We sequenced 35 Salmonella enterica isolates carrying incompatibility group I1 (IncI1) plasmids from different serotypes to study their genotypic characteristics. The isolates originated from food animals (n = 32) and human patients (n = 3). All isolates carried IncI1 plasmids, and many had additional plasmids detected along with virulence and antimicrobial resistance genes.
ABSTRACT
We sequenced 35 Salmonella enterica isolates carrying incompatibility group I1 (IncI1) plasmids from different serotypes to study their genotypic characteristics. The isolates originated from food animals (n = 32) and human patients (n = 3). All isolates carried IncI1 plasmids, and many had additional plasmids detected along with virulence and antimicrobial resistance genes.
ANNOUNCEMENT
Bacterial foodborne pathogens such as Salmonella enterica contribute to significant morbidity and mortality worldwide (1). Patients with more severe manifestations of illness often require antimicrobial treatment for resolution of disease (2). Unfortunately, several Salmonella strains are resistant to antimicrobial therapy, and often, this resistance is encoded on plasmids (3). Several plasmid types, including members of the incompatibility group I1 (IncI1), have been implicated in carrying antimicrobial resistance genes (4). Additionally, IncI1 plasmids have been reported to carry genes that may increase the virulence of strains where they reside (4). The aim of this study was to assess the antimicrobial resistance and virulence gene content of IncI1-positive Salmonella. We sequenced 35 Salmonella enterica isolates originating from cattle (n = 12, 34%), swine (n = 4, 11%), turkey (n = 6, 17%), chicken (n = 6, 17%), a chicken farm environment (n = 4, 11%), and human patients (n = 3, 9%). These isolates were collected from different locations in the United States during the period from 1999 to 2009. Isolates selected belong to Salmonella enterica serovars Heidelberg (n = 12, 34%), Typhimurium (n = 10, 29%), Newport (n = 6, 17%), Kentucky (n = 3, 9%), Anatum (n = 1, 3%), Dublin (n = 1, 3%), Cerro (n = 1, 3%), and Montevideo (n = 1, 3%).
Isolation approaches and phenotypic characterization of the isolates were described previously (5). We sequenced 35 Salmonella enterica isolates identified as carrying IncI1 plasmids using PCR-based plasmid replicon typing in our previous study (6). Each of the 35 isolates was stored at −80°C in brain heart infusion broth (Remel, Lenexa, KS) containing 20% glycerol, and prior to sequencing, isolates were subcultured on blood agar plates (tryptic soy agar with 5% sheep’s blood; Remel). All plates were incubated at 35°C for 24 hours. Overnight bacterial growth from individual isolates was scraped from the plate with a 1-μl inoculating loop and added to 180 μl animal tissue lysis (ATL) buffer (Qiagen, Valencia, CA, USA). Next, bacterial genomic DNA was extracted using a DNeasy blood and tissue kit (Qiagen). The quality and quantity of the DNA were examined using a NanoDrop instrument (Thermo Fisher Scientific, Grand Island, NY, USA) and a Qubit broad range (BR) assay kit (Thermo Fisher Scientific). DNA libraries were generated using 1 ng of DNA from each sample using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA) and were multiplexed using combinations of two indexes of the Nextera XT index kit (Illumina). Isolates were sequenced in two batches with a maximum of 19 isolates per run. DNA sample libraries were diluted, denatured, and loaded onto the Illumina MiSeq instrument, and sequencing was performed using the v2 500-cycle kits. The two runs were monitored using a sequence analysis viewer with an emphasis on appropriate cluster densities of 1,193,000/mm2 with the final quality score (>Q30 score of 80.41) and 1,326,000/mm2 with the final quality score (>Q30 score of 80.66). FASTQ files were demultiplexed with MiSeq software, and the reads for each isolate were assembled using CLC Genomics Workbench ver. 9.0 (Qiagen, Redwood City, CA). Sequences were annotated initially using the Pathosystems Resource Integration Center (PATRIC) software version 3.5.36 (7). Subsequently, sequences were submitted to NCBI for final annotation through the Prokaryotic Genome Annotation Pipeline (PGAP) to annotate the draft genomes of these strains (8). The numbers of contigs, assembly sizes, coding sequences (CDS), and GC contents were annotated by PATRIC as shown in Table 1. The final annotations performed by the PGAP are available in NCBI under the accession numbers shown in Table 1.
