Search

Background: Streptococcus canis is a commensal bacterium in companion animals. This microorganism can infect humans who have been in deep contact with or bitten by pet dogs, suggesting that the skin/soft tissue is one of infection entry sites. To understand pathological process in human cells, we aimed to determine S. canis transcriptomic changes in invasive environments of human keratinocytes., Methods: We selected one isolate from candidates with whole-genome sequences, based on re-obtained cell invasion ability (CIA) data into human keratinocytes along with bacterial cytotoxicity. RNA-sequencing was conducted for the samples at baselines and 2 h/5 hr post-inoculation using NovaSeq 6000. Global/differential gene expression analyses [principal component analysis (PCA)/k-means clustering analysis/differentially expressed gene (DEG) analyses] were performed. We classified DEGs into their functional categories. To validate transcriptomic results, we did quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays., Results: FU1 isolate was selected from seven candidates, based on re-obtained CIA data with less cytotoxicity. Total read bases of 6.17-9.02 Gbp were obtained by RNA-sequencing. PCA and k-means clustering analysis indicated clustering according to their inoculation times. Volcano plots and Venn diagrams revealed that S. canis invasion into keratinocytes produced altered distributions of many genes. Gene ontology enrichment analysis showed most of the gene expressions were downregulated. DEG functional analysis showed the downregulated DEGs belonging to energy production and conversion/carbohydrate transport and metabolism/amino acid transport and metabolism/nucleotide transport and metabolism, with the upregulated DEGs belonging to transcription. qRT-PCR assays for downregulated/upregulated expressions of four genes (pgk-slo/opuAA-kdpB) validated transcriptomic results., Conclusion: Our observations suggest that S. canis can downregulate its metabolism-associated gene expressions in human keratinocyte environments. The observed gene expression changes can imply the latent infection in human cells. Further investigation is needed to elucidate the underlying mechanisms for the latent infection., (© 2024. The Author(s).)

Objective: The purpose of this study was to identify the M protein trans-acting positive regulator (Mga) orthologue and its adjacent M-like protein (SCM) alleles in Streptococcus canis., Results: Using the 39 SCM allele isolates and polymerase chain reaction-based amplification and sequencing, we obtained the deduced Mga amino acid (AA) sequences. The 22 Mga sequences in whole-genome sequences were obtained by searching the National Collection of Type Cultures 12,191(T) Mga sequence into the database. The percentage identity to the type-strain Mga sequence was examined along with its size. The presence of the Mga-specific motifs was confirmed. Of the 62 strains, we identified 59 Mga sequences with an AA size of 509 (except for four different sizes). Percentage identity ranged from 96.66 to 100% with the confirmed Mga-specific motifs and diverse SCM allele populations. Our findings support the presence of an Mga orthologue and diverse SCM allele populations., (© 2024. The Author(s).)

Streptococcus uberis is one of major pathogens causing bovine mastitis. However, there is poor information on antimicrobial resistance (AMR) among the Japanese isolates. To provide treatment information for the mastitis caused by S. uberis in Japan, we aimed to clarify AMR patterns of the isolates from bovine milk mainly in Chiba. AMR phenotyping/genotyping [blaZ-erm(A)-erm(B)-mef(A)-linB-lnuD-tet(M)-tet(O)-tet(K)-tet(L)-tet(S)] and multilocus sequence typing were performed to analyze relationships between AMR patterns and clonal complexes (CCs). Resistance to tetracycline-, macrolide-, and lincosamide-classes was mainly associated with possession of tet(O), tet(S), erm(B), linB, and lnuD genes. CC996 was significantly associated with multidrug resistance (P<0.0001). These findings will aid Chiba farm animal clinics in treating bovine mastitis.

We evaluated the cell invasion ability (CIA) of non-invasive Streptococcus dysgalactiae subsp. equisimilis using human keratinocytes and determined the association of CIA populations with their hosts and microbiological traits. Forty-two isolates from humans and companion animals were selected with host information. In addition to CIA, virulence-associated gene (VAG, spegg-ska-scpA-inlA-sicG-brpA-prtF1-prtF2-lmb-cbp-srtp1-srtp2) profiling, emm genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping were performed. We designated CIA values higher than the mean of all isolates as high-frequency and those lower than the mean as low-frequency. Differences in the CIA between the different sources and Lancefield groups were assessed. We analyzed the association between high- and low-frequency CIA and VAG, emm genotype, sequence type/clonal complex, and AMR phenotype/genotype. Based on the mean (19.368 colony-forming units/100 cells) of 42 isolates, eight isolates had high-frequency CIA, whereas 34 had low-frequency CIA. We found an association between low-frequency CIA population and group G isolates, as well as a link between high-frequency CIA population and group C isolates. We also observed associations between low-frequency CIA population and oral/respiratory tract origin, ska, scpA, and lmb detection, and the AMR phenotype. Our observations suggest potential associations between high-/low-frequency CIA and the group, source, VAG, and AMR phenotypes.

