关键词: 多粘菌素, 质粒, 光催化, mcr-1, 活的不可培养状态

Abstract:

Antibiotics emerged in the 20th century. They are crucial because they can kill bacteria, treat many life-threatening diseases, save countless lives, and revolutionize modern medicine. The mass production of antibiotics and their use in clinical practice have significantly improved the quality of life and increased life expectancy of patients. However, antibiotic resistance has emerged as a severe global threat because of the widespread and often overuse of antibiotics in various fields, not just in human medicine but also in livestock farming, which has made bacteria increasingly resistant to these once powerful antibiotics. Polymyxins, long seen as the “last line of defense” against multidrug-resistant gram-negative bacteria because of their potent bactericidal properties, have faced a setback with the appearance of the plasmid-mediated polymyxin resistance gene (mcr-1) in recent years, which significantly reduces their therapeutic efficacy. This antibiotic resistance is widely spread across different bacterial populations in various ecological niches worldwide, including humans, animals, and the environment, thereby undermining the powerful antibacterial effects of polymyxin. Therefore, clarifying the transmission characteristics of plasmid-mediated polymyxins in the environment is crucial for effectively countering this threat. The viable but nonculturable (VBNC) dormant state is a self-protection mechanism initiated by microorganisms in response to harsh external environmental pressures. Although bacteria in the VBNC state cannot grow colonies visible to the naked eye on conventional media, they still maintain certain metabolic activities. Moreover, once the environmental conditions become suitable, these bacteria can regain their vitality and start to grow. This VBNC state is crucial as it enables bacteria to survive in adverse circumstances for extended periods. Research indicates that diverse environmental factors, such as extreme temperatures, nutrient scarcity, and high salinity, can trigger the entry of bacteria into the VBNC state. In this study, we comprehensively investigated Salmonella strains carrying two distinct plasmid types, IncI2 and IncHI2. Photocatalytic stimulation has been ingeniously employed as a technical approach to closely simulate the stress inflicted on bacteria by light and mineral interfaces in the external environment. Subsequently, bacteria carrying different plasmid types mediated by mcr-1 were effectively induced into the VBNC state, with a special focus on exploring the potential underlying mechanisms and their long-term impacts. The risk of mcr-1 proliferation after bacteria enter the VBNC state under external environmental stress was explored. Strains mediated by mcr-1 were subjected to photocatalysis in a simulated environment. Samples were collected at different time points in a time series, diluted, and spread on culture media to determine the number of culturable bacteria. Furthermore, the impact of the external environment on the culturability of mcr-1-positive bacteria was evaluated. Analysis of the number of culturable bacteria showed that the number of cultured bacteria began to decrease at 6 h of stimulation. At 9 h of stimulation, the number of culturable strains carrying the IncHI2 plasmid (10³ CFU∙mL⁻¹) was lower than that of strains carrying the IncHI2 plasmid (10⁵ CFU∙mL⁻¹). By comparing the entire stimulation process under the same stimulation time, it was found that the number of culturable strains carrying the IncHI2 plasmid was lower than that of strains carrying the IncHI2 plasmid, indicating that the bacteria carrying the IncI2 plasmid were more resistant to photocatalytic oxidation stimulation. When the bacteria were stimulated for 11 h, no colonies were observed on the agar medium. However, live/dead staining experiments revealed that the bacteria remained active under photocatalytic stimulation. The combined results of the culturable bacterial count and live/dead staining experiments clearly demonstrated that the bacteria were in the VBNC state. The results showed that after 11 h of photocatalytic stimulation, mcr-1-positive bacteria completely entered the VBNC state. In addition, the bacteria stimulated by light at 0 h (wild type) and 11 h (VBNC state) were collected to analyze the effect of photocatalysis on bacterial phenotype when bacteria enter the VBNC state, and the characterization included the phenotypic changes of bacteria in the VBNC state on growth rate, stability of mcr-1 plasmid in bacteria, and changes in bacterial flagellar motility. The results showed that after stimulation, the growth curve of the bacteria was a straight line parallel to the X-axis (bacterial culturability was completely lost), resulting in a line where plasmid stability was close to 0. In addition, plasmid stability and flagellar motility of mcr-1 bacteria decreased simultaneously. By comparing the flagellar motility of IncI2-carrying plasmid bacteria and IncHI2-carrying plasmid bacteria, it was found that in both the WT state and VBNC state, the flagellar motility of IncI2-carrying plasmid bacteria was stronger, but photocatalysis had a greater impact on the flagellar motility of IncI2-carrying plasmid bacteria. To thoroughly explore the mechanism of different plasmid types of bacteria, such as analyzing the influence of plasmid size and copy number, entering the VBNC state mediated by mcr-1, the differentially expressed genes of mcr-1 bacteria in the VBNC state and WT state were further quantitatively analyzed resistance gene (mcr-1) and reactive oxygen species (ROS) related genes (oxyR, rpoS), SOS response genes (lexA, umuD) using quantitative polymerase chain reaction (qPCR). Molecular mechanism studies further showed that after entering the VBNC state, the expression of the mcr-1 gene was upregulated, indicating that in the VBNC state, the bacteria were more resistant to polymyxin. When comparing ROS- and SOS-related genes, detailed analysis revealed that mcr-1 bacteria in the VBNC state were upregulated to various degrees compared with WT state bacteria. Notably, the degree of upregulation of bacteria carrying the IncI2 plasmid was smaller than that of bacteria carrying the IncHI2 plasmid, clearly indicating that photocatalytic stimulation, through its specific reactive oxygen species generation and interaction mechanisms, caused greater damage to bacteria carrying the IncHI2 plasmid. This study clarifies the changes in the VBNC state of diverse plasmid-mediated mcr-1 bacteria when stimulated by photocatalysis. This study offers novel insights into the risk of antibiotic resistance transmission in mcr-1-mediated dormant-state bacteria, enhancing our understanding of this complex issue.

Key words: polymyxin, plasmid, photocatalysis, mcr-1, viable but nonculturable state