Clonal Distribution of Clindamycin-Resistant Erythromycin-Susceptible (CRES) Streptococcus agalactiae in Korea Based on Whole Genome Sequences

Background@#The clindamycin-resistant erythromycin-susceptible (CRES) phenotype is rare in Streptococcus agalactiae (group B streptococci). We aimed to determine the molecular characteristics of CRES S. agalactiae using whole genome sequencing (WGS). @*Methods@#Sixty-six S. agalactiae isolates obtained from blood (N=26), cerebrospinal fluid (N=10), urine (N=17), and vaginal discharge (N=13) between 2010 and 2017 in Korea were subjected to WGS. Based on the WGS data, we analyzed antimicrobial resistance (AMR) determinants, sequence types (STs), capsular polysaccharide (CPS) genotypes, and virulence gene profiles, and constructed a phylogenetic tree. We included the clindamycin-susceptible erythromycin-resistant (CSER) phenotype for comparison. @*Results@#We identified seven CRES S. agalactiae isolates from urine (N=5) and vaginal discharge (N=2) collected between 2010 and 2011. All CRES isolates harbored AMR determinants of lnu(B), lsa(E), and aac(6’)-aph(2’’), revealed ST19 and CPS genotype III, and had a virulence gene profile of rib-lmb-cylE. Phylogenetic tree analysis revealed that all CRES isolates belonged to the same cluster, suggesting a clonal distribution. In contrast, seven CSER isolates showed a diverse distribution and clustered separately from the CRES isolates. @*Conclusions@#CRES isolates collected between 2010 and 2011 showed a unique cluster with ST19 and CPS genotype III in Korea. This is the first report on WGS-based characteristics of S. agalactiae in Korea.

Comparison of Characteristics of Streptococcus dysgalactiae subsp. equisimilis Isolates Causing Repetitive vs Single Infections

No study has described Streptococcus dysgalactiae subsp. equisimilis (SDSE) isolates that cause repetitive infections (recurrence and reinfection). We compared the microbiological characteristics of SDSE causing repetitive infections with those causing single infections. Three patients with invasive infections were identified based on their medical records, and multiple SDSE isolates were collected at intervals over three weeks, using a laboratory repository. Isolates from 12 patients with single-episode infections served as controls. Six isolates were collected from three patients with first and second episodes of infection. All isolates causing either repetitive or single-episode infection were subjected to emm typing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and random amplified polymorphic DNA (RAPD) analyses. Amplification of five virulence genes (sicG, prtF1, prtF2, lmb, and cbp), biofilm formation (BF), and cell invasion abilities (CIAs) were measured as virulent phenotypes. We observed close genetic similarities in the data obtained by emm typing, MLST, PFGE, and RAPD in four isolates from two patients, suggesting recurrence, whereas two isolates from one patient indicated genetic differences in these data, suggesting re-infection. The presence of the five virulence genes and the BF and CIA measurements appeared not to contribute to repetitive infections, compared with isolates causing single-episode infection. In conclusion, clinicians encountering patients with repetitive infections should be aware of both possibilities: recurrence with closely related strains and reinfection with different strains.

Prevalence of blaZ Gene and Performance of Phenotypic Tests to Detect Penicillinase in Staphylococcus aureus Isolates from Japan

Guidelines recommend that clinical laboratories perform phenotypic tests (nitrocefin-based test and penicillin 10-U [P10] or 1-U [P1] zone edge tests) to detect penicillinase in Staphylococcus aureus isolates. This study aimed to assess the prevalence of blaZ encoding penicillinase and perform various phenotypic tests in S. aureus isolates from Japan. We prospectively collected 200 methicillin-susceptible S. aureus isolates from June 2015 to January 2016 and performed six phenotypic tests (nitrocefin-based test, P10 zone edge test/P10 diffusion test, penicillin 2-U [P2] zone edge test/P2 diffusion test, and cloverleaf test) on each sample. We confirmed the presence of blaZ (two blaZ-positive isolates) using PCR. Using blaZ PCR as a standard, we observed a low sensitivity (50%) and positive predictive value (PPV, 50%) of the nitrocefin-based test, low PPV (18.2%) of the P10 zone edge test, low sensitivity (50%) of the P10 diffusion test, low PPV (50% and 22.2%) of the P2 zone edge test and P2 diffusion test, respectively, and low sensitivity (50%) of the cloverleaf test. These data suggest a low performance (sensitivity and PPV) of these six phenotypic tests because of the low prevalence (1%) of blaZ in S. aureus isolates from Japan.

