Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.

MORPHOLOGIC OBSERVATION AND FIRST MOLECULAR CHARACTERIZATION OF ENCYCLOMETRA JAPONICA (DIGENEA: ENCYCLOMETRIDAE) IN THE DIGESTIVE TRACT OF WILD TIGER KEELBACKS (RHABDOPHIS TIGRINUS) IN JAPAN.

Encyclometra japonica Yoshida and Ozaki, 1929 is a fluke that parasitizes the digestive tract of snakes. When the species was first reported, it was originally characterized on the basis of morphologic features, namely, the characteristics of the 2 testes located obliquely in the anterior-posterior direction, and the position of the ventral sucker one-fourth to one-third from the anterior extremity. Thereafter, more specimens with other morphologic variations were reported, and a new morphologic feature for species discrimination was proposed. However, the proposal is uncertain, and the criteria for morphologic discrimination between E. japonica and other Encyclometra species remain debatable. Thus, morphologically discriminable characteristics on the basis of a sufficient number of specimens are required, and molecular methods for species identification that allow for a more objective assessment need to be established. Therefore, in the present study, nucleotide sequences of 18S, 28S ribosomal deoxyribonucleic acid and cytochrome c oxidase subunit I (COI) of E. japonica were determined along with detailed morphologic observations for the first time. Key characteristics were observed in all specimens, whereas some morphologic variations were observed in the symmetry of the cecum. The COI sequences, which are known to be variable genetic regions, were identical among all specimens; therefore, all of them were considered the same species. This result clarifies the high morphologic variation in E. japonica. The 18S and 28S sequences were 99.78% and 99.11-99.19% similar to those of Encylometra colubrimurorum. The high degree of homology between these genes indicates that E. japonica and E. colubrimurorum are closely related but independent species. The present data will be used for the identification of E. japonica and to evaluate the relationships within the genus Encyclometra.

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean.

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.

Detection and molecular characteristics of Rhytidodoides sp. (Digenea: Rhytidodidae) from the gall bladder of green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan.

Trematodes of the genus Rhytidodoides are parasitic in marine turtles. Of the already known species, Rhytidodoides similis Price, 1939, occurs especially in the gall bladder. In this study, we surveyed 73 green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan, and detected Rhytidodoides sp. from the gall bladders of 18 turtles. A detailed morphological analysis revealed that the forebody of Rhytidodoides sp. differed slightly in shape from that of R. similis. There has been no information on DNA sequences of the family Rhytidodidae. A molecular phylogeny based on 28S rDNA sequences of Rhytidodoides sp. and related taxa suggested that the Rhytidodidae is sister to the other families of Echinostomatoidea. The intraspecific diversity of Rhytidodoides sp. was examined by using DNA sequences of mitochondrial cytochrome c oxidase subunit 1 gene (COI). The population genetic features of the COI haplotypes demonstrated that Rhytidodoides sp. is highly diverse in the Ogasawara Islands. The DNA sequences determined in this study will contribute to the species identification of congeners and the taxonomic reconsideration of the Echinostomatoidea.

Morphological observation and first molecular characterization of Grassenema procaviae Petter, 1959 (Cosmocercoidea: Atractidae) in the stomach of Cape hyrax (Procavia capensis) raised in a zoo in Japan.

The superfamily Cosmocercoidea comprises three families: Cosmocericidae, Kathlaniidae and Atractidae. Information on the nucleotide sequences of the Cosmocercoidea is quite limited, and the molecular classification of the whole superfamily has been slow to progress. The genus Grassenema of the family Atractidae is a parasitic nematode group that occurs in the digestive tract of hyraxes and includes three species: Grassenema procaviae, G. dendrohyraci, and G. hyracis. The type species of the genus, G. procaviae, was isolated from the digestive tract of Cape hyraxes (Procavia capensis) and has the potential to cause gastric ulcers. Although G. procaviae is a common parasite of Cape hyraxes, no genetic information for the parasite is currently available. In this study, we obtained the first genomic sequences of G. procaviae and performed detailed morphological observations. Furthermore, molecular phylogenetic analysis was performed, and the taxonomic position of the parasite was evaluated using 18S and 28S rDNA sequences. Those data will be useful for molecular identification of G. procaviae and future phylogenetic analysis within the Atractidae.

