PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture.

Ruijie Huang,X Frank Yang,Junjie Zhang,Richard L Gregory,Hauke Smidt

doi:10.1371/journal.pone.0126628

Abstract

Changes of bacterial profiles in microbial communities are strongly associated with human health. There is an increasing need for multiple species research in vitro. To avoid high cost or measurement of a limited number of species, PCR-based multiple species cell counting (PCR-MSCC) has been conceived. Species-specific sequence is defined as a unique sequence of one species in a multiple species mixed culture. This sequence is identified by comparing a random 1000 bp genomic sequence of one species with the whole genome sequences of the other species in the same artificial mixed culture. If absent in the other genomes, it is the species-specific sequence. Species-specific primers were designed based on the species-specific sequences. In the present study, ten different oral bacterial species were mixed and grown in Brain Heart Infusion Yeast Extract with 1% sucrose for 24 hours. Biofilm was harvested and processed for DNA extraction and q-PCR amplification with the species-specific primers. By comparing the q-PCR data of each species in the unknown culture with reference cultures, in which the cell number of each species was determined by colony forming units on agar plate, the cell number of that strain in the unknown mixed culture was calculated. This technique is reliable to count microorganism numbers that are less than 100,000 fold different from other species within the same culture. Theoretically, it can be used in detecting a species in a mixed culture of over 200 species. Currently PCR-MSCC is one of the most economic methods for quantifying single species cell numbers, especially for the low abundant species, in a multiple artificial mixed culture in vitro.

Highlights

There are over 700 microbial species that live together in the oral cavity
Fluorescence labeling costs time and is limited to less than five different species [8], antibiotic-resistance gene labeling is time-consuming [9], and mass amplicon sequencing is expensive for quantifying several species cell numbers in a lab with an average supply budget
S. epidermidis has been reported in endodontic lesions [10] and it is the fifth most abundant species observed in used tooth brushes [11]

Summary

Introduction

There are over 700 microbial species that live together in the oral cavity. They facilitate or compete with each other dynamically [1,2]. For in vivo multiple-species study, 16S rRNA clone analysis and recently mass amplicon sequencing are the major methods used to count the microbial cell number of each species within a mixed culture. For in vitro multiple species study, many current techniques can be used, such as fluorescence, antibiotic-resistance gene labeling, or mass amplicon sequencing. Fluorescence labeling costs time and is limited to less than five different species [8], antibiotic-resistance gene labeling is time-consuming [9], and mass amplicon sequencing is expensive for quantifying several species cell numbers in a lab with an average supply budget. The purpose of the present study was to introduce a new PCR-based multiple species cell counting (PCR-MSCC) technique that meets these two criteria

Objectives

Methods

Results