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Symptoms of Staphylococcus lugdunensis infection are often similar to those of Staphylococcus aureus infection, including skin and soft-tissue lesions, bacteremia and infective endocarditis. Despite the severity of these infections, S. lugdunensis is regarded as a less important pathogen than drug-resistant S. aureus. To investigate its ability to cause infectious diseases, a methicillin-resistant S. lugdunensis (MRSL) strain JICS135 was isolated from a patient with bacteremia and subjected to whole genome sequencing. Similar to most strains of methicillin-resistant S. aureus (MRSA), this MRSL strain possessed the staphylococcal cassette chromosome mec (SCCmec) located close to the origin of replication. However, the SCCmec in this MRSL strain, with three ccr complexes, was structurally unique and currently untypable. Moreover, the SCCmec of this MRSL strain was found to carry two genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM)-like proteins accompanied by glycosyl transferases, one of which may have been derived from S. aureus and the other from S. epidermidis, indicating that this MRSL evolved to carry virulence factors from other staphylococci. The emergence of this strain, the first MRSL strain whose genome has been sequenced completely, may be of public concern.

Methicillin-susceptible S. aureus (MSSA) changes to methicillin-resistant S. aureus upon the acquisition of Staphylococcal Cassette Chromosome mec (SCCmec), a genomic island that encodes methicillin resistance. All SCCmec elements reported to date share four common characteristics: (1) carrying the mec gene complex (mec); (2) carrying the ccr gene complex (ccr); (3) being flanked by characteristic nucleotide sequences, inverted repeats, and direct repeats, at both ends; and (4) being integrated at the integration site sequence (ISS) for SCC, which is located at the 3'-end of orfX or at the extremity of the SCC element. SCCmec elements in S. aureus are classified into different types based on the combination of mec and ccr, which share variations, five classes in mec and eight in ccr. To date, at least 11 types of SCCmec elements have been identified. Regions other than mec and ccr within the SCCmec element are designated as "joining regions" (J-regions), which are classified into three subgroups, J1-3. Many J-region variants have been identified among the SCCmec elements of types I-V. We herein describe PCR methods to type SCCmec elements by first identifying the mec and ccr type, and then identifying genes in the J-regions.

Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.

The BD GeneOhm MRSA assay could identify methicillin-resistant Staphylococcus aureus (MRSA) strains at a high ratio (97.8%). Analysis of 11 assay-negative MRSA strains suggested that insertion of non-mec staphylococcal cassette chromosome elements (SCCs) downstream of orfX, and carriage of SCCmecs with a left extremity that cannot be detected by the kit, might lead to their being given an incorrect negative status.

In clinical microbiology, bacterial identification is labor-intensive and time-consuming. A solution for this problem is the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In this study, we evaluated a modified protein extraction method of identification performed on target plates (on-plate extraction method) with MALDI-TOF (Bruker Microflex LT with Biotyper version 3.0) and compared it to 2 previously described methods: the direct colony method and a standard protein extraction method (standard extraction method). We evaluated the species of 273 clinical strains and 14 reference strains of staphylococci. All isolates were characterized using the superoxide dismutase A sequence as a reference. For the species identification, the on-plate, standard extraction, and direct colony methods identified 257 isolates (89.5%), 232 isolates (80.8%), and 173 isolates (60.2%), respectively, with statistically significant differences among the three methods (P < 0.05). In conclusion, the on-plate extraction method is at least as good as standard extraction in identification rate and has the advantage of a shorter processing time.

The clinical vancomycin-intermediate Staphylococcus aureus (VISA) strain Mu50 carries two mutations in the vraSR and graRS two-component regulatory systems (TCRSs), namely, vraS(I5N) and graR(N197S) (hereinafter designated graR). The clinical heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3 shares with Mu50 the mutation in vraS that encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR, carrying the mutated two-component response regulator graR, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy of graR into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50 rpoB gene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR with rifampin gave rise to rpoB mutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR were found to possess rpoB mutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneous rpoB mutants with various degrees of increased vancomycin resistance.

Vancomycin-intermediate Staphylococcus aureus (VISA) is generated from vancomycin-susceptible Staphylococcus aureus by multiple spontaneous mutations. We previously reported that sequential acquisition of mutations in the two-component regulatory systems vraSR and graRS was responsible for the VISA phenotype of strain Mu50. Here we report on the identification of a novel set of regulator mutations, a deletion mutation in two-component regulatory system walRK (synonyms, vicRK and yycFG), and a truncating mutation in a proteolytic regulatory gene, clpP, responsible for the raised vancomycin resistance in a laboratory-derived VISA strain, LR5P1-V3. The contributory effect of the two mutations to vancomycin resistance was confirmed by introducing the walK and clpP mutations into the vancomycin-susceptible parent strain N315LR5P1 by a gene replacement procedure. The vancomycin MIC of N315LR5P1 was raised from 1 to 2 mg/liter by the introduction of the walK or clpP mutation, but it was raised to 4 mg/liter by the introduction of both the walK and clpP mutations. The vancomycin MIC value of the double mutant was equivalent to that of strain LR5P1-V3. Like VISA clinical strains, LR5P1-V3 and the double mutant strain LR5P1walK*clpP* exhibited a thickened cell wall, slow growth, and decreased autolytic activity. Transcriptional profiles of the mutants with gene replacements demonstrated that introduction of both the walK and clpP mutations could alter expression of dozens or hundreds of genes, including those involved in cell envelope and cellular processes, intermediary metabolism, and information pathway. A mutation prevalence study performed on 39 worldwide clinical VISA strains showed that 61.5, 7.7, 10.3, and 20.5% of VISA strains harbored mutations in walRK, clpP, graRS, and vraSR, respectively. The mutation of walRK was most frequently carried by VISA strains. Together, these results suggested that the mutations of walK and clpP identified in LR5P1-V3 constitute a new combination of genetic events causing vancomycin resistance in Staphylococcus aureus.

