Background: Salmonella enterica subspecies enterica serovar Saintpaul (S. Saintpaul) is an important gut pathogen which causes salmonellosis worldwide. Although intestinal salmonellosis is usually self‑ limiting, it can be life‑ threatening in children, the elderlies and immunocompromised patients. Appropriate antibiotic treatment is therefore required for these patients. However, the efficacy of many antibiotics on S. enterica infections has been greatly compromised due to spreading of multidrug resistance (MDR) plasmids, which poses serious threats on public health and needs to be closely monitored. In this study, we sequenced and fully characterized an S. enterica MDR plasmid pSGB23 isolated from chicken. Results: Complete genome sequence analysis revealed that S. Saintpaul strain SGB23 harbored a 254 kb megaplas‑ mid pSGB23, which carries 11 antibiotic resistance genes responsible for resistance to 9 classes of antibiotics and quaternary ammonium compounds that are commonly used to disinfect food processing facilities. Furthermore, we found that pSGB23 carries multiple conjugative systems, which allow it to spread into other Enterobacteriaceae spp. by self‑ conjugation. It also harbors multiple types of replicons and plasmid maintenance and addictive systems, which explains its broad host range and stable inheritance. Conclusions: We report here a novel MDR plasmid pSGB23 harboured by S. enterica. To our knowledge, it carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. enterica MDR plasmids identified so far. The isolation of pSGB23 from food sources is worrisome, while surveillance on its further spreading will be carried out based on the findings reported in this study. that food processing facilities are one of the main sources Background for the dissemination of S. Saintpaul [2, 4]. Salmonella enterica serovar Saintpaul (referred to as S. Although intestinal salmonellosis is normally self- Saintpaul hereafter) is a foodborne gut pathogen and a limiting and does not require antibiotic treatment in major cause of salmonellosis worldwide . S. Saintpaul healthy adults, it can be life-threatening among chil- was reported to be responsible for several recent large dren, the elderlies, and immunocompromised patients outbreaks of salmonellosis, which affected hundreds of . Therefore, appropriate antimicrobial treatments people across multiple cities and states [2, 3]. These out - are required for these patients . It was suggested that breaks were mainly due to contamination of food such as fluoroquinolones such as ciprofloxacin, the third-gener - unpasteurized fruit juice and raw products, suggesting ation cephalosporins such as ceftriaxone, and trimetho- prim–sulfamethoxazole can be beneficial for patients *Correspondence: firstname.lastname@example.org; email@example.com 2 with Salmonella infections . However, the emergence Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551, Singapore of multidrug resistant Salmonella strains worldwide has Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ding et al. Gut Pathog (2018) 10:20 Page 2 of 5 challenged the effectiveness of these antibiotics . In HGAP2 pipeline. The low-quality ends of the assembled Salmonella, the acquisition of antibiotic resistance genes contigs were trimmed using CLC Genomic Workbench is usually mediated by MDR plasmids, many of which can v10.0 with an estimated error rate of lower than 0.1% spread among Salmonella and other Enterobacteriaceae as the cut-off. The overlapping regions of the trimmed spp. by conjugation . The surveillance on novel MDR contigs were further identified and resolved by BLASTn plasmids in Salmonella is therefore important for the search assisted with manual curation. The plasmid understanding of their epidemiology and transmission, sequence was uploaded to the rapid annotations using which can provide clues for the design of effective thera - subsystem technology (RAST) server for initial gene pies and the control of their further spreading. prediction and annotation, followed by manual BLASTp In this study, we sequenced and characterized a novel search to ensure accurate annotation . Antibiotic MDR plasmid carried by a S. Saintpaul strain isolated resistance genes carried by pSGB23 were predicted using from chicken in a food market in Singapore. This plas - the ResFinder 2.1 server , whereas the IS26 sequences mid displays a mosaic backbone structure and encodes were identified by the ISfinder . Comparative sequence resistance mechanisms to 9 classes of antibiotics and analysis of pSGB23 with pEC2–4 was performed by the commonly used disinfectant quaternary ammonium BLASTn search using BLAST Ring Image Generator 0.95 compounds (QACs). The further spreading of this plas - , with antibiotic resistance genes and IS elements mid among Salmonella and other Enterobacteriaceae labeled in the figure as instructed by the manual. spp. may pose serious threats to public health. Quality assurance Methods Genomic DNA used for sequencing was isolated from a Conjugation and antibiotic susceptibility test single colony of SGB23 to avoid contamination caused The S. Saintpaul strain SGB23 was previously isolated by allochthonous microorganisms. In addition, PacBio from a local food market. Conjugation between S. Saint- assembly only generated two contigs, which are the chro- paul SGB23 and the azide-resistant E. coli J53 strain was mosome and plasmid sequences of SGB23, whereas no performed on LB agar plates at 25 and 37 °C for 48 h. other assemblies were identified. Low-quality regions Briefly, overnight culture of the two strains was washed at both ends of the assembled contigs were trimmed to with 0.9% NaCl for three times. Bacterial suspension of ensure high-accuracy of the genome as described in the two strains was mixed at 1:1 ratio with approximately “Methods”. 