Human norovirus (HuNoV) GII.P17-GII.17 (Kawasaki2014 variant) reportedly emerged in 2014 and caused gastroenteritis outbreaks worldwide. To clarify the evolution of both VP1 and RNA-dependent RNA polymerase (RdRp) regions of GII.P17-GII.17, we analyzed both global and novel Japanese strains detected during 2013-2017. Time-scaled phylogenetic trees revealed that the ancestral GII.17 VP1 region diverged around 1949, while the ancestral GII.P17 RdRp region diverged around 2010. The evolutionary rates of the VP1 and RdRp regions were estimated at ~2.7 × 10-3 and ~2.3 × 10-3 substitutions/site/year, respectively. The phylogenetic distances of the VP1 region exhibited no overlaps between intra-cluster and inter-cluster peaks in the GII.17 strains, whereas those of the RdRp region exhibited a unimodal distribution in the GII.P17 strains. Conformational epitope positions in the VP1 protein of the GII.P17-GII.17 strains were similar, although some substitutions, insertions and deletions had occurred. Strains belonging to the same cluster also harbored substitutions around the binding sites for the histo-blood group antigens of the VP1 protein. Moreover, some amino acid substitutions were estimated to be near the interface between monomers and the active site of the RdRp protein. These results suggest that the GII.P17-GII.17 virus has produced variants with the potential to alter viral antigenicity, host-binding capability, and replication property over the past 10 years.

In the context of the coronavirus disease 2019 (COVID-19) pandemic, environmental surveillance for the detection of SARS-CoV-2 has become increasingly important. Studies have demonstrated that the SARS-CoV-2 RNA is present in the feces of infected individuals; further, its presence in wastewater has been reported. However, an optimized method for its detection in sewage has not yet been adequately investigated. Therefore, in this study, the efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater was investigated using two quantitative PCR assays. In particular, wastewater samples were collected from a manhole located in the commercial district of a metropolitan region in Japan, where COVID-19 is highly prevalent, and two wastewater treatment plants (WWTPs). The samples were concentrated using four separate methods, namely, electronegative membrane adsorption, polyethylene glycol precipitation, ultrafiltration, and solid precipitation. Each method revealed a significant concentration of pepper mild mottle virus (PMMoV) RNA, which is an indicator virus for wastewater. As expected, non-enveloped PMMoV RNA was enriched in the supernatant fraction such that relatively low concentrations were detected in the solid fraction of the wastewater samples. In contrast, higher SARS-CoV-2 RNA concentrations were consistently detected in the solid fractions compared with the supernatant fractions based on the other methods that were investigated in this study. Spearman's correlation tests showed that the SARS-CoV-2 RNA concentrations in wastewater samples from the WWTP were significantly correlated with the number of COVID-19 cases recorded during the data collection period. These results demonstrate that viral recovery from the solid fraction is an effective method for SARS-CoV-2 RNA surveillance in an aqueous environment.

After the first case of coronavirus disease 2019 (COVID-19) in Japan on 15 January 2020, multiple nationwide COVID-19 clusters were identified by the end of February. The Japanese government focused on mitigating the emerging COVID-19 clusters by conducting active nationwide epidemiological surveillance. However, an increasing number of cases continued to appear until early April 2020, many with unclear infection routes and no recent history of travel outside Japan. We aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from the COVID-19 cases that appeared until early April 2020 and to characterize their genealogical networks in order to demonstrate possible routes of spread in Japan. Nasopharyngeal specimens were collected from patients, and reverse transcription-quantitative PCR tests for SARS-CoV-2 were performed. Positive RNA samples were subjected to whole-genome sequencing, and a haplotype network analysis was performed. Some of the primary clusters identified during January and February 2020 in Japan descended directly from the Wuhan-Hu-1-related isolates from China and other distinct clusters. Clusters were almost contained until mid-March; the haplotype network analysis demonstrated that the COVID-19 cases from late March through early April may have created an additional large cluster related to the outbreak in Europe, leading to additional spread within Japan. In conclusion, genome surveillance has suggested that there were at least two distinct SARS-CoV-2 introductions into Japan from China and other countries.IMPORTANCE This study aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from COVID-19 cases and to characterize their genealogical networks to demonstrate possible routes of spread in Japan. We found that there were at least two distinct SARS-CoV-2 introductions into Japan, initially from China and subsequently from other countries, including Europe. Our findings can help understand how SARS-CoV-2 entered Japan and contribute to increased knowledge of SARS-CoV-2 in Asia and its association with implemented stay-at-home/shelter-in-place/self-restraint/lockdown measures. This study suggested that it is necessary to formulate a more efficient containment strategy using real-time genome surveillance to support epidemiological field investigations in order to highlight potential infection linkages and mitigate the next wave of COVID-19 in Japan.

