Human Keratinocyte Entry of Noninvasive Streptococcus dysgalactiae Subsp. equisimilis from Humans and Companion Animals: Relatedness with Lancefield Group, Source, Virulence-Associated Genes, and Antimicrobial Resistance Phenotype.

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.

Biofilm Production Ability of Streptococcus dysgalactiae Subsp. equisimilis: Associations with Host Species, Lancefield Group, Source, Clonal Complex, and Virulence-Associated Genes.

We assessed the biofilm production ability (BPA) of noninvasive Streptococcus dysgalactiae subsp. equisimilis (SDSE) in humans and companion animals and determined the relationship between bacterial populations with BPA and other host and microbiological features. Sixty-four isolates from companion animals and humans were collected along with host information. We measured BPA using crystal violet staining, in addition to emm typing, multilocus sequence typing, antimicrobial resistance (AMR) phenotyping/genotyping, and virulence-associated gene (VAG) detecting (prtF1-prtF2-lmb-cbp-sicG-srtp1-srtp2-brpA). Differences in the BPA of SDSE from different hosts and sources and different Lancefield groups were assessed. We analyzed the associations between populations with and without BPA (strong, moderate, weak, and no biofilm producers) and emm types, sequence types/clonal complexes (CCs), AMR phenotypes/genotypes, and VAG types. Seventeen, twenty-four, and twelve isolates were strong, moderate, and weak biofilm producers, respectively; eleven showed no BPA. There was a difference in the distribution of populations with BPA between human and animal origins and between isolates of groups G and C. We found an association between populations with BPA and the eye and ear source (vs. the pus and skin source). A relationship was observed between the populations with BPA and CC127 (vs. CC17). We observed no association between the populations with BPA and AMR phenotype/genotype. There was an association between the distribution of populations with BPA and srtp1 expression. Our observations suggest potential associations between populations with BPA and the host species, Lancefield group, source, CC, and VAG type.

Genogrouping of type II-A CRISPR array in Streptococcus dysgalactiae subsp. equisimilis from humans and companion animals compared to multilocus sequence and emm typing.

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.

Comparison between Invasive and Non-Invasive Streptococcus agalactiae Isolates from Human Adults, Based on Virulence Gene Profiles, Capsular Genotypes, Sequence Types, and Antimicrobial Resistance Patterns.

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).

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.

Characterization of azithromycin-resistant Neisseria gonorrhoeae isolated in Tokyo in 2005-2011.

A total of 122 Neisseria gonorrhoeae isolated in the Tokyo metropolitan area in 2005-2011 were collected and analyzed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and for their susceptibility to azithromycin and ceftriaxone. All 122 strains were susceptible to ceftriaxone, but 8 strains were azithromycin-resistant, defined as an azithromycin MIC ≥ 1 µg/ml. The 8 azithromycin-resistant strains were in 6 NG-MAST types, 3 strains in NG-MAST type 1407 and each of the other 5 strains in a different NG-MAST type. NG-MAST type 1407 strains are multidrug-resistant and are disseminated worldwide.

Effects of Y-39983, a selective Rho-associated protein kinase inhibitor, on blood flow in optic nerve head in rabbits and axonal regeneration of retinal ganglion cells in rats.

PURPOSE: To investigate the effects of Y-39983, a selective Rho-associated coiled coil-forming protein kinase inhibitor, on blood flow in the optic nerve head (ONH) in rabbits and axonal regeneration of retinal ganglion cells (RGCs) in rats. METHODS: Blood flow in ONH was measured by the laser speckle method after topical administration of 0.05% Y-39983 solution or its vehicle in rabbit eyes. To investigate the effects of Y-39983 on axonal regeneration of RGCs, RGCs purified from rat eyes were cultured with or without 10 µM Y-39983 and morphologically observed by phase-contrast microscopy. Moreover, the effects of intravitreal administration of Y-39983 were evaluated using an in vivo model of axotomized RGCs in peripheral nerve-grafted rats. RESULTS: Topical administration of 0.05% Y-39983 solution significantly increased blood flow in ONH compared with the vehicle group in rabbits. Maximum increase in blood flow in the 0.05% Y-39983 group was 122.84 ± 5.98 % (Mean ± S.E.) at 90 minutes after administration compared with before administration. Neurites in rat RGCs treated with 10 µM Y-39983 were extended compared with those without Y-39983 treatment of RGCs in vitro. Y-39983 dose-dependently increased the number of RGCs with regenerating axons in vivo. The numbers of RGCs with regenerating axons in 10 and 100 µM Y-39983-treated rats were 99.3 ± 10.5 and 169.5 ± 43.3 cells/mm(2) (Mean ± S.D.), respectively, and significantly increased compared with those in saline-treated rats (43.3 ± 6.0 cells/mm(2)). CONCLUSION: Y-39983 may be a candidate drug not only for lowering of IOP but also for increasing of blood flow in ONH in the treatment of glaucoma. Moreover, Y-39983 may have therapeutic potential for axonal regeneration of RGCs in the treatment of diseases with degenerating axons of RGCs including glaucoma, although improvements of formulation or route of administration are needed in order to reach an effective concentration in retina.

PACAP induces neurite outgrowth in cultured trigeminal ganglion cells and recovery of corneal sensitivity after flap surgery in rabbits.

PURPOSE: To evaluate the ability of pituitary adenylate cyclase-activating polypeptide (PACAP) to induce growth of neuronal processes in cultured trigeminal ganglion cells, and to accelerate neurite outgrowth and recovery of corneal sensitivity after creation of a corneal flap in a rabbit model of laser-assisted in situ keratomileusis (LASIK) surgery. DESIGN: Animal study. METHODS: The cDNA of rabbit PACAP was sequenced, and the expression of PACAP receptors in the trigeminal ganglia from rabbits was quantified by quantitative real-time polymerase chain reaction. Trigeminal ganglion cells were isolated from rabbits and cultured for 48 hours with or without PACAP27 (bioactive N-terminal peptide from PACAP). Cells were stained with antibody against neurofilaments, and neurite outgrowth was quantified by cell counting. In the rabbit LASIK model, a corneal flap with a planned thickness of 130 microm and 8.5 mm diameter was created with a microkeratome. The rabbits then received eyedrops containing PACAP27 four times a day for eight weeks, and corneal sensitivity was measured. Neurite outgrowth was assessed by staining histologic sections of the flap area for cholinesterase. RESULTS: The deduced amino acid sequence of PACAP in rabbit was identical to that of human. PACAP receptor, PAC1, was highly expressed in trigeminal ganglia from newborn and adult rabbits. PACAP27 at 1 microM induced growth of neuronal processes in cultured primary trigeminal ganglion cells. In the LASIK model, extensions of neuronal processes from amputated nerve trunks in cornea were observed after administration of eyedrops containing 1 or 10 microM PACAP27. The 10 microM PACAP27 treatment also greatly accelerated recovery of corneal sensitivity. CONCLUSIONS: PACAP may be a candidate drug for ameliorating dry eye after LASIK surgery.