Traces of pandemic fluoroquinolone-resistant Escherichia coli clone ST131 transmitted from human society to aquatic environments and wildlife in Japan.

Transmission of antimicrobial-resistant bacteria among humans, animals, and the environment is a growing concern worldwide. The distribution of an international high-risk fluoroquinolone-resistant Escherichia coli clone, ST131, has been documented in clinical settings. However, the transmission of ST131 from humans to surrounding environments remains poorly elucidated. To comprehend the current situation and identify the source of ST131 in nature, we analyzed the genetic features of ST131 isolates from the aquatic environment (lake/river water) and wildlife (fox, raccoon, raccoon dog, and deer) and compared them with the features of isolates from humans in Japan using accessory and core genome single nucleotide polymorphism (SNP) analyses. We identified ST131 isolates belonging to the same phylotype and genome clusters (four of eight clusters were concomitant) with low SNP distance between the human isolates and those from the aquatic environment and wildlife. These findings warn of ST131 transmission between humans and the surrounding environment in Japan.

Fourth national Japanese antimicrobial susceptibility pattern surveillance program: Bacterial isolates from patients with complicated urinary tract infections.

INTRODUCTION: Antimicrobial susceptibility patterns of bacterial pathogens isolated from patients with complicated urinary tract infections were analyzed using the national surveillance data, comprising 793 bacterial strains from eight clinically relevant species. MATERIALS AND METHODS: Data were collected for the fourth national surveillance project from July 2020 to December 2021 by the Japanese Society of Chemotherapy, the Japanese Association for Infectious Disease, and the Japanese Society of Clinical Microbiology. Surveillance was supervised with the cooperation of 43 medical institutions throughout Japan. RESULTS: Fluoroquinolone required a minimum inhibitory concentration (MIC) of 2-64 mg/L to inhibit the 330 tested Escherichia coli strains. The proportion of levofloxacin-resistant E. coli strains increased from 28.6% in 2008 to 29.6% in 2011, 38.5% in 2015, and 44.5% in 2021. The proportion of levofloxacin-resistant strains of Pseudomonas aeruginosa also increased from previous survey results, showing a continuing downward trend. Conversely, the proportion of levofloxacin-resistant strains of Enterococcus faecalis decreased relative to previous reports. Neither multidrug-resistant P. aeruginosa nor carbapenem-resistant Enterobacteriaceae were detected. For methicillin-resistant Staphylococcus aureus (MRSA), the proportion of vancomycin-susceptible strains (MIC of 2 µg/mL) decreased from 14.7% to 7.7%. DISCUSSION: Bacterial strains that produced extended-spectrum ß-lactamase included E. coli (82/330 strains, 24.8%), Klebsiella pneumoniae (11/68 strains, 16.2%), and Proteus mirabilis (4/26 strains, 15.4%). As compared to previous surveillance reports, these strains showed an increase in proportion over the years.

The performance of a Fully Automated Urine Particle Analyzer, Sysmex UF-5000, in detecting fastidious bacteria in urine samples.

Several types of fastidious bacteria can cause tract infections. We evaluated the performance of counting fastidious bacteria using a Fully Automated Urine Particle Analyzer UF-5000. The results showed that UF-5000 counts fastidious bacteria in urine without the need for culture using measurement principles based on flow cytometry.

Third nationwide surveillance of bacterial pathogens in patients with acute uncomplicated cystitis conducted by the Japanese surveillance committee during 2020 and 2021: Antimicrobial susceptibility of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus saprophyticus.

