Characterization of HcaA, a novel autotransporter protein in Helicobacter cinaedi, and its role in host cell adhesion.

IMPORTANCE: Helicobacter species are classified as gastric or enterohepatic according to their habitat. Among enterohepatic Helicobacter species, which inhabit the intestine, colon, and liver, Helicobacter cinaedi has been most frequently isolated from humans. H. cinaedi often causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on the H. cinaedi autotransporter protein A (HcaA), a novel virulence factor in H. cinaedi. We discovered that HcaA contributes to cell adhesion via its Arg-Gly-Asp motif. Furthermore, in animal experiments, bacterial colonization was reduced in mice infected with HcaA-knockout strains, supporting the hypothesis that HcaA contributes to H. cinaedi adhesion to host cells. Our study provides a novel mechanism for the establishment of H. cinaedi infections and provides new insights into the role of autotransporter proteins in the establishment of Helicobacter infection.

Protocol for detecting Helicobacter suis infection in gastric biopsies and serum by PCR and ELISA.

Infection with Helicobacter suis, which causes many cases of gastric disease, is not reliably diagnosed. Here, we present a protocol for detecting H. suis infection. We describe steps for collecting gastric biopsies and sera from patients, preparing DNA for PCR, and targeting the H. suis-specific gene. We then define procedures for inoculating biopsies onto primary agar plates and transferring colonies to secondary agar plates. Finally, we detail whole-genome sequencing of bacteria and assess H. suis infection in sera with ELISA. For complete details on the use and execution of these protocols, please refer to Matsui et al.1.

Development of serological assays to identify Helicobacter suis and H. pylori infections.

Helicobacter suis, hosted by hogs, is the most prevalent gastric non-Helicobacter pylori Helicobacter species found in humans. Recent studies have suggested that H. suis infection has caused many cases of gastric disease, but the transmission route from hogs remains unclear. Diagnostic methods based on H. suis urease activity often yield negative results, and there is no reliable method for diagnosing H. suis infection in clinical practice without gastric biopsy specimens. This study presents the world's first use of whole-bacterial cell ELISA to simultaneously assess H. suis and H. pylori infections. The ELISAs showed high accuracy, with an area under the ROC curve of 0.96, 100% sensitivity, 92.6% specificity, 76.9% positive predictive value, and 100% negative predictive value for the H. suis test, and an area under the ROC curve of 0.92, 88.2% sensitivity, 87.5% specificity, 65.2% positive predictive value, and 96.6% negative predictive value for the H. pylori test.

Helicobacter ailurogastricus in Patient with Multiple Refractory Gastric Ulcers, Japan.

We report the isolation of Helicobacter ailurogastricus, a Helicobacter species that infects cats and dogs, from a person with multiple refractory gastric ulcers. In addition to H. suis, which infects pigs, Helicobacter species that infect cats and dogs should be considered as potential gastric pathogens in humans.

Cutibacterium acnes phylogenetic type IC and II isolated from patients with non-acne diseases exhibit high-level biofilm formation.

Cutibacterium (formerly Propionibacterium) acnes is an important for not only exacerbating factor of acne vulgaris but also pathogen of surgical site infections (SSIs) in orthopedics and plastic surgery. Although biofilm-forming (BF) C. acnes are associated with intractable SSI, characteristics of these strains were still unknown. Here, we explored detailed molecular epidemiological features of BF C. acnes isolated as causative pathogen of infectious diseases. Phylogenetic types of 205 C. acnes strains isolated between 2013 and 2018 from 18 clinical departments of a university hospital in Japan were determined by single-locus sequence type (SLST). Clade H (traditional type IC) and K (type II) which are less relevant with healthy skin and acne vulgaris, were detected in 26.8% (55/205) and 16.1% (33/205) of the strains, respectively. The incidence of them was significantly higher than that of acne patients (H and K, each 2.9%, P < 0.05). In addition, SLST distribution of C. acnes strains differed by each department and isolation site. When biofilm formation was quantified, 51 strains (24.9%) were defined as high-BF strains. Notably, most high-BF strains were classified into the strains of clade H (56.4%, 31/55) and clade K (54.4%, 18/33), and these strains were frequently found in the strains isolated from patients of medical emergency center and plastic surgery. Similarly, high-BF strains were frequently found among the isolates from blood (35.7%) and catheters (30.0%), with a high proportion belonging to clades H and K. Compared to C. acnes strains isolated from acne patients, antimicrobial-resistant strains were less identified in non-acne patients. Our findings showed that pathogenicity of C. acnes strains differs by their phylogenetic types. Furthermore, we showed clade H and K have the ability of high biofilm formation and suggest that these strains have potential to become a risk factor for SSI.

