Bloodstream infections in the elderly Japanese population: Current reality and countermeasures.

We reviewed bloodstream infections in the elderly in Japan, referring to data recently reported from the National Center for Global Health and Medicine in Tokyo. We divided the locations of bloodstream infections into Hospital-onset (HO), healthcare-associated (HCA), and CA (community-acquired), as the elderly reside in different places. The study focused on the fact that the general condition and underlying diseases of the elderly differ by age group. And thus, we divided them into three groups: Pre-old (65-74 years), Old (75-89 years), and Super-old (≥ 90 years), and compared their characteristics of bloodstream infections. HO bacteremia was most common in the pre-old group. On the other hand, HCA bloodstream infections tended to increase as the population aged, and it was most prevalent in super-old group. According to the study results, early intervention through infectious diseases (ID) consultation may improve the prognosis of bloodstream infections even in the elderly. Since the rate of ID consultation is lower in the super-old group than in other groups, this group may be a significant target. In conclusion, a study of a cohort of elderly patients with bloodstream infections in Japan indicates that bloodstream infections in patients over 65 years is not uniform.

Identification of key yeast species and microbe-microbe interactions impacting larval growth of Drosophila in the wild.

Microbiota consisting of various fungi and bacteria have a significant impact on the physiological functions of the host. However, it is unclear which species are essential to this impact and how they affect the host. This study analyzed and isolated microbes from natural food sources of Drosophila larvae, and investigated their functions. Hanseniaspora uvarum is the predominant yeast responsible for larval growth in the earlier stage of fermentation. As fermentation progresses, Acetobacter orientalis emerges as the key bacterium responsible for larval growth, although yeasts and lactic acid bacteria must coexist along with the bacterium to stabilize this host-bacterial association. By providing nutrients to the larvae in an accessible form, the microbiota contributes to the upregulation of various genes that function in larval cell growth and metabolism. Thus, this study elucidates the key microbial species that support animal growth under microbial transition.

Systematic analysis of plasmids of the Serratia marcescens complex using 142 closed genomes.

Plasmids play important roles in bacterial genome diversification. In the Serratia marcescens complex (SMC), a notable contribution of plasmids to genome diversification was also suggested by our recent analysis of >600 draft genomes. As accurate analyses of plasmids in draft genomes are difficult, in this study we analysed 142 closed genomes covering the entire complex, 67 of which were obtained in this study, and identified 132 plasmids (1.9-244.4 kb in length) in 77 strains. While the average numbers of plasmids in clinical and non-clinical strains showed no significant difference, strains belonging to clade 2 (one of the two hospital-adapted lineages) contained more plasmids than the others. Pangenome analysis revealed that of the 28 954 genes identified, 12.8 % were plasmid-specific, and 1.4 % were present in plasmids or chromosomes depending on the strain. In the latter group, while transposon-related genes were most prevalent (31.4 % of the function-predicted genes), genes related to antimicrobial resistance and heavy metal resistance accounted for a notable proportion (22.7 %). Mash distance-based clustering separated the 132 plasmids into 23 clusters and 50 singletons. Most clusters/singletons showed notably different GC contents compared to those of host chromosomes, suggesting their recent or relatively recent appearance in the SMC. Among the 23 clusters, 17 were found in only clinical or only non-clinical strains, suggesting the possible preference of their distribution on the environmental niches of host strains. Regarding the host strain phylogeny, 16 clusters were distributed in two or more clades, suggesting their interclade transmission. Moreover, for many plasmids, highly homologous plasmids were found in other species, indicating the broadness of their potential host ranges, beyond the genus, family, order, class or even phylum level. Importantly, highly homologous plasmids were most frequently found in Klebsiella pneumoniae and other species in the family Enterobacteriaceae, suggesting that this family, particularly K. pneumoniae, is the main source for plasmid exchanges with the SMC. These results highlight the power of closed genome-based analysis in the investigation of plasmids and provide important insights into the nature of plasmids distributed in the SMC.

Virulence of Shigatoxigenic and Enteropathogenic Escherichia coli O80:H2 in Galleria mellonella Larvae: Comparison of the Roles of the pS88 Plasmids and STX2d Phage.

The invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 µL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties.

Clinical outcomes and epidemiological characteristics of bacteremia in the older Japanese population.

