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The eye (including the cornea) and ear canal are the major sources of Streptococcus canis in companion animal practice. In this study, we aimed to clarify the genotypic and phenotypic features of eye-origin isolates collected in 2021 compared to ear-origin isolates collected in 2021 and eye-origin isolates collected in 2017. Of the 102 isolates in 2021, 9 eye-origin isolates were enrolled. Twenty ear-origin isolates in 2021 and 13 eye-origin isolates in 2017 were included as controls. Genotypic analyses included profiling of virulence-associated genes (VAGs; inl, sagA, slo, scp, lbp, fbp, gbp, ap1, fp1, and brp), S. canis M-like protein (SCM) allele typing, multilocus sequence typing, and antimicrobial resistance (AMR) genotyping and phenotyping analyses including hemolytic activity (HA) measurement and AMR phenotyping. One 2017-eye-origin isolate displayed high-level HA; the others displayed low-level HA. No association was evident between the 2021-eye-origin population and the detection rate of each VAG. There was no association between the 2021-eye-origin population and the main SCM allele 2. A significant association was evident between the 2021-eye-origin population and the main clonal complex (CC) 46 containing sequence type (ST) 46/ST2. A significant association was also detected between the 2021-eye-origin population and AMR phenotypes/genotypes. Our observations suggest unique microbiological features (CC46 with AMR phenotypes/genotypes) among the 2021-eye-origin population.

This study assessed biofilm production ability (BPA) and other microbiological features of Streptococcus canis strains. Companion animal-origin 40 strains from each year (2015/2017) were randomly selected with the host information, and three blood-origin strains from 2 humans/1 dog were included. We measured BPA using crystal violet staining, along with S. canis M-like protein (SCM) allele typing, sequence type (ST) determination, antimicrobial resistance (AMR) phenotyping/genotyping, and virulence-associated gene profiling (gbp-ap1-fp1-brp). BPA measurements revealed that 35 strains with BPA and 48 strains without BPA. There was association of the producer with isolation year (2017). We found association between the non-producer and SCM allele 1/ST9: there was association of the producer with SCM allele 10/ST21. We observed correlation between the producer and presence of AMR genotypes. There was association between the producer and ap1 detection and between non-producer and gbp detection. Our observations suggest the correlation between the producer and other microbiological features (isolation year/SCM allele type 10/ST21/presence of AMR genotypes/ap1 detection).

Objective We evaluated biofilm production ability (BPA) of Streptococcus agalactiae isolates from companion animals/humans and clarified the relationship between BPA populations and other microbiological features. Methods Companion animal-/human-origin isolates were collected with host information. We measured BPA using crystal violet staining, via virulence-associated gene profiling (hylB-pavA-pilB-spb1-srtC1-brpA), capsular genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping. Significant difference in BPA of isolates from different hosts was assessed. We analyzed the association between BPA populations and the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes. Inhibitory effect of berberine on BPA was evaluated. Results Five, twenty-six, and twenty-six isolates belonged to strong, moderate, and weak biofilm producers, whereas seventeen showed no biofilm production. We defined strong, moderate, or weak biofilm producers as the producer group (n = 57) to conduct a comparative analysis between the producer and non-producer populations. There was a significant correlation between the producer population and vaginal specimen. We found significant associations between the producer group and presence (57.9%) of pilB and between the non-producer population and presence (70.6%) of spb1. There was no association between the producer group and capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes (except for a significant correlation between the producer group and AMR to minocycline). We confirmed inhibitory effect of berberine at sub-minimum inhibitory concentrations (MICs) against the type strain on BPA. Conclusion Our observations suggest that S. agalactiae harboring pilB is more capable of producing biofilms, with berberine inhibitory effect at sub-MICs on BPA.