We have previously reported the establishment of a Staphylococcus aureus laboratory strain, 10 3d1, having reduced susceptibility to daptomycin and heterogeneous vancomycin-intermediate S. aureus (VISA) phenotype. The strain was generated in vitro by serial daptomycin selection (Camargo, I. L., H. M. Neoh, L. Cui, and K. Hiramatsu, Antimicrob. Agents Chemother. 52:4289-4299, 2008). Here we explored the genetic mechanism of resistance in the strain by whole-genome sequencing and by producing gene-replaced strains. By genome comparison between 10 3d1 and its parent methicillin-resistant Staphylococcus aureus (MRSA) strain N315ΔIP, we identified five nonsynonymous single nucleotide polymorphisms (SNPs). One of the five mutations was found in the rpoB gene encoding the RNA polymerase β subunit. The mutation at nucleotide position 1862 substituted the 621st alanine by glutamic acid. The replacement of the intact rpoB with the mutated rpoB, designated rpoB(A621E), conferred N315ΔIP with the phenotypes of reduced susceptibility to daptomycin and hetero-VISA. The rpoB(A621E)-mediated resistance conversion was accompanied by a thickened cell wall and reduction of the cell surface negative charge. Being consistent with these phenotypic changes, microarray data showed that the expression of the dlt operon, which increases the cell surface positive charge, was enhanced in the rpoB(A621E) mutant. Other remarkable findings of microarray analysis of the rpoB(A621E) mutant included repression of metabolic pathways of purine, pyrimidine, arginine, the urea cycle, and the lac operon, enhancement of the biosynthetic pathway of vitamin B2, K1, and K2, and cell wall metabolism. Finally, mutations identified in rplV and rplC, encoding 50S ribosomal proteins L22 and L3, respectively, were found to be associated with the slow growth, but not with the phenotype of decreased susceptibility to vancomycin and daptomycin, of 10 3d1.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant pathogens around the world. MRSA is generated when methicillin-susceptible S. aureus (MSSA) exogenously acquires a methicillin resistance gene, mecA, carried by a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec), which is speculated to be transmissible across staphylococcal species. However, the origin/reservoir of the mecA gene has remained unclear. Finding the origin/reservoir of the mecA gene is important for understanding the evolution of MRSA. Moreover, it may contribute to more effective control measures for MRSA. Here we report on one of the animal-related Staphylococcus species, S. fleurettii, as the highly probable origin of the mecA gene. The mecA gene of S. fleurettii was found on the chromosome linked with the essential genes for the growth of staphylococci and was not associated with SCCmec. The mecA locus of the S. fleurettii chromosome has a sequence practically identical to that of the mecA-containing region (∼12 kbp long) of SCCmec. Furthermore, by analyzing the corresponding gene loci (over 20 kbp in size) of S. sciuri and S. vitulinus, which evolved from a common ancestor with that of S. fleurettii, the speciation-related mecA gene homologues were identified, indicating that mecA of S. fleurettii descended from its ancestor and was not recently acquired. It is speculated that SCCmec came into form by adopting the S. fleurettii mecA gene and its surrounding chromosomal region. Our finding suggests that SCCmec was generated in Staphylococcus cells living in animals by acquiring the intrinsic mecA region of S. fleurettii, which is a commensal bacterium of animals.

Streptococcus intermedius is an opportunistic pathogen of humans that causes purulent infections, including brain and liver abscesses. This pathogen secretes a human-specific cytolysin, intermedilysin, which has been recognized as a major virulence factor. However, most of the expressional control mechanisms of ily are still unknown. To determine these mechanisms, we analyzed the nucleotide sequence of the ily promoter region. We found a highly homologous region to the catabolite-repressible element (cre) in the ily promoter region and observed a considerable decrease in the amount of secreted intermedilysin when cells were grown in a culture medium containing high concentrations of glucose/utilizable carbohydrates. Disruption of the ccpA gene, which encodes catabolite control protein A, did not induce catabolite repression of ily by glucose/utilizable carbohydrates. In cre mutants, catabolite repression of ily was partially restored, and purified catabolite control protein A bound to an oligonucleotide containing the cre consensus sequence in the ily promoter region. In addition, a prolonged lag phase and slower doubling time of the ccpA mutant cells were observed. Our data show that S. intermedius can modulate ily expression and growth rate through catabolite control protein A-mediated monitoring of the extracellular glucose/utilizable carbohydrate concentration.

Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide. It is a major cause of hospital-acquired infections in most hospitals for nearly half century. The present study was conducted to examine the antimicrobial susceptibilities and staphylococcal cassette chromosome mec (SCCmec)-type for MRSA isolates from 237 patients treated at Srinagarind Hospital between September 2002 and August 2003. Antimicrobial susceptibility testing for all isolates was performed using an agar dilution method and SCCmec-types of 81 representatives from 237 isolates were determined using multiplex PCR. The minimum inhibitory concentration (MIC) ranges for the MRSA isolates were as follows: cefazolin 8 to > or =64; erythromycin < or = 0.5 to > or =64; gentamicin < or = 0.5 to > or =64; imipenem < or = 0.5 to >16; ofloxacin < or = 0.5 to > or =64; oxacillin 16 to > or =64; tetracycline 2 to > or =64 and vancomycin < or = 0.5 to 2 microg/ml. All MRSA isolates were susceptible to vancomycin, but only 0.4% to 8.9% was susceptible to the remaining antimicrobial agents. Of the 81 isolates tested, 2 types of SCCmec were found (76 with type III and 2 with type II) and no mecA gene was detected in 3 isolates. Sixty-seven of the 78 isolates carried the mercury-resistant operon. The multilocus sequence type in isolates with type III SCCmec was ST239 and in isolates with type II SCCmec was ST5.