TABLE 1.
Whole-genome sequencing analyses of 35 Salmonella enterica isolates from food animal and clinical sources
| Isolate | Serotype | Source | Yr of isolation | No. of sequence reads | N50 (bp) | No. of contigs | Genome length (bp) | No. of CDS | G+C content (%) | Accession no. |
|---|---|---|---|---|---|---|---|---|---|---|
| 67 | Newport | Cattle | 2002 | 5,019,561 | 25,424 | 485 | 4,486,820 | 4,678 | 52.15 | VCBN00000000 |
| 74 | Newport | Cattle | 2002 | 4,989,252 | 25,085 | 420 | 4,328,188 | 4,481 | 52.22 | VCBO00000000 |
| 76 | Newport | Chicken | 2001 | 4,926,523 | 32,358 | 343 | 4,349,537 | 4,475 | 52.15 | VCBP00000000 |
| 89 | Newport | Swine | 2001 | 4,977,554 | 26,443 | 407 | 4,620,558 | 4,755 | 52.18 | VCIK00000000 |
| 93 | Newport | Swine | 2002 | 5,027,143 | 32,187 | 320 | 4,403,717 | 4,586 | 52.10 | VCIL00000000 |
| 100 | Newport | Turkey | 2001 | 4,837,174 | 42,117 | 294 | 4,355,301 | 4,419 | 52.15 | VCIM00000000 |
| 111 | Heidelberg | Cattle | 2001 | 5,182,383 | 29,076 | 363 | 4,895,560 | 5,076 | 51.95 | VCIN00000000 |
| 114 | Heidelberg | Cattle | 2002 | 5,138,950 | 22,618 | 517 | 4,917,806 | 5,123 | 52.00 | VCIO00000000 |
| 115 | Heidelberg | Cattle | 2002 | 5,191,943 | 45,514 | 263 | 4,657,342 | 4,803 | 51.96 | VCIP00000000 |
| 116 | Heidelberg | Cattle | 2002 | 5,248,272 | 30,402 | 385 | 4,895,176 | 5,115 | 51.82 | VCIQ00000000 |
| 159 | Heidelberg | Turkey | 2002 | 5,111,403 | 31,495 | 310 | 4,737,325 | 4,924 | 51.78 | VCIR00000000 |
| 470 | Typhimurium | Swine | 1999 | 8,612,134 | 7,828 | 425 | 4,407,658 | 4,610 | 52.42 | VCSK00000000 |
| 471 | Typhimurium | Swine | 1999 | 5,001,947 | 20,290 | 490 | 4,718,137 | 4,882 | 52.32 | VCIT00000000 |
| 482 | Typhimurium | Turkey | 1999 | 4,960,480 | 24,216 | 417 | 4,670,043 | 4,763 | 52.22 | VCIU00000000 |
| 695 | Heidelberg | Turkey | 2000 | 5,071,071 | 28,231 | 380 | 4,907,961 | 5,026 | 51.96 | VCIV00000000 |
| 706 | Heidelberg | Turkey | 2000 | 4,897,846 | 25,101 | 405 | 4,522,077 | 4,649 | 52.13 | VDBX00000000 |
| 715 | Heidelberg | Turkey | 2000 | 4,848,412 | 36,230 | 279 | 4,613,597 | 4,714 | 52.15 | VCPT00000000 |
| 121 | Heidelberg | Cattle | 2002 | 4,846,037 | 36,611 | 379 | 4,843,682 | 5,006 | 52.23 | VCSL00000000 |
| N134 | Typhimurium | Chicken farm | Unknown | 5,086,478 | 92,540 | 130 | 4,508,660 | 4,683 | 52.00 | VCQG00000000 |
| N53 | Typhimurium | Chicken | Unknown | 5,049,701 | 19,622 | 577 | 4,912,152 | 5,091 | 52.23 | VCQH00000000 |
| 849 | Dublin | Cattle | 2005 | 5,047,829 | 42,899 | 236 | 4,599,915 | 4,802 | 51.99 | VCPU00000000 |
| 855 | Typhimurium | Cattle | 2006 | 5,014,625 | 49,906 | 226 | 4,499,875 | 4,632 | 52.05 | VCPV00000000 |
| 856 | Cerro | Cattle | 2006 | 4,568,478 | 21,626 | 437 | 4,470,137 | 4,521 | 52.42 | VCPW00000000 |
| N865 | Kentucky | Chicken | 2008 | 4,879,802 | 23,260 | 503 | 4,523,952 | 4,615 | 52.08 | VCQJ00000000 |
| 880 | Montevideo | Cattle | 2006 | 4,694,168 | 43,514 | 282 | 4,251,545 | 4,350 | 52.34 | VCIS00000000 |
| 891 | Anatum | Cattle | 2006 | 4,741,580 | 28,637 | 300 | 4,601,255 | 4,657 | 52.18 | VCPY00000000 |
| 990 | Heidelberg | Human | 2008 | 5,098,170 | 33,395 | 303 | 4,892,063 | 5,061 | 51.85 | VCQK00000000 |
