Noriko Urushibara

Molecular Characteristics of Community-Acquired Methicillin-Resistant Staphylococcus aureus in Hokkaido, Northern Main Island of Japan: Identification of Sequence Types 6 and 59 Panton-Valentine Leucocidin–Positive Community-Acquired Methicillin-Resistant Staphylococcus aureus

Prevalence and molecular characteristics of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) were studied in Hokkaido, the main northern island of Japan. Among the 1,015 S. aureus isolates derived from clinical specimens of outpatients collected in 2009, methicillin resistance gene mecA was detected in 189 isolates (18.6%). The most frequent staphylococcal cassette chromosome mec (SCCmec) type in MRSA was II (83.1%), followed by IV (6.9%) and V (3.2%). MRSA with type II-SCCmec showed multiple drug resistance and harbored various toxin and virulence factor genes except for Panton-Valentine leucocidin (PVL) gene. These isolates were mostly classified into sequence type 5 (ST5) (or other STs in CC5) and coagulase genotype II and were thus genetically similar to hospital-acquired MRSA, which have been predominating in Japan (New York/Japan clone). PVL gene was detected in three MRSA strains belonging to ST6 (two strains) and ST59 (one strain), having type IVa- and Vt-SCCmec, respectively, and also in two methicillin-susceptible S. aureus ST121 and ST188. The arcA gene within the arginine catabolic mobile element (ACME) was detected in the two PVL-negative ST5 MRSA strains, which had type IIa- or V-SCCmec. The PVL gene-positive ST6 and ST59 CA-MRSA strains were susceptible to more antimicrobials and had less virulence factor genes than the PVL-negative ST5 MRSA, including the ACME-arcA-positive strains. In the present study, ST6 was identified as a lineage of PVL-positive CA-MRSA, the ACME-arcA was first detected in ST5 MRSA with type V-SCCmec, and ST59 Taiwanese CA-MRSA strain was isolated in Hokkaido for the first time. These findings suggest a potential spread of these emerging CA-MRSA clones in Japan.

Full genomic analysis of a G8P[1] rotavirus strain isolated from an asymptomatic infant in Kenya provides evidence for an artiodactyl-to-human interspecies transmission event.

Group A rotavirus (GAR) G8P[1] strains, found sometimes in cattle, have been reported rarely from humans. Therefore, analysis of the full genomes of human G8P[1] strains are of significance in the context of studies on interspecies transmission of rotaviruses. However, to date, only partial‐length nucleotide sequences are available for the 11 genes of a single human G8P[1] strain, while the partial sequences of two other strains have been reported. The present study reports the first complete genome sequence of a human G8P[1] strain, B12, detected from an asymptomatic infant in Kenya in 1987. By nucleotide sequence identities and phylogenetic analyses, the full‐length nucleotide sequences of VP7‐VP4‐VP6‐VP1‐VP2‐VP3‐NSP1‐NSP2‐NSP3‐NSP4‐NSP5 genes of strain B12 were assigned to G8‐P[1]‐I2‐R2‐C2‐M2‐A3‐N2‐T6‐E2‐H3 genotypes, respectively. Each of the 11 genes of strain B12 appeared to be more related to cognate genes of artiodactyl (ruminant and/or camelid) and/or artiodactyl‐derived human GAR strains than those of most other rotaviruses. Strain B12 exhibited low levels of genetic relatedness to canonical human GAR strains, such as Wa and DS‐1, ruling out the possibility of its origin from reassortment events between artiodactyl‐like human and true human strains. These observations suggest that strain B12 might have been directly transmitted from artiodactyls to humans. Unhygienic conditions and close proximity of humans to livestock at the sampling site might have facilitated this rare event. This is the first report on a full genomic analysis of a rotavirus strain from Kenya. To our knowledge, strain B12 might be the oldest G8 strain characterized molecularly from the Africa continent. J. Med. Virol. 83:367–376, 2011.