Methicillin-resistant Staphylococcus aureus (MRSA) arises when methicillin-susceptible S. aureus (MSSA) acquires the staphylococcal cassette chromosome mec (SCCmec). Most pvl-positive MRSA in Taiwan belong to ST59 lineage and carry SCCmec V. The genetic profiles of 51 MSSA were compared with those of 80 MRSA from the same hospitals. Nine pvl-positive MSSA (oxacillin MIC < or = 2 microg/mL) shared >80% similarity in pulsed field gel electrophoresis pattern with 17 pvl-positive SCCmec V MRSA. Further investigation found that 5 of these 9 isolates were MRSA by cefoxitin and carried SCCmec V. All 26 pvl-positive isolates had very similar genetic profile (ST59, protein A clonal complex [spa-CC] c2:441/437, and agr group I). The success of the ST59:SCCmec V MRSA may be due in part to its heterogeneous and borderline resistance to methicillin, which may be missed by testing only oxacillin, with subsequent exposure to beta-lactams causing the emergence of more resistant subpopulations.

We describe here the genetic analysis of a vancomycin-susceptible Staphylococcus aureus (VSSA) strain, Mu50Omega, a strain related to vancomycin-intermediate S. aureus (VISA) strain Mu50. Using a combination of Mu50Omega whole-genome sequencing and genome engineering, we observed a stepwise evolution of vancomycin resistance from VSSA to VISA after the mutated vraS and graR genes of Mu50 were engineered into Mu50Omega.

The DNA sequence of the genome of Staphylococcus haemolyticus JCSC1435 revealed a putative capsule operon composed of 13 genes in tandem. The first seven genes (capABCDEFG(Sh)) showed > or = 57% similarity with the Staphylococcus aureus cap5 or cap8 locus. However, the capHIJKLM(Sh) genes are unique to S. haemolyticus and include genes encoding a putative flippase, an aminotransferase, two glycosyltransferases, and a transcriptional regulator. Capsule-like material was readily apparent by immunoelectron microscopy on bacteria harvested in the postexponential phase of growth. Electron micrographs of a JCSC1435 mutant with a deleted cap region lacked the capsule-like material. Both strains produced small amounts of surface-associated material that reacted with antibodies to polyglutamic acid. S. haemolyticus cap genes were amplified from four of seven clinical isolates of S. haemolyticus from humans, and three of these strains produced a serologically cross-reactive capsular polysaccharide. In vitro assays demonstrated that the acapsular mutant strain showed greater biofilm formation but was more susceptible to complement-mediated opsonophagocytic killing than the parent strain. Structural characterization of capsule purified from S. haemolyticus strain JCSC1435 showed a trisaccharide repeating unit: -3-alpha-L-FucNAc-3-(2-NAc-4-N-Asp-2,4,6-trideoxy-beta-D-Glc)-4-alpha-D-GlcNAc-. This structure is unique among staphylococcal polysaccharides in that its composition includes a trideoxy sugar residue with aspartic acid as an N-acyl substituent.

Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR, designated graR*, but not intact graR, designated graRn, could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR* expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR* increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.

SaeRS is a two-component system that has been characterized as a positive regulatory system for the expression of several virulence factors, including coagulase, alpha-, beta- and gamma-haemolysins, nuclease, and fibronectin-binding proteins in Staphylococcus aureus. Previously, the SaeRS system was found to be induced at the transcriptional level by beta-lactam. Here, we found that subinhibitory concentrations of beta-lactam induce haemolytic activity in the S. aureus N315 strain but not in the saeRS null mutant KSA. Comparison of the transcriptional profile of the N315 and KSA strains by microarray analysis reveals that the SaeRS system modulates the regulation of coagulase (coa), alpha-, beta- and gamma-haemolysins (hla, hlb and hlg), nuclease (SA0746), fibrinogen-binding proteins (emp, efb, SA1000 and SA1004), fibronectin-binding protein B (fnbB), and 13 other genes. Further, the use of cefoxitin as a signal inducer reveals that the SaeRS system appears to modulate 22 additional genes as a secondary regulon, including the staphylococcal accessory regulators SarA and SarT and the Clp protease ATPase subunits ClpB and ClpL. These observations suggest that beta-lactam is able to induce the SaeRS system, which acts as a crucial signal transduction system for S. aureus pathogenicity rather than antimicrobial resistance.

The assembly of ring-like structures, composed of FtsZ proteins (i.e. the Z ring), is the earliest and most essential process in bacterial cytokinesis. It has been shown that this process is directly regulated by the FtsZ-binding proteins, FtsA, ZapA, and EzrA, in Bacillus subtilis. In this study, protein complexes that are involved in Z-ring formation were chemically cross-linked in vivo, purified by affinity chromatography, and analysed by mass spectrometry. Analysis of the results identified YlmF as a new component of the FtsZ complex. Yeast two-hybrid analysis and fluorescence microscopy of YFP-YlmF in B. subtilis cells indicated YlmF localizes to the division site in an FtsZ-dependent manner. A single disruption of YlmF resulted in a slight elongation of cells; however, simultaneous inactivation of both YlmF and FtsA showed synthetic lethality caused by complete blockage of cell division due to the defect in Z-ring formation. In contrast, the ftsA-null mutant phenotype, caused by inefficient Z-ring formation, could be complemented by overexpression of YlmF. These results suggest that YlmF has an overlapping function with FtsA in stimulating the formation of Z rings in B. subtilis.

Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to approximately 128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.

Many antiseptic agents are used in hygienic handwashes in the prevention of nosocomial infections by methicillin-resistant Staphylococcus aureus (MRSA). The plasmid-borne genes qacA/B and smr confer resistance to cationic antiseptic agents in S. aureus. In this study, the susceptibilities for dyes and antiseptic agents (e.g. acriflavine, acrinol, benzalkonium chloride, benzethonium chloride, chlorhexidine digluconate and alkyldiaminoethylglycine hydrochloride) of 894 isolates of MRSA collected from 11 Asian countries (South Korea, China, the Philippines, Singapore, Vietnam, Thailand, Indonesia, India, Sri Lanka, Saudi Arabia and Japan) between 1998 and 1999 were examined. In addition, the distributions of the antiseptic-resistance genes qacA/B and smr were studied by PCR. Among the Asian MRSA isolates 57.7% (516/894) were acriflavine-resistant. The smr gene was detected in 31.6% (12/38) of MRSA isolates from India but only in 1.9% (16/856) of all the isolates from other Asian countries. MRSA with qacA/B comprised 41.6% (372/894) of the isolates across Asia. In addition, PFGE was performed to type the MRSA and grouped the tested 30 MRSA isolates with qacA/B into 21 PFGE types. The results indicated that qacA/B is functionally the most important gene mediating antiseptic resistance in the MRSA strains of Asia and that a specific MRSA with qacA/B was not prevalent in Asia but qacA/B were widely spread among MRSA of Asia, while the geographical distribution of smr is more limited.

Staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element composed of the mec gene complex, which encodes methicillin resistance, and the ccr gene complex, which encodes the recombinases responsible for its mobility. The mec gene complex has been classified into four classes, and the ccr gene complex has been classified into three allotypes. Different combinations of mec gene complex classes and ccr gene complex types have so far defined four types of SCCmec elements. Now we introduce the fifth allotype of SCCmec, which was found on the chromosome of a community-acquired methicillin-resistant Staphylococcus aureus strain (strain WIS [WBG8318]) isolated in Australia. The element shared the same chromosomal integration site with the four extant types of SCCmec and the characteristic nucleotide sequences at the chromosome-SCCmec junction regions. The novel SCCmec carried mecA bracketed by IS431 (IS431-mecA-DeltamecR1-IS431), which is designated the class C2 mec gene complex; and instead of ccrA and ccrB genes, it carried a single copy of a gene homologue that encoded cassette chromosome recombinase. Since the open reading frame (ORF) was found to encode an enzyme which catalyzes the precise excision as well as site- and orientation-specific integration of the element, we designated the ORF cassette chromosome recombinase C (ccrC), and we designated the element type V SCCmec. Type V SCCmec is a small SCCmec element (28 kb) and does not carry any antibiotic resistance genes besides mecA. Unlike the extant SCCmec types, it carries a set of foreign genes encoding a restriction-modification system that might play a role in the stabilization of the element on the chromosome.

A specific phenotype of Staphylococcus aureus strains Mu50 and Mu3 is characterized by thickened cell wall and moderate resistance to vancomycin. The N315 strain is a prototype of methicillin-resistant S. aureus (MRSA), but it is methicillin susceptible, despite carrying the mecA resistance gene. Here, we revised differences in the sequences of Mu50 and N315, referencing that of Mu3 which were assumed to be of one lineage. The 362 ORFs diverse between Mu50 and N315 were picked up, and the corresponding ones in three strains were re-sequenced. This defined 213 ORFs diverse between Mu50 and N315, and 9 between Mu50 and Mu3. The fixed diversities of 174 ORFs (except for 39 silent ORFs from 213), including nucleotide substitution (NSs), frame shift, and truncation were grouped into three major functional categories, which were transport (14.9% in the 174 diverse ORFs), metabolism of carbohydrates (5.7%), and RNA synthesis (9.6%). The other gene categories had small diversities. These gene categories seemed to be functionally decisive for the Mu50-specific characters, the thickened cell wall and moderate vancomycin resistance. All of the diverse genes and the high quality sequence of Mu50 can be viewed at the web site (http://133.5.48.239/VRSA/).

DNA microarray covering the whole genome of Staphylococcus aureus strain N315 was prepared to investigate transcription profiles. The microarray analyses revealed that vancomycin induces transcription of 139 genes. Forty-six genes among them failed to be induced in the vraSR null mutant KVR. Part of the genes regulated by VraSR system is associated with cell-wall biosynthesis, such as PBP2, SgtB and MurZ. Other cell-wall synthesis inhibitors also induced VraSR, suggesting that the sensor kinase VraS responds to the damage of cell-wall structure or inhibition of cell-wall biosynthesis. Additionally, the vraSR null mutants derived from hetero- and homo-methicillin-resistant S. aureus showed significant decrease of resistance against teicoplanin, beta-lactam, bacitracin and fosfomycin but not of D-cycloserine and levofloxacin. The observation strongly indicates that VraSR constitutes a positive regulator of cell-wall peptidoglycan synthesis, and that is deeply involved in the expression of beta-lactam and glycopeptide resistance in S. aureus.

