Novel Method For Estimating Bactericidal Dynamics Of Antimicrobial Agents And Distinguishing Bacterial Tolerance From Resistance To Antibiotics

Antibiotic resistance is a great challenge to global medical safety that we must face and overcome. The most research work of resistance and new antibiotics discovery focused on the targets resulted by resistance mutation or the new targets, which never eradicated the antibiotic resistance from scale to degree; however, the containment in different ways to overcome resistance is attracting more and more attention (1). Historically the confusion of resistance — resulted from gene mutation, and tolerance — caused by global physiological response of cells, is not clarified because they are equivalent sources for phenotype variation of antibiotic susceptivity and the routine antibiotic susceptivity tests are unable to distinguish them effectively (2-4). Focusing on this difficult and usually neglected key issue, we have successfully established concentration-killing curve (CKC) method to selectively estimate bacterial tolerance, and further discussed the variation of antibiotic tolerance of E.coli population in different physiological states. Finally, several strategies are validated to overcome resistance. The three innovative research results acquired in the dissertation showed that the distinction between resistance and tolerance would be of great value in rational use of antibiotics and decrease of resistance occurrence and spread.â… . Concentration-killing curve method was established to selectively estimate bacterial tolerance and provided novel valid parameters in antibiotic pharmaco-dynamics.The interaction of bacterial population and antibiotics is a dynamic process. Antibiotic susceptivity test, based on the principle of drug dilution or diffusion, is the main instrument in antibiotic pharmacodynamics, in which the MBC (Minimum bactericidal concentration) is the accepted standard parameter for threshold point of antibiotic concentration. However, MBC is unable to focus on the exact concentration point of entire eradication of the tested bacterial population. The common modals such as Dose-response curve, E_max are used to give a complete overview of the bactericidal dynamic course versus drug concentration, but the tested inoculum is always regarded as a uniform and stable bacterial population, ignoring the heterogeneity of individual heredity and physiology, say nothing of distinguishing resistant strain from tolerant strain (5,6). Based on the equation analysis of bacterial growth dynamics and pharmacodynamics, concentration-killing curve (CKC) method is established to describe the dynamic function between the drug concentration and bactericidal potency.When a susceptive population of 800-1000 bacteria (N₀), much less than the base of random spontaneous mutation rate of 10^-8, was selected as test sample, the probability of resistant strain preexisted in it was too small to occur. After the sampled population wasinoculated onto the Luria-Bertani plates containing a series concentration of antibiotic and incubated at 37°C for 24h, the persistent colonies of the same size of inoculum, to the utmost of genetic homogeneity, exposed to different drug concentration x were enumerated as N. A plot ofN versus x generates a sigmoidal curve (Fig. 1).With certain concentrations of antibiotics in the agar medium, the more effects from the antibiotics from which each cell suffers, the higher the rate of mortality is, the less the1 dNrate of viability is, and the less N is; and this reflects the specific viability rate,---------,N dxN which would be negatively correlated with the instantaneous mortality rate, (1------).1 dN N.For given values ofNand x, T7'⁷ -^r\\^<sup>), where the constant r >0. T77 rU rrND By integration, N =-------frIt is straightforward to define BC$q as the antibiotic concentration that provokes a bactericidal response equivalent to half of A'o. Thus, the constant C is equal to rBCso, and so the function of N and x was proposed to quantify the concentration-killing curve and fitted with the modal:N0/2,x = J N 0 ------J No -\,x = 5Q, = BCK -h\(N0 -l)/rN0/(l+e-HiC?),x = 0 N0/(l+erBC?),x = 2BC5OAs shown in figure 1, the sigmoidal CKC is point symmetrical towards its inflexiono, No/2), monotonously decreasing between the asymptotes of N=No and #=0, and the bactericidal rate versus concentration dN/dx accord with normal distribution. The tangent slope on inflexion is - rAV4. When A'o was limited, the variate r could estimate the curve slope, viz. bactericidal intensity of an antibiotic. The median bactericidal concentration BC$o represents the average of susceptiblity (or scilicet tolerance), and BC\, BCy) is drug concentration where only one bacterium persisted or be killed, respectively, therefore, the susceptiblity threshold is defined as (BC99, BC\) to selectively estimate bacterial tolerance. The entire bactericidal concentration BC\ is more valid, accurate, and reproducible for estimating bactericidal effect than was the endpoint MBC method as well as the traditional Dose-response sigmoidal curve and ï¿¡max model. CKC is suitable to measure bactericidal potency of both antibiotics such as gentamicin, penicillin, enoxacin, and Chinese medicine such as sanhuang and Coptis Root. In conclusion, CKC was an effective method for selectively estimating bacterial tolerance and provided novel validparameters in antibiotic pharmacodynamics. The main research result gained affirmative response from researcher abroad and was published in Antimicrobial Agents and Chemotherapy, 2004.48(10):3884-3891 (SCI. IF 4.2) and The American Journal of Chinese Medicine, 2005. (SCI. IF 0.7, Accepted) (7,8).II. The novel growth curve phases of population in batch culture was proposed to distinguish its physiological states and the variety of antibiotic susceptibility of E.coli population in different physiological states was compared by CKC method (9).Physiology and genetics of microbiology developed by turns all the 20th century. It is almost the same time at the end of 19th century that both the inhibition between microorganisms and drug-fast of bacteria to dye were discovered, however, this desensitization (drug-fast) was usually imputed to physiological adaptation or acclimatization, because the forepart of microbiology research was badly limited in morphologies and physiology. Along with the discovery of the inherited resistance and determination of genetic material, especially the world-shaking proposal of the double helix structure of DNA, the variation was summed so more up to genetics that most researchers believe that the resistance could be explained and even resolved by the identification of resistant mutation gene. In act, gene