Intestinal Microflora and Antibiotic Resistance

Basic Attributes of Gut Microflora

The microflora of the human gastrointestinal (GI) tract is so extensive and integral to the proper functioning of the digestive system that it has been characterized as an additional organ of the human body (1). It is estimated that the microfloral community consists of 500 -1000 distinct species of bacteria in a single person; collectively, their population is 10 times the number of their host’s body cells (2). The intestine provides a suitable niche for many species of bacteria, as it remains at a stable temperature and is replete with bioavailable carbon, nitrogen, and solute sources of nutrition (7). The suite of microbes is acquired primarily during infancy, since the GI tract of a fetus is sterile during development. Bacteria are initially transferred to the infant during the delivery process, then continually from the immediate environment and from contact with its mother and other adults. Escherichia and Streptococcus are the first to colonize the GI tract, typically followed by bifidobacteria, staphylococci, lactobacilli, micrococci, and propionibacteria. As the infant matures, it is continually exposed to bacteria, principally by the digestion of food, and the makeup of its intestinal microbiome changes dramatically (2, 5). The dynamic nature of the microfloral community entails shifting of community species composition over time and variation in composition among individuals. However, the predominant taxa of bacteria in the gut are fairly consistent. Firmicutes and Bacteroidetes are the most prevalent phyla by far; populations of Proteobacteria, Actinobacteria, Fusobacteria, and Verrucobacteria are frequently established as well (2, 6). The functions performed by gut bacterial include regulation of intestine epithelial development, contributions to the innate immune system, improved absorption of nutrients from food, and maintenance of mucosol homeostasis and repair (1, 2, 3). These functions are all highly relevant to medicine, as a deeper understanding of how bacteria influence these systems will yield more sophisticated solutions to conditions affecting the body. An important factor to the effective functioning of the human microbiome is the movement of genetic material. Genes are exchanged both among bacteria species, via lateral gene transfer, and among bacteria and their host. The presence of 233 documented human proteins that have homologues only in bacteria hints at the extent to which human development and function has historically been strongly influenced by bacteria (2). The transfer of genetic material among bacterial species affects human hosts in more immediate and clinically relevant ways, including, notably, influencing response to antibiotics (4).

Studying Human Microflora

Characterizing the diversity and function of the microbiome is notoriously difficult. Most gut bacteria cannot be cultured due to their very particular nutritional requirements and the obligate anaerobic nature of bacteria in the distal regions of the gut (2); for this reason, previous studies that may be significantly biased in their analyses. The use of phenotypic characteristics to classify bacteria (which was utilized until only recently) also leads to an incomplete and oversimplified resolution of species. More modern techniques to evaluate bacterial communities and their evolutionary lineages involve whole-genome sequencing and a strong emphasis on rRNA (1, 4, 6). The ubiquity of 16S strands in prokaryotic lineages makes them a natural choice for elucidating diversity and evolutionary histories of gut microbiota. rRNA 16S, which is located on the 30S subunit of bacterial ribosomes, contains regions that are highly conserved, variable, and highly variable; the conserved “signature” sequences indicate evolutionary history of a particular taxonomic group with precision. A number of techniques have been employed to sequence 16S rRNA, including sequencing of cloned amplicons, direct sequencing, TRFLP, dot-blot hybridization, shotgun sequencing, and quantitative PCR (or real-time PCR) (9). Cloned amplicon sequencing is common, due to its moderately high resolution and ability to clone the full length of the 16S strand. The 16S sequences are treated with primers that bind to these conserved regions and amplified with standard PCR techniques, so that the resulting clones may be identified and catalogued (8). Though all of these sequencing techniques are powerful, they are inherently limited by the method of sampling. Fecal samples are commonly used to assess bacterial composition, but do not accurately represent the flora community. Mucosal tissue samples directly from the intestine are preferable, but are more invasive and expensive to obtain (1). Studies utilizing these molecular techniques consistently identify great numbers of novel species (with benchmark similarity values of 95% or greater), indicating efforts to characterize the gut flora have been insufficient. It is estimated in review literature that only 19% of the adult flora has been identified, and only 8% of the adult flora (9). Due to the density of the microbial system and the complex interactions among species and between bacteria and the host, many researchers consider the gut to be its own ecosystem. This perspective has influenced the analysis of the data output from these sequencing projects: many ecological measures of richness, abundance, and growth are used to predict bacterial dynamics (1, 17).

The Gut Microbiome and the Defenses of the Intestine

Since the intestine is such an attractive habitat for microbes, it must employ many types of defenses to protect the body from invasion of pathogen microbes. Many of these defense mechanisms are intricately related to a normal population of gut microflora. Microflora inhabit the mucosal layer of the intestine, which is a gel-like substance secreted by goblet cells onto the lumen-facing surface. Compared in function to a biofilm, this mucus layer can either trap or prevent the adhesion of potentially pathogenic organisms (depending on the pathogen's physiology and adaptive ability). The invasion of exogenous bacteria, both Gram-positive and Gram-negative, stimulates the secretion of mucus, strengthening this form of defense (16). Indigenous bacteria must be specially adapted to dwell in this layer. The intestine constantly “checks on” the population of microflora using toll-like receptors. Two pathways have been identified as ways TLRs protect microflora: either TLRs constitutively express protective factors in response to concentration of microbial products, or protection factors are produced in response to physical or chemical damage to the epithelium (15). Evidence that intestinal microbiota have a distinct and crucial effect on the stimulation of mucus production is presented in a study that compared goblet cells of germ-free mice to conventional mice. The size of the goblet cells were significantly augmented in conventional specimens, but more importantly the mucosal layer was up to two times as thick. The structural components of gut flora bacteria that trigger this thickening effect remain unknown (17). Resistance to antibiotics is a necessary attribute of bacterial populations dwelling in the human gut. The epithelium of the intestine expresses a variety of antimicrobial agents as a first-line defense of the immune system. These peptides are either expressed constitutively or induced by inflammatory mediators (10). Thus, in order to flourish in the human gut commensal bacteria must be recognized by toll-like receptors and other mediators as well as be resistant to these comparatively non-specific antimicrobial agents. Pathogenic bacteria are recognized by their structural attributes by means of complex pathways, then are destroyed by inflammation or the antimicrobial peptides (11). The ability to distinguish between commensal bacteria and potentially pathogenic bacteria is crucial to the continued function of the microflora “organ” as well as the immune system as a whole.