Monoclonal antibody therapy is a promising option for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and a cocktail of antibodies (REGN-COV) has been administered to infected patients with a favorable outcome. However, it is necessary to continue generating novel sets of monoclonal antibodies with neutralizing activity because viral variants can emerge that show resistance to the currently utilized antibodies. Here, we isolated a new cocktail of antibodies, EV053273 and EV053286, from peripheral blood mononuclear cells derived from convalescent patients infected with wild-type SARS-CoV-2. EV053273 exerted potent antiviral activity against the Wuhan wild-type virus as well as the Alpha and Delta variants in vitro, whereas the antiviral activity of EV053286 was moderate, but it had a wide-range of suppressive activity on the wild-type virus as well as the Alpha, Beta, Delta, Kappa, Omicron BA.1, and BA.2 variants. With the combined use of EV053273 and EV053286, we observed similar inhibitory effects on viral replication as with REGN-COV in vitro. We further assessed their activity in vivo by using a mouse model infected with a recently established viral strain with adopted infectious activity in mice. Independent experiments revealed that the combined use of EV053273 and EV053286 or the single use of each monoclonal antibody efficiently blocked infection in vivo. Together with data showing that these two monoclonal antibodies could neutralize REGN-COV escape variants and the Omicron variant, our findings suggest that the EV053273 and EV053286 monoclonal antibody cocktail is a novel clinically applicable therapeutic candidate for SARS-CoV-2 infection.

The Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo.

Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.

We recently detected a novel variant of an IMP-type metallo-β-lactamase gene (blaIMP-68) from meropenem-resistant but imipenem-susceptible Klebsiella pneumoniae TA6363 isolated in Tokyo, Japan. blaIMP-68 encodes a Ser262Gly point mutant of IMP-11, and transformation experiments showed that blaIMP-68 increased the MIC of carbapenems in recipient strains, whereas the MIC of imipenem was not greatly increased relative to that of other carbapenems, including meropenem. Kinetics experiments showed that IMP-68 imipenem-hydrolyzing activity was lower than that for other carbapenems, suggesting that the antimicrobial susceptibility profile of TA6363 originated from IMP-68 substrate specificity. Whole-genome sequencing showed that blaIMP-68 is harbored by the class 1 integron located on the IncL/M plasmid pTMTA63632 (88,953 bp), which was transferable via conjugation. The presence of plasmid-borne blaIMP-68 is notable, because it conferred antimicrobial resistance to carbapenems, except for imipenem, on Enterobacteriaceae and will likely affect treatment plans using antibacterial agents in clinical settings.IMPORTANCE IMP-type metallo-β-lactamases comprise one group of the "Big 5" carbapenemases. Here, a novel blaIMP-68 gene encoding IMP-68 (harboring a Ser262Gly point mutant of IMP-11) was discovered from meropenem-resistant but imipenem-susceptible Klebsiella pneumoniae TA6363. The Ser262Gly substitution was previously identified as important for substrate specificity according to a study of other IMP variants, including IMP-6. We confirmed that IMP-68 exhibited weaker imipenem-hydrolyzing activity than that for other carbapenems, demonstrating that the antimicrobial susceptibility profile of TA6363 originated from IMP-68 substrate specificity, with this likely to affect treatment strategies using antibacterial agents in clinical settings. Notably, the carbapenem resistance conferred by IMP-68 was undetectable based on the MIC of imipenem as a carbapenem representative, which demonstrates a comparable antimicrobial susceptibility profile to IMP-6-producing Enterobacteriaceae that previously spread in Japan due to lack of awareness of its existence.