is relatively steady while protein is metabolic and mutative momently, and the latter maintains the former with high fidelity while makes microevolution up to genome slowly. The adaptive variation is the course of gene-environment interaction mediated by protein, including two kinds: spontaneous or induced sequence mutation of genome (involving chromosome, plasmids and transposable elements)—resistance; expression of genome regulated by two-component signal transduction system of cell—physiological tolerance, also named as persistence reported in Science. Persistence is linked to preexisting heterogeneity in bacterial populations because phenotypic switching occurred between normally growing cells and persister cells having reduced growth rate (3,4).In microbial physiology, the growth curve phases of population in batch culture is usually divided according to Logistic equation, which is only a rough drawing of population growth dynamics (10). In the dissertation in order to be in conformity with the corresponding physiological states of the batch culture population, instantaneous change rate dN/dt was proposed as index to distinguish the growth curve of population in batch culture into five phases: the first one terms as lag phase in which dN/dt —0, then accelerative exponential phase in which dN/dt increased from 0, decelerative exponential phase in which dN/dt declined, stationary phase in which dN/dt returned towards 0 and at last decline phase in which dN/dt <0 (Figure 2).The susceptibility of bacterial cells to antibiotics lies on the regulation and catalytic activity of enzyme related with growth and division and the repair of DNA damage that is different in each growth phase. The heterogenous individuals in the population even sampled at the same time point present various susceptibility to an antibiotic as normal distribution. CKC measurement revealed the course and degree of antibiotic tolerance ofN a susceptive population in a same sample with equation N = -—r|r"./,,ti) : the medianbactericidal concentration BC$o represented the mean value of susceptibility, and the2 bactericidal span BC^_W = BCi-BC99-—\nN0 indicated the bactericidal capacity frombeginning to maximum of an antibiotic to a given bacterial population, i.e. the variety of a population susceptibility.The proportion of cells at each physiological state in the population is changing with growth curve phases, and the individual heterogeneity of the populations will certainly result in variation of antibiotic tolerance, therefore the median bactericidal concentration BCso and bactericidal span BCuw are different with growth curve phases. Generally the enzyme regulation and the DNA repair are more effective in multiplication phase than lag or stationary phase, thus the population susceptibility in multiplication phase is weaker (Fig. 3, Fig. 4). The antibiotic potency is the combined result of cell physiological state and bactericidal mechanism of the antibiotics.The exact delimitation of growth curve and application of CKC measurement help further understanding of the dynamic interaction of drug and bacteria in different condition and help us rationally select and use antibiotics.III. Probed into the inhibition mechanism and resistance evolution of Chinese medicine, and validated the combination effect of antibiotics and Chinese medicine on mixed infection model involving multi-resistant Staphyococcus aureus (MRSA).From the point of view of adaptive evolution, there are every kind of resistant strains in the animal body and the environment that keep their balance and steady between each other though bacteriostastic activity or competition of nutrition and space (11). For clinic example, infections occur in human or animal are frequently caused by mixed bacteria population, involving various species, specie of various pathogenicity or susceptibility. Only in the selective pressure of antibiotics, the resistant strains, maybe in possession of more powerful pathogenicity, could be free to multiply exponentially and inactivate the antibiotics in regular use. Aiming at this impolitic administration of antibiotics, containment to resistance was validated here:1. Chinese medicine is applied for thousands years with no visible resistance depending on the inhibition model of mixed-component versus multi-target. It approved in the dissertation that sanhuang, composed of Rhizoma coptidis, Rhizoma rhubarb, Cortex phellodendri sanhuang, could kill Ecoli of various susceptibilities and MRSA without selection.2. Based on antibacterial characteristics of antibiotics and Chinese medicine, the combinational inhibition was tested. As mentioned before, the physiological state of the Ecoli population was altered by sanhuang in certain concentration, then the susceptibility to gentamicin was enhanced, whereas gentamicin killed most of the sensitive cells of the population, then sanhuang inhibited the persister strains or possible resistant cells.3. Besides pure population, a mixed infection model of "sensitive E.coli + MRSA" was designed as table 1. After the most sensitive E.coli cells were killed by enoxacin, the tolerant E.coli cells and resistant MRSA persisted and multiplied rapidly so as to inactivate enoxacin. This is the consequence of routine chemotherapy. However, when combined with sanhuang, the persisted E.coli and MRSA were cut down greatly until eradicated. To eradicate the mixed population, it need at least 50ug/ml enoxacin or 62.5mg/ml sanhuang alone, whereas only 0.8ug/ml enoxacin and 62.5mg/ml sanhuang when combined (Figure 5). The results provided experimental support for combinational therapy of antibiotic and Chinese medicine.Antibiotic resistance is a global disaster for medical safety, which need deep understanding. The coexisting of animal, plant and their commensal microorganisms is necessary for each existing of them, which is the result of long term co-evolution, so we never attempt to eradicate pathogen with an "almighty" drug. The various spontaneous resistant mutation of bacteria population is doomed to occur at any moment and space, therefore, at the utmost the "gene strategy" explained part of resistance mechanism, and new antibiotic discovery is only an important way rather than surefire strategy. The rational use of antibiotics is vital because two factors are essential for resistant gene to transform into dominance: the persister base of tolerance cells and selective killing of antibiotics.In the dissertation, we have made primary research in distinguishing the resistance mutation and physiological tolerance. The research of the transition from tolerance to resistance will make epochal progress in pharmacology, genetics, physiology, ecology, genomics and proteomics, and even evolutionism.