Characterization Of Microorganisms Beyond Identification

Situations exist in which characterizing a microbial pathogen beyond identification provides important information for patient management and public health. In such situations, knowledge regarding an organism's virulence, resistance to antimicrobial agents, or related-ness to other strains of the same species can be extremely important. Although various phenotypic methods have been able to provide some of this information, the development of molecular technologies has greatly expanded our ability to generate this information in the diagnostic setting. This is especially true with regard to antimicrobial resistance and strain relatedness.

Detection of Antimicrobial Resistance

Like all phenotypic traits, those that render microorganisms resistant to antimicrobial agents are encoded on specific genes (for more information regarding antimicrobial resistance mechanisms, see Chapter 11). Therefore, molecular methods for gene amplification or hybridization can be used to detect antimicrobial resistance. In many ways, phenotypic methods for resistance detection are reliable and are the primary methods for antimicrobial susceptibility testing (see Chapter 12). However, the complexity of emerging resistance mechanisms often challenges the ability of commonly used susceptibility testing methods to detect clinically important resistance to antimicrobial agents.

Methods such as PCR play a role in the detection of certain resistance profiles that may not always readily be detected by phenotypic methods. Two such examples include detection of the van genes, which mediate vancomycin resistance among enterococci (see Figure 8-8), and the mec gene, which encodes resistance among staphylococci to all currently available drugs of the beta-

144 Part II GENERAL PRINCIPLES IN CLINICAL MICROBIOLOGY Table 8-4 Examples of Methods to Determine Strain Relatedness



Plasmid analysis

Simple to implement but cannot often discriminate because many bacterial species have few or no plasmids

Multilocus enzyme electrophoresis

Provides only an estimate of overall genetic relatedness and diversity (protein-based)

Muliocus sequence typing

Data are electronically portable and used as non-culture-based typing method; labor Intensive and expensive

Pulsed-field gel electrophoresis

Highly discriminatory but it is difficult to resolve bands of similar size and interlaboratory reproducibility is limited

Randomly amplified polymorphic DNA

High discriminatory power but poor laboratory interlaboratoiy and intralaboratory reproducibility due to short random primer sequences and low PCR annealing temperatures

Repetitive sequence-based PCR; manual and automated

Manual system—useful for strain typing but low rates of Interlaboratory reproducibility; suboptimal turn-around times (TATs).

Automated system—Increases reproducibility and decreased TATs

Bibotyping and PCR ribotyplng

Difficult to distinguish among different subtypes

lactam class (see Figure 8-9). Undoubtedly, conventional and molecular methods will both continue-to play key roles in the characterization of microbial resistance to antimicrobial agents.

Investigation of Strain Relatedness/ Pulsed-Field Gel Electrophoresis

An important component to recognizing and controlling disease outbreaks inside or outside of a hospital is identification of the reservoir and mode of transmission of die infectious agents , involved. This often requires establishing relatedness among the pathogens isolated during the outbreak. For example, if all the microbial isolates thought to be associated with a nosocomial infection outbreak are shown to be identical, or at least very closely related, then a common source or reservoir for those isolates should be sought. If the etiologic agents are not the same, other explanations for the outbreak must be investigated (see Chapter 64). Because each species of a microorganism comprises an almost limitless number of strains, identification of an organism to the species level is not sufficient for establishing relatedness. Strain typing, the process used to establish the relatedness among organisms belonging to the same species, is required.

Although phenotypic characteristics (e.g., biotyping, serotyping, antimicrobial susceptibility profiles) historically have been used to type strains, these methods often are limited by their inability to consistently discriminate between different strains, their labor intensity, or their lack of reproducibility. In contrast, certain molecular methods do not have these limitations and have enhanced strain-typing capabilities. The molecular typing methods either direcdy compare nucleotide sequences between strains or produce results that indirectly reflect similarities in nucleotide sequences among "outbreak" organisms. Indirect methods usually involve enzymatic digestion and electrophoresis of microbial DNA to produce RFLPs for comparison and analysis.

Several molecular methods have been investigated for establishing strain relatedness. Examples of these methods are in Table 8-4. The method chosen primarily depends on the extent to which the following four criteria proposed by Maslow and colleagues are met:

  • Typeability: the method's capacity to produce clearly interpretable results with most strains of the bacterial species to be tested
  • Reproducibility: the method's capacity to repeatedly obtain the same typing profile result with the same bacterial strain
  • Discriminatory power: the method's ability to produce results that clearly allow differentiation between unrelated strains of the same bacterial species
  • Practicality: the method should be versatile, relatively rapid, inexpensive, technically simple, and provide readily inteipretable results

The last criterion, practicality, is especially important for busy clinical microbiology laboratories that provide support for infection control and hospital epidemiology.

Among the molecular methods used for strain typing, pulsed-field gel electrophoresis (PFGE) meets most of MasloWs criteria for a good typing system and is frequently referred to as the microbial typing "gold standard" for the present. This method is applicable to most of the commonly encountered bacterial pathogens, particularly those frequently associated with nosocomial infections and outbreaks such as staphylococci (MRSA), enterococd (vancomydn-resistant enterococd), and gram-negative pathogens induding Escherichia coli, Klebsiella spp., Enterobader spp., and Acinetobacter spp. For

  1. Culture of Bacterial cells
  2. Cell lysis and release of DNA
  3. Enzymatic digestion of DNA
  4. Gel electrophoresis and restriction fragment length polymorphism (RFLP) analysis

Isolate 1

Isolate 2

Isolate 1

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