Search

Methicillin-resistant Staphylococcus aureus (MRSA) is a major multidrug-resistant bacterial pathogen. The evolution of MRSA is dynamic posing an ongoing threat to humans. The evolution of MRSA includes horizontal gene transfer, which is mediated by mobile genetic elements, plasmids, and bacteriophages, and also mutations. In this review, we clarify the recent trends in MRSA from the perspectives of drug-resistance transfer and uncontrollable infections, particularly those occurring in community settings. We first address the role of MRSA as a disseminator of multidrug resistance. We have studied the cell-to-cell transfer of drug resistance, in which transfer frequencies range from 10-3 to 10-8. The mechanisms of drug-resistance transfers include the self-transmission of large plasmids, the mobilization of small nonconjugative plasmids, the generalized transduction of phages, and the transfer of transposons with circular intermediates. We then discuss uncontrollable infections. Although several anti-MRSA agents have been developed, uncontrollable cases of MRSA infections are still reported. Examples include a case of uncontrollable sepsis arising from a community-associated MRSA (CA-MRSA) with the ST8/SCCmecIVl genotype, and a relapsing severe invasive infection of ST30/SCCmecIVc CA-MRSA in a student athlete. Some of these cases may be attributable to unique adhesins, superantigens, or cytolytic activities. The delayed diagnosis of highly adhesive and toxic infections in community settings may result in CA-MRSA diseases that are difficult to treat. Repeated relapse, persistent bacteremia, and infections of small-colony variants may occur. To treat MRSA infections in community settings, these unique features of MRSA must be considered to ensure that diagnostic delay is avoided.

Anti-acid drugs, proton pump inhibitor (PPI) and histamine-2 blocker (H2-blocker), are commonly prescribed to treat gastrointestinal disorders. These anti-acid drugs alter gut microbiota in the general population, but their effects are not known in hemodialysis patients. Hence, we investigated the microbiota composition in hemodialysis patients treated with PPIs or H2-blocker. Among 193 hemodialysis patients, we identified 32 H2-blocker users, 23 PPI users, and 138 no anti-acid drug subjects. Fecal samples were obtained to analyze the gut microbiome using 16S RNA amplicon sequencing. Differences in the microbial composition of the H2-blocker users, PPI users, and controls were assessed using linear discriminant analysis effect size and the random forest algorithm. The species richness or evenness (α-diversity) was similar among the three groups, whereas the inter-individual diversity (β-diversity) was different between H2-blocker users, PPI users, and controls. Hemodialysis patients treated with H2-blocker and PPIs had a higher microbial dysbiosis index than the controls, with a significant increase in the genera Provetella 2, Phascolarctobacterium, Christensenellaceae R-7 group, and Eubacterium oxidoreducens group in H2-blocker users, and Streptococcus and Veillonella in PPI users. In addition, compared to the H2-blocker users, there was a significant enrichment of the genera Streptococcus in PPI users, as confirmed by the random forest analysis and the confounder-adjusted regression model. In conclusion, PPIs significantly changed the gut microbiota composition in hemodialysis patients compared to H2-blocker users or controls. Importantly, the Streptococcus genus was significantly increased in PPI treatment. These findings caution against the overuse of PPIs.

Sequence type 59 (ST59) is the dominant type of community-associated methicillin-resistant Staphylococcus aureus (MRSA) in Taiwan. Previously, we reported that ST59 MRSA harbors enterococcal IS1216V-mediated multidrug-resistant composite transposons MESPM1 or MES6272-2. The MES were found to have a mosaic structure, largely originating in enterococci and partly native to S. aureus. The current study aimed to track the origin of the MES and how they disseminated from enterococci to ST59 S. aureus. A total of 270 enterococcal isolates were analyzed, showing that two ST64 Enterococcus faecalis isolated in 1992 and 11 clonal complex 17 Enterococcus faecium harbored MESPM1-like and MES6272-2-like structures, respectively. Sequence analysis revealed that ST64 E. faecalis strain N48 acquired the MESPM1-like structure on the plasmid pEflis48. The pEflis48 harbored the enterococci-originated region (erythromycin, kanamycin, and streptomycin resistances) and the S.aureus-originated region (chloramphenicol resistance) of MESPM1 but was separated by the replication region of the plasmid. Homologous recombination between the two direct repeats of IS1216V resulted in excision of the replication region of the plasmid to regenerate MESPM1. The p4780-1 and pV19 of E. faecium carried MES6272-2-like structures with IS1216V, albeit with multiple insertions by other insertion sequences. The findings show that IS1216V plays important roles in bidirectional gene transfer of multidrug resistance between enterococci and S. aureus.

Objectives This study aimed to provide detailed genetic characterization of Tn6636, a multidrug-resistant and composite mobile element, in clinical isolates of Staphylococcus aureus. Methods A total of 112 ermB-positive methicillin-susceptible S. aureus (MSSA) and 224 ermB-positive methicillin-resistant S. aureus (MRSA) isolates collected from 2000 to 2015 were tested for the presence of Tn6636. Detection of the plasmids harboring Tn6636 was performed by S1 nuclease digestion pulsed-field gel electrophoresis (PFGE) analysis, conjugation test, and whole genome sequencing (WGS). Results Prevalence of Tn6636 in MSSA is higher than that in MRSA. Ten MSSA isolates and 10 MRSA isolates carried Tn6636. The 10 MSSA isolates belonged to three sequence types (ST), including ST7 (n = 6), ST5 (n = 3), and ST59 (n = 1). The 10 MRSA isolates belonged to ST188 (n = 8) and ST965 (n = 2). Analysis of plasmid sequences revealed that Tn6636 was harbored by six different mosaic plasmids. In addition to resistance genes, some plasmids also harbored toxin genes. Conclusion The presence of multi-resistant Tn6636 in plasmids of both MSSA and MRSA with various STs suggests its broad dissemination. Results indicate that Tn6636 has existed for at least 16 years in Taiwan. The mosaic plasmids harboring Tn6636 can be transferred by conjugation. Ongoing surveillance of Tn6636 is essential to avoid continued spreading of resistant plasmids.

