Antibiotic resistance is attributed to the misuse or overuse of antibiotics in agriculture, and antibiotic resistance genes can also be transferred to bacteria under environmental stress. In this study, we report a unidirectional alteration in antibiotic resistance from susceptibility to increased resistance. Highly sensitive Salmonella enterica serovar Typhimurium isolates from organic farm systems quickly acquired tetracycline resistance under antibiotic pressure in simulated farm soil environments within 2 weeks, with expression of antibiotic resistance-related genes that was significantly upregulated. Conversely, originally resistant S. Typhimurium isolates from conventional farm systems lost little of their resistance when transferred to environments without antibiotic pressure. Additionally, multidrug-resistant S. Typhimurium isolates genetically shared relevancy with pathogenic S. Typhimurium isolates, whereas susceptible isolates clustered with nonpathogenic strains. These results provide detailed discussion and explanation about the genetic alterations and simultaneous acquisition of antibiotic resistance in S. Typhimurium in agricultural environments.
KEYWORDS: Salmonella, acquired resistance, virulence, soil environment, horizontal gene transfer
ABSTRACT
Microbial horizontal gene transfer is a continuous process that shapes bacterial genomic adaptation to the environment and the composition of concurrent microbial ecology. This includes the potential impact of synthetic antibiotic utilization in farm animal production on overall antibiotic resistance issues; however, the mechanisms behind the evolution of microbial communities are not fully understood. We explored potential mechanisms by experimentally examining the relatedness of phylogenetic inference between multidrug-resistant Salmonella enterica serovar Typhimurium isolates and pathogenic Salmonella Typhimurium strains based on genome-wide single-nucleotide polymorphism (SNP) comparisons. Antibiotic-resistant S. Typhimurium isolates in a simulated farm environment barely lost their resistance, whereas sensitive S. Typhimurium isolates in soils gradually acquired higher tetracycline resistance under antibiotic pressure and manipulated differential expression of antibiotic-resistant genes. The expeditious development of antibiotic resistance and the ensuing genetic alterations in antimicrobial resistance genes in S. Typhimurium warrant effective actions to control the dissemination of Salmonella antibiotic resistance.
IMPORTANCE Antibiotic resistance is attributed to the misuse or overuse of antibiotics in agriculture, and antibiotic resistance genes can also be transferred to bacteria under environmental stress. In this study, we report a unidirectional alteration in antibiotic resistance from susceptibility to increased resistance. Highly sensitive Salmonella enterica serovar Typhimurium isolates from organic farm systems quickly acquired tetracycline resistance under antibiotic pressure in simulated farm soil environments within 2 weeks, with expression of antibiotic resistance-related genes that was significantly upregulated. Conversely, originally resistant S. Typhimurium isolates from conventional farm systems lost little of their resistance when transferred to environments without antibiotic pressure. Additionally, multidrug-resistant S. Typhimurium isolates genetically shared relevancy with pathogenic S. Typhimurium isolates, whereas susceptible isolates clustered with nonpathogenic strains. These results provide detailed discussion and explanation about the genetic alterations and simultaneous acquisition of antibiotic resistance in S. Typhimurium in agricultural environments.
INTRODUCTION
More than 20,000 potential antibiotic resistance genes of nearly 400 different types are predicted or identified in available bacterial genome sequences, and this number is expected to increase with further advancements in microbiome analysis (1, 2). Several recent independent studies found that many antibiotic resistance genes that are integrated into the bacterial genomes carry their origins in disparate reservoirs (3, 4). Furthermore, the acquisition or alteration of those multidrug resistance genes indicates the widespread dissemination of distinct antibiotic resistance genes in various microbial ecosystems, including the intestinal tracts of birds and other species of wildlife (5, 6). It is now well known that horizontal gene transfer is a continuous process and that it shapes bacterial genomes. The influences of microbial ecological niches and the human introduction of synthetic antimicrobials to the microbial ecosystem on the evolution of biological networks are not completely understood.
Animal agriculture, particularly poultry and swine farming in developed countries, accounts for more than half of the antibiotics used in the world (7, 8). Antibiotics are used in animal farming both for treating diseases (therapeutic) and for growth promotion (subtherapeutic) (9). Subtherapeutic dosages of antibiotics in animal feed/water induce faster weight gain via inhibition/modulation of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs of the gastrointestinal (GI) system due to decreased inflammation (10, 11). Furthermore, subtherapeutic dosage of antibiotics makes additional contributions to animal agriculture by improving animal health and welfare and control of human foodborne infectious diseases by reducing cross-contamination and extending the shelf life of products (9, 12).
In addition to subtherapeutic dosages of antibiotics, significant amounts of therapeutic antibiotics are used in food animals to treat clinical diseases or, prophylactically, to prevent common diseases (9). So far, very little attention, other than the data generated from in vitro laboratory studies, has been paid to how dosages of antibiotics used in farm animal production contribute to the overall problem of antibiotic resistance (8, 13). Furthermore, farmers also use various drugs for prevention and treatment of animal parasitic disease; for instance, amprolium (Corid) and sulfadimethoxine are commonly used to control coccidiosis (caused by protozoa of the genus Eimeria) in poultry production (14). However, the mechanisms by which the residues of these therapeutic and subtherapeutic antibiotics and other antimicrobials affect horizontal gene transfer have not been evaluated yet.
As current animal farming practices lead to an increase in antibiotic-resistant fecal bacteria in animals, including on carcasses (15–18), and as there is growing concern over antibiotic resistance in human-pathogenic bacteria, the U.S. Food and Drug Administration (FDA) has announced its plan to gradually withdraw nontherapeutic/growth-promoting antibiotics from farm animal production (19–21). Furthermore, consumer desire to avoid synthetic antibiotics has led to increased marketing of meat and poultry from animals reared on organic farms. However, the lower growth rate of organically reared farm animals and their higher mortality rates cause high production costs and nonprofitable practices, as well as higher rates of contamination with zoonotic pathogens that cause foodborne infections (22–24). It is well known that pasture/organic farming cannot meet the demand for animal products for seven billion people. Furthermore, solely blocking or withdrawing subtherapeutic antibiotic use in animal farming without controlling therapeutic or higher doses of antibiotics and use of other chemicals might not be enough to mitigate the threat of rising rates of antibiotic-resistant zoonotic pathogenic bacteria (9, 25, 26). According to published data from the Centers for Disease Control and Prevention (CDC), multidrug resistance in Salmonella accounts for approximately 6,200 cases (10% of total) of Salmonella infections each year in the United States. Salmonella enterica serovar Typhimurium is one of the primary serotypes responsible for antibiotic resistance and among the predominant isolates responsible for multidrug resistance in Salmonella infections (27). In the current study, we investigate induced alterations in tetracycline (TET) and sulfamethoxazole-trimethoprim (SXT) resistance and antibiotic resistance-related gene expression for Salmonella enterica serovar Typhimurium, both in vitro and in soil environments under pressure imposed by these antibiotics, and further characterize derived S. Typhimurium isolates based on genome-wide single-nucleotide polymorphism (SNP) comparisons with published pathogenic/nonpathogenic S. Typhimurium strains.
