Introduction

Bovine mastitis, mainly caused by bacterial pathogens, is the most prevalent and costly diseases of dairy industry worldwide.1 Current control and prevent programs based on post-milking teat disinfection, culling of chronically affected cows and antibiotic therapy have successfully reduced the incidence of contagious pathogens mastitis.2 Antibiotic treatment may lead to an increase in resistance of some environmental pathogens.3Trueperella pyogenes (formerly Arcanobacterium pyogenes) is an important environmental pathogen causing mastitis, which is often described as an opportunistic pathogen in domestic animals worldwide.4–7 This bacterium commonly cause persistent infection and pyogenic lesions in mammary gland, thus it is more often linked to the mastitis with a high severity of symptoms.8–10

Antimicrobial therapy is the primary measure of treatment for T. pyogenes infections,11 According to reports, plant secondary metabolites, antimicrobial peptides, and bacteriophages are currently widely studied alternatives to antibiotics and have made significant progress.12–14 Unfortunately, these antibiotic substitutes still need time to follow suit in drug development. However, the therapeutic effectiveness has been attenuated by emerging resistant strains result of the widespread use of antimicrobials in food animals.15 Antimicrobial resistance of T. pyogenes is mainly attributed to different resistant genes, such as tetW (tetracyclines resistance), ermB and ermX (macrolides resistance) and aacC, aadA1, aadA9 and aadA11 (aminoglycosides resistance), which pose a potential threat to both animal and human medicine due to their dissemination through mobile genetic elements.7,16 Integrons are the common bacterial genetic elements that can capture, rearrange, and express mobile gene cassettes.17T. pyogenes integrons, especially classes 1 integrons, play important roles in the horizontal transfer of resistance genes.18 Moreover, several known and putative virulence factors that may contribute to the pathogenicity of T. pyogenes, including pyolysin (Plo) with cytolytic activity on immune cells and adhesion-related factors collagen-binding protein (CbpA), neuraminidases (NanH and NanP), and fimbriae (FimA, FimC, FimE and FimG).19 The genotypic profiles of these virulence factors varied greatly among T. pyogenes isolates in different infection.20

Although investigations on T. pyogenes were reported worldwide, little is known about the isolates originated exclusively from bovine mastitis in China. The aim of the current study was to investigate the resistance determinants and virulence genes of T. pyogenes isolated from clinical bovine mastitis cases in Gansu, China. To the best of our knowledge, this is the first description of integron gene cassettes of T. pyogenes isolated from bovine mastitis in China.

Materials and Methods Bacterial Isolates

The 45 T. pyogenes strains investigated in this study were isolated from clinical bovine mastitis cases in 16 commercial dairy herds located in Hexi Corridor of Gansu in China during July 2000 to Aug 2022 and preserved in our laboratory. Mastitis infection was confirmed by the California Mastitis Test. After transportation to the laboratory, mastitic milk samples were inoculated onto blood agar plates supplemented with 5% defibrinated sheep blood and incubated with 5% CO2 at 37°C for 48 h. Smooth and glistening bacterial colonies surrounded by a conspicuous β-hemolytic zone were further identified by PCR and sequencing as described in our previous study21,22 (Figure 1).

Figure 1 Experimental method flowchart.

The 16S rRNA gene was amplified by the 16S rDNA Bacterial Identification PCR Kit (Takara, Shiga, Japan) in accordance with the manufacturer’s recommendations (https://www.takarabiomed.com.cn/DownLoad/RR176.pdf). The PCR products were purified and sequenced by Sanger sequencing by Sangon Biotech (Shanghai) Co., Ltd. in China. Nucleotide sequences were analyzed with the program NCBI-BLAST (http://www.ncbi.nlm.nih.gov).

