The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

Discovery of a new volcanic soil material, "Akahoya," as an adsorbent for bacterial and viral pathogens and its application to environmental purification.

Akahoya is a volcanic soil rich in alumina, primarily deposited in Kyushu, Japan. We have found that Akahoya adsorbs bacteria in the water surrounding cattle grazing areas, suggesting a potential for environmental purification. This study investigated the spectrum of microorganisms adsorbed by Akahoya using a column filled with Akahoya through which a suspension of microorganisms was passed. Shirasu soil, another volcanic soil with a different chemical composition, was used as a control. Akahoya effectively adsorbed a diverse range of microorganisms including Escherichia coli, Campylobacter jejuni, Vibrio parahaemolyticus, Salmonella Enteritidis, Staphylococcus aureus, Clostridium perfringens, spores of Bacillus subtilis and Bacillus anthracis, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), murine norovirus, and avian influenza virus (H3N2), whereas Shirasu soil did not adsorb any of the organisms examined. Moreover, bacteria naturally present in river water, such as aerobic bacteria, total coliforms, and Enterobacteriaceae as indicators of river contamination, as well as E. coli added artificially to sterilized river water, were reduced to below the detection limit (<1 CFU/mL) after being passed through Akahoya. Additionally, the number of viable E. coli continued to decrease after contact with Akahoya for 1 month, suggesting bactericidal effects. Notably, the adsorption of E. coli to Akahoya was influenced by the concentration of phosphate and the pH of the suspension due to the interaction between the surface phosphorylation of organisms and Al2O3, the major chemical component of Akahoya. The present results demonstrate the remarkable ability of Akahoya to remove phosphate and microbes, suggesting that Akahoya could be used for water purification processes.IMPORTANCEAlthough a safe and sufficient water supply is essential for the maintenance of hygienic conditions, a major challenge is to develop a comprehensive effective, sustainable, and cost-effective technological approach for the treatment and purification of contaminated water. In this study, we demonstrated that a novel volcanic soil, Akahoya, which has unlimited availability, is a highly effective adsorbent for a wide range of bacterial and viral pathogens, suggesting its potential as a sustainable resource for this purpose. It was suggested that the adsorption of microorganisms on Akahoya was mediated by phosphate groups present on the surface structures of microorganisms, which bind to the alumina component of Akahoya according to the phosphate concentration and pH of the liquid phase. The present findings highlight the exceptional ability of Akahoya to eliminate or reduce phosphate and microorganisms effectively in water purification processes, thus contributing to the development of efficient and sustainable solutions for addressing water pollution challenges.

The growth-stimulating factor of Treponema phagedenis from bovine digital dermatitis lesions.

Bovine digital dermatitis (BDD) is an infectious skin disease of the hoof characterized by painful ulcerations that cause lameness in dairy cattle. Cell-free supernatants (CFS) of Falsiporphyromonas endometrii predominantly isolated from BDD lesions had the highest growth-stimulating effect on Treponema phagedenis among BDD-associated bacteria. Butyric acid was detected at a concentration of 45.4 mM in CFS of F. endometrii, and the growth of T. phagedenis was significantly promoted by butyric acid supplementation.

A surge in respiratory syncytial virus infection-related hospitalizations associated with the COVID-19 pandemic: An observational study at pediatric emergency referral hospitals in Tokushima Prefecture.

