Identification and Classification
There may be nothing the physician encounters on a routine basis that provides more consternation than the expanding array of medically important bacteria and the changing names of existing ones. An oft-posed question to me is “Why do you laboratory guys keep changing the names of bacteria?” It is a good question, but not one that is easily answered.
Large, multi-celled organisms are relatively easily classified because of their wide array of physical features. It seems intuitive that a horse and a zebra are placed in the same group while a crocodile is placed in another. Complex organisms lend themselves to a natural hierarchal classification scheme and the well-known Linnaean – Kingdom / Phylum / Class / Order / Family / Genus / Species system has been very successful and made sense in the context of Darwin’s essential observations. That is, the system was Natural and genetic relatedness was reflected in well recognizable physical properties.
There is much less difference between bacteria that is immediately obvious. However, phenotypic differences have formed the basis of classifying bacteria. Some of the features exploited are:
- Gram stain reaction (reflecting cell wall structure)
- Growth temperature
- Ability to form heat stable spores
- Cell shape
- Ability to metabolize various substrates
- Ability to metabolize various substrates
- Special nutritional requirements (e.g., vitamins)
This means of classifying bacteria is referred to as determinative – the characteristics determining the name and not the ancestry. This system is not Natural in that bacteria with very similar characteristics may be very unrelated in the genetic sense.
Despite the shortcomings of this means of naming and classifying bacteria, this “biochemical” characterization is most practical and available to the diagnostic medical microbiology laboratory and is the basis for most routine identification of common human pathogenic organisms.
Microorganisms are made up of complex proteins and carbohydrates of foreign origin and are, therefore antigenic. The highly specific nature of an animal’s immune response to microorganisms can be used to identify or differentiate between bacteria in the laboratory. Differences among bacteria of the same biochemical species may result in the designation Serotype or “strain”. This is often useful when trying to determine if an outbreak of bacterial disease was caused by a common source of identical bacteria.
Genetic Identification and Classification
The ability to easily identity, amplify, and categorize microbial genetic material is revolutionizing the study of microorganisms. One of the main reasons for changes in nomenclature is the ability to determine genetic (Natural) relatedness through genomic analysis that is in marked contrast to phenotypic (Determinative) features. An example of recent name changes for this reason is in a bacterium known to older physicians as Pseudomonas maltophilia. Its name was changed to Xanthomonas maltophilia and subsequently to Stenotrophomonas maltophilia. You can see why clinicians and taxonomists sometimes don’t get along!!
The ability to identify “strains” of the same species by detecting small differences in their genomes has developed into a very useful tool in epidemiology. “Fingerprinting” of bacterial genomes by various techniques is used to determine whether bacteria recovered from multiple sources had a common “point source”.
Another use of genomic identification that is rapidly expanding is in the direct detection of microorganisms in clinical samples. Amplification techniques such as the Polymerase Chain Reaction (PCR) are employed in many ways to determine if microorganisms are present. This is particularly important when dealing with microorganisms that do not grow well in culture.
Diagnostic Laboratory Techniques
The principle aim of the diagnostic microbiology laboratory is to help with patient care. As such, speed and communication are essential. The absolute identity of the infecting agent is less important as long as further investigation and therapy can be directed.
Microscopic examination of clinical material provides rapid initial information. Not only can bacteria be tentatively identified, the cellular response of the patient can be evaluated. Several techniques are used, the most common of which is simple light microscopy after staining with Gram’s technique.
Christian Gram described this differential stain in 1884. While not understood at the time, the ability of certain bacterial cells to resist decolorization with alcohol defines cell walls into their two primary types. The first phenotypic characteristic considered in determining the identity of a bacterium is still whether it is Gram Positive or Gram Negative. The results of Gram stain analysis are available soon after the specimen is received.
Culture and Sensitivity
Clinical material is placed on solid media or into liquid media that supports the growth of bacteria. After an incubation period (usually one day) media is examined for the presence of pathogenic bacteria. Biochemical or serological tests are then performed to confirm the identity of the organism and testing for susceptibility to antibiotics is initiated. A preliminary report on identity is usually issued the day after receipt of the specimen and a susceptibility report one day later.
Virulent adj — able to overcome bodily defensive mechanisms. This definition from Webster succinctly describes the properties of microorganisms that enable them to cause disease. Any substance of bacterial origin that aids in attachment, the resisting of phagocytosis or other immunologic processes, or that interferes with normal host cellular function is a virulence factor.
- Polysaccharide capsules that inhibit phagocytosis e.g. Streptococcus pneumoniae
- Pili that enable attachment to mucosal surfaces e.g. Neisseria gonorrhoeae
- Toxins that damage local cells e.g. Bordetella pertussis
- Toxins that act remotely e.g. Clostridium botulinum
Microbes and Man — A symbiotic relationship
Microbes came first! All multi-celled organisms have, therefore, coexisted with microbes from their evolutionary inception. Our continued coexistence is ample evidence that the relationship between man and microorganism is, to a very large extent, symbiotic — a fact that is often overlooked in this age of vaccines and antibiotics. An appreciation for the ecological relationships between microbes and higher life forms is essential in the understanding of infectious diseases.
There are, basically, two kinds of infectious diseases:
- Those caused by “our” (endogenous) organisms – microbes that are only found in and on humans and coexist with us peacefully at most times causing disease only intermittently, and
- Those caused by outside (exogenous) agents. These may be microbes of other animals (zoonotic disease) or of environmental origin.
There are some types of diseases in which the boundary between endogenous and exogenous is rather blurry, however it is a useful way of thinking about infections in the context of overall ecology and brings us to the concept of “Normal” or “Usual” Flora.
Normal or Usual Flora
There are many more bacterial cells in and on the human organism than there are human cells! Bacteria are absolutely essential for our well being and are responsible for many physiologic processes, many of which are little understood. An appreciation and respect for this circumstance is critical when making decisions about antimicrobial therapy that have profound effect on this symbiotic relationship for both the individual and people as a whole. Another, more pragmatic, reason to be familiar with the usual flora is to aid in the interpretation of clinical culture results in samples where members of the usual flora may be present but not necessarily causing disease.
Long lists of organisms that can be found colonizing various parts of the human body are published but are impossible to remember and therefore are of little practical use. Suffice it to say that hundreds of different types of microorganisms can inhabit the human body without causing disease. There are several practical generalizations that can be remembered which aid the clinician in making assessment about importance of microbiologic findings:
- Skin is colonized with Coagulase Negative Staphylococci and Propionibacterium acnes. The finding of either of these organisms in samples obtained on or through skin must be interpreted with caution.
- The oropharynx is heavily colonized with viridans streptococci and many anaerobic bacteria.
- The colon is heavily colonized with Enterobacteriaciae (esp. E. coli), Enterococci, and many anaerobic organisms (esp. Bacteroides fragilis).
- The vagina in child-bearing age women is heavily colonized with lactobacilli.
There are no “normals” in microbiology. Repeated isolation of an organism especially in the context of signs of inflammation should increase the suspicion that an organism commonly considered a “contaminant” is causing disease.
A call to the microbiologist will often be of great help when in doubt.