Global Journal of Medical Research, K: Interdisciplinary, Volume 22 Issue 3

dilution by de-ionized water, as described previously (Watanabe et al., 2015b, 2016). d) Reference database used for the phylogenetic estimation The reference database used for this research included 30,844 post-amplification sequence files of 16S rDNA amplified by 41f/1066r primers (Watanabe et al., 2016), which were mainly re-edited from small subunit rRNA files in the Ribosomal Database Project (RDP) II release 9_61 (Cole et al., 2007) under 5-bases mismatches in both primer annealing sites, and consisted of 1,379 bacterial genera, including uncultured and unidentified bacteria (Watanabe et al., 2016). From post-amplification sequence files, fragment size for each restriction enzyme was calculated and save in the restriction fragment database and used for similarity search as described previously (Watanabe et al., 2008; Watanabe and Koga 2009). e) Data processing to select homogenous 16S rDNA and phylogenetic estimation f) Enumeration of antibiotic resistant bacterial groups by MPN Based on the results of phylogenetic estimation, each 16S rDNA was differentiated into the following 12 groups: Actinobacteria (A), Bacillus group (bF), Staphylococcus sp. (sF), other Firmicutes (F), Sphingomonadaceae (sP), other α -Proteobacteria (aP), β -Proteobacteria (bP), γ -Proteobacteria (rP), δ - Proteobacteria (dP), ε -Proteobacteria (eP), Cytophaga (C), other bacteria (O), and unidentified or uncultured bacterial (U), as shown in Table S1 and Table S2. By using MPN score for each groups (Table 1 and Table 2) and a table for a five-tube and three-decimal-dilution experiment (Blodgett 2010), the MPN of each bacterial group and MRB group were estimated (Table 1, 2). Using the FDA’s Bacterial Analytical Manual (Blodgett 2010), confidence limits were obtained and shown in Table 1 and Table 2. III. R esults and D iscussion a) Phylogenetic estimation and enumeration of general bacteria There was a large difference in the total bacteria numbers included in the tested composts (from 7.08x109 MNP g-1 dry matter to 316.2x109 MNP g-1) (Table 1), which were higher than those of the reported numbers by plate count (Rebollido et al., 2008; Vishan et al., 2014). Although there was no report of a bacterial number by the culture-independent method (Schloss et al., 2005), the higher bacterial numbers by the method might be caused by included unculturable bacteria (Watanabe et al., 2015b). In composts originating from chicken droppings and pig feces (compost A, B, BB, C, and D), the major bacteria were gram-positive bacterial groups, such as Actinobacteria and Firmicutes, which occupied 79.7% to 98.3% of the total bacterial number when unidentified bacterial numbers were subtracted (Table 1, and Figure 1). Extremely high numbers of total bacteria in compost D (316.2x109 MNP g-1) are attributed to the higher number of Staphylococcus sp. (297x109 MNP/g), where Staphylococcus aureus occupied most of them (95.7%) (Table 1). Staphylococcus sp. was also the numerically dominant bacteria in compost BB (55.2x109 MNP g-1), which occupied 67.9% of the total bacterial number (86.03 x109 MNP g-1) (Table 1, Figure 1). The higher number of total bacteria in compost C (146.4x x109 MNP g-1) is attributed to the number of spore-forming bacteria group, such as Bacillus sp.(21.0 x109 MNP), Paenibacillus sp., and Clostiridium sp. (43.6 x109 MNP g-1; 65.6%) (Table 1 and S1). The number of spore- forming bacteria was also higher in compost B (25.7 x109 MNP g-1), which occupied 65.8% of the total bacterial number (41.2 x109 MNP g-1) (Table 1, Figure 1). As our former results about bacterial compositional changes during each composting process indicated 21 Year 2022 Global Journal of Medical Research Volume XXII Issue III Version I ( D ) K © 2022 Global Journals Dispersion of Multidrug Resistant Bacteria and Fecal Bacteria into Field Soils of Japan through Compost Application For precise phylogenetic estimation by MERFL, the measured MERFL originating from homogeneous 16S rDNAs had to be selected among the mixed MERFLs by data processing (Watanabe et al., 2015a, 2015b). Because all the reference MERFLs were calculated from the homogeneous 16S rDNA sequence in the RDP II database, while the measured MERFL was obtained by restriction digestions of a mixture of 16S rDNAs, which were amplified using DNAs from different bacteria in each MPN tube as described previously (Watanabe et al., 2015a, 2015b). The selected restriction fragments (RFs) with the highest relative mole concentrations (ratio of fluorescent intensity to fragment size) was summed up until to leach the 16S rDNA size before restriction digestion, which was treated as the major RFLP (represented as H in Table S1 and S2) originated from a the major homogenous 16S rDNA in a MPN vial. The 2nd major RFs (represented as M in Table S1 and S2), and the 3rd major RFs (represented as L in Table S1 and S2) were similarly selected as described in the former manuscript (Watanabe et al., 2015a, 2015b). If the completely identical theoretical MERFL was not found out by using all the measured MERFL data, combinations of restriction enzymes used for the analysis was changed (Table 1, and Table 2) (Watanabe et al., 2015a, 2015b). Because measured RFs with near DNA length could not always be separated by electrophoresis, which resulted in lower similarity in similarity search for RFLP (Watanabe et al., 2015a, 2015b). As to the measured MERFL which had not completely identical theoretical MERFL, the theoretical MERFL having the highest similarity to the measured MERFL was indicated in Table 1 and Table 2.