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

Genotyping of Mycoplasma pneumoniae strains isolated in Japan during 2019 and 2020: spread of p1 gene type 2c and 2j variant strains

We characterized 118 Mycoplasma pneumoniae strains isolated from three areas of Japan (Saitama, Kanagawa, and Osaka) during the period of 2019 and 2020. Genotyping of the p1 gene in these strains revealed that 29 of them were type 1 lineage (29/118, 24.6%), while 89 were type 2 lineage (89/118, 75.4%), thereby indicating that type 2 lineage was dominant in this period. The most prevalent variant of type 2 lineage was type 2c (57/89, 64%), while the second-most was type 2j, a novel variant identified in this study (30/89, 33.7%). Type 2j p1 is similar to type 2 g p1, but cannot be distinguished from reference type 2 (classical type 2) using the standard polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) with HaeIII digestion. Thus, we used MboI digestion in the PCR-RFLP analysis and re-examined the data from previous genotyping studies as well. This revealed that most strains reported as classical type 2 after 2010 in our studies were actually type 2j. The revised genotyping data showed that the type 2c and 2j strains have been spreading in recent years and were the most prevalent variants in Japan during the time-period of 2019 and 2020. We also analyzed the macrolide-resistance (MR) mutations in the 118 strains. MR mutations in the 23S rRNA gene were detected in 29 of these strains (29/118, 24.6%). The MR rate of type 1 lineage (14/29, 48.3%) was still higher than that of type 2 lineage (15/89, 16.9%); however, the MR rate of type 1 lineage was lower than that found in previous reports published in the 2010s, while that of type 2 lineage strains was slightly higher. Thus, there is a need for continuous surveillance of the p1 genotype and MR rate of M. pneumoniae clinical strains, to better understand the epidemiology and variant evolution of this pathogen, although M. pneumoniae pneumonia cases have decreased significantly since the COVID-19 pandemic

Rhabdomyolysis Associated with Antimicrobial Drug–Resistant Mycoplasma pneumoniae

We describe a case of rhabdomyolysis in a patient infected with antimicrobial drug–resistant Mycoplasma pneumoniae The patient’s acute-phase serum levels of interleukin-18 and tumor necrosis factor–α were high, which suggests a pathogenic role for M. pneumoniae. In an era of increasing antimicrobial drug resistance, a system for rapidly identifying resistant M. pneumoniae would be beneficial

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-7363N. 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 mosaicpenA-34) and ST-7363(harboring mosaicpenA-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 Neisseriaspecies, 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 withinNeisseriaspeciesintersect in driving the emergence and spread of drug-resistant gonorrhea. Keywords: Recombination, Horizontal gene transfer, Genomic epidemiology, Antimicrobial resistance, Phylogeny,Surveillance, Evolution,Neisseria gonorrhoea

Characterization and Molecular Analysis of Macrolide-Resistant Mycoplasma pneumoniae Clinical Isolates Obtained in Japan

In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, ≥256 μg/ml) and 1 was weakly resistant (MIC, 8 μg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan