Figure 11 Left disk, optochin; right disk, oxacillin.
Bacteria are typically identified on the basis of colonial morphology, Gram stain reaction, the primary isolation media on which the organism is growing, and biochemical and serologic tests of various degrees of complexity. Figures 12 and 13 are flow charts that give fairly simple means of distinguishing commonly encountered human pathogens. Yeasts are identified in much the same way that bacteria are, while molds are generally identified on the basis of the arrangement of microscopic reproductive structures called conidia. It is important to accurately identify bacteria and fungi because certain organisms (e.g., B. pertussis) are the cause of certain clinical syndromes (in this case, whooping cough). Other bacteria (e.g., Staphylococcus epidermidis) may represent contamination in a clinical specimen (e.g., a wound culture). The accurate identification of a bacterium or fungus may help determine what role a particular microbe may be having in the patient’s disease process.
Antimicrobial susceptibility typically is performed on rapidly growing bacteria if the organism is believed to play a role in the patient’s illness and if the profile of antimicrobial agents to which the organism is susceptible is not predictable. Let’s take three clinical scenarios to explain this concept.
First, a patient with a “strep throat” has group A streptococci recovered from his throat. Although the organism is clearly playing a role in the illness of this patient, antimicrobial susceptibility testing is not warranted. This organism is uniformly susceptible to first-line therapy—penicillin—and is susceptible more than 98% of the time to second-line therapy—the macrolide antibiotics such as erythromycin—although recent reports suggest that erythromycin resistance is becoming more frequent in this organism.
Second, a patient presents with a leg abscess from which S. aureus is recovered. Susceptibility testing is indicated because some strains are resistant to the first-line drugs used to treat this infection—semisynthetic penicillins, including oxacillin and dicloxacillin—and the second-line drug, clindamycin. In this situation, the patient may be started on empiric antimicrobial therapy until the susceptibility of the organism is known. If the organism is resistant to the agent used for empiric therapy, then the patient should be treated with an alternative antimicrobial agent to which the organism is susceptible.
The third scenario is more subtle. A patient comes to the hospital with a high fever. He has two sets of blood cultures drawn in the emergency department. Two days later, S. epidermidis is recovered from one of these blood culture sets. As with S. aureus, this organism may show resistance to a variety of antimicrobial agents that are used to treat infected patients. However, no susceptibility testing is done by the laboratory, and this practice is acceptable to the clinician caring for the patient. Why? S. epidermidis is a component of skin microbiota and may have contaminated the culture. If the laboratory had performed the susceptibility testing without considering that this isolate was a potential contaminant, they would be validating that the isolate was clinically significant. In this setting, the laboratory should only do susceptibility testing if instructed to by the caregiver, who is in a better position to know if this organism is clinically important.
There are several approaches to antibacterial susceptibility testing. All the approaches are highly standardized to ensure that the susceptibility results will be consistent from laboratory to laboratory. Screening of selected organisms for resistance to specific antimicrobial agents is one strategy that is frequently used, especially with the emergence of resistance in three organisms: S. aureus to cefoxitin to predict oxacillin resistance, S. pneumoniae to penicillin, and Enterococcus faecium and Enterococcus faecalis to vancomycin. Other strategies are to determine susceptibility to a preselected battery of antimicrobial agents using automated or manual systems that determine the MIC of antibiotics to the organism being tested or by using the disk diffusion susceptibility testing technique.
A novel approach to susceptibility testing is to perform MIC determinations using the E-test. The E-test is a plastic strip that contains a gradient of a specific antimicrobial agent. This strip is applied to a lawn of bacteria on an agar plate. Where the zone of inhibition intersects with the strip is the MIC value of that antibiotic for the organism tested. This test has many applications but is used most frequently for determining penicillin MIC values for S. pneumoniae isolates that show resistance to penicillin in the screening test previously described (Fig. 14).
Susceptibility testing is performed with increasing frequency on Candida spp. other than C. albicans but is rarely done on other yeasts and almost never on molds. Because of their slow growth, special susceptibility testing techniques are used for the mycobacteria.
Tissue culture for Chlamydia and viruses
Both Chlamydia, a bacterium, and the viruses are obligate intracellular parasites. As such, they do not grow on artificial media, as fungi and other bacteria do. Rather, they can only grow by parasitizing living animal cells (including human cells) that are maintained by continuous tissue culture. Animals such as mice, or chicken eggs, can be inoculated in an attempt to isolate certain viruses, but this approach is rarely done. Tissue culture for Chlamydia may still be attempted, especially in situations where the detection of C. trachomatis is at issue in a legal proceeding, such as a case of sexual abuse of a child. However, molecular detection has become the standard method for diagnosis of C. trachomatis infection.
Tissue culture is still an important technique for the detection of viruses in many laboratories, though laboratories are converting to molecular methods for viral detection at an increasing rate. Herpes simplex virus can be isolated from skin and genital tract lesions, often within the first 24 hours of incubation. Another herpesvirus, varicella-zoster virus, the etiologic agent of chicken pox and herpes zoster, can also be isolated from skin lesions, but it typically requires 3 to 7 days to grow. The enteroviruses are the major etiologic agents of aseptic meningitis and can be isolated from cerebrospinal fluid, but at a significantly reduced rate compared with molecular detection.
A modification of the tissue culture technique is done to detect cytomegalovirus and several respiratory viruses in clinical specimens called rapid centrifugation cultures or shell vial cultures. In this method, the specimen is centrifuged onto tissue culture cells that are growing on a round glass coverslip inside a vial referred to as a shell vial. The cells are incubated for a brief period of time (24 to 72 hours) and then stained with fluorescent antibodies to detect the virus. This technique is much more rapid and sensitive than conventional tissue culture but is still less sensitive than molecular detection.
SEROLOGY
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