The outbreak of coronavirus disease (COVID-19) resulted in implementation of social distancing and other public health measures to control the spread of infection and improve prevention, resulting in a decrease in respiratory syncytial virus (RSV) and pediatric respiratory tract infection rates. However, there was a rapid and large re-emergence of RSV infection in Japan. Notably, we were faced with a difficult situation wherein there was a shortage of hospital beds. This study aimed to examine the epidemiological patterns of RSV-related hospitalizations among children before and after the COVID-19 pandemic onset at two pediatric emergency referral hospitals covering the entire Tokushima Prefecture. Data were extracted from electronic medical records of children hospitalized with RSV infection between January 1, 2018, and December 31, 2021. All patients meeting the eligibility criteria were included in this study. The rates of study outcomes were documented annually during 2018-2021 and compared between the 2018-2020 and 2021 periods. In 2020, there was no RSV infection outbreak. Hospitalizations at the peak week in 2021 were 2.2- and 2.8-fold higher than those in 2018 and 2019, respectively. Hospitalizations in 2021 were concentrated within a short period. In addition, there was a significant increase in hospitalizations among children aged 3-5 months and those older than 24 months. The high-flow nasal cannula (HFNC) use rate nearly doubled in 2021. A new pandemic in the future may cause an outbreak of RSV infection that can result in an intensive increase in the number of hospitalizations of pediatric patients requiring respiratory support, especially in infants aged <6 months. There is an urgent need to improve the preparedness of medical systems, particularly in terms of the number of inpatient beds and the immediate availability of HFNC.

Fine Particle Adsorption Capacity of Volcanic Soil from Southern Kyushu, Japan.

"Akahoya" is a volcanic soil classified as a special soil deposited in Kyushu, Japan. Many of its properties are not yet clearly understood. We found that Akahoya had the potential to adsorb bacteria in cattle feces, which prompted us to investigate its material properties and perform experiments to comprehensively evaluate its adsorption performance for various fine particles such as acidic and basic dyes, NOx/SOx gas, and phosphoric acid ions, in addition to bacteria. Akahoya had a very high specific surface area owing to the large number of nanometer-sized pores in its structure; it exhibited a high adsorption capacity for both NO2 and SO2. Regarding the zeta potential of Akahoya, the point of zero charge was approximately pH 7.0. The surface potential had a significant effect on the adsorption of acidic and basic dyes. Akahoya had a very high cation exchange capacity when the sample surface was negatively charged and a high anion exchange capacity when the sample surface was positively charged. Akahoya also exhibited a relatively high adsorption capacity for phosphoric acid because of its high level of Al2O3, and the immersion liquid had a very high Al ion concentration. Finally, filtration tests were performed on Escherichia coli suspension using a column filled with Akahoya or another volcanic soil sample. The results confirmed that the Escherichia coli adhered on the Akahoya sample. The results of the Escherichia coli release test, after the filtration test, suggested that this adhesion to Akahoya could be phosphorus-mediated.

Comprehensive Analyses of the Bacterial Population in Non-Healing Claw Lesions of Dairy Cattle.

Non-healing claw lesions (NHCLs) are a newly characterized disorder affecting the deep dermis of the hoof in dairy cattle. Although NHCLs are thought to be associated with bovine digital dermatitis (BDD), their precise etiology is not yet understood. To investigate the bacterial populations present in each type of NHCL (toe necrosis: TN, non-healing white line disease: nhWLD, and a non-healing sole ulcer: nhSU), and the newly added entity non-healing verrucous-like lesions (nhVLL), 16S rRNA-based metagenomic analysis with next-generation sequencing (NGS) was employed. Twelve cases of NHCLs (3 TN, 3 nhWLD, 4 nhSU, and 2 nhVLL) were collected from five dairy farms in two prefectures in Japan. Three samples of healthy hoof dermis collected from two farms and a slaughterhouse were used as controls. Furthermore, culture-dependent and -independent approaches were conducted for detecting Treponema species and Fusobacterium necrophorum. As reported in BDD, Treponema species and F. necrophorum were detected frequently from NHCLs by PCR and immunohistochemistry, but NGS showed that these bacterial genera were not predominant in NHCLs. The predominant bacterial genera in NHCLs differed among the lesions examined, suggesting that Treponema species present predominantly in BDD were not predominant in NHCLs and that the bacterial population in NHCLs may vary among individual cattle and/or farms.

Detection and characterization of new mangromicin analogs by tandem mass spectrometry.

Many useful natural products are usually screened based on their biological activities. On the other hand, various natural products can be detected based on their physicochemical properties. We have already reported the isolation and characterization of mangromicins from a cultural broth of Lechevalieria aerocolonigenes K10-0216 using physicochemical screening. In this report, we have conducted the mass spectrometry-based screening of new mangromicin analogs based on the neutral loss pattern originated from the unique cyclopentadecane skeleton of mangromicins. Two novel analogs were detected showing characteristic neutral loss pattern found in eight known mangromicin analogs. We propose the structures of the newly-found analogs based on the mass spectrometric as well as genomic and metabolic pathway data.

