Akihito Nishiyama

Community-acquired methicillin-resistant Staphylococcus aureus: community transmission, pathogenesis, and drug resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is able to persist not only in hospitals (with a high level of antimicrobial agent use) but also in the community (with a low level of antimicrobial agent use). The former is called hospital-acquired MRSA (HA-MRSA) and the latter community-acquired MRSA (CA-MRSA). It is believed MRSA clones are generated from S. aureus through insertion of the staphylococcal cassette chromosome mec (SCCmec), and outbreaks occur as they spread. Several worldwide and regional clones have been identified, and their epidemiological, clinical, and genetic characteristics have been described. CA-MRSA is likely able to survive in the community because of suitable SCCmec types (type IV or V), a clone-specific colonization/infection nature, toxin profiles (including Pantone-Valentine leucocidin, PVL), and narrow drug resistance patterns. CA-MRSA infections are generally seen in healthy children or young athletes, with unexpected cases of diseases, and also in elderly inpatients, occasionally surprising clinicians used to HA-MRSA infections. CA-MRSA spreads within families and close-contact groups or even through public transport, demonstrating transmission cores. Re-infection (including multifocal infection) frequently occurs, if the cores are not sought out and properly eradicated. Recently, attention has been given to CA-MRSA (USA300), which originated in the US, and is growing as HA-MRSA and also as a worldwide clone. CA-MRSA infection in influenza season has increasingly been noted as well. MRSA is also found in farm and companion animals, and has occasionally transferred to humans. As such, the epidemiological, clinical, and genetic behavior of CA-MRSA, a growing threat, is focused on in this study.

Genotypes, intrafamilial transmission, and virulence potential of nasal methicillin-resistant Staphylococcus aureus from children in the community.

Pediatric outpatients and healthy children in the community were examined for nasal methicillin-resistant Staphylococcus aureus (MRSA) in Japan. MRSA isolation frequencies were 0.7% (3/426) and 3.7% (5/136), respectively, in pediatric outpatients and healthy children in the community (overall frequency, 1.4%). The frequency of MRSA isolation was higher in children 5–9 years of age compared with the other age groups. All eight MRSA strains isolated were Panton-Valentine leukocidin-negative. Of these, three with the genotype multilocus sequence type (ST) 8/spa606/SCCmecIV (2 cases) and ST88/spa999/SCCmecIV/exfoliative toxin A gene (eta) were identical or similar to MRSA from bullous impetigo, determined by pulsed-field gel electrophoresis. One strain with ST764 (ST5 variant)/spa2/SCCmecII/staphylococcal enterotoxin B gene seb2 (seb variant) was similar to MRSA from bacteremia, and one with ST5/spa2/SCCmecII was the Pandemic New York/Japan clone. The remaining three strains, with ST22/spa998/SCCmecI, ST380/spa799/SCCmecIV, and ST857/spa416/SCCmecII, have not been identified. All MRSA strains were resistant to one or more non-β-lactam antibiotics, and the ST5 and ST764 strains were multidrugresistant. Family analysis demonstrated parent-to-child transmission (for ST8 and ST764), as well as acquisition from outside the family (for ST8 and ST380). The data suggest that young school-age children have a higher carriage rate of nasal MRSA than children of other ages, and that not only community-acquired MRSA strains but also MRSA strains with characteristics of hospital-acquired MRSA are spreading in the community.

Capture of heat-killed Mycobacterium bovis bacillus Calmette-Guérin by intelectin-1 deposited on cell surfaces

Intelectin is an extracellular animal lectin found in chordata. Although human and mouse intelectin-1 recognize galactofuranosyl residues included in cell walls of various microorganisms, the physiological function of mammalian intelectin had been unclear. In this study, we found that human intelectin-1 was a serum protein and bound to Mycobacterium bovis bacillus Calmette-Guérin (BCG). Human intelectin-1-binding to BCG was inhibited by Ca(2+)-depletion, galactofuranosyl disaccharide, ribose, or xylose, and was dependent on the trimeric structure of human intelectin-1. Although monomeric, mouse intelectin-1 bound to BCG, with its C-terminal region contributing to efficient binding. Human intelectin-1-transfected cells not only secreted intelectin-1 into culture supernatant but also expressed intelectin-1 on the cell surface. The cell surface intelectin-1 was not a glycosylphosphatidylinositol-anchored membrane protein. Intelectin-1-transfected cells captured BCG more than untransfected cells, and the BCG adherence was inhibited by an inhibitory saccharide of intelectin-1. Intelectin-1-preincubated cells took up BCG more than untreated cells, but the adhesion of intelectin-1-bound BCG was the same as that of untreated BCG. Mouse macrophages phagocytosed BCG more efficiently in medium containing mouse intelectin-1 than in control medium. These results indicate that intelectin is a host defense lectin that assists phagocytic clearance of microorganisms.

