Mobile genetic element-driven genomic changes in a community-associated methicillin-resistant Staphylococcus aureus clone during its transmission in a regional community outbreak in Japan.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are now a public health concern in both community and healthcare settings worldwide. We previously identified a suspected case of a maternity clinic-centred outbreak of CA-MRSA skin infection in a regional community in Japan by PFGE-based analysis. In this study, we performed genome sequence-based analyses of 151 CA-MRSA isolates, which included not only outbreak-related isolates that we previously defined based on identical or similar PFGE patterns but also other isolates obtained during the same period in the same region. Our analysis accurately defined 133 isolates as outbreak-related isolates, collectively called the TDC clone. They belonged to a CA-MRSA lineage in clonal complex (CC) 30, known as the South West Pacific (SWP) clone. A high-resolution phylogenetic analysis of these isolates combined with their epidemiological data revealed that the TDC clone was already present and circulating in the region before the outbreak was recognized, and only the isolates belonging to two sublineages (named SL4 and SL5) were directly involved in the outbreak. Long persistence in patients/carriers and frequent intrahousehold transmission of the TDC clone were also revealed by this analysis. Moreover, by systematic analyses of the genome changes that occurred in this CA-MRSA clone during transmission in the community, we revealed that most variations were associated with mobile genetic elements (MGEs). Variant PFGE types were generated by alterations of prophages and genomic islands or insertion sequence (IS)-mediated insertion of a plasmid or a sequence of unknown origin. Dynamic changes in plasmid content, which were linked to changes in antimicrobial resistance profiles in specific isolates, were generated by frequent gain and loss of plasmids, most of which were self-transmissible or mobilizable. The introduction of IS256 by a plasmid (named pTDC02) into sublineage SL5 led to SL5-specific amplification of IS256, and amplified IS256 copies were involved in some of the structural changes of chromosomes and plasmids and generated variations in the repertoire of virulence-related genes in limited isolates. These data revealed how CA-MRSA genomes change during transmission in the community and how MGEs are involved in this process.

An enterococcal phage-derived enzyme suppresses graft-versus-host disease.

Changes in the gut microbiome have pivotal roles in the pathogenesis of acute graft-versus-host disease (aGVHD) after allogenic haematopoietic cell transplantation (allo-HCT)1-6. However, effective methods for safely resolving gut dysbiosis have not yet been established. An expansion of the pathogen Enterococcus faecalis in the intestine, associated with dysbiosis, has been shown to be a risk factor for aGVHD7-10. Here we analyse the intestinal microbiome of patients with allo-HCT, and find that E. faecalis escapes elimination and proliferates in the intestine by forming biofilms, rather than by acquiring drug-resistance genes. We isolated cytolysin-positive highly pathogenic E. faecalis from faecal samples and identified an anti-E. faecalis enzyme derived from E. faecalis-specific bacteriophages by analysing bacterial whole-genome sequencing data. The antibacterial enzyme had lytic activity against the biofilm of E. faecalis in vitro and in vivo. Furthermore, in aGVHD-induced gnotobiotic mice that were colonized with E. faecalis or with patient faecal samples characterized by the domination of Enterococcus, levels of intestinal cytolysin-positive E. faecalis were decreased and survival was significantly increased in the group that was treated with the E. faecalis-specific enzyme, compared with controls. Thus, administration of a phage-derived antibacterial enzyme that is specific to biofilm-forming pathogenic E. faecalis-which is difficult to eliminate with existing antibiotics-might provide an approach to protect against aGVHD.

Posttransplantation cyclophosphamide mediates effective reconstitution of memory B cells after allogeneic hematopoietic cell transplantation.

OBJECTIVES: Impaired B-cell reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT) contributes to the pathogenesis of chronic graft-versus-host disease (cGVHD). Therefore, methods to consistently achieve effective B cell lymphogenesis are required. We assessed the long-term effects of posttransplantation cyclophosphamide (PTCy) use on immune reconstitution in clinical settings, an emerging strategy to suppress allogeneic immunological inflammation early after allo-HCT and prevent subsequent GVHD. METHODS: We comprehensively analyzed peripheral immune cell subsets and measured serum immunoglobulin G (IgG) or cytokine levels in 39 patients who survived for >1 year after allo-HCT. RESULTS: The absolute counts of B1 and IgM memory B cells were significantly lower in patients with severe cGVHD than in those without. The absolute count and percentage (among total CD19+ B cells) of switched memory B cells and serum IgG levels were significantly higher in patients transplanted with PTCy than in those transplanted with conventional GVHD prophylaxis. Interestingly, increased percentages of switched memory B cells and serum IgG levels were observed only in patients transplanted with PTCy and not in those transplanted with umbilical cord blood. CONCLUSIONS: PTCy administration can mediate favorable memory B-cell reconstitution long after allo-HCT and may therefore suppress cGVHD.

