Clinical outcomes of COVID-19 caused by the Alpha variant compared with one by wild type in Kobe, Japan. A multi-center nested case-control study.

OBJECTIVES: The emergence of the Alpha variant of novel coronavirus 2019 (SARS-CoV-2) is a concerning issue but their clinical implications have not been investigated fully. METHODS: We conducted a nested case-control study to compare severity and mortality caused by the Alpha variant (B.1.1.7) with the one caused by the wild type as a control from December 2020 to March 2021, using whole-genome sequencing. 28-day mortality and other clinically important outcomes were evaluated. RESULTS: Infections caused by the Alpha variant were associated with an increase in the use of oxygen (43.4% vs 26.3%. p = 0.017), high flow nasal cannula (21.2% vs 4.0%, p = 0.0007), mechanical ventilation (16.2% vs 6.1%, p = 0.049), ICU care (30.3% vs 14.1%, p = 0.01) and the length of hospital stay (17 vs 10 days, p = 0.031). More patients with the Alpha variant received medications such as dexamethasone. However, the duration of each modality did not differ between the 2 groups. Likewise, there was no difference in 28-day mortality between the 2 groups (12% vs 8%, p = 0.48), even after multiple sensitivity analyses, including propensity score analysis. CONCLUSION: The Alpha variant was associated with a severe form of COVID-19, compared with the non-Alpha wild type, but might not be associated with higher mortality.

Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli OX18 from a Fatal Hemolytic Uremic Syndrome Case.

We report a fatal case of hemolytic uremic syndrome with urinary tract infection in Japan caused by Shiga toxin-producing Escherichia coli. We genotypically identified the isolate as OX18:H2. Whole-genome sequencing revealed 3 potentially pathogenic lineages (OX18:H2, H19, and H34) that have been continuously isolated in Japan.

Two New Cases of Pulmonary Infection by Mycobacterium shigaense, Japan.

We report 2 case-patients in Japan with Mycobacterium shigaense pulmonary infections. One patient was given aggressive treatment and the other conservative treatment, according to distinctive radiologic evidence. A close phylogenetic relationship based on whole-genome sequencing was found between strain from the conservatively treated patient and a reference strain of cutaneous origin.

Acquisition of antimicrobial-resistant variants in repeated infections caused by Pseudomonas aeruginosa revealed by whole genome sequencing.

Pseudomonas aeruginosa, responsible for serious nosocomial-acquired infections, possesses intrinsic antibiotic resistance mechanisms and commonly exhibits multidrug resistance. Here, we report the evolving resistance profiles of strains isolated from the sputum of a patient being treated for repeated P. aeruginosa infections following cancer resection. Whole genome sequencing of six isolates obtained over a 2-month period revealed two key single nucleotide polymorphisms in the mexR and gyrB genes that affected efflux pump expression and antimicrobial resistance.

Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF.

The terminal step of 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis is performed by NspF, a mono-oxygenase that converts o-aminophenols to the corresponding nitroso product (hydroxyanilinase activity). Previous biochemical characterization of the resting form of NspF suggested that this enzyme belonged to the coupled binuclear copper enzyme (CBC) family. Another member of this enzyme family, tyrosinase, is able to mono-oxygenate monophenols (monophenolase activity) but not o-aminophenols. To gain insight into the unique reactivity of NspF, we have generated and characterized the oxy form of its active site. The observation of spectral features identical to those of oxy-tyrosinase indicates that oxy-NspF contains a Cu(2)O(2) core where peroxide is coordinated in a µ-η(2):η(2) mode, confirming that NspF is a CBC enzyme. This oxy form is found to react with monophenols, indicating that, like tyrosinase, NspF also possesses monophenolase activity. A comparison of the two electrophilic mechanisms for the monophenolase and hydroxyanilinase activity indicates a large geometric change between their respective transition states. The potential for specific interactions between the protein pocket and the substrate in each transition state is discussed within the context of the differential reactivity of this family of enzymes with equivalent µ-η(2):η(2) peroxy bridged coupled binuclear copper active sites.

Development of a multilocus sequence typing scheme for Streptococcus gallolyticus.