Staphylococcus aureus clinical isolate 61/5896 exhibited methicillin resistance (MIC 64 mg/L), but lacked mecA, which encodes penicillin-binding protein 2'. The strain was isolated in England in 1961, and exhibited unstable heterogeneous methicillin resistance. When cultivated in drug-free medium, the methicillin resistance of 61/5896 increased after three daily passages, then decreased and was completely lost after 12 days' passage. Electron microscopy revealed that strain 61/5896 had a thicker and rougher cell wall than its methicillin-susceptible derivatives. It produced about three times more penicillin-binding protein 2 (PBP2) than methicillin-susceptible derivatives. The strain was characteristically a non-producer of autolytic enzyme, though the phenotype, which was lost easily, was not directly correlated with methicillin resistance.

A large and growing proportion of Staphylococcus aureus clinical isolates are methicillin resistant, and are resistant to practically all beta-lactam antibiotics. Methicillin-resistant S. aureus (MRSA) strains harbor mecA, which is carried by a unique mobile genetic element, staphylococcal cassette chromosome mec (SCCmec) integrated into the S. aureus chromosome. The mecA gene encodes a methicillin-insensitive transpeptidase, the production of which confers resistance to otherwise inhibitory concentrations of beta-lactam antibiotics. Several distinct clones have been identified among MRSA that apparently have been generated by integration of distinct types of SCCmec. While MRSA are primarily nosocomial pathogens, recent observations indicate that other MRSA clones are colonizing a significant proportion of healthy individuals in the community as well. Community-acquired MRSA (C-MRSA), may become a new threat to humans, and international cooperation of researchers and clinicians will be of cardinal importance in addressing this problem.

Background: A new type of meticillin-resistant Staphylococcus aureus (MRSA), designated community-acquired MRSA, is becoming increasingly noticeable in the community, some strains of which cause fatal infections in otherwise healthy individuals. By contrast with hospital-acquired MRSA, community-acquired MRSA is more susceptible to non b-lactam antibiotics. We investigated the high virulence potential of certain strains of this bacterium., Methods: We ascertained the whole genome sequence of MW2, a strain of community-acquired MRSA, by shotgun cloning and sequencing. MW2 caused fatal septicaemia and septic arthritis in a 16-month-old girl in North Dakota, USA, in 1998. The genome of this strain was compared with those of hospital-acquired MRSA strains, including N315 and Mu50., Findings: Meticillin resistance gene (mecA) in MW2 was carried by a novel allelic form (type IVa) of staphylococcal cassette chromosome mec (SCCmec), by contrast with type II in N315 and Mu50. Type IVa SCCmec did not carry any of the multiple antibiotic resistance genes reported in type II SCCmec. By contrast, 19 additional virulence genes were recorded in the MW2 genome. All but two of these virulence genes were noted in four of the seven genomic islands of MW2., Interpretation: MW2 carried a range of virulence and resistance genes that was distinct from those displayed on the chromosomes of extant S aureus strains. Most genes were carried by specific allelic forms of genomic islands in the MW2 chromosome. The combination of allelic forms of genomic islands is the genetic basis that determines the pathogenicity of medically important phenotypes of S aureus, including those of community-acquired MRSA strains.

We detected the metallo-beta-lactamase gene blaIMP positive strains of the gram-negative rods (GNR) isolated in Oita Medical University Hospital between 1993 and 1999 and studied the clinical characteristics of patients infected or colonized with blaIMP positive GNR. 25 strains (20 Pseudomonas aeruginosa and 5 Serratia marcescens) were detected and most of them were isolated from urinary samples after 1997. In the studies of antimicrobial susceptibility, some strains had sensitivity to aztreonum or imipenem although most of the strains showed multidrug resistance. When blaIMP positive GNR were isolated from patients, these strains were thought to have caused infection in 88% of the patients. About half of the patients were over 65 years old and had malignant diseases. Most of the patients had inserted urinary tract catheters, intratracheal tube or intravernous catheters. It was suggested that the insertion of the catheters were related to infection of blaIMP positive GNRs. Two patients were not treated with any antibiotics before the isolation of blaIMP positive GNRs although more than half of the patients were administered carbapenems and cephems. Most of strains were isolated in the same department and showed the same genotype by pulsed field gel electrophoresis.

Significant advances have been made in recent years in our understanding of how methicillin resistance is acquired by Staphylococcus aureus. Integration of a staphylococcal cassette chromosome mec (SCCmec) element into the chromosome converts drug-sensitive S. aureus into the notorious hospital pathogen methicilin-resistant S. aureus (MRSA), which is resistant to practically all beta-lactam antibiotics. SCCmec is a novel class of mobile genetic element that is composed of the mec gene complex encoding methicillin resistance and the ccr gene complex that encodes recombinases responsible for its mobility. These elements also carry various resistance genes for non-beta-lactam antibiotics. After acquiring an SCCmec element, MRSA undergoes several mutational events and evolves into the most difficult-to-treat pathogen in hospitals, against which all extant antibiotics including vancomycin are ineffective. Recent epidemiological data imply that MRSA has embarked on another evolutionary path as a community pathogen, as at least one novel SCCmec element seems to have been successful in converting S. aureus strains from the normal human flora into MRSA.