The fusidic acid (FUS) resistance determinants fusB, fusC, fusD and fusF in coagulase-negative staphylococci (CoNS) clinical isolates were examined. Among 208 FUS-resistant isolates, the fusB gene was the most common resistance determinant in each species, except in Staphylococcus hominis subsp. hominis or in species carrying intrinsic fusD or fusF. In S. hominis subsp. hominis, the fusC gene was the major determinant responsible for FUS resistance. To understand the genetic context of fusC in S. hominis subsp. hominis, 31 fusC-positive S. hominis subsp. hominis isolates were examined. Among these isolates, 14 carried SCCfusC, 3 carried an SCC476-like element and 7 carried a new SCC structure (SCC3390). As shown by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, the S. hominis subsp. hominis clinical isolates showed limited clonality. Taken together, SCCfusC has been found in S. hominis subsp. hominis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus capitis subsp. ureolyticus and Staphylococcus aureus, suggesting its wide distribution and spread among different species of staphylococci.

Streptococcus pneumoniae, a common human pathogen, colonizes the nasopharynx and causes diseases including acute otitis media (AOM). Herein, pneumococcal serotype distributions in children before and after PCV7 vaccination and in patients with pneumococcal disease in Siberian Russia (Krasnoyarsk) are reported. Analyses included antimicrobial susceptibility testing, sequence typing (ST), pulsed field gel electrophoresis, virulence-related surface protein gene (VSG) typing with novel primers and structural analysis by scanning electron microscopy. In healthy children (HC) prior to administration of PCV7, drug-susceptible serotype23F/ST1500 was a major pneumococcal genotype. In the PCV7 trial, multidrug-resistant serotype19A/ST320 emerged in vaccinees after PCV7, exhibiting a PCV7-induced serotype replacement. Multidrug-resistant serotype19A/ST320 was evident in patients with AOM. Community-acquired pneumonia (CAP) isolates showed genetic similarities to the AOM (ST320) genotype, constituting a common non-invasive AOM-CAP group. In contrast, meningitis isolates were more divergent. Overall, 25 ST types were identified; five (20%) of which were Krasnoyarsk-native. Regarding VSGs, PI-1 (rlrA/rrgB), PI-2 (pitA/B), psrP and cbpA were present at 54.3%, 38.6%, 48.6%, and 95.7%, respectively, with two major VSG content types, PI-1- /PI-2- /psrP+ /cbpA+ and PI-1+ /PI-2+ /psrP- /cbpA+ , being found for HC and non-invasive diseases, respectively. A major clone of serotype19A/ST320 (PI-1+ /PI-2+ ) produced the longest pneumococcal wire (pilus) structures in colonies. ST1016 (PI-1- /PI-2- ) in HC had HEp-2 cell-adherent pili. These results suggest that serotype19A/ST320 and related genotypes, with the VSG content type PI-1+ /PI-2+ /psrP- /cbpA+ , emerged in vaccinees after PCV7 in Siberia, accompanying diseases in non-vaccinated children, and that some genotypes (serotypes19A/ST320 and 18/ST1016) produced novel pneumococcal structures, predicting their roles in colony formation and adherence.

The pathogenesis of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia in influenza-infected elderly individuals has not yet been elucidated in detail. In the present study, a 92-year-old man infected with influenza developed CA-MRSA pneumonia. His CA-MRSA was an emerging type, originated in ST121/agr4 S. aureus, with diversities of Panton-Valentine leucocidin (PVL)(-)/spat5110/SCCmecV(+) versus PVL(+)/spat159((etc.))/SCCmec (-), but with common virulence potentials of strong adhesin and cytolytic activities. Resistance to erythromycin/clindamycin (inducible-type) and gentamicin was detected. Pneumonia improved with the administration of levofloxacin, but with the subsequent development of fatal aspiration pneumonia. Hence, characteristic CA-MRSA with strong adhesin and cytolytic activities triggered influenza-related sequential complications.

Objectives Small colony variants (SCVs) of Staphylococcus aureus are associated with persistent and drug-resistant infections. We demonstrated for the first time the emergence of SCVs in a patient with vancomycin-intermediate S. aureus (VISA) infection during long-term treatment with daptomycin. Methods A 73-year-old man with septic arthritis was infected with VISA. The patient was treated with daptomycin; however, the patient remained infected with VISA, with continuous isolation of VISA from his blood during long-term treatment. Five VISA isolates were characterized by: PFGE; genotyping including staphylococcal cassette chromosome mec (SCCmec), spa and MLST; antimicrobial susceptibility testing; and scanning and transmission electron microscopy. WGS and fatty acid analysis were also performed. Results The five VISA isolates were from a single clone of ST239/spa3(t037) and, of these, the first three were SCCmecIII positive and daptomycin susceptible, whereas the last two were SCCmecIII negative and daptomycin resistant and exhibited the characteristics of SCVs. The first and last isolates showed 13 remarkable genetic differences in SCCmec and the mprF, cls2, clpX and fabF genes. Of these, mutation of fabF (encoding the fatty acid synthase) seemed to be partially responsible for the slow growth and ultrastructural features, including an abnormal intercellular substance, and for the daptomycin resistance of SCVs. Conclusions For the first time, we identified SCVs of VISA in a patient with septic arthritis during long-term treatment with daptomycin. Daptomycin-resistant SCVs of VISA were evolved in a stepwise manner and the mutation of fabF is likely responsible for the physical and ultrastructural characteristics and daptomycin resistance.