RESULTS
In vitro antibiotic resistance alterations in S. Typhimurium.
To investigate the loss of spontaneous antibiotic resistance, bacterial cells were allowed to grow in optimal culture conditions without antibiotic stress (see Tables 1 and 2 for summaries of isolate sources, distribution, and antibiotic resistance profiles). The initial TET and SXT MICs for both ST-CRC26 and ST-CRC32 isolates were 64 μg/ml and 76/4 μg/ml, respectively (Table 3). Twelve passages of both isolates were performed in nutritious blood broth in the absence of antibiotics or any other antimicrobial component. The MIC of TET for the ST-CRC26 isolate was reduced to 32 μg/ml, a level that was still above the breakpoint of sensitivity. The MIC of TET for the ST-CRC32 isolate was unchanged (Table 3). Furthermore, MICs of SXT for both isolates ST-CRC26 and ST-CRC32 remained unchanged after 12 passages in this in vitro study (Table 3).
TABLE 1.
S. Typhimurium isolates across distinct categories isolated from farms and markets
| Harvest level | Farm/market type | Total no. of isolates | No. of strains analyzede |
|---|---|---|---|
| Preharvest | Mixed crop-livestock farma | 25 | 15 |
| Conventional poultry farmb | 3 | 1 | |
| Postharvest | Farmers' marketc | 73 | 34 |
| Conventional retailerd | 7 | 4 | |
| Total | 108 | 54 |
Samples from mixed crop-livestock farms include compost, feces, feed, produce, and soil.
Samples from conventional poultry farms include chicken bedding.
Samples from farmers' markets include produce, whole chicken, and chicken parts.
Samples from conventional retailers include whole chicken and chicken parts.
The number of analyzed strains was determined based on the total number of isolates harvested.
TABLE 2.
Antimicrobial resistance profiles of S. Typhimurium isolates from various sources
| Antibiotic susceptibilitya | Farm/market type (% [no./total]) |
||
|---|---|---|---|
| Conventional | Organic | Total | |
| Ampicillin | 0.00 (0/5) | 2.04 (1/49) | 1.85 (1/54) |
| Cefazolin | 0.00 (0/5) | 6.12 (3/49) | 5.56 (3/54) |
| Sulfamethoxazole-trimethoprim | 80.00 (4/5) | 46.94 (23/49) | 50.00 (27/54) |
| Tetracycline | 60.00 (3/5) | 10.20 (5/49) | 14.81 (8/54) |
| AMR | 80.00 (4/5) | 65.31 (32/49) | 66.67 (36/54) |
| MDR | 60.00 (3/5) | 0.00 (0/49) | 5.56 (3/54) |
AMR, antimicrobial resistant (isolates were resistant to at least one antimicrobial tested); MDR, multidrug resistant (isolates were resistant to two or more antimicrobials tested).
TABLE 3.
Alterations of antibiotic resistance patterns in S. Typhimurium isolates
| Strain | MIC (μg/ml) for: |
|
|---|---|---|
| Sulfamethoxazole-trimethoprima | Tetracyclineb | |
| ST-CRC26 | 76/4 | 64 |
| ST-CRC32 | 76/4 | 64 |
| ST-FMC22 | 19/1 | 4 |
| ST-FMC46 | 19/1 | 4 |
| ST-CRC26mc | 76/4 | 32 |
| ST-CRC32m | 76/4 | 64 |
| ST-FMC22m | 76/4 | 32 |
| ST-FMC46m | 76/4 | 64 |
The resistance breakpoint of sulfamethoxazole-trimethoprim is 76/4 μg/ml.
The resistance breakpoint of tetracycline is 32 μg/ml.
m, mutant strain naturally developed in vitro.
Conversely, the non-antibiotic-resistant strains were cultured in antibiotic-stress medium with a double-concentrated dose of antibiotic per two passages to assess the acquisition of antibiotic resistance. The initial TET and SXT MICs for both ST-FMC22 and ST-FMC46 isolates were 4 μg/ml and 19/1 μg/ml, respectively (Table 4). During subsequent subculturing, the MICs of these two S. Typhimurium isolates were gradually increased until they reached the resistance breaking points of TET and SXT. For TET, ST-FMC22 and ST-FMC46 reached the resistance breakpoint (32 μg/ml) after 10 passages, and the TET MIC for ST-FMC46 increased to 64 μg/ml after 12 passages but did not tolerate higher concentrations (Table 4). Similarly, both ST-FMC22 and ST-FMC46 isolates acquired SXT resistance at the 11th passage, but the MIC did not exceed the resistance breakpoint (76/4 μg/ml) after 12 passages (Table 4).
TABLE 4.
Gradual antibiotic resilience in S. Typhimurium isolates over 12 passages
| Passage | Tetracycline MIC (μg/ml) |
Sulfamethoxazole-trimethoprim MIC (μg/ml) |
||
|---|---|---|---|---|
| FMC22 | FMC46 | FMC22 | FMC46 | |
| Original | 4 | 4 | 19/1 | 19/1 |
| 1st | 4 | 4 | 19/1 | 19/1 |
| 2nd | 4 | 4 | 19/1 | 19/1 |
| 3rd | 4 | 4 | 19/1 | 19/1 |
| 4th | 8 | 8 | 19/1 | 19/1 |
| 5th | 8 | 8 | 38/2 | 38/2 |
| 6th | 8 | 8 | 38/2 | 38/2 |
| 7th | 16 | 16 | 38/2 | 38/2 |
| 8th | 16 | 16 | 38/2 | 38/2 |
| 9th | 16 | 16 | 38/2 | 38/2 |
| 10th | 32 | 32 | 38/2 | 38/2 |
| 11th | 32 | 32 | 76/4 | 76/4 |
| 12th | 32 | 64 | 76/4 | 76/4 |
Phylogenetic inference and genomic characteristics of S. Typhimurium isolates.
