High-throughput nanopore sequencing of Treponema pallidum tandem repeat genes arp and tp0470 reveals clade-specific patterns and recapitulates global whole genome phylogeny.

Sequencing of most Treponema pallidum genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modeling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged ß-sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Loop-Mediated Isothermal Amplification Assay for Identifying Neisseria gonorrhoeae Nonmosaic penA-Targeting Strains Potentially Eradicable by Cefixime.

Treatment regimens for gonorrhea have limited efficacy worldwide due to the rapid spread of antimicrobial resistance. Cefixime (CFM) is currently not recommended as a first-line treatment for gonorrhea due to the increasing number of resistant strains worldwide. Nonetheless, Neisseria gonorrhoeae strains can be eradicated by CFM at a 400 mg/day dose, provided that the strains are CFM responsive (MIC ≤ 0.064 mg/L). To develop a nonculture test for predicting the CFM responsiveness of N. gonorrhoeae strains, we developed an assay to detect N. gonorrhoeae nonmosaic penA using loop-mediated isothermal amplification (LAMP). To avoid false-positive reactions with commensal Neisseria spp. penA, we amplified specific regions of the N. gonorrhoeae penA (NG-penA-LAMP1) and also the nonmosaic N. gonorrhoeae penA (NG-penA-LAMP3). This assay was validated using isolated N. gonorrhoeae (n = 204) and Neisseria spp. (n = 95) strains. Clinical specimens (n = 95) with confirmed positivity in both culture and real-time PCR were evaluated to validate the system. The combination of the previously described NG-penA-LAMP1 and our new NG-penA-LAMP3 assays had high sensitivity (100%) and specificity (100%) for identifying N. gonorrhoeae carrying the nonmosaic type. To determine whether CFM could be applicable for gonorrhea treatment without culture testing, we developed a LAMP assay that targets penA allele-specific nonmosaic types for use as one of the tools for point-of-care testing of antimicrobial resistance. IMPORTANCE Neisseria gonorrhoeae is among the hot topics of "resistance guided therapy," one of the top 5 urgent antimicrobial threats according to the Centers for Disease Control and Prevention (CDC). There is a need either to develop new agents or to make effective use of existing agents, with the current limited number of therapeutic agents available. Knowing the drug susceptibility information of the target microorganism prior to treating patients is very useful in selecting an effective antibiotic, especially in gonococcal infections where drug resistance is prominent, and is also important in preventing treatment failure. In this study, we developed a new method for obtaining drug susceptibility profiles of Neisseria gonorrhoeae using the loop-mediated isothermal amplification (LAMP) method. The LAMP assay does not require expensive devices. Therefore, this method is expected to be a tool for point-of-care testing of antimicrobial resistance for individualized treatment in the future.

Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar.

In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains-including 191 T. pallidum subsp. pallidum-sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543-1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

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.

Phylogenetic and genetic characterization of Treponema pallidum strains from syphilis patients in Japan by whole-genome sequence analysis from global perspectives.

Japan has had a substantial increase in syphilis cases since 2013. However, research on the genomic features of the Treponema pallidum subspecies pallidum (TPA) strains from these cases has been limited. Here, we elucidated the genetic variations and relationships between TPA strains in Japan (detected between 2014 and 2018) and other countries by whole-genome sequencing and phylogenetic analyses, including syphilis epidemiological surveillance data and information on patient sexual orientation. Seventeen of the 20 strains in Japan were SS14- and the remaining 3 were Nichols-lineage. Sixteen of the 17 SS14-lineage strains were classified into previously reported Sub-lineage 1B. Sub-lineage 1B strains in Japan have formed distinct sub-clusters of strains from heterosexuals and strains from men who have sex with men. These strains were closely related to reported TPA strains in China, forming an East-Asian cluster. However, those strains in these countries evolved independently after diverging from their most recent common ancestor and expanded their genetic diversity during the time of syphilis outbreak in each country. The genetic difference between the TPA strains in these countries was characterized by single-nucleotide-polymorphism analyses of their penicillin binding protein genes. Taken together, our results elucidated the detailed phylogenetic features and transmission networks of syphilis.

