ABSTRACT
Dissemination of extended-spectrum-cephalosporin (ESC)-resistant Salmonella, especially extended-spectrum-β-lactamase (ESBL)-producing Salmonella, is a concern worldwide. Here, we assessed Salmonella carriage by food workers in Japan to clarify the prevalence of ESC-resistant Salmonella harboring blaCTX-M. We then characterized the genetic features, such as transposable elements, of blaCTX-M-harboring plasmids using whole-genome sequencing. A total of 145,220 stool samples were collected from food workers, including cooks and servers from several restaurants, as well as food factory workers, from January to October 2017. Isolated salmonellae were subjected to antimicrobial susceptibility testing (disk diffusion method), and whole-genome sequencing was performed for Salmonella strains harboring blaCTX-M. Overall, 164 Salmonella isolates (0.113%) were recovered from 164 samples, from which we estimated that at least 0.113% (95% confidence interval [CI]: 0.096 to 0.132%) of food workers may carry Salmonella. Based on this estimation, 3,473 (95% CI = 2,962 to 4,047) individuals among the 3,075,330 Japanese food workers are likely to carry Salmonella. Of the 158 culturable isolates, seven showed resistance to ESCs: three isolates harbored blaCMY-2 and produced AmpC β-lactamase, while four ESBL-producing isolates harbored blaCTX-M-14 (n = 1, Salmonella enterica serovar Senftenberg) or blaCTX-M-15 (n = 3, S. enterica serovar Haardt). blaCTX-M-15 was chromosomally located in the S. Haardt isolates, which also contained ISEcp1, while the S. Senftenberg isolate contained an IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid plasmid carrying blaCTX-M-14 along with ISEcp1. This study indicates that food workers may be a reservoir of ESBL-producing Salmonella and associated genes. Thus, these workers may contribute to the spread of blaCTX-M via plasmids or mobile genetic elements such as ISEcp1.
IMPORTANCE Antimicrobial-resistant Salmonella bacteria arise in farm environments through imprudent use of antimicrobials. Subsequently, these antimicrobial-resistant strains, such as extended-spectrum-β-lactamase (ESBL)-producing Salmonella, may be transmitted to humans via food animal-derived products. Here, we examined Salmonella carriage among food handlers in Japan. Overall, 164 of 145,220 fecal samples (0.113%) were positive for Salmonella. Among the 158 tested isolates, four were identified as ESBL-producing isolates carrying ESBL determinants blaCTX-M-15 or blaCTX-M-14. In all cases, the genes coexisted with ISEcp1, regardless of whether they were located on the chromosome or on a plasmid. Our findings suggest that food workers may be a reservoir of ESBL-producing strains and could contribute to the spread of resistance genes from farm-derived Salmonella to other bacterial species present in the human gut.
INTRODUCTION
Nontyphoidal Salmonella is a major cause of foodborne illness worldwide as suggested by the World Health Organization (https://www.who.int/news-room/fact-sheets/detail/salmonella-(non-typhoidal), accessed 17 July 2019). Food handlers and individuals who work in food production, referred to here as food workers, have been identified as a potential source of transmission of nontyphoidal Salmonella among children (1). Thus, surveillance of Salmonella carriage by food workers is important for public health. Human-to-human transmission of nontyphoidal Salmonella is very rare. However, asymptomatic carriers can sometimes transmit Salmonella if hygiene standards are not maintained (1, 2). It is therefore important to assess rates of Salmonella carriage by food workers in developed countries such as Japan. To the best of our knowledge, there has been no recent survey of Salmonella carriage rates and serotypes in food workers in developed countries since our previous study conducted in 1999 and 2000 (3). Therefore, such an investigation is overdue.