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.

Theileria-free grazing of dairy heifers on grassland in Kyushu, Japan where T. orientalis was epidemic before a 7-year vacancy.

This study aimed to produce a Theileria-free grazing system for Holstein heifers reared on a dairy farm in the Hita area, Kyushu, Japan. In the grazing area, spreading of T. orientalis infection was confirmed in 2009. To eradicate the T. orientalis infection, four measures were conducted: 1) 7-year deferred grazing; 2) grazing only T. orientalis-uninfected heifers; 3) anemia check by red blood cell parameters at least once per month; and 4) protecting heifers from blood-sucking T. orientalis-infected ticks. Grazing was restarted in 2017 in the same area and continued to 2021. During last 2 years of pasturing (2020-2021) all of the 129 heifers were confirmed to be T. orientalis-free. In summary, it is possible to establish a T. orientalis-free grazing system by conducting appropriate measures.

Survival capability of Campylobacter upsaliensis under environmental stresses.

OBJECTIVE: Campylobacter upsaliensis has been recognized as an emerging pathogen. However, little is known about its survival in the environment. To evaluate its survival capability, we estimated the reduction in viable counts of C. upsaliensis after aerobic exposure to starvation in phosphate-buffered saline (PBS), acidity (pH = 4.3), high osmolarity (4% NaCl), and dryness in wet pulp disks at different temperatures. Also, survival in dog feces and dog food at variable temperate was assessed. RESULTS: Campylobacter upsaliensis remained culturable under starvation for 4 days at 25 °C and for 10 weeks at 4 °C. C. upsaliensis was also recoverable after exposure to high osmolality for 9 days, dryness for 5 days, and acidity for 2 days, respectively. Similarly, C. upsaliensis survived in dog feces and dog food for several days at 25 °C and weeks at 4 °C. The survival capability of the organism was dependent on the water content, and also temperature. Notably, the tested C. upsaliensis strain was less resilient under all tested conditions than a C. jejuni strain used as a control. The findings showed that C. upsaliensis is able to survive under various environmental stresses, suggesting that it could pose a potential threat to public health.

Work-related increases in titer of Campylobacter jejuni antibody among workers at a chicken processing plant in Miyazaki prefecture, Japan, independent of individual ingestion of edible raw chicken meat.

Workers in poultry abattoirs may be frequently exposed to Campylobacter jejuni, which is a leading cause of bacterial food poisoning in Japan. The present study was conducted to measure the titers of IgG and IgA antibodies against C. jejuni among 104 female workers in a chicken processing plant in Miyazaki prefecture, Japan. Information regarding habitual ingestion of raw chicken meat and potential occupational risk factors was collected using a questionnaire. Acid extracts of four C. jejuni strains representing the genotypes most dominant in Miyazaki were used as antigens. The levels of both immunoglobulins measured by ELISA were not correlated with ingestion of edible raw chicken meat, the amount consumed in one sitting, or its frequency. Although age was correlated with antibody levels, the length of employment was not. Furthermore, the IgG and IgA levels in workers at the evisceration step were significantly higher than those at other locations in the plant. To identify the bacterial proteins recognized by the workers' IgG and IgA antibodies, Western blotting followed by LC/MS was conducted. Flagellin was identified as the common protein recognized in the sera of workers for whom ELISA demonstrated both the highest and lowest antibody levels. We concluded that the titers of IgG and IgA against C. jejuni in workers at the processing plant had been increased by occupational exposure to Campylobacter, regardless of raw chicken meat ingestion.

Effects of Theileria orientalis Infection on Health Status and Productivity of Dairy Cows Reared inside Barns.

