Characterization of a three bacteria mixed culture in a chemostat: Evaluation and application of a quantitative terminal‐restriction fragment length polymorphism (T‐RFLP) analysis for absolute and species specific cell enumeration

Abstract

Growth dynamics of Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus in a batch and chemostat, were investigated as a laboratory model system for persistent infections in cystic fibrosis. Most species-specific enumeration methods for mixed cultures are laborious or only qualitative, and therefore impede generation of quantitative data required for validation of mathematical models. Here, a quantitative T-RFLP method was evaluated and applied for specific and absolute cell number enumerations. The method was tested to be unbiased by quantitative sample composition and allowed reproducible enumerations of mixed cultures. For assay validation, samples of defined concentration containing one, two or three species were quantified. Logarithmically transformed absolute cell numbers of single-species dilutions were linear within a lower working range of 10(4)-10(6) cfu/mL (species-dependent) and an upper working range of 10(10) cfu/mL. Quantifications of single species (10(6)-10(10) cfu/mL) spiked with one or two other species agreed well with single species controls. Differences between slopes of first order linear regression of spiked and pure dilution series were insignificant. Coefficient of variation of defined mixed replicates was maximum 4.39%, of a three-species chemostat it was maximum 1.76%. T-RFLP monitoring of pure cultures in parallel shake flasks and of a three-species mixed chemostat gave very consistent results. Coexistence of at least two species after a time period equivalent to more than 33 volume exchanges was found. This result was not predicted from pure cultures clearly indicating the need for quantitative mixed culture experiments to better understand microbial growth dynamics and for mathematical model validation.

Cultivation-Independent Analysis of Bacteria in IDEXX Quanti-Tray/2000 Fecal Indicator Assays

“…during incubation in Quanti-Trays increased the relative abundance of the target TRFs from nondetectable (in water samples) to dominant (in individual positive Quanti-Tray wells). The absence of these TRFs in the source water samples is not surprising, given the FIB concentrations in this study and published detection limits for TRFLP (ϳ10 7 CFU per 100 ml) (46). Therefore, it is reasonable to infer that where Vibrio and Lactobacillales/Clostridiales were the dominant nontarget TRFs in IDEXX enrichments, the cause was growth.…”

“…In this study, culture-independent bacterial community analyses were used to reduce potential biases due to selective culture media. While TRFLP can have its own biases, e.g., originating during DNA extraction, PCR amplification, enzymatic restriction, and electrophoresis (22,25,41), relative changes in community structures are reliably reflected using TRFLP (25,46). Further, dominant taxa can be detected, even if the dominant taxa are not represented by the dominant TRFs (22,40).…”

“…Consequently, at least one of the nontarget TRFs was associated with bacterial taxa causing the false-positive signals. Since DNA yields from the false-positive wells were sufficiently high for reliable PCR and TRFLP, target TRFs were dominant in other (true) positive wells, and TRFLP is generally very reproducible (25,46), the absence of target TRFs in some wells was unlikely to be caused by TRFLP biases. The falsepositive rates in this study, based on TRFLP, are very similar to those reported previously for total coliforms but slightly higher for E. coli (1, 9, 23) and higher for Enterolert (6 to 20% here versus 2.4 to 5.1% previously) (1,9,23,26).…”

Species-specific viability analysis of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus in mixed culture by flow cytometry

“…For quantitative characterization of mixed communities by T-RFLP, Trotha et al introduced an internal quantification standard [7], an 16S ribosomal RNA (rRNA) gene fragment from a known species with a defined cell number. Schmidt et al adapted this quantitative T-RFLP (qT-RFLP) method for species-specific cell enumeration of a three-species mixed model community, comprising P. aeruginosa , B. cepacia and S. aureus , relevant to infections of lung of Cystic Fibrosis (CF) patients [8]. Furthermore, the authors characterized the growth of these species in a defined mixed culture in chemostat cultivations, and a mathematical chemostat model was established to identify interspecies effects [9].…”

Quantitative Community Fingerprinting Methods for Estimating the Abundance of Operational Taxonomic Units in Natural Microbial Communities

“…However, using peak areas as an indicator for absolute abundance has been confirmed to be a fruitless exercise, even when using simplified microbial communities (4,10,55). However, when experiments are performed under well-defined laboratory conditions or on simplified communities whose rrs copy numbers per genome are known, quantitative estimates of each target organism may be successfully obtained via community fingerprinting methods (27,41). A corollary of the lack of quantitativeness in community fingerprinting methods is that they may not be appropriate to assess the richness of microbial communities or to be used with diversity metrics because of their underestimation of the actual richness and because of their limited detection threshold and dynamic range (reviewed in reference 1).…”