BACKGROUND: The characteristics and clinical consequences of bacteremia in older people, who are highly susceptible to infections, need to be clarified. This study aimed to determine the epidemiological characteristics, prognosis, and predictors of 7-day mortality in patients with community-acquired (CA), healthcare-associated (HCA), and hospital-onset (HO) bacteremia in older adults aged ≥65 years. METHODS: Patients aged ≥65 years with positive blood cultures between April 1, 2015, and March 31, 2018, were divided into three groups: pre-old (65-74 years), old (75-89 years), and super-old (≥90 years). Characteristics based on medical exposure, including CA, HCA, and HO, were also compared and factors related to mortality were identified. RESULTS: Overall, 1716 episodes of bacteremia were identified in 1415 patients. Of the 1211 episodes without contamination, 32.8%, 54.3%, and 12.9% occurred in pre-old, old, and super-old patients. Central line-associated bloodstream infections were more common in pre-old patients and urinary tract infections in the old and super-old. The 7-day mortality rates in the pre-old, old, and super-old groups were 7.4%, 5.8%, and 14.2% (P = 0.002), respectively. Multivariable logistic regression showed that super-old age (adjusted odds ratio, aOR: 2.09 [1.13-3.88], P = 0.019) and HO bacteremia (aOR: 1.97 [1.18-3.28], P = 0.010) were independent risk factors for 7-day mortality. Infectious disease consultation had a protective effect on 7-day mortality (aOR: 0.59 [0.35-0.99], P = 0.047). CONCLUSIONS: The epidemiology of bacteremia differs among older people; thus, they should not be treated as a single entity. A careful approach is needed for the optimal management of bacteremia in these vulnerable patients.

Nationwide analysis of antimicrobial resistance in pathogenic Escherichia coli strains isolated from diseased swine over 29 years in Japan.

Pathogenic Escherichia coli strains are important causes of several swine diseases that result in significant economic losses worldwide. In Japan, the use of antimicrobials in swine is much higher than that in other farm animals every year. Antimicrobial resistance in pathogenic E. coli strains also heavily impacts the swine industry due to the limited treatment options and an increase in the potential risk of the One Health crisis. In 2016, we investigated 684 Japanese isolates of swine pathogenic E. coli belonging to four major serogroups and reported the emergence and increase in the highly multidrug-resistant serogroups O116 and OSB9 and the appearance of colistin-resistant strains. In the present study, by expanding our previous analysis, we determined the serotypes and antimicrobial resistance of 1,708 E. coli strains isolated from diseased swine between 1991 and 2019 in Japan and found recent increases in the prevalences of multidrug-resistant strains and minor serogroup strains. Among the antimicrobials examined in this study that have been approved for animal use, a third-generation cephalosporin was found to be effective against the most isolates (resistance rate: 1.2%) but not against highly multidrug-resistant strains. We also analyzed the susceptibilities of the 1,708 isolates to apramycin and bicozamycin, both which are available for treating swine in Japan, and found that the rates of resistance to apramycin and bicozamycin were low (6.7% and 5.8%, respectively), and both antimicrobials are more effective (resistance rates: 2.7% and 5.4%, respectively) than third-generation cephalosporins (resistance rate: 16.2%) against highly multidrug-resistant strains.

Escherichia cryptic clade I is an emerging source of human intestinal pathogens.

BACKGROUND: Within the genus Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear. RESULTS: We defined a set of true C-I strains (n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC. CONCLUSIONS: Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.

Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

Dynamic changes in Shiga toxin (Stx) 1 transducing phage throughout the evolution of O26:H11 Stx-producing Escherichia coli.

Shiga toxin (Stx) is the key virulence factor of Stx-producing Escherichia coli (STEC). All known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages). Although the genetic diversity of Stx phages has frequently been described, systematic analyses of Stx phages in a single STEC lineage are limited. In this study, focusing on the O26:H11 STEC sequence type 21 (ST21) lineage, where the stx1a gene is highly conserved, we analysed the Stx1a phages in 39 strains representative of the entire ST21 lineage and found a high level of variation in Stx1a phage genomes caused by various mechanisms, including replacement by a different Stx1a phage at the same or different locus. The evolutionary timescale of events changing Stx1a phages in ST21 was also determined. Furthermore, by using an Stx1 quantification system developed in this study, we found notable variations in the efficiency of Stx1 production upon prophage induction, which sharply contrasted with the conserved iron regulated Stx1 production. These variations were associated with the Stx1a phage alteration in some cases but not in other cases; thus, Stx1 production in this STEC lineage was determined by differences not only in Stx1 phages but also in host-encoded factors.

