Molecular Epidemiology of Microorganisms: Methods and Protocols

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Bacterial typing of space-time clusters has identified unsuspected linkages triggering investigations, as well as demonstrating that apparent clusters were not related, ruling out need for investigation [ 10 ]. Molecular typing also facilitates the detection of chains of transmission. Molecular typing led to a reassessment of the epidemiology of tuberculosis in the United States by establishing that tuberculosis does not require prolonged contact but can be transmitted in casual settings [ 11 ].

Typing also allows us to relate clinical outcome to strain types, distinguishing recent tuberculosis infection from reactivation of disease, [ 12 ] and establishing that an individual can be infected with a second, different tuberculosis strain following initial infection [ 13 ]. When the investigator needs to identify potential outbreaks by typing surveillance isolates, or to distinguish between point source and propagated outbreaks, a more discriminatory technique is required.

In a common or point source outbreak we expect the causative agent to be similar in all infected persons. Therefore, a more discriminatory technique is necessary to determine if a space-time cluster of isolates detected via surveillance represents a potential outbreak compared to a technique for typing isolates already epidemiologically linked.

In a propagated outbreak or when tracking chains of transmission, the genetic sequence of the bacteria may be slightly different at the end compared to the beginning of the outbreak how fast this occurs depends on the bacteria, however. If the bacteria are naturally competent, i. Using a typing technique that allows classification consistent with phylogenetic relationships e. Unfortunately, many typing techniques are analogous to nominal scales, e. Even for PFGE, which can be used to assess relatedness, similarity may vary by choice or number of restriction enzymes used.

Advances in molecular genetics have facilitated the description of the genetic diversity of bacterial populations. Molecular genetic techniques have been used to distinguish if there have been independent spontaneous mutations leading to antibiotic resistance or if resistance was transmitted between strains via a mobile genetic element.

In other applications molecular genetic techniques have determined the flow of infection from one group to another. These descriptive molecular epidemiologic studies often use strains collected from disparate areas and the epidemiologic and clinical information is minimal or non-contributory. In this case the chosen bacterial typing technique must be interpretable in terms of genetic distance phylogeny for the given time period and organism.

Further, the technique should reflect whether the hypothesis is of clonal spread of a strain or of a mobile genetic element, e. Some typing techniques are based on conserved genes within the bacterial genome, e. On average, when bacterial strains are compared using a genetic typing technique, there are fewer genetic differences between bacterial strains in the conserved genes than variable genes.

Thus, typing techniques based on differences in conserved genes, such as MLST, will place strains into fewer, larger, groups, than typing techniques based on more variable genes, such as PFGE.

Molecular Epidemiology of Microorganisms

For bacterial characteristics that are dependent both on the conserved and variable portions of the genome, such as virulence, the use of multiple typing techniques may be helpful, see, for example, [ 16 ]. Selection of the appropriate typing technique and a valid interpretation of the results for studies of distribution of bacterial types and the determinants of that distribution is easiest when at least some preliminary data are available. For example, knowledge of the rarity of the observed groups in the community, propensity of the species to acquire insertion elements or phage, the timing of strain collection and the evolutionary clock of the organism, that is, how quickly mutations occur or horizontal elements are acquired provides important information for both technique selection and interpretation of resulting findings.

The identification of pathogenic factors is an exercise in identifying what is different between strains causing and not causing disease. This identification proceeds in the manner of a case-control study with the bacterial agent as the unit of analysis [see, for example, [ 17 ]]. Standard epidemiologic study design issues apply: the study population must include both disease-causing and commensal isolates.

Most disease-causing strains will predominate in a culture; non-pathogenic, or commensal organisms are often comprised of a mixture of strains of the same species. The investigator must select isolates for study accordingly. For example, E.

Advances in Chemical and Biological Methods to Identify Microorganisms—From Past to Present

Typically an individual has several E. The investigator must decide if the predominant isolate in the bowel flora is the one of interest or if several isolates should be selected for testing. If the objective were to link the bowel to the urinary tract flora, then choosing only the predominant bowel strain would not be sufficient. Identifying common elements generating pathogenicity may be the study objective: when the typing technique is unable to discriminate between pathogenic and diverse commensal isolates, epidemiologic and clinical information should be used to make that distinction, such as grouping together E.