The Japanese surveillance committee conducted a third nationwide surveillance of antimicrobial susceptibility of acute uncomplicated cystitis at 55 facilities throughout Japan between April 2020 and September 2021. In this surveillance, we investigated the susceptibility of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Staphylococcus saprophyticus (S. saprophyticus) for various antimicrobial agents by isolating and culturing bacteria from urine samples. In total, 823 strains were isolated from 848 patients and 569 strains of target bacteria, including E. coli (n = 529, 92.9 %), K. pneumoniae (n = 28, 4.9 %), and S. saprophyticus (n = 12, 2.2 %) were isolated. The minimum inhibitory concentrations of 18 antibacterial agents were determined according to the Clinical and Laboratory Standards Institute manual. In premenopausal patients, there were 31 (10.5 %) and 20 (6.8 %) fluoroquinolone (FQ)-resistant E. coli and extended-spectrum ß-lactamase (ESBL)-producing E. coli, respectively. On the other hand, in postmenopausal patients, there were 75 (32.1 %) and 36 (15.4 %) FQ-resistant E. coli and ESBL-producing E. coli, respectively. The rate of FQ-resistant E. coli and ESBL-producing E. coli in post-menopausal women was higher than that for our previous nationwide surveillance (20.7 % and 32.1 %: p = 0.0004, 10.0 % and 15.4 %; p = 0.0259). For pre-menopausal women, there was no significant difference in the rate of FQ-resistant E. coli and ESBL-producing E. coli between this and previous reports, but the frequency of FQ-resistant E. coli and ESBL-producing E. coli exhibited a gradual increase. For appropriate antimicrobial agent selection and usage, it is essential for clinicians to be aware of the high rate of these antimicrobial-resistant bacteria in acute uncomplicated cystitis in Japan.

Emergence of ciprofloxacin- and penicillin-resistant Neisseria meningitidis isolates in Japan between 2003 and 2020 and its genetic features.

Although we previously reported that some meningococcal isolates in Japan were resistant to penicillin (PCG) and ciprofloxacin (CIP), the antibiotic susceptibilities of Neisseria meningitidis isolates obtained in Japan remained unclear. In the present study, 290 N. meningitidis isolates in Japan between 2003 and 2020 were examined for the sensitivities to eight antibiotics (azithromycin, ceftriaxone, ciprofloxacin, chloramphenicol, meropenem, minocycline, penicillin, and rifampicin). All isolates were susceptible to chloramphenicol, ceftriaxone, meropenem, minocycline, and rifampicin while two were resistant to azithromycin. Penicillin- and ciprofloxacin-resistant and -intermediate isolates (PCGR, CIPR, PCGI and CIPI, respectively) were also identified. Based on our previous findings from whole genome sequence analysis, approximately 40% of PCGI were associated with ST-11026 and cc2057 meningococci, both of which were unique to Japan. Moreover, the majority of ST-11026 meningococci were CIPR or CIPI. Sensitivities to PCG and CIP were closely associated with genetic features, which indicated that, at least for Japanese meningococcal isolates, PCGR/I or CIPI/R would be less likely to be horizontally conferred from other neisserial genomes by transferring of the genes responsible (penA and gyrA genes, respectively), but rather that ancestral N. meningitidis strains conferring PCGR/I or CIPI/R phenotypes clonally disseminated in Japan.

Detection of Novel US Neisseria meningitidis Urethritis Clade Subtypes in Japan.

Neisseria meningitidis causes invasive meningococcal diseases and has also been identified as a causative agent of sexually transmitted infections, including urethritis. Unencapsulated sequence type 11 meningococci containing the gonococcal aniA-norB locus and belonging to the United States N. meningitidis urethritis clade (US_NmUC) are causative agents of urethral infections in the United States, predominantly among men who have sex with men. We identified 2 subtypes of unencapsulated sequence type 11 meningococci in Japan that were phylogenetically close to US_NmUC, designated as the Japan N. meningitidis urethritis clade (J_NmUC). The subtypes were characterized by PCR, serologic testing, and whole-genome sequencing. Our study suggests that an ancestor of US_NmUC and J_NmUS urethritis-associated meningococci is disseminated worldwide. Global monitoring of urethritis-associated N. meningitidis isolates should be performed to further characterize microbiologic and epidemiologic characteristics of urethritis clade meningococci.

The third nationwide surveillance of antimicrobial susceptibility against Neisseria gonorrhoeae from male urethritis in Japan, 2016-2017.