Emergence of the community-acquired methicillin-resistant Staphylococcus aureus USA300 clone in a Japanese child, demonstrating multiple divergent strains in Japan

In 2008 we isolated methicillin-resistant Staphylococcus aureus (MRSA) from an 11-month-old Japanese girl who lived in Saitama, Japan, and suffered from cellulitis of the lower thigh and sepsis. The MRSA (strain NN47) belonged to multilocus sequence type (ST) 8 and exhibited spa363 (t024), agr1, staphylococcal cassette chromosome mec (SCCmec) type IVa, and coagulase type III. It was positive for Panton–Valentine leukocidin (PVL) and the arginine catabolic mobile element (ACME). Pulsed-field gel electrophoresis (PFGE) demonstrated that the MRSA was the USA300 clone, which is the predominant community-acquired MRSA (CA-MRSA) in the US. Strain NN47 was divergent, in terms of the spa type and patterns of PFGE and plasmids, from the USA300-0114 type strain or USA300 strain NN36, previously isolated from a visitor (Indian girl) from the US. Strain NN47 was resistant to erythromycin, in addition to β-lactam agents (e.g., oxacillin). These data demonstrate the first emergence of the USA300 clone in Japanese children who have never been abroad and have had no contact with foreigners (and therefore, the first USA300 spread in Japan), and also emergence of multiple divergent strains of the USA300 clone in Japan. Because the USA300 clone is highly transmissible and virulent, surveillance of the USA300 clone is needed.

Chitin particles induce size-dependent but carbohydrate-independent innate eosinophilia.

Murine Mφ that phagocytose CMP develop into M1; this response depends on the size and the chemical composition of the particles. In contrast, recent studies concluded that chitin particles induce M2 and eosinophil migration, promoting acquired Th2 immune responses against chitin‐containing microbes or allergens. This study examined whether these apparently inconsistent responses to chitin could be induced by variation in the size and chemical composition of the chitin particles. We compared the responses of Mφ with CMP, LCB, and Sephadex G‐100 beads (>40 μm). Beads were given i.p. to WT mice and to mice deficient in a CRTH2, a receptor for the eosinophil chemoattractant PGD2. In contrast to the M1 activation induced by CMP, i.p. administration of LCB or Sephadex beads induced within 24 h a CRTH2‐dependent peritoneal eosinophilia, as well as CRTH2‐independent activation of peritoneal Mφ that expressed Arg I, an M2 phenotype. LCB‐induced Mφ exhibited elevated Arg I and a surface MR, reduced surface TLR2 levels, and no change in the levels of CHI3L1 or IL‐10 production. Our results indicate that the effects of chitin in vivo are highly dependent on particle size and that large, nonphagocytosable beads, independent of their chemical composition, induce innate eosinophilia and activate Mφ expressing several M2, but not M1, phenotypes.

A New Local Variant (ST764) of the Globally-Disseminated ST5 Lineage of Hospital-Associated Methicillin-Resistant Staphylococcus aureus (MRSA), Carrying the Virulence Determinants of Community-Associated MRSA

ABSTRACT 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 φ2NN54, and streptococcal Tn5251 and decreased numbers of copies of Tn554. 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.

Healthcare- and Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) and Fatal Pneumonia with Pediatric Deaths in Krasnoyarsk, Siberian Russia: Unique MRSA's Multiple Virulence Factors, Genome, and Stepwise Evolution

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.