Antimicrobial Susceptibility Testing

Minimum inhibitory concentrations (MICs) of penicillin, oxacillin, ceftiofur, erythromycin, tetracycline, streptomycin, gentamicin and ciprofloxacin against T. pyogenes were tested by E-test (Liofilchem, Roseto, Italy) method on Muller-Hinton agar supplemented with 5% defibrinated sheep blood. Antimicrobial agent concentrations ranged from 0.002 to 32 μg/mL for penicillin and ciprofloxacin, 0.016 to 256 μg/mL for oxacillin, ceftiofur, erythromycin, tetracycline and gentamicin, and 0.064 to 1024 μg/mL for streptomycin. T. pyogenes ATCC19411 was used as quality control strain. The experiments were carried out in triplicates biological replicate (three independent cultures). Currently, there are no T. pyogenes-specific breakpoints for antimicrobial susceptibility testing available in the Clinical and Laboratory Standards Institute guidelines. Thus, the susceptibility of the T. pyogenes was determined according to the breakpoints reported previously23,24 (Figure 1).

Detection of Resistance Determinants and Virulence Genes

Single PCR was used to detect resistance genes of tetracyclines (tetW, tetA33, tetL, tetM, tetO, tetK and tet32), macrolides (ermX and ermB), and aminoglycosides (aadA1, aadA9, aadA11, aacC, strA-strB, aph(3’)-IIIa and aac(6’)-aph(2”)), as well as integrase genes (intI I and intI II) and gene cassette region.7,25–27 Briefly, the genomic DNA was extracted using the Bacterial DNA Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The PCR products were analyzed using 1.0% agarose gel electrophoresis. Subsequently, DNA sequencing was carried out on gene cassette region. The data analysis of the gene cassettes was similar to that of the 16S rRNA gene. Similarly, genes encoding virulence factors pyolysin (plo), neuraminidases (nanH and nanP), collagen binding protein (cbpA) and fimbriae (fimA, fimC, fimE and fimG) were also determined by single PCR as previously described7 (Figure 1).

Statistical Analysis

All drug resistance assays were carried out in triplicate. Data were classified using the Microsoft Office Excel software.

Results Antimicrobial Susceptibility Testing

The antimicrobial susceptibility of the 45 T. pyogenes isolates against 8 antimicrobial agents were summarized in Table 1. The isolates showed high resistance to streptomycin (88.9%) and tetracycline (64.4%), followed by erythromycin (15.6%) and gentamicin (13.3%). Surprisingly, the MICs of erythromycin were greater than 256 μg/mL in 71.4% of the erythromycin-resistant isolates (data not shown). In addition, all tested isolates were susceptible to penicillin, oxacillin, ceftiofur and ciprofloxacin. It is worth noting that in this study, 20% of the isolates displayed multidrug resistance (MDR).

Table 1 Distribution of Resistance Determinants and Virulence Genes in 45 T. Pyogenes Isolates from Clinical Bovine Mastitis

Genetic Determinants for Antimicrobial Resistance

In the present investigation the T. pyogenes isolates only showed resistance to tetracycline, erythromycin, gentamicin, and streptomycin. Hence, the corresponding resistant-genes of tetracyclines, macrolides and aminoglycosides as well as integrase genes and gene cassette region were tested and shown in Table 1. The tetW, tetA33, and tetK were found in 64.4%, 8.9% and 2.2% of the T. pyogenes isolates, respectively. All tetracycline-resistant isolates harbored tetW alone or in combination with tetA33. Besides, the erythromycin-resistant gene ermX was found in 13.3% of the isolates, one erythromycin-resistant isolate was negative for this gene. In addition, aadA1, aadA9, aadA11 and strA-strB were detected in 17.8%, 88.9%, 11.1%, and 11.1% of the tested isolates, respectively. Importantly, all streptomycin-resistant isolates were positive for aadA9 alone or in combination with aadA1, aadA11 or strA-strB. However, ermB, tetM, tetO, tetL, tet32, aacC, aph(3’)-IIIa and aac(6’)-aph(2”) were not detected in any of the isolates. The results of integrase genes showed that all T. pyogenes isolates carried class 1 integron while no isolate harbored class 2. Furthermore, 17.8% of them were positive for gene cassettes, including arrays aadA1-aadB (8.9%), aadA2-aadB (4.4%), aadA24-dfrA1-ORF1 (2.2%) and aadA2 (2.2%). Notably, all gentamicin-resistant isolates contained gene cassette aadB.