Dissemination of extended-spectrum-cephalosporin (ESC)-resistant Salmonella is of global concern because these antibiotics are commonly used to treat severe cases of salmonellosis in children (4, 5). Salmonella isolates containing blaCTX-M, encoding a class A extended-spectrum β-lactamase (ESBL) have been isolated from human clinical samples in multiple countries across Europe, the Americas, and Asia, including Japan (6–11). Worryingly, there is evidence of horizontal transmission of blaCTX-M among bacterial species via transferable plasmids. For example, an ∼215-kb blaCTX-M-15-carrying the IncHI2 plasmid was translocated between Salmonella strains such as Salmonella enterica serovar Enteritidis derived from poultry meat and an S. Virchow isolate recovered from an individual in South Korea (12). Thus, a survey of the genetic features of the plasmid vehicles, including replicon types, will help us to understand how plasmids harboring bla genes are disseminated among bacteria. In addition, plasmid-borne bla genes may be translocated to other plasmids or to the bacterial chromosome with the aid of transposable elements (TEs) such as IS26 (13) and ISEcp1 (14), suggesting that the identification of TEs may also be useful for understanding bla gene transmission. For example, chromosomally located blaCTX-M-15 genes identified in several human S. Concord isolates were shown to be derived from an IncY/IncA/C2 fusion plasmid containing IS26 (13). Accordingly, determining the mechanisms of bla gene translocation through molecular analysis of plasmid vectors or TEs might lead to a better understanding of the dissemination of ESC-resistant Salmonella. Food workers may process or handle chicken meat contaminated with ESBL-producing Salmonella more often than other people (15). Therefore, the aims of this study were to clarify the prevalence of Salmonella carriage among food workers in Japan and, after identifying ESBL-producing isolates, to characterize the genetic features of blaCTX-M-harboring plasmids and resistance gene-related TEs in the isolates using whole-genome sequencing (WGS).
RESULTS
Prevalence of Salmonella isolates in human stools and serotype determination.A total of 164 (0.113%) Salmonella isolates were obtained from 164 of 145,220 human stool samples (Fig. 1). Of the 164 isolates, 158 were assigned to 50 different serotypes (Table 1), with the predominant serotypes being S. Infantis (12.7%, 20/158) and S. Schwarzengrund (9.5%, 15/158). The remaining six isolates could not be revived after storage in Casitone medium. The three isolates identified as serotype O8 (O6-positive):k:1,5 were classified as S. Haardt instead of S. Blockley because WGS analysis showed that the serotype formula was O8:k:1,5.
Numbers of Salmonella-positive fecal samples derived from food workers in eight different regions of Japan. The numbers of Salmonella-positive samples, the numbers of tested samples, the positive rate, and region names are shown.
Number and serotypes of Salmonella isolates recovered from 164 stool samples from food workers in Japan
Antimicrobial susceptibility and bla gene sequences.Antimicrobial susceptibility tests showed that 73 (46.2%) of the 158 Salmonella isolates were resistant to at least one antimicrobial. Among these 73 isolates, 6 (3.8%), 6 (3.8%), 7 (4.4%), 7 (4.4%), 3 (1.9%), 62 (39.2%), 52 (32.9%), 30 (19.0%), and 7 (4.4%) isolates were resistant to cefotaxime (CTX), ceftazidime (CAZ), cefpodoxime (CPDX), cefixime (CFIX), cefoxitin (CFX), tetracycline (TC), streptomycin (SM), kanamycin (KM), and nalidixic acid (NA), respectively. None of the isolates were resistant to norfloxacin (NFLX), meropenem (MEPM), or colistin (CL). Of the 73 isolates, four produced ESBLs and three produced AmpC β-lactamase. Among the four ESBL-producing isolates, one S. Senftenberg isolate harbored blaCTX-M-14 and three S. Haardt isolates harbored blaCTX-M-15. The AmpC β-lactamase-producing isolates harbored blaCMY-2 and consisted of one S. Agona isolate, one S. Infantis isolate, and one untypeable (O4:–:–) isolate.
WGS-based identification of antimicrobial resistance genes, plasmid replicon types, and TEs.As shown in Table 2, the ESBL-producing S. Senftenberg isolate (strain SESen3709) contained two plasmids, one of which was classified as Col440II, while the other was identified as an IncFIA(HI1)/IncHI1A/IncHI1B(R27) fusion plasmid. The fusion plasmid, designated pSESen3709_1, carried blaCTX-M-14 along with other antimicrobial resistance genes. In pSESen3709_1, blaCTX-M-14 was located downstream of ΔISEcp1 (Fig. 2). ΔISEcp1 was identical to wild-type ISEcp1 except for a point mutation (C→T) at position 94,135, resulting in a pseudogene. In comparison, no antimicrobial resistance genes were identified in the Col440II plasmid.
