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.

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.

A Salmonella enterica Serovar Oranienburg Clone Caused a Cluster of Bacteremia Cases in Persons With No Recognizable Underlying Diseases in Japan.

Background: Salmonella enterica subspecies enterica serovar Oranienburg (SO) is a foodborne pathogen but rarely causes systemic infections such as bacteremia. Between July and September 2018, bacteremia cases caused by SO were identified in 12 persons without any underlying medical conditions in the southern Kyushu area of Japan. Methods: Randomly amplified polymorphic DNA (RAPD) analysis was performed to investigate the genetic similarity of the 12 bacteremia-related strains and other Japanese isolates. Furthermore, a series of whole-genome sequence (WGS)-based phylogenetic analyses was performed with a global SO strain set (n = 1648). Results: The resolution power of RAPD was insufficient to investigate the genetic similarity between the bacteremia-related strains and other strains. WGS-based phylogenetic analyses revealed that the bacteremia-related strains formed a tight cluster along with 2 strains isolated from asymptomatic carriers in 2018 in the same area, with a maximum within-cluster single-nucleotide polymorphism (SNP) distance of 11. While several strains isolated in the United States and the United Kingdom were found to be closely related to the bacteremia-related strains, 2 strains isolated in 2016 in the southern Kyushu area were most closely related, with SNP distances of 4-11 and 5-10, and had the same plasmids as the bacteremia-related strains. Conclusions: The 12 bacteremia cases identified were caused by a single SO clone. As none of the bacteremia patients had any underlying diseases, this clone may be prone to cause bacteremia. Although further analyses are required to understand its virulence, particular attention should be given to this clone and its close relatives in the surveillance of nontyphoidal salmonellae.

Serogroup B invasive meningococcal disease (IMD) outbreak at a Japanese high school dormitory: An outbreak investigation report from the first IMD outbreak in decades.

PURPOSE: The first outbreak of invasive meningococcal disease (IMD) in decades occurred in a high school dormitory in 2011. This report aims to describe the results of the IMD outbreak investigation and to discuss current issues of IMD in Japan. METHODS: We conducted an epidemiological and microbiological investigation against the IMD outbreak of serogroup B among students and staff in a high school dormitory. Information on patients was collected to analyze risk factors for IMD. Control measures and public health actions were summarized. RESULTS: Three cases of meningitis and two cases of bacteremia were identified. Freshmen (15-16 years old) living in the dormitory with preceding cough were high-risk populations in this outbreak. Pulsed-field gel electrophoresis, multilocus sequence typing, and porA gene sequencing results revealed that all isolates were closely related to each other and had deep similarities to the domestic circulating meningococcal strain. The outbreak was terminated after promptly implementing control measures. Based on the results of our investigation, from April 2013, national infectious disease surveillance started to target meningococcal bacteremia as part of IMD, in addition to meningococcal meningitis, which was newly designated as a category II school infectious disease under the School Health and Safety Act. CONCLUSIONS: This outbreak has enhanced public health measures against IMD in Japan. The development of national guidelines for appropriate public health interventions on the IMD outbreak response including chemoprophylaxis is still needed.

Development of a simple and cost-effective gel-based duplex PCR method to identify both encapsulated and unencapsulated Neisseria meningitidis applicable under resource-limited conditions.

Tens of thousands of cases of invasive meningococcal diseases (IMD) with thousands of deaths are reported annually worldwide; however, only approximately 40 cases occur each year in Japan. Therefore, the majority of medical technologists in Japan have never performed or prepared for analyses of the causative agent, Neisseria meningitidis. Since IMD outbreaks have been reported at mass gathering events, the risk of IMD will increase in Japan in 2021 because of the Olympics. In the present study, we developed a new simple gel-based duplex PCR method that may be employed by the majority Japanese clinical laboratories. It is simple to perform and time- and cost-effectively identifies encapsulated and unencapsulated N. meningitidis by detecting the encapsulated N. meningitidis-specific ctrB and N. meningitidis-specific ggt genes. We consider this simple and cost-effective identification method to compensate for the lack of experience and resource-poor conditions in most Japanese laboratories in which N. meningitidis has rarely been examined.

