# Modern Applications of Microbial Genetics

## Visualizing and Characterizing DNA, RNA, and Protein

### Learning Objectives

By the end of this section, you will be able to:
1. Explain the use of nucleic acid probes to visualize specific DNA sequences
2. Explain the use of gel electrophoresis to separate DNA fragments
3. Explain the principle of restriction fragment length polymorphism analysis and its uses
4. Compare and contrast Southern and northern blots
5. Explain the principles and uses of microarray analysis
6. Describe the methods uses to separate and visualize protein variants
7. Explain the method and uses of polymerase chain reaction and DNA sequencing
The sequence of a DNA molecule can help us identify an organism when compared to known sequences housed in a database. The sequence can also tell us something about the function of a particular part of the DNA, such as whether it encodes a particular protein. Comparing protein signatures—the expression levels of specific arrays of proteins—between samples is an important method for evaluating cellular responses to a multitude of environmental factors and stresses. Analysis of protein signatures can reveal the identity of an organism or how a cell is responding during disease.
The DNA and proteins of interest are microscopic and typically mixed in with many other molecules including DNA or proteins irrelevant to our interests. Many techniques have been developed to isolate and characterize molecules of interest. These methods were originally developed for research purposes, but in many cases they have been simplified to the point that routine clinical use is possible. For example, many pathogens, such as the bacterium , which causes stomach ulcers, can be detected using protein-based tests. In addition, an increasing number of highly specific and accurate DNA amplification-based identification assays can now detect pathogens such as antibiotic-resistant enteric bacteria, herpes simplex virus, varicella-zoster virus, and many others.

### Molecular Analysis of DNA

In this subsection, we will outline some of the basic methods used for separating and visualizing specific fragments of DNA that are of interest to a scientist. Some of these methods do not require knowledge of the complete sequence of the DNA molecule. Before the advent of rapid DNA sequencing, these methods were the only ones available to work with DNA, but they still form the basic arsenal of tools used by molecular geneticists to study the body’s responses to microbial and other diseases.

### Nucleic Acid Probing

DNA molecules are small, and the information contained in their sequence is invisible. How does a researcher isolate a particular stretch of DNA, or having isolated it, determine what organism it is from, what its sequence is, or what its function is? One method to identify the presence of a certain DNA sequence uses artificially constructed pieces of DNA called probes. Probes can be used to identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically. For example, DNA probes are used to detect the vaginal pathogens , , and .
To screen a genomic library for a particular gene or sequence of interest, researchers must know something about that gene. If researchers have a portion of the sequence of DNA for the gene of interest, they can design a DNA probe, a single-stranded DNA fragment that is complementary to part of the gene of interest and different from other DNA sequences in the sample. The DNA probe may be synthesized chemically by commercial laboratories, or it may be created by cloning, isolating, and denaturing a DNA fragment from a living organism. In either case, the DNA probe must be labeled with a molecular tag or beacon, such as a radioactive phosphorus atom (as is used for autoradiography) or a fluorescent dye (as is used in fluorescent in situ hybridization, or FISH), so that the probe and the DNA it binds to can be seen (). The DNA sample being probed must also be denatured to make it single-stranded so that the single-stranded DNA probe can anneal to the single-stranded DNA sample at locations where their sequences are complementary. While these techniques are valuable for diagnosis, their direct use on sputum and other bodily samples may be problematic due to the complex nature of these samples. DNA often must first be isolated from bodily samples through chemical extraction methods before a DNA probe can be used to identify pathogens.

### Agarose Gel Electrophoresis

There are a number of situations in which a researcher might want to physically separate a collection of DNA fragments of different sizes. A researcher may also digest a DNA sample with a restriction enzyme to form fragments. The resulting size and fragment distribution pattern can often yield useful information about the sequence of DNA bases that can be used, much like a bar-code scan, to identify the individual or species to which the DNA belongs.
Gel electrophoresis is a technique commonly used to separate biological molecules based on size and biochemical characteristics, such as charge and polarity. Agarose gel electrophoresis is widely used to separate DNA (or RNA) of varying sizes that may be generated by restriction enzyme digestion or by other means, such as the PCR ().
Due to its negatively charged backbone, DNA is strongly attracted to a positive electrode. In agarose gel electrophoresis, the gel is oriented horizontally in a buffer solution. Samples are loaded into sample wells on the side of the gel closest to the negative electrode, then drawn through the molecular sieve of the agarose matrix toward the positive electrode. The agarose matrix impedes the movement of larger molecules through the gel, whereas smaller molecules pass through more readily. Thus, the distance of migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling a longer distance through the gel. Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run on the same gel. To separate very large DNA fragments, such as chromosomes or viral genomes, agarose gel electrophoresis can be modified by periodically alternating the orientation of the electric field during pulsed-field gel electrophoresis (PFGE). In PFGE, smaller fragments can reorient themselves and migrate slightly faster than larger fragments and this technique can thus serve to separate very large fragments that would otherwise travel together during standard agarose gel electrophoresis. In any of these electrophoresis techniques, the locations of the DNA or RNA fragments in the gel can be detected by various methods. One common method is adding ethidium bromide, a stain that inserts into the nucleic acids at non-specific locations and can be visualized when exposed to ultraviolet light. Other stains that are safer than ethidium bromide, a potential carcinogen, are now available.

### Restriction Fragment Length Polymorphism (RFLP) Analysis

Restriction enzyme recognition sites are short (only a few nucleotides long), sequence-specific palindromes, and may be found throughout the genome. Thus, differences in DNA sequences in the genomes of individuals will lead to differences in distribution of restriction-enzyme recognition sites that can be visualized as distinct banding patterns on a gel after agarose gel electrophoresis. Restriction fragment length polymorphism (RFLP) analysis compares DNA banding patterns of different DNA samples after restriction digestion ().
RFLP analysis has many practical applications in both medicine and forensic science. For example, epidemiologists use RFLP analysis to track and identify the source of specific microorganisms implicated in outbreaks of food poisoning or certain infectious diseases. RFLP analysis can also be used on human DNA to determine inheritance patterns of chromosomes with variant genes, including those associated with heritable diseases or to establish paternity.
Forensic scientists use RFLP analysis as a form of DNA fingerprinting, which is useful for analyzing DNA obtained from crime scenes, suspects, and victims. DNA samples are collected, the numbers of copies of the sample DNA molecules are increased using PCR, and then subjected to restriction enzyme digestion and agarose gel electrophoresis to generate specific banding patterns. By comparing the banding patterns of samples collected from the crime scene against those collected from suspects or victims, investigators can definitively determine whether DNA evidence collected at the scene was left behind by suspects or victims.

### Southern Blots and Modifications

Several molecular techniques capitalize on sequence complementarity and hybridization between nucleic acids of a sample and DNA probes. Typically, probing nucleic-acid samples within a gel is unsuccessful because as the DNA probe soaks into a gel, the sample nucleic acids within the gel diffuse out. Thus, blotting techniques are commonly used to transfer nucleic acids to a thin, positively charged membrane made of nitrocellulose or nylon. In the Southern blot technique, developed by Sir Edwin Southern in 1975, DNA fragments within a sample are first separated by agarose gel electrophoresis and then transferred to a membrane through capillary action (). The DNA fragments that bind to the surface of the membrane are then exposed to a specific single-stranded DNA probe labeled with a radioactive or fluorescent molecular beacon to aid in detection. Southern blots may be used to detect the presence of certain DNA sequences in a given DNA sample. Once the target DNA within the membrane is visualized, researchers can cut out the portion of the membrane containing the fragment to recover the DNA fragment of interest.
Variations of the Southern blot—the dot blot, slot blot, and the spot blot—do not involve electrophoresis, but instead concentrate DNA from a sample into a small location on a membrane. After hybridization with a DNA probe, the signal intensity detected is measured, allowing the researcher to estimate the amount of target DNA present within the sample.
A colony blot is another variation of the Southern blot in which colonies representing different clones in a genomic library are transferred to a membrane by pressing the membrane onto the culture plate. The cells on the membrane are lysed and the membrane can then be probed to determine which colonies within a genomic library harbor the target gene. Because the colonies on the plate are still growing, the cells of interest can be isolated from the plate.
In the northern blot, another variation of the Southern blot, RNA (not DNA) is immobilized on the membrane and probed. Northern blots are typically used to detect the amount of mRNA made through gene expression within a tissue or organism sample.

