Application of Genomic Epidemiology of Pathogens to Farmed Yellowtail Fish Mycobacteriosis in Kyushu, Japan.

To investigate mycobacterial cases of farmed yellowtail fish in coastal areas of western Japan (Kagoshima, Kyushu), where aquaculture fisheries are active, Mycobacterium pseudoshottsii, the causative agent, was isolated from six neighboring fishing ports in 2012 and 2013. A phylogenetic ana-lysis revealed that the strains isolated from one fishing port were closely related to those isolated from other regions of Japan, suggesting the nationwide spread of a single strain. However, strains from Japan were phylogenetically distinct from those from the Mediterranean and the United States; therefore, worldwide transmission was not observed based on the limited data obtained on the strains exami-ned in this study. The present results demonstrate that a bacterial genomic ana-lysis of infected cases, a mole-cular epidemiology strategy for public health, provides useful data for estimating the prevalence and transmission pathways of M. pseudoshottsii in farmed fish. A bacterial genome ana-lysis of strains, such as that performed herein, may play an important role in monitoring the prevalence of this pathogen in fish farms and possible epidemics in the future as a result of international traffic, logistics, and trade in fisheries.

Pathogenicity of enterotoxigenic Escherichia coli in Caenorhabditis elegans as an alternative model host.

Enterotoxigenic Escherichia coli (ETEC), one of the diarrheagenic E. coli, is the most common cause of diarrhea in developing country and in travelers to those areas. In this study, Caenorhabditis elegans was used as an alternative model host to evaluate ETEC infections. The ETEC strain ETEC1, which was isolated from a patient with diarrhea, possessed enterotoxins STh, LT1, and EAST1 and colonization factors CS2 and CS3. Live ETEC1 shortened the life span and body size of C. elegans in association with increased expression of enterotoxin genes and intestinal colonization. In contrast, heat-killed ETEC1 did not affect the life span of C. elegans. Caenorhabditis elegans infected with ETEC1 showed upregulated expression of genes related to insulin-like peptides and host defense responses. These results suggest that ETEC1 exhibits pathogenicity through intestinal colonization and enterotoxin production in C. elegans. This system is useful as an ETEC infection model.

Improvement of Locomotion Caused by Lactococcus lactis subsp. lactis in the Model Organism Caenorhabditis elegans.

Lactococcus lactis subsp. lactis exhibits probiotic properties in humans. Considering that Caenorhabditis elegans can be used to study the effects of microorganisms on animal behavior, owing to its simple nervous system, we assessed the impacts of two strains of Lactococcus lactis subsp. Lactis-a non-nisin-producing strain, NBRC 100933 (LL100933), and a nisin-producing strain, NBRC 12007 (LL12007)-on the lifespan, locomotion, reproductive capacity of, and lipid accumulation in, C. elegans. The lifespan of adult C. elegans fed a mixture (1:1) of Escherichia coli OP50 and LL100933 or LL12007 did not show a significant increase compared to that of the group fed a standard diet of E. coli OP50. However, the nematodes fed Lactococcus strains showed notable enhancement in their locomotion at all of the tested ages. Further, the beneficial effects of LL100933 and LL12007 were observed in the daf-16 mutants, but not in the skn-1 and pmk-1 mutants. The lipid accumulation in the worms of the Lactococcus-fed group was lower than that in the control group at all experimental ages. Overall, LL100933 and LL12007 enhance the locomotor behavior of C. elegans, likely by modulating the PMK-1/p38 MAPK and SKN-1/Nrf2 transcription factors.

Retrospective identification of pathogenic mycobacterial species in fish: Mycobacterium pseudoshottsii YM-3, isolated from a yellowtail fish in 1986 in Kochi, Japan.

The complete genome sequence of mycobacterial strain YM-3, isolated from cultured yellowtail in 1986, was determined. The strain was Mycobacterium pseudoshottsii, a closely related subspecies of Mycobacterium marinum, so the strain was isolated earlier than the first report of the subspecies in 2005.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

Impacts of Bacillus subtilis var. natto on the lifespan and stress resistance of Caenorhabditis elegans.