Bordetella pertussis is a common pathogen in infants hospitalized for acute lower respiratory tract infection during the winter season.

INTRODUCTION: There is some evidence that Bordetella pertussis (B. pertussis) can co-infect with viral respiratory infections in young infants. METHODS: B. pertussis infection was studied by culture, polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP) from nasopharyngeal swabs (NPSs) in 49 infants < 12 months of age, who were admitted for lower respiratory tract infections during the winter season. Seven other possible viral pathogens were documented by antigen detection or PCR in NPSs. The clinical feature of infants with mixed infection of B. pertussis and respiratory viruses were examined. RESULTS: Overall, B. pertussis infection was found in 10 (20.4%) cases, nine were less than 6 months of age and seven were unvaccinated. Viral etiology was found in 41 (84%) cases and pertussis-viral co-infection was present in eight patients, five of whom had mixed infection with respiratory syncytial virus. Only the presence of staccato coughing, cyanosis, and lymphocytosis were significantly different in B. pertussis-positive cases compared with B. pertussis-negative cases. Of the 10 pertussis cases, only the culture-positive cases showed the typical symptoms and laboratory findings of pertussis in addition to virus-associated respiratory symptoms with severe hospital course, whereas cases identified as DNA-positive lacked the characteristics of pertussis and their clinical severities were the same as B. pertussis-negative cases. CONCLUSION: In the absence of typical paroxysmal cough and lymphocytosis, we should carefully consider diagnosis of pertussis in young children hospitalized for presumed viral respiratory illness according to local epidemiological surveillance.

Differential dynamics and impacts of prophages and plasmids on the pangenome and virulence factor repertoires of Shiga toxin-producing Escherichia coli O145:H28.

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our understanding of their dynamics. Here, we performed a systematic analysis of 239 draft genomes and 7 complete genomes of Shiga toxin (Stx)-producing Escherichia coli O145:H28, the major virulence factors of which are encoded by prophages (PPs) or plasmids. The results indicated that PPs are more stably maintained than plasmids. A set of ancestrally acquired PPs was well conserved, while various PPs, including Stx phages, were acquired by multiple sublineages. In contrast, gains and losses of a wide range of plasmids have frequently occurred across the O145:H28 lineage, and only the virulence plasmid was well conserved. The different dynamics of PPs and plasmids have differentially impacted the pangenome of O145:H28, with high proportions of PP- and plasmid-associated genes in the variably present and rare gene fractions, respectively. The dynamics of PPs and plasmids have also strongly impacted virulence gene repertoires, such as the highly variable distribution of stx genes and the high conservation of a set of type III secretion effectors, which probably represents the core effectors of O145:H28 and the genes on the virulence plasmid in the entire O145:H28 population. These results provide detailed insights into the dynamics of PPs and plasmids, and show the application of genomic analyses using a large set of draft genomes and appropriately selected complete genomes.

Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Legionella pneumophila and Other Legionella Species Isolated from Legionellosis Patients in Japan between 2008 and 2016.

The Legionella Reference Center in Japan collected 427 Legionella clinical isolates between 2008 and 2016, including 7 representative isolates from corresponding outbreaks. The collection included 419 Legionella pneumophila isolates, of which 372 belonged to serogroup 1 (SG1) (87%) and the others belonged to SG2 to SG15 except for SG7 and SG11, and 8 isolates of other Legionella species (Legionella bozemanae, Legionella dumoffii, Legionella feeleii, Legionella longbeachae, Legionella londiniensis, and Legionella rubrilucens). L. pneumophila isolates were genotyped by sequence-based typing (SBT) and represented 187 sequence types (STs), of which 126 occurred in a single isolate (index of discrimination of 0.984). These STs were analyzed using minimum spanning tree analysis, resulting in the formation of 18 groups. The pattern of overall ST distribution among L. pneumophila isolates was diverse. In particular, some STs were frequently isolated and were suggested to be related to the infection sources. The major STs were ST23 (35 isolates), ST120 (20 isolates), and ST138 (16 isolates). ST23 was the most prevalent and most causative ST for outbreaks in Japan and Europe. ST138 has been observed only in Japan, where it has caused small-scale outbreaks; 81% of those strains (13 isolates) were suspected or confirmed to infect humans through bath water sources. On the other hand, 11 ST23 strains (31%) and 5 ST120 strains (25%) were suspected or confirmed to infect humans through bath water. These findings suggest that some ST strains frequently cause legionellosis in Japan and are found under different environmental conditions.IMPORTANCELegionella pneumophila serogroup 1 (SG1) is the most frequent cause of legionellosis. Our previous genetic analysis indicated that SG1 environmental isolates represented 8 major clonal complexes, consisting of 3 B groups, 2 C groups, and 3 S groups, which included major environmental isolates derived from bath water, cooling towers, and soil and puddles, respectively. Here, we surveyed clinical isolates collected from patients with legionellosis in Japan between 2008 and 2016. Most strains belonging to the B group were isolated from patients for whom bath water was the suspected or confirmed source of infection. Among the isolates derived from patients whose suspected infection source was soil or dust, most belonged to the S1 group and none belonged to the B or C groups. Additionally, the U group was discovered as a new group, which mainly included clinical isolates with unknown infection sources.

