The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Clinical manifestations and biomarkers to predict mortality risk in adults with invasive Streptococcus dysgalactiae subsp. equisimilis infections.

PURPOSE: The incidence of invasive Streptococcus dysgalactiae subsp. equisimilis (iSDSE) infections is increasing in developed countries, but studies on the risk factors for death in iSDSE infections are scant. Here, we aimed to clarify risk factors and predictors of mortality in adults with iSDSE infections. METHODS: A multicentre observational study of adults with iSDSE infections was conducted to investigate the effects of host factors, disease severity, biomarkers, and antibiotic regimens, and bacterial factors on 28-day mortality. RESULTS: The overall mortality rate of 588 patients was 10.4%, with a significant increase in those aged ≥ 60 years. Most of the patients (97.4%) had underlying diseases. The mortality rate (70.4%) of patients with severe disease was significantly higher than that of patients with mild-to-moderate disease (4.3%; p < 0.001). The risk factors for death identified using multivariable analysis were age ≥ 60 years (hazard ratio [HR], 3.4; 95% confidence interval [CI], 1.0-11.3, p = 0.042); severe disease (HR, 15.0; 95% CI 7.7-29.2, p < 0.001); bacteraemia without primary focus (HR, 20.5; 95% CI 2.8-152.3, p = 0.003); serum creatinine ≥ 2.0 mg/dL (HR, 2.2; 95% CI 1.2-4.0, p = 0.010); serum creatine kinase ≥ 300 IU/L (HR, 2.1; 95% CI 1.1-3.8, p = 0.019); and macrolide resistance (HR, 1.8; 95% CI 1.0-3.3, p = 0.048). Treatment regimens and emm types were not associated with poor outcomes. CONCLUSION: Evaluation of clinical manifestations and biomarkers on admission is important to predict invasive SDSE infection prognosis.

The Association between Transformation Ability and Antimicrobial Resistant Potential in Haemophilus influenzae.

The prevalence of quinolone low-susceptible Haemophilus influenzae has increased in Japan. Low quinolone susceptibility is caused by point mutations in target genes; however, it can also be caused by horizontal gene transfer via natural transformation. In this study, we examined whether this horizontal gene transfer could be associated with resistance to not only quinolones but also other antimicrobial agents. Horizontal transfer ability was quantified using the experimental transfer assay method for low quinolone susceptibility. Further, the association between horizontal transfer ability and resistance to ß-lactams, the first-choice drugs for H. influenzae infection, was investigated. The transformation efficiency of 50 clinical isolates varied widely, ranging from 102 to 106 colony forming unit (CFU) of the colonies obtained by horizontal transfer assay. Efficiency was associated with ß-lactam resistance caused by ftsI mutations, indicating that strains with high horizontal transfer ability acquired quinolone low-susceptibility as well as ß-lactam resistance more easily. Strains with high transformation efficiency increased the transcript level of comA, suggesting that enhanced com operon was associated with a high DNA uptake ability. Overall, this study revealed that the transformation ability of H. influenzae was associated with multiple antimicrobial resistance. Increase in the number of strains with high horizontal transformation ability has raised concerns regarding the rapid spread of antimicrobial-resistant H. influenzae.

Contribution of amino acid substitutions in ParE to quinolone resistance in Haemophilus haemolyticus revealed through a horizontal transfer assay using Haemophilus influenzae.

BACKGROUND: In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. METHODS: A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. RESULTS: By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA, parC and parE of H. influenzae were replaced by those of H. haemolyticus, suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE, as well as gyrA and parC, contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. CONCLUSIONS: These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance.

High-Level Quinolone-Resistant Haemophilus haemolyticus in Pediatric Patient with No History of Quinolone Exposure.

