Normal flora as a source of opportunistic pathogens

(S. epidermidis, S. mutans, P. aeruginosa, Candida albicans)

The Role of Biofilms in Human Disease

P = NV/R

During a lecture to a group in Kingston Ontario (Sept. 2000), Bill Costerton was asked if biofilms had always been around or is this something new? Costerton essentially answered, “Yes”. That is yes, biofilms have always been around but yes, there is in increase in their numbers indicating that there is indeed something new going on. His explanation included the following points.

There has been a shift in the age structure of the population and now a much larger proportion of our population is elderly.
Improvements in medical technology has translated into longer survival of immune or otherwise compromised patients (CF, AIDS, Autoimmune conditions).
There has been a dramatic increase in the number of operations implanting prosthetic devices (artificial joints, stents, heart valves, catheters, voice prostheses etc), which serve as favored locations for the initiation and growth of biofilms.
There has been an increase in the use of antibiotics, which may alter the normal body flora, opening niches for the invasion of biofilm formers.

The American microbiologist Theobald Smith, better known perhaps for his work on Rickettsial diseases, developed an expression for thinking about the factors that contribute to the development of disease. That is:

P = NV/R

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Figure 1. Microbiologist Theobald Smith.

In this expression, P is the probability of developing an infectious disease, N is the number of organisms encountered, V is the virulence or disease causing ability of the infectious agent and R is the intrinsic or acquired resistance of the host. Although this expression was originally formulated to explain acute or planktonic disease processes its applicability to biofilm related diseases is also intriguing. Biofilm formation alters each of the variables in this formula in such a way as to increase the probability of a prolonged or chronic disease process.

N In most habitats where the relative numbers of the planktonic and biofilm associated cells have been established, the cells in the biofilm outnumber the cells in the planktonic phase by as much as 1000 to 1. According to Bill Costerton (various reviews) this ratio holds for clinical infections as well as for non-pathogenic niches. This enormous reservoir of cells is, to be sure, encapsulated within the biofilm itself but fragments of the biofilm may slough off or planktonic cells may be released as a consequence of Quorum Sensing regulated release. These individual cells or cell masses can initiate biofilm formation in other areas of the body or may lead to episodes of acute infection (e.g. otitis media).

V Biofilm formation also influences the virulence of pathogens. Virulence includes microbial attributes that increase the organisms invasiveness or persistence in the host or increase the toxigenicity of the pathogen. While biofilms are not noted for their dramatic invasiveness, their persistence is legendary. Biofilm formation contributes in many ways to increase the persistence of pathogens at infection sites.

Virulence factors, in many pathogenic microorganisms, are produced by genes regulated by Quorum Sensing mechanisms. These mechanisms depend upon the accumulation of quorum sensing effector molecules, in Gram negative cells principally Acyl homoserine lactones (AHL). These molecules in turn typically accumulate either in the late log stage of planktonic growth or in biofilms. When their concentrations reach a critical level, they can turn on suites of genes which, may be responsible for the production of a variety of proteins resulting in dramatic changes in the characteristics of the organisms. This change has been called the biofilm phenotype by some (ref). One of the best-studied biofilm producing organisms is Pseudomonas aeruginosa, a common opportunistic pathogen responsible for pneumonia, septicemia and wound infections, especially in immune compromised patients.

Pseudomonas aeruginosa is a ubiquitous organisms which is typically found in soil, fresh water, plants and animals. Its invasive properties are rather limited so although most of us harbor this organism most of the time no infections are typically observed. In patients whose host defense mechanisms have been compromised by injury or disease this toypically commensal organism can cause life-threatening infection. Patients with cystic fibrosis, for example, are prone to infections with Pseudomonas and other opportunistic bacteria (Burkholderia cepacea). The symptoms of disease are caused predominantly by the production by the bacterium of large volumes of a slimy exopolysaccharide called alginate. This material clogs bronchioles and alveoli limiting oxygen and carbon dioxide exchange in the patient. In addition to alginate, other proteinaceous virulence factors such as proteases (elastase and alkaline protease), and a variety of exotoxins (A and S) are known to be under the control of the Las R auto induction mechanism. In addition to these soluble factors there are a number of cellular components including pili, flagellae and lipopolysaccharides that aid the organism in attachment to tissue surfaces and are thus also key elements in the virulence of Pseudomonas aeruginosa.

R The third of Professor Smith’s variables was the resistance of the host. Typically in healthy individuals there are a variety of specific and nonspecific mechanisms which enable the individual to ward off infection. The non-specific mechanisms include the skin, the mucociliary escalator mechanisms which sweeps potential pathogens from the bonchi, the low pH of the stomach and vagina which either kills pathogens or puts them under severe stress and certain chemicals produced by the body like lysozyme and lactoferrin which are bactericidal to certain groups of bacteria. In addition there is the phagocytic mechanism mediated by the polymorphonucear leucocytes of the blook and lymphatic system that engulf and destroy most invading planktonic bacterial cells.

The specific host defenses include the cells of the humoral and cell mediated immune mechanisms. Certain blood cells called B-lymphocytes when stimulated by specific antigens aociated with invading bacteria produce immunoglobins (antibodies) that bind to and target the invaders as candidates for the action of the bodies complement cascade or the cells of the host cell mediated response. The cell mediated response system is carried out by a bewildering array of cells called T-lymphocytes which when stimulated are drawn to and attack “foreign” cells and tissues by releasing protein molecules called cytokines. These cytokines act in a variety of ways to kill bacterial cellls, cancer cells and virus infected cells, terminating the disease process.