### Microarray Analysis

Another technique that capitalizes on the hybridization between complementary nucleic acid sequences is called microarray analysis. Microarray analysis is useful for the comparison of gene-expression patterns between different cell types—for example, cells infected with a virus versus uninfected cells, or cancerous cells versus healthy cells ().
Typically, DNA or cDNA from an experimental sample is deposited on a glass slide alongside known DNA sequences. Each slide can hold more than 30,000 different DNA fragment types. Distinct DNA fragments (encompassing an organism’s entire genomic library) or cDNA fragments (corresponding to an organism’s full complement of expressed genes) can be individually spotted on a glass slide.
Once deposited on the slide, genomic DNA or mRNA can be isolated from the two samples for comparison. If mRNA is isolated, it is reverse-transcribed to cDNA using reverse transcriptase. Then the two samples of genomic DNA or cDNA are labeled with different fluorescent dyes (typically red and green). The labeled genomic DNA samples are then combined in equal amounts, added to the microarray chip, and allowed to hybridize to complementary spots on the microarray.
Hybridization of sample genomic DNA molecules can be monitored by measuring the intensity of fluorescence at particular spots on the microarray. Differences in the amount of hybridization between the samples can be readily observed. If only one sample’s nucleic acids hybridize to a particular spot on the microarray, then that spot will appear either green or red. However, if both samples’ nucleic acids hybridize, then the spot will appear yellow due to the combination of the red and green dyes.
Although microarray technology allows for a holistic comparison between two samples in a short time, it requires sophisticated (and expensive) detection equipment and analysis software. Because of the expense, this technology is typically limited to research settings. Researchers have used microarray analysis to study how gene expression is affected in organisms that are infected by bacteria or viruses or subjected to certain chemical treatments.

### Molecular Analysis of Proteins

In many cases it may not be desirable or possible to study DNA or RNA directly. Proteins can provide species-specific information for identification as well as important information about how and whether a cell or tissue is responding to the presence of a pathogenic microorganism. Various proteins require different methods for isolation and characterization.

### Polyacrylamide Gel Electrophoresis

A variation of gel electrophoresis, called polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins. In PAGE, the gel matrix is finer and composed of polyacrylamide instead of agarose. Additionally, PAGE is typically performed using a vertical gel apparatus (). Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on their net charges. Alternatively, proteins can be denatured and coated with a negatively charged detergent called sodium dodecyl sulfate (SDS), masking the native charges and allowing separation based on size only. PAGE can be further modified to separate proteins based on two characteristics, such as their charges at various pHs as well as their size, through the use of two-dimensional PAGE. In any of these cases, following electrophoresis, proteins are visualized through staining, commonly with either Coomassie blue or a silver stain.

### Amplification-Based DNA Analysis Methods

Various methods can be used for obtaining sequences of DNA, which are useful for studying disease-causing organisms. With the advent of rapid sequencing technology, our knowledge base of the entire genomes of pathogenic organisms has grown phenomenally. We start with a description of the polymerase chain reaction, which is not a sequencing method but has allowed researchers and clinicians to obtain the large quantities of DNA needed for sequencing and other studies. The polymerase chain reaction eliminates the dependence we once had on cells to make multiple copies of DNA, achieving the same result through relatively simple reactions outside the cell.

### Polymerase Chain Reaction (PCR)

Most methods of DNA analysis, such as restriction enzyme digestion and agarose gel electrophoresis, or DNA sequencing require large amounts of a specific DNA fragment. In the past, large amounts of DNA were produced by growing the host cells of a genomic library. However, libraries take time and effort to prepare and DNA samples of interest often come in minute quantities. The polymerase chain reaction (PCR) permits rapid amplification in the number of copies of specific DNA sequences for further analysis (). One of the most powerful techniques in molecular biology, PCR was developed in 1983 by Kary Mullis while at Cetus Corporation. PCR has specific applications in research, forensic, and clinical laboratories, including:
1. determining the sequence of nucleotides in a specific region of DNA
2. amplifying a target region of DNA for cloning into a plasmid vector
3. identifying the source of a DNA sample left at a crime scene
4. analyzing samples to determine paternity
5. comparing samples of ancient DNA with modern organisms
6. determining the presence of difficult to culture, or unculturable, microorganisms in humans or environmental samples
PCR is an in vitro laboratory technique that takes advantage of the natural process of DNA replication. The heat-stable DNA polymerase enzymes used in PCR are derived from hyperthermophilic prokaryotes. , commonly used in PCR, is derived from the  bacterium isolated from a hot spring in Yellowstone National Park. DNA replication requires the use of primers for the initiation of replication to have free 3ʹ-hydroxyl groups available for the addition of nucleotides by DNA polymerase. However, while primers composed of RNA are normally used in cells, DNA primers are used for PCR. DNA primers are preferable due to their stability, and DNA primers with known sequences targeting a specific DNA region can be chemically synthesized commercially. These DNA primers are functionally similar to the DNA probes used for the various hybridization techniques described earlier, binding to specific targets due to complementarity between the target DNA sequence and the primer.
PCR occurs over multiple cycles, each containing three steps: denaturation, annealing, and extension. Machines called thermal cyclers are used for PCR; these machines can be programmed to automatically cycle through the temperatures required at each step (). First, double-stranded template DNA containing the target sequence is denatured at approximately 95 °C. The high temperature required to physically (rather than enzymatically) separate the DNA strands is the reason the heat-stable DNA polymerase is required. Next, the temperature is lowered to approximately 50 °C. This allows the DNA primers complementary to the ends of the target sequence to anneal (stick) to the template strands, with one primer annealing to each strand. Finally, the temperature is raised to 72 °C, the optimal temperature for the activity of the heat-stable DNA polymerase, allowing for the addition of nucleotides to the primer using the single-stranded target as a template. Each cycle doubles the number of double-stranded target DNA copies. Typically, PCR protocols include 25–40 cycles, allowing for the amplification of a single target sequence by tens of millions to over a trillion.
Natural DNA replication is designed to copy the entire genome, and initiates at one or more origin sites. Primers are constructed during replication, not before, and do not consist of a few specific sequences. PCR targets specific regions of a DNA sample using sequence-specific primers. In recent years, a variety of isothermal PCR amplification methods that circumvent the need for thermal cycling have been developed, taking advantage of accessory proteins that aid in the DNA replication process. As the development of these methods continues and their use becomes more widespread in research, forensic, and clinical labs, thermal cyclers may become obsolete.

### PCR Variations

Several later modifications to PCR further increase the utility of this technique. Reverse transcriptase PCR (RT-PCR) is used for obtaining DNA copies of a specific mRNA molecule. RT-PCR begins with the use of the reverse transcriptase enzyme to convert mRNA molecules into cDNA. That cDNA is then used as a template for traditional PCR amplification. RT-PCR can detect whether a specific gene has been expressed in a sample. Another recent application of PCR is real-time PCR, also known as quantitative PCR (qPCR). Standard PCR and RT-PCR protocols are not quantitative because any one of the reagents may become limiting before all of the cycles within the protocol are complete, and samples are only analyzed at the end. Because it is not possible to determine when in the PCR or RT-PCR protocol a given reagent has become limiting, it is not possible to know how many cycles were completed prior to this point, and thus it is not possible to determine how many original template molecules were present in the sample at the start of PCR. In qPCR, however, the use of fluorescence allows one to monitor the increase in a double-stranded template during a PCR reaction as it occurs. These kinetics data can then be used to quantify the amount of the original target sequence. The use of qPCR in recent years has further expanded the capabilities of PCR, allowing researchers to determine the number of DNA copies, and sometimes organisms, present in a sample. In clinical settings, qRT-PCR is used to determine viral load in HIV-positive patients to evaluate the effectiveness of their therapy.

