Application of molecular techniques to the study of hospital infection

Abstract

Nosocomial infections are an important source of morbidity and mortality in hospital settings, afflicting an estimated 2 million patients in United States each year. This number represents up to 5% of hospitalized patients and results in an estimated 88,000 deaths and 4.5 billion dollars in excess health care costs. Increasingly, hospital-acquired infections with multidrug-resistant pathogens represent a major problem in patients. Understanding pathogen relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of pathogen typing is to determine whether epidemiologically related isolates are also genetically related. To determine molecular relatedness of isolates for epidemiologic investigation, new technologies based on DNA, or molecular analysis, are methods of choice. These DNA-based molecular methodologies include pulsed-field gel electrophoresis (PFGE), PCR-based typing methods, and multilocus sequence analysis. Establishing clonality of pathogens can aid in the identification of the source (environmental or personnel) of organisms, distinguish infectious from noninfectious strains, and distinguish relapse from reinfection. The integration of molecular typing with conventional hospital epidemiologic surveillance has been proven to be cost-effective due to the associated reduction in the number of nosocomial infections. Cost-effectiveness is maximized through the collaboration of the laboratory, through epidemiologic typing, and the infection control department during epidemiologic investigations.

FIG. 1.

Flow chart comparison of the different procedural steps used for various molecular typing techniques. dNTPs, deoxynucleoside triphosphates.

FIG. 2.

Dendrogram of pulsed-field gel electrophoresis analysis of community-associated methicillin-resistant Staphylococcus aureus isolates (from this study).

FIG. 3.

Protein A gene map. Boxes indicate the genes coding for the initial sequence (S), the immunoglobulin G-binding regions (A to D), a region homologous to regions A to D (E), and the COOH terminus (X), which includes the short sequence repeats (Xr) and the cell wall attachment sequence (Xc). Primers are numbered from the 5′ end of the primer on the forward strand of Staphylococcus aureus. (Reprinted from reference with permission.)

FIG. 4.

Diagrammatic representation of the procedure used for MLST. The bacterial DNA sequence at the left represents different gene targets amplified using locus-specific primers (small arrows whose color matches the gene locus). The genes are amplified and sequenced from the locus-specific primers. The nucleotide sequences from the loci are compared to other sequences in an MLST database, and the allele name is assigned. The assigned alleles for each locus are combined to form the multilocus sequence type. dNTPs, deoxynucleoside triphosphates; ddNTPs, dideoxynucleoside triphosphates.

FIG. 5.

Distribution of multilocus genotypes, showing multilocus trees for Staphylococcus aureus. Trees are shown for each MLST gene and for the concatenated sequences of all seven genes (a) with a sample of 25 diverse strain types (b). Conserved division is marked with an arrow in each tree. (Reprinted from reference with permission.)