Detection of Virulence Genes

We also detected the virulence-encoding genes of the T. pyogenes isolates (Table 1). The results showed that all tested isolates carried plo and fimA. Genes fimC, fimE, nanP, nanH and cbpA were found in 88.9%, 86.6%, 75.6%, 40.0%, and 35.6% the isolates, respectively. While only 6.7% of the isolates contained fimG. In addition, a total of 14 different virulent genotypes were identified in the isolates. Plo, nanP, cbpA, fimA, fimE and fimC was the most dominant genotype and was detected in 33.3% of the isolates.

Discussion

T. pyogenes is an important opportunistic animal pathogen causing a wide variety of purulent infections and conveys significant economic losses to the animal husbandry industry. Beta-Lactams, tetracyclines and macrolides were often used to treat T. pyogenes infections, which accelerated the emergence of resistant strains.28 In this study, most of the T. pyogenes isolates displayed high resistance against streptomycin and tetracycline and a few of them were resistant to erythromycin or gentamicin. Similar phenotypic resistance to these antimicrobial agents had been frequently observed in T. pyogenes isolates result from frequent use of these antimicrobials.23,29–31 Bacterial tetracycline resistance is mainly conferred by tet genes of the ribosomal protection class, particularly the widespread determinant tetW.32 Indeed, in the current study, all tetracycline-resistant isolates carried the tetW. Although few of the resistant isolates also contained tetracycline-specific efflux pump protein-encoding gene tetA33, the positive isolates are usually not considered resistant because of the low tetracycline MIC conferred by tetA33 alone in T. pyogenes.25 These results suggesting tetracycline-resistant mechanism was closely related to ribosomal protection proteins encoded by the tetW in T. pyogenes isolated from clinical bovine mastitis cases in Hexi Corridor of Gansu, China.

Although of the low erythromycin-resistant frequency (7/45) in the tested isolates, most of the ermX-containing isolates showed high erythromycin MICs (˃256). Meanwhile, the MICs of erythromycin for the ermX-containing isolates varied considerably (data not shown). Moreover, one erythromycin-resistant isolate carried no expected resistance genes. This discrepancy could be attributed to the presence of other resistance mechanisms, such as additional erm genes, genes encoding efflux pumps, or ribosomal mutations.33 In accordance with the previous report,23 we found most of the T. pyogenes isolates were highly resistant to streptomycin but were susceptible to gentamicin. The aadA9 was the most prevalent aminoglycosides resistant-gene in the current study. This may not be surprising because aadA9 was shown to be significantly more prevalent in bovine isolates than other origin species.26 Noteworthily, all streptomycin-resistant isolates carried aadA9 alone or in combination with aadA1, aadA11, or strA-strB, which mainly conferred to resistance against streptomycin.26 Similarly, all gentamicin-resistant isolates were positive for class 1 integrons gene cassette aadB conferred to gentamicin resistance.34 These results indicating that gene ermX and determinants aadA9 and gene cassette aadB may play an important role in erythromycin and aminoglycosides resistance in the tested isolates, respectively. Recently, more than 9 classes of integrons have been described, class 1 and 2 integrons are the most predominantly associated with antibiotic resistance in clinical isolates.35,36 In current study, all T. pyogenes isolates carried class 1 integrons while no isolate harbored class 2. These data are in agreement with previous reports in China showed that class 1 was the most popular integrons in T. pyogenes isolates.23,24 Additionally, 17.8% of the tested isolates were positive for 4 types of gene cassettes. All resistance gene cassettes were conferred resistance to aminoglycosides (aadA1-aadB, aadA2-aadB, aadA2 and aadA24) expect one trimethoprim-resistant gene cassette dfrA1, coinciding with previous study reported that these aminoglycosides resistance determinants were highly prevalent among pathogens from bovine mastitis in China.37 It is worth noting that the high-level of aminoglycosides-resistant gene cassettes may facilitates horizontal transfer of these resistance genes among microorganisms,35 which means that multidrug resistance can develop.27