### DNA Sequencing

A basic sequencing technique is the chain termination method, also known as the dideoxy method or the Sanger DNA sequencing method, developed by Frederick Sanger in 1972. The chain termination method involves DNA replication of a single-stranded template with the use of a DNA primer to initiate synthesis of a complementary strand, DNA polymerase, a mix of the four regular deoxynucleotide (dNTP) monomers, and a small proportion of dideoxynucleotides (ddNTPs), each labeled with a molecular beacon. The ddNTPs are monomers missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain (). Every time a ddNTP is randomly incorporated into the growing complementary strand, it terminates the process of DNA replication for that particular strand. This results in multiple short strands of replicated DNA that are each terminated at a different point during replication. When the reaction mixture is subjected to gel electrophoresis, the multiple newly replicated DNA strands form a ladder of differing sizes. Because the ddNTPs are labeled, each band on the gel reflects the size of the DNA strand when the ddNTP terminated the reaction.
In Sanger’s day, four reactions were set up for each DNA molecule being sequenced, each reaction containing only one of the four possible ddNTPs. Each ddNTP was labeled with a radioactive phosphorus molecule. The products of the four reactions were then run in separate lanes side by side on long, narrow PAGE gels, and the bands of varying lengths were detected by autoradiography. Today, this process has been simplified with the use of ddNTPs, each labeled with a different colored fluorescent dye or fluorochrome (), in one sequencing reaction containing all four possible ddNTPs for each DNA molecule being sequenced (). These fluorochromes are detected by fluorescence spectroscopy. Determining the fluorescence color of each band as it passes by the detector produces the nucleotide sequence of the template strand.
Since 2005, automated sequencing techniques used by laboratories fall under the umbrella of next generation sequencing, which is a group of automated techniques used for rapid DNA sequencing. These methods have revolutionized the field of molecular genetics because the low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to 600 base pairs) just in one day. Although several variants of next generation sequencing technologies are made by different companies (for example, 454 Life Sciences’ pyrosequencing and Illumina’s Solexa technology), they all allow millions of bases to be sequenced quickly, making the sequencing of entire genomes relatively easy, inexpensive, and commonplace. In 454 sequencing (pyrosequencing), for example, a DNA sample is fragmented into 400–600-bp single-strand fragments, modified with the addition of DNA adapters to both ends of each fragment. Each DNA fragment is then immobilized on a bead and amplified by PCR, using primers designed to anneal to the adapters, creating a bead containing many copies of that DNA fragment. Each bead is then put into a separate well containing sequencing enzymes. To the well, each of the four nucleotides is added one after the other; when each one is incorporated, pyrophosphate is released as a byproduct of polymerization, emitting a small flash of light that is recorded by a detector. This provides the order of nucleotides incorporated as a new strand of DNA is made and is an example of synthesis sequencing. Next generation sequencers use sophisticated software to get through the cumbersome process of putting all the fragments in order. Overall, these technologies continue to advance rapidly, decreasing the cost of sequencing and increasing the availability of sequence data from a wide variety of organisms quickly.
The National Center for Biotechnology Information houses a widely used genetic sequence database called GenBank where researchers deposit genetic information for public use. Upon publication of sequence data, researchers upload it to GenBank, giving other researchers access to the information. The collaboration allows researchers to compare newly discovered or unknown sample sequence information with the vast array of sequence data that already exists.

### Key Concepts and Summary

1. Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon (typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample.
2. Agarose gel electrophoresis allows for the separation of DNA molecules based on size.
3. Restriction fragment length polymorphism (RFLP) analysis allows for the visualization by agarose gel electrophoresis of distinct variants of a DNA sequence caused by differences in restriction sites.
4. Southern blot analysis allows researchers to find a particular DNA sequence within a sample whereas northern blot analysis allows researchers to detect a particular mRNA sequence expressed in a sample.
5. Microarray technology is a nucleic acid hybridization technique that allows for the examination of many thousands of genes at once to find differences in genes or gene expression patterns between two samples of genomic DNA or cDNA,
6. Polyacrylamide gel electrophoresis (PAGE) allows for the separation of proteins by size, especially if native protein charges are masked through pretreatment with SDS.
7. Polymerase chain reaction allows for the rapid amplification of a specific DNA sequence. Variations of PCR can be used to detect mRNA expression (reverse transcriptase PCR) or to quantify a particular sequence in the original sample (real-time PCR).
8. Although the development of Sanger DNA sequencing was revolutionary, advances in next generation sequencing allow for the rapid and inexpensive sequencing of the genomes of many organisms, accelerating the volume of new sequence data.

### Multiple Choice

### Fill in the Blank

### True/False

### Short answer

### Critical Thinking


# Modern Applications of Microbial Genetics

## Whole Genome Methods and Pharmaceutical Applications of Genetic Engineering

### Learning Objectives

By the end of this section, you will be able to:
1. Explain the uses of genome-wide comparative analyses
2. Summarize the advantages of genetically engineered pharmaceutical products
Advances in molecular biology have led to the creation of entirely new fields of science. Among these are fields that study aspects of whole genomes, collectively referred to as whole-genome methods. In this section, we’ll provide a brief overview of the whole-genome fields of genomics, transcriptomics, and proteomics.

### Genomics, Transcriptomics, and Proteomics

The study and comparison of entire genomes, including the complete set of genes and their nucleotide sequence and organization, is called genomics. This field has great potential for future medical advances through the study of the human genome as well as the genomes of infectious organisms. Analysis of microbial genomes has contributed to the development of new antibiotics, diagnostic tools, vaccines, medical treatments, and environmental cleanup techniques.
The field of transcriptomics is the science of the entire collection of mRNA molecules produced by cells. Scientists compare gene expression patterns between infected and uninfected host cells, gaining important information about the cellular responses to infectious disease. Additionally, transcriptomics can be used to monitor the gene expression of virulence factors in microorganisms, aiding scientists in better understanding pathogenic processes from this viewpoint.
When genomics and transcriptomics are applied to entire microbial communities, we use the terms metagenomics and metatranscriptomics, respectively. Metagenomics and metatranscriptomics allow researchers to study genes and gene expression from a collection of multiple species, many of which may not be easily cultured or cultured at all in the laboratory. A DNA microarray (discussed in the previous section) can be used in metagenomics studies.
Another up-and-coming clinical application of genomics and transcriptomics is pharmacogenomics, also called toxicogenomics, which involves evaluating the effectiveness and safety of drugs on the basis of information from an individual’s genomic sequence. Genomic responses to drugs can be studied using experimental animals (such as laboratory rats or mice) or live cells in the laboratory before embarking on studies with humans. Changes in gene expression in the presence of a drug can sometimes be an early indicator of the potential for toxic effects. Personal genome sequence information may someday be used to prescribe medications that will be most effective and least toxic on the basis of the individual patient’s genotype.
The study of proteomics is an extension of genomics that allows scientists to study the entire complement of proteins in an organism, called the proteome. Even though all cells of a multicellular organism have the same set of genes, cells in various tissues produce different sets of proteins. Thus, the genome is constant, but the proteome varies and is dynamic within an organism. Proteomics may be used to study which proteins are expressed under various conditions within a single cell type or to compare protein expression patterns between different organisms.
The most prominent disease being studied with proteomic approaches is cancer, but this area of study is also being applied to infectious diseases. Research is currently underway to examine the feasibility of using proteomic approaches to diagnose various types of hepatitis, tuberculosis, and HIV infection, which are rather difficult to diagnose using currently available techniques.E.O. List, D.E. Berryman, B. Bower, L. Sackmann-Sala, E. Gosney, J. Ding, S. Okada, and J.J. Kopchick. “The Use of Proteomics to Study Infectious Diseases.”
A recent and developing proteomic analysis relies on identifying proteins called biomarkers, whose expression is affected by the disease process. Biomarkers are currently being used to detect various forms of cancer as well as infections caused by pathogens such as  and .Mohan Natesan, and Robert G. Ulrich. “Protein Microarrays and Biomarkers of Infectious Disease.”
Other “-omic” sciences related to genomics and proteomics include metabolomics, glycomics, and lipidomics, which focus on the complete set of small-molecule metabolites, sugars, and lipids, respectively, found within a cell. Through these various global approaches, scientists continue to collect, compile, and analyze large amounts of genetic information. This emerging field of bioinformatics can be used, among many other applications, for clues to treating diseases and understanding the workings of cells.
Additionally, researchers can use reverse genetics, a technique related to classic mutational analysis, to determine the function of specific genes. Classic methods of studying gene function involved searching for the genes responsible for a given phenotype. Reverse genetics uses the opposite approach, starting with a specific DNA sequence and attempting to determine what phenotype it produces. Alternatively, scientists can attach known genes (called reporter genes) that encode easily observable characteristics to genes of interest, and the location of expression of such genes of interest can be easily monitored. This gives the researcher important information about what the gene product might be doing or where it is located in the organism. Common reporter genes include bacterial , which encodes beta-galactosidase and whose activity can be monitored by changes in colony color in the presence of X-gal as previously described, and the gene encoding the jellyfish protein green fluorescent protein (GFP) whose activity can be visualized in colonies under ultraviolet light exposure ().

