Ivan Reva

Novel Characteristics of Community-Acquired Methicillin-Resistant Staphylococcus aureus Strains Belonging to Multilocus Sequence Type 59 in Taiwan

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains, which often produce Panton-Valentine leucocidin (PVL), are increasingly noted worldwide. In this study, we examined 42 MRSA strains (25 PVL-positive [PVL+] strains and 17 PVL-negative [PVL−] strains) isolated in Taiwan for their molecular characteristics. The PVL+ MRSA strains included CA-MRSA strains with multilocus sequence type (ST) 59 (major PVL+ MRSA in Taiwan), its variants, and worldwide CA-MRSA ST30 strains. The PVL− MRSA strains included the pandemic Hungarian MRSA ST239 strain, the Hungarian MRSA ST239 variant, MRSA ST59 (largely hospital-acquired MRSA strains) and its variants, the pandemic New York/Japan MRSA ST5 strain (Japanese type), and the MRSA ST8 strain. The major PVL+ CA-MRSA ST59 strain possessed a tetracycline resistance-conferring (tetK positive) penicillinase plasmid and a drug resistance gene cluster (a possible composite transposon) for multidrug resistance. Moreover, it carried a novel staphylococcal cassette chromosome mec (SCCmec) with two distinct ccrC genes (ccrC2-C8). This SCCmec (previously named SCCmec type VT) was tentatively designated SCCmec type VII. Sequencing of the PVL genes revealed the polymorphisms, and the PVL+ CA-MRSA ST59 strain possessed the ST59-specific PVL gene sequence. The data suggest that a significant amount of clonal spread is occurring in Taiwan and that the major PVL+ CA-MRSA ST59Taiwan strain exhibits unique genetic characteristics, such as a novel SCCmec type and an ST59-specific PVL gene sequence.

Molecular Characterization of Methicillin‐Resistant Staphylococcus aureus in Hospitals in Niigata, Japan: Divergence and Transmission

The major methicillin‐resistant Staphylococcus aureus (MRSA) distributed among hospitals in Japan is New York/Japan clone [multilocus sequence type 5 (ST5), agr type 2 and methicillin resistance locus type (SCCmec) II] which possesses both the toxic shock syndrome toxin 1 gene (tst) and staphylococcal enterotoxin C gene (sec). In this study, we collected 245 MRSA strains from four hospitals during 2001 to 2005 in Niigata, Japan, and analyzed tst and sec genes and SCCmec type among them. A total of 13 strains were further examined for their genotypes, virulence gene patterns and drug resistance. Among the 245 strains four tst sec genes patterns were observed; tst+sec+ strains represented a majority of 86.5% and 9.4% were tst−sec−. SCCmec typing revealed that 91.4% had type II, 4.1% type IV and 4.1% type I. Multilocus sequence typing (MLST) revealed that 10 of the 13 typed strains belonged to clonal complex 5 (7 had ST5 while 3 were single locus variants of ST5) with similar characteristics to the New York/Japan clone and possessed multi‐drug resistance with high virulence gene content The remaining 3 strains were ST8 (n=2) and ST91 (n=1). The ST91 strain had SCCmecIV and seemed to originate in the community, while ST8 strains exhibited SCCmec type I, which is distinct from community type IV. The data suggest that MRSA in hospitals in Niigata now mainly includes the New York/Japan clone (undergoing genomic divergence and clonal expansion) and other minor types (e.g. ST8) as well as the community type.

Comparative genomics and drug resistance of a geographic variant of ST239 methicillin-resistant Staphylococcus aureus emerged in Russia.

Two distinct classes of methicillin-resistant Staphylococcus aureus (MRSA) are spreading in hospitals (as hospital-acquired MRSA, HA-MRSA) and in the community (as community-acquired MRSA, CA-MRSA). Multilocus sequence type (ST) 239 MRSA, one of the most worldwide-disseminated lineages, has been noted as a representative HA-MRSA. Here, we isolated ST239 MRSA (spa type 3 [t037] and staphylococcal cassette chromosome mec [SCCmec] type III.1.1.1) and its novel variant with ST239/spa351 (t030)/SCCmecIII.1.1.4 (SCCmecIIIR) not only from hospitals but also from patients with urethritis in the community in Russia. The Russian variant (strain 16K) possessed a hybrid genome consisting of CC8 and CC30, similar to the ST239/spa3/SCCmecIII.1.1.1 HA-MRSA (TW20) genome, but with marked diversity. The 16K′ CC30 section had SCCmecIIIR carrying the dcs-carrying unit (which corresponded to the SCCmecIVc J3 joining region of ST30 CA-MRSA), lacked SCCmercury, and possessed a novel mobile element structure (MES16K) carrying the ccrC-carrying unit (with the recombinase gene ccrC1 allele 3) and drug resistance tranposons. The Russian variant included strains with a high ability to transfer its multiple drug resistance by conjugation; e.g., for strain 16K, the transfer frequency of a chloramphenicol resistance plasmid (p16K-1 with 2.9 kb in size) reached 1.4×10−2, followed by Tn554 conjugative transfer at 3.6×l0−4. The Russian variant, which has been increasing recently, included divergent strains with different plasmid patterns and pulsed field gel electrophoresis profiles. The data demonstrate the alternative nature of ST239 MRSA as CA-MRSA and also as a drug resistance disseminator, and its micro but dynamic evolution in Russia.

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.

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.

Molecular typing of Campylobacter jejuni and C. coli from chickens and patients with gastritis or Guillain-Barré syndrome based on multilocus sequence types and pulsed-field gel electrophoresis patterns.

Campylobacter jejuni has recently been noted as the most common cause of bacterial foodborne diseases in Japan. In the present study, we determined ST types of C. jejuni and Campylobacter coli isolated from chickens and patients with enteritis or GBS in Japan and Thailand. C. jejuni from chickens, enteritis, and GBS exhibited divergent ST types and included several novel types in addition to worldwide common types. C. coli from enteritis was also divergent. Novel ST types may represent unidentified native clones in each country. Pulsed‐field gel electrophoresis confirmed the above typing and demonstrated long‐term persistence and transmission.

Electron microscopic structures, serum resistance, and plasmid restructuring of New Delhi metallo-β-lactamase-1 (NDM-1)-producing ST42 Klebsiella pneumoniae emerging in Japan

Enterobacteriaceae, carrying the New Delhi metallo-β-lactamase-1 (NDM-1) gene (blaNDM-1), have emerged and posed a threat since 2006. In Japan, blaNDM-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 blaNDM-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 blaNDM-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 blaNDM-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 blaNDM-1-carrying plasmid.

Emergence of Panton-Valentine leukocidin-positive ST59 methicillin-susceptible Staphylococcus aureus with high cytolytic peptide expression in association with community-acquired pediatric osteomyelitis complicated by pulmonary embolism

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.

Elderly infection in the community due to ST5/SCCmecII methicillin-resistant Staphylococcus aureus (the New York/Japan clone) in Japan: Panton–Valentine leukocidin-negative necrotizing pneumonia

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/spa2(t002)/agr2/SCCmecII/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.