Clonal complex 59 (CC59) is the dominant community-associated methicillin-resistant Staphylococcus aureus (MRSA) strain in Taiwan and includes the Asian-Pacific clone with Panton-Valentine leukocidin (PVL)-negative/staphylococcal cassette chromosome mec (SCCmec) IVg and the Taiwan clone characterised as PVL-positive/SCCmec V (5C2&5). Nevertheless, data on the evolutionary history of the two dominant CC59 MRSA clones in Taiwan are scarce. In this study, a total of 258 CC59 S. aureus strains from Taiwan were classified by multiple-locus variable-number tandem repeat analysis (MLVA), which revealed two major clusters (MT1 and MT2) with distinct mobile genetic elements (MGEs). However, sequencing and PCR mapping of the β-lactamase-producing plasmid revealed no difference among all CC59 S. aureus strains. Bayesian evolutionary analysis of 18 of the CC59 S. aureus strains based on core genome alignment revealed two clades: (i) Clade A, which shared the samples with MT1, had the features of mainly harbouring gentamicin-resistant MES6272-2 or MES4578, φSA3 translocation in νSaβ and SCCmec IVg; and (ii) Clade B, which shared the samples with MT2, had the features of mainly harbouring streptomycin-resistant MESPM1, PVL phage and SCCmec V (5C2&5). Based on the time-calibrated phylogenetic tree, the estimated time of divergence of the two clades was in the 1980s. These results suggest that the CC59 S. aureus progenitor acquired a β-lactamase-producing plasmid and then developed the varied genetic backgrounds, which were associated with the acquisition and maintenance of distinct MGEs, leading to differences in antimicrobial susceptibility profiles and molecular virulence determinants.

Antimicrobial resistance inStaphylococcus aureusis a major problem and the acquisition of resistance genes may occur by horizontal gene transfer (HGT). The transposon, an important means of HGT, is recognized as a mobile genetic element that can integrate in plasmids, replicate and transfer to other strains. We have previously reported a novel structure of theEnterococcus faecium-originated Tn1546-ermBelement inS. aureus. The emergence of the Tn1546-like element is an emerging problem that requires continuous monitoring. In the present study, we expand the examination of Tn1546-ermBelement toermB-positive methicillin-susceptibleS. aureus(MSSA) (n = 116) andermB-positive methicillin-resistantS. aureus(MRSA) (n = 253) during a 16-year period, from 2000 to 2015. PCR mapping showed that 10 MSSA and 10 MRSA carried the Tn1546-ermBelement. The 10 MSSA belonged to three sequence types (ST), ST7 (n = 6), ST5 (n = 3), and ST59 (n = 1), and the 10 MRSA belonged to two STs, ST188 (n = 8) and ST965 (n = 2). Since only clonal complex 5 (including ST5, ST85, ST231, and ST371) MRSA, ST8 MRSA and ST5 MSSA have been previously reported to carry Tn1546plasmids, this is the first report describing the presence of the Tn1546-ermBelement in ST7/5/59 MSSA and ST188/965 MRSA. Plasmid sequencing revealed that the Tn1546-ermBelement was harbored by five different mosaic plasmids. In addition to resistance genes, some plasmids also harbored toxin genes.

A high prevalence of fusC (16/46, 59%) was found in fusidic acid-resistant methicillin-resistant Staphylococcus aureus isolates collected from 2008 to 2010. Nucleotide sequencing of fusC and flanking regions revealed a novel staphylococcal cassette chromosome (SCC) structure, SCC fusC , which was integrated into rlmH and located upstream from SCC mec . The SCC fusC element contained speG , which may contribute to the polyamine resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) emerged in 1961, just after the introduction of methicillin as a countermeasure against penicillinase-producing “multidrug-resistant” S. aureus, a threat at that time. Since then, MRSA has posed a continuous threat to medical care as a major multidrug-resistant pathogen in hospitals. In 1997–1999, severe invasive infection with MRSA occurred in the community, and this attracted attention as community-associated MRSA (CA-MRSA). The evolutionary steps include species-to-species transfer and salvage of key genetic structures, responsible for community spread, virulence, and resistance. The MRSA epidemic, including invasive diseases, in the community is dynamic.

Antibiotic-resistant patterns, a mecA homologue complex, and staphylococcal cassette chromosome mec (SCCmec) were analysed in samples of ready-to-eat (RTE) street food in Taiwan. RTE food samples (270) were collected in three densely populated Taiwanese cities between June and November 2014. Among 14 strains being identified as methicillin-resistant coagulase-negative staphylococci (MRCoNS), genetic diversities was determined by PFGE analysis. SCCmec types IV, V, VIII and TXG-24 were detected in 9, and mecASs (a mecA homologue) detected in 8. The mecASs gene complex from S. sciuri subsp. sciuri TXG-24 was found to be closely related to those found in both S. sciuri subsp. sciuri (ATCC29062) and S. sciuri subsp. rodentium (ATCC700061). SCCmecTXG24 carries a class A mec complex, a ccrA5B3-like gene complex, a heavy metal gene complex, and an IS1216 mobile element carrying tet(S). Matching identity to ccrA5 was 84.5% for ccrA in S. pseudintermedius KM241. Matching identify to ccrB3 was 92.1% for ccrB in S. pseudintermedius AI16. Similar ccrA and SCCmec boundary sequences suggest that SCCmec is easily transmitted to coagulase-negative staphylococci (CoNS). Based on MRCoNS strains identified in this research, Taiwanese RTE food products likely carry multiple antibiotic resistance genes that can be transmitted to hospitals and other clinical settings.