Phylogenic position and low genomic diversity of "Candidatus Rickettsia kotlanii" inferred by complete genome sequences of two Japanese isolates.

Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fever." One of the candidate SFG Rickettsia species is "Candidatus Rickettsia kotlanii," which was first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single-gene sequence-based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese "Ca. R. kotlanii" isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of "Ca. R. kotlanii" in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of "Ca. R. kotlanii" relative to the other species indicated that "Ca. R. kotlanii" is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in "Ca. R. kotlanii." While the genome of "Ca. R. kotlanii" is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in "Ca. R. kotlanii," but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to "Ca. R. kotlanii."

Escherichia coli O80 in Healthy Cattle: Absence of Shigatoxigenic and Enteropathogenic E. coli O80:H2 and (Phylo) Genomics of Non-Clonal Complex 165 E. coli O80.

The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli O80:H2 is still unknown. The aim of this study was to identify E. coli O80 in healthy cattle with an emphasis on melibiose non-fermenting E. coli O80:H2. Faecal materials collected from 149 bulls at 1 slaughterhouse and 194 cows on 9 farms were tested with O80 antigen-encoding gene PCR after overnight growth in enrichment broths. The 53 O80 PCR-positive broths were streaked on different (semi-)selective agar plates. Five E. coli colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting E. coli was isolated. However, these 16 E. coli O80 were negative with PCR targeting the fliCH2, eae, stx1, stx2 and hlyF genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to E. coli O80:H6 and O80:H45 isolated from different animal species in different countries, respectively, but neither to STEC and EPEC O80:H2/ST301, nor to other serotypes of the clonal complex 165. As a conclusion, healthy adult cattle were not identified as a source of contamination of humans and calves by STEC or EPEC O80:H2.

Insertion Sequence (IS)-Excision Enhancer (IEE)-Mediated IS Excision from the lacZ Gene Restores the Lactose Utilization Defect of Shiga Toxin-Producing Escherichia coli O121:H19 Strains and Is Responsible for Their Delayed Lactose Utilization Phenotype.

Lactose utilization is one of the general biochemical characteristics of Escherichia coli, and the lac operon is responsible for this phenotype, which can be detected on lactose-containing media, such as MacConkey agar, after 24 h of incubation. However, some Shiga toxin-producing E. coli (STEC) O121:H19 strains exhibit an unusual phenotype called delayed lactose utilization (DLU), in which lactose utilization can be detected after 48 h of cultivation but not after only 24 h of cultivation. Insertion of an insertion sequence (IS), IS600, into the lacZ gene appears to be responsible for the DLU phenotype, and exposure to lactose has been reported to be necessary to observe this phenotype, but the mechanism underlying these phenomena remains to be elucidated. Here, we performed detailed analyses of the lactose utilization abilities of a set of O121:H19 strains and their mutants and found that IS-excision enhancer (IEE)-mediated excision of IS600 reactivates the lacZ gene and that the selective proliferation of IS-cured subclones in lactose-supplemented culture medium is responsible for the expression of the DLU phenotype. In addition, we analyzed the patterns of IS insertion into the lacZ and iee genes in the global O121:H19 population and revealed that while there are O121:H19 strains or lineage/sublineages that contain the IS insertion into iee or intact lacZ and thus do not show the DLU phenotype, most currently circulating O121:H19 strains contain IS600-inserted lacZ and intact iee and thus exhibit this phenotype. IMPORTANCE Insertion sequences (ISs) can modulate gene expression by gene inactivation or activation. While phenotypic changes due to IS insertion/transposition are frequently observed, gene reactivation by precise or simple IS excision rarely occurs. In this study, we show that IS600 is excised from the lacZ gene by IS-excision enhancer (IEE) during the cultivation of Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains that show an unusual phenotype called delayed lactose utilization (DLU). This excision rescued their lactose utilization defect, and the subsequent selective proliferation of IS-cured subclones in lactose-containing medium resulted in the expression of the DLU phenotype. As we also show that most currently circulating O121:H19 strains exhibit this phenotype, this study not only provides information helpful for the isolation and identification of O121:H19 STEC but also offers novel insights into the roles of IS and IEE in the generation of phenotypic variation in bacterial populations.

Clinical performance testing of the automated haematology analyzer XN-31 prototype using whole blood samples from patients with imported malaria in Japan.

