepithelial cells (14, 29, 34, 35). The hemagglutinin was dubbed UafA in the sequenced ATCC 15305 strain, and deletion of the uafA gene resulted in reduced S. saprophyticus hemagglutination (HA) and adherence to human bladder carcinoma cells (29). Kuroda et al. noted that UafA-mediated adherence of S. saprophyticus to the T24 cell line was inhibited by the presence of the ATCC 15305 polysaccharide capsule (29).Staphylococcal species produce a variety of extracellular glycopolymers that contribute to the surface properties and virulence of the bacterium, such as capsular polysaccharides (CP), teichoic acids, and poly-N-acetylglucosamine (PNAG). CP production renders Staphylococcus aureus resistant to opsonophagocytic killing; alanine modifications of teichoic acids promote bacterial resistance to antimicrobial peptides (40); and PNAG is involved in biofilm formation (4). Recently, the secretion of another anionic polymer (poly-γ-dl-glutamic acid) by certain other coagulase-negative staphylococci was reported (28). Polyglutamic acid production is enhanced under high-salt conditions and may contribute to the survival of Staphylococcus epidermidis on human skin.S. saprophyticus strain 15305 does not produce PNAG or polyglutamic acid (28, 29), but this uropathogenic species is encapsulated. CP are lacking in isolates of S. epidermidis, the most common of the coagulase-negative species, but genomic evidence indicates that Staphylococcus haemolyticus (7, 57), S. saprophyticus (29), and Staphylococcus carnosus (47) carry capsule loci with genetic similarity to the Staphylococcus aureus cap5 (cap8) gene locus. In this study, we purified and characterized the CP produced by S. saprophyticus ATCC 15305 and investigated the CP phenotype of S. saprophyticus clinical isolates.     

Production of the Bsa Lantibiotic by Community-Acquired Staphylococcus aureus Strains

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.Staphylococcus aureus can be a human commensal bacterium, colonizing the skin and mucosal surfaces such as the nares, pharynx, and vagina in approximately 25 to 40% of the population. However, it is also a human pathogen that can cause epidemics of invasive disease. Genome sequencing of S. aureus strains has highlighted that the species is highly clonal, with approximately 78% of the genes being conserved and representing the core genome. The remaining 22% of the genes, which are variable and include those present on genomic islands, pathogenicity islands, prophages, integrated plasmids, and transposons, can in turn be regarded as an accessory genome (for a review, see reference 19) that provides a means via which S. aureus can evolve to adapt to particular niches and environmental pressures. The environmental pressure that has most strongly influenced S. aureus evolution in the past century has been the development and application of different antibiotics. These advancements have dictated that the strains that have flourished in hospitals, most notably hospital-acquired methicillin-resistant S. aureus (HA-MRSA) strains, tend to be multidrug resistant but suffer from a concomitant reduction in fitness relative to isolates from the community, due to being encumbered with staphylococcal cassette chromosome mec (SCCmec) types I to III and additional antibiotic resistance genes (48, 55). The negative consequences of this reduction in fitness are, however, mitigated by the reduction in competition from the human commensal microbiota by antibiotic exposure.Since the late 1990s, MRSA infections have been detected among the general population and among healthy individuals (typically children and young adults) who lack traditional risk factors (26). It was apparent that the S. aureus strains responsible for these community-acquired MRSA (CA-MRSA) infections were genetically distinct from their HA counterparts, possessing the more simple type IV (and to a lesser extent, type V and VII) allelic versions of SCCmec (13, 55) and fewer antibiotic resistance genes (20). While this fact indicated that these strains might represent less of a health care challenge than the HA strains, it quickly became apparent that the enhanced competitiveness of these strains, resulting in rapid growth (CA-MRSA strains grow much faster than HA-MRSA strains) (4) and increased virulence (67) of CA-MRSA, meant that any delay in switching from the β-lactam antibiotics normally used to treat infections of unknown etiology could have very serious medical implications, including death. Indeed, paradoxically, CA-MRSA strains have since spread to hospitals and have been responsible for a number of infections.In contrast to HA-MRSA strains, which by virtue of their multidrug-resistant nature, coupled with exposure to antibiotics, have a selective advantage over other microorganisms in the hospital environment, CA-MRSA strains, like commensal S. aureus strains, often face stiff competition from the natural flora of healthy individuals. It has been speculated that the production of an antimicrobial compound may provide CA-MRSA isolates with a competitive advantage in such environments (4, 14). The theory was first suggested when sequencing of strain FPR3757 (part of the virulent USA300 clonal group) revealed the presence of bsa (bacteriocin of S. aureus) genes, which resembled those associated with production of the epidermin subgroup of lantibiotics (2, 60). Lantibiotics are ribosomally produced, posttranslationally modified peptide antibiotics that are generally active against bacterial species which are closely related to the producing organism, and these antimicrobials are thought to have a role in niche competition in many natural environments (41). Lantibiotics have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications (10, 72). Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, there have also been a few examples of lantibiotic production by pathogens (11, 46, 69). In this instance, despite the identification of the bsa genes, the production of a lantibiotic by CA-MRSA isolates has remained speculative. Indeed, to date, there has been only one confirmed example of a lantibiotic, i.e., staphylococcin C55 (46), produced by S. aureus and no definitive evidence that CA- (or HA)-MRSA strains produce such compounds. There is, however, some evidence to suggest that staphylococcin Au-26, which is produced by a vaginal isolate of S. aureus and has an inhibitory spectrum encompassing lactobacilli isolated from the endocervix and representative strains of Staphylococcus hominis, Staphylococcus warneri, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus mutans, Lactococcus spp., and oral Neisseria spp., may also be a lantibiotic (63). Here, 17 years after its initial characterization, we have carried out a closer inspection of staphylococcin Au-26 and the associated producer and have established that the staphylococcin Au-26 and Bsa genetic loci are almost identical. Prompted by this finding, we employed a combination of mutagenesis and mass spectrometry (MS) to reveal that these genes are functional in a number of other staphylococci, including a large percentage of CA-MRSA isolates. We suggest that, as a consequence of eliminating competing human microbiota, this lantibiotic contributes strongly to the fitness of these community-associated isolates.     

Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.The production of biofuels from nonfood cellulosic biomass would benefit the economy, the environment, and national energy security (17, 32). The largest technological and economical obstacle is the release of soluble fermentable sugars at prices competitive with those from sugarcane or corn kernels (17, 31). One of the approaches is discovering new cellulases from cellulolytic microorganisms, followed by cellulase engineering for enhanced performance on pretreated solid substrates. However, cellulase engineering remains challenging because enzymatic cellulose hydrolysis is complicated, involving heterogeneous substrates (33, 37), different action mode cellulase components (18), synergy and/or competition among cellulase components (36, 37), and declining substrate reactivity over the course of conversion (11, 26). Directed enzyme evolution, independent of knowledge of the protein structure and the enzyme-substrate interactions (6, 34), has been conducted to generate endoglucanase mutants, such as enhanced activities on soluble substrates (14, 16, 22), prolonged thermostability (20), changed optimum pH (24, 28), or improved expression levels (21). Here, we cloned and characterized a family 5 glycoside hydrolase (Cel5A) from a cellulolytic bacterium, Clostridium phytofermentans ISDg (ATCC 700394) (29, 30), and engineered it for enhanced thermostability.     

Complete Genome Sequence of Staphylococcus aureus Strain JKD6159, a Unique Australian Clone of ST93-IV Community Methicillin-Resistant Staphylococcus aureus

Community methicillin-resistant Staphylococcus aureus (cMRSA) is an emerging issue that has resulted in multiple worldwide epidemics. We report the first complete genome sequence of an ST93-MRSA-IV clinical isolate that caused severe invasive infection and a familial outbreak of skin infection. This isolate is a representative of the most common Australian clone of cMRSA that is more distantly related to the previously sequenced genomes of S. aureus.Staphylococcus aureus is a major cause of both hospital- and community-acquired infections, with rapid emergence of antibiotic resistance, in particular methicillin resistance, adding complexity to the treatment of this organism (3). While previously a hospital problem, methicillin-resistant S. aureus (MRSA) is now being increasingly documented in healthy patients in the community, and these isolates are termed “community MRSA” (cMRSA). A number of cMRSA genomes have been sequenced; however, these are phylogenetically closely related to each other. In contrast, ST93-MRSA-IV, a unique Australian clone, is a singleton by multilocus sequence typing (MLST) eBURST analysis (4). It is now the dominant cMRSA clone in Australia and is associated with both skin infection and severe invasive infection, including necrotizing pneumonia, deep-seated abscesses, and septicemia (5, 10). JKD6159 is a representative ST93-MRSA-IV clinical isolate which caused septicemia and multifocal pulmonary and musculoskeletal abscesses in a previously well intravenous drug user and also resulted in a familial outbreak of skin infection.The genome sequence of S. aureus strain JKD6159 was determined by high-throughput whole-genome shotgun sequencing, using both Illumina GAII (Illumina, CA) and Roche GS FLX Titanium (Roche Diagnostics, Basel, Switzerland) sequencing technologies, producing approximately 164× and 32× coverage of the genome, respectively. The GS FLX Titanium reads were assembled using Newbler 2.0.01.12, resulting in 56 contigs totaling 2.8 Mbp (9). The paired GAII reads were aligned to the contigs using SHRiMP 1.3.2 to identify and correct 74 homopolymeric sequencing errors (11). Optical mapping was used to produce a high-resolution XbaI chromosome restriction map, and the contigs were ordered and oriented against this map using MapSolver 2.1.1 (Opgen). Gap closures were performed by PCR followed by Sanger sequencing of amplification products (3730S genetic analyzer sequencer; Applied Biosystems, CA). The finished sequence was validated by reference to the XbaI optical map, Roche GS FLX Titanium mate pair analysis, and Illumina paired-end-read analysis.Protein coding regions were predicted using GeneMarkS 4.6b, tRNA genes using tRNAscan-SE 1.23, and rRNA genes using RNAmmer 1.2 (2, 7, 8). 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, 6). These automated analyses were followed by manual curation, including comparison with other completed S. aureus genomes.The genome of S. aureus strain JKD6159 consists of a circular 2,811,435-bp chromosome with 33% G+C content—similar to those of other staphylococci—and one circular plasmid of 20,730 bp. A total of 2,605 coding regions, 57 tRNA genes, and 5 rRNA loci were detected. Over 67% of genes were assigned to specific Clusters of Orthologous Groups (COG) Database functional groups, and 40% were assigned an enzyme classification number (12).Initial analysis of the whole-genome sequence of JKD6159 confirms that ST93-MRSA-IV is distantly related to other previously sequenced S. aureus genomes. ST93-MRSA-IV has a distinct accessory genome. There were a number of regions of difference in JKD6159 that contain coding sequences (CDS) not present in any other published S. aureus genomes. Additionally, the ssl gene cluster in JKD6159 appears distinct from other sequenced S. aureus isolates. Comparison with other S. aureus genomes also shows that although JKD6159 carries lukSF-PV (the genes encoding Panton-Valentine leukocidin), there is a relative paucity of virulence factors such as tst-1, genes encoding staphylococcal enterotoxins A to U, and the ACME locus. Further analysis of the genome is now under way to identify factors that might explain the emergence of this MRSA strain in the community.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

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.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).