Antimicrobial resistance genes on chromosomes and plasmids from Salmonella enterica subsp. enterica serovar Senftenberg and Haardt isolates
Structural analysis of blaCTX-M-14-carrying plasmid pSESen3709_1 derived from Salmonella enterica subsp. enterica serovar Senftenberg. The structure of pSESen3709_1 was compared to those of pSa4-CIP (derived from S. Typhimurium from pork meat in Hong Kong, GenBank accession no. MG874042.1) (27) and p14E509-CTXM (derived from a clinical Escherichia coli isolate from Norway, GenBank accession no. MG764547.1) (38). (A) Overview of the three plasmids. The locations of blaCTX-M-14, other antimicrobial resistance genes, conjugal transfer systems, and other genes are shown as red, yellow, brown, and gray triangles, respectively. (B) Enlarged scale sketch of the blaCTX-M-14 regions from panel A. The ΔISEcp1 transposase was generated by a point mutation at nucleotide position 94,135, although the complete sequence was present. blaCTX-M-14, other antimicrobial resistance genes, ISEcp1 tnp, ΔISEcp1 tnp, other tnp, Δtnp, and other genes are shown as red, yellow, light green, pink, green, white, and gray arrows, respectively. Left inverted repeats (IRL) and right inverted repeats (IRR) are indicated by pink flags and green flags, respectively. The pSa4-CIP plasmid was rearranged for easier comparison, with the asterisk indicating the start position prior to rearrangement. Minimum and maximum are abbreviated as Min and Max, respectively.
On the chromosome of S. Haardt strain SEHaa3795, blaCTX-M-15 and ISEcp1 were located between genes coding for an S-formylglutathione hydrolase and GTP cyclohydrolase I (nucleotide positions 1707642 to 1710613) (Table 2 and Fig. 3). An intact ISEcp1 element was also found upstream of blaCTX-M-15 (Fig. 3). A Col440I-type plasmid was also detected in the S. Haardt isolates, but it did not contain any antimicrobial resistance genes (Table 2).
Structural analysis of the chromosome of Salmonella enterica subsp. enterica serovar Haardt carrying blaCTX-M-15. The chromosome of the S. Haardt (SEHaa3795) isolate carrying blaCTX-M-15 was compared to that of the S. Senftenberg (SESen3709) isolate and with blaCTX-M-15-carrying plasmid pEC_L46 from a clinical Escherichia coli isolate from Belgium (GenBank accession no. GU371929.1) (39). blaCTX-M-15, ISEcp1 tnp, other tnp, Δtnp, conjugate transfer systems, and other genes are shown as red, light green, green, white, brown, and gray arrows, respectively. Left inverted repeats (IRL) and right inverted repeats (IRR) are indicated by pink flags and green flags, respectively. Minimum and maximum are abbreviated as Min and Max, respectively.
Comparison of plasmid replicon types in S. Senftenberg and other serotypes.The replicon type of pSESen3709_1 was compared to those of plasmids derived from S. Typhi, S. Paratyphi A, and S. Typhimurium isolates in other studies (see Table S1 in the supplemental material). The IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid replicon type was in accordance with other previously described plasmids, as shown in Table S1.
Comparison with reference plasmid sequences.pSESen3709_1 was compared with reference plasmids pSa4-CIP and p14E509-CTXM (Fig. 2). The complete sequence of pSESen3709_1 was similar to that of pSa4-CIP (coverage, 90.6%) (Fig. 2A), and the two plasmids shared the same replicon type. However, the region containing blaCTX-M-14 and ΔISEcp1 (nucleotides 93526 to 98137) in pSESen3709_1 was not present in pSa4-CIP (Fig. 2B), whereas a highly similar region (>99% nucleotide sequence identity, 4,611/4,612 bp) was identified in p14E509-CTXM. Analyses of the S. Haardt chromosomal sequences showed that the blaCTX-M-15- and ISEcp1-containing region shares 100% nucleotide sequence identity (across 2,972 bp) with a fragment from plasmid pEC_L46.
DISCUSSION
Our study had three main findings. First, the rate of Salmonella carriage among food workers in Japan between January and October, 2017, was about 0.113%, with the recovered isolates belonging to 50 different serotypes. Second, we recovered an ESC-resistant S. Senftenberg isolate harboring a blaCTX-M-14-carrying plasmid, named pSESen3709_1. The replicon type of this plasmid was determined to be IncFIA(HI1)/IncHI1A/IncHI1B(R27), which is in accordance with other Salmonella-derived plasmids. Finally, both the translocation of blaCTX-M-14 into pSa4-CIP-like plasmid pSESen3709_1 and the integration of blaCTX-M-15 into the S. Haardt chromosome likely involved ISEcp1.
