Haruno Yoshida

CD46 Transgenic Mouse Model of Necrotizing Fasciitis Caused by Streptococcus pyogenes Infection

ABSTRACT We developed a human CD46-expressing transgenic (Tg) mouse model of subcutaneous (s.c.) infection into both hind footpads with clinically isolated 11 group A streptococcus (GAS) serotype M1 strains. When the severity levels of foot lesions at 72 h and the mortality rates by 336 h were compared after s.c. infection with 1 × 107 CFU of each GAS strain, the GAS472 strain, isolated from the blood of a patient suffering from streptococcal toxic shock syndrome (STSS), induced the highest severity levels and mortality rates. GAS472 led to a 100% mortality rate in CD46 Tg mice after only 168 h postinfection through the supervention of severe necrotizing fasciitis (NF) of the feet. In contrast, GAS472 led to a 10% mortality rate in non-Tg mice through the supervention of partial necrotizing cutaneous lesions of the feet. The footpad skin sections of CD46 Tg mice showed hemorrhaging and necrotic striated muscle layers in the dermis, along with the exfoliation of epidermis with intracellular edema until 48 h after s.c. infection with GAS472. Thereafter, the bacteria proliferated, reaching a 90-fold or 7-fold increase in the livers of CD46 Tg mice or non-Tg mice, respectively, for 24 h between 48 and 72 h after s.c. infection with GAS472. As a result, the infected CD46 Tg mice appeared to suffer severe liver injuries. These findings suggest that human CD46 enhanced the progression of NF in the feet and the exponential growth of bacteria in deep tissues, leading to death.

Mouse Models for Assessing the Protective Efficacy of Lactobacillus gasseri SBT2055 against Helicobacter suis Infection Associated with the Development of Gastric Mucosa-Associated Lymphoid Tissue Lymphoma

Helicobacter suis strain TKY infection has been strongly associated with the development of gastric mucosa‐associated lymphoid tissue (MALT) lymphoma in a C57BL/6J mouse model.

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.

Comparative Genomics of the Mucoid and Nonmucoid Strains of Streptococcus pyogenes, Isolated from the Same Patient with Streptococcal Meningitis.

ABSTRACT Mucoid (MTB313) and nonmucoid (MTB314) strains of group A streptococcus emm type 1 were simultaneously isolated from a single patient suffering from streptococcal meningitis. Whole-genome sequencing revealed that MTB313 carried a nucleotide substitution within rocA, which generated an amber termination codon.

Dermal mast cells reduce progressive tissue necrosis caused by subcutaneous infection with Streptococcus pyogenes in mice.

A single subcutaneous (s.c.) infection with 1×10(7) c.f.u. GAS472, a group A streptococcus (GAS) serotype M1 strain isolated from the blood of a patient suffering from streptococcal toxic shock syndrome, led to severe damage of striated muscle layers in the feet of mast cell (MC)-deficient WBB6F(1)-Kit(W)/Kit(W-v) (W/W(v)) mice 72 h after infection. In contrast, no damage was recognized in striated muscle layers in the feet of the control WBB6F(1)-Kit(+/+) (+/+) mice 72 h after infection. In addition, adoptively transferred MCs reduced progressive tissue necrosis of the feet of W/W(v) mice after infection. However, there was no significant difference in the mortality rates between the W/W(v) and +/+ mice, or between the human CD46-expressing transgenic (Tg) mouse bone marrow-derived cultured MC-reconstituted W/W(v) and non-Tg mouse bone marrow-derived cultured MC-reconstituted W/W(v) mice after infection. Consequently, although MCs can help to reduce the severity of necrosis of the feet caused by s.c. infection with GAS472, such reduction of tissue necrosis scarcely improves the mortality rates of these mice. Moreover, human CD46 does not play a crucial role in the MC-mediated innate immune defence against GAS infection.