T. pyogenes produces a number of extracellular or surface-exposed proteins involved in the infections caused by this bacterium.38 Plo is a primary virulence factor with cytolytic activity related to transmembrane pore formation and considered as an important marker in the definitive diagnosis of T. pyogenes.39,40 Similar to previous reports of bovine mastitis conducted in China and other countries,6,7,11,29plo was observed in all tested isolates, indicating it’s a critical role in establishment of T. pyogenes infections. Other putative virulence factors in this study primarily contribute to the adhesion and colonization of the host tissues. Among them, cbpA is collagen-binding protein in T. pyogenes that mediates adhesion to epithelial and fibroblast cells.41 Previous studies showed that the detection rate of cpbA ranged from 1.4% to 100.0% in T. pyogenes from bovine origins.42–44 In this study, only 35.6% of the isolates harbored cpbA, which was much lower than other study in China reported that all tested isolates from bovine mastitis contained this gene.11 Moreover, neuraminidases NanH and NanP were found to play an important role in the colonization of host tissue by cleaving the terminal sialic acid residues of host cell and reducing mucus viscosity of tissue.45 In current study, the nanH and nanP genes were detected in 75.6% and 40.0% of the isolates, respectively. These findings are in accordance with the previous results of bovine T. pyogenes isolates.39,46 The fimbriae were also involved in the cell adhesion and the colonization of host tissue. FimA is a dominating fimbria in T. pyogenes.40 Indeed, all T. pyogenes isolates carriedthe fimA in this study, and most of them simultaneously harbored the fimC (88.9%) and fimE (86.6%). On the contrary, the fimG was only found in 6.7% of the isolates. These results were in accordance with the previous study that reported a high prevalence of fimA among T. pyogenes isolates, whereas other fimbriae-encoding genes were detected with different frequencies.40 The differences of virulence factor genes detected in the current study could be explained by inherent variations between different isolates.47

Conclusions

The T. pyogenes isolates showed high frequencies of phenotypic and genotypic resistance to streptomycin and tetracycline, as well as high incidences of class 1 integrons and aminoglycosides-resistant gene cassette arrays among the cassettes, which remind the government to pay continuous attention to use antimicrobial agents in dairy industry. It is worth noting that in this study, 20% of the isolates displayed multidrug resistance. Meanwhile, the potential threat of horizontal transmission of the resistance genes cannot be ignored. In addition, frequent occurrence of plo, fimA, fimC, fimE and nanP may indicate their pathogenic potential in bovine mastitis in China, although different infections caused by T. pyogenes may be equipped with variable virulence factors. Further investigations need to be performed to explore the diversity of virulence factors combination in T. pyogenes pathogenesis. In addition, in order to address the durability of antibiotics, we should further study the antibacterial mechanisms and effects of alternative to antibiotic.

Ethics Approval and Consent to Participate

Compliance with ethical standards: This study was approved by Ethics Committee of Gansu Agricultural University (No.GASU-Eth-AST-2023-008.) and was conducted in compliance with ethical, legal, and regulatory norms. The animal owners were informed about the purpose of the study, and consent of each animal owner was obtained before the physical examination of cows for clinical mastitis and the collection of milk samples.

Funding

This work was financially supported by the program for Gansu Academy of Agricultural Sciences Scientific Research Conditions Construction and Achievement Transformation Project Key R&D Programme (No. 2020GAAS28); Gansu Provincial Science and Technology Department in the Field of Social Development Key R&D Programme (No. 21YF5NA150); National Natural Science Foundation of China (No. 31760482); Gansu Provincial Key Research and Development Plan (No. 18YF1NA075).

Disclosure

The authors report no conflicts of interest in this work.