Whole genomic analysis reveals the porcine origin of human G9P[19] rotavirus strains Mc323 and Mc345.

The group A rotavirus (RVA) P[19] is a rare P-genotype of the RVA VP4 gene, reported so far in humans and pigs. Whole genomic analyses of P[19] strains are essential to study their origin and evolutionary patterns. To date, all the 11 genes of only two P[19] strains, RVA/Human-wt/IND/RMC321/1990/G9P[19] and RVA/Human-wt/IND/mani-97/2006/G9P[19], have been analyzed, providing evidence for their porcine origin. In the present study, the whole genomes of the first reported human P[19] strains, RVA/Human-tc/THA/Mc323/1989/G9P[19] and RVA/Human-tc/THA/Mc345/1989/G9P[19], were analyzed. Strains Mc323 and Mc345 exhibited a G9-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotype constellation. With the exception of the NSP5 gene, both the strains were closely related to each other. Most of the genes of Mc323 (VP2-4, VP6-7, NSP1-4 genes) and Mc345 (VP2-4, VP6-7 and NSP1-5 genes) appeared to be of porcine origin, whilst the exact origin of VP1 and NSP5 genes of Mc323 and VP1 gene of Mc345 could not be ascertained. Therefore, strains Mc323 and Mc345 were found to have a porcine RVA genetic backbone, and are likely of porcine origin. Taken together, our observations corroborated the hypothesis that P[19] strains might be derived from porcine RVAs, providing important insights into the origin of P[19] strains, and on interspecies transmission of RVAs.

Molecular epidemiology and genetic evolution of the whole genome of G3P[8] human rotavirus in Wuhan, China, from 2000 through 2013.

Background Rotaviruses are a major etiologic agent of gastroenteritis in infants and young children worldwide. Since the latter of the 1990s, G3 human rotaviruses referred to as “new variant G3” have emerged and spread in China, being a dominant genotype until 2010, although their genomic evolution has not yet been well investigated. Methods The complete genomes of 33 G3P[8] human rotavirus strains detected in Wuhan, China, from 2000 through 2013 were analyzed. Phylogenetic trees of concatenated sequences of all the RNA segments and individual genes were constructed together with published rotavirus sequences. Results Genotypes of 11 gene segments of all the 33 strains were assigned to G3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, belonging to Wa genogroup. Phylogenetic analysis of the concatenated full genome sequences indicated that all the modern G3P[8] strains were assigned to Cluster 2 containing only one clade of G3P[8] strains in the US detected in the 1970s, which was distinct from Cluster 1 comprising most of old G3P[8] strains. While main lineages of all the 11 gene segments persisted during the study period, different lineages appeared occasionally in RNA segments encoding VP1, VP4, VP6, and NSP1-NSP5, exhibiting various allele constellations. In contrast, only a single lineage was detected for VP7, VP2, and VP3 genes. Remarkable lineage shift was observed for NSP1 gene; lineage A1-2 emerged in 2007 and became dominant in 2008–2009 epidemic season, while lineage A1-1 persisted throughout the study period. Conclusion Chinese G3P[8] rotavirus strains have evolved since 2000 by intra-genogroup reassortment with co-circulating strains, accumulating more reassorted genes over the years. This is the first large-scale whole genome-based study to assess the long-term evolution of common human rotaviruses (G3P[8]) in an Asian country.

Two novel arginine catabolic mobile elements and staphylococcal chromosome cassette mec composite islands in community-acquired methicillin-resistant Staphylococcus aureus genotypes ST5-MRSA-V and ST5-MRSA-II.