### Recombinant DNA Technology and Pharmaceutical Production

Genetic engineering has provided a way to create new pharmaceutical products called recombinant DNA pharmaceuticals. Such products include antibiotic drugs, vaccines, and hormones used to treat various diseases.  lists examples of recombinant DNA products and their uses.
For example, the naturally occurring antibiotic synthesis pathways of various  spp., long known for their antibiotic production capabilities, can be modified to improve yields or to create new antibiotics through the introduction of genes encoding additional enzymes. More than 200 new antibiotics have been generated through the targeted inactivation of genes and the novel combination of antibiotic synthesis genes in antibiotic-producing Streptomyces hosts.Jose-Luis Adrio and Arnold L. Demain. “Recombinant Organisms for Production of Industrial Products.”
Genetic engineering is also used to manufacture subunit vaccines, which are safer than other vaccines because they contain only a single antigenic molecule and lack any part of the genome of the pathogen (see Vaccines). For example, a vaccine for hepatitis B is created by inserting a gene encoding a hepatitis B surface protein into a yeast; the yeast then produces this protein, which the human immune system recognizes as an antigen. The hepatitis B antigen is purified from yeast cultures and administered to patients as a vaccine. Even though the vaccine does not contain the hepatitis B virus, the presence of the antigenic protein stimulates the immune system to produce antibodies that will protect the patient against the virus in the event of exposure.U.S. Department of Health and Human Services. “Types of Vaccines.” 2013. http://www.vaccines.gov/more_info/types/#subunit. Accessed May 27, 2016. The Internet Drug List.
Genetic engineering has also been important in the production of other therapeutic proteins, such as insulin, interferons, and human growth hormone, to treat a variety of human medical conditions. For example, at one time, it was possible to treat diabetes only by giving patients pig insulin, which caused allergic reactions due to small differences between the proteins expressed in human and pig insulin. However, since 1978, recombinant DNA technology has been used to produce large-scale quantities of human insulin using E. coli in a relatively inexpensive process that yields a more consistently effective pharmaceutical product. Scientists have also genetically engineered E. coli capable of producing human growth hormone (HGH), which is used to treat growth disorders in children and certain other disorders in adults. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector. Eventually, genetic engineering will be used to produce DNA vaccines and various gene therapies, as well as customized medicines for fighting cancer and other diseases.

### RNA Interference Technology

In Structure and Function of RNA, we described the function of mRNA, rRNA, and tRNA. In addition to these types of RNA, cells also produce several types of small noncoding RNA molecules that are involved in the regulation of gene expression. These include antisense RNA molecules, which are complementary to regions of specific mRNA molecules found in both prokaryotes and eukaryotic cells. Non-coding RNA molecules play a major role in RNA interference (RNAi), a natural regulatory mechanism by which mRNA molecules are prevented from guiding the synthesis of proteins. RNA interference of specific genes results from the base pairing of short, single-stranded antisense RNA molecules to regions within complementary mRNA molecules, preventing protein synthesis. Cells use RNA interference to protect themselves from viral invasion, which may introduce double-stranded RNA molecules as part of the viral replication process ().
Researchers are currently developing techniques to mimic the natural process of RNA interference as a way to treat viral infections in eukaryotic cells. RNA interference technology involves using small interfering RNAs (siRNAs) or microRNAs (miRNAs) (). siRNAs are completely complementary to the mRNA transcript of a specific gene of interest while miRNAs are mostly complementary. These double-stranded RNAs are bound to DICER, an endonuclease that cleaves the RNA into short molecules (approximately 20 nucleotides long). The RNAs are then bound to RNA-induced silencing complex (RISC), a ribonucleoprotein. The siRNA-RISC complex binds to mRNA and cleaves it. For miRNA, only one of the two strands binds to RISC. The miRNA-RISC complex then binds to mRNA, inhibiting translation. If the miRNA is completely complementary to the target gene, then the mRNA can be cleaved. Taken together, these mechanisms are known as gene silencing.

### Key Concepts and Summary

1. The science of genomics allows researchers to study organisms on a holistic level and has many applications of medical relevance.
2. Transcriptomics and proteomics allow researchers to compare gene expression patterns between different cells and shows great promise in better understanding global responses to various conditions.
3. The various –omics technologies complement each other and together provide a more complete picture of an organism’s or microbial community’s (metagenomics) state.
4. The analysis required for large data sets produced through genomics, transcriptomics, and proteomics has led to the emergence of bioinformatics.
5. Reporter genes encoding easily observable characteristics are commonly used to track gene expression patterns of genes of unknown function.
6. The use of recombinant DNA technology has revolutionized the pharmaceutical industry, allowing for the rapid production of high-quality recombinant DNA pharmaceuticals used to treat a wide variety of human conditions.
7. RNA interference technology has great promise as a method of treating viral infections by silencing the expression of specific genes

### Multiple Choice

### Fill in the Blank

### True/False

### Short answer

### Critical Thinking


# Modern Applications of Microbial Genetics

## Gene Therapy

### Learning Objectives

By the end of this section, you will be able to:
1. Summarize the mechanisms, risks, and potential benefits of gene therapy
2. Identify ethical issues involving gene therapy and the regulatory agencies that provide oversight for clinical trials
3. Compare somatic-cell and germ-line gene therapy
Many types of genetic engineering have yielded clear benefits with few apparent risks. Few would question, for example, the value of our now abundant supply of human insulin produced by genetically engineered bacteria. However, many emerging applications of genetic engineering are much more controversial, often because their potential benefits are pitted against significant risks, real or perceived. This is certainly the case for gene therapy, a clinical application of genetic engineering that may one day provide a cure for many diseases but is still largely an experimental approach to treatment.

### Mechanisms and Risks of Gene Therapy

Human diseases that result from genetic mutations are often difficult to treat with drugs or other traditional forms of therapy because the signs and symptoms of disease result from abnormalities in a patient’s genome. For example, a patient may have a genetic mutation that prevents the expression of a specific protein required for the normal function of a particular cell type. This is the case in patients with Severe Combined Immunodeficiency (SCID), a genetic disease that impairs the function of certain white blood cells essential to the immune system.
Gene therapy attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into the patient’s genome. The nonmutated gene encodes a functional protein that the patient would otherwise be unable to produce. Viral vectors such as adenovirus are sometimes used to introduce the functional gene; part of the viral genome is removed and replaced with the desired gene (). More advanced forms of gene therapy attempt to correct the mutation at the original site in the genome, such as is the case with treatment of SCID.
So far, gene therapies have proven relatively ineffective, with the possible exceptions of treatments for cystic fibrosis and adenosine deaminase deficiency, a type of SCID. Other trials have shown the clear hazards of attempting genetic manipulation in complex multicellular organisms like humans. In some patients, the use of an adenovirus vector can trigger an unanticipated inflammatory response from the immune system, which may lead to organ failure. Moreover, because viruses can often target multiple cell types, the virus vector may infect cells not targeted for the therapy, damaging these other cells and possibly leading to illnesses such as cancer. Another potential risk is that the modified virus could revert to being infectious and cause disease in the patient. Lastly, there is a risk that the inserted gene could unintentionally inactivate another important gene in the patient’s genome, disrupting normal cell cycling and possibly leading to tumor formation and cancer. Because gene therapy involves so many risks, candidates for gene therapy need to be fully informed of these risks before providing informed consent to undergo the therapy.

### Oversight of Gene Therapy

Presently, there is significant oversight of gene therapy clinical trials. At the federal level, three agencies regulate gene therapy in parallel: the Food and Drug Administration (FDA), the Office of Human Research Protection (OHRP), and the Recombinant DNA Advisory Committee (RAC) at the National Institutes of Health (NIH). Along with several local agencies, these federal agencies interact with the institutional review board to ensure that protocols are in place to protect patient safety during clinical trials. Compliance with these protocols is enforced mostly on the local level in cooperation with the federal agencies. Gene therapies are currently under the most extensive federal and local review compared to other types of therapies, which are more typically only under the review of the FDA. Some researchers believe that these extensive regulations actually inhibit progress in gene therapy research. In 2013, the Institute of Medicine (now the National Academy of Medicine) called upon the NIH to relax its review of gene therapy trials in most cases.Kerry Grens. “Report: Ease Gene Therapy Reviews.”  However, ensuring patient safety continues to be of utmost concern.

### Ethical Concerns

Beyond the health risks of gene therapy, the ability to genetically modify humans poses a number of ethical issues related to the limits of such “therapy.” While current research is focused on gene therapy for genetic diseases, scientists might one day apply these methods to manipulate other genetic traits not perceived as desirable. This raises questions such as:
1. Which genetic traits are worthy of being “corrected”?
2. Should gene therapy be used for cosmetic reasons or to enhance human abilities?
3. Should genetic manipulation be used to impart desirable traits to the unborn?
4. Is everyone entitled to gene therapy, or could the cost of gene therapy create new forms of social inequality?
5. Who should be responsible for regulating and policing inappropriate use of gene therapies?
The ability to alter reproductive cells using gene therapy could also generate new ethical dilemmas. To date, the various types of gene therapies have been targeted to somatic cells, the non-reproductive cells within the body. Because somatic cell traits are not inherited, any genetic changes accomplished by somatic-cell gene therapy would not be passed on to offspring. However, should scientists successfully introduce new genes to germ cells (eggs or sperm), the resulting traits could be passed on to offspring. This approach, called germ-line gene therapy, could potentially be used to combat heritable diseases, but it could also lead to unintended consequences for future generations. Moreover, there is the question of informed consent, because those impacted by germ-line gene therapy are unborn and therefore unable to choose whether they receive the therapy. For these reasons, the U.S. government does not currently fund research projects investigating germ-line gene therapies in humans.

