The Escherichia coli-derived thymosin ß4 concatemer promotes cell proliferation and healing wound in mice.

Thymosin ß4 (Tß4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tß4 will increase. In order to develop a new approach to produce recombinant Tß4, a 168 bp DNA (termed Tß4) was designed based on the Tß4 protein sequence and used to express a 4 × Tß 4 concatemer (four tandem copies of Tß4, termed 4 × Tß4) together with a histidine tag (6 × His) in E. coli (strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tß4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropyl ß-D-thiogalactoside (IPTG). The E. coli-derived 4 × Tß4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cells in vitro and in vivo wound healing. The results demonstrate that these activities of the E. coli-derived recombinant 4 × Tß4 were similar or even better than existing commercially obtained Tß4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tß4.

ST9 MRSA strains carrying a variant of type IX SCCmec identified in the Thai community.

BACKGROUND: Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in Thailand occur most frequently in healthcare facilities. However, reports of community-associated MRSA are limited. METHODS: We characterized 14 MRSA isolates from outpatients (O-1 to O-14) by phenotypic and genotypic methods and compared them with 5 isolates from inpatients (I-1 to I-5). Thai MRSA isolates from a healthcare worker (N-1) and a pig (P-1) were also included as ST9 MRSA strains from other sources. RESULTS: All MRSA isolates from the outpatients and inpatients were multidrug-resistant (resistant to ≥3 classes of antimicrobials). All of them except strains O-2 and I-3 carried type III SCCmec and belonged to agrI, coagulase IV, spa type t037 or t233, which related to ST239. The strain O-2 (JCSC6690) carried type IX SCCmec and belonged to agrII, coagulaseXIc, spa type t337 and ST9, whereas the strain I-3 carried a type III SCCmec and belonged to ST1429. Nucleotide sequence determination revealed that the type IX SCCmec element in strain O-2 was distinct from that in a Thai ST398 strain (JCSC6943) previously identified in 2011; nucleotide identities of ccrA and ccrB were 93 and 91%, respectively and several open reading frames (ORFs) at the joining regions were different. PCR experiments suggested that strain O-2 and N-1 carried similar SCCmec element, whereas that of strain P-1 was different, suggesting that distinct ST9-MRSA-IX clones might be spreading in this province. CONCLUSIONS: The SCCmecIX-ST9 MRSA clones of distinct SCCmec subtypes might have emerged in the Thai community and might also have disseminated into the hospital.

Analysis of Staphylococcal cassette chromosome mec in BD GeneOhm MRSA assay-negative strains.

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.

Molecular characterization of methicillin-resistant Panton-valentine leukocidin positive staphylococcus aureus clones disseminating in Tunisian hospitals and in the community.

BACKGROUND: The spread of MRSA strains at hospitals as well as in the community are of great concern worldwide. We characterized the MRSA clones isolated at Tunisian hospitals and in the community by comparing them to those isolated in other countries. RESULTS: We characterized 69 MRSA strains isolated from two Tunisian university hospitals between the years 2004-2008. Twenty-two of 28 (79%) community-associated MRSA (CA-MRSA) strains and 21 of 41 (51%) healthcare-associated MRSA (HA-MRSA) strains were PVL-positive. The PVL-positive strains belonged to predicted founder group (FG) 80 in MLST and carried either type IVc SCCmec or nontypeable SCCmec that harbours the class B mec gene complex. In contrast, very diverse clones were identified in PVL-negative strains: three FGs (5, 15, and 22) for HA-MRSA strains and four FGs (5, 15, 45, and 80) for CA-MRSA strains; and these strains carried the SCCmec element of either type I, III, IVc or was nontypeable. The nucleotide sequencing of phi7401PVL lysogenized in a CA-MRSA strain JCSC7401, revealed that the phage was highly homologous to phiSA2mw, with nucleotide identities of more than 95%. Furthermore, all PVL positive strains were found to carry the same PVL phage, since these strains were positive in two PCR studies, identifying gene linkage between lukS and mtp (major tail protein) and the lysogeny region, both of which are in common with phi7401PVL and phiSa2mw. CONCLUSIONS: Our experiments suggest that FG80 S. aureus strains have changed to be more virulent by acquiring phi7401PVL, and to be resistant to ß-lactams by acquiring SCCmec elements. These novel clones might have disseminated in the Tunisian community as well as at the Tunisian hospitals by taking over existing MRSA clones.

Identification of the third type of PVL phage in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains.