Streptococcus gallolyticus is often found as a member of the normal gut microflora in various animals. However, it has been reported to cause mastitis in cattle, septicaemia in pigeons, and meningitis, septicaemia and endocarditis in humans. However, little is known about the epidemiology and crucial virulence factors of S. gallolyticus. To help address these issues, we developed a multilocus sequence typing (MLST) scheme for S. gallolyticus. Seven housekeeping gene fragments were sequenced from each of 58 S. gallolyticus isolates collected from diverse origins and sources. The MLST scheme had good discriminatory ability. The 63 strains, including the 5 whole genome sequenced strains examined, resolved into 57 sequence types (STs), with 52 STs represented by only a single strain. With respect to the identification of S. gallolyticus subspecies (i.e. S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus and S. gallolyticus subsp. macedonicus), the results of biochemical tests and DNA-DNA hybridization were in high concordance with those of the MLST scheme. The MLST scheme developed in this study may be a useful tool capable of replacing the conventional methods used for S. gallolyticus subspecies identification. The results of this study suggest that the biology and virulence of two pathogenic S. gallolyticus subspecies (i.e. S. gallolyticus subsp. gallolyticus and S. gallolyticus subsp. pasteurianus) are very different. The MLST scheme offers researchers a valuable typing tool that will promote further investigation of the epidemiology of S. gallolyticus.

Comparison of three tannases cloned from closely related lactobacillus species: L. Plantarum, L. Paraplantarum, and L. Pentosus.

BACKGROUND: Tannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum. RESULTS: The genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917(T), respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The K(m) values of the enzymes on each galloyl ester were comparable; however, the k(cat)/K(m) values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity. CONCLUSION: Two tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases.

Metagenome and Resistome Analysis of Beta-Lactam-Resistant Bacteria Isolated from River Waters in Surabaya, Indonesia.

Antimicrobial agents are administered to humans and livestock, and bacterial antimicrobial resistance (AMR) and antimicrobial agents are released into the environment. In this study, to investigate the trend of AMR in humans, livestock, and the environment, we performed a metagenomic analysis of multidrug-resistant bacteria with CHROMagar ESBL in environmental river water samples, which were collected using syringe filter units from waters near hospitals, downtown areas, residential areas, and water treatment plants in Surabaya, Indonesia. Our results showed that Acinetobacter, Pseudomonas, Aeromonas, Enterobacter, Escherichia, and Klebsiella grew in CHROMagar ESBL; they were most frequently detected in water samples from rivers surrounding hospitals contaminated with various AMR genes (ARGs) in high levels. These results identified bacteria as ARG reservoirs and revealed that hospitals could be sources for various ARGs disseminated into the environment. In conclusion, this study details a novel metagenomic analysis of collected bacteria in environmental water samples using a syringe filter unit for an AMR epidemiological study based on the One Health approach.

Isolation and identification of hyaluronan-degrading bacteria from Japanese fecal microbiota.

Hyaluronan (HA) is a high-molecular-weight glycosaminoglycan and widely distributed in all connective tissues and organs with diverse biological functions. HA has been increasingly used as dietary supplements targeted to joint and skin health for humans. We here first report isolation of bacteria from human feces that are capable of degrading HA to lower molecular weight HA oligosaccharides (oligo-HAs). The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted feces of healthy Japanese donors were individually incubated in an enrichment medium containing HA, followed by the isolation of candidate strains from streaked HA-containing agar plates and selection of HA-degrading strains by measuring HA using an ELISA. Subsequent genomic and biochemical assays identified the strains as Bacteroides finegoldii, B. caccae, B. thetaiotaomicron, and Fusobacterium mortiferum. Furthermore, our HPLC analysis revealed that the strains degraded HA to oligo-HAs of various lengths. Subsequent quantitative PCR assay targeting the HA degrading bacteria showed that their distribution in the Japanese donors varied. The evidence suggests that dietary HA is degraded by the human gut microbiota with individual variation to oligo-HAs components, which are more absorbable than HA, thereby exerting its beneficial effects.

Purification, crystallization and preliminary X-ray analysis of SGR6054, a Streptomyces homologue of the mycobacterial integration host factor mIHF.

The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b = 69.35, c = 77.71 Å, ß = 96.63°, and diffracted X-rays to 2.22 Å resolution.