### Key Concepts and Summary

1. While gene therapy shows great promise for the treatment of genetic diseases, there are also significant risks involved.
2. There is considerable federal and local regulation of the development of gene therapies by pharmaceutical companies for use in humans.
3. Before gene therapy use can increase dramatically, there are many ethical issues that need to be addressed by the medical and research communities, politicians, and society at large.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Control of Microbial Growth

## Introduction

How clean is clean? People wash their cars and vacuum the carpets, but most would not want to eat from these surfaces. Similarly, we might eat with silverware cleaned in a dishwasher, but we could not use the same dishwasher to clean surgical instruments. As these examples illustrate, “clean” is a relative term. Car washing, vacuuming, and dishwashing all reduce the microbial load on the items treated, thus making them “cleaner.” But whether they are “clean enough” depends on their intended use. Because people do not normally eat from cars or carpets, these items do not require the same level of cleanliness that silverware does. Likewise, because silverware is not used for invasive surgery, these utensils do not require the same level of cleanliness as surgical equipment, which requires sterilization to prevent infection.
Why not play it safe and sterilize everything? Sterilizing everything we come in contact with is impractical, as well as potentially dangerous. As this chapter will demonstrate, sterilization protocols often require time- and labor-intensive treatments that may degrade the quality of the item being treated or have toxic effects on users. Therefore, the user must consider the item’s intended application when choosing a cleaning method to ensure that it is “clean enough.”


# Control of Microbial Growth

## Controlling Microbial Growth

### Learning Objectives

By the end of this section, you will be able to:
1. Compare disinfectants, antiseptics, and sterilants
2. Describe the principles of controlling the presence of microorganisms through sterilization and disinfection
3. Differentiate between microorganisms of various biological safety levels and explain methods used for handling microbes at each level
To prevent the spread of human disease, it is necessary to control the growth and abundance of microbes in or on various items frequently used by humans. Inanimate items, such as doorknobs, toys, or towels, which may harbor microbes and aid in disease transmission, are called fomites. Two factors heavily influence the level of cleanliness required for a particular fomite and, hence, the protocol chosen to achieve this level. The first factor is the application for which the item will be used. For example, invasive applications that require insertion into the human body require a much higher level of cleanliness than applications that do not. The second factor is the level of resistance to antimicrobial treatment by potential pathogens. For example, foods preserved by canning often become contaminated with the bacterium , which produces the neurotoxin that causes botulism. Because C. botulinum can produce endospores that can survive harsh conditions, extreme temperatures and pressures must be used to eliminate the endospores. Other organisms may not require such extreme measures and can be controlled by a procedure such as washing clothes in a laundry machine.

### Laboratory Biological Safety Levels

For researchers or laboratory personnel working with pathogens, the risks associated with specific pathogens determine the levels of cleanliness and control required. The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) have established four classification levels, called “biological safety levels” (BSLs). Various organizations around the world, including the World Health Organization (WHO) and the European Union (EU), use a similar classification scheme. According to the CDC, the BSL is determined by the agent’s infectivity, ease of transmission, and potential disease severity, as well as the type of work being done with the agent.US Centers for Disease Control and Prevention. “Recognizing the Biosafety Levels.” http://www.cdc.gov/training/quicklearns/biosafety/. Accessed June 7, 2016.
Each BSL requires a different level of biocontainment to prevent contamination and spread of infectious agents to laboratory personnel and, ultimately, the community. For example, the lowest BSL, BSL-1, requires the fewest precautions because it applies to situations with the lowest risk for microbial infection.
BSL-1 agents are those that generally do not cause infection in healthy human adults. These include noninfectious bacteria, such as nonpathogenic strains of  and , and viruses known to infect animals other than humans, such as baculoviruses (insect viruses). Because working with BSL-1 agents poses very little risk, few precautions are necessary. Laboratory workers use standard aseptic technique and may work with these agents at an open laboratory bench or table, wearing personal protective equipment (PPE) such as a laboratory coat, goggles, and gloves, as needed. Other than a sink for handwashing and doors to separate the laboratory from the rest of the building, no additional modifications are needed.
Agents classified as BSL-2 include those that pose moderate risk to laboratory workers and the community, and are typically “indigenous,” meaning that they are commonly found in that geographical area. These include bacteria such as  and Salmonella spp., and viruses like hepatitis, mumps, and measles viruses. BSL-2 laboratories require additional precautions beyond those of BSL-1, including restricted access; required PPE, including a face shield in some circumstances; and the use of biological safety cabinets for procedures that may disperse agents through the air (called “aerosolization”). BSL-2 laboratories are equipped with self-closing doors, an eyewash station, and an autoclave, which is a specialized device for sterilizing materials with pressurized steam before use or disposal. BSL-1 laboratories may also have an autoclave.
BSL-3 agents have the potential to cause lethal infections by inhalation. These may be either indigenous or “exotic,” meaning that they are derived from a foreign location, and include pathogens such as , , West Nile virus, and human immunodeficiency virus (HIV). Because of the serious nature of the infections caused by BSL-3 agents, laboratories working with them require restricted access. Laboratory workers are under medical surveillance, possibly receiving vaccinations for the microbes with which they work. In addition to the standard PPE already mentioned, laboratory personnel in BSL-3 laboratories must also wear a respirator and work with microbes and infectious agents in a biological safety cabinet at all times. BSL-3 laboratories require a hands-free sink, an eyewash station near the exit, and two sets of self-closing and locking doors at the entrance. These laboratories are equipped with directional airflow, meaning that clean air is pulled through the laboratory from clean areas to potentially contaminated areas. This air cannot be recirculated, so a constant supply of clean air is required.
BSL-4 agents are the most dangerous and often fatal. These microbes are typically exotic, are easily transmitted by inhalation, and cause infections for which there are no treatments or vaccinations. Examples include Ebola virus and Marburg virus, both of which cause hemorrhagic fevers, and smallpox virus. There are only a small number of laboratories in the United States and around the world appropriately equipped to work with these agents. In addition to BSL-3 precautions, laboratory workers in BSL-4 facilities must also change their clothing on entering the laboratory, shower on exiting, and decontaminate all material on exiting. While working in the laboratory, they must either wear a full-body protective suit with a designated air supply or conduct all work within a biological safety cabinet with a high-efficiency particulate air (HEPA)-filtered air supply and a doubly HEPA-filtered exhaust. If wearing a suit, the air pressure within the suit must be higher than that outside the suit, so that if a leak in the suit occurs, laboratory air that may be contaminated cannot be drawn into the suit (). The laboratory itself must be located either in a separate building or in an isolated portion of a building and have its own air supply and exhaust system, as well as its own decontamination system. The BSLs are summarized in .

### Sterilization

The most extreme protocols for microbial control aim to achieve sterilization: the complete removal or killing of all vegetative cells, endospores, and viruses from the targeted item or environment. Sterilization protocols are generally reserved for laboratory, medical, manufacturing, and food industry settings, where it may be imperative for certain items to be completely free of potentially infectious agents. Sterilization can be accomplished through either physical means, such as exposure to high heat, pressure, or filtration through an appropriate filter, or by chemical means. Chemicals that can be used to achieve sterilization are called sterilants. Sterilants effectively kill all microbes and viruses, and, with appropriate exposure time, can also kill endospores.
For many clinical purposes, aseptic technique is necessary to prevent contamination of sterile surfaces. Aseptic technique involves a combination of protocols that collectively maintain sterility, or asepsis, thus preventing contamination of the patient with microbes and infectious agents. Failure to practice aseptic technique during many types of clinical procedures may introduce microbes to the patient’s body and put the patient at risk for sepsis, a systemic inflammatory response to an infection that results in high fever, increased heart and respiratory rates, shock, and, possibly, death. Medical procedures that carry risk of contamination must be performed in a sterile field, a designated area that is kept free of all vegetative microbes, endospores, and viruses. Sterile fields are created according to protocols requiring the use of sterilized materials, such as packaging and drapings, and strict procedures for washing and application of sterilants. Other protocols are followed to maintain the sterile field while the medical procedure is being performed.
One food sterilization protocol, commercial sterilization, uses heat at a temperature low enough to preserve food quality but high enough to destroy common pathogens responsible for food poisoning, such as C. botulinum. Because C. botulinum and its endospores are commonly found in soil, they may easily contaminate crops during harvesting, and these endospores can later germinate within the anaerobic environment once foods are canned. Metal cans of food contaminated with C. botulinum will bulge due to the microbe’s production of gases; contaminated jars of food typically bulge at the metal lid. To eliminate the risk for C. botulinum contamination, commercial food-canning protocols are designed with a large margin of error. They assume an impossibly large population of endospores (1012 per can) and aim to reduce this population to 1 endospore per can to ensure the safety of canned foods. For example, low- and medium-acid foods are heated to 121 °C for a minimum of 2.52 minutes, which is the time it would take to reduce a population of 1012 endospores per can down to 1 endospore at this temperature. Even so, commercial sterilization does not eliminate the presence of all microbes; rather, it targets those pathogens that cause spoilage and foodborne diseases, while allowing many nonpathogenic organisms to survive. Therefore, “sterilization” is somewhat of a misnomer in this context, and commercial sterilization may be more accurately described as “quasi-sterilization.”