The relatedness among the S. Typhimurium isolates from this study was investigated based on genome-wide SNP comparisons, and the draft genome contigs have been submitted to the NCBI Sequence Read Archive (SRA; accession numbers presented in Table 5). SNPs were identified with the CSI Phylogeny webserver, separating the 54 S. Typhimurium isolates from one another and from selected S. Typhimurium published genome sequences available in NCBI GenBank (Fig. 1 and Table 5). According to the whole-genome SNP comparisons, 4 major lineages were identified, in which S. Typhimurium isolates from conventional chicken (ST-CRC26, ST-CRC29, ST-CRC31, and ST-CRC32) sources clustered with published pathogenic S. Typhimurium strains within lineage 1. Lineage 2 had 3 isolates; ST-FMC71 and ST-O65 (sourced from chicken from organic retailer and farmers' market) clustered with published S. Typhimurium isolate ST-U288. In lineages 3 and 4, isolates from organic retailers and farmers' markets clustered with ST-LT2. Assembled whole-genome analysis indicated that the draft genomes of the S. Typhimurium isolates from this study harbored plasmids, and the respective NCBI accession numbers of the draft genome sequences are presented in Table 5. Isolates from organic retailers and farmers' markets harbored plasmids IncFIB(S) and IncFII(S). Interestingly, ST-LT2 also harbored plasmids IncFIB(S) and IncFII(S). Isolates from conventional retail chickens, as well as a previously isolated pathogenic ST-21B isolate, harbored plasmids IncA/C2 and ColRNAI. Annotation of the assembled contigs of ST-FMC22, ST-FMC22m, ST-FMC46, ST-FMC46m, ST-CRC26, ST-CRC26m, ST-CRC32, and ST-CRC32m with the Rapid Annotation using Subsystem Technology (RAST) server identified multidrug resistance-conferring efflux pump genes mdsB, mdtK, acrA, and sdiA in all of the isolates (Table 6). Similarly, TET and sulfonamide resistance genes, tetA and sul2, were also present in all the isolates from conventional retailers.
TABLE 5.
List of S. Typhimurium isolates and their genomic information
| Isolatea | Draft genome length (bp) | No. of contigs | Coverage (×)b | N50 (bp) | Plasmid | SRA accession no. |
|---|---|---|---|---|---|---|
| ST-O340 | 4,896,263 | 85 | 334.28 | 224,483 | Ac | SRR5340795 |
| ST-O335 | 4,897,698 | 87 | 211.50 | 224,465 | A | SRR5340794 |
| ST-O332 | 4,897,999 | 97 | 185.55 | 224,085 | A | SRR5340793 |
| ST-O292 | 4,898,827 | 92 | 218.81 | 247,396 | A | SRR5340792 |
| ST-O235 | 4,900,584 | 96 | 226.13 | 247,079 | A | SRR5340791 |
| ST-O231 | 4,897,200 | 90 | 272.10 | 301,394 | A | SRR5340790 |
| ST-O147 | 4,899,383 | 97 | 234.63 | 247,079 | A | SRR5340789 |
| ST-O135 | 4,897,940 | 98 | 280.49 | 301,394 | A | SRR5340788 |
| ST-O130 | 4,899,330 | 90 | 294.09 | 301,394 | A | SRR5340787 |
| ST-O124 | 4,895,924 | 89 | 237.82 | 301,394 | A | SRR5340786 |
| ST-O115 | 4,896,552 | 88 | 466.80 | 301,394 | A | SRR5340785 |
| ST-O105 | 4,898,745 | 89 | 235.75 | 223,861 | A | SRR5340784 |
| ST-O103 | 4,897,896 | 89 | 358.07 | 301,394 | A | SRR5340783 |
| ST-O102 | 4,896,521 | 83 | 236.67 | 225,432 | A | SRR5340782 |
| ST-O65 | 4,904,722 | 96 | 184.55 | 223,976 | A | SRR5340781 |
| ST-FMC333 | 4,897,765 | 80 | 224.80 | 301,394 | A | SRR5340780 |
| ST-FMC78 | 4,899,174 | 98 | 253.07 | 301,394 | A | SRR5340779 |
| ST-FMC77 | 4,896,928 | 87 | 138.26 | 301,394 | A | SRR5340778 |
| ST-FMC75 | 4,897,929 | 95 | 218.21 | 301,210 | A | SRR5340777 |
| ST-FMC74 | 4,896,614 | 97 | 231.70 | 224,404 | A | SRR5340776 |
| ST-FMC71 | 4,896,879 | 85 | 130.61 | 301,394 | A | SRR5340775 |
| ST-FMC70 | 4,895,993 | 84 | 262.49 | 301,394 | A | SRR5340774 |
| ST-FMC69 | 4,897,859 | 84 | 260.07 | 247,477 | A | SRR5340773 |
| ST-FMC68 | 4,897,555 | 92 | 265.08 | 247,096 | A | SRR5340772 |
| ST-FMC67 | 4,897,601 | 99 | 247.50 | 178,287 | A | SRR5340771 |
| ST-FMC65 | 4,898,898 | 92 | 177.14 | 301,394 | A | SRR5340770 |
| ST-FMC64 | 4,899,081 | 96 | 198.91 | 246,879 | A | SRR5340769 |
| ST-FMC63 | 4,899,787 | 102 | 153.67 | 247,869 | A | SRR5340768 |
| ST-FMC61 | 4,898,352 | 94 | 202.66 | 247,079 | A | SRR5340767 |
| ST-FMC59 | 4,897,860 | 93 | 284.11 | 301,394 | A | SRR5340766 |
| ST-FMC58 | 4,898,554 | 103 | 183.55 | 246,879 | A | SRR5340765 |
| ST-FMC57 | 4,895,754 | 84 | 239.10 | 301,394 | A | SRR5340764 |
| ST-FMC56 | 4,898,766 | 90 | 285.94 | 246,879 | A | SRR5340763 |
| ST-FMC51 | 4,897,186 | 87 | 285.17 | 225,553 | A | SRR5340762 |