We report on the structural diversity of mecA gene complexes carried by 38 methicillin-resistant Staphylococcus aureus and 91 methicillin-resistant coagulase-negative Staphylococcus strains of seven different species with a special reference to its correlation with phenotypic expression of methicillin resistance. The most prevalent and widely disseminated mec complex had the structure mecI-mecR1-mecA-IS431R (or IS431mec), designated the class A mecA gene complex. In contrast, in S. haemolyticus, mecA was bracketed by two copies of IS431, forming the structure IS431L-mecA-IS431R. Of the 38 S. haemolyticus strains, 5 had low-level methicillin resistance (MIC, 1 to 4 mg/liter) and characteristic heterogeneous methicillin resistance as judged by population analysis. In these five strains, IS431L was located to the left of an intact mecI gene, forming the structure IS431L-class A mecA-gene complex. In other S. haemolyticus strains, IS431L was associated with the deletion of mecI and mecR1, forming the structure IS431L-DeltamecR1-mecA-IS431mec, designated the class C mecA gene complex. Mutants with the class C mecA gene complex were obtained in vitro by selecting strain SH621, containing the IS431L-class A mecA gene complex with low concentrations of methicillin (1 and 3 mg/liter). The mutants had intermediate level of methicillin resistance (MIC, 16 to 64 mg/liter). The mecA gene transcription was shown to be derepressed in a representative mutant strain, SH621-37. Our study indicated that the mecI-encoded repressor function is responsible for the low-level methicillin resistance of some S. haemolyticus clinical strains and that the IS431-mediated mecI gene deletion causes the expression of methicillin resistance through the derepression of mecA gene transcription.

The beta-lactam resistance gene mecA of Staphylococcus aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec), identified in the chromosome of a Japanese methicillin-resistant S. aureus (MRSA) strain. We now report identification of two additional types of mecA-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the elements and the SCCmec. However, new elements shared the chromosomal integration site with the SCCmec. Structural analysis of the new elements revealed that they possessed all of the salient features of the SCCmec: conserved terminal inverted repeats and direct repeats at the integration junction points, conserved genetic organization around the mecA gene, and the presence of cassette chromosome recombinase (ccr) genes responsible for the movements of SCCmec. The elements, therefore, were considered to comprise the SCCmec family of staphylococcal mobile genetic elements together with the previously identified SCCmec. Among 38 epidemic MRSA strains isolated in 20 countries, 34 were shown to possess one of the three typical SCCmec elements on the chromosome. Our findings indicated that there are at least three distinct MRSA clones in the world with different types of SCCmec in their chromosome.

The metallo-beta-lactamase producing bacterium are increasing in recent years. We characterized cases infected or colonized with blaIMP, which is metallo-beta-lactamase IMP-1 gene, positive gram-negative rods(GNRs). About half of the cases had malignant diseases as underliningg disease. When blaIMP positive GNRs were isolated, most of cases shows clinical signs of infection such as fever and elevation of C-reactive protein in serum. Most of strains were isolated from urinary samples and all patients from whom blaIMP positive bacterium were isolated from urinary samples were inserted urinary tract catheter. It was suggested that the insertion of catheters related to infection of blaIMP positive GNRs. No antibiotics were administered to several patients before the isolation of blaIMP positive GNRs although broad spectrum antibiotics such as carbapenems were administered to more than half of the patients. It is very important to prevent their dissemination in hospitals because most of blaIMP positive GNRs were isolated in same departments and showed identical pattern by pulsed field gel electrophoresis. And it is also important for treatment of the infectious disease due to blaIMP positive GNR to remove the catheter and improve general conditions of patients.

We have previously shown that the methicillin-resistance gene mecA of Staphylococcus aureus strain N315 is localized within a large (52-kb) DNA cassette (designated the staphylococcal cassette chromosome mec [SCCmec]) inserted in the chromosome. By sequence determination of the entire DNA, we identified two novel genes (designated cassette chromosome recombinase genes [ccrA and ccrB]) encoding polypeptides having a partial homology to recombinases of the invertase/resolvase family. The open reading frames were found to catalyze precise excision of the SCCmec from the methicillin-resistant S. aureus chromosome and site-specific as well as orientation-specific integration of the SCCmec into the S. aureus chromosome when introduced into the cells as a recombinant multicopy plasmid. We propose that SCCmec driven by a novel set of recombinases represents a new family of staphylococcal genomic elements.

More than 90% of methicillin-resistant Staphylococcus aureus (MRSA) isolates produce a penicillin-binding protein PBP2' (or PBP2a) with low affinity for beta-lactam antibiotics. PBP2' is encoded by the mecA gene, a foreign gene integrated into the chromosome of methicillin-susceptible S. aureus (MSSA). DNA vaccination by injection of transgene-expressing plasmids has been demonstrated to elicit an immune response against transgene-encoded protein. We hypothesized that the application of DNA vaccination with the mecA sequence would elicit protective immunity against MRSA. This immunity was evoked by injection of a mecA-expressing plasmid into BALB/c mice. Anti-PBP2' antibody was detected in the sera obtained from the DNA-vaccinated mice. These sera produced a five-fold increase in phagocytosis of MRSA compared with sera from mice treated with control plasmid. However, there was no difference in phagocytosis of MSSA among these groups. In addition, the in-vivo antibacterial effect of DNA vaccination was demonstrated in mice infected with MRSA. Eight days after iv inoculation of 10(8) cfu of MRSA into mice, the number of bacteria in the kidneys obtained from mice vaccinated with mecA-expressing plasmid (1.48 +/- 0.27 x 10(5) cfu/mg kidney; n = 18) was significantly lower than that from mice vaccinated with negative control plasmid (3.59 +/- 0.57 x 10(5) cfu/mg kidney; n = 17) (P < 0.02) or that from sham-treated mice (3. 43 +/- 0.66 x 10(5) cfu/mg kidney; n = 9) (P < 0.02). Interestingly, PBP2' was found in both the bacterial membrane fraction and the supernatant, thus being accessible to serum antibodies. Together these observations indicate that PBP2' or the mecA sequence may be eligible as a candidate molecule for vaccination against MRSA.

