Application of multiple enzyme restriction fragment length polymorphism analysis and microchip electrophoresis for estimation of antibiotic-tolerant bacterial group

Abstract

A combined method of microchip electrophoresis and multiple enzyme restriction fragment length polymorphism analysis, by which bacterial phylogeny was estimated by comparing the measured multiple enzyme restriction fragment (MERF) to that of the theoretical MERF of various kinds of bacteria, was adapted to monitor an antibiotic-resistant bacterial group in field soils. One hundred and ninety bacteria showing polymyxin B tolerance were newly isolated from experimental field soils where a large amount of liquid livestock feces (600 t/ha/year, 120 t/haրyear) has been applied annually, and neighboring area where feces were not applied (0 t area). In the 0 t area, most bacteria grown on PP agar was Bacillus spp. (80%), which decreased to 21.7% in the 120 t field, and 15.6% in the 600 t field. As the percentages gram negative bacteria, Cytophagales, and Spirochaetales increased to 10.0% in the 120 t field, and 33.3% in the 600 t field, and 7 isolates from the 120 t field and 18 isolates from the 600 t field were ordinary bacteria inhabiting in animals and feces, they originated from livestock. As polymyxin B is bactericidal for gram-negative bacteria, some of the gram-negative bacteria isolated from both the feces-applied fields were supposed to be polymyxin B-resistant bacteria. © Pesticide Science Society of Japan Keywords: polymyxin B resistant bacteria, field soil, livestock feces, microchip electrophoresis, multiple enzyme restriction fragment length polymorphism analysis.

Detection of protease genes in field soil applied with liquid livestock feces and speculation on their function and origin

“…However, the numbers of Bacillus spp. in these fields were overestimated because the bacterial numbers included a high ratio of non‐bacillus polymyxin B resistant bacteria (Watanabe and Niimi 2005; Watanabe 2008).…”

“…2004). Although we did not test the antibiotic resistance of the proteolytic S. marcescens , it is possible that the proteolytic S. marcescens is one of the polymyxin B resistant bacteria (Watanabe 2008). Lower growth suppression by chloramphenicol treatment in the 600 t field soils might also be attributed to the existence of chloramphenicol‐resistant bacteria in these field soils (Table 4).…”

“…The present results support our former suggestions that various types of non‐indigenous bacteria, such as Gram‐negative proteolytic bacteria (Table 7; Watanabe et al . 2008), non‐bacillus polymyxin B resistant bacteria (Watanabe and Niimi 2005; Watanabe 2008) and bacteria concerned with reduction (Watanabe et al . 2006) were introduced with liquid livestock feces and survived for several months.…”

“…As the selectivity of these media for Bacillus spp. was mainly attributed to the addition of polymyxin B and there were some polymyxin B resistant species in the feces‐applied field soil (Watanabe and Niimi 2005; Watanabe 2008), the microorganisms counted included some portion of non‐bacillus polymyxin B resistant bacteria (Watanabe and Niimi 2005; Watanabe 2008).…”

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

“…Fragment lengths were measured by microchip electrophoresis system (Cosmo-i SV1200; Hitachi Electronics Engineering Co., Ltd. Tokyo Japan) as described previously [16] [19]. The sample was diluted by de-ionized water (10 folds for Low salt buffer) before loading on i-tip DNA (IC-1000, Hitachi Chemical Industry Co., Ltd., Tokyo Japan).…”