Isolation and molecular characterization of methicillin-resistant Staphylococcus aureus from public transport

Methicillin‐resistant Staphylococcus aureus (MRSA) not only causes disease in hospitals, but also in the community. The characteristics of MRSA transmission in the environment remain uncertain. In this study, MRSA were isolated from public transport in Tokyo and Niigata, Japan. Of 349 trains examined, eight (2.3%) were positive for MRSA. The MRSA isolated belonged to sequence types (STs) 5, 8, 88, and 89, and included community infection‐associated ST8 MRSA (with novel type IV staphylococcal cassette chromosome mec) and the ST5 New York/Japan hospital clone. The data indicate that public transport could contribute to the spread of community‐acquired MRSA, and awareness of this mode of transmission is necessary.

Heat-killed BCG induces biphasic cyclooxygenase 2+ splenic macrophage formation—role of IL-10 and bone marrow precursors

Previous studies have shown that prostaglandin E2 (PGE2) release by splenic F4/80+ cyclooxygenase (COX)‐2+ macrophages (MØ) isolated from mice, treated with mycobacterial components, plays a major role in the regulation of immune responses. However, splenic MØ, isolated from untreated mice and treated in vitro with lipopolysaccharide and interferon‐γ, express COX‐1 and COX‐2 within 1 day but release only minimal amounts of PGE2 following elicitation with calcium ionophore A23187. For further characterization of in vivo requirements for development of PGE2‐releasing MØ (PGE2‐MØ), C57Bl/6 [wild‐type (WT)], and interleukin (IL)‐10‐deficient (IL‐10−/−) mice were treated intraperitoneally with heat‐killed Mycobacterium bovis bacillus Calmette‐Guerin (HK‐BCG). One day following injection, COX‐2 was induced in splenic MØ of both mouse strains. However, PGE2 biosynthesis by these MØ was not increased. Thus, expression of COX‐2 is not sufficient to induce PGE2 production in vivo or in vitro. In sharp contrast, 14 days after HK‐BCG treatment, PGE2 release by COX‐2+ splenic MØ increased as much as sevenfold, and a greater increase was seen in IL‐10−/− cells than in WT cells. To further determine whether the 14‐day splenic PGE2‐MØ could be derived from bone marrow precursors, we established a chimera in which bone marrow cells were transfused from green fluorescent protein (GFP)‐transgenic donors to WT mice. Donors and recipients were treated with HK‐BCG simultaneously, and marrow transfusion was performed on Days 1 and 2. On Day 14 after BCG treatment, a significant number of spleen cells coexpressed COX‐2 and GFP, indicating that bone marrow‐derived COX‐2+ MØ may be responsible for the increased PGE2 production.

A rapid screening method for Panton-Valentine leucocidin-positive community-acquired methicillin-resistant Staphylococcus aureus belonging to multilocus sequence type 30 and its related clone using a combination of multiplex PCR and pulsed-field gel electrophoresis

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), which is often positive for Panton-Valentine leucocidin (PVL), is increasingly noted as an emerging pathogen worldwide. In Japan, PVLpositive CA-MRSA belonging to multilocus sequence type (ST) 30 has spread and caused, for example, pediatric death due to community-acquired pneumonia and severe pelvic abscesses in an athlete. In this study, we investigated a new rapid screening method for PVL-positive ST30 CA-MRSA and its related clone by a combination of multiplex polymerase chain reaction (M-PCR) and pulsed-field gel electrophoresis (PFGE). For M-PCR, the targets of the assay were the five genes for PVL, collagen adhesin, bone sialoprotein adhesin, methicillin resistance, and S. aureus-specifi c thermostable nuclease. Only PVL-positive ST30 CA-MRSA strains produced all five bands in M-PCR. With PFGE, Japanese strains and most foreign strains of PVLpositive ST30 CA-MRSA shared the same pattern. Moreover, PFGE distinguished current PVL-positive CA-MRSA ST30/spa19 strains from previous PVL-positive MRSA ST30/spa43 strains (which were isolated at the time of nosocomial MRSA outbreaks in the late 1980s and early 1990s) in Japan. Thus, the M-PCR assay rapidly, and the M-PCR/PFGE combination assay more precisely, discriminated between PVL-positive ST30 CA-MRSA (or its related clone) and PVL-positive CA-MRSA belonging to other ST types such as ST1, 8, 59, and 80, PVL-negative CA-MRSA, hospital-acquired MRSA, methicillin-susceptible S. aureus, or coagulase-negative staphylococci (CNS), including MRCNS. This screening method is more useful than genotyping for routine work in many clinical laboratories.