The objective of the present study was to investigate the effects of Theileria orientalis on the severity of anemia, the prevalence of disease within 21 days after calving and productivity in cows raised inside barns. This longitudinal observational study, which was conducted on a commercial dairy farm in Japan, involved 627 Holstein cows subjected to PCR analysis for T. orientalis. In study 1, we collected blood samples from 156 sick cows within 21 days after calving, and we found the prevalence of T. orientalis infection to be 65.4%. In study 2, we randomly selected 471 cows during the dry period and collected blood samples to conduct PCR analysis for T. orientalis and determined the prevalence of T. orientalis infection to be 69.0%. Compared with the values for the T. orientalis-uninfected group, the T. orientalis-infected cows had significantly decreased hemoglobin concentrations and hematocrit, but there were no differences in the other complete blood count indexes between the two groups. In addition, there were no differences in productivity and the prevalence of major diseases between the T. orientalis-infected and uninfected cows. In summary, T. orientalis had few effects on anemia, productivity and the health of cows raised inside a barn.

Phf5a regulates DNA repair in class switch recombination via p400 and histone H2A variant deposition.

Antibody class switch recombination (CSR) is a locus-specific genomic rearrangement mediated by switch (S) region transcription, activation-induced cytidine deaminase (AID)-induced DNA breaks, and their resolution by non-homologous end joining (NHEJ)-mediated DNA repair. Due to the complex nature of the recombination process, numerous cofactors are intimately involved, making it important to identify rate-limiting factors that impact on DNA breaking and/or repair. Using an siRNA-based loss-of-function screen of genes predicted to encode PHD zinc-finger-motif proteins, we identify the splicing factor Phf5a/Sf3b14b as a novel modulator of the DNA repair step of CSR. Loss of Phf5a severely impairs AID-induced recombination, but does not perturb DNA breaks and somatic hypermutation. Phf5a regulates NHEJ-dependent DNA repair by preserving chromatin integrity to elicit optimal DNA damage response and subsequent recruitment of NHEJ factors at the S region. Phf5a stabilizes the p400 histone chaperone complex at the locus, which in turn promotes deposition of H2A variant such as H2AX and H2A.Z that are critical for the early DNA damage response and NHEJ, respectively. Depletion of Phf5a or p400 blocks the repair of both AID- and I-SceI-induced DNA double-strand breaks, supporting an important contribution of this axis to programmed as well as aberrant recombination.

Transcellular penetration of Treponema phagedenis isolated from papillomatous digital dermatitis in polarized normal human epidermal keratinocytes in vitro.

Papillomatous digital dermatitis (PDD) is a polymicrobial infection causing lameness in dairy cattle. Culture-independent analysis has shown that Treponema phagedenis is present consistently and predominantly in the lesions. However, the pathogenesis of PDD, especially the tissue penetration pathway, has not been examined. In the present study, we investigated whether T. phagedenis strains isolated from PDD produce proteolytic enzyme (s) for disruption of the epithelial cell barrier and have the ability to translocate in polarized normal human epidermal keratinocytes (NHEK) in vitro. Ten strains of T. phagedenis isolated from lesions did not show proteolytic activity on modified skim milk agar, although a human strain of T. denticola used as a control showed such activity. The integrity of tight junctions was monitored by measurement of transepithelial electrical resistance (TER). The TER values after inoculation of the T. phagedenis strains examined did not change during the experimental period; however, apical to basolateral translocation of T. phagedenis was confirmed after 24 hr by microscopy and Treponema-specific PCR. We further confirmed that translocation of T. phagedenis was accelerated by co-inoculation with live T. denticola, but not with heat-killed organisms. Furthermore, tight junction ZO-1 protein was not lost intensity after inoculation with T. phagedenis and the organism was observed in NHEK cells using a florescence microscope. These results suggest that T. phagedenis strains may translocate via a transcellular route in vitro and that the invasion is accelerated by other bacteria, such as T. denticola, producing proteolytic activity.

A 1-Year Investigation of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Bovine Mastitis at a Large-Scale Dairy Farm in Japan.