Characteristics and Immunological Roles of Surface Layer Proteins in Clostridium difficile

Clostridium difficile is a major causative agent of antibiotic-associated diarrhea and has become the most common pathogen of healthcare-associated infection worldwide. The pathogenesis of C. difficile infection (CDI) is mediated by many factors such as colonization involving attachment to host intestinal epithelial cells, sporulation, germination, and toxin production. Bacterial cell surface components are crucial for the interaction between the bacterium and host cells. C. difficile has two distinct surface layer proteins (SLPs): a conserved high-molecular-weight SLP and a highly variable low-molecular-weight SLP. Recent studies have shown that C. difficile SLPs play roles not only in growth and survival, but also in adhesion to host epithelial cells and induction of cytokine production. Sequence typing of the variable region of the slpA gene, which encodes SLPs, is one of the methods currently used for typing C. difficile. SLPs have received much attention in recent years as vaccine candidates and new therapeutic agents in the treatment of C. difficile-associated diseases. Gaining mechanistic insights into the molecular functions of C. difficile SLPs will help advance our understanding of CDI pathogenesis and the development of vaccines and new therapeutic approaches. In this review, we summarize the characteristics and immunological roles of SLPs in C. difficile.

Molecular Epidemiological Features and Antibiotic Susceptibility Patterns of Streptococcus dysgalactiae subsp. equisimilis Isolates from Korea and Japan

BACKGROUND: The molecular characterization of Streptococcus dysgalactiae subsp. equisimilis (SDSE) has not yet been performed in Korea. This study aimed to find the differences or similarities in the clinical features, molecular epidemiological findings, and antimicrobial resistance patterns of SDSE from two countries (Korea and Japan). METHODS: SDSE isolates were collected from Korea (N=69) from 2012–2016 and Japan (N=71) from 2014–2016. Clinical characteristics, emm genotypes, and sequence types (STs) were compared. Microdilution tests were performed using different antimicrobials, and their resistance determinants were screened. RESULTS: Median ages were 69 years in Korea and 76 years in Japan. The most common underlying diseases were diabetes and malignancy. Blood-derived isolates comprised 36.2% and 50.7% of Korean and Japanese isolates, respectively; mortality was not different between the two groups (5.8% vs 9.9%, P=0.53). Among Korean isolates with 20 different combined ST-emm types, ST127-stG245 (N=16), ST128-stG485 (N=10), and ST138-stG652 (N=8) were prevalent. Among Japanese isolates with 29 different combined types, ST17-stG6792 (N=11), ST29-stG485 (N=7), and ST205-stG6792 (N=6) were prevalent. Resistance rates to erythromycin, clindamycin, and minocycline were 34.8%, 17.4%, and 30.4% in Korea and 28.2%, 14.1%, and 21.4% in Japan, respectively. CONCLUSIONS: SDSE infections commonly occurred in elderly persons with underlying diseases. There was a significant difference in the distribution of ST-emm types between the two countries. Antimicrobial resistance rates were comparable with different frequencies of resistance determinants in each country.

Prevalence of Complement-Mediated Cell Lysis-like Gene (sicG) in Streptococcus dysgalactiae subsp. equisimilis Isolates From Japan (2014–2016)

BACKGROUND: Streptococcus dysgalactiae subsp. equisimilis (SDSE; a β-hemolytic streptococcus of human or animal origin) infections are emerging worldwide. We evaluated the clonal distribution of complement-mediated cell lysis-like gene (sicG) among SDSE isolates from three central prefectures of Japan. METHODS: Group G/C β-hemolytic streptococci were collected from three institutions from April 2014 to March 2016. Fifty-five strains (52 from humans and three from animals) were identified as SDSE on the basis of 16S rRNA sequencing data.; they were obtained from 25 sterile (blood, joint fluid, and cerebrospinal fluid) and 30 non-sterile (skin-, respiratory tract-, and genitourinary tract-origin) samples. emm genotyping, multilocus sequence typing, sicG amplification/sequencing, and random amplified polymorphic DNA (RAPD) analysis of sicG-positive strains were performed. RESULTS: sicG was detected in 30.9% of the isolates (16 human and one canine) and the genes from the 16 human samples (blood, 10; open pus, 3; sputum, 2; throat swab, 1) and one canine sample (open pus) showed the same sequence pattern. All sicG-harboring isolates belonged to clonal complex (CC) 17, and the most prevalent emm type was stG6792 (82.4%). There was a significant association between sicG presence and the development of skin/soft tissue infections. CC17 isolates with sicG could be divided into three subtypes by RAPD analysis. CONCLUSIONS: CC17 SDSE harboring sicG might have spread into three closely-related prefectures in central Japan during 2014–2016. Clonal analysis of isolates from other areas might be needed to monitor potentially virulent strains in humans and animals.