### Other Methods of Control

Sterilization protocols require procedures that are not practical, or necessary, in many settings. Various other methods are used in clinical and nonclinical settings to reduce the microbial load on items. Although the terms for these methods are often used interchangeably, there are important distinctions ().
The process of disinfection inactivates most microbes on the surface of a fomite by using antimicrobial chemicals or heat. Because some microbes remain, the disinfected item is not considered sterile. Ideally, disinfectants should be fast acting, stable, easy to prepare, inexpensive, and easy to use. An example of a natural disinfectant is vinegar; its acidity kills most microbes. Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to clean nonliving surfaces such as laboratory benches, clinical surfaces, and bathroom sinks. Typical disinfection does not lead to sterilization because endospores tend to survive even when all vegetative cells have been killed.
Unlike disinfectants, antiseptics are antimicrobial chemicals safe for use on living skin or tissues. Examples of antiseptics include hydrogen peroxide and isopropyl alcohol. The process of applying an antiseptic is called antisepsis. In addition to the characteristics of a good disinfectant, antiseptics must also be selectively effective against microorganisms and able to penetrate tissue deeply without causing tissue damage.
The type of protocol required to achieve the desired level of cleanliness depends on the particular item to be cleaned. For example, those used clinically are categorized as critical, semicritical, and noncritical. Critical items must be sterile because they will be used inside the body, often penetrating sterile tissues or the bloodstream; examples of critical items include surgical instruments, catheters, and intravenous fluids. Gastrointestinal endoscopes and various types of equipment for respiratory therapies are examples of semicritical items; they may contact mucous membranes or nonintact skin but do not penetrate tissues. Semicritical items do not typically need to be sterilized but do require a high level of disinfection. Items that may contact but not penetrate intact skin are noncritical items; examples are bed linens, furniture, crutches, stethoscopes, and blood pressure cuffs. These articles need to be clean but not highly disinfected.
The act of handwashing is an example of degerming, in which microbial numbers are significantly reduced by gently scrubbing living tissue, most commonly skin, with a mild chemical (e.g., soap) to avoid the transmission of pathogenic microbes. Wiping the skin with an alcohol swab at an injection site is another example of degerming. These degerming methods remove most (but not all) microbes from the skin’s surface.
The term sanitization refers to the cleansing of fomites to remove enough microbes to achieve levels deemed safe for public health. For example, commercial dishwashers used in the food service industry typically use very hot water and air for washing and drying; the high temperatures kill most microbes, sanitizing the dishes. Surfaces in hospital rooms are commonly sanitized using a chemical disinfectant to prevent disease transmission between patients.  summarizes common protocols, definitions, applications, and agents used to control microbial growth.

### Measuring Microbial Control

Physical and chemical methods of microbial control that kill the targeted microorganism are identified by the suffix -cide (or -cidal). The prefix indicates the type of microbe or infectious agent killed by the treatment method: bactericides kill bacteria, viricides kill or inactivate viruses, and fungicides kill fungi. Other methods do not kill organisms but, instead, stop their growth, making their population static; such methods are identified by the suffix -stat (or -static). For example, bacteriostatic treatments inhibit the growth of bacteria, whereas fungistatic treatments inhibit the growth of fungi. Factors that determine whether a particular treatment is -cidal or -static include the types of microorganisms targeted, the concentration of the chemical used, and the nature of the treatment applied.
Although -static treatments do not actually kill infectious agents, they are often less toxic to humans and other animals, and may also better preserve the integrity of the item treated. Such treatments are typically sufficient to keep the microbial population of an item in check. The reduced toxicity of some of these -static chemicals also allows them to be impregnated safely into plastics to prevent the growth of microbes on these surfaces. Such plastics are used in products such as toys for children and cutting boards for food preparation. When used to treat an infection, -static treatments are typically sufficient in an otherwise healthy individual, preventing the pathogen from multiplying, thus allowing the individual’s immune system to clear the infection.
The degree of microbial control can be evaluated using a microbial death curve to describe the progress and effectiveness of a particular protocol. When exposed to a particular microbial control protocol, a fixed percentage of the microbes within the population will die. Because the rate of killing remains constant even when the population size varies, the percentage killed is more useful information than the absolute number of microbes killed. Death curves are often plotted as semilog plots just like microbial growth curves because the reduction in microorganisms is typically logarithmic (). The amount of time it takes for a specific protocol to produce a one order-of-magnitude decrease in the number of organisms, or the death of 90% of the population, is called the decimal reduction time (DRT) or D-value.
Several factors contribute to the effectiveness of a disinfecting agent or microbial control protocol. First, as demonstrated in , the length of time of exposure is important. Longer exposure times kill more microbes. Because microbial death of a population exposed to a specific protocol is logarithmic, it takes longer to kill a high-population load than a low-population load exposed to the same protocol. A shorter treatment time (measured in multiples of the D-value) is needed when starting with a smaller number of organisms. Effectiveness also depends on the susceptibility of the agent to that disinfecting agent or protocol. The concentration of disinfecting agent or intensity of exposure is also important. For example, higher temperatures and higher concentrations of disinfectants kill microbes more quickly and effectively. Conditions that limit contact between the agent and the targeted cells cells—for example, the presence of bodily fluids, tissue, organic debris (e.g., mud or feces), or biofilms on surfaces—increase the cleaning time or intensity of the microbial control protocol required to reach the desired level of cleanliness. All these factors must be considered when choosing the appropriate protocol to control microbial growth in a given situation.

### Key Concepts and Summary

1. Inanimate items that may harbor microbes and aid in their transmission are called fomites. The level of cleanliness required for a fomite depends both on the item’s use and the infectious agent with which the item may be contaminated.
2. The CDC and the NIH have established four biological safety levels (BSLs) for laboratories performing research on infectious agents. Each level is designed to protect laboratory personnel and the community. These BSLs are determined by the agent’s infectivity, ease of transmission, and potential disease severity, as well as the type of work being performed with the agent.
3. Disinfection removes potential pathogens from a fomite, whereas antisepsis uses antimicrobial chemicals safe enough for tissues; in both cases, microbial load is reduced, but microbes may remain unless the chemical used is strong enough to be a sterilant.
4. The amount of cleanliness (sterilization versus high-level disinfection versus general cleanliness) required for items used clinically depends on whether the item will come into contact with sterile tissues (critical item), mucous membranes (semicritical item), or intact skin (noncritical item).
5. Medical procedures with a risk for contamination should be carried out in a sterile field maintained by proper aseptic technique to prevent sepsis.
6. Sterilization is necessary for some medical applications as well as in the food industry, where endospores of Clostridium botulinum are killed through commercial sterilization protocols.
7. Physical or chemical methods to control microbial growth that result in death of the microbe are indicated by the suffixes -cide or -cidal (e.g., as with bactericides, viricides, and fungicides), whereas those that inhibit microbial growth are indicated by the suffixes -stat or-static (e.g., bacteriostatic, fungistatic).
8. Microbial death curves display the logarithmic decline of living microbes exposed to a method of microbial control. The time it takes for a protocol to yield a 1-log (90%) reduction in the microbial population is the decimal reduction time, or D-value.
9. When choosing a microbial control protocol, factors to consider include the length of exposure time, the type of microbe targeted, its susceptibility to the protocol, the intensity of the treatment, the presence of organics that may interfere with the protocol, and the environmental conditions that may alter the effectiveness of the protocol.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer

### Critical Thinking


# Control of Microbial Growth

## Using Physical Methods to Control Microorganisms

### Learning Objectives

By the end of this section, you will be able to:
1. Understand and compare various physical methods of controlling microbial growth, including heating, refrigeration, freezing, high-pressure treatment, desiccation, lyophilization, irradiation, and filtration
For thousands of years, humans have used various physical methods of microbial control for food preservation. Common control methods include the application of high temperatures, radiation, filtration, and desiccation (drying), among others. Many of these methods nonspecifically kill cells by disrupting membranes, changing membrane permeability, or damaging proteins and nucleic acids by denaturation, degradation, or chemical modification. Various physical methods used for microbial control are described in this section.

