Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration （MIC） of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick＇s laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antirnicrobial agents, and guide clinical treatment.

Synergistic effects of cellobiose dehydrogenase and manganese-dependent peroxidases during lignin degradation

The synergistic effects of cellobiose dehydro-genase (CDH) and manganese-dependent peroxidases (MnP) on the degradation of kraft pulp cellulolytic enzyme lignin (CEL) were investigated. Addition of CDH significantly increased the amount of water-soluble products reduced from CEL by MnP. CDH facilitated the reduction of the contents of methoxyl, carboxyl, phenolic hydroxyl and total hydroxyl groups of CEL by MnP. 1H-NMR analysis showed that addition of CDH also decreased further the amount of protons of CEL degraded by MnP. The results proved for the first time that CDH could promote degradation of lignin by MnP and suggest that CDH could not only promote degradation of cellulose but also is an important part of the lignin biodeg-radation system.

Discussion on research methods of bacterial resistant mutation mechanisms under selective culture——uncertainty analysis of data from the Luria-Delbrück fluctuation experiment

Whether bacterial drug-resistance is drug-induced or results from rapid propagation of random spontaneous mutations in the flora prior to exposure, remains a long-term key issue concerned and debated in both genetics and medicinal fields. In a pio-neering study, Luria and Delbruck exposed E. coli to T1 phage, to investigate whether the number of resistant colonies fol- lowed the Poisson distribution. They deduced that the development of resistant colonies is independent of phage presence. Similar results have since been obtained on solid medium containing antibacterial agents. Luria and Delbruck＇s conclusions were long considered a gold standard for analyzing drug resistance mutations. More recently, the concept of adaptive mutation has triggered controversy over this approach. Microbiological observation shows that, following exposure to drugs of various concentrations, drug-resistant cells emerge and multiply depending on the time course, and show a process function, incon-sistent with the definition of Poisson distribution （which assumes not only that resistance is independent of drug quantity but follows no specific time course）. At the same time, since cells tend to aggregate after division rather than separating, colonies growing on drug plates arise from the multiplication of resistant bacteria cells of various initial population sizes. Thus, statisti-cal analysis based on equivalence of initial populations will yield erroneous results. In this paper, 310 data from the Lu- ria-DelbNck fluctuation experiment were reanalyzed from this perspective. In most cases, a high-end abnormal value, resulting from the non-synchronous variation of the two above-mentioned time variables, was observed. Therefore, the mean value cannot be regarded as an unbiased expectation estimate. The ratio between mean value and variance was similarly incompara-ble, because two different sampling methods were used. In fact, the Luria-Delbrtick data appear to follow an aggregated, rather than Poisson distribution. In stmnnary, the statistical analysis of Luria and Delbruck is insufficient to describe rules of resistant mutant development and multiplication. Correction of this historical misunderstanding will enable new insight into bacterial resistance mechanisms.