This study evaluated the cell invasion ability (CIA) of Streptococcus canis isolates, and clarified the relationship between high-frequency CIA and its microbiological features. Of the companion animal-origin isolates (n = 117) that were obtained in 2017, 40 isolates were randomly selected with the host information, with two human blood-origin isolates included. CIA was measured using human colon carcinoma epithelium and the hemolytic activity (HA) using sheep blood, along with S. canis M-like protein (SCM) allele typing, sequence type (ST) determination, and antimicrobial resistance (AMR) phenotyping/genotyping. CIA measurements revealed that 19 and 24 isolates had high- and low-frequencies, respectively. HA assessment revealed that 24 and 19 isolates were categorized as high- and low- level, respectively. No difference was observed in the high-/low-level HA between the high- /low-frequency CIA populations. A significant difference was found in the high-/low-frequency CIA between the SCM group I/II populations. Additionally, a significantly higher CIA was found in the SCM allele type 10/type 11 than in the others. A significant association was observed between high-frequency CIA and the ST21/ST41 populations. No difference was found in the high-/low-frequency CIA between the presence and absence of the AMR phenotype/genotype. These observations suggest a relationship between high-frequency CIA and its microbiological characteristics (SCM allele type 10/type 11 or ST21/ST41).

This study investigated quinolone nonsusceptible Streptococcus canis with point mutations in quinolone resistance-determining regions (QRDRs). After selecting targets from 185 isolates, we tested antimicrobial susceptibility using levofloxacin, ciprofloxacin, norfloxacin, and moxifloxacin. We also determined the amino acid sequences of QRDRs in gyrA/gyrB/parC/parE genes and their point mutations. Finally, we performed S. canis-derived M-like protein (SCM) allele typing, multilocus sequence typing, and antimicrobial resistance genotyping. Correlations between nonsusceptible strains and their related factors were examined. We found 13 (7.0%) nonsusceptible isolates consisting of two classes, high-level minimum inhibitory concentrations (MICs) (n = 7, 3.8%) and low-level MICs (n = 6, 3.2%). Mutations Ser81Phe/Ser81Tyr/Glu85Lys in gyrA, Ser67Phe/Ser67Tyr/Asp71Tyr in parC, Asp438Asn in parE, and Gly408Asp in gyrB were observed in these nonsusceptible strains. Common mutations included Ser81 and Ser67/Asp71; additionally, we found one strain each with Glu85, Asp438, and Gly408 mutations. There was a significant correlation between nonsusceptible isolates and the presence of SCM allele type 2, sequence type 46, tetracyclineresistance genes, and macrolide/lincosamide-resistance genes. These results could be used in future, by veterinarians while treating companion animals with clinical symptoms of streptococcal infections.

We determined the whole-genome sequence (WGS) of Streptococcus canis strain TA4 harboring the M-like protein gene (scm); the strain was isolated from a human patient presenting with bacteremia. The potential of type II-A clustered regularly interspaced short palindromic repeats (CRISPR) array-based typing was evaluated, and the genetic relation was elucidated between spacer genogroups and scm prevalence and/or polymorphisms among the isolates from 19 diseased companion animals and the human patient. CRISPRFinder and CRISPRCasFinder detected the type II-A locus with the same repeat sequences in strain TA4 and another WGS of S. canis strain, isolated from a cow with mastitis. An optimized PCR-based amplification method was used to sequence the region covering the locus around the leader and terminal repeat sequences. Among the 20 isolates sequenced, 16 strains (including TA4) were identified with the CRISPR array. We conducted comparative analysis of the homologous spacer sequences and performed grouping based on the successive common ancestral spacer types. These 16 isolates were assigned to five genogroups (A to E) with scm being absent in genogroup A. We found a relationship between genogroups C and E and allele type 1 of the deduced M-like protein. These preliminary findings suggest the feasibility of CRISPR array-based typing and a genetic relation between the spacer genogroups and scm prevalence and/or polymorphisms in the isolates.