Neisseria gonorrhoeae is one of the important pathogens of sexually transmitted infections. N. gonorrhoeae is rapidly becoming antimicrobial resistant, and there are few drugs that are effective in the initial treatment of gonorrhea. To understand the trends of antimicrobial susceptibility of N. gonorrhoeae, the Surveillance Committee of the Japanese Society of Infectious Diseases, the Japanese Society for Chemotherapy, and the Japanese Society of Clinical Microbiology conducted the third nationwide antimicrobial susceptibility surveillance of N. gonorrhoeae isolated from male urethritis. The specimens were collected from male patients with urethritis at 30 facilities from May 2016 to July 2017. From the 159 specimens collected, 87 N. gonorrhoeae strains were isolated, and 85 were tested for susceptibility to 21 antimicrobial agents. All strains were non-susceptible to penicillin G. Seven strains (8.2%) were ß-lactamase-producing strains. The rates of susceptibility to cefixime and cefpodoxime were 96.5% and 52.9%, respectively. Three strains were non-susceptible with a minimum inhibitory concentration (MIC) of 0.5 mg/L for cefixime. None of the strains were resistant to ceftriaxone or spectinomycin. The susceptibility rate for ciprofloxacin was 23.5% (20 strains), and no strains showed intermediate susceptibility. The susceptibility rate against azithromycin was 81.2%, with one strain isolated with a MIC of 8 mg/L against azithromycin. The results of this surveillance indicate that ceftriaxone and spectinomycin, which are currently recommended for gonococcal infections in Japan, appear to be effective. It will be necessary to further expand the scale of the next surveillance to understand the current status of drug-resistant N. gonorrhoeae in Japan.

Is it meaningful to apply midstream urine culture to urine specimens with negative Gram stain results?

INTRODUCTION: Gram staining is a convenient method for bacterial estimation. Urine culture is typically used to diagnose urinary tract infections. Therefore, urine culture is also performed on Gram stain-negative urine specimens. However, the frequency of uropathogen identification in these samples remains unclear. METHODS: From 2016 to 2019, we retrospectively compared the results of Gram staining and urine culture tests on midstream urine specimens submitted for the diagnosis of urinary tract infections to confirm the significance of urine culture on Gram stain-negative specimens. Analysis was performed according to the patients' sex and age, and the frequency of uropathogen identification in the culture was examined. RESULTS: A total of 1763 urine specimens (women, 931; men, 832) were collected. Of these, 448 (25.4%) were not positive on Gram staining but were positive on culture. In specimens without bacteria on Gram staining, the frequencies of specimens with uropathogens detected on culture were 20.8% (22/106) in women aged <50 years, 21.4% (71/332) in women aged ≥50 years, 2.0% (2/99) in men aged <50 years, and 7.8% (39/499) in men aged ≥50 years. CONCLUSIONS: In men aged <50 years, the frequency of uropathogenic bacteria identification by urine culture was low in Gram stain-negative specimens. Therefore, urine cultures may be excluded from this group. In contrast, in women, a small number of Gram stain-negative specimens showed significant culture results for the diagnosis of urinary tract infection. Therefore, urine culture should not be omitted in women without careful consideration.

Prediction of presence of fastidious bacteria by the Fully Automated Urine Particle Analyzer UF-1000i in the case of ineffective antimicrobial therapy for urinary tract infection.

INTRODUCTION: Recent studies have reported associations between fastidious bacteria that are difficult to grow and isolate in conventional urine culture conditions and urinary tract infections (UTIs). Because the Fully Automated Urine Particle Analyzer UF-1000i (hereinafter referred to as "UF-1000i") detects fastidious bacteria without being affected by culture conditions, owing to its flow cytometry-based principle, we evaluated the robustness of UF-1000i detection using clinical urine samples from patients with UTIs following ineffective antimicrobial therapy. METHODS: A total of 150 patients diagnosed with UTIs were enrolled, and their laboratory findings were analyzed, focusing on the discrepancy in bacterial numbers between UF-1000i and conventional culture at each antimicrobial therapy effectiveness classification. In addition, gene identification was conducted by molecular analysis using 16S ribosomal RNA gene sequencing and next-generation sequencing (NGS) to elucidate the reason for the presence of fastidious bacteria in these samples. RESULTS: The ineffective therapy cases showed more than 100-fold discrepancy in bacterial counts, with a higher proportion (30.8%) than effective therapy cases without secondary administration (5.7%) between the bacterial counts in UF-1000i and conventional culture methods. The presence rates of fastidious bacteria were 100% and 66.7% in discrepant cases of ineffective and effective without secondary administrations, respectively. CONCLUSION: This study suggests that discrepancies in bacterial numbers between the conventional culture method and UF-1000i measurement at the primary visit can predict the presence of fastidious bacteria, especially in cases of ineffective antimicrobial therapy.

Nationwide surveillance of the antimicrobial susceptibility of Chlamydia trachomatis from male urethritis in Japan: Comparison with the first surveillance report.

The Urogenital Sub-committee and the Surveillance Committee of the Japanese Society of Chemotherapy, The Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology conducted the second nationwide surveillance of the antimicrobial susceptibility of Chlamydia trachomatis. In this second surveillance study, clinical urethral discharge specimens were collected from patients with urethritis in 26 hospitals and clinics from May 2016 to July 2017. Based on serial cultures, the minimum inhibitory concentration (MIC) could be determined for 41 isolates; the MICs (MIC90) of ciprofloxacin, levofloxacin, tosufloxacin, sitafloxacin, doxycycline, minocycline, erythromycin, clarithromycin, azithromycin and solithromycin were 2 µg/ml (2 µg/ml), 1 µg/ml (0.5 µg/ml), 0.25 µg/ml (0.25 µg/ml), 0.125 µg/ml (0.063 µg/ml), 0.125 µg/ml (0.125 µg/ml), 0.25 µg/ml (0.25 µg/ml), 0.031 µg/ml (0.031 µg/ml), 0.25 µg/ml (0.125 µg/ml), and 0.016 µg/ml (0.008 µg/ml), respectively. In summary, this surveillance project did not identify any strains resistant to fluoroquinolone, tetracycline, or macrolide agents in Japan. In addition, the MIC of solithromycin was favorable and lower than that of other antimicrobial agents. However, the MIC of azithromycin had a slightly higher value than that reported in the first surveillance report, though this might be within the acceptable margin of error. Therefore, the susceptibility of azithromycin, especially, should be monitored henceforth.

AAUS guideline for chlamydial urethritis.

Urogenital chlamydial infection is the most common sexually transmitted infection. Many cases of chlamydial infection are reported worldwide every year. Genital chlamydial infection in women can also cause obstetric issues, including infertility and miscarriage. For that purpose, appropriate care should be conducted with the latest knowledge. Only few guidelines come from Asian countries. The Asian Association of Urinary Tract Infection and Sexually Transmitted Infection (AAUS) belonging to the Urological Association of Asia (UAA) had developed the guidelines regarding chlamydial urethritis. We have collected the feedback and updated the guidelines which is now submitted for consideration of publication. In addition to the levels of evidence, the recommendation grades were defined using the modified GRADE methodology. Herein, we present the new edition of the UAA-AAUS guidelines for chlamydial urethritis.

Guidelines for Infection Control in the Urological Field, including Urinary Tract Management (revised second edition).

The Committee for the Development of Guidelines for Infection Control in the Urological Field, including Urinary Tract Management of the Japanese Urological Association, together with its systematic review team and external reviewers, have prepared a set of practice guidelines, an abridged version of which is published herein. These guidelines cover the following topics: (i) foundations of infection control, standard precautions, route-specific precautions, and occupational infection control (including vaccines); (ii) the relationship between urologists and infection control; (iii) infection control in urological wards and outpatient clinics; (iv) response to hepatitis B virus reactivation; (v) infection control in urological procedures and examinations; (vi) prevention of infections occurring in conjunction with medical procedures and examinations; (vii) responses to urinary tract tuberculosis and bacillus Calmette-Guérin; (viii) aseptic handling, cleaning, disinfection, and sterilization of urinary tract endoscopes (principles of endoscope manipulation, endoscope lumen cleaning, and disinfection); (ix) infection control in the operating room (principles of hand washing, preoperative rubbing methods, etc.); (x) prevention of needlestick and blood/bodily fluid exposure and response to accidental exposure; (xi) urinary catheter-associated urinary tract infection and purple urinary bag syndrome; and (xii) urinary catheter-associated urinary tract infections in conjunction with home care. In addressing these topics, the relevant medical literature was searched to the extent possible, and content was prepared for the purpose of providing useful information for clinical practice.

