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Category: Clinical Microbiology
Molecular Methods for Epidemiologic Typing of Microorganisms, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817435/9781555815271_Chap12_4-1.gif /docserver/preview/fulltext/10.1128/9781555817435/9781555815271_Chap12_4-2.gifAbstract:
The ability to identify specific strains within a species of pathogen is an important aid in the rational development of effective measures to prevent and control nosocomial infections. The efforts of both microbiologists and hospital epidemiologists are facilitated greatly by the availability of the newer molecular epidemiologic typing techniques. The variety of molecular epidemiologic tools available at present is considerable, and based on current experience the methods that appear to be the most practical and useful for both large- and small-scale epidemiologic studies are the DNA-based methods such as pulsed-field gel electrophoresis (Table 12.1-5). Although these methods clearly have limitations, they generally are a significant improvement over the more conventional typing methods, many of which are too cumbersome, insensitive, and time-consuming to be of practical value for epidemiologic evaluations. It is important to understand that no single technique is universally applicable and that the choice for a particular application is related to the species studied, the scope of the question posed, and the convenience of the technique. The techniques of molecular epidemiology are useful in answering real clinical and infection control questions and are not limited to research uses. Examples include distinguishing between relapse and reinfection in an individual patient and in tracking the spread of an individual strain of a bacterium or fungus within the hospital environment ( 1 – 3 ).
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S. aureus plasmid DNA preparations. Uncut, undigested plasmid DNA; EcoRI, DNA digested with EcoRI; HindIII, plasmid DNA digested with HindIII. Lanes: C, control strain SM818-73; 1, 2, and 3, isolates collected from a single patient. Isolate 3 does not have detectable plasmid DNA. The report for this group of isolates stated, “Based on restriction endonuclease analysis of plasmid DNA, these strains were grouped as follows: isolates 1 and 2 are identical to each other; isolate 3 is not related to isolates 1 and 2.”
S. aureus plasmid DNA preparations. Uncut, undigested plasmid DNA; EcoRI, DNA digested with EcoRI; HindIII, plasmid DNA digested with HindIII. Lanes: C, control strain SM818-73; 1, 2, and 3, isolates collected from a single patient. Isolate 3 does not have detectable plasmid DNA. The report for this group of isolates stated, “Based on restriction endonuclease analysis of plasmid DNA, these strains were grouped as follows: isolates 1 and 2 are identical to each other; isolate 3 is not related to isolates 1 and 2.”
Ribotype profiles of S. aureus isolates. Lanes: S, molecular size standards; 1 and 6, ribotype A; 2 and 5, ribotype B; 3, ribotype C; 4 and 7, ribotype D. Figure from H. D. Isenberg (ed.). 1998. Essential Procedures for Clinical Microbiology. ASM Press, Washington, DC.
Ribotype profiles of S. aureus isolates. Lanes: S, molecular size standards; 1 and 6, ribotype A; 2 and 5, ribotype B; 3, ribotype C; 4 and 7, ribotype D. Figure from H. D. Isenberg (ed.). 1998. Essential Procedures for Clinical Microbiology. ASM Press, Washington, DC.
Restriction fragment length polymorphism of S. aureus chromosomal DNA digested with SmaI. Switch time ramped from 10 to 90 s (running condition A [ Table 12.4.5-2 ]). Lanes 1 through 7 show different patterns. Lanes 8 and 9, 10 and 11, and 12 and 13 represent three pairs of indistinguishable patterns. This photo was kindly provided by Andreas Widmer.
Restriction fragment length polymorphism of S. aureus chromosomal DNA digested with SmaI. Switch time ramped from 10 to 90 s (running condition A [ Table 12.4.5-2 ]). Lanes 1 through 7 show different patterns. Lanes 8 and 9, 10 and 11, and 12 and 13 represent three pairs of indistinguishable patterns. This photo was kindly provided by Andreas Widmer.
Restriction fragment length polymorphism of S. maltophilia chromosomal DNA digested with SpeI (running condition A [ Table 12.4.5-2 ). Lanes 3 and 4 show identical patterns, while the patterns in lanes 5 and 6 are similar but not identical and represent subtypes of a single strain.
Restriction fragment length polymorphism of S. maltophilia chromosomal DNA digested with SpeI (running condition A [ Table 12.4.5-2 ). Lanes 3 and 4 show identical patterns, while the patterns in lanes 5 and 6 are similar but not identical and represent subtypes of a single strain.
Suggested enzymes and running conditions for some species
a Size range includes the majority of fragments created after digestion with the corresponding enzyme. Some species will have several fragments under 48.5 kbp; however, it is difficult to analyze them when the 48.5-kbp lambda ladder molecular size standard is used. In order to analyze those fragments, use Hind III-cut lambda as a molecular size standard and/or a lower switch time, such as 5 to 15 s.
b See Table 12.4.5-2.
Suggested enzymes and running conditions for some species
a Size range includes the majority of fragments created after digestion with the corresponding enzyme. Some species will have several fragments under 48.5 kbp; however, it is difficult to analyze them when the 48.5-kbp lambda ladder molecular size standard is used. In order to analyze those fragments, use Hind III-cut lambda as a molecular size standard and/or a lower switch time, such as 5 to 15 s.
b See Table 12.4.5-2.
Examples of running conditions a
a All electrophoretic conditions are at 13°C.
Examples of running conditions a
a All electrophoretic conditions are at 13°C.
Rinse solution preparation
Rinse solution preparation
Conjugate and substrate preparation
Conjugate and substrate preparation