| 1000 | Heidelberg | Human | 2009 | 5,087,599 | 23,713 | 453 | 4,773,954 | 4,962 | 51.87 | VCPZ00000000 |
| 1163 | Heidelberg | Human | 2007 | 4,950,681 | 49,656 | 344 | 4,678,168 | 4,854 | 52.01 | VCQB00000000 |
| N822 | Kentucky | Chicken farm | 2008 | 4,891,568 | 23,503 | 511 | 4,533,159 | 4,691 | 52.12 | VCQI00000000 |
| N860 | Kentucky | Chicken | 2008 | 4,880,247 | 27,698 | 417 | 4,486,160 | 4,589 | 52.02 | VDBM00000000 |
| N136 | Typhimurium | Chicken farm | Unknown | 5,082,919 | 41,441 | 275 | 4,700,886 | 4,820 | 52.02 | VCQC00000000 |
| N74 | Typhimurium | Chicken | Unknown | 5,099,545 | 35,108 | 326 | 4,587,257 | 4,780 | 51.88 | VCQD00000000 |
| N82 | Typhimurium | Chicken farm | Unknown | 5,056,644 | 22,986 | 497 | 4,740,914 | 4,928 | 52.12 | VCQE00000000 |
| N97 | Typhimurium | Chicken | Unknown | 5,054,428 | 36,102 | 279 | 4,729,717 | 4,859 | 52.04 | VCQF00000000 |
Sequence data from each of the isolates were further analyzed using the PlasmidFinder (9) and ResFinder (10) tools to predict the presence of plasmids and antimicrobial resistance genes, respectively. PlasmidFinder analyses confirmed that all isolates contained IncI1 plasmids, many along with other plasmid replicon types, including IncA/C (n = 14, 40%), IncHI2 (n = 8, 23%), IncColpVC (n = 9, 26%), IncX1 (n = 7, 20%), and IncFIB (n = 6, 17%). ResFinder analyses were performed by selecting acquired antimicrobial resistance genes with default parameter settings. Multiple antimicrobial resistance genes, including tet(A) in 51% (n = 18) of the strains, followed by blaCMY-2 (n = 17, 49%), aph(3ʺ)-Ib (n = 16, 46%), sul2 (n = 14, 40%), fosA7 (n = 12, 34%), aadA1 (n = 12, 34%), floR (n = 10, 29%), sul1 (n = 9, 26%), tet(B) (n = 9, 26%), blaTEM-1B (n = 8, 23%), dfrA1 (n = 4, 11%), blaTEM-1A (n = 1, 3%), sul3 (n = 1, 3%), dfrA2 (n = 1, 3%), cml (n = 1, 3%), and cmlA1 (n = 1, 3%), were most commonly detected among the sequences. Default parameters were used for all bioinformatic software tools unless otherwise specified.
Data availability.
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers listed in Table 1, and the SRA submission of the FASTQ files has been recorded under the accession number PRJNA543125.
ACKNOWLEDGMENTS
We recognize Rajesh Nayak for the provision of some of the isolates in the study and Ashraf Khan, Jing Han, and Carl Cerniglia for their critical review of the manuscript.
This project was supported by the National Center for Toxicological Research and the U.S. Food and Drug Administration. Nesreen H. Aljahdali and Pravin R. Kaldhone are supported through the Oak Ridge Institute for Science and Education (ORISE). The opinions expressed in the manuscript are solely the responsibility of the authors and do not necessarily represent the official views and policy of the Food and Drug Administration or Department of Health and Human Services. Reference to any commercial material, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
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Data Availability Statement
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers listed in Table 1, and the SRA submission of the FASTQ files has been recorded under the accession number PRJNA543125.