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Updated: Aug 20, 2025
Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes
Published on: March 3, 2023
Prevalence, antimicrobial susceptibility, and antibiotic resistance gene transfer of Bacillus strains isolated from
Zhengyuan Zhai1, Chang Cui2, Xueli Li2
1Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and Beijing Municipality, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083 China; Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083 China.
Abstract:
Pasteurization is carried out in dairy industries to kill harmful bacteria present in raw milk. However, endospore-forming bacteria, such as Bacillus, cannot be completely eliminated by pasteurization. In this study, a total of 114 Bacillus strains were isolated from 133 pasteurized milk samples. Antibiotic susceptibility tests showed that the percentage of Bacillus with intrinsic resistance to ampicillin and penicillin were 80 and 86%, respectively. Meanwhile, some Bacillus isolates had acquired resistance, including trimethoprim-sulfamethoxazole resistance (10 isolates), clindamycin resistance (8 isolates), erythromycin resistance (2 isolates), and tetracycline resistance (1 isolate). To further locate these acquired resistance genes, the plasmids were investigated in these 16 Bacillus strains. The plasmid profile indicated that Bacillus cereus BA008, BA117, and BA119 harbored plasmids, respectively. Subsequently, the Illumina Novaseq PE150 was applied for the genomic and plasmid DNA sequencing. Notably, the gene tetL encoding tetracycline efflux protein was found to be located on plasmid pBC46-TL of B. cereus BA117. In vitro conjugative transfer indicated that pBC46-TL can be transferred into Bacillus invictae BA142, Bacillus safensis BA143, and Bacillus licheniformis BA130. The frequencies were of 1.5 × 10-7 to 1.7 × 10-5 transconjugants per donor cells. Therefore, Bacillus strains with acquired antibiotic resistance may represent a potential risk for the spread of antibiotic resistance between Bacillus and other clinical pathogens via horizontal gene transfer.
Insights
Pasteurization doesn't eliminate all Bacillus endospores in milk. Acquired antibiotic resistance genes on plasmids, like tetracycline resistance, can spread between Bacillus strains, posing a public health risk.
Area of Science:
- Microbiology
- Food Safety
- Antimicrobial Resistance
Background:
- Pasteurization is standard for dairy, but Bacillus endospores survive.
- Bacillus species in pasteurized milk can exhibit intrinsic and acquired antibiotic resistance.
- Understanding the mechanisms of acquired resistance is crucial for food safety.
Purpose of the Study:
- To isolate and characterize Bacillus strains from pasteurized milk.
- To investigate the presence and location of acquired antibiotic resistance genes.
- To assess the potential for horizontal gene transfer of these resistance genes.
Main Methods:
- Isolation of 114 Bacillus strains from 133 pasteurized milk samples.
- Antibiotic susceptibility testing and plasmid profiling.
- Whole-genome and plasmid DNA sequencing (Illumina Novaseq PE150).
- In vitro conjugative transfer experiments.
Main Results:
- High intrinsic resistance to ampicillin (80%) and penicillin (86%) observed.
- Acquired resistance detected for trimethoprim-sulfamethoxazole, clindamycin, erythromycin, and tetracycline.
- The tetracycline resistance gene tetL was identified on plasmid pBC46-TL in Bacillus cereus BA117.
- Plasmid pBC46-TL was successfully transferred to other Bacillus species via conjugation.
Conclusions:
- Surviving Bacillus strains in pasteurized milk harbor acquired antibiotic resistance genes.
- Plasmids facilitate the spread of antibiotic resistance within Bacillus populations.
- Horizontal gene transfer of antibiotic resistance from Bacillus poses a potential risk to public health.
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