We have previously reported methicillin-resistant Staphylococcus aureus clinical strains, Mu50 and Mu3, representing two categories of vancomycin resistance: Mu50 representing vancomycin-resistant S. aureus (VRSA) with MICs > or = 8 mg/L, and Mu3 representing hetero-VRSA with MICs < or = 4 mg/L using standard MIC determination methods. The mechanisms of vancomycin resistance in these strains were investigated. These strains did not carry the enterococcal vancomycin-resistance genes, vanA, vanB, or vanC1-3, as tested by PCR using specific primers. However, both strains produced three to five times the amount of penicillin-binding proteins (PBPs) 2 and 2' when compared with vancomycin-susceptible S. aureus control strains with or without methicillin resistance; the amounts of PBP2 produced in Mu3 and Mu50 were comparable to those in the vancomycin-resistant S. aureus mutant strains selected in vitro. Incorporation of 14C-labelled Nacetyl-glucosamine into the cell was three to 20 times increased in Mu50 and Mu3, and release of the radioactive cell wall material was increased in Mu3 (and also in Mu50, though to a lesser extent), compared with control strains. The amounts of intracellular murein monomer precursor in these strains were three to eight times greater than those found in control strains. Transmission electron microscopy showed a doubling in the cell wall thickness in Mu50 compared with the control strains. Mu3 did not show obvious cell wall thickening. These data indicate that activated synthesis and an increased rate of cell wall turnover are common features of Mu3 and Mu50 and may be the prerequisite for the expression of vancomycin resistance in S. aureus.

The mechanism of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA) Mu50 was investigated. More than 3 times increase of the incorporation of 14C-GluNAc into the cell wall of Mu50 was observed compared to those of vancomycin-susceptible strains FDA209P, H-1, LR5P1. The amount of cytoplasmic murein-monomer precursor increased more than 3 times in Mu50 compared to those of control strains. There was an increased production of PBP1, PBP2, and PBP2', which were 1.51, 17.2, and 7.06 times greater, respectively, in Mu50 than those in H-1, and 2.38, 4.46, and 1.96 times greater respectively, than those in LR5P1. By transmission electromicrograph, it was shown that the cell wall of Mu50 was twice thicker than that of LR5P1. Increase of tightly-bound vancomycin to the cell wall fraction was observed in Mu50 when compared to those in FDA209P and H-1 strains. From these results, the increase of the vancomycin targets, free D-Ala-D-Ala residues in the cell wall, in number, due to the activated cell wall synthesis, and/or decrease of the cross-linkage of the cell wall was suggested to be the mechanism of vancomycin resistance in the Mu50 strain.

The mechanism of methicillin susceptibility was studied in Staphylococcus aureus N315P, a pre-methicillin-resistant S. aureus strain that is susceptible to methicillin, despite the presence of mecA in the chromosome. In the presence of mec regulator genes mecI and mecR1, transcription of the mecA gene was not inducible by the addition of methicillin to the culture medium. Inactivation of the mecI gene function by replacing it with tetL made N315P express heterogeneous-type methicillin resistance. The subclone, in which the mecI gene was replaced, subclone P delta I, produced 12 times greater amounts of mecA gene transcripts and 8.5 times more PBP 2' protein than N315P. These data indicate that the mecI gene-encoded repression of mecA gene transcription is responsible for the apparent methicillin susceptibility phenotype of pre-methicillin-resistant S. aureus N315P.

Eighty percent minimum inhibitory concentrations (MIC80) of sulopenem against clinically isolated 12 to 80 strains of each of different bacteria were as follows: methicillin-susceptible Staphylococcus aureus (MSSA): 0.20 micrograms/ml, methicillin-resistant S. aureus (MRSA): 50 micrograms/ml, coagulase-negative staphylococci: 3.13 micrograms/ml, Streptococcus pyogenes: < or = 0.013 microgram/ml, Streptococcus pneumoniae: < or = 0.013 microgram/ml, beta-streptococci: 0.05 microgram/ml, Enterococcus faecalis: 12.5 micrograms/ml, Enterococcus faecium: > 100 micrograms/ml, Escherichia coli CS2(R+): 0.10 microgram/ml, Klebsiella pneumoniae: 0.05 microgram/ml, Proteus mirabilis: 0.10 microgram/ml, Proteus vulgaris: 0.20 microgram/ml, Morganella morganii: 0.39 micrograms/ml, Providencia rettgeri: 3.13 micrograms/ml, Citrobacter freundii: 0.20 microgram/ml, Enterobacter cloacae: 0.39 microgram/ml, Serratia marcescens: 1.56 micrograms/ml, Pseudomonas aeruginosa: 50 micrograms/ml, Pseudomonas cepacia: 3.13 micrograms/ml, Xanthomonas maltophilia: > 100 micrograms/ml, Acinetobacter calcoaceticus: 1.56 micrograms/ml, ampicillin-resistant Haemophilus influenzae: 0.39 microgram/ml and Bacteroides fragil is: 0.20 microgram/ml, respectively. Sulopenem possesses a stronger activity than flomoxef or cefuzonam against Gram-positive bacteria, the strongest activity among the antibiotics tested against Gram-negative bacteria except P. aeruginosa. Sulopenem has stronger affinities than imipenem to all fractions of PBPs of S. aureus, E. coli, P. vulgaris, S. marcescens, even of P. aeruginosa. Affinities of sulopenem to PBPs-1 and -3 of S. aureus, PBP-2 of E. coli were much stronger than those of imipenem (IPM). Sulopenem generally has small Ki values to all types of beta-lactamases and also has stronger permanent inactivation effect to Ia and IIb types of beta-lactamases than IPM. No synergistic bactericidal activity of sulopenem was apparent with serum complement. However, synergism of sulopenem with macrophages was prominent in bactericidal activity. The cells of E. coli were well phagocytosed and rapidly digested by cultured macrophages in the presence of a higher than 1/8 MIC of sulopenem. Moreover, sulopenem was more stable than imipenem against swine and human dehydropeptidase-Is. Sulopenem is one of the antibiotics that do not induce the appearance of subclones resistant to the drug.

