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Sequencing of most Treponema pallidum (T. 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 modelling 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 β- 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.

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.

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.Author SummaryEach year, millions of new cases of venereal and congenital syphilis, caused by the bacterium Treponema pallidum (T. pallidum) subsp. pallidum, are diagnosed worldwide, resulting in significant morbidity and mortality. Alongside endemic circulation of syphilis in low-income countries, disease resurgence in high-income nations has underscored the need for a vaccine. Due to prior technological limitations in culturing and sequencing the organism, the extent of the genetic diversity within modern strains of T. pallidum subsp. pallidum remains poorly understood, hampering development of a broadly protective vaccine. In this study, we obtained 196 near-complete T. pallidum genomes directly from clinical swabs from eight countries across six continents. Of these, 191 were identified as T. pallidum subsp. pallidum, including 90 Nichols clade genomes. Bayesian analysis revealed a high degree of variance in mutation rate among subclades. Interestingly, a Nichols subclade with a particularly high mutation rate harbors a non-synonymous mutation in a putative DNA repair helicase. Coupling sequencing data with protein structure prediction, we identified multiple novel amino acid variants in several proteins previously identified as potential vaccine candidates. Our data help inform current efforts to develop a broadly protective syphilis vaccine.

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.

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.

Additional file 5: Figure S1. Whole-genome sequence, dated phylogeny, resistance patterns of the antimicrobials, and genetic polymorphisms in the ST-1901-associated sub-lineage carrying penA-34. Figure S2. Whole-genome alignment of the ancestral strain encoding penA-5, three strains encoding penA-34 dating from 2005, and the reference WHO_Y (F89) strain encoding penA-34. Figure S3. Frequency distribution of other 21 STs in the ST-1901-associated lineage. Figure S4. Amino acid sequence alignment of penA-34 and 35. Figure S5. Nucleotide sequence alignment of penA and its downstream sequences in the ST-1901-associated lineage. Figure S6 Nucleotide sequence alignment of penA and its downstream sequences in the ST-7363-associated lineage. Figure S7. Maximum-likelihood tree of the recombined region (orange in Fig. 3).

Additional file 1. Supplementary Methods and Results.

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.

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.

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.

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.

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. 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. gonorrhoeae penA-60.001. This assay system can differentiate N. gonorrhoeae strains carrying mosaic penA-60.001 from strains carrying other penA alleles.

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.

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.

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.

In recent years, syphilis notifications have increased dramatically in Japan. We performed molecular typing and analyzed macrolide resistance ofTreponema pallidumsamples collected from four clinics and a hospital in Tokyo and Osaka prefectures in 2017.Macrolide resistant strain type 14d/f was found significantly more in heterosexual syphilis cases compared with those strains identified in men who have sex with men (MSM) syphilis cases. The proportion of 14d/f among heterosexuals was 79% (31/39) compared to 37% (7/19) among MSM [OR, 6.6 (95% CI, 1.7 to 26.7); P=0.002]. 83% (50/60) of the strains were identified as macrolide resistant with an A2058G mutation at the 23S rRNA gene. 90% (35/39) of the heterosexual strains were macrolide resistant, relative to 58% (11/19) of MSM strains; the odds of having the resistant mutation was considerably higher in heterosexuals compared with MSM [OR, 6.4 (95% CI, 1.3 to 33.5); P=0.02]. Heterosexual females and males showed similar distributions, and the results remained the same when restricted to men.The strain type distribution and frequency of macrolide resistance differed substantially between heterosexual and MSM syphilis samples, suggesting distinct epidemiologic profiles for the two communities and insight into syphilis transmission dynamics in Japan.

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.

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.