We assessed biofilm production ability (BPA) of noninvasive Streptococcus dysgalactiae subsp. equisimilis (SDSE) from humans/companion animals and determined relationships between BPA populations and other host/microbiological features. Sixty-four companion animal-/human-origin isolates were collected with host information. We measured BPA using crystal violet staining, along with doing emm typing, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping, and detection of virulence-associated genes (VAGs) (prtF1-prtF2-lmb-cbp-sicG-srtp1-srtp2-brpA). Significant difference in BPA of SDSE from different hosts/sources and with different Lancefield groups was assessed. We analyzed associations between BPA populations (strong/moderate/weak/no biofilm producers) and emm types, sequence types/clonal complexes (CCs), AMR phenotypes/genotypes, and VAG types. Seventeen, twenty-four, and twelve isolates belonged to strong, moderate, and weak biofilm producers: eleven showed no BPA. There was a difference in BPA distributions between human- and animal-origins and between groups G and C isolates. We found an association of BPA populations with eye/ear source (vs. pus/skin source). There was a relationship between BPA populations and CC127 (vs. CC17). We observed no associations of BPA populations with AMR phenotype/genotype. There was an association of BPA distributions with srtp1 detection. Our observations suggest potential associations of BPA with host, Lancefield group, source, CC, or VAG.

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).

We evaluated the feasibility of type II-A clustered regularly interspaced short palindromic repeats (CRISPR) array-based genogrouping using Streptococcus dysgalactiae subsp. Equisimilis isolates from 32 humans and 8 companion animals and compared Simpson's diversity index of this genogrouping to those of multilocus sequence typing (MLST) and emm genotyping. CRISPRCasFinder detected a type II-A CRISPR array with the same repeat sequences in three whole-genome sequences. Subsequently, optimized polymerase chain reaction-based II-A CRISPR array amplification was performed to sequence the region around the leader and terminal repeat sequences. We conducted spacer genogrouping by evaluating the spacer sequence similarities. A phylogenetic dendrogram was constructed, and spacer content and polymorphisms were illustrated. Simpson's diversity indices were calculated for the CRISPR array genogrouping, MLST, and emm genotyping. We analyzed the association between the spacer genogroup with sequence type (ST)/emm genotype for each isolate. Of the 40 isolates, 39 with the II-A CRISPR array were amplified, sequenced, and assigned to 13 genogroups (A-M). The Simpson's diversity indices for the three typing were 0.874, 0.914, and 0.924, respectively. We found genetic lineages between genogroup M and ST127/stG245.0 and between genogroup I and ST29/stG485.0. These observations suggest the feasibility of II-A CRISPR array genogrouping and the genetic relationship between spacer genogroups and STs/emm genotypes in the isolates.

This study assessed whether Streptococcus agalactiae isolates from companion animals differed from those of human origin. Beta-hemolytic S. agalactiae was collected from a veterinary laboratory center and a university hospital. Strains were identified using 16S rRNA amplicon sequencing and amplification of the species-specific dltS gene. We conducted virulence gene profiling, capsular genotyping, determination of clonal complex (CC), and antimicrobial resistance (AMR) phenotyping or genotyping. The 20 non-invasive isolates obtained from animals and 15 non-invasive isolates from adult humans were comparatively analyzed in this study. We found significant differences in the virulence gene profiles of bca-rib-lmb-cylE (40.0% vs. 93.3%) and the possession of bac (30.0% vs. 0%) between animal-origin and human-origin non-invasive strains. We observed a significant difference in the distribution of CC1 between the two non-invasive populations. There were significant differences in the prevalence of tetracycline resistance genotypes (60.0% vs. 20.0%) and absence of AMR genotypes (30.0% vs. 80.0%), and AMR rates of tetracycline (35.0% vs. 0%) and fluoroquinolone (20.0% vs. 66.7%) between the two non-invasive populations. These observations suggest that there were different features, in terms of virulence gene profile, CC, and AMR genotype/phenotype in the non-invasive isolates of animal origin compared to those of human origin.

We aimed to clarify antimicrobial susceptibility patterns of anaerobes from diseased companion animals. Bacterial identification was based on the Japanese 2012 guidelines for the testing of anaerobic bacteria. AST was performed using the broth microdilution method. The anaerobe-containing samples collected from 2014 to 2018 included blood (anaerobe recovery rate, 5.0%), bile (9.4%), joint fluids (0.6%), pleural effusions (42.6%), ascites (64.1%), cerebrospinal fluids (3.0%), and punctures (75.0%). The anaerobes identified included Bacteroides spp. (33.2%), Peptostreptococcus spp. (19.6%), Prevotella spp. (13.6%), Propionibacterium spp. (10.3%), Clostridium spp. (9.3%), and Fusobacterium spp. (7.5%). Bacteroides fragilis group isolates were resistant to penicillin G (100%), ampicillin (100%), cefmetazole (63.6%), ceftizoxime (90.0%), and clindamycin (40.0%). Our observations demonstrated antimicrobial susceptibility in anaerobes isolated from Japanese companion animals.

