Food Research International 45 (2012) 931–934
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Sequencing of plasmids from a multi-antimicrobial resistant Salmonella enterica serovar Dublin strain☆ Jing Han a,1, Aaron M. Lynne b,1, Donna E. David c, Rajesh Nayak a, Steven L. Foley a,⁎ a b c
Division of Microbiology, National Center for Toxicological Research, U.S. FDA, Jefferson, AR, 72079, USA Department of Biological Sciences, Sam Houston State Univ., Huntsville, TX, 77341, USA Core Research Laboratory, Marshfield Clinic Research Foundation, Marshfield, WI, 54449, USA
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Article history: Received 14 February 2011 Accepted 7 April 2011 Keywords: Salmonella enterica serovar Dublin Plasmid sequencing Antimicrobial resistance Virulence
a b s t r a c t Salmonella enterica serovar Dublin (S. Dublin) is a host-adapted serotype whose primary host is cattle, which can serve as a potential reservoir for human infections. S. Dublin remains one of the leading causes of severe invasive infections and deaths associated with salmonellosis. Because of their propensity to cause severe infection, antimicrobial therapy is often required, thus antimicrobial resistance is an important concern. Plasmids play a key role in facilitating drug resistance in these pathogens. This study reports the results of DNA sequencing and sequence analysis of plasmids from a highly multidrug resistant strain (resistant to 11/15 drugs tested) of S. Dublin that originated from cattle. The strain was found to contain four plasmids of approximately 8, 77, 89, and 174 kb. The 174 kb plasmid is an incompatibility group (Inc) A/C plasmid containing genes associated with resistance to at least 9 different antimicrobials, as well as disinfectants and metals. The 88.5 kb plasmid is an IncFIB plasmid containing genes associated with resistance to at least 3 antimicrobial agents and mercurial compounds. The 77 kb plasmid is a S. Dublin virulence plasmid containing multiple virulence-associated genes and the 7.9 kb plasmid encodes mobilization and replication genes. Overall, sequencing identified multiple plasmids containing antimicrobial resistance and virulence genes. The resistance genes identified correlated to the observed resistance phenotype, further indicating the importance of plasmids in antimicrobial resistance in many Salmonella. Published by Elsevier Ltd.
1. Introduction Salmonella enterica serovar Dublin (S. Dublin) is a serotype that is primarily associated with cattle. In cattle, the organism can be carried asymptomatically by adult animals, serving as a potential reservoir for human infections (Nielsen, Schukken, Grohn, & Ersboll, 2004). Human infections have been associated with the consumption of raw milk and cheese made from it, improperly handled beef and direct zoonotic transmission from cattle and other animals (Allerberger et al., 2002; Mateus et al., 2008; Vaillant et al., 1996). In the United States approximately 75 cases of human infections were reported each year from 1996–2006 (Centers for Disease Control and Prevention, 2008). Even with this low number of S. Dublin human infections, it remains a leading cause of severe invasive infections and deaths associated with salmonellosis (Vugia et al., 2004). S. Dublin human infections are often associated with septicemia and severe infections of different organ systems, which
☆ The views presented in this manuscript do not necessarily reflect those of the U.S. Food and Drug Administration or the U.S. Department of Health and Human Services. ⁎ Corresponding author at: Division of Microbiology, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, AR 72079, USA. Tel.: + 1 8705437547; fax: + 1 8705437307. E-mail address:
[email protected] (S.L. Foley). 1 Both authors contributed equally to the manuscript. 0963-9969/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.foodres.2011.04.016