Nationwide surveillance of bacterial pathogens isolated from patients with acute uncomplicated cystitis in 2018: Conducted by the Japanese Research Group for Urinary Tract Infections (JRGU).

INTRODUCTION: The aim of this study was to monitor the development of drug-resistant bacteria isolated from acute uncomplicated cystitis (AUC) and to evaluate methodology of the survey conducted by collecting only clinical data. METHODS: We enrolled female patients at least 16 years of age diagnosed with AUC in 2018. Patient information including age, menopausal status, and results of bacteriological examination were collected and analyzed regardless of bacterial identification, antimicrobial susceptibility testing or extended-spectrum ß-lactamase (ESBL) detection method. RESULTS: A total of 847 eligible cases were collected. Escherichia coli (E. coli) was the most frequently isolated bacterial species at about 70%, with proportions of fluoroquinolone-resistant E. coli (QREC) and ESBL-producing E. coli isolates at 15.6% and 9.5% of all E. coli isolates, respectively. The proportion of Staphylococcus saprophyticus (S. saprophyticus) was significantly higher in premenopausal women. Regarding the drug susceptibility of E. coli, isolates from Eastern Japan had significantly higher susceptibility to cefazolin, cefotiam and cefpodoxime and lower susceptibility to levofloxacin in postmenopausal women. ESBL-producing E. coli isolates had a high susceptibility to tazobactam-piperacillin, cefmetazole, carbapenems, aminoglycosides, and fosfomycin. In S. saprophyticus, the susceptibility to ß-lactams including carbapenems was 40-60%. CONCLUSIONS: The proportions of QREC and ESBL-producing E. coli were increasing trends and lower susceptibility to LVFX in postmenopausal women was observed. Such surveillance, consisting of the collecting only clinical data, could be conducted easily and inexpensively. It is expected to be continuously performed as an alternative survey to conventional one collecting bacterial strains.

Emergence and evolution of antimicrobial resistance genes and mutations in Neisseria gonorrhoeae.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to third-generation cephalosporins, mainly due to mosaic penA alleles. These two STs were first detected in Japan; however, the timeline, mechanism, and process of emergence and spread of these mosaic penA alleles to other countries remain unknown. METHODS: We studied the evolution of penA alleles by obtaining the complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1075 strains sampled from 35 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996 to 2005 and reconstructed a phylogeny including 88 previously sequenced genomes. RESULTS: Based on an estimate of the time-of-emergence of ST-1901 (harboring mosaic penA-34) and ST-7363 (harboring mosaic penA-10), and > 300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996-2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by recombination with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, by independent mutations rather than horizontal gene transfer. Data in the literature suggest that the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. CONCLUSIONS: Our findings highlight how antibiotic use and recombination across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.

Deuterium-labeled Raman tracking of glucose accumulation and protein metabolic dynamics in Aspergillus nidulans hyphal tips.

Filamentous fungi grow exclusively at their tips, where many growth-related fungal processes, such as enzyme secretion and invasion into host cells, take place. Hyphal tips are also a site of active metabolism. Understanding metabolic dynamics within the tip region is therefore important for biotechnology and medicine as well as for microbiology and ecology. However, methods that can track metabolic dynamics with sufficient spatial resolution and in a nondestructive manner are highly limited. Here we present time-lapse Raman imaging using a deuterium (D) tracer to study spatiotemporally varying metabolic activity within the hyphal tip of Aspergillus nidulans. By analyzing the carbon-deuterium (C-D) stretching Raman band with spectral deconvolution, we visualize glucose accumulation along the inner edge of the hyphal tip and synthesis of new proteins from the taken-up D-labeled glucose specifically at the central part of the apical region. Our results show that deuterium-labeled Raman imaging offers a broadly applicable platform for the study of metabolic dynamics in filamentous fungi and other relevant microorganisms in vivo.