OBJECTIVES The arginine catabolic mobile element (ACME) is a novel staphylococcal genetic island. ACME is located downstream of the staphylococcal cassette chromosome mec (SCCmec), forming the ACME-SCCmec composite island. Recently, ACME II (located upstream of SCCmec IV) was described from a methicillin-resistant Staphylococcus aureus (MRSA) strain M1 in Denmark (ST8-MRSA-IVa) and 15 MRSA isolates in Ireland (ST22-MRSA-IVh). We report the novel genetic characteristics of the ACME-SCCmec composite islands found in Japanese community-acquired MRSA (CA-MRSA) isolates. METHODS ACME-SCCmec composite islands from two ACME-arcA-positive CA-MRSA isolates with the genotypes ST5-MRSA-V (SR141) and ST5-MRSA-II (SR388) were characterized using long-range PCR and nucleotide sequencing. RESULTS Both isolates harboured a 12 kb DNA region primarily identified in ACME II in Staphylococcus epidermidis ATCC 12228 upstream of each SCCmec. The arcA and its flanking regions in SR141 and SR388 showed high sequence identity (99.8% at the highest) to those in MRSA M1 and M08/0126 (the representative of 15 Irish ST22-MRSA-IVh isolates), suggesting that the ACMEs of these four isolates originated from the same ancestral gene. The ACME II-like element in SR141 included an insertion sequence IS1182 at a position close to SCCmec, resulting in a new variant. SR388 contained ∼11.5 kb of the J1 region of type I SCCmec (J1 SCCmecI) between orfX and ACME (orfX-J1 SCCmecI-ACME II), unlike the homologous region in M08/0126 (orfX-ACME II-J1 SCCmecI). CONCLUSIONS This is the first report of the ACME II-like element inserted upstream of SCCmec in CA-MRSA with the genotypes ST5-MRSA-V and ST5-MRSA-II.

Whole-genome analysis reveals the complex evolutionary dynamics of Kenyan G2P[4] human rotavirus strains.

Although G2P[4] rotaviruses are common causes of acute childhood diarrhoea in Africa, to date there are no reports on whole genomic analysis of African G2P[4] strains. In this study, the nearly complete genome sequences of two Kenyan G2P[4] strains, AK26 and D205, detected in 1982 and 1989, respectively, were analysed. Strain D205 exhibited a DS-1-like genotype constellation, whilst strain AK26 appeared to be an intergenogroup reassortant with a Wa-like NSP2 genotype on the DS-1-like genotype constellation. The VP2-4, VP6-7, NSP1, NSP3 and NSP5 genes of strain AK26 and the VP2, VP4, VP7 and NSP1-5 genes of strain D205 were closely related to those of the prototype or other human G2P[4] strains. In contrast, their remaining genes were distantly related, and, except for NSP2 of AK26, appeared to originate from or share a common origin with rotavirus genes of artiodactyl (ruminant and camelid) origin. These observations highlight the complex evolutionary dynamics of African G2P[4] rotaviruses.

Analysis of Staphylococcal Cassette Chromosome mec in Staphylococcus haemolyticus and Staphylococcus sciuri: Identification of a Novel ccr Gene Complex with a Newly Identified ccrA Allotype (ccrA7)

Methicillin resistance in staphylococci is conferred by the acquisition in its chromosome of the mecA gene, which is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). Genetic type of SCCmec is defined by combination of mec gene complex class and cassette chromosome recombinase gene (ccr) allotype. In this study, we analyzed genetic diversity of the SCCmec in 11 Staphylococcus haemolyticus strains and a Staphylococcus sciuri strain, which were recently isolated from clinical specimens in Bangladesh. Among these strains, only two S. haemolyticus strains were proved to have the known types of SCCmec, that is, SCCmec V (class C2 mec-ccrC) and VII (class C1 mec-ccrC). Five S. haemolyticus strains were assigned two unique mec-ccr gene complexes combination; that is, class C1 mec-ccrA4B4 (four isolates) and class A mec-ccrC (one isolate). In the remaining four S. haemolyticus strains with class C1 mec, no known ccr allotypes could be detected. A single S. sciuri strain with class A mec complex carried a ccrA gene belonging to a novel allotype designated ccrA7, together with ccrB3. The ccrA7 gene in the S. sciuri strain showed 61.7%-82.7% sequence identity to the ccrA gene sequences published so far, and 75.3% identity to ccrA3, which is a component of the type 3 ccr complex (ccrA3-ccrB3) in methicillin-resistant Staphylococcus aureus. The results of the present study indicated that mec gene complex and ccr genes in coagulase-negative staphylococci are highly divergent, and distinct from those of common methicillin-resistant S. aureus. Identification of the novel ccrA7 allotype combined with ccrB3 suggested an occurrence of recombination between different ccr complexes in nature.

