Genetic framework sequencing analysis of Candida tropicalis in dairy cow mastitis and study of pathogenicity and drug resistance

Abstract

Candida tropicalis (C. tropicalis) is a zoonotic pathogen that is widespread in the environment and in recent years an increasing number of dairy cows have been infected with the fungus causing mastitis in cows.In this study, 37 milk samples from the udders of cows with clinical mastitis were collected from a dairy farm in Guangxi Province, China, from which C. tropicalis was isolated and identified, and then the isolated fungi were subjected to genome frame map sequencing, genome functional analysis as well as comparative genome analysis of the sequencing results, and combined with the virulence test of the fungi and drug sensitivity test of the fungi determined in infected mice, the resistance genes and pathogenicity of the fungi were Analysis of resistance genes and pathogenicity.Our study results revealed the isolation and characterisation of C. tropicalis from diseased cows, with a genome length of approximately 14.27 Mb. Functional annotation of the genome identified 4068 genes associated with C. tropicalis. The strain exhibited a chemoresistance mutation in the gene cyp51,a virulence-enhancing mutation in the gene VTC4, and mutations in genes linked to drug resistance. Pathogenicity tests demonstrated that C. tropicalis could induce damage to the internal organs of mice, leading to different levels of cyanosis in the abdominal cavity, white necrotic foci on the surface of internal organs, lung hemorrhage, and enlargement of the spleen and thymus.Histological sections also revealed varying degrees of hemorrhage and degenerative changes in the cells of different organs in the mice. Drug sensitivity tests showed that the fungus was highly sensitive to nystatin and ketoconazole, moderately sensitive to amphotericin B, and insensitive to antibiotics such as itraconazole, gentamicin, and penicillin. In conclusion, C. tropicalis isolated from dairy cows in the Guangxi region in this study was pathogenic and resistant to azoles such as itraconazole and fluconazole, and this study provides a theoretical basis for the further screening of novel resistance genes in C. tropicalis, as well as providing a certain reference for the drugs used for the treatment of fungal cow mastitis in this region.

Keywords: C. Tropicalis; Drug resistance; Genome sequence; Mastitis; Pathogenicity.

Fig. 1

Isolation and identification of pathogens a: Fungi cultured on Stachybotrys agar medium; b: Observe the results under oil lens of microscope after gram stain; c: Observe the results under oil lens of microscope after gram stain; d:Identification of fungal fluid by PCR. M: Mark; 1–3: Bacterial liquid amplification result is about 500 bp; e: Sample evolutionary tree

Fig. 2

Function annotation of the C. tropicalis strain genes. A: Statistical Chart of species Annotation in NR Database; B: Results for CAZy carbohydrate-active enzyme annotation; C: KEGG metabolic pathway classification; D: PHI database; E-F: Translocator protein annotation results; G COG function classification; H: GO function classification

Fig. 3

Comparative genomic analysis of the C. tropicalis strain genes. A: Results of two-dimensional collinear analysis. The horizontal axis represents the reference strain, the vertical axis represents the sample strain, the red line represents the forward comparison, and the blue line represents the reverse comparison. B: Results of parallel collinear analysis. The upper and lower axes represent the two genomes, yellow represents the forward strand, blue represents the reverse strand, the height of the filled color in the box indicates the similarity of the comparison, and the complete fill indicates that the similarity is 100%. Pink Collinear means synchronous alignment; blue Translocation means translocation alignment; yellow Inversion means inversion alignment; green Tran + Inver means translocation and inversion alignment. C: Result of InDel statistical. D: Result of SV analysis. The inner circle represents the genome sequence of the sample strain, and the outer circle represents the genome sequence of the reference strain

Fig. 4

Observation on pathological changes of tissues and organs in mice after challenge

Fig. 5

Histopathological observation

Fig. 6

Analysis of drug resistance in C. tropicalisA:Growth curve of C. tropicalis; B:Results of drug sensitivity test.(The No.1 and No.6 tablets are nystatin; 2, 9 tablets for penicillin; 3, 10 tablets for ciprofloxacin; The No.4 and No.11 tablet are gentamicin; Tablets 5, 7, 13 and 14 are amphotericin B; The No.8 pap sheet is ketoconazole; Slip number 12 was itraconazole)

Fig. 7

Results of a constant drug gradient sensitivity test A-D. Sensitivity of C. tropicalis to various drug concentration gradients. Ketoconazole, fluconazole, amphotericin B and itraconazole are shown in this order. The horizontal coordinate is the drug concentration(µg/ml) and the vertical coordinate is the OD₆₀₀