Mitral valve aneurysm (MVA) is a rare but life-threatening valvular pathologic entity most commonly associated with infective endocarditis (IE) of the aortic valve (AV). We describe a diabetic patient with ruptured anterior MVA secondary to capsular genotype V Streptococcus agalactiae (GBS) harboring novel ST1656 IE without AV involvement. Our patient presented with manifestations of various serious systemic and intracardiac complications, requiring early surgery, but ultimately died from non-cardiogenic causes. This case emphasizes the importance of treating MVA as a dangerous sequela of IE, of performing transesophageal echocardiography to make its accurate diagnosis and institute early surgical intervention, and of considering GBS as a rare but important causative agent of IE in elderly patients with comorbidities.

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.

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 (MIC90) 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.

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 assessed whether invasive group B Streptococcus (GBS) isolates were similar to non-invasive isolates from adult patients. Invasive and non-invasive GBS isolates were collected from three hospitals and two laboratory centers between January 2015 and October 2019. The isolates were identified by 16S rRNA amplicon sequencing and amplification of the GBS-specific dltS gene. The virulence gene profiles, capsular genotypes, sequence types (STs)/clonal complexes (CCs), and antimicrobial resistance (AMR) phenotypes/genotypes were determined for the 72 invasive and 50 non-invasive isolates that were comparatively analyzed. We observed a significantly decreased rate of rib detection in the invasive isolates compared to that in the non-invasive isolates (77.8% vs. 92.0%, P < 0.05). Additionally, we found significant differences in the prevalence of CC1 (23.6% vs. 46.0%, P < 0.05) and CC26 (12.5% vs. 2.0%, P < 0.05) between invasive and non-invasive populations. However, there were no significant differences in the comparative data of the virulence gene profiles, capsular genotypes, other STs/CCs, and AMR phenotypes/genotypes between the two populations. These findings suggest that both invasive and non-invasive isolates share similar features in terms of virulence gene profile, capsular genotype, ST/CC, and AMR genotype/phenotype (except for the rates of rib detection and CC1/CC26 prevalence).

This study evaluated the cell invasion ability (CIA) of Streptococcus agalactiae isolates from humans and companion animals and clarified the relationship between CIA populations and their microbiological features.Human-origin and companion animal-origin isolates were collected along with host information. We measured CIA using human-lineage colon cancer epithelium (Caco-2) and keratinocyte (HaCaT) cell lines, via virulence-associated gene profiling (bca-rib-bac-lmb-cylE-hylB-pavA-pilB-spb1-srtC1-brpA), capsular genotyping, multilocus sequence typing, and antimicrobial resistance (AMR) phenotyping/genotyping. Significant differences in data regarding CIA into epithelium and keratinocytes and those of isolates from different hosts were assessed. We analyzed the association of CIA populations with the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes.A comparative analysis was performed between human (n = 15) and canine (n = 17) non-invasive isolates. There was a difference in CIA data between Caco-2 and HaCaT cells using human and animal isolates. For percent invasion ability into Caco-2 cells, we designated values ≥ 0.1 as high-frequency CIA and values 0.1 as low-frequency CIA. Fourteen isolates harbored high-frequency and 18 isolates harbored low-frequency strains. There was no association between the high-frequency population and the virulence genotypes, capsular genotypes, sequence types/clonal complexes, and AMR phenotypes/genotypes.This is the first report assessing the invasion ability of S. agalactiae into HaCaT and Caco-2 cells. Our observations suggest that S. agalactiae is more capable of entering Caco-2 rather than HaCaT.

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).