10 colony forming units (CFU) of each strain and spot- ted onto a filter paper placed on LB agar plates. Transcon - Results and discussion jugants were selected using LB agar plates containing In 2016, an MDR S. Saintpaul strain SGB23 was isolated 100 μg/ml of sodium azide and 100 μg/ml of ampicillin. from chicken meat in a local food market in Singapore. Conjugation efficiency was calculated by dividing the The whole-genome of SGB23 was sequenced on a PacBio CFU of transconjugants by the CFU of the donor strain RS II platform and the sequencing reads were success- SGB23. Antimicrobial susceptibility test was performed fully assembled by HGAP 2.0 into two contigs: one chro- by broth microdilution using Mueller–Hinton Broth. The mosome contig with 89-fold coverage and one plasmid inoculum for each strain was approximately 10 CFU/ml, contig with 109-fold coverage. The close coverages of followed by incubation at 37 °C for 18 h. the chromosome and plasmid sequences suggests that pSGB23 plasmid has a low copy number. The sizes, GC Sequencing, assembly, and annotation content and coding capacity of SGB23 chromosome and The total DNA of SGB23 was purified using Blood and pSGB23 plasmid are summarized in Table 1. Cell Culture DNA Midi Kit (Qiagen) and sequenced on To further identify the MDR determinants of S. Saint- a PacBio RS II system. The full-length chromosome and paul SGB23, we analyzed the acquired antibiotic resist- plasmid contigs of SGB23 were assembled from long ance genes carried by SGB23 using the ResFinder 2.1 reads obtained from the PacBio RS II system by using server . Interestingly, the chromosome of SGB23 Table 1 Length, GC contents and coding capacity of the chromosome and plasmid of S. Saintpaul SGB23 Length (bp) GC content (%) Protein-coding genes tRNA rRNA SBG23 chromosome 4,792,385 52.2 4803 85 14 pSGB23 plasmid 254,041 47.5 313 0 0 The coding sequences were predicted and annotated by the RAST server as described in “Methods” Ding et al. Gut Pathog (2018) 10:20 Page 3 of 5 encodes no antibiotic resistance genes, whereas its plas- plasmids with single replicon, and therefore, allow it mid pSGB23 is the sole antimicrobial determinant. To to adopt a broader host range within the Enterobacte- understand this epidemically important MDR plasmid riaceae [13, 14]. In particular, the IncFIA replicon was pSGB23, we annotated its sequence on the RAST server reported to commonly present in IncHI1 plasmids and  assisted with manual BLASTp search, analyzed its is responsible for the one-way incompatibility between replicon sequences by using the PlasmidFinder 1.3 server IncHI1 and IncF plasmids . . In addition, we found that the closest sequence to The IncHI1 type plasmids have been identified from pSGB23 in GenBank is plasmid pEC2–4 (CP016184) , several S. enterica serovars and were mainly associ- which covers 77% of pSGB23 sequence with a similarity ated with multidrug or heavy metal resistance [14, 15]. of 99%. pEC2–4 is an IncHI1 plasmid recently isolated in Similarly, we found that pSGB23 carries 11 antibiotic Malaysia and carries the colistin resistance gene mcr-1 resistance genes, which are responsible for resistance . The comparison between pSGB23 and pEC2–4 with to aminoglycosides (addA1, acc(3)-Iva and aph(4)-Ia), the predicted replicons and predicted antibiotic resist- macrolides (mph(A)), quinolones (qnrS1), tetracycline ance genes in pSGB23 are summarized in Fig. 1. (tet(A)), β-lactams (bla ), chloramphenicol and TEM-1B In total, pSGB23 harbours four replicons, which florfenicol (floR), fosfomycin (fosA4), sulfamethazine include two IncHI-specific replicons designated (sul3) and trimethoprim (dfrA15) (Fig. 1). In addition, RepHI1A and RepHI1B, one IncFIA replicon, and one it also harbors a qacF gene responsible for resistance IncN replicon (Fig. 1). The multiple replicons harbored to QACs, which are a class of disinfectants commonly by pSGB23 may give it competitive advantages over used in food production industry and clinical settings Fig. 1 The sequence map of pSGB23 in comparison to pEC4‑2 (CP016184). Black circle: pSGB23; blue circle: pEC2–4. The outermost circle shows the predicted genes encoded by pSGB23 with the arrow indicates the direction of transcription. Blue: plasmid replication proteins with their respective Inc types; green: genes related to conjugative transfer; red: antimicrobial resistance genes; purple: genes involved in plasmid partition and addiction systems; grey: IS26 sequences; black: genes with other predicted functions. Other genes encoding proteins with hypothetical functions