The genes lukS-PV and lukF-PV for Panton-Valentine leukocidin (PVL) that confers high virulence to Staphylococcus aureus are located on the prophages (PVL phages) which have been classified into group 1 and 2 sfi21-like Siphoviridae. We report novel PVL phages lysogenized in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Japan (JCSC7247) and Taiwan (JCSC5967). The genomes of φ7247PVL and φ5967PVL showed more than 99% identity, and the regions containing the five genes located at both ends of the prophages, int (integrase), hol (holin), ami (amidase), lukS-PV, and lukF-PV, are highly homologous to extant PVL phages. The genes for the structural module are less homologous to these phages, but are highly homologous to non-PVL phages belonging to group 3 Sfi21-like Siphoviridae, for example φN315. Subsequent PCR identification and nucleotide sequencing of an additional 11 Taiwanese ST59 MRSA isolates suggested they all carry the same phage as φ5967PVL, which differed from φ7247PVL by a single base. This study adds evidence to the notion that novel PVL phages would be generated through illegitimate recombination events by acquiring the region at which hol, ami, luk, and int genes would line up upon lytic growth, and suggests that the PVL-positive MRSA clones that have emerged worldwide may carry distinct phages.

Novel types of staphylococcal cassette chromosome mec elements identified in clonal complex 398 methicillin-resistant Staphylococcus aureus strains.

The structures of staphylococcal cassette chromosome mec (SCCmec) elements carried by 31 clonal complex 398 (CC398) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from the participants at a conference were analyzed. The SCCmecs were classified into novel types, namely, IX, X, V(5C2&5) subtype c, and IVa. Type V(5C2&5) subtype c, IX, and X SCCmecs carried genes conferring resistance to metals. The structures of SCCmecs from CC398 strains were distinct from those normally found in humans, adding to the evidence that humans are not the original host for CC398.

Dissemination of multiple MRSA clones among community-associated methicillin-resistant Staphylococcus aureus infections from Japanese children with impetigo.

The proportion of MRSA strains that cause skin and soft infections has recently increased. In 3 months we have characterized 17 MRSA strains isolated from children with impetigo at a Japanese hospital. Seventeen MRSA strains belonged to 7 clones defined by clonal complex (CC) in MLST genotype and type of SCCmec, which were rarely identified among healthcare-associated MRSA: CC 91-SCCmecIIb (4 strains); CC91-SCCmecIIn (2 strains); CC91-SCCmecIVa (2 strains); CC91-SCCmecV (4 strains); CC88-SCCmecIVg (3 strains); CC1-SCCmecIVc (1 strain); and CC5-SCCmecIVn (1 strain). Although one strain belonged to CC5, which has been commonly identified in healthcare-associated MRSA, it did not carry type II SCCmec, but carried type IV SCCmec. Fourteen of the 17 strains carried exfoliative toxin a or b gene, and none carried Panton-Valentine leukocidine gene. Furthermore, we determined the entire nucleotide sequences of two type V SCCmec elements carried by strains JCSC5952, a CC91 strain, and TSGH17, a Taiwanese CC59 strain. The structure of SCCmecJCSC5952 was more than 99% homologous in nucleotide identity with those of Taiwanese PVL-positive ST59 MRSA strains TSGH17 and PM1, which were designated as type V (5C2&5). Identification of multiple MRSA clones distinct from those disseminating at the hospital suggests that MRSA strains might be emerging in the community from MSSA strains by acquiring SCCmec elements on various occasions. Carriage of the similar type V(5C2&5) SCCmec element by strains of distinct genetic backgrounds, CC91 and CC59, suggested horizontal transfer of the SCCmec element.

Genetic diversity of staphylocoagulase genes (coa): insight into the evolution of variable chromosomal virulence factors in Staphylococcus aureus.

BACKGROUND: The production of staphylocoagulase (SC) causing the plasma coagulation is one of the important characteristics of Staphylococcus aureus. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We compared the nucleotide sequences of 105 SC genes (coa), 59 of which were determined in this study. D1 regions, which contain prothrombin-activating and -binding domains and are presumed to be the binding site of each type-specific antiserum, were classified into twelve clusters having more than 90% nucleotide identities, resulting to create two novel SC types, XI and XII, in addition to extant 10 types. Nine of the twelve SC types were further subdivided into subtypes based on the differences of the D2 or the central regions. The phylogenetical relations of the D1 regions did not correlate exactly with either one of agr types and multilocus sequence types (STs). In addition, genetic analysis showed that recombination events have occurred in and around coa. So far tested, STs of 126 S. aureus strains correspond to the combination of SC type and agr type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively. CONCLUSION: The data suggested that the evolution of coa was not monophyletic in the species. Chromosomal recombination had occurred at coa and agr loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome.

Two different Panton-Valentine leukocidin phage lineages predominate in Japan.