Objective We aimed to determine novel alleles and their prevalence in Streptococcus canis M-like protein (SCM) and to elucidate association of their alleles with sequence types (STs)/clonal complexes (CCs) and antimicrobial resistance (AMR) phenotypes/genotypes. Methods We amplified and sequenced scm, by using primers reported by Pinho recently, for 40 isolates in 2015 and 2017, in which the sequences could not be determined with conventional primers. Isolates, for which SCM alleles, STs, and AMR phenotypes/genotypes were previously determined, were included as controls. A phylogenetic tree of SCM amino acid sequences was constructed. Alleles, based on the tree positions with their prevalence, as well as STs/CCs and AMR phenotypes/genotypes were characterized. Results Although one isolate possessed SCM allele type 1, 39 isolates had novel allele types 10–15, based on cluster analysis. The 11 and 12 allele types were firstly found in this study. We designated novel allele types as group II and non-novel allele types as group I. Prevalence of group II alleles was 29.9% and 16.2% in 2015 and 2017. Prevalent group II types were allele 10 (10.3%), allele 11 (2.7%), and allele 15 (3.3%) through both periods. There was a significant difference in distribution of STs/CCs between groups I/II SCM populations. We found significant differences in distribution of macrolide/lincosamide AMR genotype (7.7% vs. 26.8%) and AMR rates of fluoroquinolone (0% vs. 12.5%) between the two populations. Conclusion Our study presents group II scm sequences and their prevalence among diseased companion animals in Japan, with association of their alleles with STs.

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.

This study assessed whether Streptococcus agalactiae isolates from companion animals differed from those of human origin. Beta-hemolytic S. agalactiae was collected from a veterinary laboratory center and a university hospital. Strains were identified using 16S rRNA amplicon sequencing and amplification of the species-specific dltS gene. We conducted virulence gene profiling, capsular genotyping, determination of clonal complex (CC), and antimicrobial resistance (AMR) phenotyping or genotyping. The 20 non-invasive isolates obtained from animals and 15 non-invasive isolates from adult humans were comparatively analyzed in this study. We found significant differences in the virulence gene profiles of bca-rib-lmb-cylE (40.0% vs. 93.3%) and the possession of bac (30.0% vs. 0%) between animal-origin and human-origin non-invasive strains. We observed a significant difference in the distribution of CC1 between the two non-invasive populations. There were significant differences in the prevalence of tetracycline resistance genotypes (60.0% vs. 20.0%) and absence of AMR genotypes (30.0% vs. 80.0%), and AMR rates of tetracycline (35.0% vs. 0%) and fluoroquinolone (20.0% vs. 66.7%) between the two non-invasive populations. These observations suggest that there were different features, in terms of virulence gene profile, CC, and AMR genotype/phenotype in the non-invasive isolates of animal origin compared to those of human origin.

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.

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa are very difficult to treat. The aim of this study was to develop more effective treatments by investigating in vitro the effects of combinations of antibiotics against 47 MDR P. aeruginosa isolates harbouring various resistance factors. The isolates included 41 (87%) metallo-β-lactamase (MBL)-positive strains, 37 (79%) strains with mutations in OprD and 46 (98%) strains carrying the genes encoding aminoglycoside-modifying enzymes (AMEs). The quinolone resistance-determining region was mutated in all of the strains. These strains were classified into 16 groups according to amplified fragment length polymorphism and resistance factors. The effects of combinations of antibiotics on 16 representative strains were determined using a ‘Break-point Checkerboard Plate’ assay. Combinations of amikacin + aztreonam (coverage rate, 81.3%) and arbekacin + aztreonam (93.8%) inhibited growth. In contrast, combinations of ciprofloxacin + meropenem (6.3%) and ciprofloxacin + ceftazidime (12.5%) were much less effective. Aztreonam and arbekacin (or amikacin) are not substrates for MBLs and AMEs, respectively. We conclude that the combined effects of these drugs were possibly because of resistance factors.

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 -MIC90 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.

A catastrophic earthquake occurred off the Pacific coast of Japan on 11 March 2011, striking the northeastern part of the country. The earthquake was followed by huge tsunamis, which destroyed many coastal cities and towns. Many displaced people moved into shelters or temporary homes supplied by the government, not only because of disruptions to community utility services but also because of health risks associated with nuclear power plant malfunctions in Fukushima. In this review, we summarize the characteristics of illnesses that occurred in the aftermath of this earthquake, including respiratory tract infection (tsunami-related aspiration pneumonia, legionellosis and influenza), wound infection (tetanus) and other infections (food poisoning, tsutsugamushi disease and measles). Our review also outlines several activities concerning the management of illnesses and infection control.

