Course: Medical Microbiology, PAMB 650/720 Lecture 33: Culture and identification of infectious agents Faculty: Dr.

Alvin Fox, Phone: 733 3288, E-mail: afox@med.sc.edu, Suggested reading: Murray, Fifth Edition, Chapter 2, 16-18, 21 KEY WORDS Isolation (culture) Agar plate/colonies Liquid media

2008/2009

Office: C-19, Blg. 2

Identification after culture 1. Biochemical (physiological) tests 2. Genetic tests (genomics): PCR Sequencing Restriction digestion DNA-DNA homology (probes) 3. Immunological 4. Fatty acid profiling 5. Protein profiling (proteomics) Non culture based detection Polymerase chain reaction (PCR) Antigen detection Stain (e.g. Gram strain) Serology (antibody levels)

Identification & taxonomy Family Genus Species Type Isolate Taxonomy

The basis of bacterial identification is rooted in taxonomy. Taxonomy is concerned with cataloging bacterial species and nowadays generally uses molecular biology (genetic) approaches. It is now recognized that many of the classical (physiology-based) schemes for differentiation of bacteria provide little insight into their genetic relationships and in some instances are scientifically incorrect. New information has resulted in renaming of certain bacterial species and in some instances has required totally re-organizing relationships within and between many bacterial families. Genetic methods provide much more precise identification of bacteria but are more difficult to perform than physiology-based methods. Family: a group of related genera. Genus: a group of related species. Species: a group of related strains. Type: sets of strain within a species (e.g. biotypes [example, phage recognition], serotypes). Strain: one line or a single isolate of a particular species.

The most commonly used term is the species name (e.g. Streptococcus pyogenes or Streptococcus pyogenes abbreviation S. pyogenes). There is always two parts to the species name one defining the genus in this case "Streptococcus" and the other the species (in this case "pyogenes"). The genus name is always capitalized but the species name is not. Both species and genus are underlined or in italics. Nowadays underlining is rarely used. The Diagnostic Laboratory The diagnostic laboratory uses taxonomic principles to identify bacterial species from patients. When patients are suspected of having a bacterial infection, it is usual to isolate visible colonies of the organism in pure culture (on agar plates) and then speciate the organism. Physiological methods for speciation of bacteria (based on morphological and metabolic characteristics) are simple to perform, reliable and easy to learn and are the backbone of the hospital clinical microbiology laboratory. More advanced reference laboratories, or laboratories associated with larger medical schools or hospitals additionally use genetic testing. Isolation by culture and identification of bacteria from patients aids treatment. Since infectious diseases (caused by different bacteria) have a variety of clinical courses and consequences. Susceptibility testing of isolates (i.e. establishing the minimal inhibitory concentration [MIC]) can help in selection of antibiotics for therapy. Recognizing that certain species (or strains) are being isolated atypically may suggest that an outbreak has occurred e.g. from contaminated hospital supplies or poor aseptic technique on the part of certain hospital personnel. Steps in diagnostic isolation and identification of bacteria Step 1: Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are streaked on culture plates and isolated colonies of bacteria (which are visible to the naked eye) appear after incubation for one - several days. It is not uncommon for cultures to contain more than one bacterial species (mixed cultures). If they are not separated from one another, subsequent tests cant be readily interpreted. Each colony consists of millions of bacterial cells. Observation of these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. Whether the organism requires oxygen for growth is another important differentiating characteristic. Step 2: Colonies are Gram stained and individual bacterial cells observed under the microscope. Step 3: The bacteria are speciated using these isolated colonies. This often requires an additional 24 hr of growth. Step 4: Antibiotic susceptibility testing is performed (optional).

