Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism

Abstract

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

Long-term ecological impacts of antibiotic administration on the human intestinal microbiota

“…For amplification of Bacteroides spp., the reverse primer g-Bfra-R (Matsuki et al, 2002) was used together with fD1-FAM (for primer sequences, see Table 1). The PCR products were digested with HaeIII and the fluorescent terminal restriction fragments (TRFs) were separated according to size and quantified on an ABI sequencer as described previously (Jernberg et al, 2005). The threshold value for TRFs was established at a relative abundance X0.5% and TRFs differing by 70.5 bp were grouped together.…”

“…Thus they inhibit susceptible organisms and select for resistant ones. A number of different molecular fingerprinting techniques have previously been used to analyse the impact of antibiotics on the faecal microbiota such as denaturing or temperature gradient gel electrophoresis (DGGE or TGGE) (Donskey et al, 2003;Harmoinen et al, 2004), temporal TGGE (TTGE) (De La Cochetiere et al, 2005), terminal restriction fragment length polymorphism (T-RFLP) (Jernberg et al, 2005;Engelbrektson et al, 2006) and denaturing high-performance liquid chromatography (DHPLC) (Goldenberg et al, 2006). These techniques all bypass the necessity for cultivation, enabling comparative analyses of community fingerprints and the ability to monitor relative shifts in specific populations.…”

The Gut Microbiota: Ecology and Function

“…This is particularly the case for bacterial populations that are targeted by clindamycin (Lofmark et al, 2006;Jernberg et al, 2007). For example, a conventional 7-day administration of clindamycin was found to alter the ecology of the gut microbiota up to 2 years posttreatment (Jernberg et al, 2005;Lofmark et al, 2006;Jernberg et al, 2007). Our recent 454 pyrosequencing studies have also shown that the gut microbiota is dramatically perturbed for up to 4 years posttreatment after taking the common triple therapy for Helicobacter pylori (clarithromycin, metronidazole, and omeprazole) (Jakobsson et al, submitted).…”

Propiedades probióticas de Lactobacillus spp aislados de biopsias gástricas de pacientes con y sin infección por Helicobacter pylori

“…Otro género que ha concitado interés es Lactobacillus, pues está constituido por bacterias acidolácticas, que crecen en diversidad de condiciones y forman parte de la microbiota normal de la boca, tracto gastrointestinal y genitourinario humano 5,14,15 . En el estómago es posible encontrarlo en concentraciones de 0 a 10 3 UFC por ml de contenido gástrico 16 .…”