Food workers may be a significant reservoir of Salmonella and other human enteropathogens. It is difficult to accurately determine the Salmonella carriage rate among food workers in the present study because almost all of the participants provided three samples during the examination period. However, we estimate that at least 0.113% (95% CI = 0.096 to 0.132%) of food workers are likely to carry the pathogen because it is generally excreted for 3 to 92 days following infection (16, 17). Based on this estimation, 3,473 (95% CI = 2,962 to 4,047) of the 3,075,330 Japanese food workers (national census data set 2015 [https://www.stat.go.jp/data/kokusei/2015/kekka.html], accessed 12 July 2019) might carry Salmonella and could potentially be actively excreting Salmonella at any given time. The Salmonella incidence among food workers in the present study (0.113%) is inconsistent with that found in our previous study (0.032%) (3), probably because the conditions differed in these studies (Table S2). A further study to assess Salmonella prevalence between a targeted population like food workers (as in the present study) versus a control population such as non-food workers of similar age and sex, using the same detection methods, may help to address this inconsistency and provide useful information for public health purposes.
Food workers are potentially involved in ESC-Salmonella dissemination. In general, antimicrobial-resistant Salmonella strains arise in farm environments where imprudent antibiotic use selects for the development of resistance (15, 18). Subsequently, these antimicrobial-resistant strains, such as ESBL-producing Salmonella, may be transmitted to humans via animal-derived food products especially chicken meat (15). However, the results of the present study indicate that food workers might also spread animal-derived ESBL-producing Salmonella, as well as other bacterial species that have acquired ESBL-encoding genes from the Salmonella isolates in the intestines of the food workers. Because children show higher incidence rates of salmonellosis compared with adults (19), it is important that food workers at kindergartens, day care centers, and primary schools are free of the causative pathogen. Given that the dominant serovars in the present study (S. Infantis and S. Schwarzengrund) are often found in chicken meat (15, 20, 21), it is also important that chicken meat is handled carefully in these facilities to prevent infection. Food workers may act as reservoirs for antimicrobial-resistant Salmonella because they process or handle chicken meat that is potentially contaminated with ESBL-producing Salmonella (15) more often than other groups.
In silico analysis of the replicon type of blaCTX-M-14-carrying plasmid pSESen3709_1 (hybrid IncFIA(HI1)/IncHI1A/IncHI1B(R27)-type) from the S. Senftenberg isolate recovered in this study suggests that the plasmid might be related to plasmids with the same replicon type found in other Salmonella serotypes, especially S. Typhi (22, 23). Salmonella-derived plasmids with the IncFIA(HI1)/IncHI1A/IncHI1B(R27) replicon type are listed in Table S1. IncHI1-group plasmids are a major contributing factor to multidrug resistance in S. Typhi (24). However, the hybrid-type plasmids are not only found in S. Typhi isolates but are also associated with other Salmonella serotypes (22, 23, 25–28). This might indicate that R27, the prototypical IncHI1-group plasmid (23, 28), acquired various antimicrobial resistance genes, with the resulting progeny plasmids being spread and maintained among Salmonella. Furthermore, it is possible that the blaCTX-M-14-carrying plasmid pSESen3709_1 may be disseminated from S. enterica serovar Senftenberg to other Salmonella serotypes because IncHI1-group plasmids can be transferred among Enterobacteriaceae (29). The dissemination of these antimicrobial resistance plasmids should be monitored by carrying out further investigations of the replicon types of blaCTX-M-14-carrying plasmids.
The results of this study might help uncover the possible mechanisms of blaCTX-M translocation. pSESen3709_1 from S. Senftenberg showed a high degree of similarity (coverage, 90.6%) to plasmid pSa4-CIP from S. Typhimurium, with the two plasmids showing the same replicon type. However, pSa4-CIP lacked the blaCTX-M-14 region. In addition, a region sharing >99% nucleotide sequence identity (4,611/4,612 bp) with blaCTX-M-14 from pSESen3709_1 in this study was identified in p14E509-CTXM from Escherichia coli. These findings indicate that blaCTX-M-14 might be translocated from certain plasmids to pSa4-CIP-like plasmids in Salmonella by ISEcp1. One of these plasmids could then have been translocated into the S. Senftenberg strain, followed by a point mutation in the ΔISEcp1 element. Given the high level of similarity, the chromosomally located blaCTX-M-15 region in the S. Haardt isolate may have been translocated by ISEcp1 from another plasmid such as pEC_L46 from E. coli. Several other reports have identified chromosomal integrations of bla genes in association with ISEcp1 in Enterobacteriaceae species including Escherichia coli (30), Proteus mirabilis, and Morganella morganii (31) derived from humans. Overall, our findings suggest that ISEcp1 plays an important role in the dissemination of blaCTX-M among Salmonella via translocation to either plasmids or chromosomes.