Emergence and evolution of antimicrobial resistance genes and mutations in Neisseria gonorrhoeae.

BACKGROUND: Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to third-generation cephalosporins, mainly due to mosaic penA alleles. These two STs were first detected in Japan; however, the timeline, mechanism, and process of emergence and spread of these mosaic penA alleles to other countries remain unknown. METHODS: We studied the evolution of penA alleles by obtaining the complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1075 strains sampled from 35 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996 to 2005 and reconstructed a phylogeny including 88 previously sequenced genomes. RESULTS: Based on an estimate of the time-of-emergence of ST-1901 (harboring mosaic penA-34) and ST-7363 (harboring mosaic penA-10), and > 300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996-2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by recombination with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, by independent mutations rather than horizontal gene transfer. Data in the literature suggest that the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. CONCLUSIONS: Our findings highlight how antibiotic use and recombination across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.

Infection by and Molecular Features of Learedius learedi (Digenea: Schistosomatoidea) in Green Sea Turtles (Chelonia mydas) on the Ogasawara Islands, Japan.

Learedius learedi Price, 1934 , is a blood fluke found in sea turtles, and the adult fluke parasitizes the cardiovascular system of the host. In this study we surveyed 46 green sea turtles, Chelonia mydas, on the Ogasawara Islands, Japan, and blood flukes were detected in the heart and blood vessels of 26 turtles. The flukes were identified as L. learedi based on a detailed morphological description. In addition, molecular identification and characterization of the parasite were performed. The nucleotide sequences of nuclear internal transcribed spacer 2 (ITS2) regions were almost identical to those of L. learedi reported previously, but not to those of Hapalotrema spp., which is the closest related genus. The nucleotide sequences of the 28S ribosomal DNA region formed a single clade with those of the reference L. learedi in the phylogenetic tree, but not with those of Hapalotrema spp. Therefore, the nucleotide sequences of ITS2 and 28S are robust markers for distinguishing L. learedi from other species. The nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (COI) region were analyzed to evaluate the genetic variations in L. learedi. The COI haplotypes revealed the extremely high genetic diversity of the species as well as the host turtles on the Ogasawara Islands. The haplotype frequency in the mitochondrial DNA of the green sea turtles on the Ogasawara Islands is known to be significantly different from those in other Pacific rookeries. Although the number of analyzed flukes is small in this study, no haplotype was close to that in other areas; on the basis of the data, we hypothesized that L. learedi differentiated along with the host turtles on the Ogasawara Islands.

Prevalence of Salmonella enterica Subspecies enterica in Red-Eared Sliders Trachemys scripta elegans Retailed in Pet Shops in Japan.

We investigated the prevalence of Salmonella in 227 small red-eared sliders (Trachemys scripta elegans) from 2006 to 2008. A total of 130 turtles (57.3%) tested positive for S. enterica subsp. enterica. Twenty-two serotypes including S. Montevideo, S. Newport, S. Pomona, S. Braenderup, S. Sandiego, and S. Litchfield were identified. Salmonella strains with closely related pulsed field gel electrophoresis (PFGE) patterns were isolated from several shops located in different areas from 2006 to 2008. Antimicrobial resistance was detected among strains of S. Montevideo, S. Newport, S. Braenderup, S. Sandiego, and S. Litchfield. The relatedness of antimicrobial resistance and PFGE profiles was not observed. The PFGE patterns of S. Poona strains isolated in 2006 and 2008 and the causative strains of turtle-associated salmonellosis in 2006 were identical. These results revealed a high prevalence of Salmonella enterica subsp. enterica in red-eared sliders retailed in Japan. In addition, genetically closely-related strains of turtle-associated Salmonella were repeatedly introduced into Japan over the study period and were distributed widely in Japan. These Salmonella strains present a risk of a widely disseminated outbreak of turtle-associated salmonellosis.

Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages.

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

Outbreak of Enterohemorrhagic Escherichia coli O157:H7 Infection Associated with Minced Meat Cutlets Consumption in Kanagawa, Japan.

An outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection occurred in October 2016 in Kanagawa, Japan. A total of 61 patients and 17 asymptomatic cases of EHEC O157:H7 infection were confirmed by laboratory testing. Among them, 24 patients were hospitalized and 4 developed hemolytic-uremic syndrome. An epidemiological investigation revealed that this outbreak of EHEC O157:H7 infection was associated with the consumption of uncooked minced meat cutlets that were sold frozen at branches of a supermarket chain. The implicated uncooked meat cutlets were made of a mixture of minced beef, pork, onions, and eggs. All 40 meat cutlets tested from one particular batch were positive for EHEC O157:H7. The patterns observed on pulsed-field gel electrophoresis of strains isolated from the affected patients and meat cutlets were identical. The bacterial counts of EHEC O157:H7 and E. coli in meat cutlets ranged from 2.3 to 110 most-probable-number (MPN)/g and from 240 to 4,600 MPN/g, respectively. There are currently no national regulatory standards to ensure the safety of these types of meat products in Japan. Consumers should ensure that such products are cooked thoroughly and that safe food handling procedures are used to prevent infection.

Characterization of Shiga toxin-producing Escherichia coli from feces of sika deer (Cervus nippon) in Japan using PCR binary typing analysis to evaluate their potential human pathogenicity.

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.

Outbreak of Legionnaire's Disease Caused by Legionella pneumophila Serogroups 1 and 13.

In Japan, hot springs and public baths are the major sources of legionellosis. In 2015, an outbreak of Legionnaires' disease occurred among 7 patients who had visited a spa house. Laboratory investigation indicated that L. pneumophila serogroup 1 and 13 strains caused the outbreak and that these strains were genetically related.

Prevalence and Characteristics of Salmonella and Campylobacter in Retail Poultry Meat in Japan.

This study was performed to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Salmonella enterica subsp. enterica and Campylobacter spp. in poultry meat, and to analyze the association of genetic types of these bacteria with their geographical distribution and antimicrobial resistance profiles. Salmonella and Campylobacter isolates have been detected, respectively, in 54 and 71 samples out of 100 samples tested. Nine Salmonella serotypes were found, including S. enterica subsp. enterica serovar Infantis (33%), Schwarzengrund (12%), Manhattan (9%), and others. Campylobacter jejuni and C. coli were detected in 64 (64%) and 14 (14%) samples, respectively. S. enterica subsp. enterica isolates were very frequently resistant to tetracycline (78.3%) and streptomycin (68.3%). Many C. jejuni and C. coli isolates were resistant to sulfamethoxazole/trimethoprim (90.5%), nalidixic acid (47.3%), ampicillin (45.9%), and ciprofloxacin (40.5%). Cluster analysis was performed for the Salmonella isolates using pulsed-field gel electrophoresis (PFGE) data. For Campylobacter isolates, the cluster analysis was based on both PFGE and comparative genomic fingerprinting. The molecular typing results were compared with the information about antimicrobial resistance and geographical locations in which the poultry meat was produced. This analysis revealed that C. jejuni strains with a particular genotype and antimicrobial resistance profile are spreading in specific areas of Japan.

[Molecular Characterization of Salmonella enterica subsp. enterica Serovar 4: b: -].

Salmonella is a major causative agent of food borne diseases. Recently, monophasic strains of Salmonella, such as S. enterica 4: i: -, have been frequently reported. Here, we investigated the genetic background of S. enterica 4: b: - using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. A total of 10 strains of S. enterica (I) 4: b: - were examined and compared with 34 strains including serovar Paratyphi B and Paratyphi B var Java, Schleissheim, and II b: -. All I 4: b: - strains were negative for hin which encodes an invertase that converts the H phases, and six were also negative for fljB, which encodes the second phase of the H antigen. An MLST analysis identified 12 sequence types (ST) and 6 ST complexes (STC) from the 44 strains. A clustering analysis of PFGE patterns almost corresponded to the STC. The monophasic I 4: b: - strains were assigned to 3 STCs (19, 32 and 155), corresponding to those of Paratyphi B var. Java or a monophasic strain according to the data of this and previous studies. These findings suggest that the monophasic strains examined in this study might have been derived from multiple clones of Paratyphi B var Java. This study shows the usefulness of molecular typing as complementation tools of the conventional serotyping system.

