Genomic characterization of a large plasmid containing a blaNDM-1 gene carried on Salmonella enterica serovar Indiana C629 isolate from China

Background The bla NDM-1 gene in Salmonella species is mostly reported in clinical cases, but is rarely isolated from red and white meat in China. Methods A Salmonella Indiana (S. Indiana) isolate was cultured from a chicken carcass procured from a slaughterhouse in China. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing of the isolate was carried out and data was analyzed. Results A large plasmid, denoted as plasmid pC629 (210,106 bp), containing a composite cassette, consisting of IS26-bla NDM-1-ble MBL -△trpF-tat-cutA-ISCR1-sul1-qacE△1-aadA2-dfrA12-intI1-IS26 was identified. The latter locus was physically linked with bla OXA-1, bla CTX-M-65, bla TEM-1-encoding genes. A mercury resistance operon merACDEPTR was also identified; it was flanked on the proximal side, among IS26 element and the distally located on the bla NDM-1 gene. Plasmid pC629 also contained 21 other antimicrobial resistance-encoding genes, such as aac(6′)-Ib-cr, aac(3)-VI, aadA5, aph(4)-Ia, arr-3, blmS, brp, catB3, dfrA17, floR, fosA, mph(A), mphR, mrx, nimC/nimA, oqxA, oqxB, oqxR, rmtB, sul1, sul2. Two virulence genes were also identified on plasmid pC629. Conclusion To the best of our knowledge, this is the first report of bla NDM-1 gene being identified from a plasmid in a S. Indiana isolate cultured from chicken carcass in China.


Background
The New Delhi metallo-beta-lactamase (NDM) is one of the most commonly reported carbapenemase resistance mechanisms in the world [1]. The NDM-1 encoding gene (bla NDM-1 ) was first detected in Klebsiella pneumoniae recovered from a Swedish patient who was infected with an antibiotic-resistant bacterium acquired in New Delhi, India [2,3]. Thereafter, this plasmid-mediated NDM-1 resistance mechanism has been widely reported [4]. The presence of bla NDM-1 was generally associated with resistance to antimicrobial compounds, such as aminoglycosides, beta-lactams and fluoroquinolones. The bla NDM-1 was found to be located on different large plasmids, which were often readily transferable to other bacterial species [5,6]. Therefore the dissemination of the blaNDM-1-containing plasmids has reduced the therapeutic options available for the treatment of patients [7].
Salmonella species is one of the most prevalent zoonotic pathogens that cause outbreaks of gastroenteritis in the world. Recently, many researchers have isolated Salmonella from chicken meat (at farm, slaughter house and retail outlets) and its byproducts in China [8,9]. Moreover, it has been reported that Salmonella has the potential to act as a reservoir for different antimicrobial resistance-encoding genes [10]. Of note, transmission of Salmonella species from animal to humans via different food chains is well recognized [11]. Currently, NDM-1-producing Salmonella species have been reported, but the majority are linked to hospital clinics. However, this resistance mechanism is hardly ever reported in bacteria cultured from the meat of food-producing animals [12][13][14].
Here, for the first time, we report the isolation of a S. Indiana strain recovered from a chicken carcass in China. This bacterium was positive for bla NDM-1 gene, which was located on a large plasmid, along with several other antimicrobial resistance genes, that conferred an extensively-drug resistance (XDR).

Methods
Strain collection and antibiotic susceptibility testing S. Indiana C629 was cultured from a slaughtered chicken carcass sample, in Qingdao, Shandong province, in November 2014. The sample was procured from poultry meat that was ready to dispatch in the local supermarket. The slaughterhouse studied, is the largest slaughter house in Qingdao, Shandong province, with approximately 5000 tons production/month. Antimicrobial susceptibility testing (AST) using the Biofosun® Gram-negative panels (Fosun Diagnostics, Shanghai, China), containing 23 compounds, was carried out using the broth dilution method.