References

1. Polveiro RC, Vidigal PMP, de Oliveira Mendes TA, et al. Distinguishing the milk microbiota of healthy goats and goats diagnosed with subclinical mastitis, clinical mastitis, and gangrenous mastitis. Front Microbiol. 2022;13:918706. doi:10.3389/fmicb.2022.918706

2. El Garch F, Youala M, Simjee S, et al. Antimicrobial susceptibility of nine udder pathogens recovered from bovine clinical mastitis milk in Europe 2015–2016: vetPath results. Vet Microbiol. 2020;245:108644. doi:10.1016/j.vetmic.2020.108644

3. Raplee I, Walker L, Xu L, et al. Emergence of nosocomial associated opportunistic pathogens in the gut microbiome after antibiotic treatment. Antimicrob Resist Infect Control. 2021;10(1):36. doi:10.1186/s13756-021-00903-0

4. Huang T, Zhao K, Zhang Z, et al. DNA vaccination based on pyolysin co-immunized with IL-1β enhances host antibacterial immunity against Trueperella pyogenes infection. Vaccine. 2016;34(30):3469–3477. doi:10.1016/j.vaccine.2016.04.025

5. Acharya KR, Brankston G, Slavic D, Greer AL. Spatio-temporal variation in the prevalence of major mastitis pathogens isolated from bovine milk samples between 2008 and 2017 in Ontario, Canada. Front Vet Sci. 2021;8:742696. doi:10.3389/fvets.2021.742696

6. Ashrafi Tamai I, Mohammadzadeh A, Zahraei Salehi T, et al. Investigation of antimicrobial susceptibility and virulence factor genes in Trueperella pyogenes isolated from clinical mastitis cases of dairy cows. Food Sci Nutr. 2021;9(8):4529–4538. doi:10.1002/fsn3.2431

7. Rezanejad M, Karimi S, Momtaz H. Phenotypic and molecular characterization of antimicrobial resistance in Trueperella pyogenes strains isolated from bovine mastitis and metritis. BMC Microbiol. 2019;19(1):305. doi:10.1186/s12866-019-1630-4

8. Flöck M, Winter P. Diagnostic ultrasonography in cattle with diseases of the mammary gland. Vet J. 2006;171(2):314–321. doi:10.1016/j.tvjl.2004.11.002

9. Ishiyama D, Mizomoto T, Ueda C, et al. Factors affecting the incidence and outcome of Trueperella pyogenes mastitis in cows. J Vet Med Sci. 2017;79(3):626–631. doi:10.1292/jvms.16-0401

10. Amos MR, Healey GD, Goldstone RJ, et al. Differential endometrial cell sensitivity to a cholesterol-dependent cytolysin links Trueperella pyogenes to uterine disease in cattle. Biol Reprod. 2014;90(3):54. doi:10.1095/biolreprod.113.115972

11. Alkasir R, Wang J, Gao J, et al. Properties and antimicrobial susceptibility of Trueperella pyogenes isolated from bovine mastitis in China. Acta Vet Hung. 2016;64(1):1–12. doi:10.1556/004.2016.001

12. Guo Y, Liu Y, Zhang Z, et al. The antibacterial activity and mechanism of action of luteolin against Trueperella pyogenes. Infect Drug Resist. 2020;13:1697–1711. doi:10.2147/idr.S253363

13. Duarte VS, Dias RS, Kropinski AM, et al. Complete genome sequence of vB_EcoM-UFV13, a new bacteriophage able to disrupt Trueperella pyogenes biofilm. Genome Announc. 2016;4(6). doi:10.1128/genomeA.01292-16

14. Torres MDT, Sothiselvam S, Lu TK, de la Fuente-Nunez C. Peptide design principles for antimicrobial applications. J Mol Biol. 2019;431(18):3547–3567. doi:10.1016/j.jmb.2018.12.015

15. Galán-Relaño Á, Gómez-Gascón L, Luque I, et al. Antimicrobial susceptibility and genetic characterization of Trueperella pyogenes isolates from pigs reared under intensive and extensive farming practices. Vet Microbiol. 2019;232:89–95. doi:10.1016/j.vetmic.2019.04.011

16. Tomazi T, Sumnicht M, Tomazi A, et al. Negatively controlled, randomized clinical trial comparing different antimicrobial interventions for treatment of clinical mastitis caused by gram-positive pathogens. J Dairy Sci. 2021;104(3):3364–3385. doi:10.3168/jds.2020-18830

17. Ghaly TM, Geoghegan JL, Tetu SG, Gillings MR. The peril and promise of integrons: beyond antibiotic resistance. Trends Microbiol. 2020;28(6):455–464. doi:10.1016/j.tim.2019.12.002