The prevalence of antimicrobial resistance among Haemophilus spp. is a critical concern, but high-level quinolone-resistant strains had not been isolated from children. We isolated high-level quinolone-resistant H. haemolyticus from the suction sputum of a 9-year-old patient. The patient had received home medical care with mechanical ventilation for 2 years and had not been exposed to any quinolones for >3 years. The H. haemolyticus strain we isolated, 2019-19, shared biochemical features with H. influenzae. However, whole-genome analysis found this strain was closer to H. haemolyticus. Phylogenetic and mass spectrometry analyses indicated that strain 2019-19 was in the same cluster as H. haemolyticus. Comparison of quinolone resistance-determining regions showed strain 2019-19 possessed various amino acid substitutions, including those associated with quinolone resistance. This report highlights the existence of high-level quinolone-resistant Haemophilus species that have been isolated from both adults and children.

Quinolone Resistance Is Transferred Horizontally via Uptake Signal Sequence Recognition in Haemophilus influenzae.

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA. Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC, respectively, contributed to the horizontal transfer of resistance in H. influenzae. Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

Alternative quinolone-resistance pathway caused by simultaneous horizontal gene transfer in Haemophilus influenzae.

BACKGROUND: Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. OBJECTIVES: To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. METHODS: Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. RESULTS: By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1-100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. CONCLUSIONS: These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.

Molecular characterisation of carbapenem- and tigecycline-resistant Klebsiella pneumoniae strains isolated from blood and bile samples.

INTRODUCTION: The number of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains are increasing, further raising healthcare concerns worldwide. In this study, we isolated three CRKP strains from bile and blood samples of an elderly patient (90s) with acute cholangitis and characterised the features and antimicrobial resistance mechanism of CRKP isolates. METHODS: Three CRKP isolates were characterised by Pulsed-field gel electrophoresis (PFGE), whole genome sequencing using the NovaSeq 6000, and antimicrobial susceptibility testing. Transcriptional levels of resistance-associated genes were measured by real-time RT-qPCR. RESULTS: PFGE analysis revealed highly similar patterns for these isolates. Furthermore, they showed resistance to not only carbapenem but also tigecycline. Genomic analysis of the blood isolate identified the exogenous resistance genes blaCTX-M14, tet(A), tet(D), opxAB, and qnrS1 but not any carbapenemase-encoding genes. In addition, nonsense mutations were found in both the outer membrane protein K36 (ompK36) and transcriptional regulator ramR, suggesting that this isolate developed multidrug resistance by acquiring both exogenous resistance genes and nonsense mutations. The extended-spectrum ß-lactamase-producing carbapenem-susceptible K. pneumoniae isolate exhibited the same susceptibility pattern, except to ß-lactams, as prior CRKP isolates. CONCLUSIONS: Antimicrobial susceptibility to carbapenem and tigecycline should be continuously monitored, because it might change from susceptible to resistant during another antimicrobial treatment, even if an isolate initially shows susceptibility, and the patient has not been exposed to these agents.

A case of Shewanella algae-induced bacteremia in Japan: Case report and literature review.

Shewanella algae (S. algae) is a rare bacterium that causes infectious diseases in humans. Herein, we present a case of an 84-year-old man with S. algae-induced bacteremia and performed a review of 12 cases identified via a literature search and this case. Literature review of previous reports in Japan have revealed that 69.2% of patients with S. algae-induced bacteremia had a history of contact with fresh fish. Appropriate interviews of patients, especially in the hot season, and the accurate identification of the causative bacterium, by using techniques such as MALDI-TOF-MS and genetic testing, are necessary if S. algae or other bacteria from the genus Shewanella are detected in blood-culture tests.

Circulating immune-complexes and complement activation through the classical pathway in myeloperoxidase-ANCA-associated glomerulonephritis.

