Abstract
Dairy animals are reservoirs of antimicrobial-resistant Escherichia coli that are frequently resistant to tetracycline, aminoglycoside, β-lactam, sulfonamide, and macrolide–lincosamide–streptogramin B antibiotics. However, resistance to other classes of antimicrobials is less frequently observed, and resistance to fosfomycin is rarely observed in E. coli. In this study, we describe the genomic characteristics of E. coli encoding fosA7.5 that have been recovered from bovine sources in the United States. Most isolates only encoded the fosA7.5 gene, whereas 37% encoded at least one other resistance gene, and 25% were genotypically multidrug-resistant. Most (112 isolates, 93%) belonged to phylogenetic group B1 and were assigned to 19 sequence types (STs), the most frequently identified being ST1727, ST2307, and ST3234. Results of this study indicate that fosA-encoding E. coli from bovine sources is very rare in the United States with isolates demonstrating a high level of similarity across a broad geographic region.
Antimicrobial resistance is a severe public health threat due to the difficulty in treating multidrug-resistant (MDR) infections that is compounded by the difficulty of identifying novel antimicrobial therapeutics. The World Health Organization has designated suites of antibiotics as “Access,” “Reserve,” and “Watch” group antibiotics. Reserve group antibiotics should be used as “last resorts” for treatment of MDR infections and Watch group antibiotics have a high potential for resistance selection, are more frequently used in very sick humans, and should be monitored to avoid overuse (World Health Organization, 2023).
Fosfomycin, a Reserve Group (intravenous) and Watch Group (oral) member, is a broad-spectrum bactericidal antibiotic that acts by blocking cell wall synthesis through the inhibition of peptidoglycan synthesis (Dijkmans et al., 2017). In the United States, fosfomycin is indicated for the treatment of lower urinary tract infections in humans but has been used in some countries for the treatment of pneumonia, osteomyelitis, and central nervous system infections (Falagas et al., 2016; Grabein et al., 2017). It is considered a “last resort” antibiotic against carbapenemase–producing Gram negatives, because these organisms are typically resistant to all β-lactams (Meletis, 2016). It is currently not approved for use in dairy and beef cattle in the United States (U.S. Food and Drug Administration, 2023).
A large-scale analysis of antimicrobial-resistant Escherichia coli from nonredundant fecal samples collected across 80 dairy farms in Pennsylvania identified five genomes out of a total of 288 that encoded a 423 bp sequence that was 94% similar to the fosfomycin resistance glutathione transferase fosA7 nucleotide sequence in the Resfinder 4.1 database (Bortolaia et al., 2020; Haley et al., 2023). These sequences were aligned with fosA variants in the National Center for Biotechnology Information (NCBI) database and were 100% similar to the fosA7.5 gene, the variant most frequently identified to date in E. coli isolates (Milner et al., 2020).
Four genomes did not encode any other antimicrobial resistance genes (ARGs) while one encoded a tetracycline resistance gene (tetC). Four genomes were identified as phylogenetic group B1 and one was identified as group A. Two were sequence type 75 (ST75), and the others were ST342, ST5521, and ST96 (Table 1).
Characteristics of fosA-Encoding Escherichia coli Genomes Recovered from Bovine Sources
AZ, Arizona; CA, California; CO, Colorado; FL, Florida; GA, Georgia; HI, Hawaii; IA, Iowa; ID, Idaho; IN, Indiana; KS, Kansas; KY, Kentucky; MD, Maryland; MI, Michigan; MLS, macrolide–lincosamide–streptogramin B; MN, Minnesota; NC, North Carolina; NE, Nebraska; NJ, New Jersey; NY, New York; OH, Ohio; OR, Oregon; PA, Pennsylvania; SC, South Carolina; SD, South Dakota; ST, sequence type; TN, Tennessee; TX, Texas; UT, Utah; WA, Washington; WI, Wisconsin; WY, Wyoming.
In-house isolates.
Encodes the gyrA (p.S83L) point mutation.
Encodes the lnu(G) MLS gene.