18. Karthik K, Anbazhagan S, Ananda Chitra M, et al. Comparative genomics of Trueperella pyogenes available in the genome database reveals multidrug resistance genomic islands. J Glob Antimicrob Resist. 2022;31:216–221. doi:10.1016/j.jgar.2022.09.011

19. Hijazin M, Ulbegi-Mohyla H, Alber J, et al. Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. J Dairy Sci. 2011;94(4):1813–1819. doi:10.3168/jds.2010-3678

20. Rogovskyy AS, Lawhon S, Kuczmanski K, et al. Phenotypic and genotypic characteristics of Trueperella pyogenes isolated from ruminants. J Vet Diagn Invest. 2018;30(3):348–353. doi:10.1177/1040638718762479

21. Yang F, Zhang S, Shang X, et al. Short communication: antimicrobial resistance and virulence genes of Enterococcus faecalis isolated from subclinical bovine mastitis cases in China. J Dairy Sci. 2019;102(1):140–144. doi:10.3168/jds.2018-14576

22. Kasimanickam VR, Owen K, Kasimanickam RK. Detection of genes encoding multidrug resistance and biofilm virulence factor in uterine pathogenic bacteria in postpartum dairy cows. Theriogenology. 2016;85(2):173–179. doi:10.1016/j.theriogenology.2015.10.014

23. Liu MC, Wu CM, Liu YC, et al. Identification, susceptibility, and detection of integron-gene cassettes of Arcanobacterium pyogenes in bovine endometritis. J Dairy Sci. 2009;92(8):3659–3666. doi:10.3168/jds.2008-1756

24. Dong WL, Kong LC, Wang Y, et al. Aminoglycoside resistance of Trueperella pyogenes isolated from pigs in China. J Vet Med Sci. 2017;79(11):1836–1839. doi:10.1292/jvms.16-0597

25. Kwiecień E, Stefańska I, Chrobak-Chmiel D, et al. Trueperella pyogenes isolates from livestock and European bison (bison bonasus) as a reservoir of tetracycline resistance determinants. Antibiotics. 2021;10(4). doi:10.3390/antibiotics10040380

26. Kwiecień E, Stefańska I, Chrobak-Chmiel D, et al. New determinants of aminoglycoside resistance and their association with the class 1 integron gene cassettes in Trueperella pyogenes. Int J Mol Sci. 2020;21(12). doi:10.3390/ijms21124230

27. Hall CW, Mah TF. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol Rev. 2017;41(3):276–301. doi:10.1093/femsre/fux010

28. Ji Y, Song L, Zhou Z, et al. vB-ApyS-JF1, the first trueperella pyogenes phage, shows potential as an alternative treatment strategy for Trueperella pyogenes infections. Front Microbiol. 2021;12:736304. doi:10.3389/fmicb.2021.736304

29. Zastempowska E, Lassa H. Genotypic characterization and evaluation of an antibiotic resistance of Trueperella pyogenes (Arcanobacterium pyogenes) isolated from milk of dairy cows with clinical mastitis. Vet Microbiol. 2012;161(1–2):153–158. doi:10.1016/j.vetmic.2012.07.018

30. Ribeiro MG, Risseti RM, Bolaños CA, et al. Trueperella pyogenes multispecies infections in domestic animals: a retrospective study of 144 cases (2002 to 2012). Vet Q. 2015;35(2):82–87. doi:10.1080/01652176.2015.1022667

31. Dong WL, Liu L, Odah KA, et al. Antimicrobial resistance and presence of virulence factor genes in Trueperella pyogenes isolated from pig lungs with pneumonia. Trop Anim Health Prod. 2019;51(7):2099–2103. doi:10.1007/s11250-019-01916-z

32. Billington SJ, Jost BH. Multiple genetic elements carry the tetracycline resistance gene tet(W) in the animal pathogen Arcanobacterium pyogenes. Antimicrob Agents Chemother. 2006;50(11):3580–3587. doi:10.1128/aac.00562-06

