Screening Bacterial Persisters From Clinical Microbiological Samples And Studying The Persistence Mechanisms Of Klebsiella Pneumoniae

Since the use of antibiotics, it has brought a great contribution to mankind in defensing the bactericidal infection. However, the following problems of drug resistance, have brought the global anti-infection therapy a tremendous challenge. By this time, with the discovery of more in-depth studies of the mechanism of bacterial resistance, researchers are now increasingly concerned about a strange drug case, which featured with the pathogen phenotypic resistance. This phenotypic resistance does not involve mutations in the bacterial genome. But in the condition of high concentrations of antibiotics, there is still a small fraction of the bacteria surviving through antibiotic pressure. This fraction of bacteria stop the growth and proliferation and keep in a stationary state. In fact, this phenomenon has already been discovered in the early discovery of antibiotics. in 1944, when Joseph Bigger was studying the pharmacodynamic of penicillin, he first described the terms of “persister” and “bacterial persistence” phenomenon. When adding penicillin into the growing state S. aureus, he found that a small part of bacteria still survive, but these bacterias are not mutants that resistant to antibiotics. Bigger speculated that when facing the antibiotics stress, persister cells can escape killing because they are in a dormant state and not to secession growth, which has the overall down regulation of cell metabolism. Therefore, this part can avoid the antibiotics killing. Because the ratio of persister bacteria in bacterial population is very low, the research of these cells has been ignored for a long time. Not until the 1980 s, researchers have achieved a little progress in isolating and culturing the persisters. The current national scientists have successfully isolated and cultured E. coli, Mycobacterium tuberculosis, Bacillus natto bacteria from clinical samples. There are also some reports about fungi persistence, with some phenomena of Candida albicans. All these studies help the bacterial persistence back to the public view. Especially due to the challenge of chronic infection, antibiotics failure of controlling infection, clinical drug resistance, we have urgent need to confront this old problem with new studies.In this study, through analyzing the clinical microbiological data, we screened the chronic infection caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. By the specific growth curves under antibiotic stress of persistent bacteria, we eventually isolated 4 strains of persistent Escherichia coli, 23 strains of persistent Klebsiella pneumonia, 8 strains of persistent Pseudomonas aeruginosa, and 6 persistent Staphylococcus aureus strains.Analysis of patients with chronic bacterial persisters infection. In this part we concluded some characteristics of bacterial persisters, as follows: persistent bacteria is multi-drug tolerant, but different species hold different tolerant levels on different antibiotics, in which Escherichia coli holds the lowest persistent level to cefotaxime, Klebsiella pneumoniae to ciprofloxacin, Pseudomonas aeruginosa to levofloxacin, and Staphylococcus aureus to imipenem; persistent bacteria can survive in the acidic, alkaline and nutritional deficiencies conditions; daughter persisters can recovery growth in the general media. But after added with antibiotic, the growth curve of the daughter generation is consistent with the parent. Thus, persistence is a self-protection strategy for the overall population. Once facing some lethal factors, most of the population would be killed, while there are still a fraction of bacterias survive and make sure the genomic information of the population reserved and the entire colony would recovery.Using the transposon mutagenesis method to research the persistence-related genes in Klebsiella pneumoniae strains. After reviewing the published literature results, our group found that there has no reports about the mechanism of Klebsiella pneumonia persistence. So in this study we selected a special persistent strain of Klebsiella pneumonia and constructed a mutant library using transposon-induced mutagenesis method. The library contained 3282 mutants, and were screened to search a specific mutant which totally lost the persistence characteristics. Then we located the persistence-related genes by inverse PCR and other methods, and eventually determined gene NC₀16845.1. Gene NC₀16845.1 encodes phospholipase D, which takes part in the metabolism of phospholipase in bacteria. Thus we speculate that, by regulating the metabolism, bacteria alters membrane permeability and surface charge, making itself survive through the killing of antibiotics, acid, alkali and other lethal factors.