Whole genome sequencing, assembly, and annotation
The complete genome of S. Indiana C629 was isolated as described by Wang et al. [15]. Briefly, whole-genome sequencing was performed using the Pacific Biosciences RS II platform (SMRT® Pacific Biosciences, Menlo Park, CA, USA). De novo assembly of the reads obtained was carried out using continuous long reads (CLR) following the Hierarchical Genome Assembly Process (HGAP) workflow (PacBioDevNet; Pacific Biosciences) as available in the SMRT® Analysis v2.3 program [16]. The predicted functions of proteins identified were annotated based on homologs when compared to SwissProt (http://www.uniprot.org/uniprot/) clusters of orthologs groups (COG) (http:// www.ncbi.nlm.nih.gov/COG/), and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) based on the Bestplaced reference protein set and GeneMarkS+. The complete genome of S. Indiana plasmid pC629 was deposited in the NCBI database with the fallowing accession number CP015725 (plasmid pC629).

Sequence analysis of the plasmid pC629 genome
Antimicrobial resistance genes in the genome were predicted using the Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/analyze) [17]. Virulence factor database (VFDB) (http://www.mgc.ac.cn/ VFs/main.htm) was used to predict the presence of virulence factors in the genome of plasmid pC629 [18]. Finally, the antibacterial Biocide and Metal Resistance Genes Database (BacMet) (http://bacmet.biomedicine.gu.se/) was used to predict the presence of any metal-resistance genes contained in the genome of plasmid pC629 [19].

Susceptibility to a panel of antimicrobial compounds
In this study, it was found that S. Indiana C629 isolate was only susceptible to colistin (MIC 0.5 mg/L) and resistant to 18 compounds representing 10 different antimicrobial classes ( Table 1). Of note the S. Indiana C629 isolate was found to be resistant to imipenem (MIC 4 mg/L) and meropenem (MIC 8 mg/L), both belong to carbapenem antibiotics. Although there are no standards available for antimicrobial agents of mequindox (MIC >64 mg/L), streptomycin (MIC >32 mg/L), ceftiofur (MIC >32 mg/L) and tigecycline (MIC <0.5 mg/L) in the CLSI guideline, MIC measurements for these antimicrobials have shown that the activity of these antimicrobial agents may also be compromised when being considered either for treatment of infection cases or growth promotion applications ( Table 1).

Analysis of the genome to identify antimicrobial resistance-encoding genes
Whole genome of plasmid pC629 was circular. The total size of plasmid pC629 was determined to be 210,106-bp. The average GC content was 48.6% and it was predicted to encode 223 open reading frames (ORFs) which covered almost 83.4% of the structure. The CARD database was queried to identify resistance related genotypes in the genome of plasmid pC629.
A total of 30 antimicrobial resistance genes (two genes were identified as sul1) were identified and which encoded resistance to 19 antimicrobials. The resistance-encoding region in plasmid pC629 was bracketed by several IS26 elements that were located in different orientations.

Analysis of the genome to identify virulence-encoding genotypes
To identify potential virulence genes in the plasmid pC629 genome, the virulence factors listed in the Virulence Factors Database (VFDB) were aligned to the ORF protein sequences using BLASTP and filtered with 50% identity and 90% match length. By using this approach, two virulence genes, such as dDE_Tnp_1, matched abzi_00085 and abzi_00086, were found on plasmid pC629 (Fig. 1).

Analysis of the genome to identify metal resistance-encoding genotypes
In this study we used the antibacterial biocide and metal resistance genes database (BacMet) to predict the presence of antibacterial biocides and metal-resistance genes on the genome of plasmid pC629. These results predicted the presence of mercury and tellurium resistance genes on plasmid pC629.

