A New Evaluation Method for Antibiotic-Resistant Bacterial Groups in Environment

Abstract

In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg•L −1 ) were included in row cattle feces (1.78 × 10 4 MPN g −1 ) and cattle feces manure (>3.84 × 10 4 MPN g −1 ). Compost originated from leftover food (>44.8 × 10 4 MPN g −1 ) and shochu lee (>320 × 10 4 MPN g −1 ) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg•L −1 ), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 10 4 MPN g −1 ), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg•L −1 of ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 10 4 MPN g −1 ), followed by cattle feces manure (4.19 × 10 4 MPN g −1 ), and shochu lee (0.36 × 10 4 MPN g −1 ), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.

A novel culture-independent method to search unknown dominant bacterial groups and its application to microbial analysis of membrane bioreactors

“…Using the V2 forward primer (41f), and the V6 reverse primer (1066r) (44), 16S rDNA was amplified, as described previously (36, 37). Their restriction fragment lengths were measured by microchip electrophoresis systems (MCE-202 MultiNA; Shimadzu Co., Ltd. Kyoto, Japan) after digestion of the PCR product (10μl) using each of the following 4 restriction enzymes: Hae III or Hha I or Rsa I or Alu I (10 units, Takara Bio Co. Ltd. Shiga, Japan) in buffer solution (10xLow salt buffer, Takara Bio Co. Ltd.) and 5 folds dilution by de-ionized water, as described previously (36-43).…”

“…The reference database used for this research included 30,844 post-amplification sequence files for the 41f/1066r primers, which were mainly re-edited from small subunit rRNA files in RDP II release 9_61 (45) under 5 - bases mismatches in the both in primer annealing sites (36, 37), and consisted of 1,379 bacterial genera, including uncultured and unidentified bacteria (40-43).…”

“…After subtraction of the second major RFs from the remaining heterogeneous RFs, the 3rd major RFs were similarly selected (represented as L in Table 1-3). The similarity between the measured RFLP (A) and the reference RFLP (B) was calculated as described previously (36-43), based on the pairwise distance ( D AB ) according to Nei and Li (46). The pairwise distance of the MERFLPs ( D ABME ) was an average of all the D ABs for used restriction enzymes.…”

“…When the identical theoretical MERFL was not found using three restriction enzymes, combinations of the two restriction enzymes was used. If the identical theoretical MERFL (100% similarity) was not found using the two restriction enzymes, the theoretical MERFL having the highest similarity (over 90%) to the measured MERFL was indicated in most cases (Table 1-3) (38-43).…”

“…As microbial numbers might become a simple and useful index to show bacterial activity in the environment, the author has developed an original method to specify and trace unknown dominant microorganisms, in which taxonomies of each bacterial groups were elucidated from the multiple enzyme restriction fragment length polymorphism (MERFLP) of 16S rDNA (36, 37), and each number of the bacterial groups were estimated by most probable number (MPN) (38). Although the method had mostly been applied as a culture-based method (39-43), we presented the results of our application as culture-independent analyses of three industrial membrane bioreactors (MBR) situated in the Hokkaido area. The results were compared with those by the culture-based same method, and those by culture-independent PCR-DGGE.…”