THE GRAM STAIN, a colony is dried on a slide and treated as follows: 2

Step 1: Staining with crystal violet. Step 2: Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue. Step 3: Extraction with alcohol or other solvent. Gram negative bacteria are generally decolorized but Gram positive are not. Step 4: Counterstaining with safranin. Gram positive bacteria are already stained with crystal violet and remain purple or blue. Gram negative bacteria are stained pink. Under the microscope the appearance of bacteria are observed including: Are they Gram positive or negative? What is the morphology (rod, coccus, spiral, pleomorphic [variable form] etc)? Do cells occur singly or in chains, pairs etc? How large are the cells? There are other less commonly employed stains available (e.g. for spores and capsules). Another similar colony from the primary isolation plate is then examined for biochemical properties (e.g. will it ferment a sugar such as lactose). In some instances the bacteria are identified (e.g. by aggregation) with commercially available antibodies recognizing defined surface antigens. As noted above genetic tests are now widely used. Genetic characterization of bacteria The coding DNA of a representative strain of most major human pathogens has been sequenced, and this is referred to as genomics. This huge data-base of sequences is highly useful in helping design diagnostic tests. There can be substantial variability in sequences among individual strains. Rarely are more than a few representative genomes sequenced; it is simply too expensive and time consuming. Genetic tests are designed to detect individual genes that are conserved in sequence among strains of a species; multiple strains can be easily compared. Thus for practical reasons diagnostic tests are at the gene not genomic level. Metagenomics refers to genomic characterization of a population (e.g. the human flora, or microbiome). 1. Sequencing of 16S ribosomal RNA molecules (16S rRNA) has become the "gold standard" in bacterial taxonomy for differentiating bacterial species or populations. The molecule is approximately sixteen hundred nucleotides in length. 2. Once the sequence is known, specific genes (e.g. 16S rRNA) are detected by amplification using the polymerase chain reaction, PCR. The amplified product is then detected most simply by fluorescence (real time PCR) or by gel electrophoresis (molecular weight) of the product). 3. Restriction enzymes recognize and cut at specific DNA sequences; differentiate species by producing different size DNA fragments. 4. DNA-DNA homology (how well two strands of DNA from different bacteria bind [hybridize] is employed to compare the genetic relatedness of bacterial strains/species. If the DNA from two bacterial strains display a high degree of homology (i.e. they bind well) the strains are 3

considered to be members of the same species. DNA-DNA homology is rarely commonly used nowadays. However, probe-based diagnostic assays work on the same principle. 5. The guanine (G) + cytosine (C) content usually expressed as a percentage (% GC) is now only of historical value. Fatty acid profiling: a. The chain length (and other characteristics) of structural (long chain) fatty acid monomers present in the membranes of bacteria are determined. b. Characterization of secreted metabolic products (e.g. volatile alcohols and short chain fatty acids) is also occasionally employed. Proteomics and protein profiling: Proteomics refers to characterization of the entire set of proteins expressed (from the genome) under specific conditions. This is technically complex and time consuming and not used in routine bacterial diagnostics. A few proteins can be selected and identified (e.g. molecular weight and sequence) to identify bacterial species. Rapid diagnosis without prior culture: Certain human pathogens (including the causative agents of tuberculosis, Lyme disease and syphilis) either cant be isolated in the laboratory or grow extremely poorly. Successful isolation can be slow and in some instances currently impossible. Direct detection of bacteria without culture is possible in some cases; some examples are given below: Bacterial DNA sequences can be amplified directly from human body fluids using PCR. For example, great success has been achieved in rapid diagnosis of tuberculosis. A simple approach to rapid diagnosis (as an example of antigen detection) is used in many doctor's offices for the group A streptococcus. The patient's throat is swabbed and streptococcal antigen extracted directly from the swab (without prior bacteriological culture). The bacterial antigen is most simply detected by aggregation (agglutination) of antibody coated latex beads. A more current example of the test is described in powerpoint/lecture 44). Direct microscopic observation of certain clinical samples for the presence of bacteria can be helpful (e.g. detection of M. tuberculosis in sputum). However, sensitivity is poor and many false negatives occur. Serologic identification of an antibody response (in patient's serum) to the infecting agent can only be successful several weeks after an infection has occurred. This is commonly used in the diagnosis of Lyme disease. Epidemics: Defining that a particular strain is being isolated from multiple sources (e.g. tomatoes or spinach) suggests the occurrence of an outbreak. However this is not the usual function of the local hospital clinical laboratory but the state or federal laboratory system.