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

virulence genes, such as the Vero toxin gene (Kudo et al., 2007), or the Shiga toxin gene (Parsons et al., 2016), which could differentiate pathogenic bacteria from the other harmless bacteria, but harmless genes, which were widely distributed into indigenous bacteria in natural environments (D’Costaet al.,2011; Nesme et al., 2014), and into natural mammalian intestines (Stanton et al.2011; Zhang et al., 2011) before the modern selective pressure of clinical antibiotic use (D’Costa et al.,2011), and the hazard level of the samples could not be evaluated by the detection of ARGs. MRB groups in the sample were found to be rapidly identified and quantified by analyzing the bacteria that proliferated under antibiotics (Watanabe et al., 2016). In this manuscript, each MRB group included in compost had been identified by multiple enzyme restriction fragment length polymorphism (MERFLP) (Watanabe et al., 2008; Watanabe & Koga 2009) and quantified by the most probable number method by using an originally developed method (Watanabe et al., 2015a, 2015b, 2016). The author had explored MRB and fecal bacteria in nine composts that originated from diverse livestock feces and had annually been applied on soils of organic farms in various regions of Japan. The purposes of this experiment were 1) to speculate how widely MRBs and the other fecal bacteria had spread into the field soil of Japanese organic farms through compost application, 2) to know what kinds of MRB and fecal bacteria had introduced into field soil though compost application, and 3) to find out a way to reduce MRB and fecal bacteria during composting process. In order to speculate composting conditions of the tested composts, which might had affected the residual MRB and fecal bacteria, the composition and numbers of numerically dominant bacteria were also searched. II. M aterials And M ethods a) Samples The nine tested composts had been used on organic farms in various regions of Japan. Compost A, which was a marketable good originating from chicken droppings, has been applied on organic farm A in Nagano Prefecture in the Chubu region, where organic rice has been cultivated. Compost B, which was handmade from chicken droppings, has been applied on organic farm B in Niigata Prefecture in the Hokuriku region, where organic rice has been cultivated. Compost BB was a so-called “Bokashi-compost”, which was a handmade from several kinds of organic waste through fermentation, and has also been applied on farm B, where organic rice has been cultivated. Compost C, which was a marketable good originating from pig feces, has been applied on organic farm C in Chiba Prefecture in the Kanto region, where organic vegetables have been cultivated. Compost D, which was a marketable good originating from pig feces, has been applied on organic farm D in Ibaraki Prefecture in the Kanto region, where organic vegetable has been cultivated. Compost E, which was a marketable good made from cattle feces, has been applied on organic farm E in Fukushima Prefecture in the Tohoku region, where organic vegetables have been cultivated. Compost F1, F2, and F3, which were made from cattle feces by the National Agricultural Research Center for the Kyushu-Okinawa region in Kumamoto Prefecture in the Kyushu region, have been applied on experimental fields in the research center, where vegetables and rice have been cultivated. Although there was not such a large difference in the composting process among these three composts (Watanabe et al., 2015b), recycled paper was added to adjust the moisture content (60%) of the starting material in the composting process of compost F1, rice straw was added for composting to compost F2, and wood chips were added to compost F3. b) MPN and used antibiotics For analysis of general bacteria (B), serial 10- fold dilutions (10-8 to 10-12) prepared from samples (1g fresh wt.) were inoculated to centrifuge tubes (5 replicates) including an LB medium. After 5 days of incubation at 30 ℃ , the bacterial DNA in each tube was extracted as described previously and purified by conventional methods (Watanabe et al., 2015a, 2015b). For analysis of MRB (M), the following antibiotics were simultaneously added to the LB medium: streptomycin (25 mgl-1), chloramphenicol (25 mgl-1), and ampicillin (25 mgl-1). Serial 10-fold dilutions (10-4 to 10-7) prepared from samples (1g fresh wt.) were inoculated to centrifuge tubes (5 replicates) including an LB medium and the antibiotics. As the MRB detected by the method was bacteria that proliferated under a mixture of 25 ppm each of three antibiotics, they had higher resistance to those detected by conventional susceptibility tests such as the disk diffusion test, where the resistance of each antibiotic was separately tested. Until now, the MRB had been exceptionally detected in limited samples only, such as livestock feces, composts (Watanabe et al., 2016), feces applied to field soils, activated sludges, a few fresh meats, river water, and fresh vegetables (Watanabe unpublished results). c) MERFLP of the amplified 16S rDNA Using the V2 forward primer (41f), and the V6 reverse primer (1066r)(Weidner et al., 1996), 16S rDNA was amplified, as described previously (Watanabe et al., 2008). Their restriction fragment lengths were measured by microchip electrophoresis systems (MCE-202 MultiNA; Shimadzu Co., Ltd. Kyoto Japan) after digestion of the PCR product (10 μ l) using each restriction enzyme, HaeIII or HhaI or Rsa I (10 units, Takara Bio Co. Ltd. Shiga Japan) in a buffer solution (10xLow salt buffer, Takara Bio Co. Ltd.) and 5 folds 20 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