| ST-FMC47 | 4,897,330 | 88 | 230.24 | 301,394 | A | SRR5340761 |
| ST-FMC46 | 4,897,764 | 84 | 218.10 | 301,394 | A | SRR5340760 |
| ST-FMC45 | 4,898,904 | 93 | 246.49 | 247,096 | A | SRR5340759 |
| ST-FMC44 | 4,898,789 | 99 | 188.28 | 224,012 | A | SRR5340758 |
| ST-FMC43 | 4,896,545 | 90 | 132.52 | 301,394 | A | SRR5340757 |
| ST-FMC42 | 4,897,826 | 93 | 190.90 | 247,079 | A | SRR5340756 |
| ST-FMC40 | 4,898,599 | 106 | 179.32 | 246,879 | A | SRR5340755 |
| ST-FMC34 | 4,898,862 | 98 | 243.88 | 296,746 | A | SRR5340754 |
| ST-FMC31 | 4,900,808 | 103 | 158.35 | 247,079 | A | SRR5340753 |
| ST-FMC28 | 4,898,294 | 86 | 265.12 | 301,394 | A | SRR5340752 |
| ST-FMC27 | 4,897,893 | 97 | 220.03 | 301,394 | A | SRR5340751 |
| ST-FMC24 | 4,895,878 | 89 | 217.42 | 301,394 | A | SRR5340750 |
| ST-FMC23 | 4,897,529 | 94 | 264.76 | 223,976 | A | SRR5340749 |
| ST-FMC22 | 4,897,495 | 87 | 253.85 | 225,432 | A | SRR5340748 |
| ST-FMC20 | 4,897,325 | 100 | 148.25 | 301,394 | A | SRR5340747 |
| ST-FMC14 | 4,898,908 | 111 | 105.89 | 246,576 | A | SRR5340746 |
| ST-FM76 | 4,898,908 | 104 | 130.47 | 224,270 | A | SRR5340745 |
| ST-CRC32 | 4,988,203 | 108 | 147.88 | 246,785 | Bd | SRR5340744 |
| ST-CRC31 | 4,944,634 | 108 | 148.20 | 283,521 | B | SRR5340743 |
| ST-CRC29 | 4,948,215 | 150 | 143.24 | 208,466 | B | SRR5340742 |
| ST-CRC26 | 4,944,228 | 115 | 183.55 | 185,300 | B | SRR5340741 |
| ST-C32 | 5,001,673 | 159 | 171.41 | 229,563 | B | SRR5340739 |
| ST-LT2 | 4,897,621 | 104 | 99.02 | 301,394 | A | SRR5340738 |
| ST-21B | 5,009,502 | 154 | 162.30 | 235,881 | B | SRR5340737 |
Assembled draft genomes were analyzed with the tools available from the Center for Genomic Epidemiology web service, namely, ResFinder-2.1, KmerFinder-2.1, ContigAnalyzer-1.0, PlasmidFinder-1.2, MLST-1.6, pMLST-1.4, PathogenFinder-1.1, SeqSero-1.1, SalmonellaTypeFinder-1.2, and PathogenFinder-1.1.
Coverage per base pair was calculated using BBMap (https://sourceforge.net/projects/bbmap/) and expressed as the mean. Assembled contigs were set as a reference, and trimmed pair end reads were mapped.
A, IncFIB(S) and IncFII(S).
B, IncA/C2 and ColRNAI.
FIG 1.
SNP-based phylogenetic tree of S. Typhimurium isolates and selected S. Typhimurium genome sequences from GenBank. SNP detection and phylogenetic inference were performed by the CSI Phylogeny webserver; branch lengths correspond to numbers of nucleotide substitutions per site. Isolates from conventional retail chickens clustered with selected pathogenic S. Typhimurium strains (lineage 1) whereas most of the organic retailer and farmers' market chicken isolates clustered separately in lineages 3 and 4.
TABLE 6.
Genes involved in antibiotic resistance in S. Typhimurium isolates
| Function(s)a | Geneb | Presence (+) or absence (−) of gene in isolate: |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| ST-CRC26 | ST-CRC26m | ST-CRC32 | ST-CRC32m | ST-FMC22 | ST-FMC22m | ST-FMC46 | ST-FMC46m | ||
| Multidrug efflux | tolC | + | + | + | + | ||||
| Multidrug efflux | mdsB | + | + | + | + | + | + | + | + |
| Multidrug efflux | mdtK | + | + | + | + | + | + | + | + |
| Multidrug efflux | acrA | + | + | + | + | + | + | + | + |
| Multidrug efflux | sdiA | + | + | + | + | + | + | + | + |
| Sulfonamide | sul2 | + | + | + | + | − | − | − | − |
| Tetracycline | tetA | + | + | + | + | − | − | − | − |
| Resistance to antibiotics and toxins | vapB | + | − | + | + | + | + | + | + |
| Resistance to antibiotics and toxins | Membrane-bound lysozyme inhibitor of c-type lysozyme | + | − | − | − | + | + | + | + |
| Restriction modification | Type III restriction-modification system DNA endonuclease res | − | − | + | − | − | − | − | − |
| Cofactors | Dihydrolipoamide dehydrogenase | + | + | − | − | − | − | − | + |
Gene activity.
Both antibiotic resistance and functional genes.
Alterations of S. Typhimurium antibiotic resistance in soil environment.
The acquisition/loss of antibiotic resistance in S. Typhimurium isolates ST-CRC26 and ST-FMC46 was investigated in soil environments under a biosafety level 2 (BSL-2) greenhouse facility, and an alteration of TET resistance was observed (Table 7). In both conventional and organic soil environments, isolate ST-CRC26 failed to lose TET resistance, whereas isolate ST-FMC46 in both types of soil environment gradually acquired TET resistance, with higher MICs, over periods of time (Table 7).
TABLE 7.