Increased prevalence of doxycycline low-susceptible Cutibacterium acnes isolated from acne patients in Japan caused by antimicrobial use and diversification of tetracycline resistance factors.

The prevalence of antimicrobial-resistant Cutibacterium acnes is an important concern for the antimicrobial treatment of acne vulgaris. We hypothesized that antimicrobial treatment regimens for acne vulgaris would change following the revisions in the Japanese acne treatment guidelines, which added a statement regarding appropriate antimicrobial usage. Here, we studied the changes in antimicrobial use and antimicrobial-resistant C. acnes isolated from acne patients. A total of 127 C. acnes isolates collected from 212 patients with acne between 2013 and 2018 were used. Roxithromycin and clindamycin resistance rates were approximately 50% and 40%, respectively. In contrast, the prevalence of low doxycycline-susceptible strains (minimum inhibitory concentration [MIC] ≥8 µg/mL) in 2018 (17.4%) was 5.6-fold higher than that in 2013 (3.1%). Although the number of patients with severe and moderate acne did not change, the number of patients with a history of oral tetracycline use increased. The incidence of low doxycycline-susceptible strains was high in patients with a history of oral tetracycline use. The prevalence of strains with a 16S rRNA mutation, which confers reduced susceptibility to tetracyclines, increased by 8.6-fold (12.1%) from 2016 to 2018 in comparison with the previously revised guidelines (1.4%). Furthermore, the prevalence of low susceptibility strains with two resistance factors, 16S rRNA mutation and ribosomal S10 protein substitution, also increased. Approximately 10% of strains had the exogenous resistance gene, tet(W) (2013 to 2015, 10.1%; 2016 to 2018, 8.6%), and these strains showed different susceptibility to doxycycline dependent on the expression of tet(W) (MIC range 0.5-8 µg/mL). Our data show that the antimicrobial resistance pattern in C. acnes changes according to the trend of antimicrobial usage for acne treatment. Therefore, we should pay heed to the rapid dissemination of tetracycline resistance in C. acnes owing to acquisition of 16S rRNA mutation and tet(W).

[Evaluation of the Antimicrobial Effects of a Novel Visible Light-driven Photocatalyst in Vitro and in the Environment].

Environmental microorganisms can cause several infections in humans, especially in compromised hosts. Since there are many compromised hosts in a hospital setting, it is important to control environmental pathogens in such scenarios. To disinfect the environment, photocatalysts that produce reactive oxygen in response to light have attracted attention. In the present study, the effects of a visible-light-driven antimicrobial photocatalyst, silver (I) iodide and benzalkonium complex, on bacteria, viruses, and fungi were evaluated in vitro. In addition, uncoated panels and panels coated with the photocatalyst were set up at 11 points in a university campus for 6 months, and the adherent bacteria and fungi were measured. Bacteria, bacterial spores, viruses, and fungi were completely inactivated within 45 min on the photocatalyst-coated surface exposed to approximately 700-lux fluorescent light. In the university setting, there were fewer viable adherent bacteria and fungi on the coated plates. Our findings indicate that the silver (I) iodide and benzalkonium complex photocatalyst can decrease environmental bacteria in vitro and in actual environmental settings, and thus highlight its potential in controlling and disinfecting environmental pathogens.

Characterization of acne patients carrying clindamycin-resistant Cutibacterium acnes: A Japanese multicenter study.

Use of antimicrobials for acne treatment is correlated with an increased occurrence of antimicrobial-resistant Cutibacterium acnes. To clarify the role of antimicrobial use on the resistance and to investigate the characteristics of resistant strains, we conducted a multicenter study in dermatological clinics frequently visited by new patients with acne vulgaris. We collected specimens in 264 acne patients and tested 164 C. acnes strains isolated from 164 patients visiting 13 dermatological clinics. Antimicrobial susceptibility testing showed that the rates of resistance for tetracyclines, macrolides and clindamycin were significantly higher in C. acnes strains isolated from patients using antimicrobials for acne treatment than patients not using them. In particular, clindamycin-resistant strains were frequently isolated from patients with older median age (≥24 years) and severe/moderate acne. After investigating the resistance mechanism of 15 high-level clindamycin-resistant strains, the transposable clindamycin resistance genes, erm(X) or erm(50), were detected in 14 strains. Using single-locus sequence typing for C. acnes, the strains with erm(X) or multidrug resistance plasmid pTZC1 coding erm(50) and tetracycline resistance gene tet(W) were classified into clade F, which were specifically isolated from Japanese patients with acne, except for one strain. Our data showed that patients' information, such as antimicrobial use, age and acne severity, are valuable in estimating whether a patient carries antimicrobial-resistant C. acnes. Additionally, our results suggest that the clade F strains have a high risk of acquiring multidrug resistance.