In conclusion, we confirmed that some food workers (approximately 0.1%) in Japan carry Salmonella, including ESC-resistant Salmonella harboring blaCTX-M-14 or blaCTX-M-15, and that the resistance genes, which are translocated by ISEcp1, are present on both transferable plasmids and the chromosome. Food workers may therefore transmit Salmonella harboring blaCTX-M to other individuals, increasing the likelihood of horizontal transmission of the resistance genes to other bacteria via plasmids. This study suggests that food workers may be a reservoir of antimicrobial-resistant Salmonella and antimicrobial resistance determinants in Japan.
MATERIALS AND METHODS
Human stools.A total of 145,220 stool samples were collected from food workers in Japan between January and October 2017 (Fig. 1). The samples were provided by approximately 48,400 individuals, almost all of whom provided three samples over the sampling period and did not report any symptoms of salmonellosis. However, food workers who tested positive for Salmonella at one sampling point did not provide any subsequent samples. The food workers ranged in age from 18 to 65 years and included cooks and servers from several restaurants, as well as food factory workers. We were not provided with the exact number of individuals who submitted the fecal samples or the number of facilities involved in the sampling. Samples were collected from eight different regions of Japan, including 1,619, 8,045, 69,488, 23,057, 25,989, 5,612, 3,391, and 8,019 samples from Hokkaido, Tohoku, Kanto, Chubu, Kansai, Chugoku, Shikoku, and Kyushu, respectively (Fig. 1). Each sample was examined in one of 51 trials conducted during the sampling period.
Detection of invA and isolation of Salmonella from human stool samples.DNA was extracted from the human stool samples for detection of Salmonella-specific gene invA as follows. A 0.5-g aliquot from each stool sample was suspended in 1 ml of distilled water and heated at 95°C for 10 min. The heated suspensions were then centrifuged at 21,500 × g for 5 min. The resulting supernatants contained extracted DNA. DNA extracts were pooled into combined samples containing 50 extracts per sample and used for PCR assays targeting invA. For each invA-positive combined DNA sample, the corresponding samples were inoculated onto modified Salmonella-Shigella agar (product no. E-MA55; Eiken Chemical Co., Tokyo, Japan) and cultured at 37°C for 24 h. Putative Salmonella colonies from these plates were confirmed using biochemical identification assays and serotyping, as described previously (15, 32, 33). Isolates identified as Salmonella were maintained on Casitone agar (product no. E-MP77; Eiken Chemical Co.) at room temperature until further antimicrobial susceptibility testing.
Antibiotic susceptibility testing and detection and sequencing of ESBL and AmpC β-lactamase genes.Antibiotic susceptibility tests were carried out using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (34, 35). The following 12 antimicrobials were tested: CTX (30 μg), CAZ (30 μg), CPDX (10 μg), CFIX (5 μg), CFX (30 μg), TC (30 μg), SM (10 μg), KM (30 μg), NA (30 μg), NFLX (10 μg), MEPM (10 μg), and CL (10 μg). Antimicrobial disks were obtained from Becton, Dickinson, and Co. (Franklin Lakes, NJ). Phenotypic confirmatory tests for ESBL production were conducted to further examine the resistant isolates using CTX (30 μg)-clavulanic acid (CVA; Becton, Dickinson, and Co.), CAZ (30 μg)-CVA (Becton, Dickinson, and Co.), and CPDX (30 μg)-CVA (Nissui Pharmaceutical Co., Tokyo, Japan) disks, as previously described (15, 20). A boronic acid test to confirm AmpC β-lactamase production was also performed using CTX, CPDX, and CFX disks as described previously (15). E. coli strain ATCC 25922 was used as a control in all assays in accordance with CLSI guidelines (34, 35). Klebsiella pneumoniae strain ATCC 700603 was used as an additional reference strain.
A PCR assay was performed to detect the presence of β-lactamase genes blaCTX-M, blaTEM, blaSHV, or blaCMY in the resistant isolates as previously described (15, 20). The resistance genes were sequenced using the DNA sequencing primers shown in Table 3. Resulting sequence data were analyzed using the Basic Local Alignment Search Tool (BLAST) available from the DNA Data Bank of Japan (DDBJ) (https://www.ddbj.nig.ac.jp/index.html, accessed 1 May 2019).