Tetracycline resistance profiles of ST-FMC46 and ST-CRC26 in soil environment
| Soil no.a | Strainb (tetracycline in μg/ml) | Isolate | MIC (μg/ml) at: |
||||
|---|---|---|---|---|---|---|---|
| 0 days | 14 days | 28 days | 3 mo | 6 mo | |||
| 1 | ST-FMC46 (4) | A5 | 4 | 32 | 64 | 128 | NAc |
| B3 | 4 | 4 | 64 | 128 | 128 | ||
| D1 | 4 | 4 | 4 | 16 | 64 | ||
| F1 | 4 | 4 | 4 | 128 | 128 | ||
| F3 | 4 | 4 | 4 | 128 | NA | ||
| F5 | 4 | 4 | 4 | 128 | NA | ||
| 2 | ST-FMC46 (4) | A6 | 4 | 4 | 64 | 128 | 128 |
| D4 | 4 | 4 | 64 | 128 | NA | ||
| E1 | 4 | 32 | 128 | 128 | 128 | ||
| E3 | 4 | 4 | 4 | 128 | 128 | ||
| E7 | 4 | 32 | 128 | 128 | NA | ||
| F2 | 4 | 4 | 4 | 128 | 128 | ||
| 1 | ST-CRC26 (128) | A3 | 64 | 64 | 64 | 128 | NA |
| A7 | 64 | 64 | 64 | 64 | 128 | ||
| A8 | 64 | 64 | 64 | 64 | 64 | ||
| B7 | 64 | 64 | 64 | 64 | 64 | ||
| C2 | 64 | 64 | 64 | 128 | 128 | ||
| D5 | 64 | 64 | 64 | 64 | NA | ||
| 2 | ST-CRC26 (128) | B1 | 64 | 64 | 64 | 64 | 64 |
| B2 | 64 | 64 | 64 | 128 | 128 | ||
| C3 | 64 | 64 | 64 | 128 | 128 | ||
| C6 | 64 | 64 | 64 | 128 | 128 | ||
| F4 | 64 | 64 | 64 | 128 | NA | ||
| F8 | 64 | 64 | 64 | 64 | 128 | ||
Soil 1 was collected from a conventional farm; soil 2 was collected from an organic farm.
ST-FMC46, farmer's market chicken S. Typhimurium isolates susceptible to tetracycline. ST-CRC26, conventional retailer chicken S. Typhimurium isolates resistant to tetracycline. The resistance breakpoint of tetracycline is 32 μg/ml.
NA, not applicable; S. Typhimurium isolates could not be recovered from soil samples.
More specifically, 100% of ST-CRC26 (12 of 12) isolates maintained their resistance potential against TET at a MIC of 64 μg/ml during the first month in a TET-free soil environment. After the second month, one of the recovered ST-CRC26 isolates raised its MIC against TET to 128 μg/ml, and 5 other isolates of ST-CRC26 recovered from soil samples also reached a MIC of 128 μg/ml against TET after the third month. Eventually, at least 2 ST-CRC26 isolates recovered from conventional farm soil and 4 isolates recovered from organic farm soil (in total, at least 6 out of 12 isolates) stabilized their MICs against TET at 128 μg/ml, which is double the TET resistance breaking point.
On the other hand, with 4 mg TET per kg soil supplement, only 1 out of 6 ST-FMC46 (16.6%) isolates recovered from conventional soil culture environment and 2 out of 6 ST-FMC46 (33.3%) isolates recovered from organic soil acquired TET resistance (MIC = 32 μg/ml) within 2 weeks. Additionally, another 3 ST-FMC46 isolates from soil samples reached the TET resistance breakpoint after 1 month. After 3 months, 11 out of 12 (91.6%) recovered isolates had MIC values of 128 μg/ml. Within 6 months in the soil environments, all of the recovered cultivable ST-FMC46 isolates were TET resistant.
However, no significant alteration in SXT resistance was observed within 6 months of sublethal SXT supplement or non-antibiotic-resistance induction (data not shown). All 12 susceptible ST-FMC46 isolates recovered from soil environments retained their SXT MIC at 19/1 μg/ml after 14 days and after 1, 2, 3, and 6 months. Similarly, all 12 recovered resistant ST-CRC26 isolates retained their SXT MIC at 76/4 μg/ml after 14 days and after 1, 2, 3, and 6 months. In other words, none of the 12 recovered ST-FMC46 isolates developed SXT resistance in a soil environment, and all of the 12 ST-CRC26 isolates recovered from soil environments maintained their SXT resistance.
Differential expression of genes involved in antibiotic resistance.
Differential expression of genes involved in antibiotic resistance in S. Typhimurium isolates that were artificially inoculated into soil under antibiotic pressure was determined with quantitative real-time PCR (qRT-PCR) and is presented in Table 8. Two S. Typhimurium isolates, ST-FMC46 (TET sensitive) and ST-CRC26 (TET resistant), were inoculated in the presence or absence of TET, respectively. We observed, with TET pressure, that expression of the tetA gene was upregulated by 803-, 1,310-, 3,296-, and 653-fold in isolates E1, A6, B3, and F1, respectively, compared to that in the wild-type control isolate, ST-FMC46. Collateral damage of TET in soil was noticed, and significant upregulation of sul2 was also observed in these isolates (by 1,348-, 1,692-, 13,375-, and 42-fold in E1, A6, B3, and F1, respectively) compared to expression in ST-FMC46. Expression levels of vapB in E1, F6, and A1 were upregulated by 2.24-, 4.54-, and 2.31-fold, respectively, compared to that in the wild-type control isolate, ST-FMC46. On the other hand, antibiotic pressure was associated with downregulation of the dihydrolipoamide dehydrogenase and emrA genes in these isolates, while expression of res, mdfA, tolC, acrA, mdtK, and macA remained unaltered. The presence of TET also resulted in the downregulation of sdiA in all of the isolates compared to expression in ST-FMC46. In the absence of TET, expression of tetA and vapB genes was downregulated in isolates A8, F8, and B7 compared to that in the wild-type control, ST-CRC26. Alterations in the expression levels of tetA in the absence or presence of TET were highly noticeable, i.e., addition of TET resulted in a more than 40-fold alteration of expression, but its absence resulted in a less than 2-fold alteration.
TABLE 8.
