Cow mastitis is a serious disease in dairy industry worldwide, which is mainly caused by pathogenic bacteria infection caused by inflammation of cow breast, resulting in the decline of milk quality and quantity[23]. In recent years, there are more and more reports about the isolation of fungi from cow mastitis, and the isolation rate of fungi is higher and higher[24]. Antibiotics are the most effective treatment for bovine mastitis, but with the abuse of antibiotics leading to the emergence of drug resistance of pathogenic bacteria, the therapeutic effect is gradually reduced[5]. In our trials, we isolated the fungus from the milk of diseased cows, identified it by a series of methods and the results indicated that it was C. tropicalis. The fungus is an opportunistic yeast pathogen that causes mastitis infections that are well known in animals, and the use and abuse of antimicrobial agents, as well as the treatment of contaminated antibiotic solutions, and catheters or other materials in contact with the mammary glands also facilitate colonization in the breast of the cow[6, 25]. At present, the research on C. tropicalis mainly focuses on human diseases, and the research on bovine mastitis is rare. Moreover, most of them describe the clinical hazards and drug resistance, and the research on its genome is even less. Therefore, understanding the genomic structure and molecular biological function of C. tropicalis is of great importance to our study of fungal-type diseases of bovine mastitis.
Whole genome sequencing enables accurate identification of pathogens and also provides a comprehensive characterisation of AMR markers, plasmid replicon types and virulence factors, and the genomic information obtained from sequencing can also be used for WGS predictive antimicrobial susceptibility testing (WGS-AST)[26].We sequenced the whole genome of the sample strain and obtained a genomic sequence measuring 14.27Mb. Compared with the reference strain, the total genome number of the sample strain was more, totaling8,168, and the sample strain predicted 166 tRNAs, indicating that there might be differences in protein synthesis and structure between the two, but the specific need for further research. Studies have shown that in higher animals, there are many different genomic rearrangements associated with sequence duplication and functionalization, all of which imperceptibly alter transcriptional activity and thus affect basic biological functions such as development and differentiation[27]. In our study, the repetitive sequences of the sample strains of the sample strains were 209,319 bp, accounting for 1.4659% of the genome. A large number of repetitive sequences mean that the genome is constantly self-replicating, which is an inevitable process of evolution. In addition, repetitive sequences protect certain structure-coding genes from damage. In addition, comparative genomic analysis showed that there were more translocations between the two, and the translocation was likely to cause structural changes of genetic material.
Through the functional prediction of the genome of the sample strain by each database, we can find that these genes are prominent in cellular processes and metabolic processes. At the same time, through comparison with the PHI database, we found that the gene cyp51 underwent chemical resistance mutation, and the gene VTC4 underwent virulence-enhancing mutation. Cyp51 proteins, as a member of cytochrome P450 family, are important targets for the action of antifungal drugs. When CYP51 is mutated, it may affect the action mechanism of antifungal drugs in the body[28]. However, whether the Cyp51 mutation is associated with the increased drug resistance of C. tropicalis needs further investigation. In yeast, polyPs are involved in the control of the cell cycle, stress response, and virulence, while Vtc4 is the main enzyme involved in the synthesis of polyPs in fungi, suggesting that Vtc4 is involved in the alteration of fungal virulence mechanisms[29]. In addition, the structural variations of SNP, InDel and SV were identified by comparing with the reference genome. The results showed that the coding region of the sample strain was rich in SNP, including drug-resistant genes CDR1, CDR2, CDR3, CDR4 and ABC family proteins. At the same time, a large number of InDel are located in the coding region, which has a direct impact on the protein produced by the coding gene, but whether it has an impact on the pathogenicity and drug resistance of the strain needs further study.
Candida tropicalis has been widely considered the second most virulent Candida species, only preceded by Candida albicans[30].In the pathogenicity test, the isolates could cause mouse death and different degrees of damage to the mouse liver, kidney, lung and thymus. It has been reported in the literature that the infection rate of C. tropicalis is higher than that of other Candida species, and C. tropicalis spreads faster in the body than other Candida species, with a higher mortality rate[31, 32].In the interaction between pathogen and host, a mutation site with enhanced virulence was found in the gene VTC4, but whether the mutation of VTC4 can enhance the pathogenicity of C. tropicalis needs further study.VTC4 is the main enzyme that performs polyP synthesis in fungi, which is a part of the Vacuole Transporter Chaperone (VTC) complex, which plays an important role in vacuolar membrane fusion and has physical relations with vacuolar H+-ATPase (V-ATPase),influencing vacuolar H + uptake[33, 34].The virulence-enhancing mutation of VTC4 indicated that the stress resistance of the strain was enhanced, but the mechanism between them required further study.
It has been reported that compared with Candida albicans, Candida tropicalis can produce more biofilms which is highly adherent to epithelial and endothelial cells and be more resistant to antifungal drugs[30, 35] .In studies recently presented by Sasani, C. tropicalis strains isolated from patients with candidemia and characterized by increased metabolic activity and high biofilm mass were related to higher mortality rates, and biofilms formed by these strains showed increased resistance to azole drugs[36]. Our susceptibility results showed that C. tropicalis isolated in the test was not sensitive to itraconazole, but sensitive to ketoconazole. Also in the drug gradient sensitivity test, when the concentration of ketoconazole reached 8 µg/ml, the inhibitory effect on C. tropicalis suddenly increased, while the other three drugs did not change significantly. The results showed that C. tropicalis developed resistance to azole drugs, which may be related to long-term use of drugs, or to the mutation or overexpression of drug-resistant genes related to C. tropicalis. At the same time, through the detection of drug resistance genes of the samples, we found that the sample strains had azole drug-related resistance genes CDR1, CDR2, CDR3, CDR4 and ABC protein family, and the SNP site mutation sense existed in the comparison of these genes with the reference sequence, and the mutation could change the type of amino acids, which might cause changes in protein expression of the resistance genes, thereby enhancing the resistance of the strains to azole drugs. Studies have shown the predominant mechanism of azole resistance Candida species is mutation/ overexpression of different genes[37, 38]. The drug inducible overexpression of transporter genes like the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) can cause active efflux of cellular azole antifungal drugs thereby contributing to antifungal resistance [37–39]. Different drug efflux transporters (CDR and MDR) are a well-documented mechanism of resistance in C. tropicalis [40]. In our study, CDR and ABC protein family were detected, which indicated that the isolated strains had drug resistance, but the specific mechanism needs to be further tested by detecting the expression of drug resistance genes and proteins.
Mastitis of dairy cattle is a major problem puzzling the dairy industry. How to reduce the incidence of mastitis and establish a more effective treatment are the most important issues. In this study, we isolated and identified C. tropicalis, and conducted pathogenicity test, drug sensitivity test, and whole genome sequencing on it, which will help us to better understand the biological characteristics of C. tropicalis and provide ideas for the treatment of fungal bovine mastitis. For the treatment of mastitis, we should first identify the cause of the disease, targeted medication, prevention and treatment of antibiotic abuse and unreasonable use, or the use of traditional Chinese medicine therapy, the use of Chinese herbal medicine for treatment. In addition, the total ban on the addition of antibiotics in feed may have a certain effect on reducing the yeast infection in dairy cow mastitis.