AIM: Bacillus subtilis var. natto is used in the production of natto, a typical Japanese fermented soybean food. Although the probiotic attributes and health-related effects of B. subtilis var. natto have been reported, the effect on longevity remains unknown. In the present study, the effects of B. subtilis var. natto strains on lifespan extension and the molecular mechanisms governing the prolongevity were examined using Caenorhabditis elegans as a model animal. METHODS AND RESULTS: Synchronized 3-day-old (young adult) worms were fed Escherichia coli OP50 (control) or a subcloned isolate of B. subtilis var. natto Miyagino strain (MI-OMU01) and subjected to lifespan, survival against pathogens and abiotic stress resistance assays. Notably, the lifespan of worms fed MI-OMU01 was significantly longer than that of the animals fed OP50. Moreover, MI-OMU01 increased the resistance of C. elegans to several stressors, including UV irradiation, H2O2, and Cu2+. CONCLUSIONS: Genetic and gene expression analyses using mutant animals suggested that MI-OMU01 extended the lifespan of worms in TIR-1/SARM, p38 MAPK, and insulin/IGF-1 signaling pathway-dependent manners.

Social dilemma in the excess use of antimicrobials incurring antimicrobial resistance.

The emergence of antimicrobial resistance (AMR) caused by the excess use of antimicrobials has come to be recognized as a global threat to public health. There is a 'tragedy of the commons' type social dilemma behind this excess use of antimicrobials, which should be recognized by all stakeholders. To address this global threat, we thus surveyed eight countries/areas to determine whether people recognize this dilemma and showed that although more than half of the population pays little, if any, attention to it, almost 20% recognize this social dilemma, and 15-30% of those have a positive attitude toward solving that dilemma. We suspect that increasing individual awareness of this social dilemma contributes to decreasing the frequency of AMR emergencies.

Oral cleaning habits and the copy number of periodontal bacteria in pregnant women and its correlation with birth outcomes: an epidemiological study in Mibilizi, Rwanda.

BACKGROUND: Since 1996, many studies have reported that periodontal disease during pregnancy may be a risk factor for preterm birth and low birth weight; however, in Africa, periodontal disease is considered a non-high-priority disease. In addition, there are few dental facilities in rural Rwanda; thus, the oral condition of pregnant women has not been investigated. The objective of this study was to assess the tooth brushing habits of pregnant women in rural Rwanda and evaluate whether periodontal bacteria in the oral cavity of pregnant women are related to birth outcomes or oral cleaning habits. METHODS: A questionnaire survey and saliva collection were conducted for pregnant women in the catchment area population of Mibilizi Hospital located in the western part of Rwanda. Real-time PCR was performed to quantitatively detect total bacteria and 4 species of periodontal bacteria. The relationship of the copy number of each bacterium and birth outcomes or oral cleaning habits was statistically analyzed. RESULTS: Among the participants, high copy numbers of total bacteria, Tannerella forsythia, and Treponema denticola were correlated with lower birth weight (p = 0.0032, 0.0212, 0.0288, respectively). The sex ratio at birth was higher in women who had high copy numbers of Porphyromonas gingivalis and T. denticola during pregnancy (p = 0.0268, 0.0043). Furthermore, regarding the correlation between oral cleaning habits and the amount of bacteria, the more frequently teeth were brushed, the lower the level of P. gingivalis (p = 0.0061); the more frequently the brush was replaced, the lower the levels of P. gingivalis and T. forsythia (p = 0.0153, 0.0029). CONCLUSIONS: This study suggested that improving tooth brushing habits may reduce the risk of periodontal disease among pregnant women in rural Rwanda. It also indicated that the amount of bacteria is associated with various birth outcomes according to the bacterial species. Both access to dental clinics and the oral cleaning habits of pregnant women should be important considerations in efforts to alleviate reproductive-related outcomes in rural Africa.

Periodontal disease is known to cause many complications. For instance, pregnant women with periodontal disease are at increased risk of preterm birth and delivering low-birth-weight infants. However, the importance of oral care during pregnancy is not an important consideration in rural Africa, where preterm birth rates and low-birth-weight rates are particularly high. Moreover, even the oral hygiene status of pregnant women has not been assessed in such areas. In this study, we focused on the amount of periodontal bacteria that cause periodontal disease and investigated the relationship between the amount of bacteria and birth outcomes. Our findings indicate that tooth brushing guidance for pregnant women and improved access to dental clinics in rural Africa may contribute to reduced rates of preterm birth and low birth weight.

Analytical estimation of maximum fraction of infected individuals with one-shot non-pharmaceutical intervention in a hybrid epidemic model.