During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

To assess the prevalence and antibiotic resistance profile of Mycoplasma genitalium detected from urogenital/rectal swab samples obtained from MSM in Tokyo, Japan.

The plasmid is a very well-known mobile genetic element that participates in the acquisition of virulence genes, such as staphylococcal enterotoxins (SEs), via horizontal transfer. SEs are emetic toxins and causative agents in staphylococcal food poisoning (SFP). We herein identified the types of plasmids harbored by seven SFP isolates and examined their production of plasmid-related SE/SEl to determine whether the new types of plasmid-related SE or SE-like (SEl) toxins (i.e. SElJ and SER) were involved in SFP. These isolates harbored pIB485-like plasmids, and all, except for one isolate, produced SElJ and SER. The amount of SER produced by each isolate accounted for the highest or second highest percentage of the total amount of SE/SEl produced. These new types of plasmid-related SE/SEls as well as classical SE may play a role in SFP. The seven isolates were classified into two SED-production types; a high SED-production type (>500 ng/ml) and no SED-production type. A nucleotide sequencing analysis revealed that three plasmids harbored by the SED-non-producing isolates had a single-base deletion in the sed gene with a resulting stop codon (from 233 amino acids of the intact SED to 154 amino acids of the mutant SED (mSED)). A real-time reverse transcription-PCR analysis showed that the mRNA of the msed gene was transcribed in the isolates. If the msed gene was translated as a protein, mSED may act as an emetic toxin instead of intact SED.

Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.

The continuous emergence of carbapenemase-producing Enterobacteriaceae (CPE) presents a great public health challenge. Mitigation of CPE spread in the environment is crucial, particularly from a One Health perspective. Here we describe the isolation of CPE strain SNI47 from influent water of a sewage treatment plant in Japan. SNI47 was identified as Klebsiella quasipneumoniae subsp. quasipneumoniae by phylogenetic analysis and was resistant to β-lactams, including carbapenems. Of four plasmids detected from SNI47, the 185,311-bp IncA/C2 plasmid (pTMSNI47-1), which carried 10 drug resistance genes, including genes for four β-lactamases (blaCTX-M-2, blaDHA-1, blaKHM-1, and blaOXA-10), was transferred to Escherichia coli J53 via conjugation. The MICs of all tested β-lactams for the transconjugant were higher than for the recipient. We constructed recombinant plasmids, into which each β-lactamase gene was inserted, and used them to transform E. coli DH5α cells, demonstrating that KHM-1 enhanced carbapenem resistance. In addition, these β-lactamases were responsible for a wide-spectrum β-lactam resistance acquisition with mutual compensation. KHM-1, recognized as a rare type of metallo-β-lactamase, was detected in a transferable plasmid, from a sewage treatment plant, involved in horizontal gene transfer. The detection of such plasmids raises a health risk alarm for CPE dissemination.IMPORTANCE In our investigation of urban wastewater in Japan, carbapenem-resistant Klebsiella quasipneumoniae subsp. quasipneumoniae was isolated that carried the pTMSNI47-1 plasmid, which carries four β-lactamase genes and has transferability among Enterobacteriaceae pTMSNI47-1 was found to encode a rarely reported carbapenemase, KHM-1. Cooperative effects of β-lactamases encoded by pTMSNI47-1 appeared to have broad-spectrum resistance to β-lactams. The detection of the KHM-1 gene in urban wastewater suggests that such a rare antimicrobial resistance (AMR) gene can be pooled in the environment, potentially emerging as an AMR determinant in a pathogen. When the number of β-lactamase resistance genes is increased in one plasmid, the transfer of this plasmid can confer broad-spectrum resistance to β-lactams, even if the individual gene confers narrow-spectrum resistance. The present study adds important information about the potential risk of sewage treatment plants as reservoirs and environmental suppliers of AMR genes, contributing to the public health from a One Health perspective.