Introduction Gonorrhoea is a global health concern because N. gonorrhoeae has now acquired resistance to antibiotics including the 3 rd generation cephalosporin, ceftriaxone. Mosaic structure in penicillin-binding protein 2 gene ( penA ) is the main cause of ceftriaxone resistance which is formed by natural transformation of DNA of ceftriaxone-resistant (CRO R ) commensal Neisseria spp. However, none of the previous studies has fully elucidated the source of transformed DNA. In this study, we show that genetic comparison between CRO R - N. gonorrhoeae strains (GU140106 and FC428) and CRO R – Neisseria commensals. Methods CRO R - Neisseria commensals were isolated from pharyngeal swabs. Genome sequence was obtained by MiSeq, Illumina, and the sequence of penA flanking region ( mraW-NGO 1540- penA-murE-dcaA ) was obtained using the N. gonorrhoeae FA1090 as a reference sequence. Alignment of the compared sequences were prepared by CLUSTALW. Species identification of Neisseria commensals was performed using phylogeny constructed by 53 ribosomal proteins ( rps ) sequences. Results Based on the sequence similarity of penA gene, two CRO R - Neisseria commensals were speculated to be the origin of each mosaic penA of GU140106 and FC428. Both of the candidates were N. cinerea according to the rps phylogeny. The DNA region including penA originated from N. cinerea which consist the part of mosaic penA in the CRO R - N. gonorrhoeae was about 1.3 kb including of parts of penA and murE . Further analysis revealed that mosaic penA of GU140106 occurred by recombination between CRO R - N. cinerea and CRO S - N. gonorrhoeae which the NG-MAST type was same with GU140106. The mosaic penA of FC428 was also thought to emerged in the same way with GU140106. However, there was a limitation because of the lack of CRO S -N. gonorrhoeae that has the same NG-MAST type with FC428. Conclusion The two CRO R -N. gonorrhoeae , GU140106 and FC428, might acquire resistance by homologous recombination with Neisseria commensals, and N. cinerea might be the donor of mosaic penA which causes resistance to ceftriaxone in N. gonorrhoeae .

Objectives Antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally. We report the first verified treatment failure of gonorrhoea with 2 g of azithromycin (extended-release formulation) in Japan and characteristics of the corresponding N. gonorrhoeae isolates. Methods Pre- and post-treatment isolates (n = 4) were investigated by Etest for antimicrobial susceptibility. The isolates were examined for molecular epidemiology by multilocus sequence typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multiple-locus variable-number tandem repeat analysis (MLVA), and for the presence of azithromycin resistance determinants (23S rRNA gene mutations, erm genes and mtrR mutations). Results All isolates were resistant to azithromycin (MIC 4 mg/L) and ciprofloxacin, but remained susceptible to cefixime, ceftriaxone and spectinomycin. All isolates were assigned to MLST ST1901 and NG-MAST ST1407 and three of four isolates possessed MLVA profile 8-3-21-16-1. All isolates contained the previously described C2599T mutation (N. gonorrhoeae numbering) in all four 23S rRNA alleles and the previously described single-nucleotide (A) deletion in the mtrR promoter region. Conclusions This verified treatment failure occurred in a patient infected with an MLST ST1901/NG-MAST ST1407 strain of N. gonorrhoeae. While this international strain commonly shows resistance or decreased susceptibility to multiple antimicrobials, including extended-spectrum cephalosporins, the strain reported here remained fully susceptible to the latter antimicrobials. Hence, two subtypes of azithromycin-resistant gonococcal MLST ST1901/NG-MAST ST1407 appear to have evolved and to be circulating in Japan. Azithromycin should not be recommended as a single antimicrobial for first-line empirical treatment of gonorrhoea.

We have characterized in detail a new ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain (FC428) isolated in Japan in 2015. FC428 differed from previous ceftriaxone-resistant strains and contained a novel mosaic penA allele encoding a new mosaic penicillin-binding protein 2 (PBP 2). However, the resistance-determining 3′-terminal region of penA was almost identical to the regions of two previously reported ceftriaxone-resistant strains from Australia and Japan, indicating that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread.

Recently, the first Neisseria gonorrhoeae strain (H041) that is highly resistant to the extended-spectrum cephalosporin (ESC) ceftriaxone, the last remaining option for empirical first-line treatment, was isolated. We performed a detailed characterization of H041, phenotypically and genetically, to confirm the finding, examine its antimicrobial resistance (AMR), and elucidate the resistance mechanisms. H041 was examined using seven species-confirmatory tests, antibiograms (30 antimicrobials), porB sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and sequencing of ESC resistance determinants ( penA , mtrR , penB , ponA , and pilQ ). Transformation, using appropriate recipient strains, was performed to confirm the ESC resistance determinants. H041 was assigned to serovar Bpyust, MLST sequence type (ST) ST7363, and the new NG-MAST ST4220. H041 proved highly resistant to ceftriaxone (2 to 4 μg/ml, which is 4- to 8-fold higher than any previously described isolate) and all other cephalosporins, as well as most other antimicrobials tested. A new penA mosaic allele caused the ceftriaxone resistance. In conclusion, N. gonorrhoeae has now shown its ability to also develop ceftriaxone resistance. Although the biological fitness of ceftriaxone resistance in N. gonorrhoeae remains unknown, N. gonorrhoeae may soon become a true superbug, causing untreatable gonorrhea. A reduction in the global gonorrhea burden by enhanced disease control activities, combined with wider strategies for general AMR control and enhanced understanding of the mechanisms of emergence and spread of AMR, which need to be monitored globally, and public health response plans for global (and national) perspectives are important. Ultimately, the development of new drugs for efficacious gonorrhea treatment is necessary.