To further evaluate the presence of the fosA gene in E. coli isolates recovered from other bovine sources (dairy and beef cows and calves), a search of genomes in the NCBI database from the United States that encoded any variant of the fosA gene yielded 115 genomes of strains isolated between 1978 and 2021. The majority of these were recovered between 2018 and 2021 (61%).
The 115 publicly available bovine-associated genomes were from isolates recovered from 29 states including 9 from the east coast, and all 3 west coast states and Hawaii, indicating a broad geographic range of fosA-positive E. coli from bovine sources in the United States. Interestingly, all but seven isolates were identified as phylogenetic group B1 and these seven were all identified as group A (Table 1; Supplementary Data). Of the 115 genomes, only 17 STs were identified, with the most common being ST1727 (ST Cplx 446), ST2307, and ST3234 (Table 1).
Within the phylogenetic group A strains, all were ST342 and either serotype O145:H25 or O5:H9, indicating a high level of clonality among the seven non-B1 strains. Some of the STs have been associated with human gastrointestinal infections, particularly ST678 and ST342 (Zhou et al., 2020). No major extraintestinal pathogenic E. coli STs were identified.
The virulomes of the 120 strains (5 in-house and 115 from NCBI) were highly diverse with the number of identified virulence genes ranging between 2 and 27 (mean = 8, median = 6) (Supplementary Data). Seven strains were Shiga-toxigenic E. coli (STEC), encoding Shiga toxin genes (stx) known to be responsible for severe infections. Six of these were phylogenetic group A-ST342-O5:H9 isolates, and were isolated in Oregon, California, and Pennsylvania, indicating a broad geographic range of this variant.
The other STEC genome was a phylogenetic group B1-ST1727 (ST Cplx 446) isolate recovered in Ohio. Strains of both STs have been isolated from human clinical cases and food animal sources globally (Zhou et al., 2020), indicating that they are potential pathogens and their presence in food production systems should be further evaluated.
Of the 120 genomes, 75 encoded only the fosA resistance gene, whereas 14 encoded fosA along with a second ARG known to confer resistance to another class of antibiotics, and 31 genomes had an MDR genotype. The number of antibiotic classes to which a genome encoded resistance genes ranged between 1 and 8. Among these genomes, tetracycline resistance genes were the most frequently identified (n = 30), followed by aminoglycoside (n = 27) and sulfonamide (n = 25). Besides fosA, the most frequently identified ARGs were tetA (n = 26), aph(3″)-Ib and aph(6)-Id (n = 21), and sul2 (n = 20).
β-lactamase genes were identified in 18 genomes; bla TEM-1B was detected the most frequently (n = 7), followed by bla TEM-1A (n = 5), and bla CMY-2 (n = 1) and bla CARB-1 (n = 1). The phenicol resistance gene, floR, was identified in 12 genomes, and folate pathway antagonists (trimethoprim resistance) dfrA1, dfrA12, and dfrA17 were each identified in single genomes. None of these ARGs were identified on the same contigs as those encoding the fosA gene, indicating that they were most likely acquired during separate events.
Results of this study demonstrated that fosA-encoding E. coli are rarely isolated from bovine sources in the United States. Furthermore, strains that have been isolated are highly similar. Fosfomycin is a Reserve group and Watch group antibiotic that is used to treat infections caused by MDR organisms (World Health Organization, 2023), and the presence of this resistance gene should be further evaluated with an aim to better understand the human health impacts. Future study should focus on the prevalence of fosfomycin-resistant strains in food animals and the potential pathogenicity of these strains.
Footnotes
Acknowledgments
We thank Jakeitha Sonnier and Laura Del Collo for their assistance with laboratory work.
Authors' Contributions
B.J.H. designed and coordinated the study, analyzed the data, and prepared the article. S.W.K. carried out sequencing and analyzed data. J.V.K. edited and critically revised the article. All authors read and approved the submitted article.
Disclaimer
Mention of trade names or commercial products in this article is solely for providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Disclosure Statement
The authors declare no conflicts of interest.
Funding Information
This research was funded by internal USDA/ARS funding.
Supplementary Material
Supplementary Data
References
Supplementary Material
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