A CD46 transgenic mouse model for studying the histopathology of arthritis caused by subcutaneous infection with Streptococcus dysgalactiae subspecies equisimilis

Ankle arthritis was induced by a single subcutaneous (s.c.) infection of 1×10(7) c.f.u. of the Streptococcus dysgalactiae subspecies equisimilis strain RE378, which was isolated from a patient suffering from multiple organ failure due to septicaemia, into both hind footpads of human CD46-expressing transgenic (Tg) mice. In contrast, in non-Tg mice, the incipient foot lesions (swelling and redness) resolved before arthritis developed. The number of viable bacteria in tissue samples and the arthritis frequency on days 3 and 28 after infection were higher in CD46 Tg mice than in non-Tg mice. The histopathological findings in the hind ankle sections of CD46 Tg mice showed the stimulation of osteoclast formation associated with inflammation of the synovial membrane and the development of aggressive granulation tissue (pannus). In addition, increased expression levels of interleukin (IL)-6, receptor activator of NF-κB ligand, IL-1β and tumour necrosis factor alpha were detected in the foot bones of CD46 Tg mice but not in those of non-Tg mice. These observations suggest that the s.c. infection with S. dysgalactiae subsp. equisimilis induced arthritis in the ankle joints of CD46 Tg mice as a consequence of the prolonged inflammation associated with focal bone loss.

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.

Genetic diversity in Streptococcus dysgalactiae subsp. equisimilis isolates from patients with invasive and noninvasive infections in a Japanese university hospital (2014–2015)

Streptococcus dysgalactiae subsp. equisimilis (SDSE) isolates with β-hemolysis and carbohydrate groups G or C are increasingly recovered from invasive infections in Japan. The aim of this study was to determine the epidemiological characteristics of SDSE isolates circulating locally among patients with invasive and noninvasive infections. We selected groups G/C β-hemolytic streptococci from a repository at the Clinical Laboratory of Kitasato University Medical Center, from May 2014 through April 2015. Thirteen isolates were identified as SDSE based on the data from API-20 Strep and 16S rRNA sequencing. The samples were from 7 sterile specimens (blood) and 6 non-sterile specimens (pus/sputum/vaginal secretion). Information about the patients with invasive or noninvasive SDSE infections was retrieved from their medical charts. We performed emm genotyping, multilocus sequence typing, a dendrogram analysis of the samples using pulsed-field gel electrophoresis (PFGE), and amplifications of the streptococcal inhibitor of a complement-mediated cell lysis-like gene (sicG) and antimicrobial resistance determinants. We identified 8 different emm genotypes, 8 different sequence types, including 4 novel types, 9 different groups in the PFGE dendrogram, the presence or absence of sicG, and 4 different resistance genotypes. Our observations indicate genetic diversity in SDSE isolates from patients with invasive and noninvasive infections in a Japanese university hospital (2014-2015).

Flesh‐eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor‐κB ligand

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock‐like syndrome. Human CD46‐expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast‐mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 105 colony‐forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor‐κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro‐computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4+ and CD8+ T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte‐derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast‐like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.

Prevalence and diversity of M-like protein (SCM) gene in Streptococcus canis isolates from diseased companion animals in Japan: Implication of SCM allele

Streptococcus canis (Sc)-origin M-like protein (SCM) binds to plasminogen and immunoglobulin G and facilitates anti-phagocytic properties. We aimed to determine the prevalence and diversity of the scm gene in Sc isolates from diseased companion animals in Japan and to propose potential SCM alleles of amino acid (AA) sequences. We collected β-hemolytic streptococci from diseased animals with host information nationwide in 2015 and 2017. After Sc identification and scm gene amplification and sequencing, the genes prevalence and relationship between its presence and host information were determined. Furthermore, phylogenetic trees of AA sequences were constructed, and classification and distribution of SCM alleles based on variations of AA sequences were conducted. The scm detection rates were 70.6% (n = 48, 2015) and 82.9% (n = 97, 2017). There was a relationship between scm presence and Tokyo in 2015 and 2017. We found an association between scm detection and dogs in 2017 alone. Major sequence sizes were 1311 bp, 1308 bp, and 1305 bp. Using the phylogenetic trees of AA sequences, we confirmed shared positions of five identical sequence patterns in both periods. Nine SCM alleles were determined with six signal-peptide types. Most prevalent alleles were type 1, type 2, and type 4 in both periods. Our observations suggest prevalence and diversity of scm in animal-origin Sc isolates in Japan.