Virulence-associated Genome Sequences of Pasteurella canis and Unique Toxin Gene Prevalence of P. canis and Pasteurella multocida Isol

Yoshida Haruno,Kim Jung-Min,Maeda Takahiro,Goto Mieko,Tsuyuki Yuzo,Shibata Sachiko,Shizuno Kenichi,Okuzumi Katsuko,Kim Jae-Seok,Takahashi Takashi 대한진단검사의학회 2023 Annals of Laboratory Medicine Vol.43 No.3

Background: Comparative analysis of virulence factors (VFs) between Pasteurella canis and Pasteurella multocida are lacking, although both cause zoonotic infections. We determined the virulence-associated genome sequence characteristics of P. canis and assessed the toxin gene prevalence unique to P. canis among clinical isolates of P. canis and P. multocida. Methods: We selected 10 P. canis and 16 P. multocida whole-genome sequences (WGSs) from the National Center for Biotechnology database. The VFanalyzer tool was used to estimate P. canis-characteristic VFs. Amino acid sequences of VFs were compared with multiple-aligned sequences. The genome structure containing P. canis-characteristic and adjacent loci was compared to the corresponding P. multocida genome structure. After designing primer sequences and assessing their accuracy, we examined the gene prevalence of the P. canis-characteristic VFs using PCR among clinical isolates of P. multocida and P. canis. Results: Using VFanalyzer, we found virulence-associated cytolethal distending toxin (cdt)A–cdtB–cdtC loci common to all P. canis WGSs that were not found in P. multocida WGSs. Similarities in the multiple alignments of CdtA–CdtB–CdtC amino acid sequences were found among the 10 P. canis WGSs. Shared or similar loci around cdtA–cdtB–cdtC were identified between the P. canis and P. multocida genome structures. The PCR-based cdtA–cdtB–cdtC prevalence differed for P. canis and P. multocida clinical isolates. Conclusions: P. canis-specific cdtA–cdtB–cdtC prevalence was identified among clinical isolates. These three loci may be unique toxin genes and promising targets for the rapid identification of P. canis in clinical settings.

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

Tomohiro Fujita,Haruno Yoshida,Shunsuke Osaka,Yoneji Hirose,Mieko Goto,Noriyuki Nagano,Takashi Takahashi 대한진단검사의학회 2019 Annals of Laboratory Medicine Vol.39 No.5

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.

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

김선주,변정현,박현웅,이재현,Haruno Yoshida,Akiyoshi Shibayama,Tomohiro Fujita,Yuzo Tsuyuki,Takashi Takahashi 대한진단검사의학회 2018 Annals of Laboratory Medicine Vol.38 No.3

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.

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

Takashi Takahashi,Kazuaki Arai,이동현,고은하,Haruno Yoshida,Hisakazu Yano,Mitsuo Kaku,김선주 대한진단검사의학회 2016 Annals of Laboratory Medicine Vol.36 No.1

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.

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

Takashi Takahashi,Tomohiro Fujita,Akiyoshi Shibayama,Yuzo Tsuyuki,Haruno Yoshida 대한진단검사의학회 2017 Annals of Laboratory Medicine Vol.37 No.4

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.