contribute to the observed high level of mortality. Confounding the problem is that many S. Dublin isolates are also known to be multidrug resistant (MDR), leading to potential treatment failure (Lynne, Dorsey, David, & Foley, 2009). Many of the factors known to be associated with the increased virulence and antimicrobial resistance are encoded on plasmids. In Salmonella many virulence plasmids carry the spv operon which likely also plays a role in systemic infections (Baumler, Tsolis, Ficht, & Adams, 1998). Additionally, many MDR Salmonella are known to carry resistance plasmids that carry a number of genes associated with the observed resistance phenotype. Therefore, increased knowledge of plasmid genetics will aid in our understanding of the factors associated with increased antimicrobial resistance and virulence in serovar Dublin isolates. The objective of this study was to carry out DNA sequencing of the plasmids from a MDR S. Dublin isolate containing multiple plasmids and compare the results with those of previous plasmid sequencing studies. 2. Materials and methods 2.1. Bacterial strains and plasmid isolation In order to examine the potential impact of plasmids on antimicrobial resistance in Salmonella enterica serovar Dublin, an isolate (MCRF
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853) containing multiple plasmids and resistant to 11 antimicrobials (ampicillin, amoxicillin/clavulonic acid, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole and tetracycline) was selected from the culture collection at the Marshfield Clinic Research Foundation, Marshfield, WI. The isolate originated from a bovine veterinary diagnostic sample. Total plasmid DNA was extracted using previously described methods for the isolation of large and small plasmids (Wang & Rossman, 1994). Additionally, OneShot Top 10 competent Escherichia coli (Invitrogen, Carlsbad, CA) were used in plasmid transfer experiments. 2.2. Sequencing, assembly and annotation Isolated total plasmid DNA was sent to Roche 454 LifeSciences Sequencing Facility (Bradford, CT) for pyrosequencing using the FLX system and chemistry. The sequencing reads were quality checked and initially assembled using the Newbler program (Roche 454 LifeSciences). The initial assembled contigs were further analyzed with SeqMan Pro (DNAStar, Madison, WI) and the ends of each contig were mapped to the ends of the remaining contigs using BLAST (National Center for Biotechnology Institute (NCBI), Bethesda, MD) to detect overlapping complementary sequences. The end-matching process was repeated until individual plasmids were closed. The putative plasmids were compared to sequences in GenBank (NCBI) using BLAST to determine similarity of assembly to previous projects. Following plasmid assembly, the plasmid sequences were submitted to the RAST annotation pipeline (Argonne National Laboratory, Argonne, IL) to identify putative coding sequences for initial annotation. The annotations were manually evaluated and updated using Artemis (Sanger Institute, Cambridge, UK); such that sequence locus tags were automatically assigned for each predicted coding sequence and their gene and protein identity verified and updated, if needed, by comparison to GenBank sequences using BLAST. The completed plasmid sequences were visualized using the DNAplotter program (Sanger Institute) and the sequences deposited in GenBank with accession numbers JF267651-JF267654. 2.3. Comparative genetic analysis Assembled plasmids were evaluated for the presence of genes associated with antimicrobial and disinfectant resistance, virulence and plasmid transfer, as well as mobile genetic elements such as integrons and transposons, based on the annotations and BLAST searching. Plasmid incompatibility (Inc) grouping was done in silico, by mapping the PCR primers described Carattoli et al. (2005) to the assembled plasmid sequences with BLAST configured for short reads. The GC skew was determined for the plasmids using Artemis (Grigoriev, 1998). 2.4. Transfer of antimicrobial resistance plasmids Isolated plasmid DNA was transferred into OneShot Top 10 competent cells using heat shock transformation according to the manufacturer's instructions (Invitrogen) and transformants were selected on LB agar plates containing tetracycline (16 μg/ml). The putative positive transformants were screened for antimicrobial susceptibility as previously described (Lynne et al., 2009) with E. coli ATCC 25922 as a quality control strain. 3. Results and discussion The sequencing resulted in 104,108 reads with an average read length of 209 bases. The assembly of the sequencing reads revealed the presence of 4 plasmids of 173,673 bp (pSD853_174, GenBank accession number JF267651, Fig. 1), 88,505 bp (pSD853_89, JF267652, Fig. 2), 77,331 bp (pSD853_77, JF267653, Fig. 3) and 7,860 bp (pSD853_7.9,