BACKGROUND: Facing a global epidemic of new infectious diseases such as COVID-19, non-pharmaceutical interventions (NPIs), which reduce transmission rates without medical actions, are being implemented around the world to mitigate spreads. One of the problems in assessing the effects of NPIs is that different NPIs have been implemented at different times based on the situation of each country; therefore, few assumptions can be shared about how the introduction of policies affects the patient population. Mathematical models can contribute to further understanding these phenomena by obtaining analytical solutions as well as numerical simulations. METHODS AND RESULTS: In this study, an NPI was introduced into the SIR model for a conceptual study of infectious diseases under the condition that the transmission rate was reduced to a fixed value only once within a finite time duration, and its effect was analyzed numerically and theoretically. It was analytically shown that the maximum fraction of infected individuals and the final size could be larger if the intervention starts too early. The analytical results also suggested that more individuals may be infected at the peak of the second wave with a stronger intervention. CONCLUSIONS: This study provides quantitative relationship between the strength of a one-shot intervention and the reduction in the number of patients with no approximation. This suggests the importance of the strength and time of NPIs, although detailed studies are necessary for the implementation of NPIs in complicated real-world environments as the model used in this study is based on various simplifications.

Comparative genomic analysis of Leptospira spp. isolated from Rattus norvegicus in Indonesia.

Leptospirosis is one of the most prevalent zoonotic diseases caused by pathogenic spirochetes of Leptospira spp. The disease has become a public health concern in urban localities in the tropics, where rats serve as significant reservoir animals for leptospirosis transmission. In Indonesia, the occurrence of leptospirosis is underestimated, and information on the causative Leptospira genotypes and serotypes is limited. This study reports the isolation of Leptospira spp. from rats captured in urban areas of Bogor, Indonesia. Serogroups and genotypes, sequence types (STs), or multiple-locus variable-number tandem repeat analysis (MLVA) types using 11 loci, were determined for the isolates. Furthermore, whole genome sequencing (WGS) was performed on 11 Indonesian isolates and 24 isolates from other Asian countries. Ninety small mammals, including 80 Rattus norvegicus and ten Suncus murinus, were captured and, 25 Leptospira spp. were isolated solely from R. norvegicus (31.3%). The isolates were identified as Leptospira borgpetersenii serogroup Javanica with ST 143 (four strains) and Leptospira interrogans serogroup Bataviae with the same MLVA type as isolates from other Asian countries (19); the serogroup of the two L. interrogans with ST252 could not be identified. The core genome SNP-based phylogenetic tree revealed that Indonesian isolates were genetically related to L. borgpetersenii serogroup Javanica strains widely distributed in Asian countries but formed a different cluster from other strains. The phylogenetic tree of L. interrogans serogroup Bataviae isolates from Indonesia, the Philippines, Taiwan, and Vietnam revealed that isolates were grouped into five clusters based on their geographic locations. This study discovered a high carriage rate of Leptospira spp. among R. norvegicus in Bogor, Indonesia, indicating a potential risk of rat-borne leptospirosis in the area. Besides L. borgpetersenii serogroup Javanica, WGS on L. interrogans serogroup Bataviae illustrated the geographical structuring of genetic diversity in Leptospira spp.

Suprapancreatic nodal dissection should not be uniformly selected in additional gastrectomy for the patients who diagnosed as pT1b gastric cancer by endoscopic resection.

BACKGROUND: Surgery for T1b gastric cancer requires suprapancreatic nodes (SPA nodes) and perigastric nodes (PG nodes), however, SPA nodal dissection can cause pancreatic complications. If endoscopic treatment followed by additional surgery is planned, it may be possible to predict SPA nodal metastasis by analyzing the pathological information of the primary tumor in addition to the clinical findings. METHODS: Patients who underwent D1+ or D2 and who were pathologically diagnosed with pT1b were retrospectively analyzed. The stations were divided into the nodes located at the perigastric area (PG nodes; #1-7) and the nodes located at the suprapancreatic area (SPA nodes; #8a-12a). The patients were classified into those with and without metastasis to the SPA nodes. Clinicopathological factors were investigated for their possible association with metastasis to the SPA nodes. RESULTS: A total of 2017 patients were examined in this study. Metastasis to the SPA nodes was observed in 80 patients (4%). In a multivariate analysis, undifferentiated histology, lower third, and lymphovascular invasion were independent risk factors for metastasis to the SPA nodes. The risk of metastasis to the SPA nodes was <2% in upper/middle tumors of differentiated type with no lymphovascular invasion and in lower tumors of undifferentiated type with no lymphovascular invasion. On the other hand, the risk of metastasis to the SPA nodes was >10% in lower tumors of undifferentiated type with lymphovascular invasion. CONCLUSION: SPA nodal metastasis can be predicted when endoscopic treatment is initially planned. SPA nodal dissection should not be uniformly selected for T1b gastric cancer.