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.

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.

A glycerol dehydrogenase gene was selected as a multicopy suppressor rescuing the reduced hilA expression in the Salmonella enterica serovar Typhimurium cpxA mutant. A substrate of the enzyme, 1,2-propanediol, repressed hilA expression. The 1,2-propanediol-mediated repression at 150 mM, but not that at 300 mM, was abrogated by blocking the catabolism producing propionate from 1,2-propanediol.

The InvE protein positively regulates the expression of virulence genes ipaBCD in Shigella sonnei . The InvE has significant homology with ParB of plasmid P1, which is known as a plasmid partitioning factor with DNA binding ability. Although the DNA binding activity of InvE has been predicted, it is not known whether the DNA binding activity is necessary for type III secretion system-associated gene expression. In this study, we determined the transcription start site of the icsB-ipaBCD operon ( ipa operon) and constructed a series of deletions of the icsB promoter region in the Escherichia coli K-12 background. The deletion study revealed that an 86-bp region upstream of the icsB transcription start site was essential for expression of the ipa operon, where the ParB binding motif (ParB BoxA-like sequence) was observed. Purified glutathione S -transferase-InvE fusion protein bound directly to the −93 to −54 region (designating the icsB transcription start site as nucleotide +1) containing the ParB BoxA-like sequence. These results indicated that InvE bound directly to the promoter region.

virF is the master regulator which activates the virulence determinant genes of Shigella spp. such as ipaBCD and virG . We previously reported that expression of virF itself is regulated in a pH-dependent manner and that cpxA , a sensor of a two-component regulatory system, is involved in this regulation (S. Nakayama and H. Watanabe, J. Bacteriol. 177:5062–5069, 1995). Disruption of cpxR , which has been thought to be the cognate response regulator of cpxA (J. Dong, S. Iuchi, H.-S. Kwan, Z. Lue, and E. C. C. Lin, Gene 136:227–230, 1993), abolished virF expression almost completely. Purified CpxR bound directly to the upstream region of virF . Binding capacity was enhanced when CpxR was phosphorylated by coincubation with acetyl phosphate in vitro. Furthermore, we observed that phosphorylated CpxR could activate virF transcription in vitro. These results clearly indicated that CpxR was an essential activator for virF expression and strongly suggested that the binding of phosphorylated CpxR to the target site upstream of the virF gene induced a direct activation of virF transcription.

A total of 122 Neisseria gonorrhoeae isolated in the Tokyo metropolitan area in 2005-2011 were collected and analyzed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and for their susceptibility to azithromycin and ceftriaxone. All 122 strains were susceptible to ceftriaxone, but 8 strains were azithromycin-resistant, defined as an azithromycin MIC ≥ 1 μg/ml. The 8 azithromycin-resistant strains were in 6 NG-MAST types, 3 strains in NG-MAST type 1407 and each of the other 5 strains in a different NG-MAST type. NG-MAST type 1407 strains are multidrug-resistant and are disseminated worldwide.

In 2009, the first high-level ceftriaxone-resistant Neisseria gonorrhoeae strain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) of Neisseria gonorrhoeae isolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193 N. gonorrhoeae isolates were collected and the MICs (μg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 μg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic type penA alleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of the penA mosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistant N. gonorrhoeae strain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.

Yersinia enterocolitica produces heat-stable enterotoxin (Y-ST) as one of its virulence factors. The yst gene, however, frequently and spontaneously becomes inactive (silent) during storage, which is accompanied by concurrent changes in some biological properties such as colony morphology, growth rate, carbon fermentation and ornithine decarboxylase activity. Northern blot analysis revealed that the level of mRNA for yst was repressed. To investigate the regulatory region, we transformed a yst-silent strain with a chromosomal gene library of Y-ST producing an isogenic counterpart. Out of 3604 clones, one clone resumed the Y-ST production and concurrently other biological properties. An open reading frame in this clone was designated as yrp, yersinia regulator for pleiotropic phenotype. Deduced from the nucleotide sequence, Yrp was a small protein of I01 amino acids with no similarity with any regulatory factor described, but showed high homology with an Escherichia coli host factor 1 required for Q beta-replicase, and with an Azorhizobium caulinodans NrfA required for the expression of nifA. The yrp gene mutation caused decreased negative supercoiling of plasmids, as did hfq. The yrp gene could similarly complement Y-ST production in two other silent strains of Y. enterocolitica. In all three silent strains examined, we found various mutations in the yrp region.

Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM) gene variant, bla TEM-135 , that may be a precursor in the transitional stage of a traditional bla TEM-1 gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins in N. gonorrhoeae , was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of the bla TEM-135 gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the bla TEM-135 allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a bla TEM variant ( bla TEM-135 ) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

In Neisse ria gonorrhoeae , the mosaic type of penA , which encodes penicillin-binding protein 2 (PBP 2), is associated with reduced susceptibility to oral cephalosporins. To investigate the relatedness of N. gonorrhoeae clinical isolates with reduced susceptibility, we sequenced the penA genes of 32 isolates. Five different amino acid sequence types of PBP 2 were identified, but all seemed to be derivatives of pattern X of PBP 2 (PBP 2-X). However, multilocus sequence typing of the isolates showed that the isolates belonged to six different sequence types. As PBP 2-X was identified in three different sequence types, horizontal transfer of the penA allele encoding PBP2-X was suggested. We demonstrated that the penA gene could be transferred from an isolate with reduced susceptibility to a sensitive isolate by natural transformation. Comparison of the sequence of the penA -flanking regions of 12 transformants with those of the donor and the recipient suggested that at least a 4-kb DNA segment, including the penA gene, was transferred. During horizontal transfer, some of the penA alleles also acquired variations due to point mutations and genetic exchange within the allele. Our results provide evidence that the capacity for natural transformation in N. gonorrhoeae plays a role in the spread of chromosomal antibiotic resistance genes and the generation of diversity in such genes.