Characterization of 2 Neisseria gonorrhoeae Strains With High-Level Azithromycin Resistance Isolated in 2015 and 2018 in Japan.

ABSTRACT: We identified and characterized the first 2 Neisseria gonorrhoeae strains with high-level azithromycin resistance isolated in Japan. These were in the clade of ceftriaxone- and azithromycin-resistant strains isolated in Australia and the United Kingdom. The multilocus sequence typing, N. gonorrhoeae multiantigen sequence typing, and N. gonorrhoeae sequence typing for antimicrobial resistance types of these strains were found in gonococci from eastern Asia.

A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum.

Introduction. Macrolides could be a potential alternative treatment for Treponema pallidum infections in patients; however, macrolide-resistant T. pallidum is spreading rapidly worldwide.Hypothesis/Gap Statement. There are presently no alternatives to serological tests for syphilis that can be used to evaluate therapeutic effects due to the fact that T. pallidum cannot be cultured in vitro.Aim. In this study, we constructed a method for rapidly identifying T. pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs).Methodology. A set of LAMP primers was designed to span nucleotide positions 2058 and 2059 in 23S rRNA. A PNA clamping probe was also designed to be complementary to the wild-type sequence (A2058/A2059) and positioned to interfere with both the annealing of the 3' end of the backward inner primer and the concomitant extension. Prior to the LAMP assay, swab samples from suspected syphilitic lesions were boiled for DNA extraction.Results. The assay had an equivalent detection limit of 1.0×101 copies/reaction and showed specificity against 38 pathogens. In the presence of a 4 µM PNA probe, LAMP amplified up to 1.0×101 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1.0×103 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays.Conclusion. We developed a PNA-mediated LAMP method that enabled us to rapidly identify T. pallidum and determine its macrolide susceptibility via a culture-independent protocol.

Epidemiology, molecular strain types, and macrolide resistance of Treponema pallidum in Japan, 2017-2018.

BACKGROUND: Japan has seen a substantial increase in syphilis cases since 2013 and Tokyo and Osaka prefectures accounted for about 40% of all cases in Japan. Therefore, focusing on these 2 prefectures, we assessed syphilis cases detected during 2017-2018, combining epidemiological information with molecular typing data. METHODS: Using data from surveillance reports, we described syphilis cases by gender, age, transmission route, and stage of syphilis. Clinical specimens were collected from syphilis patients in Tokyo and Osaka prefectures. Molecular typing was performed by analyzing Treponema pallidum arp, tpr, and tp0548 genes, with partial sequencing of the 23S rRNA genes for macrolide resistance. RESULTS: Between 2017 and 2018, the number of syphilis cases increased from 3934 to 4588 among males and 1895 to 2414 among females, with similar age and gender distributions during the period. The predominant strain type was 14d/f (71%, 73/103), found more frequently in women who have sex with men (86%, 25/29) and men who have sex with women (83%, 39/47) than in men who have sex with men (MSM) (33%, 9/27). The majority of the strains from heterosexuals (97%, 76/78) were macrolide-resistant, considerably higher than those from MSM (59%, 20/34). The molecular profiles in each sexual-transmission group remained similar during the 2 years. CONCLUSIONS: The epidemiological and molecular features of syphilis remained similar throughout the period, with consistent differences in strain type and macrolide resistance distributions between MSM and heterosexual cases. These findings suggest a predominantly heterosexual epidemic where the dynamics of syphilis transmission remained unchanged during 2017-2018.

A Loop-Mediated Isothermal Amplification Assay Targeting Neisseria gonorrhoeae penA-60.001.