### Heat

Heating is one of the most common—and oldest—forms of microbial control. It is used in simple techniques like cooking and canning. Heat can kill microbes by altering their membranes and denaturing proteins. The thermal death point (TDP) of a microorganism is the lowest temperature at which all microbes are killed in a 10-minute exposure. Different microorganisms will respond differently to high temperatures, with some (e.g., endospore-formers such as C. botulinum) being more heat tolerant. A similar parameter, the thermal death time (TDT), is the length of time needed to kill all microorganisms in a sample at a given temperature. These parameters are often used to describe sterilization procedures that use high heat, such as autoclaving. Boiling is one of the oldest methods of moist-heat control of microbes, and it is typically quite effective at killing vegetative cells and some viruses. However, boiling is less effective at killing endospores; some endospores are able to survive up to 20 hours of boiling. Additionally, boiling may be less effective at higher altitudes, where the boiling point of water is lower and the boiling time needed to kill microbes is therefore longer. For these reasons, boiling is not considered a useful sterilization technique in the laboratory or clinical setting.
Many different heating protocols can be used for sterilization in the laboratory or clinic, and these protocols can be broken down into two main categories: dry-heat sterilization and moist-heat sterilization. Aseptic technique in the laboratory typically involves some dry-heat sterilization protocols using direct application of high heat, such as sterilizing inoculating loops (). Incineration at very high temperatures destroys all microorganisms. Dry heat can also be applied for relatively long periods of time (at least 2 hours) at temperatures up to 170 °C by using a dry-heat sterilizer, such as an oven. However, moist-heat sterilization is typically the more effective protocol because it penetrates cells better than dry heat does.

### Autoclaves

Autoclaves rely on moist-heat sterilization. They are used to raise temperatures above the boiling point of water to sterilize items such as surgical equipment from vegetative cells, viruses, and especially endospores, which are known to survive boiling temperatures, without damaging the items. Charles Chamberland (1851–1908) designed the modern autoclave in 1879 while working in the laboratory of Louis Pasteur. The autoclave is still considered the most effective method of sterilization (). Outside laboratory and clinical settings, large industrial autoclaves called retorts allow for moist-heat sterilization on a large scale.
In general, the air in the chamber of an autoclave is removed and replaced with increasing amounts of steam trapped within the enclosed chamber, resulting in increased interior pressure and temperatures above the boiling point of water. The two main types of autoclaves differ in the way that air is removed from the chamber. In gravity displacement autoclaves, steam is introduced into the chamber from the top or sides. Air, which is heavier than steam, sinks to the bottom of the chamber, where it is forced out through a vent. Complete displacement of air is difficult, especially in larger loads, so longer cycles may be required for such loads. In prevacuum sterilizers, air is removed completely using a high-speed vacuum before introducing steam into the chamber. Because air is more completely eliminated, the steam can more easily penetrate wrapped items. Many autoclaves are capable of both gravity and prevacuum cycles, using the former for the decontamination of waste and sterilization of media and unwrapped glassware, and the latter for sterilization of packaged instruments.
Standard operating temperatures for autoclaves are 121 °C or, in some cases, 132 °C, typically at a pressure of 15 to 20 pounds per square inch (psi). The length of exposure depends on the volume and nature of material being sterilized, but it is typically 20 minutes or more, with larger volumes requiring longer exposure times to ensure sufficient heat transfer to the materials being sterilized. The steam must directly contact the liquids or dry materials being sterilized, so containers are left loosely closed and instruments are loosely wrapped in paper or foil. The key to autoclaving is that the temperature must be high enough to kill endospores to achieve complete sterilization.
Because sterilization is so important to safe medical and laboratory protocols, quality control is essential. Autoclaves may be equipped with recorders to document the pressures and temperatures achieved during each run. Additionally, internal indicators of various types should be autoclaved along with the materials to be sterilized to ensure that the proper sterilization temperature has been reached (). One common type of indicator is the use of heat-sensitive autoclave tape, which has white stripes that turn black when the appropriate temperature is achieved during a successful autoclave run. This type of indicator is relatively inexpensive and can be used during every run. However, autoclave tape provides no indication of length of exposure, so it cannot be used as an indicator of sterility. Another type of indicator, a biological indicator spore test, uses either a strip of paper or a liquid suspension of the endospores of  to determine whether the endospores are killed by the process. The endospores of the obligate thermophilic bacterium G. stearothermophilus are the gold standard used for this purpose because of their extreme heat resistance. Biological spore indicators can also be used to test the effectiveness of other sterilization protocols, including ethylene oxide, dry heat, formaldehyde, gamma radiation, and hydrogen peroxide plasma sterilization using either G. stearothermophilus, Bacillus atrophaeus, B. subtilis, or B. pumilus spores. In the case of validating autoclave function, the endospores are incubated after autoclaving to ensure no viable endospores remain. Bacterial growth subsequent to endospore germination can be monitored by biological indicator spore tests that detect acid metabolites or fluorescence produced by enzymes derived from viable G. stearothermophilus. A third type of autoclave indicator is the Diack tube, a glass ampule containing a temperature-sensitive pellet that melts at the proper sterilization temperature. Spore strips or Diack tubes are used periodically to ensure the autoclave is functioning properly.

### Pasteurization

Although complete sterilization is ideal for many medical applications, it is not always practical for other applications and may also alter the quality of the product. Boiling and autoclaving are not ideal ways to control microbial growth in many foods because these methods may ruin the consistency and other organoleptic (sensory) qualities of the food. Pasteurization is a form of microbial control for food that uses heat but does not render the food sterile. Traditional pasteurization kills pathogens and reduces the number of spoilage-causing microbes while maintaining food quality. The process of pasteurization was first developed by Louis Pasteur in the 1860s as a method for preventing the spoilage of beer and wine. Today, pasteurization is most commonly used to kill heat-sensitive pathogens in milk and other food products (e.g., apple juice and honey) (). However, because pasteurized food products are not sterile, they will eventually spoil.
The methods used for milk pasteurization balance the temperature and the length of time of treatment. One method, high-temperature short-time (HTST) pasteurization, exposes milk to a temperature of 72 °C for 15 seconds, which lowers bacterial numbers while preserving the quality of the milk. An alternative is ultra-high-temperature (UHT) pasteurization, in which the milk is exposed to a temperature of 138 °C for 2 or more seconds. UHT pasteurized milk can be stored for a long time in sealed containers without being refrigerated; however, the very high temperatures alter the proteins in the milk, causing slight changes in the taste and smell. Still, this method of pasteurization is advantageous in regions where access to refrigeration is limited.

### Refrigeration and Freezing

Just as high temperatures are effective for controlling microbial growth, exposing microbes to low temperatures can also be an easy and effective method of microbial control, with the exception of psychrophiles, which prefer cold temperatures (see Temperature and Microbial Growth). Refrigerators used in home kitchens or in the laboratory maintain temperatures between 0 °C and 7 °C. This temperature range inhibits microbial metabolism, slowing the growth of microorganisms significantly and helping preserve refrigerated products such as foods or medical supplies. Certain types of laboratory cultures can be preserved by refrigeration for later use.
Freezing below −2 °C may stop microbial growth and even kill susceptible organisms. According to the US Department of Agriculture (USDA), the only safe ways that frozen foods can be thawed are in the refrigerator, immersed in cold water changed every 30 minutes, or in the microwave, keeping the food at temperatures not conducive for bacterial growth.US Department of Agriculture. “Freezing and Food Safety.” 2013. http://www.fsis.usda.gov/wps/portal/fsis/topics/food-safety-education/get-answers/food-safety-fact-sheets/safe-food-handling/freezing-and-food-safety/CT_Index. Accessed June 8, 2016. In addition, halted bacterial growth can restart in thawed foods, so thawed foods should be treated like fresh perishables.
Bacterial cultures and medical specimens requiring long-term storage or transport are often frozen at ultra-low temperatures of −70 °C or lower. These ultra-low temperatures can be achieved by storing specimens on dry ice in an ultra-low freezer or in special liquid nitrogen tanks, which maintain temperatures lower than −196 °C ().