This study aimed to identify the species and assess the antimicrobial resistance (AMR) of β-hemolytic streptococci isolated from companion animals in Japan. Strains were isolated from clinical specimens of 131 companion animals that exhibited symptoms in April-May 2017. We identified strains by 16S rRNA sequencing and assessed their antimicrobial susceptibility using the broth microdilution method. AMR genes erm(A)-erm(B)-mef(A) and tet(M)-tet(O)-tet(K)-tet(L)-tet(S) in all isolates were amplified by PCR. 16S rRNA sequencing identified β-hemolytic streptococcal species as Streptococcus canis (n = 117, 89.3%), S. agalactiae (n = 7), S. dysgalactiae subsp. equisimilis (n = 5), S. dysgalactiae subsp. dysgalactiae (n = 1), and S. equi subsp. zooepidemicus (n = 1). Overall AMR rates were 39.7% for minocycline, 19.8% for erythromycin, and 17.6% for clindamycin, with a minimum inhibitory concentration (MIC 90 ) of > 4, > 2, and > 1 μg/mL, respectively. AMR genotyping showed the presence of single or mixed types: erm(B)-mef(A) and tet(M)-tet(O)-tet(L)-tet(S). There was a significant relationship between tetracycline-resistance genotypes and open pus/skin-derived specimens. These observations identify some unique features of β-hemolytic streptococcal isolates from companion animals in Japan, such as the dominant isolation of S. canis and resistance to tetracycline, macrolide, and lincosamide antibiotics, in terms of species identification and AMR properties.

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 gene's 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., Competing Interests: No potential conflicts of interest relevant to this article were reported., (© The Korean Society for Laboratory Medicine)

In this study, we conducted a species-level identification of group G streptococcal (GGS) isolates from companion animals in Japan and analyzed antimicrobial resistance (AMR) patterns. Strains were isolated from sterile and non-sterile specimens collected from 72 animals with clinical signs or symptoms in April-May, 2015. We identified the strain by 16S rRNA sequencing, mass spectrometry (MS), and an automated method based on their biochemical properties. Antimicrobial susceptibility was determined using the broth microdilution method and E-test. AMR determinants (erm(A), erm(B), mef(A), tet(M), tet(O), tet(K), tet(L), and tet(S)) in corresponding resistant isolates were amplified by PCR. The 16S rRNA sequencing identified the GGS species as Streptococcus canis (n = 68), Streptococcus dysgalactiae subsp. equisimilis (n = 3), and S. dysgalactiae subsp. dysgalactiae (n = 1). However, there were discrepancies between the sequencing data and both the MS and automated identification data. MS and the automated biochemical technique identified 18 and 37 of the 68 sequencing-identified S. canis strains, respectively. The AMR rates were 20.8% for tetracycline and 5.6% for clarithromycin, with minimum inhibitory concentrations (MIC) 50 -MIC 90 of 2-64 and ≤ 0.12-0.25μg/mL, respectively. AMR genotyping showed single or combined genotypes: erm(B) or tet(M)-tet(O)-tet(S). Our findings show the unique characteristics of GGS isolates from companion animals in Japan in terms of species-level identification and AMR patterns.

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., Competing Interests: No potential conflicts of interest relevant to this article were reported., (© The Korean Society for Laboratory Medicine)

Priming with tumor antigens is one of the most important strategies in cancer immunotherapy. To enhance tumor antigenicity, OK-432, a streptococcal preparation, was coupled to squamous cell carcinoma (KLN-205) by means of a 0.2% glutaraldehyde method. The purpose of this study was to investigate whether OK-432-conjugated tumor vaccines could induce tumor-specific immunity. Our originally developed mouse tongue cancer model was used throughout this work for the analysis of antitumor effects. Prepared OK-432-conjugated KLN-205 vaccines were immunized 3 times to DBA/2 mice. The results showed that the KLN-205 vaccines induced cytolytic activity and strongly suppressed both KLN-205 tumor incidence and growth, and survival of the mice was improved. Moreover, the histological results showed that a greater number of lymphocytes had infiltrated around tumor cells by 24 hours after tumor inoculation in the vaccine group. These results suggest that immunizations with KLN-205 vaccines increase the antitumor effects against tongue cancer. [ABSTRACT FROM AUTHOR]