Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model.

Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size, 2,006,877 bp; GC content, 37.6%; 2,020 predicted open reading frames [ORFs]) isolated from a brain abscess in an infant. Comparative analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long repeat regions, and 19 ORFs for cell wall-anchored proteins (CWAPs). To elucidate the genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model. The results suggest that the levels of expression of small hypothetical proteins similar to phenol-soluble modulin ß1 (PSMß1), a staphylococcal virulence factor, significantly increased in the abscess model. In addition, an experiment in a murine subcutaneous abscess model with random transposon (Tn) mutant attenuation suggested that Tn mutants with mutations in 212 ORFs in the Tn mutant library were attenuated in the murine abscess model (629 ORFs were disrupted in total); the 212 ORFs are putatively essential for abscess formation. Transcriptome analysis identified 37 ORFs, including paralogs of the T7SS and a putative glucan-binding CWAP in long repeat regions, to be upregulated and attenuated in vivo This study provides a comprehensive characterization of S. intermedius pathogenicity based on the complete genome sequence and a murine subcutaneous abscess model with transcriptome and Tn mutagenesis, leading to the identification of pivotal targets for vaccines or antimicrobial agents for the control of S. intermedius infections.

Prevalence of Colistin Resistance Gene mcr-1 and Absence of mcr-2 in Escherichia coli Isolated from Healthy Food-Producing Animals in Japan.

We screened mcr-1 and mcr-2 genes in 9,306 Escherichia coli strains isolated from healthy animals in the Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) system. mcr-1 was detected in 39 strains (5, 20, and 14 strains isolated from cattle, swine, and broilers, respectively), whereas mcr-2 was not detected. mcr-2 was also not detected with the investigation sequence homology search against our curated GenEpid-J database.

Distribution and Relationships of Antimicrobial Resistance Determinants among Extended-Spectrum-Cephalosporin-Resistant or Carbapenem-Resistant Escherichia coli Isolates from Rivers and Sewage Treatment Plants in India.

To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The blaNDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: blaNDM-1 in 2 isolates, blaNDM-5 in 7 isolates, and blaNDM-7 in 5 isolates. The blaCTX-M gene was detected in 112 isolates (66.3%): blaCTX-M-15 in 108 isolates and blaCTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the blaNDM-positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

Two different dengue virus strains in the Japanese epidemics of 2014.

In late August 2014, dengue cases were reported in Japan, and a total of 162 cases were confirmed. In the present study, the envelope (E) gene sequences of 12 specimens from the dengue patients were determined. A dengue virus serotype 1 (DENV1) strain (D1/Hu/Shizuoka/NIID181/2014), which was clearly different from the first reported strain (D1/Hu/Saitama/NIID100/2014), was identified, although the other 11 specimens showed the same nucleotide sequences as D1/Hu/Saitama/NIID100/2014. The E gene sequences of two different strains were compared with those of nine DENV1 strains of imported cases in Japan in 2014. Phylogenetic analysis based on the E gene sequences showed that the D1/Hu/Saitama/NIID100/2014 strain was closely related to a strain isolated from an imported case from Singapore. Although no strain closely related to D1/Hu/Shizuoka/NIID181/2014 was found in these imported strains, the strain was closely related to isolates in Thailand and Taiwan in 2009. These data indicate that the dengue cases in Japan were caused by two different dengue virus strains that entered Japan through different means.

Human monoclonal antibodies broadly neutralizing against influenza B virus.

Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.

Induction of anti-influenza immunity by modified green fluorescent protein (GFP) carrying hemagglutinin-derived epitope structure.

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel ß-sheet structure. Our previous study identified this ß-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this ß-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of ß-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses.

Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication.

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC50=0.74-4.92 µM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC50=29.81 µM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3.

The type III Secretion System of Bradyrhizobium japonicum USDA122 mediates symbiotic incompatibility with Rj2 soybean plants.

The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners.

Co-existence of major and minor viral populations from two different origins in patients secondarily infected with dengue virus serotype 2 in Bangkok.