Helicobacter cinaedi has being recognized as an important human pathogen which causes bloodstream infections. Although the first case of bacteremia with this pathogen in Japan was reported in 2003, the true prevalence of H. cinaedi as a pathogen of bloodstream infections in this country is not yet known. Therefore, the aim of our study was to assess the incidence of bacteremia with H. cinaedi in Japan. We conducted a prospective, multicenter analysis in 13 hospitals during 6 months in Tokyo, Japan. Among positive blood cultures from 1 October 2003 to 31 March 2004, isolates suspected of being Helicobacter species were studied for further microbial identification. Identification of the organisms was based on their biochemical traits and the results of molecular analysis of their 16S rRNA gene sequences. A total of 16,743 blood culture samples were obtained during the study period, and 2,718 samples (17.7%) yielded positive culture results for coagulase-negative staphylococci. Among nine isolates suspected to be Helicobacter species, six isolates were finally identified as H. cinaedi . The positivity rate for H. cinaedi in blood culture was 0.06% of total blood samples and 0.22% of blood samples with any positive culture results. All patients with bacteremia with H. cinaedi were found to have no human immunodeficiency virus (HIV) infection, but many of them had complications with either malignancy, renal failure, or a history of surgical operation. Therefore, our results suggest that bacteremia with H. cinaedi is rare but can occur in compromised hosts other than those with HIV infection in Japan.

To determine the status of biosafety in clinical laboratories in Japan, we conducted a survey using questionnaires on the biosafety of laboratory personnel in 2004. We obtained data from 431 hospitals (response: 59.5%). Respondents were 301 institutions (70%) having biological safety cabinets (BSCs). BSCs were held in 78% of microbiological laboratories, 7.9% of genetic laboratories, 2.7% of histopathological laboratories, and 1% or less at other laboratories. A clean bench in examination rooms for acid-fast bacilli was applied at 20 hospitals. We found 28 cases of possible laboratory-associated tuberculosis infection, 25 of which were associated with lack of BSC. Other risk factors were immature skills and insufficiently skilled eguipment operation. The frequency of rupture accidents during specimen centrifugation was 67% in dealing with blood and 9.7% in collecting acid-fast bacilli. Half or more accidents were related to inadequate sample tube materials. Technologists were shown to be working on blood collection in many hospitals (75%), and 1,534 events of self-inflicted needle puncture developed in the last 5 years. These results suggest that biosafety systems are woefully lacking or inadequate in clinical laboratories in Japan and must be established at the earliest possible opportunity.

Human herpesvirus 8 (HHV-8) can be classified into distinct subtypes on the basis of sequence polymorphisms in several open reading frames (ORFs). We analyzed the subtypes of HHV-8 in 59 human immunodeficiency virus-infected Japanese patients by using polymorphisms in ORF26 and found that over two-thirds of the HHV-8 isolates fell into major subtype A. We also found that single nucleotide polymorphisms (SNPs) at nucleotide positions 1032 (C-to-A substitution) and 1055 (G-to-T substitution) in HHV-8 ORF26 were correlated with increased susceptibility to Kaposi's sarcoma, compared to the results obtained with HHV-8 with wild-type nucleotides at these positions ( P = 0.0106). This observation suggests that molecular heterogeneity of the HHV-8 genome affects the biological properties of HHV-8, resulting in different clinical phenotypes of HHV-8 infection. Since sensitive PCR of ORF26 allowed us to analyze the SNPs by using peripheral blood from HHV-8-infected patients, the ORF26 SNPs will be a potent tool for investigating the pathogenesis of HHV-8 infection.

We report the isolation of Helicobacter cinaedi (previously called " Campylobacter-like organism") from the blood of an immunosuppressed Japanese patient receiving immunosuppressive therapy after renal transplantation. The identification of H. cinaedi was based on the findings of microscopic examinations, biochemical properties, and 16S rRNA gene nucleotide sequences. H. cinaedi bacteremia should be considered as one of the critical infectious diseases in immunocompromised patients, and the sequencing of 16S rRNA may be a useful method to confirm the identification of this organism.

