Detecting the performance of methicillin-resistant Staphylococcus aureus by a molecular diagnostic assay in positive blood culture: Influence of coexistence of mecA-positive bacteria and diversity in orfX-SCCmec junction region in methicillin-susceptible S. aureus.

BACKGROUND: In blood cultures that test positive for staphylococcal bacteria, rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-susceptible Staphylococcus aureus (MSSA) by molecular assay is useful for appropriate antimicrobial treatment of bloodstream infections. Although the Xpert MRSA/SA BC assay is widely available in clinical settings in Japan, its efficacy has not yet evaluated thoroughly. METHODS: We retrospectively studied 100 blood culture cases positive for S. aureus at Sapporo Medical University Hospital between March 2019 and May 2022. Cycle threshold (CT) values for target genes from the Xpert MRSA/SA BC assay were compared to phenotypic results. Genotyping and genetic analysis of the orfX-SCCmec junction region was performed for selected isolates. RESULTS: We analyzed 25 and 75 isolates assigned to MRSA and MSSA, respectively, using the Xpert MRSA/SA BC assay. Of these, 99 isolates from agar cultures showed compatible susceptibility to oxacillin. One genetically misidentified case of MRSA was found to be caused by the mixed growth of MSSA and methicillin-resistant S. hominis on agar culture. Of the 73 MSSA with pure growth on agar culture, 45 (61.6%) were found to be orfX-SCCmec-positive, spa-positive, and mecA-negative in this assay. These MSSA belong to diverse spa and coa types. CONCLUSION: The Xpert MRSA/SA BC assay accurately identified MRSA and MSSA in positive blood cultures. However, over half of the MSSA isolates showed positive results for orfX-SCCmec, presumably due to genetic diversity in the orfX-associated region of MSSA. Therefore, the coexistence of MSSA and mecA-harboring coagulase-negative staphylococci may cause confusion about identification of MRSA.

Surveillance of Human Rotaviruses in Wuhan, China (2019-2022): Whole-Genome Analysis of Emerging DS-1-like G8P[8] Rotavirus.

Group A rotaviruses (RVAs) are major etiologic agents of gastroenteritis in infants and young children worldwide. To study the prevalence and genetic characteristics of RVAs, a hospital-based surveillance study was conducted in Wuhan, China from June 2019 through May 2022. The detection rates of RVAs were 19.40% (142/732) and 3.51% (8/228) in children and adults, respectively. G9P[8] was the predominant genotype, followed by G8P[8] and G3P[8]. G8P[8] emerged and was dominant in the 2021-2022 epidemic season. The genome constellation of six G8P[8] strains was assigned to G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that the VP7, VP4, VP2, VP3, NSP1, NSP2, NSP3, and NSP5 genes of these G8P[8] strains clustered closely with those of the G8P[8] strains in Asia and were distant from those of the P[8] and G2P[4] strains simultaneously detected in Wuhan. In contrast, the VP1, VP6, and NSP4 genes were closely related to the typical G2P[4] rotavirus, including those of G2P[4] strains simultaneously detected in Wuhan. The detection rate of RVAs decreased in the COVID-19 pandemic era. It was deduced that the G8P[8] rotaviruses that emerged in China may be reassortants, carrying the VP6, VP1, and NSP4 genes derived from the G2P[4] rotavirus in the backbone of the neighboring DS-1-like G8P[8] strains represented by CAU17L-103.

A reverse genetics system for human rotavirus G2P[4].

Rotaviruses (RVs) are an important cause of acute gastroenteritis in young children. Recently, versatile plasmid-based reverse genetics systems were developed for several human RV genotypes; however, these systems have not been developed for all commonly circulating human RV genotypes. In this study, we established a reverse genetics system for G2P[4] human RV strain HN126. Nucleotide sequence analysis, including that of the terminal ends of the viral double-stranded RNA genome, revealed that HN126 possessed a DS-1-like genotype constellation. Eleven plasmids, each encoding 11 gene segments of the RV genome, and expression plasmids encoding vaccinia virus RNA capping enzyme (D1R and D12L), Nelson Bay orthoreovirus FAST, and NSP2 and NSP5 of HN126, were transfected into BHK-T7 cells, and recombinant strain HN126 was generated. Using HN126 or simian RV strain SA11 as backbone viruses, reassortant RVs carrying the outer and intermediate capsid proteins (VP4, VP7 and VP6) of HN126 and/or SA11 (in various combinations) were generated. Viral replication analysis of the single, double and triple reassortant viruses suggested that homologous combination of the VP4 and VP7 proteins contributed to efficient virus infectivity and interaction between other viral or cellular proteins. Further studies of reassortant viruses between simian and other human RV strains will contribute to developing an appropriate model for human RV research, as well as suitable backbone viruses for generation of recombinant vaccine candidates.

Clonal diversity of Clostridium perfringens human clinical isolates with various toxin gene profiles based on multilocus sequence typing and alpha-toxin (PLC) typing.