Human Sarcocystis infections are known to be caused by the ingestion of raw or undercooked beef or pork containing mature sarcocysts of Sarcocystis hominis or S. suihominis, respectively. In addition, several cases of parasitic food poisoning in Japan have recently been reported after consumption of raw horsemeat containing sarcocysts of S. fayeri. In this study, the presence of sarcocysts in 28 horsemeat and 121 beef samples collected in Tokyo was investigated. Sarcocysts of S. fayeri were found in 16 horsemeat samples. Sarcocysts of S. hominis were not detected in beef samples, while sarcocysts of S. cruzi were detected in 60 beef samples. In addition, S. hirsuta and S. bovini were isolated only from New Zealand beef samples. Bradyzoites in sarcocysts collected from 62/73 sarcocyst-positive refrigerated horsemeat and beef samples were determined to be viable. Molecular analysis of S. fayeri 18S rRNA gene sequences revealed that intraspecific variation among eight individual bradyzoites from a single sarcocyst was as high as 9.8%. In contrast, mitochondrial cytochrome c oxidase subunit 1 (mtDNA cox1) gene sequences from the six fragments of a single sarcocyst were 100% identical. Sarcocysts of S. bovini isolated from beef also exhibited intraspecific variation in 18S rRNA gene sequences and had to be cloned before sequencing, while mtDNA cox1 gene sequences were obtained by direct sequencing. Therefore, we conclude that molecular analysis of the mtDNA cox1 gene is the most useful for identification of Sarcocystis species. This study provides the first published partial sequence of the S. fayeri mtDNA cox1 gene.

Dissemination of blaNDM, which is carried on the IncX3 plasmid, among Enterobacterales has been reported worldwide. In particular, blaNDM-5-carrying IncX3 plasmids can spread among several hosts, facilitating their dissemination. Other variants, such as blaNDM-17-, blaNDM-19-, blaNDM-20-, blaNDM-21-, and blaNDM-33-carrying IncX3 plasmids, have also been reported. Here, we characterized, using whole-genome sequencing (WGS), a blaNDM-16b-carrying IncX3 plasmid harbored by Escherichia coli strain TA8571, which was isolated from a urine specimen of a hospital inpatient in Tokyo, Japan. The blaNDM-16b differed in sequence from blaNDM-5 (C > T at site 698, resulting in an Ala233Val substitution). This blaNDM-16b-carrying IncX3 plasmid (pTMTA8571-1) is 46,161 bp in length and transferred via conjugation. Transconjugants showed high resistance to β-lactam antimicrobials (except for aztreonam). Because pTMTA8571-1, which carries the Tn125-related region containing blaNDM and conjugative transfer genes, was similar to the previously reported IncX3 plasmids, we performed phylogenetic analysis based on the sequence of 34 shared genes in 142 blaNDM-carrying IncX3 plasmids (22,846/46,923 bp). Comparative analysis of the shared genes revealed short branches on the phylogenetic tree (average of 1.08 nucleotide substitutions per shared genes), but each blaNDM variant was divided into separate groups, and the structure of the tree correlated with the flowchart of blaNDM nucleotide substitutions. The blaNDM-carrying IncX3 plasmids may thereby have evolved from the same ancestral plasmid with subsequent mutation of the blaNDM. Therefore, pTMTA8571-1 likely emerged from a blaNDM-5-carrying IncX3 plasmid. This study suggested that the spread of blaNDM-carrying IncX3 plasmids may be a hotbed for the emergence of novel variants of blaNDM. IMPORTANCE blaNDM-carrying IncX3 plasmids have been reported worldwide. Harbored blaNDM variants were mainly blaNDM-5, but there were also rare variants like blaNDM-17, blaNDM-19, blaNDM-20, blaNDM-21, and blaNDM-33, including blaNDM-16b detected in this study. For these plasmids, previous reports analyzed whole genomes or parts of sequences among a small number of samples, whereas, in this study, we performed an analysis of 142 blaNDM-carrying IncX3 plasmids detected around the world. The results showed that regardless of the blaNDM variants, blaNDM-carrying IncX3 plasmids harbored highly similar shared genes. Because these plasmids already spread worldwide may be a hotbed for the emergence of rare or novel variants of blaNDM, increased attention should be paid to blaNDM-carrying IncX3 plasmids in the future.