and transposases are not shown in the figure Ding et al. Gut Pathog (2018) 10:20 Page 4 of 5 . Most of the antimicrobial resistance genes except Table 2 MICs of E. coli J53/pSGB23 and S. Saintpaul SGB23 to various antibiotics (unit: μg/ml) for fosA4 are located in an antibiotic resistance gene (ARG) cluster. Furthermore, the acquisition of antimi- E. coli J53 E. coli J53/pSGB23 S. crobial resistance genes by pSGB23 probably involves Saintpaul SGB23 IS26-mediated integration, as 10 of the 11 IS26 ele- ments identified by ISfinder [9 ] were found to flank the Ampicillin 32 > 1024 > 1024 antimicrobial resistance genes (Fig. 1). Interestingly, Carbenicillin 16 > 1024 > 1024 the IncN replicon is located in the ARG cluster and is Ceftriaxone 1 8 8 also flanked by two IS26 elements, suggesting that it is Azithromycin 8 32 64 probably acquired by IS26-mediated integration. Ciprofloxacin 0.03 0.5 1 The IncHI1 type plasmids were previously reported to Chloramphenicol 16 > 64 > 64 have a thermosensitive mode of conjugation, in which Fosfomycin 1 > 64 > 64 optimal conjugative transfer occurs between 22 and Gentamicin 2 128 128 28 °C, whereas inhibition of conjugation was observed Streptomycin 8 32 32 at 37 °C . In pSGB23, we identified 23 conjuga - Tetracycline 2 64 64 tive genes, which include 10 tra genes, 7 trh genes and Trimethoprim 0.5 > 256 > 256 6 htd genes that are located in two clusters (Fig. 1). To Sulfadimethoxine 64 > 256 > 256 verify if pSGB23 is self-transmissible by conjugation, we Cetrimonium bromide 16 64 64 performed mating experiments between SGB23 and the Cetylpyridinium chloride 8 16 16 azide-resistant recipient strain Escherichia coli J53 on Colistin 1 1 2 solid agar surface for 48 h. The conjugative efficiency of E. coli J53/pSGB23 and S. Saintpaul SGB23 shared similar resistance profiles −3 −5 pSGB23 is 3.1 ± 1.4 × 10 at 25 °C and 3.3 ± 0.8 × 10 at with increased resistance to nine classes of antibiotics and QACs, whereas they 37 °C (n = 4), suggesting that pSGB23 is self-conjugative remain sensitive to colistin and the conjugation activity is partially inhibited at 37 °C. We further determined the minimal inhibitory con- centrations (MICs) of E. coli J53/pSGB23 and S. Saint- The large and low-copy-number plasmids such as paul SGB23 to different antimicrobial agents by broth pSGB23 are usually under the risk of plasmid loss dur- microdilution. It was found that E. coli J53/pSGB23 ing cell division. Therefore, many of these plasmids have showed increased resistance to 12 antibiotics belonging evolved active maintenance mechanisms to ensure their to 9 different classes, and at-least 2-fold increase in the stable inheritance by the daughter cells during cell divi- MICs of two tested QAC compounds cetrimonium bro- sion . For instance, the plasmid partition systems mide and cetylpyridinium chloride compared to E. coli such as ParA/ParB actively segregate plasmid copies to J53 (Table 2). This resistance profile is consistent with the daughter cells, whereas the addiction systems such the antimicrobial resistance genes carried by pSGB23 as StbA/StbB act like toxin/antitoxin that eliminates (Table 2, Fig. 1). Among these tested antibiotics, ampi- plasmid-free daughter cells . In pSGB23, we found cillin, ceftriaxone, chloramphenicol, ciprofloxacin and two types of partition systems designated ParA/ParB trimethoprim–sulfamethoxazole are the first-line drugs and ParM/ParR that are embedded among the conjuga- to treat severe Salmonella infections, whereas azithro- tive genes, and the toxin/antitoxin system StbD/StbE mycin has been increasingly used to manage both Typhi that are at downstream of the RepHI1B replicon (Fig. 1). and non-Typhi Salmonella infections that showed poor The presence of plasmid maintenance and addiction sys - response to the recommended first-line drugs . tems can ensure the stable inheritance of pSGB23 in the Therefore, the spread of pSGB23 to other S. salmonella absence of selection pressure posed by antibiotics. serovars can greatly compromise the efficacies of these pSGB23 also carries genes that were previously antibiotics in treating Salmonella infections. In addition, reported to be implicated in stress response, such as resistance to QACs may allow the host bacteria to bet- ybaK, uspA, dsbC, umuC and a gene encoding ATP- ter adapt to the food processing facilities, where QACs dependent protease La, and genes involved in specific are extensively used as disinfectants to inhibit microbial metabolic functions such as the dmsABC operon, napF, growth and prevent foodborne illness . It was also and torD (Fig. 1). Their functional implications on the noted that the resistance patterns between E. coli J53/ host remains unclear. pSGB23 and S. Saintpaul SGB23 were very similar, which In summary, we report here a novel plasmid pSGB23 confirmed that pSGB23 is the only resistance determi - isolated from S. enterica. The plasmid harbors both nant of S. Saintpaul SGB23. IncHI1 and IncN type replicons and carries 12 antimi- crobial resistance genes, which can confer resistance to Ding et al. 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Published: Jun 4, 2018
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