Eighty-one bronchoalveolar lavage (BAL) specimens obtained from 26 HIV-infected, 45 non-HIV immunosuppressed and 10 immunocompetent patients with primary pulmonary diseases were analysed for the presence of Pneumocystis carinii by staining and by P. carinii 5S rDNA determined by PCR. P. carinii was observed by staining of BAL specimens from HIV-infected patients significantly more frequently than those from immunocompromised hosts without HIV infection (57.7% versus 20.0%, respectively). P. carinii 5S rDNA was detected by PCR assay in seven (26.9%) HIV-infected individuals, which was significantly more frequent than for four (8.9%) immunosuppressed patients without HIV infection, for whom staining was negative. None of these patients developed P. carinii pneumonia (PCP) within the follow-up period. BAL specimens from 10 immunocompetent patients with pulmonary disorders were negative for PCP by both staining and PCR assay.

To assess the characteristics of CD4 and CD8 T cells specific for HIV-1 and cytomegalovirus (CMV) antigens in untreated and treated HIV-1-infected patients.Antigen-specific T cell frequencies were determined by flow cytometric detection of antigen-induced intracellular cytokines.In untreated patients, HIV-1-specific CD4 T cell counts in peripheral blood were less than one tenth of CMV-specific CD4 T cell counts, while the number of specific CD8 T cells was approximately the same for both HIV-1 and CMV. In patients treated with highly active antiretroviral therapy (HAART) for less than 1.5 years, HIV-1-specific CD4 and CD8T cell counts were significantly lower than those in untreated patients. Perforin expression in HIV-1-specific CD8 T cells was significantly lower than that in CMV-specific CD8 T cells.These data indicate that HIV-1-specific T cells in HIV-1-infected patients have quantitative and qualitative abnormalities.

Background and Aim: It has been a conflicting issue whether TT virus (TTV), a newly isolated DNA virus from a patient with liver injury of unknown cause, is a causative agent of acute and/or chronic hepatitis. TT Virus DNA titers were shown to be 10–100-fold greater in liver tissue than in serum, whereas the majority of TTV-positive cases had no biochemical or histological evidence of significant liver damage. We therefore attempted in situ hybridization to investigate whether TTV is hepatotropic. Methods: Because of the marked divergence in TTV genome types, a template for TTV-DNA (coding region for N22 clone) was amplified and labeled with digoxigenin-dUTP by using hemi-nested PCR from the serum, then DNA probes were applied to the liver sections of the same case. After hybridization, the probes were visualized immunohistochemically. Besides TTV-DNA-negative cases, competitive inhibition experiments with unlabeled probes were performed to confirm the specificity. Results There were no positive signals in the negative controls, and the intensity of positive signals was markedly diminished in the competitive inhibition experiments. No cross-hybridization with different genotype probes also confirms the specificity. Under the optimal conditions, the positive signals were located in the cytoplasm of the hepatocytes in eight of nine TTV-DNA-positive cases. The signals were not seen in non-parenchymal cells of the liver. Conclusion: TT Virus is proved to be hepatotropic by in situ hybridization.

The emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4+cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 −589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.

HIV-1 infection induces the expression of high level of GM2 ganglioside on infected cells and IgM antibody (Ab) against GM2 can cause complement (C)-mediated cytolysis of HIV-1-infected cells. Since GM2 is immunogenic in human, we proposed that an anti-GM2 IgM Ab may be produced by some HIV-1-infected patients and the titer of this Ab might provide some insight into the progress of the disease. On this premise, the amount of IgM Ab against GM2 was determined in 124 HIV-1-infected patients and 111 seronegative donors. As expected, the anti-GM2 IgM Ab titers of the patients was significantly higher than that of the seronegative donors while the total IgM levels remained unchanged. In addition, we determined the CD4+ cell count and the HIV-RNA load in the HIV-1-infected patients. The results showed a positive correlation between the anti-GM2 IgM Ab titer and CD4+ cell count but a negative correlation between the anti-GM2 IgM Ab titer and HIV-RNA load. These suggest that anti-GM2 IgM Ab induced and/or enhanced by HIV-1 infection causes C-mediated cytolysis of HIV-1-infected cells in vivo to a certain extent, and may help lower the plateau level of the HIV-RNA load. Therefore, the amount of IgM Ab against GM2 may be related to the prognosis of HIV-1 infected patients.