Generally, RNA viruses exhibit significant genetic diversity that sometimes effect viral fitness in infected hosts and probably also pathogenesis. Dengue viruses (DENVs) consist of four antigenically distinct serotypes. All the serotypes of DENV can cause mild to severe dengue illnesses. In this study, we examined the sequence variation of DENV in plasma obtained from four patients living in Bangkok who had been secondarily infected with serotype 2 (DENV-2) in 2010. The plasma-derived RNA was directly subjected to reverse transcriptase (RT)-polymerase chain reaction (PCR) at a region including most of domain III of the envelope (E) protein gene, and the PCR products obtained were subjected to clonal sequencing. Using 19-20 clones sequenced from each patient (78 total) plus 601 corresponding sequences from a public database, phylogenetic analysis revealed that the nucleic acid sequences fell into two clusters with clearly different origins. Interestingly, all patients gave sequences indicating that they carried viral populations containing 2, 3 or 5 genetic variants that consisted of one major variant plus one or more minor variants. Three patients showed a major variant from one cluster plus one or more minor components from the other while one showed major and minor variants from a single cluster. Thus, it can be concluded that DENV belonging to two different genetic lineages were co-circulated in Bangkok in 2010. For these two genotype clusters there was also a clear difference in H or Y at the deduced amino acid position 346 (i.e. H346Y) that was consistent for our sequences and 601 sequences from the public database. Thus, one among the mixed viral genotypes introduced into human individuals seems to be variably selected as the predominant component of the carried viral population, and it is possible that the dynamics of this process could influence virus evolution and disease severity.

Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition.

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel beta-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the beta-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n=7955), swine H1N1 (n=360) and swine H3N2 (n=235); and highly conserved in human H1N1 (n=2722), swine-origin pandemic H1N1 (n=1474), human H5N1 (n=319) and avian H5N1 (n=2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.

Conventional culture methods with commercially available media unveil the presence of novel culturable bacteria.

Recent metagenomic analysis has revealed that our gut microbiota plays an important role in not only the maintenance of our health but also various diseases such as obesity, diabetes, inflammatory bowel disease, and allergy. However, most intestinal bacteria are considered 'unculturable' bacteria, and their functions remain unknown. Although culture-independent genomic approaches have enabled us to gain insight into their potential roles, culture-based approaches are still required to understand their characteristic features and phenotypes. To date, various culturing methods have been attempted to obtain these 'unculturable' bacteria, but most such methods require advanced techniques. Here, we have tried to isolate possible unculturable bacteria from a healthy Japanese individual by using commercially available media. A 16S rRNA (ribosomal RNA) gene metagenomic analysis revealed that each culture medium showed bacterial growth depending on its selective features and a possibility of the presence of novel bacterial species. Whole genome sequencing of these candidate strains suggested the isolation of 8 novel bacterial species classified in the Actinobacteria and Firmicutes phyla. Our approach indicates that a number of intestinal bacteria hitherto considered unculturable are potentially culturable and can be cultured on commercially available media. We have obtained novel gut bacteria from a healthy Japanese individual using a combination of comprehensive genomics and conventional culturing methods. We would expect that the discovery of such novel bacteria could illuminate pivotal roles for the gut microbiota in association with human health.

Whole-genome sequencing-based epidemiological analysis of anti-tuberculosis drug resistance genes in Japan in 2007: Application of the Genome Research for Asian Tuberculosis (GReAT) database.

We investigated the lineages of Mycobacterium tuberculosis (Mtb) isolates from the RYOKEN study in Japan in 2007 and the usefulness of genotypic drug susceptibility testing (DST) using the Genome Research for Asian Tuberculosis (GReAT) database. In total, 667 isolates were classified into lineage 1 (4.6%), lineage 2 (0.8%), lineage 2/Beijing (72.1%), lineage 3 (0.5%), and lineage 4 (22.0%). The nationality, gender, and age groups associated with the isolates assigned to lineage 1 were significantly different from those associated with other lineages. In particular, isolates of lineage 1.2.1 (EAI2) formed sub-clusters and included a 2,316-bp deletion in the genome. The proportion of the isolates resistant to at least one anti-tuberculosis (TB) drug was 10.8%, as determined by either the genotypic or phenotypic method of DST. However, the sensitivities to isoniazid, streptomycin, and ethambutol determined by the genotypic method were low. Thus, unidentified mutations in the genome responsible for drug resistance were explored, revealing previously unreported mutations in the katG, gid, and embB genes. This is the first nationwide report of whole-genome analysis of TB in Japan.

Overcoming the pitfalls of automatic interpretation of whole genome sequencing data by online tools for the prediction of pyrazinamide resistance in Mycobacterium tuberculosis.