Characterization of 2 Neisseria gonorrhoeae Strains With High-Level Azithromycin Resistance Isolated in 2015 and 2018 in Japan.

ABSTRACT: We identified and characterized the first 2 Neisseria gonorrhoeae strains with high-level azithromycin resistance isolated in Japan. These were in the clade of ceftriaxone- and azithromycin-resistant strains isolated in Australia and the United Kingdom. The multilocus sequence typing, N. gonorrhoeae multiantigen sequence typing, and N. gonorrhoeae sequence typing for antimicrobial resistance types of these strains were found in gonococci from eastern Asia.

Iodine staining as a useful probe for distinguishing insulin amyloid polymorphs.

It is recently suggested that amyloid polymorphism, i.e., structural diversity of amyloid fibrils, has a deep relationship with pathology. However, its prompt recognition is almost halted due to insufficiency of analytical methods for detecting polymorphism of amyloid fibrils sensitively and quickly. Here, we propose that iodine staining, a historically known reaction that was firstly found by Virchow, can be used as a method for distinguishing amyloid polymorphs. When insulin fibrils were prepared and iodine-stained, they exhibited different colors depending on polymorphs. Each of the colors was inherited to daughter fibrils by seeding reactions. The colors were fundamentally represented as a sum of three absorption bands in visible region between 400 and 750 nm, and the bands showed different titration curves against iodine, suggesting that there are three specific iodine binding sites. The analysis of resonance Raman spectra and polarization microscope suggested that several polyiodide ions composed of I3- and/or I5- were formed on the grooves or the edges of ß-sheets. It was concluded that the polyiodide species and conformations formed are sensitive to surface structure of amyloid fibrils, and the resultant differences in color will be useful for detecting polymorphism in a wide range of diagnostic samples.

Human Adenovirus B7d-Associated Urethritis after Suspected Sexual Transmission, Japan.

Outbreaks of acute respiratory disease associated with human adenovirus (HAdV) B7d have been reported, including fatal cases in the United States. In 2018, we detected HAdV-B7d in a patient with urethritis, probably transmitted through sexual contact. Infectious HAdV-B7d was excreted in urine and gargle for >10 days after the disappearance of symptoms.

Theoretical Modeling of Electronic Structures of Polyiodide Species Included in α-Cyclodextrin.

The molecular mechanism of blue color formation in an iodine-starch reaction is studied by employing the iodine-α-cyclodextrin (α-CD) complex as a practical model system that resembles the structural properties of the blue amylose-iodine complex. To this end, we construct, using the quantum chemistry method, a molecular model of the complex (I5-/Li+/2α-CD) that consists of one I5-, two molecules of α-CD, and a lithium cation, and this model is employed as a basic unit in constructing the structural models of polyiodide ions (I5-)n. The initial structure in the geometry optimization is adopted from the α-CD-iodine complex structure obtained from the X-ray crystallography study. The structural models of (I5-)n are built by adding the basic unit n times along the crystal axis and by optimizing the structure using quantum mechanics/molecular mechanics (QM (iodine)/MM (α-CD)) calculations. The electronic absorption spectra of the resulting model structures are calculated by time-dependent density functional theory (TD-DFT). We find that I5- acts as a basic unit of coloration in the visible region. The visible color originates from the electronic transition within the I5- molecule, and any charge transfer between the I5- ion and either of α-CD or a coexisting counter cation is not involved. We also reveal that the electronic transitions of (I5-)n are delocalized, which accounts for the well-known observation that the color of the iodine-starch reaction becomes bluish with an increase in the chain length of amylose. Furthermore, the preresonance Raman spectra calculated from the model suggest that the vibrational motions are localized in the I5- subunit dominantly. A comparison between an experimental absorption spectrum feature of the α-CD-iodine complex and the calculated ones of (I5-)n ions with various n values suggests that (I5-)4 polyiodide ions tend to be populated dominantly in the α-CD-iodine complex under aqueous conditions.