Ceftriaxone (CRO) is widely used as the first-line treatment for gonococcal infections. However, CRO-resistant Neisseria gonorrhoeae strains carrying mosaic penA-60.001 have emerged recently and disseminated worldwide. To meet the urgent need to detect these strains, we report here a loop-mediated isothermal amplification (LAMP) assay system that targets N. gonorrhoeaepenA-60.001. This assay system can differentiate N. gonorrhoeae strains carrying mosaic penA-60.001 from strains carrying other penA alleles.

Clonal expansion and spread of the ceftriaxone-resistant Neisseria gonorrhoeae strain FC428, identified in Japan in 2015, and closely related isolates.

OBJECTIVES: Ceftriaxone resistance in Neisseria gonorrhoeae is a major public health concern globally because a high-dose (1 g) injection of ceftriaxone is the only remaining option for empirical monotherapy of gonorrhoea. The ceftriaxone-resistant gonococcal strain FC428, cultured in Osaka in 2015, is suspected to have spread nationally and internationally. We describe the complete finished genomes of FC428 and two closely related isolates from Osaka in 2015, and examine the genomic epidemiology of these isolates plus three ceftriaxone-resistant gonococcal isolates from Osaka and Hyogo in 2016-17 and four ceftriaxone-resistant gonococcal isolates cultured in 2017 in Australia, Canada and Denmark. METHODS: During 2015-17, we identified six ceftriaxone-resistant gonococcal isolates through our surveillance systems in Kyoto, Osaka and Hyogo. Antimicrobial susceptibility testing (six antimicrobials) was performed using Etest. Complete whole-genome sequences of the first three isolates (FC428, FC460 and FC498) from 2015 were obtained using PacBio RS II and Illumina MiSeq sequencing. The three complete genome sequences and draft genome sequences of the three additional Japanese (sequenced with Illumina MiSeq) and four international ceftriaxone-resistant isolates were compared. RESULTS: Detailed genomic analysis suggested that the Japanese isolates (FC428, FC460, FC498, KU16054, KM383 and KU17039) and the four international MLST ST1903 isolates from Australia, Canada and Denmark formed four linked subclades. CONCLUSIONS: Using detailed genomic analysis, we describe the clonal expansion of the ceftriaxone-resistant N. gonorrhoeae strain FC428, initially identified in 2015 in Japan, and closely related isolates. FC428 and its close relatives show some genomic diversity, suggesting multiple genetic subclades are already spreading internationally.

A real-time PCR assay for detecting a penA mutation associated with ceftriaxone resistance in Neisseria gonorrhoeae.

OBJECTIVES: Ceftriaxone (CRO) resistance is spreading worldwide, and hindering the effective treatment of gonococcal infections. This study developed a detection system for the genomic DNA of CRO-resistant Neisseria gonorrhoeae (N. gonorrhoeae) strains, in order to improve the surveillance of antimicrobial resistance. METHODS: A real-time PCR assay targeting the penA gene of recently isolated CRO-resistant N. gonorrhoeae strains was designed. Primer and probe sequence information was obtained from sequence comparisons between penA of Neisseria spp. and penA of CRO-resistant N. gonorrhoeae strains. RESULTS: Using this assay, a positive reaction was observed using the genomic DNA of three strains (GU140106, FC428, and A8806). The assay was evaluated using genomic DNA of 204 N. gonorrhoeae and 95 Neisseria spp. isolates with known minimum inhibitory concentrations of CRO. Following PCR assays for these strains, three FC428-related strains were positively identified, which possessed penA-60.001, whereas the remaining 201 N. gonorrhoeae strains and 95 Neisseria spp. strains were negative. CONCLUSIONS: A real-time PCR-based assay was designed to detect the genomic DNA of strains harbouring mosaic penA-59.001 (GU140106), penA-60.001 (FC428), and penA-64.001 (A8806) alleles and to discriminate them from N. gonorrhoeae and Neisseria spp. strains harbouring other genes.