JF267654), respectively. This combination of plasmids is unique compared to previously sequenced bacteria. However each individual plasmid showed a degree of similarity to previously sequenced plasmids. Figs. 1–3 provide information on the presence of predicted antimicrobial and disinfectant resistance, virulence and transferassociated genes as well as the GC skew of the sequence in different regions of the plasmids. Plasmid pSD853_174 was predicted to have 198 coding sequences, of which 110 had unknown (hypothetical) function and had a 52.5% GC content. This plasmid is an incompatibility group (Inc) A/C multidrug resistance plasmid, with similarity to those of other enteric bacteria, including from S. Newport (GenBank accession number CP000604.1) (Welch et al., 2007) and Escherichia coli (FJ621588) (Call et al., 2010), to which the overall coverage is at least 95%. The main difference between pSD853_174 and the other plasmids is a deletion of accC, groLS and insertion sequence within a Tn21 transposable element. The overall Tn21 element (bases 126,637-146,358) includes genes associated with a class 1 integron with genes for sulfonamide (sul1), streptomycin (aadA) and quaternary ammonium compound (qacEΔ) resistance and mercury (mer operon) reduction and has greater similarity to the Tn21 element detected in Yersinia pestis (CP000603.1) (Welch et al., 2007). Additional genes identified on the plasmid correspond to the observed resistance to extended spectrum cephalosporins (2 copies of blaCMY), chloramphenicol (floR), tetracycline (tetA and tetR), streptomycin (strA and strB), and sulfonamides (sul2) and quaternary ammonium compound resistance (2 copies of sugE). The pSD853_89 plasmid (Fig. 2) was predicted to have 117 coding sequences, including 63 hypothetical proteins and a 51.6% GC content. pSD853_89 is an IncFIB plasmid that contains antimicrobial resistance, mercury reduction and virulence genes. Genes identified correspond to the observed resistance to tetracycline (tetA and tetR), kanamycin (aph(3')-I) and certain β-lactam drugs (blaTEM). This plasmid has the highest degree of similarity to the pU302L plasmid (NC_006816) from S. Typhimurium (Chen, Nace, Solow, & Fratamico, 2007), with the exception of a stretch that has similarity with E. coli pETN49 (FQ482074.1) from bases 16,217–28,874 and a region from 50,945–67,787 that is most similar to S. Typhimurium plasmid pYT1 (AB576781.1). The pSD853_89 plasmid contains potential virulence genes associated with iron acquisition (iutA) and plasmid maintenance (vagC and vagD) also (Mnif et al., 2010; Rodriguez-Siek, Giddings, Doetkott, Johnson, & Nolan, 2005). pSD853_77 (Fig. 3) is a S. Dublin virulence plasmid that is very similar to DQ115388.2 and CP001143.1 in GenBank (Hong, Chiu, Chu, Feng, & Ou, 2008). The sequences vary in two small regions with insertion sequence elements. The plasmid was predicted to have 112 coding sequences, with 45 identified as hypothetical proteins and a 48.8% GC content. The plasmid also contains multiple virulence genes, including the Salmonella plasmid virulence (spv) operon, vagC, vagD, ccdA and ccdB. The presence of the spv genes in S. Dublin can contribute to invasive infections (Libby et al., 1997), while the vag and ccd genes are likely associated with plasmid maintenance (Mnif et al., 2010). The pSD853_7.9 plasmid was predicted to have 11 coding sequences and is most similar to the 8.7 kb pTA1060 plasmid from Bacillus subtilis (U32380.1) and contains genes (mob, rep, rap and phr) that may play a role in plasmid mobilization (Meijer, Venema, & Bron, 1995). When the plasmids were transferred into an E. coli strain they were able to encode resistance to each of the 11 antimicrobials that the original isolate displayed. The presence of multiple resistance plasmids in a single strain is potentially worrisome. This is especially true among isolates of S. Dublin that typically cause severe, often life threatening infections in humans (Kennedy et al., 2004), which would require antimicrobial therapy. In a study by Jones et al. (2008) examining the outcomes of human Salmonella infections in the U.S. by different serotypes, S. Dublin had the highest percentage of cases that resulted in invasive disease, hospitalization and death among serotypes with at 50 infection from 1996 to 2006.