Rat trade and leptospirosis: Molecular epidemiology of Leptospira species in rats exported from Cambodia to Vietnam.

Rats are an important maintenance host of Leptospira spp., the causative agents of leptospirosis, a zoonotic disease with worldwide distribution. Rats are traded as food in the Mekong Delta, where Cambodia exports tons of rats to Vietnam. Handling wild rats is a potential health risk, but the information on Leptospira spp. carried by rats traded in the region remains limited. In this study, we investigated the carriage of Leptospira spp. in rats exported from Cambodia to Vietnam using bacterial culture, nested PCR and DNA sequencing. Isolates were then assessed using serological analysis and whole-genome sequencing (WGS), and Leptospira DNA detected in rat kidney tissues was also analysed by multilocus sequence typing (MLST). Seventy-two rats (2 Bandicota indica, 57 Rattus argentiventer, 11 R. losea, 1 R. norvegicus and 1 R. rattus) were subjected to bacterial culture, and three L. borgpetersenii serogroup Javanica isolates were obtained from R. argentiventer (5.3%). WGS revealed that although Cambodian isolates were genetically related to L. borgpetersenii serogroup Javanica strains widely distributed in East and South-East Asian countries, they formed a different cluster from other strains. In addition to the three L. borgpetersenii sequences, the flaB sequence of L. interrogans was detected in 18 R. argentiventer and 7 R. losea kidney tissue samples (38.9%) using nested PCR followed by DNA sequencing. The L. interrogans flaB-positive samples were further analysed by MLST, revealing that seven housekeeping genes (glmU, pntA, sucA, tpiA, pfkB, mreA and caiB) contained novel sequences with distinct lineages from other sequence types. This study revealed a high prevalence of Leptospira spp. among rats exported from Cambodia to Vietnam, indicating a potential risk to people engaging in rat trade and demonstrating that a fastidious L. interrogans strain circulates among Cambodian rats.

Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

High production of egc2-related staphylococcal enterotoxins caused a food poisoning outbreak.

Staphylococcal enterotoxins (SEs) produced by enterotoxigenic Staphylococcus aureus in food cause staphylococcal food poisoning. We recently reported a foodborne outbreak due to S. aureus harboring new SE/SE-like (SEl) genes (seg, sei, sem, sen, seo, and selu) related to enterotoxin gene cluster (egc) 2 as with other research groups. However, the pathogenicity of SEs production remains unclear. Therefore, we herein investigated egc2-related SEs production from S. aureus isolates and leftover food items during a foodborne outbreak using a sandwich enzyme-linked immunosorbent assay suitable for the quantification of SEs. S. aureus isolates produced markedly high levels of egc2-related SEs, and the leftover food item "Sushi" contained SEs over the toxin dose that causes food poisoning symptoms. A representative isolate was subjected to whole-genome sequencing. The isolate was homologous with previously reported ST45 strains, particularly the unique genomic island νSaß structure mostly consisting of egc2. The present study indicates that egc2-related SEs are food poisoning causative agents based on high SE production levels within an actual foodborne outbreak.

INTENSIVE TREATMENT OF A CAPTIVE BORNEAN ELEPHANT (ELEPHAS MAXIMUS BORNEENSIS) INFECTED WITH MYCOBACTERIUM CAPRAE IN JAPAN.

In 2015, an estimated 17-year-old female Bornean elephant (Elephas maximus borneensis) at Fukuyama Zoo in Japan exhibited anorexia and significant weight loss. Pan-susceptible Mycobacterium tuberculosis complex (MTBC) was isolated from vaginal discharge, oral mucus, urine, and fecal samples by culture. The isolate was identified as Mycobacterium caprae by genetic analysis. Isoniazid, pyrazinamide, and levofloxacin were administered rectally. Body weight increased to normal, but subsequently decreased again. Elevation of liver enzymes occurred, likely related to the increase in isoniazid dosage. After recovery from side effects, the elephant's weight increased further. However, isoniazid-resistant M. caprae was isolated from oral mucus after anti-tuberculosis drug treatment for 9 mo. The regimen was changed to rifampicin, pyrazinamide, ethambutol, and levofloxacin, administered orally or rectally. The 18-mo treatment was completed in October 2018. This elephant has shown no clinical sign since. No MTBC-positive sample had been obtained as of March 2020.