BACKGROUND: Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (AAGN) is the fulminant glomerular diseases with poor renal prognosis. Activation of the complement system has recently been reported in the pathogenesis of AAGN, but it remains to be clarified as to which complement pathway is mainly involved. METHODS: 20 patients with myeloperoxidase (MPO)-AAGN were retrospectively evaluated. Using serum samples, circulating immune-complexes (CICs) were assessed by the monoclonal rheumatoid factor assay, and C5a and C5b-9 were assessed by ELISA. Complement activation through the classical pathway was further evaluated by the WIESLAB® Complement System Classical Pathway kit. The affinities of ANCAs were evaluated by a competitive inhibition method using ELISA, and were classified into the high, and low-affinity group. Deposition of complement components, such as C3, C5, C4d, C5b-9, factor Bb, mannan-binding lectin serine peptidase (MASP)-1, MASP-2, and mannose/mannan-binding lectin (MBL), in frozen renal sections were analyzed by immunofluorescence staining. RESULTS: CICs were found to be positive in 65% of the patients. All CIC-positive patients belonged to the high-affinity group. Furthermore, serum C5a and C5b-9 were significantly increased in MPO-AAGN patients, and these levels positively correlated with CIC levels. A significant negative correlation was also found between levels of WIESLAB® classical pathway kit and CICs. By immunofluorescence staining, glomerular deposition of C4d, C5, and C5b-9 were observed in similar distributions in MPO-AAGN patients, whereas the deposition of MASP-1, MASP-2, MBL, and factor Bb were minimal. CONCLUSIONS: These results suggest the involvement of immune-complex induced complement activation through the classical pathway in the pathogenesis of MPO-AAGN.

Dissemination of quinolone low-susceptible Haemophilus influenzae ST422 in Tokyo, Japan.

INTRODUCTION: Haemophilus influenzae with a reduced susceptibility to quinolones (quinolone low-susceptible H. influenzae) has recently emerged in Japan. In addition, the regional outbreak of the quinolone low-susceptible H. influenzae ST422 clone has been reported. In this study, we isolated this clone from an acute care hospital located in a geographically different area from the previous outbreak and characterised the nature of this clone. METHODS: Eighty-nine H. influenzae isolated between 2017 and 2019 were tested. The antimicrobial susceptibility was determined by the broth dilution method. The genetic background was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Growth ability and ß-lactamase acquisition were evaluated by growth curve analysis and conjugative transfer experiments, respectively. RESULTS: Quinolone low-susceptible isolates accounted for 4.2% (1/24) in 2018 and 13.9% (5/36) in 2019. Most of the quinolone low-susceptible strains (83.3%) were classified as ST422 and had amino acid substitutions in quinolone resistance-determining regions in both GyrA and ParC. The patients' backgrounds were highly diverse. In addition, these isolates showed the same PFGE pattern as outbreak strains. The growth of ST422 clone was relatively faster than other clones. Furthermore, ST422 clone was able to acquire ß-lactamase from a ß-lactamase positive strain by horizontal transfer, becoming highly resistant to ß-lactams. CONCLUSION: Our study indicated that the quinolone low-susceptible H. influenzae ST422 clone has been spreading in the community undetected. In addition, this clone has the potential to grow faster and become more resistant through exogenous gene transfer. Therefore, ST422 clone should be monitored attention throughout Japan.

Molecular epidemiological characterization in mucoid-type Streptococcus pneumoniae isolates obtained from invasive pneumococcal disease patients in Japan.

INTRODUCTION: Streptococcus pneumoniae with a mucoid-type capsule is associated with invasive pneumococcal diseases (IPDs). Despite the introduction of pneumococcal vaccines, IPDs caused by mucoid-type isolates are still prevalent. The present study aimed to characterize mucoid-type S. pneumoniae isolated from IPD patients throughout Japan in 2017 (post-vaccination era). METHODS: A total of 225 mucoid-type isolates were collected. The serotype, antimicrobial susceptibility, and multilocus sequence type of these isolates were determined. RESULTS: The prevalence of IPDs caused by mucoid-type isolates was high in adults, especially in the elderly (≥65 years of age), and prognosis in these patients was significantly poor. Of the mucoid-type isolates, the predominant serotype was serotype 3 (84.4%), and the remaining were serotypes 37 (15.1%) and 8 (0.4%). Antimicrobial susceptibility showed that most mucoid isolates exhibited the penicillin-intermediate resistant S. pneumoniae genotype (gPISP). However, the serotype 3 isolate exhibited the penicillin-resistant S. pneumoniae genotype (gPRSP). This gPRSP isolate was classified into ST166, which is related to serotypes 9 V and 11 strains. Sequence analysis of the capsule-coding regions and its flanking regions indicated that recombination occurred upstream and downstream of the capsule-coding region, suggesting that gPRSP (serotype 9 V/ST166) obtaining the type-3 capsule gene cluster resulted in the emergence of gPRSP (serotype 3/ST166). CONCLUSIONS: Our findings indicated that IPDs caused by mucoid-type S. pneumoniae are still a serious concern and mucoid-type S. pneumoniae with novel phenotype could emerge via capsular switching in response to environmental changes such as introduction of vaccines and improper use of antimicrobial agents.

