Mitsuyo Kawaguchiya

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.

Detection and full genomic analysis of G6P(9) human rotavirus in Japan

A rare genotype G6P[9] was identified in two human group A rotavirus strains designated as KF14 and KF17, that were detected in stool specimens from children with diarrhea in Japan. VP7 gene sequences of these two strains were identical and genetically closely related to G6 human rotavirus strains reported in European countries and the United States. To our knowledge, this is the first report of detection of a G6 human rotavirus in Japan. For further genetic analysis to elucidate the origin of the G6 rotavirus, nearly full-length sequences of all 11 RNA segments were determined for the KF17 strain. The complete genomic constellation of KF17 was determined as G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3, a novel genotype constellation for human rotavirus. Phylogenetic analysis indicated that VP6, VP1-3, and NSP2 genes of KF17 clustered with bovine-like G6 human strains and some animal strains into sub-lineages distinct from those of common DS-1-like G2 human rotaviruses. On the other hand, KF17 genes encoding VP4, NSP1, and NSP3-5 showed high sequence identities to the human G3P[9] strain AU-1, and clustered with AU-1 and some feline strains within the same lineage. These findings suggested that the G6P[9] human rotavirus detected in Japan may have occurred through reassortment among uncommon bovine-like human rotaviruses and human/feline AU-1-like rotaviruses.

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.

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.

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.

Genetic diversity of emerging Panton–Valentine leukocidine/arginine catabolic mobile element (ACME)-positive ST8 SCCmec-IVa meticillin-resistant Staphylococcus aureus (MRSA) strains and ACME-positive CC5 (ST5/ST764) MRSA strains in northern Japan

Panton-Valentine leukocidine (PVL) is a distinctive virulence factor of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), and arginine catabolic mobile element (ACME) is a staphylococcal genomic island that enhances fitness and the ability of bacterial cells to colonize on skin and mucous membranes. ACME is characteristically found in USA300, which is a predominant CA-MRSA clone [sequence type (ST) 8] in the USA and is spreading globally, and has also been detected in non-ST8 MRSA at low frequency. In Japan, spread of MRSA with PVL and/or ACME and their genetic traits have not yet been well characterized. In the present study, the prevalence and genetic diversity of PVL(+)/ACME(+) MRSA were investigated for 422 MRSA clinical isolates collected from outpatients in northern Japan over a period of 1 year. All the isolates were genotyped for the staphylococcal cassette chromosome mec (SCCmec) and coagulase genes (coa), and screened for PVL and ACME genes. The PVL(+)/ACME(+) isolates were studied further by genetic analysis, including single-nucleotide polymorphism (SNP) analysis based on PVL genes (lukS-PV-lukF-PV), ACME (arc and opp3 clusters) and the sarU promoter region. Among all the isolates examined, PVL genes and ACME were detected in eight (SCCmec-II, n = 1; SCCmec-IV, n = 6; SCCmec-V, n = 1) and 20 (SCCmec-II, n = 14; SCCmec-IV, n = 5; SCCmec-V, n = 1) isolates, respectively. Five isolates were found to have both PVL genes and ACME (type I), and were classified into ST8/spa-t008/agr-I/coa-IIIa, which is the same genetic traits as USA300. Fifteen PVL(-)/ACME(+) isolates had type ΔII-ACME, belonging to either ST5 or ST764 [clonal complex (CC) 5], and spa-t001, -t002 or -t3557. All the ST8 PVL(+)/ACME-I(+) MRSA had identical sequences of PVL genes (haplotype R) and ACME arc/opp3 clusters as those of USA300. In contrast, in the CC5 PVL(-)/ACME-ΔII(+) MRSA, SNPs in the arc cluster were detected in 11 sites (four haplotypes), with some different profiles of virulence/resistance factors. These results indicated single clonality of ST8 PVL(+)/ACME-I(+) MRSA and heterogeneity of CC5 PVL(-)/ACME-ΔII(+) MRSA, and suggest their potential spread in northern Japan.

Emerging non-PCV13 serotypes of noninvasive Streptococcus pneumoniae with macrolide resistance genes in northern Japan

In Japan, the 7-valent pneumococcal conjugate vaccine (PCV7) was introduced to the nations routine immunization program in April 2013 and was replaced by the 13-valent pneumococcal conjugate vaccine (PCV13) in November 2013. Distribution of serotypes and macrolide resistance genotypes was investigated for a total of 1097 (975 children, 122 adults) and 960 (873 children, 87 adults) clinical isolates of Streptococcus pneumoniae from noninvasive infections in Hokkaido (northern main island of Japan) in the routine immunization periods for PCV7 and PCV13 (April–October 2013 and November 2013–November 2014, respectively). Serotype was determined by sequential multiplex PCR and additional genetic analyses. Macrolide resistance genes erm(B) and mef(A/E) were detected by multiplex PCR. Although the most prevalent serotypes in children were 23A and 6C in the PCV7 period, after replacement with PCV13, 19A became the most common, followed by 6C, 15A and 23A. Among adults, serotype 3 was consistently the most frequent throughout the study periods. Compared with values from the pre-PCV7 routine immunization period, PCV7 serotypes decreased from 48.3 to 3.3% in the PCV13 period among children, while the rates of non-PCV13 serotypes (particularly 15A, 23A, 11A, 10A and 35B) increased from 39.7 to 75.1% (p < 0.001). In the PCV13 period, erm(B), mef(A/E) and both of these genes were detected in 75.8, 31.6 and 11.3% of all isolates, respectively. Serotype 19A accounted for 76.9% of the isolates with both the macrolide resistance genes, and emerging non-PCV13 serotypes 15A, 15C and 23A mostly harboured erm(B).