We determined the entire nucleotide sequence of phiSa2958-carrying Panton-Valentine leukocidin (PVL) gene, which was lysogenized in a sequence type 5 staphylococcal cassette chromosome mec (SCCmec) type II strain of methicillin-resistant Staphylococcus aureus (MRSA). Based on the nucleotide sequences of PVL phages, we developed PCRs to discriminate among five PVL phages, with a preliminary classification into two morphological groups (elongated-head type and icosahedral-head type) with four PCRs, including two PCRs for identifying the gene lineage between lukS-PV and the tail gene. The phages were then classified into five types by four PCRs identifying each phage-specific structure. With these PCRs, we examined the PVL phage types of 67 MRSA strains isolated in Japan from 1979 through 1985 and since 2000 and found that two morphologically distinct phages were predominant in Japan. The icosahedral-head-type phage, represented by the phi108PVL type, was identified for 39 of 53 strains isolated from 1979 through 1985. Of 26 other Japanese isolates, 25 belonged either definitively or presumably to elongated-head types as follows: 3 belonged to the phiSa2958 type; 8 were determined to belong to an elongated-head type, but a determination of greater specificity was not made; and 14 belonged to a phiSa2958-like phage of unknown type. We induced prophages by treatment with mitomycin C from six strains of the phiSa2958 type or of phiSa2958-like unknown-type phages; five of six strains carried intact PVL-carrying phages, which can infect other S. aureus strains and might generate novel PVL-positive strains of S. aureus. That various SCCmec elements were carried by different strains of the same phage type suggests that S. aureus strains might independently acquire PVL phages before they acquire various SCCmec elements.

Protective effects of ischemic postconditioning against hypoxia-reoxygenation injury and hydrogen peroxide-induced damage in isolated rat hearts.

OBJECTIVE: Ischemic preconditioning (PR) protects hearts from ischemia-reperfusion injury. The purpose of the present study was to examine the protective effect of PR and postconditioning (PT) against hypoxia-reoxygenation injury and H(2)O(2)-induced damage in isolated rat hearts. METHODS AND RESULTS: Hearts from male Sprague-Dawley rats were perfused with Krebs-Henseleit solution by Langendorff methods and subjected to two protocols. In protocol A, control hearts underwent 45 min of hypoxia and 30 min of reoxygenation. Three PT cycles of 10 s of ischemia and 10 s of reperfusion after 45 min of hypoxia increased the recovery of the pressure-rate product. Three PR cycles of 3 min of ischemia and 5 min of reperfusion before hypoxia were also protective, and decreased the release of glutamic oxaloacetic transaminase. A combination of PR and PT resulted in greater protection than either alone. In protocol B, control hearts underwent perfusion with H(2)O(2) (120 muM) until the left ventricular end-diastolic pressure was elevated to 50 mmHg, and then H(2)O(2) was washed out for 30 min. Three PT cycles of 30 s of ischemia and 30 s of reperfusion before the 30 min washout increased the level of recovery of the pressure-rate product and decreased left ventricular end-diastolic pressure to baseline levels. CONCLUSIONS: The results of the present study indicate that PT protects hearts from hypoxia-reoxygenation injury and H(2)O(2)-induced damage. In addition, PR combined with PT offers more effective protection than PR or PT alone.

Mechanisms of cardioprotective effects of magnesium on hypoxia-reoxygenation-induced injury.

During cardiac ischemia or hypoxia, increased levels of extracellular Mg show cardioprotective effects. The mechanisms of high level Mg-induced cardioprotection were examined in Langendorff perfused rat hearts. In the control group (1.2 mM Mg during hypoxia), the recovery of the left ventricular developed pressure (LVDP) after 30 min of reoxygenation was 57.6+/-3.0% of the level observed before hypoxia. In the high Mg group (12 mM Mg during hypoxia), the time course of recovery was faster than in the control group; the recovery level of LVDP improved to 78.4+/-4.2%. This protective effect of high levels of Mg decreased to 69.0+/-3.6% with the application of 5-hydroxydecanoic acid (100 muM), a specific mitochondrial ATP-sensitive potassium channel (K(ATP)) blocker. In the low Ca group (0.2 mM Ca during hypoxia), the recovery of LVDP did not reach the level observed in the high Mg group (64.7+/-5.9%), but with application of diazoxide, a specific mitochondrial K(ATP) channel opener, the LVDP recovery improved to 81.8+/-11.1%, similar to the level observed in the high Mg group. These results suggest that cardioprotective effects of high levels of extracellular Mg during hypoxia occur not only due to energy conservation and/or by intracellular prevention of Ca(2+) over-load, but also by opening of the mitochondrial K(ATP) channel.