To the Editor: Spread of multidrug-resistant Neisseria gonorrhoeae is a major public health concern. Effective antimicrobial therapy is a key element in gonorrhea control. However, N. gonorrhoeae has developed resistance to multiple classes of antimicrobial drugs, including β-lactams, tetracyclines, and fluoroquinolones (1–3). Even an extended-spectrum oral cephalosporin-resistant, cefixime-resistant N. gonorrhoeae has emerged, and cefixime has now been withdrawn from use in Japan. Best practice treatment is limited to injectable extended-spectrum cephalosporins, such as ceftriaxone and spectinomycin. The emergence of ceftriaxone-resistant N. gonorrhoeae threatens effective disease control. We identified a novel ceftriaxone-resistant N. gonorrhoeae isolated from a 31-year-old female commercial sex worker; MIC of ceftriaxone for this isolate was high (2 µg/mL). The woman visited a clinic in Kyoto for a routine examination for sexually transmitted infections in January 2009. Although she had no obvious symptoms or signs, a throat sample collected on her first visit yielded a positive result for N. gonorrhoeae by the strand displacement amplification test (ProbeTec ET, Becton Dickinson, Franklin Lakes, NJ, USA), but a vaginal sample taken at the same time was negative. After 2 weeks, another throat sample was positive for N. gonorrhoeae when cultured on Thayer-Martin medium, and the patient subsequently received 1 g ceftriaxone intravenously. Her pharyngeal sample was also N. gonorrhoeae positive by strand displacement amplification test on the third visit 2 weeks later, and further ceftriaxone treatment was prescribed. However, a culture for test of cure was not conducted because reinfection was considered. A negative result was finally obtained in April 2009. The culture showed positive reactions in oxidase and catalase tests. Gram staining showed gram-negative diplococci. The ID-test HN-20 Rapid system (Nissui, Tokyo, Japan) classified the bacterium as N. gonorrhoeae. Susceptibility was determined by the agar dilution method (4). For this strain, named H041, MIC of ceftriaxone was high (2 µg/mL), and the strain was highly resistant to penicillin G (4 µg/mL), cefixime (8 µg/mL), and levofloxacin (32 µg/mL). However, it demonstrated susceptibility to spectinomycin (16 µg/mL) and reduced susceptibility to azithromycin (0.5 µg/mL). To characterize the ceftriaxone-resistant N. gonorrhoeae H041, multilocus sequence typing characterized the strain as ST7363 (5), which is the predominant sequence type (ST) among cefixime-resistant clones (6). N. gonorrhoea multiantigen sequence typing (NG-MAST) was also performed (7). The NG-MAST strategy uses 2 genes, por and tbpB, for porin and a transferrin-binding protein, respectively. NG-MAST indicated that the strain H041 was ST4220 and contained the por2594 allele and the tbpB10 allele. NG-MAST 4220 is a novel ST. However, the tbpB10 allele is the most frequently observed allele (76.5%) among multilocus sequence typing-ST7363 N. gonorrhoeae strains (n = 81) (M. Ohnishi, unpub. data). Molecular typing suggested that the novel ceftriaxone-resistant N. gonorrhoeae, H041, is closely related to the ST7363 cefixime-resistant N. gonorrhoeae. Therefore, we compared SpeI-digested genomic DNA banding patterns of strain H041 with those of other N. gonorrhoeae strains by using pulsed-field gel electrophoresis as described (8). Four ST7363 strains, including N. gonorrhoeae H041, and 4 ST1901 strains (another major ST among cefixime-resistant N. gonorrhoeae strains) (6) were analyzed. The banding pattern of SpeI digested H041 genomic DNA was similar to that of other ST7363 strains and indistinguishable from that of cefixime-resistant but ceftriaxone-susceptible NG0207 (Figure). Figure Pulsed-field gel electrophoresis patterns of ceftriaxone-resistant Neisseria gonorrhoeae strain H041 and other multilocus sequence typing (MLST) ST7363 and ST1901 strains. SpeI-digested genomic DNA from ceftriaxone-resistant N. gonorrhoeae H041, 3 of ... We describe the emergence of ceftriaxone-resistant N. gonorrhoeae, isolated from a pharyngeal specimen from a female commercial sex worker. At 2 µg/mL, the MIC was 4-fold higher than that of the previously reported ceftriaxone nonsusceptible strain (9). Our susceptibility testing suggests that only azithromycin and spectinomycin are effective drugs for treating this strain. In this case, eradication was successful, although N. gonorrhoeae colonization of the pharynx may just be tempory because the pharynx is not an ideal site for N. gonorrhoeae growth. From the routine examinations of commercial sex workers during January–March 2009, 40 N. gonorrhoeae were isolated in the clinic, but no other ceftriaxone-resistant strains were isolated. There is no evidence of dissemination of this strain in Kyoto. Three independent molecular subtyping methods indicated that the ceftriaxone-resistant H041 strain was N. gonorrhoeae, and it might originate from an ST7363 cefixime-resistant N. gonorrhoeae clone. There are several possible mechanisms for the acquisition of resistance, including formation of a new mosaic type penA allele as penA-X cefixime resistance and acquisition of an extended-spectrum β-lactamase gene. The H041 strain did not produce β-lactamase in a nitrocephin test. Further molecular analysis is needed to elucidate the precise mechanism of the ceftriaxone resistance of the H041 strain. The emergence of ceftriaxone-resistant N. gonorrhoeae raises concerns for controlling gonorrhea because ceftriaxone is widely recommended and the first-line treatment for gonorrhea around the world. N. gonorrhoeae has a potential to gain an extraordinarily high MIC to ceftriaxone. Surveillance for ceftriaxone-resistant N. gonorrhoeae should be strengthened.

Pulsed-field gel electrophoresis (PFGE) analysis revealed that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains had considerable variations in their genomes. This study investigated whether or not the molecular profile of Shiga toxin (Stx) 1- and Stx2-converting phages isolated from EHEC O157:H7 strains, derived from various sources in the USA and Japan, corresponded to the variations of host strains' genotypes as determined by PFGE. A total of 51 Stx-converting phages including 12 Stx1-converting phages and 37 Stx2-converting phages was isolated from seven USA isolates and 20 Japanese isolates. The average Dice coefficient values showed 44% similarity between phage DNAs in Stx2-converting phages digested with SmaI and 55% in Stx1-converting phages digested with HindIII, indicating considerable variation among phage DNA. In particular, restriction fragment length polymorphism (RFLP) patterns of Stx2-converting phage DNA varied according to the PFGE type of their host strain, which suggests that the phage genomes have altered their genotypic characteristics with those of host genomes. However, there are several exceptions: the RFLP patterns of some Stx2-converting phages were quite similar irrespective of the different genotypes of the host strains, indicating that horizontal transfer of Stx2-converting phage may also occur under some circumstances.