Author(s): Deanna L Gibson [[dagger]] 1 , Bruce A Vallance 2
16S rRNA technology; dysbacteriosis; enteric pathogen; enteritis; fluorescence in situ hybridization; GI tract; intestinal microbiota; inflammation; mucosal immunity; Salmonella virulence
Barman et al. examined the intestinal microbiota of the GI tract during salmonellosis utilizing a sensitivity to Friend leukemia virus B strain (FvB) murine model of enteritis  . Salmonella enterica is a gram-negative pathogen that can infect a diverse array of hosts. For example, in humans, Salmonella typhimurium causes gastroenteritis, while in mice it normally causes a systemic typhoid-like disease with little colonization of the intestine. By contrast, S. typhimurium . can heavily colonize the murine GI tract and cause enteritis normally only following streptomycin pretreatment  . However, Barman et al. demonstrate in this study that S. typhimurium . can heavily colonize the FvB mouse even without streptomycin treatment. This provides an ideal model for analysis of the indigenous intestinal microbiota during enteritis without confounding antibiotic-induced microbial alterations. In this study, all FvB mice were bred in-house, alleviating environmental or dietary differences in microbiota diversity. In addition, maternal effects were controlled for by using animals from a single litter for each experiment. To further account for false bacterial diversity, control groups received vehicle alone while infected groups received a low (LD50 = 107 colony-forming units [CFU]) or high dose (10 × LD50 = 108 CFU) of S. typhimurium .
The majority of intestinal microbes cannot be cultured and therefore are not represented in studies using culture-based microbiology. Information concerning the microbial diversity within intestinal microbiota has only recently been revealed using nonculture-based techniques such as 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH)  . 16S rRNA gene sequencing is highly useful with regards to classification and comparison of bacteria since it is the most common bacterial housekeeping genetic marker and differences in gene sequences often allow for confident identification down to the species level, since the rRNA gene has not changed functionally over time  . Another approach for identification of microbial groups is FISH, a DNA-DNA hybridization technique using fluorescent probes designed to bind the 16S rRNA region of the bacterial chromosome. Fluorescence microscopy can be used to quantify specific populations of bacteria.
To examine the intestinal microbiota during enteritis, Barman et al. utilized a modified version of the 16S rRNA technique that incorporated reverse transcriptase (RT) quantitative (q)PCR on homogenates of the distal small intestine, cecum and colon excised from day 3 and 7 postinfected (p.i.) mice. This convenient system allowed accurate and sensitive enumeration of subdominant bacterial populations. Using bacterial group-specific primers for 16S rRNA, analytical curves were constructed for Eubacterium rectale/Clostridium coccoides , Lactobacillus sp. , Bacteroides sp. , mouse-specific Bacterioides , Enterobacteriaciae , S. typhimurium , Clostridium perfringens , Helicobacter sp. and Eubacteria (all phyla of bacteria) to determine total bacterial amounts. It has been shown that rRNA-targeted RT-qPCR can determine the number of bacteria with a detection limit of 10° CFU  ; which is 100- to 1000-fold more sensitive than DNA-targeted qPCR, indicating that this method is an efficient choice...