OBJECTIVES: Automated online software tools that analyse whole genome sequencing (WGS) data without the need for bioinformatics expertise can motivate the implementation of WGS-based molecular drug susceptibility testing (DST) in routine diagnostic settings for tuberculosis (TB). Pyrazinamide (PZA) is a key drug for current and future TB treatment regimens; however, it was reported that predictive power for PZA resistance by the available tools is low. Therefore, this low predictive power may make users hesitant to use the tools. This study aimed to elucidate why and to uncover the real performance of the tools when taking into account their variation calling lists (manual inspection), not just their automated reporting system (default setting) that was evaluated by previous studies. METHODS: WGS data from 191 datasets comprising 108 PZA-resistant and 83 susceptible strains were used to evaluate the potential performance of the available online tools (TB Profiler, TGS-TB, PhyResSE, and CASTB) for predicting phenotypic PZA resistance. RESULTS: When taking into consideration the variation calling lists, 73 variants in total (47 non-synonymous mutations and 26 indels) in pncA were detected by TGS-TB and PhyResSE, covering all mutations for the 108 PZA-resistant strains. The 73 variants were confirmed by Sanger sequencing. TB Profiler also detected all but three complete loss, two large deletion at the 3'-end, and one relatively large insertion of pncA. On the other hand, many of the 73 variants were lacking in the automated reporting systems except by TGS-TB; of these variants, CASTB detected only 20. By applying the 'non-wild type sequence' approach for predicting PZA resistance, accuracy of the results significantly improved compared with that of the automated results obtained by each tool. CONCLUSION: Users can obtain more accurate predictions for PZA resistance than previously reported by manually checking the results and applying the 'non-wild type sequence' approach.

Computational search for over-represented 8-mers within the 5'-regulatory regions of 634 mouse testis-specific genes.

Accumulation of microarray data has enabled the computational analysis of gene expressions in various tissues. Although the genes showing testis-specific expression are most abundant among the genes exhibiting tissue-specific expression, no systematic study has been conducted for over-represented motifs within their regulatory regions. We have identified 117 over-represented 8-mers that appeared 2648 times within the regulatory regions of 634 testis-specific genes. Of these, 64 over-represented 8-mers were significantly more frequent in the regulatory regions of testis-specific genes than in those of non-testis-specific genes. In this group of 8-mers, 4 8-mers differed from the canonical cAMP response element (CRE) 8-mer by 1 letter, but the canonical CRE was not included in this group. We consider that CRE-like 8-mers participate in the regulatory expression of testis-specific genes to a greater extent than the canonical CRE 8-mer.

In silico study of a novel gene evolved from an ancestral SVIP gene and highly expressed in the adult mouse testes.

We found that a cDNA clone isolated from a mouse testis cDNA library, 1700015G11 (Mmu_15G11), corresponded to the most highly expressed testis-specific mRNA in the adult mouse. Although the Mmu_15G11 cDNA is predicted to encode a small protein consisting of 67 amino acid residues, it has not yet been functionally annotated and has been designated as an unclassifiable clone. Since the Mmu_15G11 protein possibly has a pivotal role in spermatogenesis, we initiated an in silico study of this clone, and revealed that an ancestral gene of 15G11 genes evolved from an ancestral gene for mammalian small valosin-containing protein-interacting protein (SVIP) genes by gene duplication. Although SVIP protein reportedly participates in endoplasmic reticulum-related protein degradation, 15G11 protein is predicted to be a nuclear protein and possibly participates in the interaction between proteins and nuclear DNA.

In silico study of whey-acidic-protein domain containing oral protease inhibitors.

Since whey-acidic-protein domain (WAP) containing protease inhibitors such as SLPI (secretory leukocyte protease inhibitor) and elafin (elastase-specific inhibitor) have antimicrobial activities and are thought to play critical roles in mucosal defenses, we are interested in these protease inhibitors. By accessing the Novartis mouse expression database, we found that the four WAP family members, SLPI, WFDC2, WFDC5, and WFDC12, are highly expressed in the oral organs, such as the trachea, tongue, and salivary glands. Since their WAP domains play pivotal roles in the antimicrobial and/or antiprotease activities and their application in therapeutics are expected to have practical value, we collected 98 WAP homologues and tried to predict their physiological functions by analyzing their amino acid sequence structures. From the multiple alignments of amino acid sequences, we predicted that most of the mammalian N-terminal WAP domains derived from SLPIs and the WAP domains derived from WFDC12s have antimicrobial activities, whereas most of the mammalian C-terminal WAP domains derived from SLPIs and the WAP domains derived from elafins have antiprotease activities. From the phylogenetic tree, it was revealed that an ancestral WAP protein initially diverged into the WFDC5-C WAP domain and the ancestral protein for the other WAP domains. Subsequently, the ancestral protein for the other WAP domains diverged into two ancestral proteins, one for elafin and SLPI-C WAP domains and the other, for SLPI-N, WFDC15b, WFDC12, and WFDC5-N WAP domains, respectively. Moreover, the tree indicated that the WFDC5-N and WFDC12 WAP domains share a common ancestral protein.