BACKGROUND: The automated haematology analyzer XN-31 prototype (XN-31p) is a new flow cytometry-based device developed to measure the number and the ratio of malaria-infected red blood cells (MI-RBC) with a complete blood count (CBC). The XN-31p can provide results in about one minute and also can simultaneously provide information on the malaria parasite (Plasmodium) species. In this study, clinical testing of the XN-31p was performed using blood samples from patients with imported malaria in Japan. METHODS: Blood samples were collected from 80 patients who visited the hospital of the National Center for Global Health and Medicine, Tokyo, Japan, for malaria diagnosis from January 2017 to January 2019. The test results by the XN-31p were compared with those by other standard methods, such as microscopic observation, rapid diagnostic tests and the nested PCR. RESULTS: Thirty-three patients were diagnosed by the nested PCR as being malaria positive (28 Plasmodium falciparum, 2 Plasmodium vivax, 1 Plasmodium knowlesi, 1 mixed infection of P. falciparum and Plasmodium malariae, and 1 mixed infection of P. falciparum and Plasmodium ovale), and the other 47 were negative. The XN-31p detected 32 patients as "MI-RBC positive", which almost matched the results by the nested PCR and, in fact, completely matched with the microscopic observations. The ratio of RBCs infected with malaria parasites as determined by the XN-31p showed a high correlation coefficient of more than 0.99 with the parasitaemia counted under microscopic observation. The XN-31p can analyse the size and nucleic acid contents of each cell, and the results were visualized on a two-dimensional cytogram termed the "M scattergram". Information on species and developmental stages of the parasites could also be predicted from the patterns visualized in the M scattergrams. The XN-31p showed a positive coincidence rate of 0.848 with the nested PCR in discriminating P. falciparum from the other species. CONCLUSIONS: The XN-31p could rapidly provide instructive information on the ratio of MI-RBC and the infecting Plasmodium species. It was regarded to be of great help for the clinical diagnosis of malaria.

Omics profiles of fecal and oral microbiota change in irritable bowel syndrome patients with diarrhea and symptom exacerbation.

BACKGROUND: Irritable bowel syndrome (IBS) is a disorder of gut-brain interaction, including dysregulation of the hypothalamic-pituitary-adrenal axis with salivary cortisol changes. However, the role of gastrointestinal microbiota during IBS symptom exacerbation remains unclear. We tested the hypothesis that the microbial species, gene transcripts, and chemical composition of fecal and oral samples are altered during the exacerbation of IBS symptoms. METHODS: Fecal, salivary, and dental plaque samples were collected at baseline from 43 men with IBS with diarrhea (IBS-D) and 40 healthy control (HC) men. Samples in the IBS-D patients were also collected during symptom exacerbation. The composition of the fecal microbiota was determined by analyzing the 16S rRNA gene, RNA-based metatranscriptome, and metabolites in samples from HC and IBS patients with and without symptom exacerbation. Oral samples were also analyzed using omics approaches. RESULTS: The fecal microbiota during IBS symptom exacerbation exhibited significant differences in the phylogenic pattern and short-chain fatty acid compared with fecal samples during defecation when symptoms were not exacerbated. Although there were no significant differences in the phylogenic pattern of fecal microbiota abundance between HCs and IBS-D patients, significant differences were detected in the expression patterns of bacterial transcriptomes related to butyrate production and neuroendocrine hormones, including tryptophan-serotonin-melatonin synthesis and glutamine/GABA. The composition of plaque microbiota was different between HC and IBS-D patients during normal defecation. CONCLUSIONS: Our findings suggest that colonic host-microbial interactions are altered in IBS-D patients during exacerbation of symptoms. There were no overlaps between feces and oral microbiomes.

Helicobacter cinaedi is a human-adapted lineage in the Helicobacter cinaedi/canicola/'magdeburgensis' complex.

Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi-like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola, little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola-like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi, H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I-IV) in H. cinaedi/canicola/'magderbugensis' (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex.

A case of severe COVID-19 with pulmonary thromboembolism related to heparin-induced thrombocytopenia during prophylactic anticoagulation therapy.