Primers used for detection and sequence determination of bla genes in this study
WGS analysis.WGS analysis was performed on one blaCTX-M-14-harboring S. Senftenberg isolate (SESen3709) and on blaCTX-M-15-harboring S. Haardt isolates, as previously described (36). Of the three blaCTX-M-15-harboring S. Haardt isolates, SEHaa3795 was selected as a representative isolate. The WGS data were analyzed using the Database of Pathogen Genomics and Epidemiology, GenEpid-J (37), and by using the ResFinder and VirulenceFinder tools available from the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/, accessed 18 July 2019).
In silico comparative analysis of plasmid replicon types.To compare the replicon types of other Salmonella-derived plasmids classified as IncH1, IncHI1, or IncH1/IncF types in previous studies with that of pSESen3709_1, nucleotide sequence data for these plasmids were obtained from the GenBank database (accession numbers AF250878, AL513383, AM412236, KP899804, KP899805, KP899806, and MG874042) (22, 23, 25–27) (Table S1). The nucleotide data were obtained by BLASTN and PubMed searches using the keywords “IncHI1 and plasmid,” “IncHI1 and Salmonella,” “plasmid and Salmonella,” and “IncHI1, plasmid, and Salmonella.” All sequence data were reanalyzed using GenEpid-J.
Comparative genome sequence analysis.The nucleotide sequence of the blaCTX-M-14-carrying plasmid pSESen3709_1 from the S. Senftenberg isolate recovered in the present study was compared to that of plasmid pSa4-CIP (GenBank accession no. MG874042.1) (27). pSa4-CIP was selected because it shared the highest nucleotide sequence similarity with pSESen3709_1 in BLASTN analysis. In addition, the blaCTX-M-14 sequence from pSESen3709_1 was compared to the corresponding region of p14E509-CTXM (GenBank accession no. MG764547.1) (38), which showed a high level of nucleotide sequence similarity in the BLASTN analysis. The chromosomal sequence of the S. Senftenberg isolate was then compared to those of the S. Haardt isolates identified in this study. The blaCTX-M-15 region of the S. Haardt chromosome was then compared to the complete sequence of plasmid pEC_L46 (GenBank accession no. GU371929.1) (39), which showed a high level of sequence similarity in the BLASTN analysis. The sequences of the reference plasmids, pSa4-CIP, p14E509-CTXM, and pEC_L46, were also reanalyzed using GenEpid-J. Comparative plasmid sequence analysis was performed using BLASTN and the Artemis Comparison Tool (40), and the results were visualized using Easyfig (41). Insertion sequences were confirmed in the analyzed plasmids using ISfinder (https://www-is.biotoul.fr/index.php, accessed 1 May 2019) (42).
Ethics statement.This study was performed in accordance with the guidelines of the Ethics Regulations Related to Epidemiological Research at the Fukuoka Institute of Health and Environmental Sciences under permit number 30-4.
Data availability.All bla sequence data obtained in the present study have been deposited in the DDBJ under the following accession numbers: blaCMY-2, LC383362 to LC383364; blaCTX-M-14, LC383361; blaCTX-M-15, LC383365 to LC383367. The complete, annotated genomic sequences of the S. Senftenberg and S. Haardt isolates have been deposited in the DDBJ under accession numbers AP020332 to AP020334 and AP020330 and AP020331, respectively. The short- and long-read sequences obtained via transcriptome sequencing (DNA-Seq) were deposited in the DDBJ, as follows: S. Senftenberg (SESen3709; BioProject PRJDB8208, BioSample SAMD00168437, DRA accession numbers DRR174990 and DRR174991); S. Haardt (SEHaa3795; BioProject PRJDB8208, BioSample SAMD00168438, DRA accession numbers DRR174992 and DRR174993).
ACKNOWLEDGMENTS
This study was supported in part by a grant-in-aid (grant JP19K19428) from the Japan Society for the Promotion of Sciences (KAKENHI), and grants from the Japan Agency for Medical Research and Development (AMED; grants JP19fk0108103 and JP20fk0108120). The sponsors played no role in the study design, in the collection, analysis, or interpretation of data, in the writing of the report, or in the decision to submit the article for publication.
We thank S. Katsuki, M. Hamasaki, S. Nakayama, and K. Hirano of the Fukuoka Institute of Health and Environmental Sciences for their invaluable advice. We are also grateful to T. Doi and T. Yamada of the National Institute of Infectious Diseases, Japan, for their assistance. We thank Tamsin Sheen and Sandra Cheesman from Edanz Group for editing a draft of the manuscript.
FOOTNOTES
- Received 9 January 2020.
- Accepted 7 April 2020.
- Accepted manuscript posted online 10 April 2020.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
