Antibiotic-Resistant Bacterial Group in Field Soil Evaluated by a Newly Developed Method Based on Restriction Fragment Length Polymorphism Analysis

Abstract

Spreading of antibiotic resistant bacteria into environment is becoming a major public health problem, implicating affair of the indirect transmission of antibiotic resistant bacteria to human through drinking water, or vegetables, or daily products. Until now, the risk of nosocomial infection of antibiotic resistant bacteria has mainly been evaluated using clinical isolates by phenotypic method. To evaluate a risk of community-acquired infection of antibiotic resistant bacteria, a new method has been developed based on PCR-RFLP without isolation. By comparing restriction fragment lengths of the 16S rDNA gene from bacterial mixture grown under antibiotic treatment to those simulated from the DNA sequence, bacterial taxonomies were elucidated using the method of Okuda and Watanabe [1] [2]. In this study, taxonomies of polymyxin B resistant bacteria group in field soils, paddy field with organic manure and upland field without organic manure were estimated without isolation. In the both field soils, the major bacteria grown under the antibiotic were B. cereus group, which had natural resistance to this antibiotic. In field applied with organic manure, Prevotella spp., and the other Cytophagales, which were suggested to be of feces origin and to acquire resistance to the antibiotic, were detected. When numbers of each bacterial group were roughly estimated by the most probable number method, B. cereus group was enumerated to be 3.30 × 10 6 MPN/g dry soil in paddy field soil and 1.32 × 10 6 MPN/g dry soil in upland filed. Prevotella spp. and the other Cytophagales in paddy field were enumerated to be 1.31 × 10 6 MPN, and 1.07 × 10 6 MPN•g −1 dry soil.

Bacterial Groups Concerned with Maturing Process in Manure Production Analyzed by a Method Based on Restriction Fragment Length Polymorphism Analysis

“…In the major MERFL, ratio of the MERFLs having 100% similarity to the corresponding theoretical MERFLs (62.9%; Table 3) was lower than that of the former study (90.5%) [1], and the ratio in the 2nd major MERFLs (13.5%; Table 3) having 100% similarity to the corresponding theoretical MERFLs was also lower than that of the former study (50.0%) [1]. This might be caused from a difference of used incubation media as the following; NB medium used in this study was not a selective medium, more diverse microorganisms were proliferated in the each MPN vials than that used in the former study, which made it more difficult to select the MERFLP digested from the homogeneous 16S rDNA among the mixed MERFL.…”

“…Although a number of Actinobacteria (E) was high after thermophilic phase, they were only detected in the two higher dilution vials (T10 −9 1H, M10 −11 2M, Table 1). Actinobacteria was also detected only in two vials in the former study [1]. As the recent research indicated that 16S rDNAs of some Actinobacteria and Firmicutes were not amplified by the used PCR condition, a numbers of Actinobacteria (E) in this study might be underestimated.…”

“…Combined use of the most probable number method and PCR-RFLP of the 16S rDNA was found effective to provide numbers and taxonomies of polymyxin B resistant bacterial groups in the former paper [1]. In this manuscript, changes of numerically dominant microorganisms by a turn of the phase of manure composting were determined using this method.…”

“…The same theoretical MERFL database as that described previously [1] [10] [11] was used for this research, which was consisted from 4370 sequence files of 576 bacterial genera, and 143 uncultured and 34 unidentified bacteria.…”

“…Whereas all the theoretical MERFLs were originated from the homogeneous 16S rDNA sequence, the measured MERFLP digested from the homogeneous 16S rDNA was selected as described previously [1]. The restriction fragments (RFs) with the highest relative mole concentration (ratio of fluorescent intensity to fragment size) were selected and used as the major RFs (represented as H in Table 1).…”