Generation and maintenance of the circularized multimeric IS26-associated translocatable unit encoding multidrug resistance.

In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.

Development of an improved colonization system for human-derived Bifidobacterium longum subsp. longum in conventional mice through the feeding of raffinose or 1-kestose.

How bifidobacteria colonize and survive in the intestine is not fully understood. The administration of bifidobacteria to conventional mice can be used to evaluate their ability to colonize the intestine in the presence of endogenous gut microbiota. However, human-derived bifidobacteria do not readily colonize the intestines of conventional mice, and although colonization by Bifidobacterium breve UCC2003 has been achieved, the viability of such populations requires improvement. Therefore, we aimed to establish a colonization system with human-derived bifidobacteria of high viability in conventional mice using Bifidobacterium longum subsp. longum 105-A. Lactose, raffinose, and 1-kestose were identified as the preferred carbohydrate sources for the growth of this strain in culture. The administration of B. longum 105-A to conventional BALB/c mice fed these carbohydrates showed that diets containing 6% (w/w) raffinose or 1-kestose facilitated colonization with >108 colony-forming units/g feces for 2 weeks. The population of this strain was more stable in the raffinose-fed group than in the 1-kestose-fed group. The ingestion of these prebiotics had a greater impact on the composition of the microbiota than the administration of B. longum 105-A. The ingestion of these prebiotics also increased the fecal concentrations of organic acids, which was indicative of greater intestinal fermentation. Collectively, we established a colonization system for B. longum 105-A with high viability in conventional mice by feeding the mice raffinose or 1-kestose. This system should be useful for elucidation of the mechanisms of colonization and survival of bifidobacteria in the intestines in the presence of the endogenous gut microbiota.

Evaluating the indicators of a heart rate variability analysis in dogs using Poincaré plots.

Heart rate variability analyses using Poincaré plots can be useful for evaluating the autonomic nervous system function. However, the interpretation of the quantitative indicators of Poincaré plots remains controversial. Thus, few studies have verified the effectiveness of the quantitative indicators in veterinary medicine. This study aimed to verify the reliability of Poincaré plot indicators using pharmacological models in dogs. Four healthy beagles were used in this study. Each dog was treated with propranolol, atropine, and propranolol-atropine to block the sympathetic, parasympathetic, and sympathetic-parasympathetic functions, respectively. The quantitative indicators of the Poincaré plots were calculated based on data from 300 electrocardiogram beats collected before and after the administration of each drug and statistically analysed. The quantitative indicators of the Poincaré plots, such as the standard deviation perpendicular to the major axis (SD1), standard deviation along the major axis (SD2), and SD1 × SD2, significantly decreased after the drug administration in both the parasympathetic and sympathetic-parasympathetic blockade models. However, no significant differences were observed in SD1/SD2 between the groups. The Poincaré plots reflected the changes in the autonomic nervous system of dogs. In dogs, SD1, SD2, and SD1 × SD2 can detect a state in which parasympathetic nerve activity is suppressed.

A comprehensive list of genes required for the efficient conjugation of plasmid Rts1 was determined by systematic deletion analysis.

While conjugation-related genes have been identified in many plasmids by genome sequencing, functional analyses have not yet been performed in most cases, and a full set of conjugation genes has been identified for only a few plasmids. Rts1, a prototype IncT plasmid, is a conjugative plasmid that was originally isolated from Proteus vulgaris. Here, we conducted a systematic deletion analysis of Rts1 to fully understand its conjugation system. Through this analysis along with complementation assays, we identified 32 genes that are required for the efficient conjugation of Rts1 from Escherichia coli to E. coli. In addition, the functions of the 28 genes were determined or predicted; 21 were involved in mating-pair formation, three were involved in DNA transfer and replication, including a relaxase gene belonging to the MOBH12 family, one was involved in coupling, and three were involved in transcriptional regulation. Among the functionally well-analysed conjugation systems, most of the 28 genes showed the highest similarity to those of the SXT element, which is an integrative conjugative element of Vibrio cholerae. The Rts1 conjugation gene set included all 23 genes required for the SXT system. Two groups of plasmids with conjugation systems nearly identical or very similar to that of Rts1 were also identified.