Epidemiological Study of Erythromycin-Resistant Streptococcus pyogenes From Korea and Japan by emm Genotyping and Multilocus Sequence Typing

BACKGROUND: We determined the epidemiological characteristics of erythromycin (EM)-resistant Streptococcus pyogenes (group A streptococci, GAS) strains isolated from Korea and Japan, using emm genotyping and multilocus sequence typing (MLST). METHODS: Clinical isolates of GAS had been collected from 1992 to 2012 in Korea and from 2004 to 2009 in Japan. EM resistance was determined by the microdilution method, and resistance genotypes were assessed by PCR. The emm genotyping and MLST were performed by DNA sequencing. RESULTS: The emm genotypes and sequence types (STs) were concordant in 143 (85.1%) of 168 EM-resistant GAS strains from Korea. ST36/emm12 (35.1%), ST52/emm28 (22.6%), and ST49/emm75 (16.1%) were the most common types. Most of the ST36 (93.9%) and ST52 (95.8%) strains harbored erm(B), whereas strains ST49, ST42, and ST15 contained mef(A). The concordance between emm genotypes and STs was 41 (93.2%) among 44 EM-resistant GAS strains from Japan. ST36/emm12 (34.1%), ST49/emm75 (18.2%), and ST28/emm1 (15.9%) were the major types. ST36 isolates harbored either erm(B) (56.3%) or mef(A) (37.5%), whereas isolates ST28, ST49, and ST38 carried only mef(A). The proportion of erm(B) and mef(A) was 66.1% and 33.3% in Korea and 22.7% and 68.2% in Japan, respectively. CONCLUSIONS: The common STs in Korea and Japan were ST36 and ST49, whereas ST52 was present only in Korea and ST28 only in Japan. Genotype erm(B) was predominant in Korea, whereas mef(A) was frequent in Japan. There were differences between Korea and Japan regarding the frequencies of emm genotypes, STs, and EM resistance genes among the EM-resistant GAS.

Efficacy of a Virtual Reality Simulator for Evaluating the Aptitude of Medical Students / General Medicine

OBJECTIVE: Our goal was to develop a system using virtual reality (VR) technology to test the haptic skills of medical students. Currently, surgical skills are learned on live patients in a clinical environment in which the student practices under the close supervision of an experienced surgeon. We are interested in using haptic feedback devices to enhance surgical skills, because simulated touch in a virtual world improves the performance of trainee surgeons. In this study, we evaluated the efficacy of a test that evaluates the surgical skill of medical students by using a VR simulator.<BR>METHODS: We used a microsurgical simulator with a force-feedback system. Its effectiveness in helping 36 medical students to acquire the tactile skills used in microscopic surgery was evaluated experimentally. Operating time and the number of sites of hemorrhage were measured to evaluate surgical aptitude. We also evaluated system performance with respect to reality, immersiveness, and operability as secondary measures. Data were analyzed using descriptive methods.<BR>RESULTS: The operating time and number of hemorrhagic sites were positively correlated. Subject students were clustered into three groups: dexterous, awkward, or clumsy. The relation between the number of hemorrhages in the retina and immersion and operability differed between the group of would-be surgeons and those of would-be internists and pediatricians. All the students commented that the simulator was a useful tool for medical education.<BR>CONCLUSIONS: The VR simulator can be used not only to teach and evaluate subtle tactile and surgical skills relevant to the surgical profession, but also to test the aptitude of medical students. The training transfer from a haptic simulator to actual practice methodology should be quantifiable in the near future. This work has steered medical informatics research into a new type of medical education.