Periodontal tissue shows various morphological changes with ageing. A typical example of these changes is the apical migration of the junctional epithelium. The distribution of fibronectin and laminin was investigated by immunofluorescent and immunoelectron-microscope methods in mice to clarify any possible functional roles of these proteins in the apical migration of junctional epithelium. Apical migration begins in 20-week-old mice, and then progresses with increasing age until the mice reach 80 weeks. In the apical tip of the junctional epithelium, fibronectin was demonstrated in the sub-epithelial fibrillar matrix, preceding the progression of apical migration. Fibronectin was also demonstrated in association with the stromal side of focal contacts between epithelial cells and basement membrane. Therefore, these focal contacts are assumed to be fibronectin receptors. There was no apparent relation between the localization of laminin and the migration of the junctional epithelium. These results suggest that the fibronectin provides a provisional matrix for the apical migration of junctional epithelium, but laminin does not appear to play a major part in that migration.

Accuracy amplified Mycobacterium Tuberculosis Direct Test (MTD), Amplicor Mycobacteria kit (Amplicor) and PCR method routinely used in the University of Tokyo Hospital (J. Clin. Microbiol. 31: 446-450 1991) were evaluated and compared with the same samples. The detection limits of MTD, Amplicor and PCR method (University of Tokyo) were 0.01-0.1 CFU/tube, 0.625 CFU/tube and 0.2 CFU/tube respectively, which were almost the same. These were shown to be at least as sensitive as the conventional culture techniques. The Tokyo Univ. method using radioisotope, takes up to 3 days, on the other hand 2 kits take 4-5 hours. These kits become useful tools for the early and rapid detection of M. tuberculosis in uncultured clinical specimens. But the risk of laboratory contamination and false-positive results remain. These must be further improved.

The synergic activity of imipenem/cilastatin combined with cefotiam was studied in a mouse bacteraemia model. Combinations of imipenem plus cefotiam in ratios from 1:5 to 1:160 were more effective than either imipenem alone or cefotiam alone (P < 0.05). Synergy was observed against both beta-lactamase producing and beta-lactamase non-producing MRSA. Staggered combinations of imipenem with cefotiam (each drug was administered at a different time) were studied in an in-vitro pharmacokinetic system to clarify relationships between killing kinetics and pharmacodynamics of the combinations. In the in-vitro system, cefotiam (1 g over 30 min) administered 2 h after imipenem administration (250 mg over 30 min) reduced viable cell counts to an undetectable level and maintained this for 4 h, while the simultaneous administration of imipenem and cefotiam maintained an undetectable cell count for only 2 h. Furthermore, imipenem administered after cefotiam showed no synergy. These results indicate that the timing of dosing of each antibiotic influences synergy, and administration of cefotiam 2 h after imipenem is more effective than the other regimens.

Human macrophage inflammatory protein-1 alpha (MIP-1alpha) is a chemotactic cytokine, which binds to macrophages, T cells, and B cells affecting their activation. We found novel polymorphisms at four sites within MIP-1alpha gene in Japanese population: C to T in exon 2; A to G in intron 2; C to G and A to G in exon 3. They occurred on the same allele. Although MIP-1alpha effectively suppresses the replication of HIV-1 in vitro, we observed no statistically significant difference in the allele frequency of this polymorphism between HIV-1-infected and uninfected individuals in Japanese population. Since an increased transcription level of MIP-1alpha has been reported to be associated with inflammatory diseases such as atopic dermatitis, we also investigated the frequency of these polymorphisms among patients with atopic dermatitis, HIV-1-infected individuals (with a normal IgE level), and healthy donors. A small increase in ratio of homozygotes to other genotypes was observed in patients with atopic dermatitis (P = 0.04).

Human parvovirus B19 (B19) has been described as a causative agent of chronic anemia in human immunodeficiency virus type-1 (HIV-1)-infected patients. We report an HIV-1 infected patient who had been receiving anti-retroviral therapy who showed sudden pancytopenia. Primary B19 infection was confirmed by the detection of plasma viremia and seroconversion. Although clearance required a prolonged period of time, the patient eventually cleared the B19 viral DNA from the plasma. More than likely, highly active anti-retroviral therapy (HAART), including a protease inhibitor, played a role in clearing the virus.