The proline residue at position 319 of BvgS is essential for BvgAS activation in Bordetella pertussis.

Bordetella pertussis is the etiological agent of pertussis and produces various virulence factors, including pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN), most of which are positively regulated by the BvgAS two-component sensory transduction system. Here, we describe a B. pertussis isolate not expressing PT, FHA and PRN recovered from a pertussis patient. Sequencing revealed that the bvgS gene of this isolate contains a spontaneous mutation (C>A at position 955) causing the proline residue at position 319 of the BvgS protein to be substituted by threonine. Moreover, loss of PT, FHA and PRN expression was completely restored by complementation with a wild-type bvgAS locus, indicating that this non-synonymous substitution in bvgS leads to impaired BvgS function. Our findings indicate that the proline residue at position 319 in this protein plays an essential role in activation of the BvgAS system and, therefore, subsequent expression of Bvg-regulated virulence factors in B. pertussis.

Defining the Genome Features of Escherichia albertii, an Emerging Enteropathogen Closely Related to Escherichia coli.

Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified. Here, we sequenced the genome of 29 E. albertii strains (3 complete and 26 draft sequences) isolated from multiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1 Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors and a possibly active second T3SS known as ETT2 (E. coli T3SS 2) that is inactivated in E. coli. Although this organism has been observed to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based on these results, we have developed a nested polymerase chain reaction system to directly detect E. albertii. Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen.

Effects of connective tissue grafting on the facial gingival level following single immediate implant placement and provisionalization in the esthetic zone: a 1-year randomized controlled prospective study.

PURPOSE: This 1-year randomized controlled prospective study evaluated the implant success rate and peri-implant tissue response following single immediate implant placement and provisionalization (IIPP) with and without subepithelial connective tissue graft (SCTG) in the esthetic zone. MATERIALS AND METHODS: Implants were placed either IIPP with SCTG (test group) or IIPP without SCTG (control group). The implants were evaluated both clinically and radiographically before surgery, immediately after implant placement, and 3, 6, and 12 months after implant placement. Data were analyzed using Friedman, Wilcoxon signed-rank, and Mann-Whitney U tests at the significance level of α = .05. RESULTS: Twenty implants (10 test, 10 control) were placed in 20 patients (7 men, 13 women) between the ages of 27 and 87 (mean age, 52.6 years). At 1 year, all implants remained osseointegrated, with overall mean marginal bone changes of -0.01 mm and -0.14 mm for the test and control groups, respectively. Mean facial gingival level change was significantly more pronounced in the control group (-0.70 mm) than in the test group (-0.25 mm). The modified Plaque Index scores showed that patients were able to maintain a good level of hygiene throughout the study. At 1 year, the Papilla Index indicated that more than 50% papilla fill was observed in 75% of the test sites and 80% of the control sites. CONCLUSIONS: Within the limitations of this study, SCTG was shown to be beneficial in maintaining facial gingival level when performed in conjunction with IIPP procedures.

Immediate implant placement and provisionalization with and without a connective tissue graft: an analysis of facial gingival tissue thickness.