Discussion
To the best of these authors knowledge, we report on the successful isolation and characterization of the metallo-betalactamase encoding bla NDM-1 gene harboring S. Indiana strain, cultured from a chicken carcass in China. This bacterium expressed a diverse resistant phenotype. In addition to its antimicrobial resistance-encoding gene repertoire, this bacterium also possessed several virulence genes along with various metal-encoding resistance genes. Generally, the poultry production operation demonstrated good pathogen control and reduction strategies; however, the isolation of Salmonella from slaughtered meat that has already been passed by processing checks is very striking. Interestingly, all of these resistant antimicrobial agents tested in this study are widely used at human and veterinary clinics.
The bla NDM-1 gene was embedded in an ISCR1 complex class 1 integron of 11.8-kb ( Figs. 1 and 2). This region was bracketed by two IS26 elements that were positioned in the same orientation. These observations suggested that the gene may have been acquired as a composite transposon. Similar structures had also been reported in E. coli (CP016035) and Citrobacter freundii (JX182975) (Fig. 2) [20,21]. Interestingly, Klebsiella pneumoniae (KT725789) also harbored a bla NDM-5 , that is located on plasmid pCC1409-1 (Fig. 2) and whose structural arrangement aligns with our observation here [22]. Downstream of the bla NDM-1 , a ble MBL gene is located which encodes resistance to bleomycin and this is followed by a truncated phosphoribosyl anthranilate isomerase gene (denoted as △trpF), a twin-arginine translocation pathway signal protein gene (encoded by tat) and a dihydroorotate dehydrogenase gene (cutA). Further downstream, an ISCR1 element was identified which was located proximally to a class 1 integron; in addition to a gene cassette containing dfrA12-aadA2 in the classical heal-to-tail arrangement. The similar genetic arrangement have also been reported in an E. coli (CP016035), in which a complex class 1 integron harboring an ISCR1 element was identified [23]. The insertion elements ISCR1 and IS26 are known to mediate the mobilization of bla NDM-1 in Enterobacteriaceae and non-Enterobacteriaceae species [21,24]. Moreover, the intI1 gene was followed by a copy of insertion element IS26 located on the distal side. A similar genetic arrangement has also been reported in pNDM-CIT (JX182975) [21].

26
]. Moreover, several other resistance genes encoded on plasmids pS414 and pSTA155 have been reported which are colocated on the IncHI2 plasmid. It has already been suggested that these genes are associated with a multidrug resistant in S. Indiana isolated in China [26]. While, in the downstream region of these resistance genes a chromate transporter protein (encoded by chrA) and a putative DNA-binding protein (ywzG) were identified. However, this locus was structured differently in the plasmids pSJ_255, pS414, and pSTA155 compared to plasmid pC629.
In this study, we identified a oqxRAB operon on plasmid pC629 and this locus was flanked by IS26 elements (Figs. 1  and 2). This gene confers low level resistance to fluoroquinolones. Additionally, this gene has 100% sequence similarity at the nucleotide level with other structures identified in in E. coli such as plasmids pHNSHP45-2 (KU341381) [33]. pSJ_255 (CP011062, unpublished), and p42-2 (KT990220, unpublished). Furthermore, it has also been reported that transconjugants carrying oqxA and oqxB exhibited a 4-to 16-fold high MIC to (fluoro) quinolones, and a 16-to 64fold high MIC against quinoxalines [34]. A fosfomycin resistance encoding gene fosA flanked with IS26 (Fig. 2) and further it was associated with bla CTX-M-65 . We suspected that bla CTX-M-65 may play role in the maintenance and dissemination of fosA gene. Moreover, both fosA and bla CTX-M-65 were separated by only six genes as shown in Fig. 1. Therefore, we suggest that these genes may be transmitting together [35].
Interestingly, we found that all of the multi-drug resistance clusters identified on plasmid pC629 and all of these multi-drug resistance clusters were flanked by IS26. On the basis of this genetic structure we suggested that pC629 has the potential to disseminate antibiotic resistance genes to other species and the host. In this study we also found two virulence genes named as dDE_Tnp_1, matched abzi_00085 and abzi_00086 on plasmid pC629 (Fig. 1), both have already been characterized in Acinetobacter baumannii MDR-ZJ06 which is a multidrugresistant bacterium detected and isolated from patient in China [37]. It has already been suggested that these two genes were participated in composition of the capsule gene cluster, which plays an important role in protecting bacteria from the host innate immune response [38].
Our results predicted the existence of mercury and tellurium resistance gene on plasmid pC629 genome (Fig. 1). Later, we identified 7 mercury resistance genes, and all of these mercury resistance genes flanked by IS26 elements (IS26-int-urf2-merE-merD-merA-merC-merP-merT-merR-IS26) (Fig. 3). Similar physical map has also been reported on plasmids pHSO91147 (KX236178, unpublished); pBK34397 (KU295132, unpublished), and pKPHS2 (CP003224) [39], with some insertion sequence variations. Levels of mercury have been elevated in the environment due to industrial pollution. Therefore, many bacterial species have developed detoxification strategies to combat its deleterious effects [40]. The gene merA is one of the components contained within the mer operon, and this is often associated with mobile genetic elements, including transposable elements and plasmid [41]. Therefore, mercury resistance is transferable among bacterial species via horizontal gene transfer (HGT) mechanisms. Several researchers have suggested that the mer operon is physically (See figure on previous page.) Fig. 2 Major structural features of plasmid pC629 compared with several plasmids from NCBI. a. Major structural features of plasmid pC629 compared with plasmids pC06114_1, pNDM-CIT, and pCC1409-1 identified in Escherichia coli, Citrobacter freundii, and Klebsiella pneumoniae, respectively. ORFs are shown as arrowheads (blue arrows, IS26, ISCR1, and tnpM; red arrows, antibiotic resistance genes; pink arrows, membrane protein, MP; white arrows, hypothetical proteins, HP; green arrows, integrase related genes -see the key to the right hand side of the panel). b. Major structural features of plasmid pC629 compared with plasmids pS414, pSJ_255, pSTA155, p112298-KPC, pEK499, and pUUH239.2 identified in Salmonella Indiana, Escherichia coli, Salmonella Indiana, Citrobacter freundii, Escherichia coli and Klebsiella pneumoniae, respectively. ORFs are shown with arrowheads (blue arrows, IS26, IS6100 and other transposase related genes; red arrows, antibiotic resistance genes; pink arrows, membrane protein, MP; white arrows, hypothetical proteins, HP; green arrows, integrase related genes -see the key to the right hand side of the panel). c. Major structural features of plasmid pC629 plasmid compared with plasmids pHNSHP45-2, pSJ_255, and p42-2 all identified in Escherichia coli. ORFs are shown with arrows (blue arrows, IS26; red arrows, antibiotic resistance genes -see the key to the right hand side of the panel). d. Major structural features of plasmid pC629 compared with plasmids p397Kp, pCT-KPC, and p1205p1 identified in Klebsiella pneumoniae, Klebsiella pneumoniae, and Shigella flexneri, respectively. ORFs are shown with arrows (blue arrows, IS26; red arrows, antibiotic resistance genes; white arrows, hypothetical proteins, HP -see the key to the right hand side of the panel). e. Major structural features of plasmid pC629 plasmid compared with plasmids pGD0503Z13, pHK0653, and pCN061p6 identified in Escherichia coli, Salmonella species and Escherichia coli, respectively. ORFs are shown with arrows (blue arrows, IS26; red arrows, antibiotic resistance genes; white arrows, hypothetical proteins, HP -see the key to the right hand side of the panel) linked with one or more antimicrobial resistanceencoding genes [42]. In this study, the mercury resistance genes were found to be linked to the bla NDM-1 gene as follows, IS26-int-urf2-merE-merD-merA-merC-merP-merT-merR-IS26-bla NDM-1 -ble MBL -△trpF-tat-cutA-ISCR1-sul1-q acE△1-aadA2-dfrA12-intI1-IS26. This observation provides further evidence of the potential role of mer genes in the dissemination of resistance genes.