OBJECTIVES: Clostridium perfringens is a common anaerobic pathogen causing enteritis/enterocolitis and wound infections in humans. We analyzed clonal diversity and toxin gene prevalence in C. perfringens clinical isolates from humans in northern Japan. METHODS: Prevalence of nine toxin genes was analyzed for 585 C. perfringens isolates from patients collected for 20-month period between May 2019 and December 2020 by molecular methods. Sequence type (ST) based on multilocus sequence typing (Xiao's scheme) and alpha-toxin (PLC) sequence type were determined for a total of 124 isolates selected in the present study along with those in our previous study (2017-2018). RESULTS: Toxinotypes A (68.2%) was the most frequent, followed by F (31.6%), and G (0.2%), while additional toxin genes encoding binary enterotoxin (BEC/CPILE) and beta2 toxin were identified in one and six isolates, respectively. Among the 124 isolates with various toxin gene profiles, 62 STs including 53 novel types were identified, revealing the presence of six clonal complexes (CCs) consisting of 27 STs. Most of enterotoxin gene (cpe)-positive isolates belonged to CC36, CC41, and CC117. Based on 22 key amino acids in alpha toxin sequence, four PLC types (I-IV) including 21 subtypes were classified, and their relation to individual STs/CCs was clarified. Two isolates harboring bec/cpile belonged to different STs (ST95, ST131) and PLC types (If, IVb), indicating distribution of this toxin gene to distinct lineages. CONCLUSIONS: The present study revealed the diversity in C. perfringens clones of human origin with various toxin gene profiles represented by ST/CC and PLC type.

Novel staphylococcal cassette chromosome mec (SCCmec) type XIV (5A) and a truncated SCCmec element in SCC composite islands carrying speG in ST5 MRSA in Japan.

BACKGROUND: Staphylococcal cassette chromosome mec (SCCmec) elements are highly diverse and have been classified into 13 types. The arginine catabolic mobile element (ACME) is an SCC-like element harbouring an arginine deiminase pathway gene cluster (ACME-arc). ACME type I (ACME I), additionally including a spermidine/spermine-N1-acetyltransferase gene (speG), is considered to have contributed to the rapid spread of the most successful MRSA clone, USA300. OBJECTIVES: To characterize the SCC composite islands (SCC-CIs) in ST5 MRSA positive for both ACME-arc and speG. METHODS: Three ST5 MRSA strains (SC640, SC792 and SC955) collected in Hokkaido, Japan were subjected to WGS and the SCC-CIs were determined. RESULTS: The SCC-CIs consisted of four (SC640 and SC792) or three (SC955) SCC/SCC-like elements and commonly harboured both an ACME type II' and an SCC encoding speG. These SCC-CIs appear to mimic ACME I in USA300, in that they are equipped with ACME-arc and speG. The SCC-CIs of SC640 and SC792 contained novel SCCmec/SCCmec-like elements at the 3' end, whereas SC955 contained SCCmec type V. The SCCmec of SC792 carried mec complex A and ccrC1, which was determined to be novel and designated as SCCmec type XIV (5A). SC640 harboured an SCCmec-like element derived from SCCmec type XIV. It lacked most of the downstream region of the mec complex, including the left chromosomal attachment site (SCCmec XIV Δkdp/DR-L), and lost its capability for chromosomal excision, suggesting that the mecA gene is immobilized on the chromosome. CONCLUSIONS: These findings provide evidence for increasing complexity of SCC-CIs.

Role of meteorological conditions in reported chickenpox cases in Wuhan and Hong Kong, China.

BACKGROUND: Chickenpox is a common contagious disease that remains an important public health issue worldwide. Over 90% of unvaccinated individuals become infected, but infection occurs at different ages in different parts of the world. Many people have been infected by 20 to 30 years of age in China, and adults and pregnant women who become infected often develop severe infection. Furthermore, a mortality rate of 2-3 per 100,000 infected persons has been reported. In this study, we explore the temperature-dependent transition of patterns of reported chickenpox cases in two large subtropical climate cities, Wuhan and Hong Kong, China, to aid in the prediction of epidemics and preparation for the effects of climatic changes on epidemiology of chickenpox in China. METHODS: We used a time series analysis comprising a spectral analysis based on the maximum entropy method in the frequency domain and the nonlinear least squares method in the time domain. Specifically, the following time series data were analyzed: data of reported chickenpox cases and meteorological data, including the mean temperature, relative humidity and total rainfall in Wuhan and Hong Kong from January 2008 to June 2015. RESULTS: The time series data of chickenpox for both Wuhan and Hong Kong have two peaks per year, one in winter and another in spring, indicating a bimodal cycle. To investigate the source of the bimodal cycle of the chickenpox data, we defined the contribution ratio of the 1-year cycle, Q 1, and the 6-month cycle, Q 2, as the contribution of the amplitude of a 1-year cycle and a 6-month cycle, respectively, to the entire amplitude of the time-series data. The Q 1 values of Wuhan and Hong Kong were positively correlated with the annual mean temperature and rainfall of each city. Conversely, the Q 2 values of Wuhan and Hong Kong were negatively correlated with the annual mean temperature and rainfall of Wuhan and Hong Kong. CONCLUSION: Our results showed that the mean temperature and rainfall have a significant influence on the incidence of chickenpox, and might be important predictors of chickenpox incidence in Wuhan and Hong Kong.

Novel Structures and Temporal Changes of Arginine Catabolic Mobile Elements in Methicillin-Resistant Staphylococcus aureus Genotypes ST5-MRSA-II and ST764-MRSA-II in Japan.

Twenty-two of 1,103 methicillin-resistant Staphylococcus aureus (MRSA) isolates containing the type II staphylococcal cassette chromosome mec element (SCCmec) (collected in Hokkaido, Japan, from 2008 to 2011) harbored the arginine catabolic mobile element (ACME). Five genetic variations were identified in the ACME-staphylococcal cassette chromosome mec composite islands, 66 to 79 kb in size. The percentage of ACME carriage temporally increased from 0.85% to 4.5% in parallel with the emergence of shorter variants (66 to 72 kb). Shorter variants may have a selective advantage and accelerate the dissemination of ACME in Japanese MRSA.