High prevalence of blaOXA-23 in Acinetobacter spp. and detection of blaNDM-1 in A. soli in Cuba: report from National Surveillance Program (2010–2012)

As a first national surveillance of Acinetobacter in Cuba, a total of 500 Acinetobacter spp. isolates recovered from 30 hospitals between 2010 and 2012 were studied. Acinetobacter baumannii–calcoaceticus complex accounted for 96.4% of all the Acinetobacter isolates, while other species were detected at low frequency (A. junii 1.6%, A. lwoffii 1%, A. haemolyticus 0.8%, A. soli 0.2%). Resistance rates of isolates were 34–61% to third-generation cephalosporins, 49–50% to β-lactams/inhibitor combinations, 42–47% to aminoglycosides, 42–44% to carbapenems and 55% to ciprofloxacin. However, resistance rates to colistin, doxycycline, tetracycline and rifampin were less than 5%. Among carbapenem-resistant isolates, 75% harboured different blaOXA genes (OXA-23, 73%; OXA-24, 18%; OXA-58, 3%). The blaNDM-1 gene was identified in an A. soli strain, of which the species was confirmed by sequence analysis of 16S rRNA gene, rpoB, rpoB–rpoC and rpoL–rpoB intergenic spacer regions and gyrB. The sequences of blaNDM-1 and its surrounding genes were identical to those reported for plasmids of A. baumannii and A. lwoffi strains. This is the first report of blaNDM-1 in A. soli, together with a high prevalence of OXA-23 carbapenemase for carbapenem resistance in Acinetobacter spp. in Cuba.

Emergence of Klebsiella pneumoniae clinical isolates producing KPC‐2 carbapenemase in Cuba

The emergence of Klebsiella pneumoniae producing carbapenemase (KPC) has now become a global concern. As a part of a nationwide multicentre surveillance study in Cuba, three K. pneumoniae clinical isolates resistant to carbapenems were detected for a 1-month period (September to October 2011). PCR and sequence analysis revealed that the three strains harboured blaKPC-2. They showed resistance or intermediate susceptibility to expanded-spectrum cephalosporins, other β-lactams, a β-lactam/β-lactamase inhibitor combination, and gentamicin. Two strains were susceptible only to colistin, whereas the other strain showing colistin resistance was susceptible to fluoroquinolones. These blaKPC-2-positive K. pneumoniae strains were classified into ST1271 (CC29), a novel clone harbouring blaKPC-2, and were revealed to be genetically identical by PCR-based DNA fingerprinting. The three patients infected with the KPC-producing K. pneumoniae had common risk factors, and had no overseas travel experience outside Cuba, suggesting local acquisition of the resistant pathogen. This is the first report of a KPC-producing K. pneumoniae in Cuba. Although detection of KPC in Enterobacteriaceae is still rare in Cuba, our finding indicated that KPC-producing bacteria are a global concern and highlighted the need to identify these microorganisms in clinical laboratories.

Characterization of Enterococcus faecium with Macrolide Resistance and Reduced Susceptibility to Quinupristin/Dalfopristin in a Japanese Hospital: Detection of Extensive Diversity in erm(B)-Regulator Regions

Cross-resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics is mainly mediated by the erm (erythromycin ribosome methylation) genes that encode 23S rRNA methylases in enterococi, and various mechanisms are involved in the streptogramin B resistance. Prevalence of MLSB resistance and its genetic mechanisms were analyzed for a total of 159 strains of Enterococcus faecium isolated from clinical specimens in a university hospital in Japan from 1997 to 2006. Resistance to erythromycin (EM) and clindamycin was detected in 88.1% and 89.9% of all the strains examined, respectively, and expression of resistance was totally constitutive. Although none of the strain was resistant to quinupristin/dalfopristin (Q/D), 28 strains (17.6%) showed intermediate resistance to Q/D (MIC: 2 μg/ml). The erm(B) gene was detected in 139 strains (87.4%), and msrC was found in all the strains examined, whereas no other known MLSB resistance genes were identified. The erm(B) regulator region (RR) containing a coding region of the leader peptide was classified into 13 genetic variations (L1-L3, M, S1-S7, D, and R genotypes) in 56 strains. However, no relatedness was identified between the erm(B) RR genotype and EM resistance, or reduced susceptibility to Q/D, although most of Q/D-intermediate strains were assigned to the L1, L2, and S1 genotypes. Q/D-intermediate strains were classified into five multiple-locus variable-number tandem-repeat analysis (MLVA) types, including four types of clonal complex (CC)-C1, five sequence types (STs), including four STs of CC-17, and several resistance gene/virulence factor profiles. The present study revealed the occurrence of Q/D-intermediate E. faecium, which are composed of heterogeneous strains in Japan, and more genetic diversity in the erm(B) RRs than those reported previously.