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Fig. 1. DNAPlotter (Sanger Institute) diagram of pSD853_174 from S. enterica serovar Dublin isolate MCRF 853. The key for the predicted functions for each of the CDSs is provided in the figure and the corresponding antimicrobial resistance genes, disinfectant resistance genes and locations of transfer-related genes are provided. The interior ring indicates the relative GC skew of the sequence region, with those areas colored in light grey having a greater that 50% GC content and those in dark grey than 50%.
In conclusion, plasmid sequencing of a multidrug resistant strain of S. Dublin identified plasmids containing antimicrobial resistance and virulence genes. Each plasmid shared a high degree of similarity to previously sequenced plasmids, yet each had unique sequence
regions. This diversity was likely due to horizontal gene transfer events, because these unique regions, as well as most resistance genes observed were flanked by insertion sequence/transposable elements. Additionally, the distinct regions were also found in other previously
Fig. 2. DNAPlotter diagram of theof pSD853_89 from S. enterica serovar Dublin isolate MCRF 853. The key for the predicted functions for each of the CDSs is provided in the figure and the corresponding antimicrobial resistance genes, disinfectant resistance genes and virulence genes are provided. The interior ring indicates the relative GC content of the sequence region, with those areas colored in light grey having a greater that 50% GC content and those in dark grey than 50%.
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Fig. 3. DNAPlotter diagram of pSD853_77 from S. enterica serovar Dublin isolate MCRF 853. The key for the predicted functions for each of the CDSs is provided in the figure and the corresponding virulence genes and locations of transfer-related genes are provided. The interior ring indicates the relative GC content of the sequence region, with those areas colored in light grey having a greater that 50% GC content and those in dark grey than 50%.
sequenced plasmids indicating the potential for DNA transfer among the enteric plasmids. Overall, of the four plasmids identified, two (pSD853_174 and pSD853_89) likely contributed to the observed multidrug resistance, since their genes correlated to the observed resistance phenotype. In several instances, common sets of resistance genes were located on both plasmids (tetA and tetR and mer genes). In addition, pSD853_89 also contained likely virulence genes, indicating the potential for the co-selection of virulence and antimicrobial resistance. pSD853_77 contained multiple genes known to be associated with S. Dublin virulence and likely contributes to the invasive phenotype often associated with S. Dublin infections. Therefore, plasmid encoded factors likely play an important role in antimicrobial resistance and virulence in S. Dublin isolates. Acknowledgements The authors would like to thank Drs. Carl Cerniglia, Bashar Shaheen and Sangeeta Khare for their critical review of the manuscript, and the Marshfield Clinic Research Foundation and U.S. Food and Drug Administration for their support of the research. Dr. Jing Han is supported through the Oak Ridge Institute for Science and Education. References Allerberger, F., Liesegang, A., Grif, K., Prager, R., Danzl, J., Hock, F., et al. (2002). Occurrence of Salmonella enterica serovar Dublin in Austria. Eurosurveillance, 7(4), 65–70. Baumler, A. J., Tsolis, R. M., Ficht, T. A., & Adams, L. G. (1998). Evolution of host adaptation in Salmonella enterica. Infection and Immunity, 66, 4579–4587. Call, D. R., Singer, R. S., Meng, D., Broschat, S. L., Orfe, L. H., Anderson, J. M., et al. (2010). blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids. Antimicrobial Agents and Chemotherapy, 54, 590–596. Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K. L., & Threlfall, E. J. (2005). Identification of plasmids by PCR-based replicon typing. Journal of Microbiological Methods, 63, 219–228. Centers for Disease Control and Prevention (2008). Salmonella Surveillance: Annual Summary, 2006. Atlanta, GA: U.S. Department of Health and Human Services, CDC.
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