Transferable Multidrug-Resistance Plasmid Carrying a Novel Macrolide-Clindamycin Resistance Gene, erm(50), in Cutibacterium acnes.

Antimicrobial-resistant Cutibacterium acnes strains have emerged and disseminated throughout the world. The 23S rRNA mutation and erm(X) gene are known as the major resistance determinants of macrolides and clindamycin in C. acnes We isolated eight high-level macrolide-clindamycin-resistant C. acnes strains with no known resistance determinants, such as 23S rRNA mutation and erm(X), from different acne patients in 2008 between 2013 and 2015. The aim of this study was to identify the novel mechanisms of resistance in C. acnes Whole-genome sequencing revealed the existence of a plasmid DNA, denoted pTZC1 (length, 31,440 bp), carrying the novel macrolide-clindamycin resistance gene erm(50) and tetracycline resistance gene tet(W). pTZC1 was detected in all C. acnes isolates (eight strains) exhibiting high-level macrolide-clindamycin resistance, with no known resistance determinants (MIC of clarithromycin, ≥256 µg/ml; clindamycin, ≥256 µg/ml). Transconjugation experiments demonstrated that the pTZC1 was horizontally transferred among C. acnes strains and conferred resistance to macrolides, clindamycin, and tetracyclines. Our data showed, for the first time, the existence of a transferable multidrug-resistant plasmid in C. acnes Increased prevalence of this plasmid will be a great threat to antimicrobial therapy for acne vulgaris.

Transconjugation of erm(X) conferring high-level resistance of clindamycin for Cutibacterium acnes.

Cutibacterium acnes (C. acnes) can become an exacerbating factor in acne vulgaris. Clindamycin has been most frequently used for the treatment of inflammatory acne vulgaris. We studied clindamycin susceptibility and resistance determinants of C. acnes isolated from acne patients in Japan. The isolation rate of clindamycin-resistant C. acnes had significantly increased from 20.3 % in 2009-2010 to 44.1 % in 2016-2017. Strains carrying erm(X), which confers high-level resistance to clindamycin, had significantly increased from 1.4 to 11.8 %. Sequence analysis of the resistance determinant showed that erm(X) was coded on transposon Tn5432. A transconjugation experiment showed that erm(X) can be transferred between C. acnes strains with high frequency and the transconjugants harboured transposon Tn5432 encoding erm(X). Our data show the transconjugation of erm(X) in C. acnes and strongly suggest that the transmission of erm(X) between C. acnes contributes to the increase and spread of clindamycin-resistant C. acnes strains in acne patients.

Long-term administration of oral macrolides for acne treatment increases macrolide-resistant Propionibacterium acnes.

Macrolide-resistant Propionibacterium acnes are frequently isolated from patients with acne vulgaris, and the most resistant isolates (>90% resistance) have the 23S rRNA mutation. An increase in resistant P. acnes with this mutation is thought to be caused by the inappropriate use of antimicrobials. Therefore, we studied the mutation frequency of macrolide resistance in P. acnes in vitro. When P. acnes mutants were exposed to clarithromycin after being incubated in broth without antimicrobials, resistant mutants with the 23S rRNA mutation were not isolated. However, the mutants were obtained at the frequency of 10-6 after being pre-incubated with 0.03 µg/mL of antimicrobials. This is the estimated epidermal concentration of clarithromycin after p.o. administration. The resistant mutants had the 23S rRNA mutations A2058G, A2059G and C2611G. When pre-incubated with clarithromycin, C2611G mutants which showed resistance to clarithromycin were obtained 32.1% more often than pre-incubated with clindamycin (P < 0.01). By contrast, when pre-incubated with clindamycin, A2058G mutants, which show high-level resistance to both clarithromycin and clindamycin, were more frequently obtained than pre-incubated with clarithromycin (87.5%, P < 0.01). No difference in the isolation rate of A2059G mutants, which show high-level resistance to macrolides but low-level resistance to clindamycin, was found with either treatment. These results indicate the possibility that long-term use of oral macrolides for acne treatment facilitate the increase of macrolide-resistant P. acnes.