Pathogenicity determinants are often present on transferable genetic material, such as plasmids, pathogenicity islands, phages, etc. Transferable genetic material has a genetic history distinct from the rest of the host bacterial genome. In this case, phylogenetic analyses of these elements can provide useful information.

For example, pathogenicity islands PAIs have been associated with a variety of conditions, including diarrhea and urinary tract infection [ 18 - 20 ]; specific virulence factor genes found on the PAIs encode for proteins that contribute directly to disease. The application and interpretation of bacterial typing tools in epidemiologic studies requires understanding of both the strengths and limitations of the chosen bacterial typing technique as well as the epidemiologic study design to answer the research question.

Beyond standard reliability, validity and cost considerations, key characteristics of a typing technique are 1 the ability to discriminate between strains and 2 a biologic basis for grouping strains with apparently different types. The level of discrimination required and need to be able to group strains depends on the research question.

Similar to the desirability of including a statistician in the design phase so that the study design will result in appropriate data for the desired analysis, integrating an expert in the different typing techniques during the design phase will improve how well the research protocol fits the question s of interest. All authors contributed to discussions leading to the manuscript, critiqued multiple drafts and approved the final manuscript. The authors thank the members of the Center for Molecular and Clinical Epidemiology of Infectious Diseases faculty discussion group for their insights into this topic.

National Center for Biotechnology Information , U. Journal List Epidemiol Perspect Innov v. Epidemiol Perspect Innov. Published online Nov Author information Article notes Copyright and License information Disclaimer. Corresponding author. Betsy Foxman: ude. Received Jun 13; Accepted Nov This article has been cited by other articles in PMC. Abstract A wide variety of bacterial typing systems are currently in use that vary greatly with respect to the effort required, cost, reliability and ability to discriminate between bacterial strains.

Keywords: molecular epidemiology, methods, bacteria, typing.

1 Introduction

Introduction Ever since Koch discovered how to grow bacteria in pure culture, the laboratory has been an integral component of epidemiologic studies of bacterial diseases. Analysis Bacterial typing systems Traditional typing systems for discriminating between bacteria from a single species have been based on phenotype, such as serotype, biotype, phage typing, or antibiogram susceptibility to one or more antibiotics. Species specific. Open in a separate window. In Molecular Epidemiology of Microorganisms: Methods and Protocols , readers will find readily reproducible methods for determining the molecular epidemiology of microorganisms, all of which provide effective discrimination of unrelated strains.

This volume covers a wide range of techniques which can be easily applied to the investigation of transmissible diseases, directing researchers towards the most effective methods based on the particular information to be obtained. Describing both traditional and novel techniques, expert researchers present a series of methods-based chapters with applications to some of the most important microbes. Composed in the highly successful Methods in Molecular Biology series format, each chapter contains a brief introduction, step-by-step methods, a list of necessary materials, and a Notes section which shares tips on troubleshooting and avoiding known pitfalls.

It is intended for researchers in the field of molecular epidemiology of microorganisms.

Molecular Epidemiology of Microorganisms: Methods and Protocols

Students as well as experts in the field will find this book quite informative and useful. JavaScript is currently disabled, this site works much better if you enable JavaScript in your browser. Biomedical Sciences Medical Microbiology.

1. Introduction

To name a few examples, genotypic methods have enabled the identification of a large diversity of previously unknown taxa, the characterization of uncultivable bacteria, and facilitated metagenomics studies on large and diverse bacterial communities [ ]. Both clinical and research setting have provided in depth insights into bacterial virulence, pathogenesis, antibiotic resistance, and epidemiological typing, as well as identification of novel, emerging, and re-emerging species [ ].

In addition, the widespread use and availability of molecular tools for bacterial genotyping has resulted in high throughput analysis, more sensitive and discriminatory results, and rapid turn-around-times, which are only likely to get better with automated tools and data analysis pipelines. Most molecular methods for bacterial identification are based on some variation of DNA analysis, either amplification or sequencing based. While the advantages and limitations of these approaches vary, the choice of the technology employed depends on several factors including sample type clinical or research, single-species or mixed-species , depth and accuracy of results generated, resources and cost factors, as well as the turn-around-times expected.