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non-motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 μm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup-like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup-like structure composed of two membranes.

We determined the resistance determinants in 274 erythromycin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) isolates during a 13-year period, 2000 to 2012. The resistance phenotypes, inducible macrolide-lincosamide-streptogramin (iMLS), constitutive MLS (cMLS), and macrolide-streptogramin (MS) resistance phenotypes, were examined by a double-disk diffusion D test. The ermB gene was more frequent (35%; 97/274) than ermC (27%; 75/274) or ermA (21%; 58/274). All 97 ermB -positive isolates harbored Tn 551 and IS 1216V . The majority (89/97) of ermB -positive isolates displayed the cMLS phenotype and carried mobile element structure (MES)-like structures, which has been previously reported in sequence type 59 (ST59) methicillin-resistant S. aureus (MRSA). The remaining 8 ermB -carrying isolates, belonging to ST7 ( n = 4), ST5 ( n = 3), and ST59 ( n = 1), were sasK intact and did not carry MES-like structures. Unlike a MES-like structure that was located on the chromosome, the ermB elements on sasK -intact isolates were located on plasmids by S1 nuclease pulsed-field gel electrophoresis (PFGE) analysis and conjugation tests. Sequence data for the ermB -containing region (14,566 bp) from ST59 NTUH_3874 revealed that the best match was a Tn 1546 -like element in plasmid pMCCL2 DNA (GenBank accession number AP009486 ) of Macrococcus caseolyticus . Tn 1546 is recognized as an enterococcal transposon and was known from the vancomycin resistance gene cluster in vancomycin-resistant Enterococcus (VRE). So far, acquisitions of Tn 1546 in S. aureus have occurred in clonal complex 5 (CC5) MRSA, but not in MSSA. This is the first report that MSSA harbors an Enterococcus faecium -originated ermB -positive Tn 1546 -like element located on a plasmid.

Methicillin-resistant Staphylococcus aureus (MRSA) includes hospital-acquired MRSA (HAMRSA) and community-acquired MRSA (CA-MRSA). Panton-Valentine leukocidin (PVL)-positive multilocus sequence type 30 (ST30) MRSA is one of worldwide CA-MRSA, which has also persisted in Japan since the 1980s. However, unexpectedly, it was not the same ST30 clone throughout. Before 2000, it was HA-MRSA with spa43 and ψSa3sea (phage Sa3 carrying the sea gene) and only one PVL-positive MRSA in Japan; in the 1980s, ST30 MRSA accounted for 23.5% of HA-MRSA, showed multidrug resistance, had high MICs for oxacillin and imipenem, and caused decubitus and pneumonia in hospitalized patients. A dynamic clonal change (spa43/ψSa3sea→ spa19) occurred around 2000-2002. A rare spa43/ψSa3sea/SCCmecI-IE25923 genotype also emerged. After 2002, the prevalent spa19 clone was CA-MRSA; it accounted for only 0.3% (or less) of MRSA in hospitals but 7.6% of CA-MRSA. Since 2007, PVL-positive CA-MRSA with other ST types (such as ST8, ST22, and ST59) also emerged in Japan, albeit at a low frequency. ST30/spa19 CA-MRSA occasionally caused severe invasive infections and a novel ST1335/spa19 genotype emerged. These ST30/spa19 CA-MRSA and variants were identified by pulsed field gel electrophoresis. Further analysis revealed that PVL-positive ST30/spa19 CA-MRSA is a highlyvirulent, successful clone, having a potential of clonal expansion.

To understand the high prevalence of fusB genes in fusidic acid-resistant Staphylococcus epidermidis , analysis of resistance elements in 34 isolates was performed. First, sequence analysis of the aj1 -LP- fusB region indicated that at least three types were present. Type I contained full-length aj1 , type II contained a partial aj1 truncated from nucleotide position 93 to 421, and type III contained a more truncated aj1 that retained only the last 37 bp. Isolates with type I or type II aj1 displayed slightly higher levels of resistance to fusidic acid (MICs, 8 to 32 μg/ml) than did those with type III aj1 (MICs, 4 to 16 μg/ml). Subsequent sequencing of the flanking regions of fusB from four selected isolates carrying different types of aj1 -LP- fusB regions revealed that the fusB genes were all located on phage-related resistance islands (RIs), referred to as SeRI fusB -2793 , SeRI fusB -704 , SeRI fusB -5907 , and SeRI fusB -7778 , respectively. Among them, three islands (SeRI fusB -2793 , SeRI fusB -704 , and SeRI fusB -5907 ) were located downstream of groEL (corresponding to the 44-min position based on Staphylococcus aureus whole genomic sequences), and one (SeRI fusB -7778 ) was located downstream of rpsR (corresponding to the 8-min position). All of the RIs were inserted into integrase-recognized att sites. Among 34 isolates, the insertion sites of fusB RIs were mostly (28/34, 82%) located downstream of groEL and two were located downstream of rpsR , but four remained unidentified. The pulsotype distribution indicated that fusB -containing S. epidermidis isolates were heterogeneous. In conclusion, the fusB resistance determinant in S. epidermidis was highly associated with phage-related RIs. This is the first report of fusB RI in S. epidermidis .

