A convenient and versatile culturomics platform to expand the human gut culturome of Lachnospiraceae and Oscillospiraceae

Acknowledgements N.P. was supported by a grant from the Graduate School of Medical Sciences of the University of Groningen, the Netherlands. L.L. was supported by a joint fellowship from the University Medical Center Groningen and the China Scholarship Council (CSC) (grant number CSC201908320432). A.W.W. and the Rowett Institute receive core funding support from the Scottish Government’s Rural and Environmental Science and Analytical Services (RESAS) division. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.Peer reviewe

A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS

Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network for the Rapid Identification of Anaerobes (ENRIA). On arrival the strains were cultured and identified using MALDI-TOF MS. The spectra derived were compared with two different Biotyper MALDI-TOF MS databases, the db5627 and the db6903. The results obtained using the db5627 shows a reasonable variation between the different laboratories. However, when a more optimized database is used, the variation is less pronounced. In this study we show that an optimized database not only results in a higher number of strains which can be identified using MALDI-TOF MS, but also corrects for differences in performance between laboratories. (C) 2017 Elsevier Ltd. All rights reserved

Antimicrobial susceptibility profile of clinically relevant Bacteroides, Phocaeicola, Parabacteroides and Prevotella species, isolated by eight laboratories in the Netherlands

Objectives: Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories. Methods: Each laboratory collected 20–25 Bacteroides (including Phocaeicola and Parabacteroides) and 10–15 Prevotella isolates for 3 months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS. Results: Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16 mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally. Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems. Conclusions: Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Comparison of two matrix-assisted laser desorption ionisation-time of flight mass spectrometry methods for the identification of clinically relevant anaerobic bacteria

Two commercially available MALDI-TOF MS systems, Bruker MS and Shimadzu MS, were compared for the identification of clinically relevant anaerobic bacteria. A selection of 79 clinical isolates, representing 19 different genera, were tested and compared with identification obtained by 16S rRNA gene sequencing. Correct genus identification was achieved for 71% of isolates by Shimadzu MS and for 61% by Bruker MS. Correct identification at the species level occurred in 61% and 51%, respectively. Shimadzu showed markedly better results for identification of Gram-positive anaerobic cocci. In contrast, the Bruker system performed better than Shimadzu for the Bacteroides fragilis group. When strains not present in the database were excluded from the analyses for each database, both systems performed equally well, with 76.7% and 75.0% correct genus identification for Shimadzu and Bruker, respectively. Similarly, when the most recently updated Bruker database was applied, no difference was observed. We conclude that the composition and quality of the database is crucial for a correct identification. The databases currently available for both systems need to be optimized before MS can be implemented for routine identification of anaerobic bacteria.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Comparison of viable cell counts and fluorescent in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria

Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared from fecal samples of three healthy volunteers. To introduce variation between the samples, they were incubated for 48 h in batch culture (anaerobic) fermenters at 37 degrees C, and pure cultures of Bifidobacterium infantis, Clostridium perfringens, or Lactobacillus acidophilus were added. Samples were taken from the fermenters at different times. Total anaerobes, bifidobacteria, bacteroides, clostridia, and lactobacilli were enumerated by both plating and FISH. The results showed that plate counts of total anaerobes, bifidobacteria, lactobacilli and bacteroides were approximately ten-fold lower than the corresponding FISH counts. Numbers of clostridia were higher using the plating method, probably because the clostridia probe used in FISH analyses was designed to only detect part of the genus Clostridium. The introduced variation in the methods could be detected by both methods and was comparable