In a large-scale dairy farm, it is important to take countermeasure of prevention against mastitis of dairy cows, and it is especially important to establish hygiene and risk management to prevent the emergence and spread of antibiotic-resistant bacteria. In this study, we have performed bacteriological testing of clinical and subclinical mastitis and investigation of antimicrobial resistance bacteria in a large-scale farm for 1 year. The bacteria isolated most frequently from 1,549 samples of 952 cow, including cows with recurring mastitis were Staphylococcus non-aureus (SNA) (27.6%), followed by Escherichia coli (18.9%), Klebsiella pneumoniae (12.3%). Although Staphylococcus aureus was isolated at 7.7% from milk sample, no methicillin-resistant S. aureus was found. The incidence of extended-spectrum ß-lactamase (ESBL)-producing E. coli was 1.4% and ESBL-producing K. pneumoniae was 1.4% of all samples, even though third- and fourth-generation cephalosporins were not used for antimicrobial treatment of mastitis in this farm. Although these genotypes of ESBL-producing E. coli and K. pneumoniae were mainly composed of CTX-M-15 and TEM-1 and CTX-M-2 and TEM-116, respectively, there was no spread and persist of predominant clonal type. Appropriate farm management, such as segregation and culling of infected animals and monitors of trends in antimicrobial resistance among mastitis pathogens, may have contributed these results.

Detection and identification of adhesins involved in adhesion of Campylobacter jejuni to chicken skin.

Campylobacter jejuni is the leading cause of bacterial food poisoning worldwide. Chickens are considered to be one of the major reservoirs of Campylobacter infection in humans due to colonization of their intestinal tract. When the chickens are slaughtered and processed, the entire skin of the carcass becomes contaminated with campylobacters. We observed that the number of C. jejuni attached to chicken skin was reduced significantly after treatment of the skin with sodium hydroxide followed by washing with PBS, implying that adhesion factors involved in binding to C. jejuni may exist on skin. Such potential binding-related proteins present in alkaline extracts of the skin surface were detected by a two-dimensional overlay assay and identified by liquid chromatography mass spectrometry (LC-MS). Chicken serum albumin (CSA) was identified as a major protein in these alkaline extracts and confirmed by ELISA to bind specifically to C. jejuni. Moreover, using the same approach, flagellar hook protein E (FlgE) and major outer membrane protein (MOMP) in C. jejuni were identified as bacterial adhesins that bound to the CSA. The ability to bind CSA was also confirmed using recombinant FlgE and MOMP of C. jejuni expressed in Escherichia coli. The present findings suggest that adhesins expressed on C. jejuni cells may bind specifically via proteins present on the skin, as well as by physical attachment.

Detection of Mycoplasma wenyonii and "Candidatus Mycoplasma haemobos" from Japanese Black breeding cows in Kyushu and Okinawa region, southern part of Japan.

This study aimed to elucidate the epidemiological status of hemoplasma infection and investigate the interaction between Theileria orientalis and hemoplasmas in Japanese Black breeding cows raised in the Kyushu and Okinawa regions. Blood samples were collected from 400 cows from 80 different farms in eight prefectures (five samples per farm and 10 farms per prefecture). Mycoplasma wenyonii (Mw), "Candidatus Mycoplasma haemobos" (CMh), and T. orientalis were examined by polymerase chain reaction (PCR) assay using whole blood samples. PCR results showed that 91.5% (366/400) of cows were positive for bovine hemoplasma: 40.3% were infected with Mw only, 9.5% with CMh only, and 41.8% with both species. T. orientalis was detected in 36% (144/400) of cows. The infection rate of T. orientalis was higher in the grazing group (P<0.001) than in the housed group, while the rate of CMh infection was higher (P<0.05) in the housed group than in the grazing group, suggesting that not only the tick but also other arthropod vectors may contribute to hemoplasma transmission. Although the cows with hemoplasma dual infection showed higher (P<0.05) white blood cell counts compared with hemoplasma-negative cows, there was no difference in hematologic parameters related to the anemia between the hemoplasma-positive and -negative animals. This may indicate that Japanese Black cattle could have resistance to the anemia caused by infection with hemoplasma.

A matricellular protein fibulin-4 is essential for the activation of lysyl oxidase.