Practical high-dimensional quantum key distribution protocol over deployed multicore fiber.

Quantum key distribution (QKD) is a secure communication scheme for sharing symmetric cryptographic keys based on the laws of quantum physics, and is considered a key player in the realm of cyber-security. A critical challenge for QKD systems comes from the fact that the ever-increasing rates at which digital data are transmitted require more and more performing sources of quantum keys, primarily in terms of secret key generation rate. High-dimensional QKD based on path encoding has been proposed as a candidate approach to address this challenge. However, while proof-of-principle demonstrations based on lab experiments have been reported in the literature, demonstrations in realistic environments are still missing. Here we report the generation of secret keys in a 4-dimensional hybrid time-path-encoded QKD system over a 52-km deployed multicore fiber link forming by looping back two cores of a 26-km 4-core optical fiber. Our results indicate that robust high-dimensional QKD can be implemented in a realistic environment by combining standard telecom equipment with emerging multicore fiber technology.

Rhizoviticin is an alphaproteobacterial tailocin that mediates biocontrol of grapevine crown gall disease.

Tailocins are headless phage tail structures that mediate interbacterial antagonism. Although the prototypical tailocins, R- and F-pyocins, in Pseudomonas aeruginosa, and other predominantly R-type tailocins have been studied, their presence in Alphaproteobacteria remains unexplored. Here, we report the first alphaproteobacterial F-type tailocin, named rhizoviticin, as a determinant of the biocontrol activity of Allorhizobium vitis VAR03-1 against crown gall. Rhizoviticin is encoded by a chimeric prophage genome, one providing transcriptional regulators and the other contributing to tail formation and cell lysis, but lacking head formation genes. The rhizoviticin genome retains a nearly intact early phage region containing an integrase remnant and replication-related genes critical for downstream gene transcription, suggesting an ongoing transition of this locus from a prophage to a tailocin-coding region. Rhizoviticin is responsible for the most antagonistic activity in VAR03-1 culture supernatant against pathogenic A. vitis strain, and rhizoviticin deficiency resulted in a significant reduction in the antitumorigenic activity in planta. We identified the rhizoviticin-coding locus in eight additional A. vitis strains from diverse geographical locations, highlighting a unique survival strategy of certain Rhizobiales bacteria in the rhizosphere. These findings advance our understanding of the evolutionary dynamics of tailocins and provide a scientific foundation for employing rhizoviticin-producing strains in plant disease control.

Complete genome sequences of 11 Streptococcus pneumoniae strains isolated from acute infectious purpura fulminans, sepsis, and pneumonia.

The complete genome sequences of 11 Japanese Streptococcus pneumoniae isolates were determined by hybrid assembly of long and short reads, including two strains isolated from patients with acute infectious purpura fulminans, six strains from patients with sepsis, and three strains from patients with pneumonia.

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.

Distributed fiber optic shape sensing with simultaneous interrogation of multiple fibers based on Rayleigh-signature domain multiplexing.

We propose a method for shape sensing that employs Rayleigh-signature domain multiplexing to simultaneously probe the fibers or cores of a shape sensing setup with a single optical frequency-domain reflectometry scan. The technique enables incrementing the measurement speed by a factor equal to the number of multiplexed fibers at the expense of an increased noise floor in accordance with the Cramér-Rao lower bound. Nonetheless, we verify that the shape reconstruction performance of the proposed method is in very good agreement with that of conventional sequential core interrogation.

Aortic valve insufficiency caused by a hypoplastic aortic valve in a calf.

A 1-month-old crossbred calf was referred for examination due to marked systolic heart murmurs and poor growth. The heart murmur was most audible on the right side of the cranial thorax. Cardiomegaly was evident on chest radiography, and echocardiography demonstrated aortic regurgitation and decreased fractional shortening. Cardiomegaly, aortic root dilation and cardiac displacement were confirmed by computed tomography. At necropsy, the heart was enlarged, and all three aortic valve leaflets were irregularly shaped. In calves with chronic aortic insufficiency, remodeling displacement of the heart and aorta causes changes in the location and timing of heart murmurs. Therefore, aortic insufficiency cannot be ruled out when a systolic heart murmur can be observed in the right chest wall.

Isolation and Genomic Characterization of a Heat-Labile Enterotoxin 1-Producing Escherichia fergusonii Strain from a Human.

