[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.

ß-Lactamase-non-producing ampicillin-resistant Haemophilus influenzae is acquiring multidrug resistance.

BACKGROUND: Haemophilus influenzae strains with reduced susceptibilities to antimicrobial agents have emerged in Japan. Here, we aimed to investigate H. influenzae non-susceptibility to ß-lactams and non-ß-lactams. METHODS: A total of 260 H. influenzae isolates from patients in 2013-2016 were analysed. Antimicrobial susceptibilities were assessed by determining the minimum inhibitory concentration. Additionally, isolates with reduced susceptibility were analysed by both genetic and statistical methods. RESULTS: ß-Lactamase-non-producing ampicillin-resistant H. influenzae (BLNAR) strains increased significantly and accounted for more than 50% of all isolates from 2014. Additionally, the proportion of quinolone-low-susceptibility isolates increased significantly (P<0.05). Among these, three quinolone-non-susceptible isolates showed minimum inhibitory concentrations higher than the susceptibility breakpoint of levofloxacin. Moreover, one of the three isolates showing multidrug resistance was resistant to macrolides, ß-lactams, and quinolones. Low susceptibilities to non-ß-lactams were significantly associated with BLNAR. CONCLUSIONS: The present study indicates that BLNAR strains are increasing and tend to show multidrug resistance. Additionally, multidrug-resistant H. influenzae (MDRHI) has emerged. To prevent the further spread of MDRHI, the proportions of BLNAR strains should be evaluated.

Emergence of Haemophilus influenzae with low susceptibility to quinolones and persistence in tosufloxacin treatment.

BACKGROUND: The use of non-ß-lactam agents has increased in Japan due to the prevalence of ß-lactam-resistant pathogens. This study aimed to clarify the recent trend of antimicrobial susceptibility and molecular epidemiological features in Haemophilus influenzae. METHODS: Fifty-seven Haemophilus influenzae isolated from a Japanese teaching hospital in 2017 were characterised, and the data were compared with those of a previous study. The MICs were determined using the broth dilution method. Genetic backgrounds were compared by multilocus sequence typing. The bactericidal activity of tosufloxacin at, or near, the therapeutic Cmax was determined in vitro, with susceptible isolates and quinolone low-susceptible isolates by time-kill assay. RESULTS: The results of the susceptibility tests showed that >90% of isolates were susceptible to cephalosporins and carbapenems, whereas ampicillin-susceptible and clarithromycin-susceptible isolates decreased. Regarding quinolones, low-susceptible isolates were noted in 2017, although all isolates were judged as susceptible. All low-susceptible isolates had an amino acid substitution in GyrA, and two isolates had an additional substitution in ParC. These isolates had different genetic backgrounds. Furthermore, the time-kill kinetic assay using the Cmax of tosufloxacin indicated that the low-susceptible isolates could persist for at least 8hours. CONCLUSIONS: This study revealed that Haemophilus influenzae has demonstrated multidrug low-susceptibility in recent years. The low-susceptible isolates had genetic diversity, meaning that resistance occurred independently.

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.

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.

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.

Rise in Haemophilus influenzae With Reduced Quinolone Susceptibility and Development of a Simple Screening Method.

BACKGROUND: ß-Lactamase-nonproducing ampicillin-resistant Haemophilus influenzae are prevalent in Japan. Resistance has increased as a consequence of the expanded use of antimicrobial agents, raising concerns about the rise of multidrug (macrolide and fluoroquinolone)-resistant H. influenzae. METHODS: In this study, we investigated susceptibility to fluoroquinolones in H. influenzae clinical isolates from 2013 to 2014 and identified the amino acid substitutions in quinolone resistance-determining regions of gyrA and parC. RESULTS: All isolates (n = 145) were susceptible to fluoroquinolones; however, some showed reduced susceptibility. The minimum inhibitory concentration of levofloxacin for these strains was 0.063-0.5 µg/mL, and the strains harbored the amino acid substitution S84L in GyrA. Such strains have seen a significant increase. Importantly, all mutants from 2014 were isolated from pediatric patients. In addition, we developed a simple polymerase chain reaction-based screening method for detecting isolates with reduced fluoroquinolone susceptibility. CONCLUSIONS: The mutation in GyrA is important as a first step in the development of fluoroquinolone resistance. Hence, detection of reduced susceptible strains may influence the choice of antimicrobial treatment.

Prevalence of macrolide-non-susceptible isolates among ß-lactamase-negative ampicillin-resistant Haemophilus influenzae in a tertiary care hospital in Japan.

ß-Lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae account for a large portion of H. influenzae clinical isolates in Japan. The aim of this study was to clarify the antimicrobial susceptibility of BLNAR H. influenzae clinical isolates as well as the annual changes in susceptibility. BLNAR H. influenzae isolates were collected from a tertiary care hospital from 2007 to 2012. Antimicrobial susceptibility testing was performed and resistance mechanisms were analysed. All of the isolates (n=304) had amino acid substitutions in penicillin-binding protein 3 (PBP3) and isolates were classified by these amino acid substitutions: R517H or N526K (class I); S385T and R517H (class II); and S385T and N526K (class III). Classes I, II and III represented 8.2% (n=25), 9.5% (n=29) and 81.6% (n=248) of the isolates, respectively; 2 isolates could not be classified because they had a PBP3 with a substantially mutated FtsI transpeptidase domain. All of the isolates were highly susceptible to fluoroquinolones and carbapenems. The number of clarithromycin (CAM)-non-susceptible [minimum inhibitory concentration (MIC) ≥16µg/mL] H. influenzae isolates increased significantly between 2010 and 2012. Moreover, CAM-non-susceptible H. influenzae isolates were prevalent among class II and class III BLNAR H. influenzae. Multilocus sequence typing (MLST) of the CAM-resistant (MIC ≥32µg/mL) H. influenzae isolates showed that they were not specific sequence types, suggesting that CAM resistance may occur in any isolates. These results raise concern regarding the occurrence of multidrug-resistant BLNAR H. influenzae.

Clarithromycin Resistance Mechanisms of Epidemic ß-Lactamase-Nonproducing Ampicillin-Resistant Haemophilus influenzae Strains in Japan.

The aim of this study was to clarify the clarithromycin resistance mechanisms of ß-lactamase-nonproducing ampicillin-resistant Haemophilus influenzae strains. In all clarithromycin-resistant strains, the transcript level of acrB was significantly elevated, and these strains had a frameshift mutation in acrR Introduction of the acrR mutation into H. influenzae Rd generated a clarithromycin-resistant transformant with the same MIC as the donor strain. Our results indicate that the acrR mutation confers clarithromycin resistance by the increasing the transcription of acrB.