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), which often produces Panton-Valentine leucocidin (PVL), has emerged worldwide as a life-threatening pathogen. Herein, we describe molecular characteristics of MRSA isolated from abdominal cellulitis in a 7-year-old Japanese boy. This MRSA was PVL-positive and belonged to the Taiwanese multiple drug-resistant CA-MRSA clone with the genotype of ST59, staphylococcal cassette chromosome mec (SCCmec) VII (SCCmecV, according to recent reclassification), agr1a (a novel agr1 subtype), and SaPI (which carried seb1, a newly designated variant seb gene). This study demonstrates the first isolation of the Taiwanese PVL-positive ST59 MRSA clone in Japan. The data also demonstrate novel subtypes in agr1 and seb and suggest that a combination of agr1a, seb1, and PVL could contribute to cellulitis (and its recurrence). Recently, a variety of PVL-positive MRSA clones are accumulating in Japan.

Background and Objectives Multidrug-resistant (MDR) Pseudomonas aeruginosa infection is becoming a critical problem worldwide. Currently, only limited therapeutic options are available for the treatment of infections caused by MDR P. aeruginosa, therefore, the development of new alternative treatments is needed. Toluidine blue O (TBO) is an effective antibacterial photosensitizing agent against various bacteria. However, reports on antibacterial photosensitization of MDR bacteria are limited. This study aims to determine the in vitro photobactericidal activity of TBO against MDR P. aeruginosa. Study Design/Materials and Methods The efficacy of antibacterial photodynamic inactivation, DNA fragmentation and protein carbonylation of three MDR P. aeruginosa strains and one susceptible strain was compared using TBO as the photosensitizer followed by red light irradiation (630 nm, 90 J/cm2) from a light-emitting diode light source. Subsequently, the efficacy of TBO photodynamic inactivation (TBO-PDI) on 60 MDR strains, including 11 with the efflux pump phenotype and 49 with no pump activity, was tested using the minimum lethal drug concentration (MLC) assay. Results TBO-PDI caused similar bactericidal effect (6–7 logs of killing effect), DNA fragmentation and protein carbonylation in three MDR and one susceptible P. aeruginosa strains. Although the TBO accumulation assay indicated that TBO is a substrate for the efflux pump, TBO-PDI produce similar photobactericidal activity against 60 MDR P. aeruginosa strains, either with or without efflux-pump phenotype, and 19 susceptible strains. Conclusion MDR did not affect the susceptibility of P. aeruginosa strains to TBO-PDI. The efflux pump played an insignificant role in TBO-PDI of MDR P. aeruginosa. Lasers Surg. Med. 41:391–397, 2009. © 2009 Wiley-Liss, Inc.

In this study, Escherichia coli LPS dose-dependently (100-500 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human and rabbit platelets stimulated by agonists. LPS also dose-dependently inhibited the intracellular Ca2+ mobilization in human platelets stimulated by collagen. In addition, LPS (200 and 500 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 microg/ml) significantly increased the production of nitrate within a 10-min incubation period. Furthermore, LPS also dose-dependently inhibited platelet aggregation induced by PDBu (30 nmol/l), a protein kinase C activator. These results indicate that the antiplatelet activity of E. coli LPS may be involved in the activation of a nitric oxide/cyclic GMP pathway in platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients.

ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaβ split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256’s strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a major concern worldwide. In the United States, ST8 CA-MRSA with SCCmecIVa (USA300) has been predominant, affecting the entire United States. In this study, we investigated Japanese ST8 CA-MRSA with new SCCmecIVl (designated ST8 CA-MRSA/J), which has emerged in Japan since 2003. Regarding community spread and infections, ST8 CA-MRSA/J spread in 16.2-34.4% as a major genotype in the community in Japan, and was associated with skin and soft tissue infections (SSTIs), colitis, and invasive infections (sepsis, epidural abscesses, and necrotizing pneumonia), including influenza prodrome cases and athlete infections, similar to USA300. It spread to even public transport and Hong Kong through a Japanese family. Regarding genetic diversity, ST8 CA-MRSA/J included ST and spa variants and was classified into at least three pulsed-field gel electrophoresis types, ST8 Jα to γ. Of those, ST8 Jβ was associated with severe invasive infections. As for genomics, ST8 CA-MRSA/J showed high similarities to USA300, but with marked diversity in accessory genes; e.g., ST8 CA-MRSA/J possessed enhanced cytolytic peptide genes of CA-MRSA, but lacked the Panton-Valentine leukocidin phage and arginine catabolic mobile element, unlike USA300. The unique features of ST8 CA-MRSA/J included a novel mosaic SaPI (designated SaPIj50) carrying the toxic shock syndrome toxin-1 gene with high expression; the evolution included salvage (through recombination) of hospital-acquired MRSA virulence. The data suggest that ST8 CA-MRSA/J has become a successful native clone in Japan, in association with not only SSTIs but also severe invasive infections (posing a threat), requiring attention.

Objectives To determine MICs of fusidic acid for and identify genetic determinants of resistance in Staphylococcus cohnii isolates. Methods Susceptibility to fusidic acid was determined by the standard agar dilution method in 24 S. cohnii subsp. urealyticus clinical isolates, 7 S. cohnii subsp. cohnii clinical isolates and 2 reference strains. Sequencing of a novel resistance determinant, fusF, and its flanking regions was performed by long and accurate PCR and inverse PCR. To evaluate the function of fusF, the MIC of fusidic acid was determined for recombinant Staphylococcus aureus carrying a plasmid expressing fusF. Results A total of 25 S. cohnii subsp. urealyticus (24 clinical isolates and 1 reference strain) and 2 S. cohnii subsp. cohnii displayed low-level resistance to fusidic acid (MICs 2-16 mg/L). Sequencing of a 4259 bp fragment from S. cohnii subsp. urealyticus ATCC 49330 revealed a novel resistance gene, designated fusF, which displayed 70.5% nucleotide and 67.3% amino acid identity to fusD. Expression of fusF in S. aureus confers resistance to fusidic acid. Conclusions A novel FusB-family gene, fusF, was identified as a major resistance determinant in S. cohnii clinical isolates resistant to fusidic acid.