Unique hemoglobin dynamics in female Tibetan highlanders.

BACKGROUND: Tibetan highlanders have adapted to hypoxic environments through the development of unique mechanisms that suppress an increase in hemoglobin (Hb) concentration even in high-altitude areas. Hb concentrations generally decrease with increasing age. However, in the highlands, chronic altitude sickness is known to occur in the elderly population. To investigate how aging in a hypoxic environment affects Hb levels in Tibetan highlanders, we focused on the Mustang people, who live above 3500 m. We tried to clarify the pure relationship between aging and Hb levels in a hypoxic environment. RESULTS: We found that the Hb concentration increased with increasing age in females but not in males. Multivariate analysis showed that age, pulse pressure, the poverty index, and vascular diameter were strongly correlated with the Hb concentration. CONCLUSIONS: We found unique Hb dynamics among female Tibetan highlanders. As seen in these Hb dynamics, there may be sex-based differences in the adaptive mechanism in Tibetan highlanders.

Genomic features of Mycobacterium avium subsp. hominissuis isolated from pigs in Japan.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in humans and pigs. There have been advances in genome analysis of MAH from human isolates, but studies of isolates from pigs are limited despite its potential source of infection to human. Here, we obtained 30 draft genome sequences of MAH from pigs reared in Japan. The 30 draft genomes were 4,848,678-5,620,788 bp in length, comprising 4652-5388 coding genes and 46-75 (median: 47) tRNAs. All isolates had restriction modification-associated genes and 185-222 predicted virulence genes. Two isolates had tRNA arrays and one isolate had a clustered regularly interspaced short palindromic repeat (CRISPR) region. Our results will be useful for evaluation of the ecology of MAH by providing a foundation for genome-based epidemiological studies.

Identification, characterization and expression of A-mating type genes in monokaryons and dikaryons of the edible mushroom Mycoleptodonoides aitchisonii (Bunaharitake).

Identifying the mating-type in Mycoleptodonoides aitchisonii is important for enhancing breeding and cultivation of this edible mushroom. To clarify the molecular mechanisms of the bipolar mating system in M. aitchisonii, the homeodomain protein gene 2 (Mahd2) was characterized. A genomic DNA fragment of Mahd2 in M. aitchisonii 50005-18 strain was 1,851 bp long and encoded a protein of 614 amino acids. Transcriptional analysis revealed that the expression of Mahd2 was higher in monokaryotic strains that produced clamp cells than in those that did not. The highest relative expression level of Mahd2 was observed in monokaryon TUFC 50005-4, which was capable of forming a true clamp. These results suggested that the formation of clamp cells is regulated by A-mating type homeodomain proteins, and the frequency of clamp cell formation might be promoted by high expression of the Mahd2 gene.

Antenatal Care Visits and Adverse Pregnancy Outcomes at a Hospital in Rural Western Province, Rwanda.

In many economically developing countries, and especially in the rural regions of sub-Saharan African coun-tries, there have been only limited investigations into the association between antenatal care (ANC) and adverse pregnancy outcomes. We obtained information on ANC and pregnancy outcomes between 2011 and 2016 from hospital files of pregnant women (n = 4,960) served at a rural hospital in Rwanda, and we examined the associa-tions between their ANC visits and the adverse pregnancy and neonatal outcomes by using univariate and mul-tivariate logistic regression models to estimate the odds ratios (ORs) and 95% confidence intervals (CIs). Most of the pregnant women had ≥ 4 ANC visits, but 39% (n = 1,911) did not have ≥ 3 visits before delivery. The prev-alence of low birth weight (LBW) and that of preterm birth (PTB) were 12% and 9.9%, respectively. Compared to the women who attended only one ANC visit, those who attended ≥ 4 ANC visits had lower risks of LBW (OR 0.20; 95%CI: 0.11-0.36) and PTB (OR 0.28; 95%CI: 0.11-0.76). Frequent ANC visits were also associ-ated with better postnatal outcomes of the newborns. Encouraging women to attend ANC visits before delivery can markedly reduce PTB-related and LBW-related complications, especially in resource-limited settings.