The distributions of the mec regulator genes mecI and mecR1, which were identified on the chromosome of mecA-carrying Staphylococcus aureus N315, in methicillin-resistant staphylococci isolated in Japan and various countries were studied. Screening by dot blot hybridization by using polymerase chain reaction (PCR)-amplified probes revealed that at least the 5'-end region of the mecR1 gene was present in all strains tested, whereas about 40% of the strains were negative for the mecI gene. The data suggested that these regulator genes were the original components of the additional mec region DNA of methicillin-resistant S. aureus as well as methicillin-resistant, coagulase-negative staphylococci of seven staphylococcal species (S. epidermidis, S. haemolyticus, S. hominis, S. sciuri, S. capitis, S. caprae, and S. warneri). The mecI gene, which presumably codes for the repressor protein of the mecA gene, was found to harbor a point mutation in all six mecI-positive methicillin-resistant S. aureus strains, and their basal level of mecA gene transcription was elevated compared with that of strain N315, which harbors a presumably intact counterpart of the mecI gene. The data suggested that the mecI gene encodes for a strong repressor function on mecA gene transcription and is deleted or mutated in clinical methicillin-resistant S. aureus strains with high levels of resistance to methicillin.

In cultures of Vibrio cholerae strains of Ogawa serotype, variant strains which had undergone serotype conversion from Ogawa to Inaba were identified. The rfbT genes cloned from the parent strains were found to produce a 31-kDa protein in the maxicell system, and to cause serotype conversion when introduced into E. coli cells expressing Inaba serotype specificity. On the other hand, rfbT genes cloned from the variant strains neither produced the 31-kDa protein nor caused serotype conversion. Nucleotide sequence of these rfbT genes as well as those of two clinical Vibrio cholerae strains of Inaba serotype revealed that mutations causing premature termination of their rfbT genes were invariably present in strains expressing Inaba serotype specificity. The results strongly suggested that genetic alteration of the rfbT gene is responsible for serotype conversion of Vibrio cholerae O1.

Molecular cloning and nucleotide sequence analysis were performed for the identification of the regulator genes of methicillin resistance in the genome of a MRSA strain N315. Two open reading frames (orfs) were identified in the 5'-flanking region of the mecA gene. Predicted amino acid sequences of these orfs showed extensive homology to the co-inducer and the repressor protein of the penicillinase (PCase) production in Staphylococcus aureus as well as in Bacillus licheniformis. These orfs are considered to encode putative co-inducer and repressor proteins specific for the regulation of methicillin resistance in MRSA.

A total number of 125 methicillin-resistant (MIC, greater than or equal to 16) coagulase-negative Staphylococcus strains isolated in Japan were surveyed for the distribution of the mecA gene, the structural gene for penicillin-binding protein 2', which is the causative genetic element for the intrinsic resistance of methicillin-resistant Staphylococcus aureus. Screening with colony hybridization by using a cloned mecA gene probe revealed that 121 strains (96.8%) belonging to the nine coagulase-negative Staphylococcus species (S. epidermidis, S. haemolyticus, S. saprophyticus, S. sciuri, S. simulans, S. hominis, S. capitis, S. warneri, and S. caprae) carried mecA in their genome, indicating wide distribution of the gene among coagulase-negative Staphylococcus species. Most (93.4%) of the mecA-carrying strains were producers of penicillinase. Four strains, including two S. haemolyticus and two S. saprophyticus strains, did not carry mecA in spite of their resistance to methicillin. One of them was of low-level resistance (MIC, 16), but three of them had moderate- to high-level resistance to methicillin (MIC, 64). Analysis of gel electrophoretic banding patterns of penicillin-binding proteins of these strains showed absence of penicillin-binding protein 2' but some alterations in signal intensities of the other penicillin-binding proteins. The result indicated that about 3% of methicillin-resistant coagulase-negative staphylococci in these hospitals had a resistance mechanism different from that associated with the production of penicillin-binding protein 2', as has been reported in the case of a borderline methicillin-resistant strain of S. aureus.

The analysis of gel banding patterns of penicillin-binding proteins was used to identify two clinical isolates of a coagulase-negative Staphylococcus species as Staphylococcus caprae, a species originally isolated from goat's milk. One of the isolates was further shown to carry mecA, the structural gene for methicillin resistance.

We analyzed gel electrophoretic banding patterns of penicillin-binding proteins (PBPs) of 16 type strains of coagulase-negative staphylococci (CNS). S. epidermidis, S. haemolyticus, S. saprophyticus, S. hominis, S. xylosus, S. simulans, S. warneri, S. capitis, S. saccharolyticus, S. auricularis, S. caseolyticus, S. gallinarum, S. hycus subsp. hycus, S. cohnii, S. caprae, and S. sciuri subsp. sciuri. The PBP profile of each CNS species was found to be unique and was clearly distinguishable from those of the rest of the species. Together with the previous work of other researchers, this study substantiates the applicability of the PBP profile analysis to the identification of clinical CNS strains.