After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.

Phytobacter diazotrophicus is an Enterobacterales species that was originally identified as a plant growth-promoting, Gram-negative bacterium. Recently, this species has been recognized as relevant to opportunistic human and nosocomial infections in clinical settings. Its frequent misidentification as other Enterobacterales species from clinical examination occasionally causes a delay in the identification of nosocomial outbreaks. Here, we report the emergence of New Delhi metallo-β-lactamase (NDM)-producing P. diazotrophicus isolated from hospitalized pediatric patients and hospital environments in Tokyo, Japan. In our case, these isolates were found during an investigation of carbapenem-resistant Enterobacterales in relation to nosocomial infections. Whole-genome sequencing is useful for overcoming the difficulty of species identification. Furthermore, we found that bla NDM-1 was carried by an IncA/C2 plasmid (approximately 170 kbp), which was transferrable from the clinical isolates to the recipient strain Escherichia coli J53. Our study demonstrated that P. diazotrophicus behaves as a carrier of bla NDM-harboring plasmids, potentially disseminating resistance to carbapenems among Enterobacterales. IMPORTANCE Early detection of nosocomial outbreaks is important to minimize the spread of bacteria. When an outbreak is caused by multidrug-resistant bacteria such as carbapenem-resistant Enterobacterales, a delay in findings makes it difficult to control it because such bacteria often spread not only among human patients but also in hospital environments. Phytobacter diazotrophicus, an Enterobacterales species that has recently been found to be relevant to clinical settings, is often misidentified as other bacteria in clinical laboratories. Here, we found NDM-producing P. diazotrophicus in hospitalized pediatric patients and their environment in Tokyo, Japan. Given that the isolates carried bla NDM-1-harboring transferrable plasmids, the influence of such bacteria could be greater with the mediation of horizontal transfer of carbapenem resistance. Our findings suggest that P. diazotrophicus should be recognized as an NDM-carrier, for which more attention should be paid in clinical settings.

In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.

In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.

Streptococcus suis is a gram-positive bacterium that causes meningitis, septicemia, endocarditis, and other disorders in pigs and humans. We obtained 42 and 50 S. suis isolates from lesions of porcine endocarditis and palatine tonsils, respectively, of clinically healthy pigs in Japan; we then determined their sequence types (STs) by multilocus sequence typing (MLST), cps genotypes, serotypes, and presence of classical major virulence-associated marker genes (mrp, epf, and sly). The 42 isolates from endocarditis lesions were assigned to a limited number of STs and clonal complexes (CCs). On the other hand, the 50 isolates from tonsils were diverse in these traits and seemingly in the degree of virulence, suggesting that tonsils can accommodate a variety of S. suis isolates. The goeBURST full algorithm using tonsil isolates obtained in this study and those retrieved from the database showed that major CCs as well as many other clusters were composed of isolates originating from different countries, and some of the STs were very similar to each other despite the difference in country of origin. These findings indicate that S. suis with not only different but also similar mutations in the genome have survived in tonsils independently across different geographical locations. Therefore, unlike the lesions of endocarditis, the tonsils of pigs seemingly accommodate various S. suis lineages. The present study suggests that S. suis acquired its diversity by natural mutations during colonization and persistence in the tonsils of pigs.