Fibulin-4 is a matricellular protein required for extracellular matrix (ECM) assembly. Mice deficient in fibulin-4 (Fbln4-/- ) have disrupted collagen and elastin fibers and die shortly after birth from aortic and diaphragmatic rupture. The function of fibulin-4 in ECM assembly, however, remains elusive. Here, we show that fibulin-4 is required for the activity of lysyl oxidase (LOX), a copper-containing enzyme that catalyzes the covalent cross-linking of elastin and collagen. LOX produced by Fbln4-/- cells had lower activity than LOX produced by wild-type cells due to the absence of lysine tyrosyl quinone (LTQ), a unique cofactor required for LOX activity. Our studies showed that fibulin-4 is required for copper ion transfer from the copper transporter ATP7A to LOX in the trans-Golgi network (TGN), which is a necessary step for LTQ formation. These results uncover a pivotal role for fibulin-4 in the activation of LOX and, hence, in ECM assembly.

Aerotolerance and multilocus sequence typing among Campylobacter jejuni strains isolated from humans, broiler chickens, and cattle in Miyazaki Prefecture, Japan.

Campylobacter jejuni is one of the leading causes of human gastroenteritis in Japan. As chickens and cattle are common reservoirs for C. jejuni, this microaerophilic, stress-sensitive bacterium can overcome and survive various stress conditions during zoonotic transmission, particularly foodborne, to humans. How C. jejuni overcomes stress conditions is, however, unclear. In the present study, 70 C. jejuni strains isolated from various sources (26 human, 20 broilers, and 24 cattle isolates) in Miyazaki, Japan, from 2010 to 2012, were subjected to multilocus sequence typing (MLST) and aerotolerance testing (aerobic shaking at 200 rpm). The results demonstrated that C. jejuni strains from Miyazaki belonged to 12 clonal complexes (CCs) and 43 sequence types (STs). CC-21 and CC-460 were mainly detected in human clinical strains. Most tested strains were aerotolerant, and only one (1.4%) was deemed sensitive to aerobic stress. Approximately 40% strains survived the 24-hr vigorous aerobic shaking at 200 rpm, and these hyper-aerotolerant strains were more prevalent in broiler and cattle isolates than in human isolates. Phylogenetic analysis divided the strains into five clusters, each showing a different pattern of host association. Thus, we have demonstrated for the first time that C. jejuni strains with increased tolerance to aerobic stress are highly prevalent in broilers and cattle in Miyazaki, Japan, and that certain clonal populations are frequently implicated in human infection in this area.

Genome Sequencing Verifies Relapsed Infection of Helicobacter cinaedi.

BACKGROUND: Recurrent infections of Helicobacter cinaedi are often reported, and long-term antimicrobial treatment is empirically recommended to prevent such infections. However, there have been no studies examining whether recurrent infections are relapses of former infections or reinfections with different clones. METHODS: A 69-year-old woman presented with recurrent H cinaedi bacteremia-associated cellulitis after a 51-day interval. We isolated 10 colonies from the blood cultures obtained during each of the 2 episodes and subjected them to whole-genome sequencing (WGS). High-confidence single-nucleotide polymorphisms (SNPs) were identified by an assembly based method. Heterogeneous SNP sites were identified by read mapping. The susceptibility of a representative isolate to 14 antimicrobials was also examined. RESULTS: Whole-genome sequence analysis revealed only 6 SNP sites among the 20 isolates at the whole-genome level. Based on the 6 SNPs, 5 within-host variants (referred to as genotypes) were identified. All 5 genotypes were detected in the first infection; however, only 2 genotypes were detected in the second infection. Although the H cinaedi clone showed a higher minimum inhibitory concentration to fluoroquinolones and macrolides and responsible mutations were identified, none of the 6 SNPs appeared related to additional resistance. CONCLUSIONS: The second infection analyzed here was a relapse of the first infection. A certain level of within-host genomic heterogeneity of the H cinaedi clone was already present in the first infection. Our results suggest the importance of longer treatment courses to eradicate H cinaedi for preventing the relapse of its infection.