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.

Glyceraldehyde-3-phosphate dehydrogenase of Mycoplasma pneumoniae induces infection-related glomerulonephritis
.

Infection-related glomerulonephritis (IRGN) was previously thought to be due mostly to Streptococcus species, but is now known to be caused by a variety of other pathogens. Nephritis-associated plasmin receptor (NAPlr) was originally isolated from group A streptococci as the protein responsible for acute poststreptococcal glomerulonephritis, and was shown to be identical to streptococcal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Here, we describe a 7-year-old boy diagnosed with Mycoplasma pneumoniae IRGN presenting with acute nephritic syndrome. Laboratory data revealed a significant increase in serum anti-M. pneumoniae antibody titer. Renal biopsy revealed diffuse global endocapillary proliferation and cellular crescents in 5/43 glomeruli examined. Although antistreptolysin O antibody titer and serum complement C3 level were within the respective normal ranges, glomeruli showed positive staining for NAPlr and upregulation of plasmin activity. In addition, positive staining for NAPlr in the glomeruli was abolished by preabsorption of anti-NAPlr antibody with recombinant M. pneumoniae GAPDH. Western blotting analysis revealed anti-NAPlr antibody reactivity with a band at around the predicted size of GAPDH in the protein isolate of M. pneumoniae (37 kDa). Furthermore, immobilized M. pneumoniae GAPDH bound to anti-NAPlr antibody as well as plasmin in vitro. These data suggest that M. pneumoniae GAPDH has a function similar to streptococcal GAPDH (NAPlr) and may induce plasmin-related glomerular damage in M. pneumoniae IRGN. NAPlr could be a marker of glomerulonephritis related to infection not only by streptococci but also by &M. pneumoniae.

Evaluation of in Vitro Bactericidal Activity of 1.5% Olanexidine Gluconate, a Novel Biguanide Antiseptic Agent.

Recently, 1.5% olanexidine gluconate, a biguanide compounds, was launched as a new antiseptic agent in Japan. However, the comprehensive bactericidal spectrum of olanexidine gluconate is still unknown. In this study, we evaluated in vitro bactericidal activity of olanexidine gluconate using time-kill assay against various bacteria, mycobacteria, and fungi. With the exception of Burkholderia cepacia and Mycobacterium spp., 1.5% olanexidine gluconate exhibited fast-acting (≤60 s) bactericidal activity against all tested Gram-positive and Gram-negative bacteria, including vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, extended spectrum ß-lactamase producing Klebsiella pneumoniae, and multidrug-resistant Pseudomonas aeruginosa. Furthermore, 1.5% olanexidine gluconate eradicated Candida albicans, Microsporum canis, and Malassezia furfur within 3 min. Our findings indicate that olanexidine gluconate has broad spectrum bactericidal activity; therefore, it may be useful for the prevention of a wide range of infectious diseases.

Isolation of multidrug-resistant Haemophilus influenzae harbouring multiple exogenous genes from a patient diagnosed with acute sinusitis.