Escherichia fergusonii strains have been isolated from patients with diarrhea, but their virulence determinant has not been well elucidated. Here, we report the first isolation of a heat-labile enterotoxin 1 (LT1)-producing E. fergusonii strain (strain 30038) from a patient in Japan. The complete genome sequence of strain 30038 was determined and subjected to comparative genomics and phylogenetic analyses with 195 publicly available genomes of E. fergusonii. In addition to strain 30038, the elt1 gene was also identified in an E. fergusonii strain that is phylogenetically distinct and which was isolated from poultry in the United Kingdom. Fine genomic comparison revealed that these two strains share comparable elt1-bearing plasmids. However, an intriguing distinction arises in strain 30038, wherein the plasmid has integrated into the chromosome via a recombination process mediated by an insertion sequence. The production of active LT1 toxin by strain 30038 was verified through an in vitro assay using cultured cells. A large plasmid carrying 11 antimicrobial resistance genes was also identified in strain 30038. Our results indicate that extensive surveillance of elt1-positive E. fergusonii strains as diarrheagenic pathogens is needed. IMPORTANCE Escherichia fergusonii, a species closely related to Escherichia coli, is known to cause sporadic conditions in humans, including diarrhea. However, the critical virulence factors in E. fergusonii clinical isolates remain to be identified. This study shows the first isolation of an E. fergusonii strain carrying the elt1 gene, which encodes heat-labile enterotoxin 1, from a patient with diarrhea. Our analysis of public databases also revealed the presence of elt1-positive E. fergusonii strains isolated from poultry in the United Kingdom. Interestingly, while the elt1 gene in the poultry isolate was present on a large plasmid, in the human isolate it was integrated into the chromosome, which may confer stability on the elt1-carrying genetic element. Our findings highlight the need for extensive surveillance of elt1-positive E. fergusonii strains in livestock animals.

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.

Molecular characterization of SrSTP14, a sugar transporter from thermogenic skunk cabbage, and its possible role in developing pollen.

In floral thermogenesis, sugars play an important role not only as energy providers but also as growth and development facilitators. Yet, the mechanisms underlying sugar translocation and transport in thermogenic plants remain to be studied. Asian skunk cabbage (Symplocarpus renifolius) is a species that can produce durable and intense heat in its reproductive organ, the spadix. Significant morphological and developmental changes in the stamen are well-characterized in this plant. In this study, we focused on the sugar transporters (STPs), SrSTP1 and SrSTP14, whose genes were identified by RNA-seq as the upregulated STPs during thermogenesis. Real-time PCR confirmed that mRNA expression of both STP genes was increased from the pre-thermogenic to the thermogenic stage in the spadix, where it is predominantly expressed in the stamen. SrSTP1 and SrSTP14 complemented the growth defects of a hexose transporter-deficient yeast strain, EBY4000, on media containing 0.02, 0.2, and 2% (w/v) glucose and galactose. Using a recently developed transient expression system in skunk cabbage leaf protoplasts, we revealed that SrSTP1 and SrSTP14-GFP fusion proteins were mainly localized to the plasma membrane. To dig further into the functional analysis of SrSTPs, tissue-specific localization of SrSTPs was investigated by in situ hybridization. Using probes for SrSTP14, mRNA expression was observed in the microspores within the developing anther at the thermogenic female stage. These results indicate that SrSTP1 and SrSTP14 transport hexoses (e.g., glucose and galactose) at the plasma membrane and suggest that SrSTP14 may play a role in pollen development through the uptake of hexoses into pollen precursor cells.

Fission yeast Srr1 and Skb1 promote isochromosome formation at the centromere.

Rad51 maintains genome integrity, whereas Rad52 causes non-canonical homologous recombination leading to gross chromosomal rearrangements (GCRs). Here we find that fission yeast Srr1/Ber1 and Skb1/PRMT5 promote GCRs at centromeres. Genetic and physical analyses show that srr1 and skb1 mutations reduce isochromosome formation mediated by centromere inverted repeats. srr1 increases DNA damage sensitivity in rad51 cells but does not abolish checkpoint response, suggesting that Srr1 promotes Rad51-independent DNA repair. srr1 and rad52 additively, while skb1 and rad52 epistatically reduce GCRs. Unlike srr1 or rad52, skb1 does not increase damage sensitivity. Skb1 regulates cell morphology and cell cycle with Slf1 and Pom1, respectively, but neither Slf1 nor Pom1 causes GCRs. Mutating conserved residues in the arginine methyltransferase domain of Skb1 greatly reduces GCRs. These results suggest that, through arginine methylation, Skb1 forms aberrant DNA structures leading to Rad52-dependent GCRs. This study has uncovered roles for Srr1 and Skb1 in GCRs at centromeres.

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.