Differential expression of genes involved in antibiotic resistance in S. Typhimurium isolates
| Salmonella isolate | Antibiotic resistance-related gene expression (fold) for: |
||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| sdiA | tetA | sul2 | GeneAa | GeneBb | GeneCc | vapB | mdfA | emrA | tolC | acrA | mdtA | mdsB | mdtK | macA | |
| ST-FMC46d | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| ST-FMC46m | 0.35 | 0.18 | 0.15 | 0.91 | 0.96 | 0.40 | 2.32 | 1.25 | 0.55 | 1.40 | 1.54 | 0.39 | 0.45 | 0.83 | 1.66 |
| A6 | 0.52 | 1,310.60 | 1,692.78 | 0.67 | 2.20 | 0.04 | 4.54 | 1.92 | 0.51 | 1.21 | 0.73 | 0.62 | 0.62 | 1.52 | 1.41 |
| B3 | 0.52 | 3,296.49 | 13,375.23 | 1.50 | 1.71 | 0.31 | 1.00 | 0.44 | 0.37 | 2.06 | 2.20 | 1.84 | 1.22 | 0.91 | 1.15 |
| E1 | 0.32 | 803.75 | 1,348.70 | 0.45 | 1.59 | 0.02 | 2.24 | 1.29 | 0.34 | 1.00 | 0.53 | 0.33 | 0.41 | 1.07 | 0.93 |
| F1 | 0.44 | 653.92 | 42.45 | 0.64 | 2.08 | 2.31 | 0.47 | 0.41 | 0.78 | 0.89 | 0.28 | 0.51 | 1.14 | 1.38 | |
| ST-CRC26e | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| ST-CRC26m | 0.74 | 1.92 | 2.46 | 1.19 | 0.37 | 0.40 | 0.34 | 0.40 | 0.88 | 1.67 | 1.53 | 0.70 | 1.08 | 0.48 | 1.22 |
| A8 | 0.64 | 0.36 | 0.26 | 1.00 | 0.91 | 0.53 | 1.11 | 0.55 | 1.18 | 0.82 | 0.97 | 1.21 | 0.77 | 0.85 | |
| B1 | 1.24 | 1.60 | 1.83 | 1.42 | 1.14 | 1.93 | 1.17 | 1.19 | 1.17 | 1.11 | 1.47 | 1.89 | 2.06 | 1.21 | 2.20 |
| B7 | 0.78 | 1.03 | 5.31 | 1.29 | 0.27 | 0.36 | 0.26 | 0.44 | 1.09 | 1.79 | 0.81 | 0.78 | 0.65 | 0.78 | |
| F8 | 1.29 | 0.96 | 3.86 | 1.52 | 1.03 | 1.01 | 0.88 | 1.38 | 1.01 | 1.23 | 1.59 | 2.43 | 2.27 | 0.89 | 1.33 |
GeneA, membrane-bound lysozyme inhibitor of c-type lysozyme gene.
GeneB, type III restriction-modification system DNA endonuclease res.
GeneC, dihydrolipoamide dehydrogenase gene.
Gene expression levels of Salmonella isolates FMC46m, E1, A6, B3, and F1 were normalized against the expression level of FMC46.
Gene expression levels of Salmonella isolates CRC26m, A8, F8, B1, and B7 were normalized against the expression level of CRC26.
DISCUSSION
Risks of Salmonella prevalence and drug resistance in farm systems.
Our previous study demonstrated the potential for Salmonella risks with both conventional and mixed crop-livestock (MCL) farm systems. A higher overall prevalence of Salmonella contamination was detected in the MCL system, whereas multiantibiotic resistance in Salmonella was found predominantly in conventional farms, where farmers are allowed to use synthetic antibiotics, and in their related postharvest products in retail stores (25). In this study, we further investigated phylogenetically inferred relatedness, genomic characteristics, and the alteration of antibiotic resistance-related genes under environmental pressures of the designated S. Typhimurium isolates. Surprisingly, we found that conventional farm S. Typhimurium isolates were closely related to pathogenic S. Typhimurium strain 14028S (GenBank accession number CP001363), while MCL or organic retailer S. Typhimurium isolates were highly genetically similar to the isolates from nonpathogenic S. Typhimurium strain ST-LT2 (GenBank accession number NC_003197). Additionally, previous epidemiological studies suggested that patients infected with multidrug-resistant Salmonella strains commonly suffered prolonged illnesses of greater severity than patients infected with susceptible Salmonella, suggesting a possible association between the antibiotic resistance and the virulence/pathogenicity of Salmonella strains (28–30), which also supported our findings.
Rapid acquisition of antibiotic resistance in susceptible Salmonella isolates.
Several groups of researchers have previously claimed that the rise in resistance came not only from clinical/veterinary therapeutic uses but also from subtherapeutic application of antibiotics in farm animals, as well as chemical contamination under certain environmental conditions (31). Through supplementation of subtherapeutic doses of antibiotics in natural soil environments, we identified antibiotic resistance gene accumulation in several S. Typhimurium isolates at different time points. Under short-term (less than 11 in vitro passages or 14 days of soil cultivation) antibiotic pressure, pansusceptible S. Typhimurium isolates gradually developed TET resistance. However, multidrug-resistant S. Typhimurium isolates barely lost or sacrificed their drug resistance, even after 12 passages in vitro or 6 months in soil environments in the absence of antibiotics. On the other hand, we also observed continuous expansion of TET resistance among several pansusceptible S. Typhimurium isolates in an unadulterated soil environment (data not shown). Generally, bacteriophages and mobile genetic elements serve as the antibiotic resistome in soil environments (32). Consequently, pathogenic bacteria like Salmonella enterica could potentially acquire resistance genes against specific antibiotics through horizontal gene transfer from this microbial resistome, which serves as the resistance gene reservoir.
The efflux pump system encoded by tet genes (e.g., tetA) serves as the main TET resistance mechanism in various bacterial pathogens, including Salmonella (33). Accordingly, in our study, we found a substantial association between TET pressure and the upregulated expression level of tetA in S. Typhimurium. Similarly, in the absence of TET, we also observed significant downregulation of tetA in Salmonella. Additionally, the upregulation under sublethal TET induction of vap genes in Salmonella, which encode small secreted virulence-associated proteins in bacterial pathogens, further supports our SNP-based phylogenetic inference results. In addition to proximity-dependent horizontal gene transfer, extra environmental factors—specifically, antibiotic and/or chemical pressure—could play a crucial role in the transfer of antibiotic resistance genes among members of the bacterial/microbial community. For example, an acidic or alkaline incubation environment was capable of stimulating horizontal transfer of TET resistance genes located on plasmids (34). In our study, environmental factors (e.g., temperature, air condition, humidity, pH, and time of cultivation) influence the metabolic rate and growth productivity of soil bacteria, potentially decreasing the cultivability of S. Typhimurium after 6 months (35). Furthermore, the existence of a TET resistome in natural soil environments might further explain the difficulty in losing TET resistance observed in resistant strains, even in the absence of antibiotic pressure.