Facial gingival tissue thickness (FGTT) is important for an esthetically pleasing anterior restoration since it determines the soft tissue's ability to conceal the underlying restorative material. The purpose of this study was to investigate the change in FGTT after immediate implant placement and provisionalization with and without a connective tissue graft. Patients with a failing maxillary anterior tooth planned for immediate implant placement and provisionalization with (CT group) or without (NCT group) a subepithelial connective tissue graft were included in this study. After tooth extraction, direct measurement of the FGTT was performed; subsequent measurements were performed at the time of definitive prosthesis placement. Data were analyzed using independent and paired t tests at a significance level of α = .05. There was no statistically significant difference in the mean FGTT at tooth extraction between the CT and NCT groups. At prosthesis delivery, the mean FGTT for the CT group was significantly greater than that of the NCT group. The mean FGTT of both groups at prosthesis delivery was significantly higher than that at tooth extraction. The mean change in FGTT in the CT group was also significantly greater than that in the NCT group. Immediate implant placement and provisionalization in conjunction with a connective tissue graft is more likely to result in sufficient peri-implant tissue thickness to conceal underlying implant restorative materials than when performed without a connective tissue graft.

Transmission of Bordetella holmesii during pertussis outbreak, Japan.

We describe the epidemiology of a pertussis outbreak in Japan in 2010-2011 and Bordetella holmesii transmission. Six patients were infected; 4 patients were students and a teacher at the same junior high school. Epidemiologic links were found between 5 patients. B. holmesii may have been transmitted from person to person.

Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay.

A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.

In vitro production of bilirubin photoisomers by light irradiation using neoBLUE.

BACKGROUND: The light-emitting diode is used as one of the new light sources for phototherapy. NeoBLUE (Atom Medical, Tokyo, Japan) incorporates blue light-emitting diodes for the treatment of neonatal hyperbilirubinemia. The authors compared the in vitro efficacy of neoBLUE with conventional phototherapy devices. METHODS: The three light devices used included neoBLUE and two conventional phototherapy devices with six blue-white (BW) or six green (GR) fluorescent tubes. A bilirubin/human serum albumin solution (15 mg/dL) in 200 x 300 mm elliptical bag was irradiated with each three light device. The average light intensity of neoBLUE, BW and GR was 22.5, 10.2 and 2.6 microW/cm(2) per nm, respectively, for the irradiated area. Bilirubin photoisomers and native bilirubin were measured by high-performance liquid chromatography. RESULTS: In neoBLUE, BW and GR, the respective production rate of cyclobilirubin was 6.0, 3.7 and 3.9 x 10(-2) mg/dL/min, and the respective (4Z, 15E)-bilirubin/(4Z, 15Z)-bilirubin ratio after irradiation was 0.44, 0.33 and 0.12; the (4Z, 15Z)-bilirubin reduction rate at 20 min after irradiation was 60, 68 and 82%, respectively. The reduction rate of (4Z, 15Z)-bilirubin correlated with the (4Z, 15E)-bilirubin/(4Z, 15Z)-bilirubin ratio. CONCLUSION: Phototherapy using the neoBLUE under high level may be clinically more effective than therapy using the conventional light source from the results of the production rate of cyclobilirubin.

Advanced renal cell carcinoma in which a combination of IFN-alpha and meloxicam was thought to be effective.

An 83-year-old man with left renal cell carcinoma (RCC; pT4N0M0) was treated with postoperative combined subcutaneous injection therapy of alpha interferon (IFN-alpha) and IFN gamma-1a (IFN-gamma-1a). Metastasis to the pleura occurred 3 months after surgery. The metastatic lesion grew while the treatment was changed to intramuscular injection of IFN-alpha-2b due to the presence of severe general malaise, which seemed to be caused by IFN-alpha and IFN-gamma therapy. As melosalgia associated with sciatica was also severe, treatment with meloxicam, which is known as a potent cyclooxigenase-2 inhibitor among commercially available non-steroidal anti-inflammatory drugs, was combined, resulting in significant improvement in activity of daily life, 43.2% decrease in the size of the pleural metastasis and complete regression of retroperitoneal residual tumor.