The rapid amplification of nucleic acid targets from relatively lower starting material, makes PCR one of the most sensitive techniques available for detection of bacterial targets. PCR-based identification of bacterial DNA through amplification and sequencing of the 16S rRNA gene has become a standard molecular method, both in the laboratory as well as in clinical settings. The 16S rRNA gene is highly specific to each bacterial species and this makes it an ideal target for identification. The standard method involves PCR amplification of the 16S rRNA gene, followed by sequencing and comparison to known databases for identification.

PCR-based methods are not only faster than conventional culture-based methods but are also helpful in identification of bacteria that are difficult to grow in laboratory conditions. In one study, universal primers for the 16S rRNA gene were designed to identify bacteria in the root canals of patients with necrotic pulp tissue [ ]. The primers included ten putative bacterial pathogens commonly found in root canals with necrotic pulp.

After DNA extraction from the necrotic pulp, a PCR was run using universal primers, as well as species specific primers, and the products were analyzed using gel electrophoresis. Twenty-two of the 24 specimens tested positive with the universal bacterial primers. As expected, certain bacterial species such as Fusobacterium spp. Of these, PCR analysis revealed two samples that showed a product with the universal primers, but not with any of the 10 species-specific primer sets tested.

Sequencing of these PCR products revealed the presence of a close relative of the Olsenella genus, previously not associated with such infections. Though the 16S rRNA gene has emerged as a popular target for PCR-based identification, in cases where the 16S rRNA gene is identical in two closely related species, other conserved genes, such as rpoB, tuf, gyrA, gyrB, and heat shock proteins are used as targets [ , , ].

In research laboratories, PCR-based identification is a straightforward procedure with reliable results. However, when applied to clinical settings, various factors come into play that can influence PCR results. Clinical samples often have very few bacteria to begin with, they also require various preprocessing steps before the PCR is carried out, to remove PCR inhibitors and enable extraction of maximum bacteria from the sample without contamination [ , ].

Despite these concerns, PCR-based identification has been successfully and widely employed to detect and identify bacteria in clinical samples [ , , ]. RT-PCR can also be quantitative or semi-quantitative, using the Cq value cycle number at which fluorescence intensity rises above the detectable level to quantify the amount of DNA. It is also applied to numerous kinds of samples, right from identification of bacteria in milk, which are otherwise non-culturable [ ], to the identification of bacteria within soil ecosystems [ ].

Using universal primers that target conserved regions of 16S rRNA gene, an assay has been developed that can detect Acinetobacter baumannii , Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa [ ].

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The primers were able to successfully detect less than genomic DNA copies. This real-time based 16S rRNA PCR has also been used to identify or quantify bacterial loads in clinical infections such as chronic wound tissue [ ] and gastrointestinal mucosal biopsies [ ], and has also been applied in forensic investigations of saliva specimens [ ]. In addition, high resolution melting HRM is a rapid, reliable, accurate, and cost-effective emerging tool for genotyping bacteria, such as from the Lactobacillus casei group and both Gram-positive and Gram-negative bacterial pathogens [ , ].

This results in amplification of random, repetitive regions of template DNA, thereby providing a unique profile for bacterial identification [ ]. RAPD-PCR reactions can start from isolated DNA or crude bacterial lysates, which are then subject to amplification in the presence of a RAPD primer or set of primers and low levels of magnesium to enhance non-specific annealing [ ].

Amplified products are then subjected to standard agarose gel electrophoresis to generate unique RAPD fingerprints. This means it can be used to identify and type a diverse range of bacterial species that have either not been identified or for which no prior sequence data is available. Furthermore, it can be performed from whole bacteria directly, without the need for DNA isolation, and can be applied on Gram-positive and Gram-negative species [ ].

In India, 20 alkaline protease producing bacterial strains isolated from soil samples from various geographic regions were subjected to RAPD screening with a set of random primers [ ].

Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols Molecular Epidemiology of Microorganisms: Methods and Protocols
Molecular Epidemiology of Microorganisms: Methods and Protocols

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