33. Jost BH, Field AC, Trinh HT, et al. Tylosin resistance in Arcanobacterium pyogenes is encoded by an erm X determinant. Antimicrob Agents Chemother. 2003;47(11):3519–3524. doi:10.1128/aac.47.11.3519-3524.2003

34. Wüthrich D, Brilhante M, Hausherr A, et al. A novel trimethoprim resistance gene, dfrA36, characterized from Escherichia coli from calves. mSphere. 2019;4(3). doi:10.1128/mSphere.00255-19

35. Jones-Dias D, Manageiro V, Ferreira E, et al. Architecture of class 1, 2, and 3 integrons from gram negative bacteria recovered among fruits and vegetables. Front Microbiol. 2016;7:1400. doi:10.3389/fmicb.2016.01400

36. Odetoyin BW, Labar AS, Lamikanra A, et al. Classes 1 and 2 integrons in faecal Escherichia coli strains isolated from mother-child pairs in Nigeria. PLoS One. 2017;12(8):e0183383. doi:10.1371/journal.pone.0183383

37. Li L, Zhao X. Characterization of the resistance class 1 integrons in Staphylococcus aureus isolates from milk of lactating dairy cattle in Northwestern China. BMC Vet Res. 2018;14(1):59. doi:10.1186/s12917-018-1376-5

38. Zhang Z, Guo Y, Guo Y, et al. Molecular basis for luteolin as a natural TatD DNase inhibitor in Trueperella pyogenes. Int J Mol Sci. 2022;23(15). doi:10.3390/ijms23158374

39. Risseti RM, Zastempowska E, Twarużek M, et al. Virulence markers associated with Trueperella pyogenes infections in livestock and companion animals. Lett Appl Microbiol. 2017;65(2):125–132. doi:10.1111/lam.12757

40. Rzewuska M, Kwiecień E, Chrobak-Chmiel D, et al. Pathogenicity and virulence of Trueperella pyogenes: a review. Int J Mol Sci. 2019;20(11). doi:10.3390/ijms20112737

41. Esmay PA, Billington SJ, Link MA, et al. The Arcanobacterium pyogenes collagen-binding protein, CbpA, promotes adhesion to host cells. Infect Immun. 2003;71(8):4368–4374. doi:10.1128/iai.71.8.4368-4374.2003

42. Silva E, Gaivão M, Leitão S, et al. Genomic characterization of Arcanobacterium pyogenes isolates recovered from the uterus of dairy cows with normal puerperium or clinical metritis. Vet Microbiol. 2008;132(1–2):111–118. doi:10.1016/j.vetmic.2008.04.033

43. Rzewuska M, Stefańska I, Osińska B, et al. Phenotypic characteristics and virulence genotypes of Trueperella (Arcanobacterium) pyogenes strains isolated from European bison (Bison bonasus). Vet Microbiol. 2012;160(1–2):69–76. doi:10.1016/j.vetmic.2012.05.004

44. Nagib S, Rau J, Sammra O, et al. Identification of Trueperella pyogenes isolated from bovine mastitis by Fourier transform infrared spectroscopy. PLoS One. 2014;9(8):e104654. doi:10.1371/journal.pone.0104654

45. Zhang Z, Liang Y, Yu L, et al. TatD DNases contribute to biofilm formation and virulence in Trueperella pyogenes. Front Microbiol. 2021;12:758465. doi:10.3389/fmicb.2021.758465

46. Ashrafi Tamai I, Mohammadzadeh A, Zahraei Salehi T, Mahmoodi P. Genomic characterisation, detection of genes encoding virulence factors and evaluation of antibiotic resistance of Trueperella pyogenes isolated from cattle with clinical metritis. Antonie Van Leeuwenhoek. 2018;111(12):2441–2453. doi:10.1007/s10482-018-1133-6

47. Santos TM, Caixeta LS, Machado VS, et al. Antimicrobial resistance and presence of virulence factor genes in Arcanobacterium pyogenes isolated from the uterus of postpartum dairy cows. Vet Microbiol. 2010;145(1–2):84–89. doi:10.1016/j.vetmic.2010.03.001