Unexpectedly, the induction of SXT declined in building up resistance in pansusceptible S. Typhimurium isolates. This might be due to the intricacy of the mechanism of action of sulfonamides, which operates by interfering with bacterial folate synthesis (36). In such cases, the synergistic effects in combination with supplemented sulfamethoxazole and trimethoprim might play an important role (37). Although several sulfonamide resistance genes were recognized in soils fertilized with animal manure or organic matter (38), the buildup of their resistance in Salmonella remains continuous and moderate, which may generally require years of interventions (39). Correspondingly, stagnant progression or the lack of sulfonamide resistance genetic elements might explain the failure in this study of SXT resistance development via sublethal dose of antibiotic induction either within 12 passages of in vitro culture or after months of natural soil incubation. However, similarly to the TET-resistant isolates, the original SXT-resistant ST-CRC isolate did not show decreased resistance toward sulfonamides. Indeed, we expected that sulfonamide resistance could be even more rigid than TET resistance, since most bacteria have to alter their metabolic pathway for nucleic acid and folic acid synthesis, which is hardly reversible even in the absence of sulfonamide induction (39, 40).
Association of drug resistance and relatedness with virulence.
As increases in antibiotic resistance and virulence properties were usually observed concurrently (41, 42), their genetic connection and global genetic regulation are inevitably important for this simultaneous phenomenon (43). Regulation of antibiotic resistance and virulence genes is intertwined and highly complex, being influenced by multiple environmental factors, quorum sensing, and two-component systems that are believed to act as the connection in coincident upregulation (43, 44). In this study, we demonstrated the rapid acquisition of drug resistance, as well as the upregulation of antibiotic resistance-related genes in S. Typhimurium isolates, and the close relatedness between drug-resistant and pathogenic S. Typhimurium strains. However, further study of the mechanisms of their coordinate regulators and coregulation system networks is necessary to uncover the key interaction/connection between antibiotic resistance and virulence, which might induce novel insights into drug invention against persistent bacterial infections.
Conclusions.
This study demonstrated alterations of antibiotic resistance in S. Typhimurium isolates from conventional farm systems under environmental pressures. Although multiantibiotic-resistant S. Typhimurium isolates exhibited barely reduced resistance, the pansusceptible isolates managed to develop TET resistance quickly in both in vitro and soil environments. Additionally, the relative bacterial genetic alterations withstanding the rapid physical buildup of antibiotic resistance in S. Typhimurium indicated their expeditious acquisition of resistance genes from environmental reservoirs. Tracking and understanding the phenotypical transitions of antibiotic resistance in the microbial ecosystem will offer us more specific and directed strategies in combating dissemination of antibiotic resistance in Salmonella and minimizing its virulence.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
A total of 54 S. Typhimurium isolates were used and analyzed in this study. Sources, identification, and characterization of isolates were reported in our previous article (25). Briefly, all samples were collected in either organic or conventional poultry farms and retail markets, including organic and conventional markets and local farmers' markets in Maryland (MD) and the District of Columbia (DC) metropolitan areas. The sources and distribution of these isolates are listed in Table 1. The antibiotic resistance profiles of the 54 S. Typhimurium isolates are summarized in Table 2. All of the S. Typhimurium isolates were recovered from stock culture (Luria-Bertani [LB] broth with 20% glycerol stored in a −80°C freezer) on LB agar overnight (18 h) at 37°C under aerobic conditions. The recovered isolates were consecutively subcultured three times on LB agar overnight at 37°C under aerobic conditions for further investigation.
In vitro investigation of S. Typhimurium antibiotic resistance shifts.
Two S. Typhimurium isolates with TET and SXT resistance, ST-CRC26 and ST-CRC32, and two pansusceptible isolates, ST-FMC22 and ST-FMC46, were used for this phase of the study. The antibiotic-resistant isolates were used for in vitro examination of their antibiotic resistance shifts with and without antibiotic pressure in a nutritious environment. ST-CRC26 and ST-CRC32 were consecutively passaged 12 times (24 h per passage) in LB broth supplemented with 10% sheep blood and then incubated for 24 h at 37°C under aerobic condition.
Approximately 106 CFU/ml of ST-FMC22 and ST-FMC46 isolates in phosphate-buffered saline (PBS), measured at an optical density at 600 nm of around 0.01 with a LAMBDA BIO/BIO+ spectrophotometer (PerkinElmer, Beaconsfield, UK), were sequentially cultured in LB broth containing gradually increasing concentrations of antibiotic, as follows: TET from 2 to 128 μg/ml and SXT from 9.5/0.5 to 152/8 μg/ml. The surviving bacteria were subcultured into double-concentrated, fresh, antibiotic-containing LB broth for 24 h at 37°C under aerobic conditions. After final passage, the isolated strains were labeled ST-CRC26m, ST-CRC32m, ST-FMC22m, and ST-FMC46m and stored in −80°C glycerol-containing stock culture for future use.
Whole-genome sequencing of S. Typhimurium isolates.
The S. Typhimurium isolates were cultured on LB agar, and a single colony was transferred to 10 ml of LB broth and incubated aerobically for 24 h at 37°C. After incubation, the liquid cultures were centrifuged and decanted, and the pellets were processed for DNA extraction using a Qiagen DNeasy kit (Qiagen, Valencia, CA). Nextera XT libraries were prepared separately for each of the samples and pooled into equimolar concentrations, following the instructions of the manufacturer (Illumina, San Diego, CA). A high-output flow cell was used for paired-end sequencing (2 × 151 bp) with an Illumina NextSeq 500 sequencing platform. Data were demultiplexed and trimmed to remove adaptor sequences using the BCL2FastQ program, and PhiX reads were removed using DeconSeq (45). Reads were further cleaned using Trimmomatic V 0.36 (leading, 20; trailing, 20; sliding, 4:20; minimum length [minlen], 36) (46). Only paired data were further analyzed and assembled using SPAdes 3.6.2 with paired-end libraries (–pe) and pipeline options (–careful) (47). Assembled contigs were analyzed with the following Center for Genomic Epidemiology pipelines: ResFinder-2.1, KmerFinder-2.1, ContigAnalyzer-1.0, MLST-1.6, PlasmidFinder-1.6, and SalmonellaTypeFinder-1.2. Single nucleotide polymorphisms (SNPs) were identified using CSI Phylogeny-1.4, available from the Center for Genomic Epidemiology (http://www.genomicepidemiology.org) (48), where the paired-end reads from each isolate were aligned against the reference genome, ST-LT2 (ATCC 700720). The qualified SNPs were designated based on the following criteria: (i) a minimum coverage of 10×; (ii) a minimum of 10 bp between SNPs; (iii) a minimum quality score of 30; (iv) a minimum read mapping quality of 25; and (v) a minimum Z-score of 1.96 with the altered FastTree option activated for more accuracy. The chromosome of ST-LT2 was selected as the reference genome for SNP identification. To infer the phylogeny of the S. Typhimurium isolates, the SNP matrices in Newick format were imported to FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Assembled contigs of S. Typhimurium isolates ST-FMC22, ST-FMC22m, ST-FMC46, ST-FMC46m, ST-CRC26, ST-CRC26m, ST-CRC32, and ST-CRC32m were further annotated with the Rapid Annotation using Subsystem Technology (RAST) server (http://rast.nmpdr.org/rast.cgi).