Background/objectives Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized as a leading pathogen and has been shown to be genetically different from the health care-associated MRSA (HA-MRSA). Photodynamic therapy (PDT) is considered a potential alternative method for the treatment of resistant bacterial infections, but the effect of PDT on CA-MRSA is unknown. The purpose of this study was to compare the bactericidal effects of toluidine blue O (TBO) on CA-MRSA and HA-MRSA and investigate the photodynamic inactivation effects of TBO (TBO-PDI) against bacterial virulence factors. Materials and methods TBO-PDI effects were determined by measuring the survival fractions for four strains and bactericidal activities for 26 CA-MRSA isolates and 26 HA-MRSA isolates. The influences of TBO-PDI on DNA fragmentation and the activities of protease, lipase, staphylococcal α-hemolysin, and enterotoxin were studied. Results TBO-PDI has effective bactericidal activity against both CA- and HA-MRSA. However, the bactericidal activity of TBO-PDI was significantly higher against HA-MRSA than CA-MRSA isolates. In addition, TBO-PDI treatment using a sublethal TBO concentration led to reduced production of several virulence factors, including protease, lipase, staphylococcal α-hemolysin, and enterotoxin. Conclusion Although TBO-PDI is slightly less effective against CA-MRSA than HA-MRSA isolates, TBO-PDI could reduce the production of virulence factors at a sublethal TBO concentration, which would be beneficial for treating CA-MRSA infections.

A 15-year-old boy, who had had a furuncle on his femur, developed femoral pyomyositis and osteomyelitis complicated by septic pulmonary embolism. Panton-Valentine leukocidin-positive (PVL(+)) ST59 methicillin-susceptible Staphylococcus aureus (MSSA) was isolated from pus and blood. Chemotherapy was started with cefazolin, followed by combination therapy with meropenem/vancomycin with surgery. The MSSA (strain KS1) was positive for increased levels of cytolytic peptide (psmα and hld) and staphylococcal enterotoxin B (SEB), and manifested IS1216V-mediated multidrug resistance (to erythromycin, clindamycin, kanamycin, streptomycin, and chloramphenicol), similar to a genome-analyzed reference strain (PM1) of ST59/SCCmecV(5C2&5) community-associated methicillin-resistant S. aureus (Taiwan CA-MRSA), but unlike another reference strain (M013) of Taiwan CA-MRSA in terms of resistance. The data suggest that CA-MSSA KS1, characterized by PVL, increased levels of cytolytic peptide, SEB, and multidrug resistance, is a possible ancestral strain of Taiwan CA-MRSA and causes the unique association of osteomyelitis and septic pulmonary embolism, requiring complicated management.

The ST5 lineage of methicillin-resistant Staphylococcus aureus (MRSA) is one of the most globally disseminated hospital-associated MRSA (HA-MRSA) lineages. We isolated a new local variant (designated ST764) over at least 5 years that causes invasive infections, including necrotizing fasciitis, and is carried by medical students, as well as household members. Analysis of the genome sequence of one isolate compared to that of the reference ST5 strain revealed that ST764 had acquired virulence traits similar to those of community-associated MRSA (CA-MRSA) through the acquisition of two new mobile genetic elements, ACMEII and SaPInn54, which carried ACME arcA and the staphylococcal enterotoxin B gene ( seb ), respectively, and through enhanced expression of cytolytic peptide genes, although ST764 was negative for Panton-Valentine leukocidin. Other differences between ST764 and ST5 included the acquisition of an ACMEII-related cassette (cJR1), prophage φ2 NN54 , and streptococcal Tn 5251 and decreased numbers of copies of Tn 554 . As for superantigen genes, although the two possessed seg , sei , sem , sen , and seo , ST764 lacked tst , sec , sel , and sep . The data suggest that ST764 MRSA is a novel hybrid variant of ST5 HA-MRSA with the characteristics of CA-MRSA and that the evolution of ST764 includes multiple steps, e.g., acquisition of novel or nonstaphylococcal mobile elements.

Clonal complex 59 (CC59) Staphylococcus aureus in Taiwan includes both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). As the most prominent community-associated MRSA (CA-MRSA) in Taiwan, CC59 has two major clones characterized as PVL-negative SCCmec IV (carrying the staphylococcal cassette chromosome mec IV but Panton-Valentine leukocidin-negative) and PVL-positive SCCmec V (5C2&5). We investigated the drug resistance, phylogeny and the distribution and sequence variation of SCCmec, staphylococcal bacteriophage φSA3, genomic island νSaβ and MES (an enterococcal mobile genetic element conferring multidrug resistance) in 195 CC59 S. aureus. Sequencing and PCR mapping revealed that all of the CC59/SCCmec V (5C2&5) MRSA strains had acquired MESPM1 or its segregants, and obtained a φSA3-related fragment in νSaβ. In contrast, MES6272-2 and MES4578, which showed gentamicin resistance that was not encoded by MESPM1, were dominant in SCCmec IVg MRSA. Translocation of a whole φSA3 into νSaβ instead of only a φSA3-related fragment was common in SCCmec IVg MRSA. However, the non-subtype-g SCCmec IV MRSA (SCCmec IVa is the major) still carried MES and νSaβ structures similar to those in SCCmec V (5C2&5) MRSA. A minimum spanning tree constructed by multiple-locus variable-number tandem repeat analysis revealed that SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA grouped respectively in two major clades. The CC59 MSSA was equally distributed among the two clades, while the non-subtype-g SCCmec IV MRSA mostly clustered with SCCmec V (5C2&5) MRSA. Our findings strongly suggest that CC59 MSSA acquired divergent mobile genetic elements and evolved to SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA/non-subtype-g SCCmec IV MRSA independently. The evolutionary history of CC59 S. aureus explains how mobile genetic elements increase the antimicrobial resistance and virulence and contribute to the success of CA-MRSA in Taiwan.

