Isolation of anaerobic bacteria of the Bacteroides fragilis group from environmental samples

Monika Harnisz,Sebastian Niestępski,Adriana Osińska,Ewa Korzeniewska

doi:10.1051/e3sconf/201910000058

Monika Harnisz, Sebastian Niestępski

Open Access

https://doi.org/10.1051/e3sconf/201910000058

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Abstract

The aim of this study was to analyze the effectiveness of identification of Bacteroides fragilis group (BFG) strains isolated from human and rat feces, hospital wastewater, and untreated and treated sewage from a wastewater treatment plant (WWTP). BFG strains were plated on Bacteroides Bile Esculin (BBE) agar. Characteristic colonies were isolated from the culture medium, tested for antibiotic resistance and identified by PCR and MALDI-TOF MS. A total of 319 strains that formed characteristic colonies were isolated from BBE agar, of which 250 were resistant to kanamycin, colistin and vancomycin. PCR revealed that only 135 strains harbored the bfr gene specific to the BFG. In MALDI-TOF MS analysis, only 123 isolates were classified as members of the BFG. The most frequently isolated species was Parabacteroides distasonis (51.22% of strains). B. fragilis, B. ovatus and B. thetaiotaomicron were less frequently encountered in the analyzed environmental samples (30.01%, 8.13% and 5.69%, respectively). The strains isolated from human and rat feces on BBE agar were most reliably identified, and 100% of the isolated strains were classified as members of the BFG. The effectiveness of isolation of BFG strains from hospital wastewater and untreated and treated sewage from the WWTP was relatively low (20%, 19%, 40%, respectively). The results of this study suggest that the method for isolating BFG strains from environmental samples on BBE agar requires modification. Additional methods, such as PCR and MALDI-TOF MS, can be used to more effectively identify BFG strains isolated from different environmental samples.

Highlights

Bacteria of the genus Bacteroides and the closely related genus Parabacteroides are Gramnegative obligate anaerobes
The analysis was carried out in four consecutive stages: bacteria were cultured on selective and differential Bacteroides Bile Esculin (BBE) agar, the isolated strains were tested for resistance to colistin, kanamycin and vancomycin, the presence of the bfr gene specific to Bacteroides fragilis group (BFG) was determined by polymerase chain reaction (PCR), and the strains were identified to the species level by MALDI-TOF MS
All strains isolated from samples of human and rat feces on BBE agar were identified as members of the BFG

Summary

Introduction

Bacteria of the genus Bacteroides and the closely related genus Parabacteroides are Gramnegative obligate anaerobes. These immobile and non-sporulating bacteria are part of normal gut microbiota in humans and animals [1]. Clinical BFG strains are presumptively identified on Bacteroides Bile Esculin (BBE) agar, a selective and differential culture medium [5, 6]. Due to its specific composition, BBE agar stimulates the growth of BFG strains only which are identified based on the blackening of the medium around the formed colonies. Most BFG species are resistant to gentamycin and to colistin, kanamycin and vancomycin. This trait is used to differentiate BFG strains from other anaerobic bacteria.

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