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Once organisms are isolated, they may be identified, and in some cases susceptibility testing needs to be performed. Bacteria and fungi grow as colonies on agar plates. The appearance of these colonies is often useful in determining the identity of the organism. Colonies may appear flat or raised, smooth or rough; may pit the agar; or may hemolyze red blood cells in blood-containing agar. Molds, for example, have very characteristic “fuzzy” growth on agar. Colonies of organisms such as S. aureus may be pigmented or may secrete a diffusible pigment, as seen with Pseudomonas aeruginosa. Skilled microbiologists often have a very good idea of the identification of a microorganism based solely on its colonial appearance.

In specimens that come from an area of the body with a resident microbiota, it is important to separate the colonies of organisms that may represent the resident microbiota from the colonies of organisms that may be pathogens. Much of the time, this can be done on the basis of colonial appearance. However, some potential pathogens, such as S. pneumoniae, a common cause of bacterial pneumonia, cannot be readily differentiated from viridans group streptococci, a member of the resident oropharyngeal microbiota. In patients with suspected bacterial pneumonia, a sputum specimen may be obtained. Sputum consists of secretions coughed up from the lower airways that are expectorated through the oropharynx and submitted for culture. Because they pass through the oropharynx, sputum specimens almost always contain viridans group streptococci. The appearance of colonies produced by viridans group streptococci is very similar to that produced by S. pneumoniae. To determine whether or not these colonies are S. pneumoniae, one must do tests based on the phenotypic characteristics of the organism; these are referred to as biochemical tests. The biochemical test that is done most often to distinguish between these two organisms is the disk diffusion test, in which the organism’s susceptibility to the compound optochin is examined. S. pneumoniae (Fig. 11) is susceptible to optochin, while the viridans group streptococci are not. On the basis of this easily performed test, the identity of S. pneumoniae can be determined from a sputum specimen.

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.

Figure 12

Figure 13

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.

Figure 14