Complete Genome Sequences of Four Streptococcus parasuis Strains Obtained from Saliva of Domestic Pigs in Japan.

Streptococcus parasuis is a close relative of Streptococcus suis, an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of four strains, including the type strain of S. parasuis, isolated from the saliva of healthy pigs in Japan.

Complete Genome Sequences of Three Streptococcus ruminantium Strains Obtained from Endocarditis Lesions of Cattle in Japan.

Streptococcus ruminantium is a close relative of Streptococcus suis, an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of three S. ruminantium strains isolated from bovine endocarditis in Japan.

Investigation of a Legionella pneumophila Outbreak at a Bath Facility in Japan Using Whole-Genome Sequencing of Isolates from Clinical and Environmental Samples.

Exposure to aerosols containing Legionella from artificially made water systems has been established as a primary cause of Legionnaires' disease. In this study, we investigated an outbreak of L. pneumophila serogroup 1 sequence type 138 which occurred at a bath facility in 2022. The whole-genome sequencing of isolates revealed that the colonization of L. pneumophila at the bath facility had occurred before 2013, and the patients had been exposed to multiple genetic lineages of the strain. Our study demonstrates the importance of performing a careful comparative genetic analysis of clinical and environmental isolates from LD outbreaks in order to effectively investigate and prevent future LD outbreaks.

SARS-CoV-2 RNA in Wastewater Was Highly Correlated With the Number of COVID-19 Cases During the Fourth and Fifth Pandemic Wave in Kobe City, Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the current coronavirus disease 2019 (COVID-19) pandemic and associated respiratory infections, has been detected in the feces of patients. Therefore, determining SARS-CoV-2 RNA levels in sewage may help to predict the number of infected people within the area. In this study, we quantified SARS-CoV-2 RNA copy number using reverse transcription quantitative real-time PCR with primers and probes targeting the N gene, which allows the detection of both wild-type and variant strain of SARS-CoV-2 in sewage samples from two wastewater treatment plants (WWTPs) in Kobe City, Japan, during the fourth and fifth pandemic waves of COVID-19 between February 2021 and October 2021. The wastewater samples were concentrated via centrifugation, yielding a pelleted solid fraction and a supernatant, which was subjected to polyethylene glycol (PEG) precipitation. The SARS-CoV-2 RNA was significantly and frequently detected in the solid fraction than in the PEG-precipitated fraction. In addition, the copy number in the solid fraction was highly correlated with the number of COVID-19 cases in the WWTP basin (WWTP-A: r = 0.8205, p < 0.001; WWTP-B: r = 0.8482, p < 0.001). The limit of capturing COVID-19 cases per 100,000 people was 0.75 cases in WWTP-A and 1.20 cases in WWTP-B, respectively. Quantitative studies of RNA in sewage can be useful for administrative purposes related to public health, including issuing warnings and implementing preventive measures within sewage basins.

Quantitative detection and genetic characterization of thermotolerant Campylobacter spp. in fresh chicken meats at retail in Japan.

Campylobacter jejuni and C. coli are one of the leading causes of gastrointestinal illnesses, and which are considered to be transmitted to humans mainly from chicken meats. Considering the less availability of quantitative contamination data in the retail chicken meats in Japan, 510 fresh chicken meats retailed at five distinct regions in Japan between June 2019 and March 2021 were examined. The quantitative testing resulted that 45.7% of the samples (254/510) were positive at mean ± standard deviation of 1.15 ± 1.03 logCFU/g, whereas 43 samples (8.4%) exceeded 3.0 logCFU/g. Seasonal comparison revealed increased bacterial counts in fall compared with spring and summer. As for the chicken slaughter age, those slaughtered at >75 days old were less contaminated than those at <75 days old. Genome sequencing analyses of 111 representative C. jejuni isolates resulted in the detection of three antimicrobial resistance genes (gyrA substitution T86I, tetO and blaOXA-61) at 25.2, 27.9 and 42.3%, respectively. In silico MLST analysis revealed the predominance of sequence types (ST)-21 clonal complex (CC), followed by ST-45CC and ST-464CC. The single nucleotide polymorphism (SNP)-based phylogenetic tree largely classified the sequenced C. jejuni isolates into two clusters (I and II), where all C. jejuni from highly contaminated samples (STs-21CC, -22CC and -45CC) belonged to cluster I, independent of both season and slaughter age. To our knowledge, this is the first example to study the current status of Campylobacter contamination levels in fresh chicken meats retailed in Japan. Our data would be contributable to future quantitative microbial risk assessment, to establish effective control measures for campylobacteriosis.

Genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae collected in Hyogo, Japan during 2015-2019.

Introduction. Azithromycin (AZM) is a therapeutic drug for sexually transmitted infections and is used for Neisseria gonorrhoeae when first- and second-line drugs are not available. Recently, the susceptibility of N. gonorrhoeae against AZM has been decreasing worldwide.Hypothesis/Gap Statement. Azithromycin-resistance (AZM-R) rates among N. gonorrhoeae in Japan are increasing, and the gene mutations and epidemiological characteristics of AZM-R in N. gonorrhoeae have not been fully investigated.Aim. We determined the susceptibility to AZM and its correlation with genetic characteristics of N. gonorrhoeae.Methodology. We investigated the susceptibility to AZM and genetic characteristics of N. gonorrhoeae. Mutations in domain V of the 23S rRNA gene and mtrR were examined in 93 isolates, including 13 AZM-R isolates. Spread and clonality were examined using sequence types (STs) of multi-antigen sequence typing for N. gonorrhoeae (NG-MAST), and whole genome analysis (WGA) to identify single nucleotide polymorphisms.Results. The number of AZM-R isolates increased gradually from 2015 to 2019 in Hyogo (P=0.008). C2599T mutations in 23S rRNA significantly increased in AZM-R isolates (P<0.001). NG-MAST ST4207 and ST6762 were frequently detected in AZM-R isolates, and they had higher MICs to AZM from 6 to 24 µg/ml. The phylogenic tree-based WGA showed that all isolates with ST4207 were contained in the same clade, and isolates with ST6762 were divided into two clades, AZM-S isolates and AZM-R isolates, which were different from the cluster containing ST1407.Conclusion. Our study showed yearly increases in AZM-R rates in N. gonorrhoeae. NG-MAST ST4207 and ST6762 were not detected in our previous study in 2015 and were frequently identified in isolates with higher MICs to AZM. WGA confirmed that isolates with these STs are closely related to each other. Continued surveillance is needed to detect the emergence and confirm the spread of NG-MAST ST4207 and ST6762.

Occurrence of Carriage of Multidrug Resistant Enterobacteriaceae among Pregnant Women in the Primary Health Center and Hospital Setting in Surabaya, Indonesia.

Objectives: The incidence of healthy individuals carrying multidrug resistant Enterobacteriaceae, including extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBL-E), especially extended-spectrum ß-lactamase producing Escherichia coli (ESBL-EC) and extended-spectrum ß-lactamase producing Klebsiella pneumoniae (ESBL-KP), is increasing worldwide. Although ESBL-E causes early or late onset of neonatal sepsis, the prevalence of ESBL-E carriage among pregnant women in Indonesia is not clear. In the present study, we compared the occurrence of carriage of ESBL-E among pregnant women in a primary health center (PHC) versus two hospitals. Materials and Methods: We collected rectal swab samples from 200 pregnant women who visited a PHC or were admitted to two hospitals in Surabaya, Indonesia from July to October 2018. The ESBL-E strains were isolated from the samples and phenotypically and genotypically analyzed. Results: ESBL-E strains were isolated from 25 (24.8%) pregnant women who visited the PHC and 49 (49.5%) pregnant women who were admitted to the hospitals. The rate of ESBL-E carriage of pregnant women in the hospitals was significantly higher than that in the PHC. Among the 74 isolated ESBL-E strains, ESBL-EC was most frequently isolated (62 strains), followed by ESBL-KP (12 strains). In addition, blaCTX-M-15 was the most frequent ESBL gene type of the isolated ESBL-E strains. Conclusions: Our results revealed the high occurrence of ESBL-E carriage in pregnant women, especially those who were admitted to the hospitals.