A 53-year-old male Japanese patient with COVID-19 was admitted to our hospital after his respiratory condition worsened on day 9 of the disease. With the diagnosis of severe COVID-19, treatment with remdesivir, dexamethasone, and unfractionated heparin was started for the prevention of thrombosis. Although the patient's respiratory status data improved after treatment, severe respiratory failure persisted. Thrombocytopenia and D-dimer elevation were observed on day 8 after heparin therapy initiation. Heparin-induced thrombocytopenia (HIT) antibody measured by immunological assay was positive, and contrast computed tomography showed pulmonary artery thrombus. The patient was diagnosed with HIT because the pre-test probability score (4Ts score) for HIT was 7 points. Heparin was changed to apixaban, a direct oral anticoagulant, which resulted in a reduction of the pulmonary thrombus and improvement of the respiratory failure. In patients with COVID-19, anticoagulant therapy with heparin requires careful monitoring of thrombocytopenia and elevated D-dimer as possible complications related to HIT. (151/250 words).

Global population structure of the Serratia marcescens complex and identification of hospital-adapted lineages in the complex.

Serratia marcescens is an important nosocomial pathogen causing various opportunistic infections, such as urinary tract infections, bacteremia and sometimes even hospital outbreaks. The recent emergence and spread of multidrug-resistant (MDR) strains further pose serious threats to global public health. This bacterium is also ubiquitously found in natural environments, but the genomic differences between clinical and environmental isolates are not clear, including those between S. marcescens and its close relatives. In this study, we performed a large-scale genome analysis of S. marcescens and closely related species (referred to as the 'S. marcescens complex'), including more than 200 clinical and environmental strains newly sequenced here. Our analysis revealed their phylogenetic relationships and complex global population structure, comprising 14 clades, which were defined based on whole-genome average nucleotide identity. Clades 10, 11, 12 and 13 corresponded to S. nematodiphila, S. marcescens sensu stricto, S. ureilytica and S. surfactantfaciens, respectively. Several clades exhibited distinct genome sizes and GC contents and a negative correlation of these genomic parameters was observed in each clade, which was associated with the acquisition of mobile genetic elements (MGEs), but different types of MGEs, plasmids or prophages (and other integrative elements), were found to contribute to the generation of these genomic variations. Importantly, clades 1 and 2 mostly comprised clinical or hospital environment isolates and accumulated a wide range of antimicrobial resistance genes, including various extended-spectrum ß-lactamase and carbapenemase genes, and fluoroquinolone target site mutations, leading to a high proportion of MDR strains. This finding suggests that clades 1 and 2 represent hospital-adapted lineages in the S. marcescens complex although their potential virulence is currently unknown. These data provide an important genomic basis for reconsidering the classification of this group of bacteria and reveal novel insights into their evolution, biology and differential importance in clinical settings.

Comorbidities as Risk Factors for Severe Disease in Hospitalized Elderly COVID-19 Patients by Different Age-Groups in Japan.

INTRODUCTION: Old age is an independent risk factor (RF) for severe COVID-19; evidence for clinico-epidemiological characteristics among elderly COVID-19 patients is scarce. We aimed to analyze clinical and epidemiological characteristics and comorbidities associated with COVID-19 inpatients in age-stratified populations of an elderly COVID-19 cohort. METHODS: We conducted a retrospective cohort study, using nationwide registry data of COVID-19 patients hospitalized before October 31, 2020 (major information entered in the registry as of December 28, 2020). Participants were divided by age according to the Japan Geriatrics Society and the Japan Gerontological Society: pre-old (65-74 years), old (75-89 years), and super-old (≥90 years). Multivariable logistic regression (MLR) analyses were conducted to identify stratified risk and relationships with comorbidities associated with worse outcomes in different age-groups of elderly patients. Demographics and supportive care were evaluated by category. RESULTS: Data of 4,701 patients from 444 hospitals were included. Most patients (79.3%) had at least one comorbidity; the proportion of patients with hypertension was high in all categories. The proportion of patients with dementia, cardiovascular disease, and cerebrovascular disease increased with age. The percentage of patients who underwent invasive mechanical ventilation/extracorporeal membrane oxygenation was lower in the super-old group. In total, 11.5% of patients died (5.3%, pre-old; 15.2%, old; and 22.4%, super-old). MLR showed that the risk of critical illness differed among age-groups. Male sex was a significant RF in all ages. Collagen disease, moderate to severe renal disorder, and dialysis were significant RFs in older patients, while hematological malignancies and metastatic tumors were more important RFs for severe disease in relatively younger patients. Most of the RFs for critical illnesses were associated with death. CONCLUSION: Differences in the epidemiological and clinical characteristics among the different age-groups were found.