- Based on whole-genome sequencing (WGS) and PCR, a multidrug-resistant (MDR) phenotype of S. enterica was strongly associated with carriage of ISVsa3, an IS91-like Family transposase mainly carried on plasmids.
- A Fourier transform infrared spectroscopy (FTIR) test was developed and found to have potential as a rapid and cost-effective diagnostic tool for Salmonella AMR typing.
- Further research is needed to better understand the role of ISVsa3 in dissemination of MDR Salmonella and the translatability of FTIR to broader Salmonella serotypes and genotypes.
) is estimated to cause 1.35 million infections, 26,500 hospitalizations, and 420 deaths each year in the United States (U.S.). Beef is a significant attributable source of S. enterica
. In the human patient, systemic Salmonella
infection requires antibiotic therapy, and when the strain is multidrug resistant (MDR [resistant to ≥3 antibiotic classes]), the case is particularly problematic. S. enterica
is also a primary pathogen in cattle, mainly causing enteritis in calves between 2 and 6 weeks of age, but can also cause enteritis, pneumonia, and abortions in adult animals, with most clinical infections associated with S
. Dublin and S
. Typhimurium. However, more than 143 serotypes have been found in cattle lacking clinical signs of illness, indicating a large reservoir of diversity in populations. S. enterica
is often associated with subclinical infections, but may have a morbidity of over 50% in calves, with a case–fatality rate approaching 100% without treatment. The most common resistance pattern in S
. Typhimurium is ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT). This MDR pattern has, historically, most often corresponded to a clone of S
. Typhimurium known as bacteriophage definitive type (DT) 104 (DT104), which often causes severe disease in both animals and humans.
In bacteria, the spread of AMR genes is mainly the result of mobile genetic elements (MGE), which enable intracellular and intercellular movement of DNA, e.g., plasmids, integrative conjugative elements (ICE), and integrative mobilizable elements (IME). DNA containing AMR genes is also spread intercellularly via transduction and transformation mechanisms. Many MDR Salmonella such as S. Typhimurium DT104 contain an IME known as a Salmonella Genomic Island (SGI). SGI1 variants contain different combinations of genes responsible for the ACSSuT phenotype, and, in addition, those for resistance to florfenicol, gentamycin, spectinomycin, tobramycin, and trimethoprim. Recent studies have shown that ICEMh1 and -like elements in respiratory pathogens of the Pasteurellaceae family readily spread among each other in cases of bovine respiratory disease (BRD) and have the potential to spread into Salmonella. Spread of these ICEs is especially evident in outbreaks of BRD in high-risk stocker and feedlot calves following metaphylactic and therapeutic administration of antibiotics. ICEMh1 in Mannheimia haemolytica and ICEMh1-like elements such as ICEPmu1 in Pasteurella multocida are fully transmissible and proven to integrate into some bacterial recipients. They potentially integrate into Salmonella based on DNA analytical evidence of the target integration site in the chromosome. Each of these ICEs transmits a potent arsenal of antibiotic resistance.
Antibiotics for metaphylaxis (control) of BRD could potentially select for AMR Salmonella secondary to selection for respiratory pathogens carrying ICEMh1 and ICEMh1-like elements. In addition, they could directly select for AMR Salmonella strains that contain SGI1 variants. The objectives of this study were to determine, in a set of Salmonella enterica isolates from cattle or their environments, the frequency of: (1) AMR genes typically associated with ICEMh1 and ICEMh1-like integrative conjugative elements; (2) Salmonella Genomic Island 1 (SGI1) variants and their associated AMR genes; and (3) other mobile genetic elements and their potential association with MDR.
A subset of S. enterica
strains (19 of 530) isolated from feedlot beef cattle feces or their pen environments in the summers of 2000 or 2001 were used in this study. All 530 strains were serotyped and tested for antimicrobial susceptibility phenotype in 2006 using the National Animal Resistance Monitoring System (NARMS) protocol at that time, and also were tested 6 by PCR for class 1 integron genes. Of the S. enterica
strains from that study, none were positive for class 1 integron genes by PCR; however, 13 were MDR. These 13 MDR strains were included in the present study; six other strains resistant to only one antimicrobial that originated from the same sample or pen of cattle were also included in the present study for whole genome sequence (WGS) and/or other test comparisons. Eleven MDR strains and six non-MDR strains were selected for WGS. A second source of S. enterica
strains isolated from cattle were accessions from cattle systems to the Nebraska Veterinary Diagnostic Center (NVDC) during the period of 2011-2020. Of 98 Salmonella
isolates from cattle systems in cases submitted to the NVDC from 2011-2020, 92 were viable from frozen stocks and included in the present study. Antimicrobial susceptibility testing (Sensititre) was conducted on all NVDC isolates at the time of the accession or as a part of this study. Of the 92 isolates, at least 17 were MDR. Culture of bacterial strains and DNA preparation for multiplex qPCR assays including targets, reagents, and primers were conducted.
Bacterial genomes were sequenced using the Illumina MiSeq platform (Illumina) at the Iowa State University Veterinary Diagnostic Center followed by Nanopore sequencing using Oxford Nanopore GridION X5 at the DNA facility of Iowa State University. Genomic DNA sequence similarity searches were conducted using BLAST+2.11.0. Genomes were annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 2021-07-01.build5508. Sequence visualization and analysis was conducted using Geneious Prime 2021.2.2. AMR gene identification and verification was conducted using CARD 3.1.3 and ResFinder 4.1 2021-08-16. Maximum-likelihood phylogenies were estimated using PhyML 3.2. Sequence alignments were conducted using Muscle 3.8.425. Pan-genome analyses were conducted using Roary 3.13.0. Core phylogeny with metadata analysis was conducted using Phandango 1.3.0. The Salmonella
strain collection tested by IR Spectroscopy included 103 of 105 isolates which excluded six non-MDR strains and two other strains. Composition of the strain collection utilized for Fourier transform infrared spectrometry (FTIR) included multiple serogroups (B, C1, C2, D, E, G, and L), serotypes, and AMR genotypes (+/- for CMY and ISVsa3
Of 92 Salmonella
strains isolated from cattle in cases submitted to the Nebraska Veterinary Diagnostic Center from 2011-2020, 17 (18.5%) were MDR. Genetic testing through PCR and whole genome sequencing of 24 of these diagnostic isolates and 17 isolates from the feces and pen environments of healthy feedlot cattle in a previous study revealed MDR was predominantly associated with ISVsa3
, an IS91
-like Family transposase, which was mainly carried on plasmids. An FTIR test was developed and found to have potential as a rapid and cost-effective diagnostic tool for Salmonella
is an important cause of foodborne illness with beef ranking fourth as an attribution source. In the human patient, bloodborne Salmonella
infection requires antibiotic therapy, and when the causative organism is resistant to multiple antibiotics, the situation is life-threatening. This study found a novel genetic mechanism used by Salmonella
to acquire antibiotic resistance. A rapid detection test was developed that, with further optimization, has the potential for use in the detection of these drug-resistant microorganisms.