Infective Endocarditis Due to Treponema pallidum: A Case Diagnosed Using Polymerase Chain Reaction Analysis of Aortic Valve.

Syphilis is a sexually transmitted disease caused by Treponema pallidum. Syphilitic aortitis might coexist in a dysfunctional aortic valve, but the etiology remains unclear, because microbiological diagnosis is difficult. A 62-year-old man with low-grade fever was diagnosed with aortitis and infective endocarditis, due to Treponema pallidum infection, using polymerase chain reaction analysis. This case suggests that syphilis might cause infective endocarditis.

Molecular Typing and Macrolide Resistance Analyses of Treponema pallidum in Heterosexuals and Men Who Have Sex with Men in Japan, 2017.

In recent years, syphilis notifications have increased dramatically in Japan. We carried out molecular typing and macrolide resistance analyses of Treponema pallidum subsp. pallidum samples collected from patients at four clinics and a hospital in Tokyo and Osaka prefectures in 2017. The macrolide resistant strain type 14d/f (SS14-like clade) was found in significantly more cases of syphilis among heterosexuals than in those among men who have sex with men (MSM); i.e., 79% (31/39) of the strains from heterosexuals were 14d/f compared to 37% (7/19) of those from MSM (odds ratio [OR], 6.6; 95% confidence interval [CI], 1.7 to 26.7; P = 0.002). In addition, 83% (50/60) of the strains were identified as macrolide resistant with an A2058G mutation in the 23S rRNA gene; 90% (35/39) of the strains from heterosexuals were macrolide resistant compared to 58% (11/19) of those from MSM. The odds of having the resistant mutation were considerably higher in the former (OR, 6.4; 95% CI, 1.3 to 33.5; P = 0.02). Heterosexual women and heterosexual men showed similar distributions, and the association remained the same when restricted to men. The strain type distribution and the prevalence of macrolide resistance differed substantially between syphilis strains from heterosexual cases and from MSM cases, suggesting distinct epidemiologic profiles for the two communities and providing important insight into the dynamics of syphilis in Japan.

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.

Cooperative Recognition of Internationally Disseminated Ceftriaxone-Resistant Neisseria gonorrhoeae Strain.

Ceftriaxone remains a first-line treatment for patients infected by Neisseria gonorrhoeae in most settings. We investigated the possible spread of a ceftriaxone-resistant FC428 N. gonorrhoeae clone in Japan after recent isolation of similar strains in Denmark (GK124) and Canada (47707). We report 2 instances of the FC428 clone in Australia in heterosexual men traveling from Asia. Our bioinformatic analyses included core single-nucleotide variation phylogeny and in silico molecular typing; phylogenetic analysis showed close genetic relatedness among all 5 isolates. Results showed multilocus sequence type 1903; N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) 233; and harboring of mosaic penA allele encoding alterations A311V and T483S (penA-60.001), associated with ceftriaxone resistance. Our results provide further evidence of international transmission of ceftriaxone-resistant N. gonorrhoeae. We recommend increasing awareness of international spread of this drug-resistant strain, strengthening surveillance to include identifying treatment failures and contacts, and strengthening international sharing of data.

Neisseria cinerea with High Ceftriaxone MIC Is a Source of Ceftriaxone and Cefixime Resistance-Mediating penA Sequences in Neisseria gonorrhoeae.

Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae, but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 µg/ml) caused ceftriaxone resistance (MIC, 1 µg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3'-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.

Cerebral Syphilitic Gumma Can Arise Within Months of Reinfection: A Case of Histologically Proven Treponema pallidum Strain Type 14b/f Infection With Human Immunodeficiency Virus Positivity.

A 44-year-old man with human immunodeficiency virus positivity developed cerebral gumma 6 months after appropriate therapy for secondary syphilis. It was surgically resected and histologically, Treponema pallidum (14b/f, a relatively rare strain type) was proven. A complete set of modern techniques was performed to depict rare complication of this classic disease.