Antimicrobial susceptibility and phylogenetic analysis of Propionibacterium acnes isolated from acne patients in Japan between 2013 and 2015.

The prevalence of antimicrobial-resistant Propionibacterium acnes strains isolated from acne patients has been increasing in Japan. Here, to estimate the current resistance rate, we tested antimicrobial susceptibility among P. acnes from acne patients having visited a specialized dermatology clinic between 2013 and 2015. Rates of resistance to macrolides and clindamycin were 44.3 (31/70) and 38.6% (27/70), respectively. erm(X), which confers high-level clindamycin resistance (minimum inhibitory concentration ≥256 µg/mL), was detected in six isolates, whereas no resistance determinants were identified in eight strains showing high-level resistance to clindamycin. Using single-locus sequence typing, the P. acnes isolates were classified into five clades (A, E, F, H and K), with all high-level clindamycin-resistant strains lacking known clindamycin resistance determinants being grouped together (in clade F). P. acnes isolates from patients previously treated with macrolides and clindamycin showed a macrolide resistance rate (55.3%) significantly higher than that of those from patients not having received these treatments (21.7%, P < 0.05). Furthermore, strains of clade F, which were very rarely isolated from healthy individuals, were more frequently recovered from patients with severe acne (40.0%) than those with mild acne (23.3%). Our data showed an increase in macrolide-resistant P. acnes prevalence in Japan due to the use of antimicrobial agents for acne treatment. Furthermore, we identified strains of specific phylogenetic groups frequently associated with severe acne patients.

Prevalence of skin infections caused by Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus in Japan, particularly in Ishigaki, Okinawa.

The prevalence of Panton-Valentine leukocidin gene (pvl)-positive community-acquired methicillin-resistant Staphylococcus aureus USA300 clone, which is designated as the ST8-staphylococcal cassette chromosome (SCC) mec type IV (ST8-IV) lineage, is a major public health concern worldwide. Thus, to elucidate the prevalence and characteristics of pvl-positive community-onset MRSA in Japan, we conducted a molecular epidemiological analysis for 854 S. aureus isolates obtained from outpatients with skin infections during 2013 and 2014. The isolation rate of MRSA was 25.6% (219 isolates), and the ratio of pvl-positive MRSA was 13.2% (29 isolates). Notably, the proportion (93.8%) of pvl-positive isolates was particularly high among MRSA isolates from Ishigaki island in Okinawa. Pulsed-field gel electrophoresis and multilocus sequence typing showed that the pulsotype C isolates (11 isolates) were typical USA300 clones with arginine catabolic mobile element (ACME) type I-CC8-IV lineages and prevalent on the main island of Japan (Honshu). Pulsotypes A (11 isolates) and B (four isolates) consisted of ACME-negative CC8-IV clones and were specific for Ishigaki island. Both USA300 and Okinawa-Ishigaki specific clones were associated with deep-seated skin infections, such as furuncle and cellulitis. Pulsotypes D (two isolates) and E (one isolate) were ACME-negative clonal complex (CC) 59-IV clones and were related to superficial skin infections, such as impetigo. Our findings revealed that pvl-positive MRSA associated with deep-seated skin infections are spreading in Japanese communities, particularly in Ishigaki, Okinawa.

Charade of the SR K+-channel: two ion-channels, TRIC-A and TRIC-B, masquerade as a single K+-channel.

The presence of a sarcoplasmic reticulum (SR) K+-selective ion-channel has been known for >30 years yet the molecular identity of this channel has remained a mystery. Recently, an SR trimeric intracellular cation channel (TRIC-A) was identified but it did not exhibit all expected characteristics of the SR K+-channel. We show that a related SR protein, TRIC-B, also behaves as a cation-selective ion-channel. Comparison of the single-channel properties of purified TRIC-A and TRIC-B in symmetrical 210 mM K+ solutions, show that TRIC-B has a single-channel conductance of 138 pS with subconductance levels of 59 and 35 pS, whereas TRIC-A exhibits full- and subconductance open states of 192 and 129 pS respectively. We suggest that the K+-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, can be explained by the gating of both TRIC-A and TRIC-B channels suggesting that the SR K+-channel is not a single, distinct entity. Importantly, TRIC-A is regulated strongly by trans-membrane voltage whereas TRIC-B is activated primarily by micromolar cytosolic Ca2+ and inhibited by luminal Ca2+. Thus, TRIC-A and TRIC-B channels are regulated by different mechanisms, thereby providing maximum flexibility and scope for facilitating monovalent cation flux across the SR membrane.