Antibiotic Resistance in Non-Typhoidal Salmonella enterica Strains Isolated from Chicken Meat in Indonesia.

The increase in antibiotic resistance in non-typhoidal Salmonella enterica (NTS) has been confirmed in Indonesia by this study. We confirmed the virulence genes and antimicrobial susceptibilities of clinical NTS (n = 50) isolated from chicken meat in Indonesia and also detected antimicrobial resistance genes. Of 50 strains, 30 (60%) were non-susceptible to nalidixic acid (NA) and all of them had amino acid mutations in gyrA. Among 27 tetracycline (TC) non-susceptible strains, 22 (81.5%) had tetA and/or tetB. The non-susceptibility rates to ampicillin, gentamicin or kanamycin were lower than that of NA or TC, but the prevalence of blaTEM or aadA was high. Non-susceptible strains showed a high prevalence of virulence genes compared with the susceptible strains (tcfA, p = 0.014; cdtB, p < 0.001; sfbA, p < 0.001; fimA, p = 0.002). S. Schwarzengrund was the most prevalent serotype (23 strains, 46%) and the most frequently detected as multi-antimicrobial resistant. The prevalence of virulence genes in S. Schwarzengrund was significantly higher than other serotypes in hlyE (p = 0.011) and phoP/Q (p = 0.011) in addition to the genes above. In conclusion, NTS strains isolated from Indonesian chicken had a high resistance to antibiotics and many virulence factors. In particular, S. Schwarzengrund strains were most frequently detected as multi-antimicrobial resistant and had a high prevalence of virulence genes.

A discernable increase in the severe acute respiratory syndrome coronavirus 2 R.1 lineage carrying an E484K spike protein mutation in Japan.

Three COVID-19 waves in Japan have been characterized by the presence of distinct PANGO lineages (B.1.1. 162, B.1.1.284, and B.1.1.214). Recently, in addition to the B.1.1.7 lineage, which shows 25% abundance, an R.1 lineage carrying the E484K mutation in the spike protein was found to show up to 40% predominance. E484K could be a pivotal amino acid substitution with the potential to mediate immune escape; thus, more attention should be paid to such potential variants of concern to avoid the emergence of mutants of concern. Such comprehensive real-time genome surveillance has become essential for the containment of COVID-19 clusters.

A Genome Epidemiological Study of SARS-CoV-2 Introduction into Japan.

After the first case of coronavirus disease 2019 (COVID-19) in Japan on 15 January 2020, multiple nationwide COVID-19 clusters were identified by the end of February. The Japanese government focused on mitigating the emerging COVID-19 clusters by conducting active nationwide epidemiological surveillance. However, an increasing number of cases continued to appear until early April 2020, many with unclear infection routes and no recent history of travel outside Japan. We aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from the COVID-19 cases that appeared until early April 2020 and to characterize their genealogical networks in order to demonstrate possible routes of spread in Japan. Nasopharyngeal specimens were collected from patients, and reverse transcription-quantitative PCR tests for SARS-CoV-2 were performed. Positive RNA samples were subjected to whole-genome sequencing, and a haplotype network analysis was performed. Some of the primary clusters identified during January and February 2020 in Japan descended directly from the Wuhan-Hu-1-related isolates from China and other distinct clusters. Clusters were almost contained until mid-March; the haplotype network analysis demonstrated that the COVID-19 cases from late March through early April may have created an additional large cluster related to the outbreak in Europe, leading to additional spread within Japan. In conclusion, genome surveillance has suggested that there were at least two distinct SARS-CoV-2 introductions into Japan from China and other countries.IMPORTANCE This study aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from COVID-19 cases and to characterize their genealogical networks to demonstrate possible routes of spread in Japan. We found that there were at least two distinct SARS-CoV-2 introductions into Japan, initially from China and subsequently from other countries, including Europe. Our findings can help understand how SARS-CoV-2 entered Japan and contribute to increased knowledge of SARS-CoV-2 in Asia and its association with implemented stay-at-home/shelter-in-place/self-restraint/lockdown measures. This study suggested that it is necessary to formulate a more efficient containment strategy using real-time genome surveillance to support epidemiological field investigations in order to highlight potential infection linkages and mitigate the next wave of COVID-19 in Japan.