Antibiotic Resistance Profiles and Genomic Analysis of Endophytic Bacteria Isolates from Wild Edible Fungi in Yunnan

Abstract

The use of antibiotics has led to the emergence of antibiotic resistance, posing significant challenges in the prevention, control, and treatment of microbial diseases, while threatening public health, the environment, and food safety. In this study, the antibiotic resistance phenotypes and genotypes of 56 endophytic bacteria isolates from three species of wild edible fungi in Yunnan were analyzed using the Kirby-Bauer disk diffusion method and PCR amplification. The results revealed that all isolates were sensitive to ofloxacin, but resistance was observed against 17 other antibiotics. Specifically, 55, 53, and 51 isolates exhibited resistance to amoxicillin, penicillin, and vancomycin, respectively. Antibiotic resistance gene (ARG) detection indicated that the sulfonamide sul1 gene had the highest detection rate (53.57%). Excluding the ARG that was not detected, the lowest detection rates were the sulfonamide sul2 and sul3 genes, both at 1.79%. Among six tetracycline resistance genes, only tetK and tetM were detected. For β-lactam antibiotics, blaTEM, blaVIM, and blaSHV genes were present, while blaOXA was absent. In aminoglycoside resistance genes, aadB was not detected, while detection rates for aac(3')-IIa, acrB, and aadA1 were 3.57%, 1.79%, and 37.5%, respectively. The chloramphenicol Cat gene was detected at a rate of 14.29%, whereas floR was absent. For polypeptide resistance, VanC was detected at 3.57%, with EmgrB not detected. All three quinolone genes were detected, with detection rates of 8.92% for GyrA, 39.29% for GyrB, and 37.5% for ParC. Through phylogenetic analysis, 12 isolates that are closely related to ten common foodborne pathogenic bacteria were further selected for whole-genome sequencing and assembly. Gene annotations revealed that each isolate contained more than 15 ARGs and over 30 virulence factors. Notably, the detection rate of antibiotic resistance phenotypes was higher than that of genotypes, highlighting the importance of studying phenotypic antibiotic resistance that lacks identifiable ARGs. This study enriches the research on endophytes in wild edible fungi and provides new data for microbial ecology and antibiotic resistance research. It also offers critical insights for monitoring microbial antibiotic resistance in wild edible fungi and potentially other food sources, contributing to more effective strategies for ecological protection, sustainable agricultural development, and public health security.

Keywords: ARG; antibiotics resistance; endophytic bacteria; foodborne pathogens; genome assembly; wild edible fungi.

Figure 1

DNA extraction and 16S rRNA amplification of isolates. (A) Electrophoretic map of genomic DNA of partial isolates. M: DNA marker DL2000; lanes 1–16: genomic DNA of 16 isolates. (B) Electrophoretic map of 16S rRNA amplification products of partial isolates. M: DNA marker DL2000; lane 0: negative control; lanes 1–16: 16S rRNA amplification products of partial isolates.

Figure 2

Phylogenetic tree among solates and common foodborne pathogens (A) Relationship between endophytic isolates of Cantharellus cibarius and foodborne pathogens. (B) Relationship between endophytic isolates of Lactarius deliciosus and foodborne pathogens. (C) Relationship between endophytic isolates of Russul avirescens and foodborne pathogens.

Figure 3

Distribution of antibiotics resistance phenotype of 56 isolates. (A) Antibiotics types; (B) antibiotics category.

Figure 4

PCR results of some resistant genes. M: DL2000 marker; 0: negative control; (A) partial electrophoresis of tetK gene; (B) partial electrophoresis of tetM gene; (C) partial electrophoresis of blaTEM gene; (D) partial electrophoresis of bla-vim gene; (E) partial electrophoresis of blaSHV gene; (F) partial electrophoresis of sul1 gene; (G) partial electrophoresis of sul2 gene; (H) partial electrophoresis of sul3 gene.

Figure 5

PCR results of some resistant genes. M: DL2000 marker; 0: negative control; (A) partial electrophoresis of aac(3′)—IIa gene; (B) partial electrophoresis of acrB gene; (C) partial electrophoresis of aadA1 gene; (D) partial electrophoresis of Cat gene; (E) partial electrophoresis of GyrA gene; (F) partial electrophoresis of GyrB gene; (G) partial electrophoresis of ParC gene; (H) partial electrophoresis of VanC gene.

Figure 6

Multi-antibiotic resistance numbers of 56 isolates.