In paediatric patients, ß-lactams and macrolides are widely used to treat acute otitis media and sinusitis, which are often caused by either Streptococcus pneumoniae or Haemophilus influenzae. However, resistant isolates have emerged and are becoming more prevalent. H. influenzae generally acquires antimicrobial resistance by mutation or by expression of ß-lactamase. In this study, we isolated H. influenzae from a paediatric patient diagnosed with acute sinusitis. This strain harboured multiple exogenous resistance genes: blaTEM-1, mef(A) and tet(M). DNA sequencing suggested that both mef(A) and tet(M) had been transferred from S. pneumoniae or another Streptococcus. This typical outpatient had not been exposed to excessive levels of antibiotics and had no underlying diseases, strongly suggesting that this type of resistant isolate could become more prevalent.

Panax Notoginseng Extract Possesses Significant Antibacterial Activity against Pathogenic Streptococci.

BACKGROUND: In traditional Chinese medicine, Panax notoginseng is used to treat inflammation and bleeding but has not been shown to affect bacterial pathogens. OBJECTIVES: Our aim was to assess the antibacterial potential of Panax notoginseng extract (PNE) against bacterial pathogens. METHODS: PNE was dissolved in autoclaved distilled water. Antimicrobial activity was measured by the disc diffusion test and bacterial growth curve assays, in which the concentration of bacterial colony forming units was monitored at several time points in the presence or absence of PNE. RESULTS: Disc diffusion and growth curve assays demonstrated that PNE significantly inhibited the growth of Streptococcus pyogenes, Streptococcus pneumoniae, and Haemophilus influenzae (p < 0.05). In contrast, the growth of the oral commensal bacteria Streptococcus intermedius, Streptococcus salivarius, and Streptococcus anginosus was not inhibited. Therefore, S. pyogenes clinical isolates were analyzed. PNE had antimicrobial effects on all tested isolates in both aerobic and anaerobic conditions. In addition, when S. pyogenes was co-cultured with S. intermedius in the presence of PNE, PNE inhibited the growth of S. pyogenes, but did not inhibit the growth of S. intermedius. CONCLUSION: Our findings suggested that PNE inhibited S. pyogenes without affecting oral commensal bacteria. Therefore, PNE could be used for the treatment of S. pyogenes infections.

Effects of Pneumococcal Conjugate Vaccine on Genotypic Penicillin Resistance and Serotype Changes, Japan, 2010-2017.

To clarify year-to-year changes in capsular serotypes, resistance genotypes, and multilocus sequence types of Streptococcus pneumoniae, we compared isolates collected from patients with invasive pneumococcal disease before and after introductions of 7- and 13-valent pneumococcal conjugate vaccines (PCV7 and PVC13, respectively). From April 2010 through March 2017, we collected 2,856 isolates from children and adults throughout Japan. Proportions of PCV13 serotypes among children decreased from 89.0% in fiscal year 2010 to 12.1% in fiscal year 2016 and among adults from 74.1% to 36.2%. Although nonvaccine serotypes increased after introduction of PCV13, genotypic penicillin resistance decreased from 54.3% in 2010 to 11.2% in 2016 among children and from 32.4% to 15.5% among adults. However, genotypic penicillin resistance emerged in 9 nonvaccine serotypes, but not 15A and 35B. Multilocus sequence typing suggested that resistant strains among nonvaccine serotypes may have evolved from clonal complexes 156 and 81. A more broadly effective vaccine is needed.

Amino Acid Substitution in the Major Multidrug Efflux Transporter Protein AcrB Contributes to Low Susceptibility to Azithromycin in Haemophilus influenzae.

Clarithromycin-resistant Haemophilus influenzae strains with a nonsense mutation in acrR generally exhibited susceptibility to azithromycin, although one strain was found to be nonsusceptible; we aimed to clarify the differences. This strain had an amino acid substitution, Arg327Ser, in AcrB. Introduction of this substitution into H. influenzae Rd caused an increase in the MIC of azithromycin, suggesting that this substitution contributed to nonsusceptibility. These findings indicate that azithromycin-nonsusceptible isolates could occur through stepwise mutation in the acr region.

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.