Methicillin-resistant Staphylococcus aureus (MRSA) with ST59/SCCmecV and Panton-Valentine leukocidin gene is a major community-acquired MRSA (CA-MRSA) lineage in Taiwan and has been multidrug-resistant since its initial isolation. In this study, we studied the acquisition mechanism of multidrug resistance in an ST59 CA-MRSA strain (PM1) by comparative genomics. PM1's non-β-lactam resistance was encoded by two unique genetic traits. One was a 21,832-bp composite mobile element structure (MES(PM1)), which was flanked by direct repeats of enterococcal IS1216V and was inserted into the chromosomal sasK gene; the target sequence (att) was 8 bp long and was duplicated at both ends of MES(PM1). MES(PM1) consisted of two regions: the 5'-end side 12.4-kb region carrying Tn551 (with ermB) and Tn5405-like (with aph[3']-IIIa and aadE), similar to an Enterococcus faecalis plasmid, and the 3'-end side 6,587-bp region (MES(cat)) that carries cat and is flanked by inverted repeats of IS1216V. MES(cat) possessed att duplication at both ends and additional two copies of IS1216V inside. MES(PM1) represents the first enterococcal IS1216V-mediated composite transposon emerged in MRSA. IS1216V-mediated deletion likely occurred in IS1216V-rich MES(PM1), resulting in distinct resistance patterns in PM1-derivative strains. Another structure was a 6,025-bp tet-carrying element (MES(tet)) on a 25,961-bp novel mosaic penicillinase plasmid (pPM1); MES(tet) was flanked by direct repeats of IS431, but with no target sequence repeats. Moreover, the PM1 genome was deficient in a copy of the restriction and modification genes (hsdM and hsdS), which might have contributed to the acquisition of enterococcal multidrug resistance.

Iliopsoas abscesses (IPAs) from methicillin-resistant Staphylococcus aureus (MRSA) are rare; however, IPAs from community-associated MRSA (CA-MRSA) may be increasing. In Japan, we previously described an adolescent athlete case of Panton–Valentine leukocidin (PVL)-positive ST30 CA-MRSA (strain NN12). In this study, we describe an IPA and discitis case from a variant of the successful PVL-negative CA-MRSA clone (ST8 CA-MRSA/J) in Japan. The patient was a 62-year-old man with intractable eczema, who had been diagnosed with IPAs and discitis (L1–L2). CA-MRSA (strain NN55) was isolated from blood, pus, and joint fluid. The invasive infections seemed to have originated in his intractable eczema, and the characteristics of this case, systemic myalgia and marked thrombocytopenia, seemed to have been caused by an exotoxin. Molecular genetic analysis revealed that NN55 possessed genotype ST8/spa606(t1767)/agr1/CoaIII and SCCmecIV of a novel subtype (encoding new cell-wall-anchored surface protein/J [CWASP/J]), exhibited enhanced expression of the cytolytic peptide genes, psmα and hld, and was resistant to gentamicin (caused by aacA-aphD), similar to ST8 CA-MRSA/J; however, NN55 lacked pathogenicity island SaPIj50 [carrying tst, encoding toxic shock syndrome toxin-1 (TSST-1)] of ST8 CA-MRSA/J, suggesting a variant (ST8 CA-MRSA/Jv). Strains NN12 and NN55 both caused bacteremia, IPAs, and adjacent musculoskeletal infections, preceded by intractable skin infections, and possessed high potential for adherence and enhanced expression of psmα and hld. The data suggest the role of a combination of CA-MRSA adhesin/cytolytic peptides (not PVL or TSST-1) in the pathogenesis of IPAs (and perhaps of systemic myalgia and marked thrombocytopenia).

An 89-year-old man suffered from and died of necrotizing pneumonia with rapid progression and cavity formation due to methicillin-resistant Staphylococcus aureus (MRSA). He was at no risk for hospital-acquired MRSA infection. His MRSA exhibited genotype ST5/ spa 2(t002)/ agr 2/SCC mec II/coagulaseII and was negative for Panton–Valentine leukocidin, indicating the New York/Japan clone (the predominant epidemic hospital-acquired MRSA clone in Japan). However, this strain expressed the cytolytic peptide (phenol-soluble modulin or δ-hemolysin) genes at high level, similar to USA300 (the most common community-acquired MRSA in the United States), indicating a variant of the New York/Japan clone with an important feature of community-acquired MRSA.