Isolation and Complete Genome Sequencing of Zika Virus Imported from the Dominican Republic to Japan.

Zika virus (ZIKV) infection has been documented within Central and South America, Asia, and Africa. Here we report the isolation of virus from a patient infected with ZIKV returning to Japan from the Dominican Republic. The ZIKV strain was imaged by electron microscopy and its complete genome sequence was analyzed. Phylogenetic analysis and molecular characterization revealed that the strain was of Asian lineage, and carried 2 unique mutations in its NS5 region. These mutations are characteristic of strains that originated in the Dominican Republic and the USA in 2016.

A summary of the imported cases of Chikungunya fever in Japan from 2006 to June 2016.

Background: Due to the huge 2-way human traffic between Japan and Chikungunya (CHIK) fever-endemic regions, 89 imported cases of CHIK fever were confirmed in Japan from January 2006 to June 2016. Fifty-four of 89 cases were confirmed virologically and serologically at the National Institute of Infectious Diseases, Japan and we present the demographic profiles of the patients and the phylogenetic features of 14 CHIK virus (CHIKV) isolates. Methods: Patients were diagnosed with CHIK fever by a combination of virus isolation, viral RNA amplification, IgM antibody-, IgG antibody-, and/or neutralizing antibody detection. The whole-genome sequences of the CHIKV isolates were determined by next-generation sequencing. Results: Prior to 2014, the source countries of the imported CHIK fever cases were limited to South and Southeast Asian countries. After 2014, when outbreaks occurred in the Pacific and Caribbean Islands and Latin American countries, there was an increase in the number of imported cases from these regions. A phylogenetic analysis of 14 isolates revealed that four isolates recovered from three patients who returned from Sri Lanka, Malaysia and Angola, belonged to the East/Central/South African genotype, while 10 isolates from 10 patients who returned from Indonesia, the Philippines, Tonga, the Commonwealth of Dominica, Colombia and Cuba, belonged to the Asian genotype. Conclusion: Through the phylogenetic analysis of the isolates, we could predict the situations of the CHIK fever epidemics in Indonesia, Angola and Cuba. Although Japan has not yet experienced an autochthonous outbreak of CHIK fever, the possibility of the future introduction of CHIKV through an imported case and subsequent local transmission should be considered, especially during the mosquito-active season. The monitoring and reporting of imported cases will be useful to understand the situation of the global epidemic, to increase awareness of and facilitate the diagnosis of CHIK fever, and to identify a future CHIK fever outbreak in Japan.

Decrease in the prevalence of extended-spectrum cephalosporin-resistant Salmonella following cessation of ceftiofur use by the Japanese poultry industry.

Extended-spectrum cephalosporin (ESC)-resistant Salmonella in chicken meat is a significant food safety concern. We previously reported that the prevalence of ESC-resistant Salmonella in chicken meat, giblets, and processed chicken (chicken meat products) increased in Japan between 2005 and 2010, with 27.9% (17/61) of Salmonella isolated from chicken meat products in 2010 showing resistance to ESC. The aims of the present study were to clarify trends in the prevalence of ESC-resistant Salmonella in chicken meat products in Japan between 2011 and 2015, and to determine the genetic profiles of bla-harboring plasmids, including replicon types, using next-generation sequencing. Our results showed that the prevalence of ESC-resistant Salmonella, mainly consisting of AmpC ß-lactamase CMY-2-producing isolates, in chicken meat products had increased to 45.5% (10/22) by 2011. However, following the voluntary cessation of ceftiofur use by the Japanese poultry industry in 2012, the prevalence of ESC-resistant Salmonella steadily decreased each year, to 29.2% (7/24), 18.2% (4/22), 10.5% (2/19), and 10.5% (2/19) in 2012, 2013, 2014, and 2015, respectively. Furthermore, no AmpC ß-lactamase CMY-2-producing isolates were identified in 2014 and 2015. However, the prevalence of Salmonella enterica subspecies enterica serovar Manhattan isolates harboring a blaTEM-52-carrying IncX1 plasmid remained steady even after the cessation of ceftiofur use. Therefore, continuous monitoring of ESC resistance amongst Salmonella isolates from chicken meat products is required for food safety.