Emergence and evolution of internationally disseminated cephalosporin-resistant Neisseria gonorrhoeae clones from 1995 to 2005 in Japan.

BACKGROUND: Neisseria gonorrhoeae strains with resistance to extended-spectrum cephalosporins (ESCs), last options for first-line monotherapy of gonorrhoea, likely emerged and initially disseminated in Japan, followed by international transmission. In recent years, multi-locus sequence typing (MLST) ST1901 and N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 isolates with the mosaic penicillin-binding protein (PBP) 2 XXXIV have accounted for most ESC resistance globally. Our aim was to elucidate the initial emergence and transmission of ESC-resistant strains by detailed examination of N. gonorrhoeae isolates from 1995 to 2005 in Kanagawa, Japan. METHODS: N. gonorrhoeae isolates were examined phenotypically (n = 690) and genetically (n = 372) by agar dilution method (cefixime, ceftriaxone and ciprofloxacin), penA gene sequencing, MLST and NG-MAST. RESULTS: Already in 1995, one cefixime-resistant (CFM-R) isolate was found, which is the first CFM-R isolate described globally. After 1996, the prevalence of CFM-R and CFM-decreased susceptibility (CFM-DS) isolates significantly increased, with the peak resistance level in 2002 (57.1% CFM-R). In 1997-2002, the CFM-R MLST ST7363 strain type with the mosaic PBP 2 X was predominant among CFM-R/DS isolates. The first CFM-R/DS MLST ST1901 clone(s), which became the predominant CFM-R/DS strain type(s) already in 2003-2005, possessed the mosaic PBP 2 X, which was possibly originally transferred from the MLST ST7363 strains, and subsequently acquired the mosaic PBP 2 XXXIV. The first MLST ST1901 and NG-MAST ST1407 isolate was identified in Kanagawa already in 2003. CONCLUSIONS: The two main internationally spread cefixime-resistant gonococcal clones, MLST ST7363 and ST1901 (NG-MAST ST1407 most frequent internationally) that also have shown their capacity to develop high-level ceftriaxone resistance (superbugs H041 and F89), likely emerged, evolved and started to disseminate in the metropolitan area, including Kanagawa, in Japan, which was followed by global transmission.

Turtle-Associated Salmonella Infections in Kanagawa, Japan.

In this paper, we examine 2 case reports for different reptile-related Salmonella enterica subspecies enterica serotypes. In case 1, a 5-year-old boy presented with gastroenteritis caused by S. enterica subspecies enterica serovar Poona. The suspected source of infection was a turtle kept at the patient's home. In case 2, a 4-year-old boy presented with gastroenteritis caused by S. enterica subspecies enterica serovar Abony. The Pulsed-field gel electrophoresis analysis suggested that a tortoise kept at the patient's home was the source of infection. This paper presents a review of the literature and an examination of cases regarding turtle-associated salmonellosis in Japan.

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined. METHODS: Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation. RESULTS: Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates. CONCLUSIONS: Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children.

Prevalence of Salmonella in wild snakes in Japan.

A total of 87 wild snakes of 6 species in 2 families collected in Japan were examined for the presence of Salmonella. The prevalence of Salmonella was 58.6%, and that of Salmonella enterica subspecies I, which includes most human pathogenic serotypes, accounted for 12.6%. S. enterica subspecies I was isolated from Japanese grass snakes and Japanese four-striped snakes, and the isolates belonged to 6 serotypes: S. enterica subspecies enterica serotypes Eastbourne, Mikawashima, Narashino, Newport, Saintpaul, and Thompson. The prevalence of S. enterica subspecies IIIb was higher (41.4%) than that of S. enterica subspecies I, and it was isolated from 4 snake species. The prevalence of Salmonella enterica subspecies and isolation of serotypes that are commonly detected in reptiles and human salmonellosis suggest that wild snakes may become a source of Salmonella infection.