Enterobacteriaceae, carrying the New Delhi metallo-β-lactamase-1 (NDM-1) gene (bla (NDM-1)), have emerged and posed a threat since 2006. In Japan, bla (NDM-1)-carrying Escherichia coli was first described in 2010. In this study, we characterized NDM-1-positive Klebsiella pneumoniae strain 419 in Japan, which was isolated from the urine of a 90-year-old Japanese patient who had never been to the Indian subcontinent. K. pneumoniae 419 belonged to ST42. It possessed a surface capsule (with untypeable capsular PCR types) and was resistant to serum killing. K. pneumoniae 419 cells were occasionally flagellated or piliated and autoaggregated. K. pneumoniae 419 was resistant to β-lactams (including carbapenems), aminoglycosides, and fluoroquinolones, and was susceptible to imipenem (or biapenem), aztreonam, polymixin B, and colistin. It possessed at least eight plasmids; of those, a 74-kb plasmid (pKPJ1) of the replicon FIIA carried bla (NDM-1) and was conjugally transferred to E. coli strains, with a 71-kb transferable azithromycin-resistant (mphA (+)) plasmid of the replicon F (pKPJ2), as a large (145-kb) plasmid (pKPJF100) through a transposition event. In addition to bla (NDM-1), pKPJ1 carried arr-2, pKPJ2 carried mphA, and pKPJF100 carried both. They were negative for the 16S rRNA methylase gene, e.g., which is frequently associated with bla (NDM-1). The data demonstrate that K. pneumoniae 419 possessed virulence- and fitness-associated surface structures, was resistant to serum killing, and possessed a unique (or rare) genetic background in terms of ST type and bla (NDM-1)-carrying plasmid.

A total of 71 fusidic acid-resistant Staphylococcus aureus (45 methicillin-resistant and 26 methicillin-susceptible) isolates were examined for the presence of resistance determinants. Among 45 fusidic acid-resistant methicillin-resistant S. aureus (MRSA), isolates, 38 (84%) had fusA mutations conferring high-level resistance to fusidic acid (the MIC was ≥128 μg/ml for 22/38), none had fusB , and 7 (16%) had fusC . For 26 fusidic acid-resistant methicillin-susceptible S. aureus (MSSA), only 3 possessed fusA mutations, but 15 (58%) had fusB and 8 (31%) had fusC . Low-level resistance to fusidic acid (MICs ≤ 32 μg/ml) was found in most fusB - or fusC -positive isolates. For 41 isolates (38 MRSA and 3 MSSA), with fusA mutations, a total of 21 amino acid substitutions in EF-G ( fusA gene) were detected, of which R76C, E444K, E444V, C473S, P478S, and M651I were identified for the first time. The nucleotide sequencing of fusB and flanking regions in an MSSA isolate revealed the structure of partial IS 257 - aj1 -LP- fusB - aj2 - aj3 -IS 257 -partial blaZ , which is identical to the corresponding region in pUB101, and the rest of fusB -carrying MSSA isolates also show similar structures. On the basis of spa and staphylococcal cassette chromosome mec element (SCC mec ) typing, two major genotypes, spa type t037-SCC mec type III (t037-III; 28/45; 62%) and t002-II (13/45; 29%), were predominant among 45 MRSA isolates. By pulsed-field gel electrophoresis analysis, 45 MRSA isolates were divided into 12 clusters, while 26 MSSA isolates were divided into 15 clusters. Taken together, the distribution of fusidic acid resistance determinants ( fusA mutations, fusB , and fusC ) was quite different between MRSA and MSSA groups.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common multidrug-resistant (MDR) pathogen. We herein discussed MRSA and its infections in Krasnoyarsk, Siberian Russia between 2007 and 2011. The incidence of MRSA in 3,662 subjects was 22.0% and 2.9% for healthcare- and community-associated MRSA (HA- and CA-MRSA), respectively. The 15-day mortality rates for MRSA hospital- and community-acquired pneumonia (HAP and CAP) were 6.5% and 50%, respectively. MRSA CAP cases included pediatric deaths; of the MRSA pneumonia episodes available, ≥27.3% were associated with bacteremia. Most cases of HA-MRSA examined exhibited ST239/spa3(t037)/SCCmecIII.1.1.2 (designated as ST239Kras), while all CA-MRSA cases examined were ST8/spa1(t008)/SCCmecIV.3.1.1(IVc) (designated as ST8Kras). ST239Kras and ST8Kras strongly expressed cytolytic peptide (phenol-soluble modulin α, PSMα; and δ-hemolysin, Hld) genes, similar to CA-MRSA. ST239Kras pneumonia may have been attributed to a unique set of multiple virulence factors (MVFs): toxic shock syndrome toxin-1 (TSST-1), elevated PSMα/Hld expression, α-hemolysin, the staphylococcal enterotoxin SEK/SEQ, the immune evasion factor SCIN/SAK, and collagen adhesin. Regarding ST8Kras, SEA was included in MVFs, some of which were common to ST239Kras. The ST239Kras (strain OC3) genome contained: a completely unique phage, φSa7-like (W), with no att repetition; S. aureus pathogenicity island SaPI2R, the first TSST-1 gene-positive (tst +) SaPI in the ST239 lineage; and a super copy of IS256 (≥22 copies/genome). ST239Kras carried the Brazilian SCCmecIII.1.1.2 and United Kingdom-type tst. ST239Kras and ST8Kras were MDR, with the same levofloxacin resistance mutations; small, but transmissible chloramphenicol resistance plasmids spread widely enough to not be ignored. These results suggest that novel MDR and MVF+ HA- and CA-MRSA (ST239Kras and ST8Kras) emerged in Siberian Russia (Krasnoyarsk) associated with fatal pneumonia, and also with ST239Kras, a new (Siberian Russian) clade of the ST239 lineage, which was created through stepwise evolution during its potential transmission route of Brazil-Europe-Russia/Krasnoyarsk, thereby selective advantages from unique MVFs and the MDR.