Full genomic analysis of a simian SA11-like G3P[2] rotavirus strain isolated from an asymptomatic infant: Identification of novel VP1, VP6 and NSP4 genotypes

We report here the full genomic analysis of a simian SA11-like G3P[2] group A rotavirus (GAR) strain, B10, isolated from an asymptomatic infant in Kenya in 1987. By nucleotide sequence identities and phylogenetic analyses, the VP7-VP4-VP2-VP3-NSP1-NSP2-NSP3-NSP5 genes of strain B10 exhibited maximum genetic relatedness to those of the different isolates of simian strain SA11, and were assigned to the G3-P[2]-C5-M5-A5-N5-T5-H5 genotypes, respectively. On the other hand, the VP1, VP6 and NSP4 genes of strain B10 did not belong to any of the established GAR genotypes, and therefore, were assigned to new genotype numbers R8, I16 and E13, respectively, by the Rotavirus Classification Working Group. These observations suggested that strain B10 might have originated from reassortment event/s involving simian SA11-like strains and GAR strains from unknown animal host species (possibly other wild animals) preceding transmission to humans. Alternatively, considering the lack of data on simian GARs, it might be also possible that the VP1, VP6 and NSP4 genes of strain B10 are those of unknown simian strains, and that strain B10 might be a typical simian strain that was directly transmitted to humans. Therefore, either hypothesis pointed towards a rare instance of possible direct transmission of GARs from an animal host (possibly a monkey or some other wild animal) to humans. This was corroborated by the presence of different species of wild animals including non-human primates, and unhygienic conditions at the sampling site. To our knowledge, the present study is the first report on the detection of a simian SA11-like G3P[2] GAR strain in humans.

Virulence Factors and Genetic Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus aureus Isolates in Myanmar

Staphylococcus aureus produces virulence factors, including various exotoxins and adhesins, which are associated with a variety of symptoms caused by its infections. In the present study, the prevalence of these virulence factors was analyzed for 23 S. aureus strains isolated from wound infections in hospitals, nasal swabs, or vomit from patients and cooks in a food poisoning case and from healthy adults in Yangon, Myanmar. Among these strains, five were methicillin-resistant S. aureus (MRSA) derived from pus (four strains, SCCmec III, ST239) and a healthy adult (one strain, SCCmec-IVa, ST5). The Panton-Valentine leukocidine (PVL) gene was detected in five methicillin-susceptible S. aureus (MSSA) clinical strains belonging to ST121 (CC121). The MRSA clinical strains had only a few or no staphylococcal enterotoxin (SE) genes, whereas PVL-positive MSSA and an MRSA strain from a healthy adult possessed an enterotoxin gene cluster (seg, sei, sem, sen, seo, and selu). Strains from the food poisoning case had either SE genes or only etd and edin-B. Adhesin genes, which are associated with binding to fibronectin, fibrinogen, and elastin, were detected in all the MRSA and most of the MSSA strains examined. However, the bone sialoprotein-binding protein gene (bbp) and the variant form of the elastin-binding protein gene (ebpS-v) with an internal 180 bp deletion were identified only in the MSSA strains harboring the PVL gene. These findings suggest that those genetic traits are characteristic of PVL-positive ST121 S. aureus strains in Myanmar.

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells.

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.