Laboratory Methods in Molecular Epidemiology: Bacterial Infections

Abstract

In infectious disease epidemiology, the laboratory plays a critical role in diagnosis, outbreak investigations, surveillance, and characterizing biologic properties of microbes associated with their transmissibility, resistance to anti-infectives, and pathogenesis. The laboratory can inform and refine epidemiologic study design and data analyses. In public health, the laboratory functions to assess effect of an intervention. In addition to research laboratories, the new-generation molecular microbiology technology has been adapted into clinical and public health laboratories to simplify, accelerate, and make precise detection and identification of infectious disease pathogens. This technology is also being applied to subtype microbes to conduct investigations that advance our knowledge of epidemiology of old and emerging infectious diseases. Because of the recent explosive progress in molecular microbiology technology and the vast amount of data generated from the applications of this technology, this Microbiology Spectrum Curated Collection: Advances in Molecular Epidemiology of Infectious Diseases describes these methods separately for bacteria, viruses, and parasites. This review discusses past and current advancements made in laboratory methods used to conduct epidemiologic studies of bacterial infections. It describes methods used to subtype bacterial organisms based on molecular microbiology techniques, following a discussion on what is meant by bacterial "species" and "clones." Discussions on past and new genotyping tests applied to epidemiologic investigations focus on tests that compare electrophoretic band patterns, hybridization matrices, and nucleic acid sequences. Applications of these genotyping tests to address epidemiologic issues are detailed elsewhere in other reviews of this series. *This article is part of a curated collection.

FIGURE 1

Repetitive element PCR (rep-PCR). The repeat double-stranded DNA sequences (light grey segments) are targeted as annealing sites for outwardly oriented complementary oligonucleotide primers, which amplify spaces located between these repeats (indicated as dark segments). Only one set of primers is needed to amplify the different segments, which generates amplicons of different sizes, shown at the end of each arrow, that can then be resolved by AGE for comparison of band patterns. (Illustrated by Paolo Harris Paz.) (Photo inset) AGE of band patterns generated by ERIC2 PCR analysis (a type of rep-PCR) of E. coli strains isolated from patients with community-acquired urinary tract infection. (Photo by Reina Yamaji.)

FIGURE 2

Conventional gel electrophoresis (left) and pulsed-field gel electrophoresis (PFGE) (right). In conventional gel electrophoresis, negatively charged DNA fragments migrate in one direction towards the positively charged electrode. In PFGE, the electrical field is applied along a contour of a hexagonal array of electrodes powered to generate two alternating electric field vectors, which allows linear pieces of DNA to “snake through” the path of least resistance along its migration in the agarose gel matrix. This allows large DNA fragments to be resolved to generate band patterns. (Illustrated by Paolo Harris Paz.) (Photo inset) PFGE of extraintestinal pathogenic E. coli isolates. (Photo by Meena Ramchandani.)

FIGURE 3

Multilocus sequence typing (MLST) of Staphylococcus aureus. A 400- to 600-bp region of housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) is amplified by PCR and the amplified products are sequenced. The sequences of the 7 loci are then concatenated, and all the concatenated sequences representing different genotypes are aligned to generate a tree, which can be used to depict strain relatedness. The analysis of similarity by comparison of concatenated sequence is called multilocus sequence analysis. The allele sequences can be uploaded onto an automated submission system (e.g., http://pubmlst.org/ or http://www.mlst.net/), which allows the database curators to provide an ST designation (ST number). Pairwise comparison of allele profiles can be performed by minimal spanning tree analysis using algorithms such as eBURST (http://eburst.mlst.net/default.asp). (Illustrated by Paolo Harris Paz.)