Soil environment simulation of S. Typhimurium antibiotic resistance development.
To verify our in vitro finding of Salmonella antibiotic resistance shifts under antibiotic pressure or absence of antibiotic, the same study was performed in a soil environment. For this study, natural soil samples were collected from both conventional and organic farms and designated soil 1 and soil 2, respectively. Both soil samples were stored in plastic buckets at room temperature and then air-dried by spreading on separate aluminum trays in a growth chamber for 3 days. The dried soil samples were sealed and stored in separate sterilized plastic bags.
Antibiotic liquid stock solutions contained 4 mg TET per ml distilled water (dH2O) and 19/1 mg SXT per ml dH2O. An aliquot of 1 ml of both antibiotic stocks was sprayed and mixed well in 1 kg soil sample labeled as Ab+ with antibiotic supplement in a final sublethal concentration of 4 mg TET per kg soil and 19/1 mg SXT per kg soil. The rest of the soil was supplemented with the same amount of dH2O, labeled as Ab− for control. An aliquot of 2 ml of either ST-CRC26 or ST-FMC46 bacterial suspension stock (108 CFU per ml PBS) was inoculated and mixed well into 1 kg soil sample labeled separately as ST-CRC26 or ST-FMC46 group, for a final bacterial concentration of 107 CFU per 50 g soil. Then, each 250-g supplemented soil sample was weighed and filled up in labeled matching clusters following the random predesigned experimental soil panel shown in Fig. 2. Each antibiotic supplementation was replicated a total of 6 times (6 clusters), and each cluster was subdivided into 5 cells for 5 time period samplings for further processing and antibiotic resistance tests as per standard procedure. The supplemented soil cells were cultured in a growth chamber (Thermo Fisher Scientific Inc., Waltham, MA) and gently irrigated with tap water every 2 days.
FIG 2.
S. Typhimurium inoculation panel in soil environment. Soil samples were collected from two types of farms, with soil 1 as conventional farm soil and soil 2 as organic farm soil. ST-CRC26 or ST-FMC46 isolate was applied for inoculation. Ab+ indicates antibiotic supplement in a final sublethal concentration of 4 mg per kg soil for tetracycline and 19/1 mg per kg soil for SXT. Ab− indicates no antibiotic supplement.
Soil sampling, processing, and enrichment for recovering S. Typhimurium isolates.
Soil samples containing S. Typhimurium isolates were collected and processed on day 0 and at 14 days, 28 days, and 2 months for the short-term investigation, plus at 3 months and 6 months for the long-term investigation. Each sample was processed separately, following “Salmonella” in the Bacteriological Analytical Manual (49). Briefly, 1 g soil sample from each soil panel cell was weighed, collected in a sterilized Eppendorf tube, and mixed into 9 ml of buffered peptone water for overnight incubation at 37°C under aerobic conditions, followed by Rappaport-Vassiliadis medium and tetrathionate broth overnight subculture at 37°C under aerobic conditions. The final enriched bacteria in tetrathionate broth were plated on xylose-lysine-tergitol 4 (XLT-4) agar at 37°C under aerobic conditions for Salmonella isolation. Triple colonies from each sample were subcultured on Mueller-Hinton (MH) agar overnight at 37°C under aerobic conditions for both −80°C glycerol stock and further MIC determination.
Determination of TET and SXT resistance of S. Typhimurium isolates recovered from soil samples.
TET and SXT resistance of Salmonella isolates was tested with a standard agar dilution method according to the Clinical and Laboratory Standards Institute (50). In total, 5 different concentrations (2-fold serial dilution) of antimicrobial solutions were mixed into freshly prepared MH agar. The isolated Salmonella colonies were picked up using sterile inoculation loops and suspended in PBS for McFarland standard (0.5) adjustment. The standardized bacterial suspension was diluted by 10-fold with PBS, and then 2 μl of each bacterial suspension containing approximately 105 CFU was spotted on antibiotic-containing MH agar for overnight incubation at 37°C under aerobic conditions. The MIC of each antibiotic was recorded as the lowest concentration that completely inhibited the visible growth of the Salmonella isolate.
Quantitative RT-PCR assay.
All isolates of ST-FMC22, ST-FMC22m, ST-FMC46, ST-FMC46m, ST-CRC26, ST-CRC26m, ST-CRC32, and ST-CRC32m were cultured in LB broth at 37°C overnight. The bacterial cells were harvested; RNA extraction was carried out, followed by cDNA synthesis; and qRT-PCR was performed in an Eco RT-PCR system (Illumina, CA), according to the protocol previously described (51, 52). The PCR cycle was 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 55°C for 15 s, and 72°C for 10 s. The custom-synthesized oligonucleotides (Erofins MWG Operon, Huntsville, AL) were used as primers to target conserved regions of S. Typhimurium (see Table S1 in the supplemental material). The relative transcriptional expression levels of target genes were estimated by the comparative fold change. The housekeeping gene was used as the reference gene for normalization of target gene expression. Quantitative RT-PCR assays were carried out in triplicate and the results were averaged.
Accession number(s).
Accession numbers of draft genome contigs have been deposited in the NCBI Sequence Read Archive and are listed in Table 5.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by an SCRI grant (award 2011-51181-30767).
We also thank Shirley Micallef and Devkumar Govindaraj in the Department of Plant and Soil Architectures, University of Maryland, College Park, for their support and for providing the growth chamber facility.
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01173-18.
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