Conclusion
To the best of our knowledge, this is the first report describing the characterization of a large XDR expressing plasmid with the metallo-beta-lactamase encoding blaNDM-1 gene cultured from a S. Indiana strain isolated from chicken carcass in China. Several multi-drug resistance gene clusters were identified and flanked by IS26 elements. Metal-encoding genes and various metalencoding resistance determinants were also identified. These data could be used proactively to assist the poultry industry in China to develop food safety measures, designed to limit the transmission of these XDR bacteria and other biological hazards from food-producing animals.
Abbreviations VFDB: Virulence factor database; CARD: Comprehensive Antibiotic Resistance Database; COG: clusters of orthologs groups; HGAP: Hierarchical Genome Assembly Process; CLR: Continuous long reads; AST: Antimicrobial susceptibility testing; CLSI: Clinical and Laboratory Standards Institute; MIC: minimum inhibitory concentration Fig. 3 Major structural features of plasmid pC629 compared with plasmids pHSO91147, pBK34397, and pKPHS2 identified in Klebsiella pneumoniae, Escherichia coli and Klebsiella pneumoniae, respectively. ORFs are shown with arrows (blue arrows, transposase; red arrows, metal resistance genes; pink arrows, membrane protein, MP; green arrows, integrase related genes --see the key to the right hand side of the panel)