### Pressure

Exposure to high pressure kills many microbes. In the food industry, high-pressure processing (also called pascalization) is used to kill bacteria, yeast, molds, parasites, and viruses in foods while maintaining food quality and extending shelf life. The application of high pressure between 100 and 800 MPa (sea level atmospheric pressure is about 0.1 MPa) is sufficient to kill vegetative cells by protein denaturation, but endospores may survive these pressures.C. Ferstl. “High Pressure Processing: Insights on Technology and Regulatory Requirements.” Food for Thought/White Paper. Series Volume 10. Livermore, CA: The National Food Lab; July 2013.US Food and Drug Administration. “Kinetics of Microbial Inactivation for Alternative Food Processing Technologies: High Pressure Processing.” 2000. http://www.fda.gov/Food/FoodScienceResearch/SafePracticesforFoodProcesses/ucm101456.htm. Accessed July 19, 2106.
In clinical settings, hyperbaric oxygen therapy is sometimes used to treat infections. In this form of therapy, a patient breathes pure oxygen at a pressure higher than normal atmospheric pressure, typically between 1 and 3 atmospheres (atm). This is achieved by placing the patient in a hyperbaric chamber or by supplying the pressurized oxygen through a breathing tube. Hyperbaric oxygen therapy helps increase oxygen saturation in tissues that become hypoxic due to infection and inflammation. This increased oxygen concentration enhances the body’s immune response by increasing the activities of neutrophils and macrophages, white blood cells that fight infections. Increased oxygen levels also contribute to the formation of toxic free radicals that inhibit the growth of oxygen-sensitive or anaerobic bacteria like as , a common cause of gas gangrene. In C. perfringens infections, hyperbaric oxygen therapy can also reduce secretion of a bacterial toxin that causes tissue destruction. Hyperbaric oxygen therapy also seems to enhance the effectiveness of antibiotic treatments. Unfortunately, some rare risks include oxygen toxicity and effects on delicate tissues, such as the eyes, middle ear, and lungs, which may be damaged by the increased air pressure.
High pressure processing is not commonly used for disinfection or sterilization of fomites. Although the application of pressure and steam in an autoclave is effective for killing endospores, it is the high temperature achieved, and not the pressure directly, that results in endospore death.

### Desiccation

Drying, also known as desiccation or dehydration, is a method that has been used for millennia to preserve foods such as raisins, prunes, and jerky. It works because all cells, including microbes, require water for their metabolism and survival. Although drying controls microbial growth, it might not kill all microbes or their endospores, which may start to regrow when conditions are more favorable and water content is restored.
In some cases, foods are dried in the sun, relying on evaporation to achieve desiccation. Freeze-drying, or lyophilization, is another method of dessication in which an item is rapidly frozen (“snap-frozen”) and placed under vacuum so that water is lost by sublimation. Lyophilization combines both exposure to cold temperatures and desiccation, making it quite effective for controlling microbial growth. In addition, lyophilization causes less damage to an item than conventional desiccation and better preserves the item’s original qualities. Lyophilized items may be stored at room temperature if packaged appropriately to prevent moisture acquisition. Lyophilization is used for preservation in the food industry and is also used in the laboratory for the long-term storage and transportation of microbial cultures.
The water content of foods and materials, called the water activity, can be lowered without physical drying by the addition of solutes such as salts or sugars. At very high concentrations of salts or sugars, the amount of available water in microbial cells is reduced dramatically because water will be drawn from an area of low solute concentration (inside the cell) to an area of high solute concentration (outside the cell) (). Many microorganisms do not survive these conditions of high osmotic pressure. Honey, for example, is 80% sucrose, an environment in which very few microorganisms are capable of growing, thereby eliminating the need for refrigeration. Salted meats and fish, like ham and cod, respectively, were critically important foods before the age of refrigeration. Fruits were preserved by adding sugar, making jams and jellies. However, certain microbes, such as molds and yeasts, tend to be more tolerant of desiccation and high osmotic pressures, and, thus, may still contaminate these types of foods.

### Radiation

Radiation in various forms, from high-energy radiation to sunlight, can be used to kill microbes or inhibit their growth. Ionizing radiation includes X-rays, gamma rays, and high-energy electron beams. Ionizing radiation is strong enough to pass into the cell, where it alters molecular structures and damages cell components. For example, ionizing radiation introduces double-strand breaks in DNA molecules. This may directly cause DNA mutations to occur, or mutations may be introduced when the cell attempts to repair the DNA damage. As these mutations accumulate, they eventually lead to cell death.
Both X-rays and gamma rays easily penetrate paper and plastic and can therefore be used to sterilize many packaged materials. In the laboratory, ionizing radiation is commonly used to sterilize materials that cannot be autoclaved, such as plastic Petri dishes and disposable plastic inoculating loops. For clinical use, ionizing radiation is used to sterilize gloves, intravenous tubing, and other latex and plastic items used for patient care. Ionizing radiation is also used for the sterilization of other types of delicate, heat-sensitive materials used clinically, including tissues for transplantation, pharmaceutical drugs, and medical equipment.
In Europe, gamma irradiation for food preservation is widely used, although it has been slow to catch on in the United States (see the Micro Connections box on this topic). Packaged dried spices are also often gamma-irradiated. Because of their ability to penetrate paper, plastic, thin sheets of wood and metal, and tissue, great care must be taken when using X-rays and gamma irradiation. These types of ionizing irradiation cannot penetrate thick layers of iron or lead, so these metals are commonly used to protect humans who may be potentially exposed.
Another type of radiation, nonionizing radiation, is commonly used for disinfection and uses less energy than ionizing radiation. It does not penetrate cells or packaging. Ultraviolet (UV) light is one example; it causes thymine dimers to form between adjacent thymines within a single strand of DNA (). When DNA polymerase encounters the thymine dimer, it does not always incorporate the appropriate complementary nucleotides (two adenines), and this leads to formation of mutations that can ultimately kill microorganisms.
UV light can be used effectively by both consumers and laboratory personnel to control microbial growth. UV lamps are now commonly incorporated into water purification systems for use in homes. In addition, small portable UV lights are commonly used by campers to purify water from natural environments before drinking. Germicidal lamps are also used in surgical suites, biological safety cabinets, and transfer hoods, typically emitting UV light at a wavelength of 260 nm. Because UV light does not penetrate surfaces and will not pass through plastics or glass, cells must be exposed directly to the light source.
Sunlight has a very broad spectrum that includes UV and visible light. In some cases, sunlight can be effective against certain bacteria because of both the formation of thymine dimers by UV light and by the production of reactive oxygen products induced in low amounts by exposure to visible light.

### Sonication

The use of high-frequency ultrasound waves to disrupt cell structures is called sonication. Application of ultrasound waves causes rapid changes in pressure within the intracellular liquid; this leads to cavitation, the formation of bubbles inside the cell, which can disrupt cell structures and eventually cause the cell to lyse or collapse. Sonication is useful in the laboratory for efficiently lysing cells to release their contents for further research; outside the laboratory, sonication is used for cleaning surgical instruments, lenses, and a variety of other objects such as coins, tools, and musical instruments.

### Filtration

Filtration is a method of physically separating microbes from samples. Air is commonly filtered through high-efficiency particulate air (HEPA) filters (). HEPA filters have effective pore sizes of 0.3 µm, small enough to capture bacterial cells, endospores, and many viruses, as air passes through these filters, nearly sterilizing the air on the other side of the filter. HEPA filters have a variety of applications and are used widely in clinical settings, in cars and airplanes, and even in the home. For example, they may be found in vacuum cleaners, heating and air-conditioning systems, and air purifiers.

### Biological Safety Cabinets

Biological safety cabinets are a good example of the use of HEPA filters. HEPA filters in biological safety cabinets (BSCs) are used to remove particulates in the air either entering the cabinet (air intake), leaving the cabinet (air exhaust), or treating both the intake and exhaust. Use of an air-intake HEPA filter prevents environmental contaminants from entering the BSC, creating a clean area for handling biological materials. Use of an air-exhaust HEPA filter prevents laboratory pathogens from contaminating the laboratory, thus maintaining a safe work area for laboratory personnel.
There are three classes of BSCs: I, II, and III. Each class is designed to provide a different level of protection for laboratory personnel and the environment; BSC II and III are also designed to protect the materials or devices in the cabinet.  summarizes the level of safety provided by each class of BSC for each BSL.
Class I BSCs protect laboratory workers and the environment from a low to moderate risk for exposure to biological agents used in the laboratory. Air is drawn into the cabinet and then filtered before exiting through the building’s exhaust system. Class II BSCs use directional air flow and partial barrier systems to contain infectious agents. Class III BSCs are designed for working with highly infectious agents like those used in BSL-4 laboratories. They are gas tight, and materials entering or exiting the cabinet must be passed through a double-door system, allowing the intervening space to be decontaminated between uses. All air is passed through one or two HEPA filters and an air incineration system before being exhausted directly to the outdoors (not through the building’s exhaust system). Personnel can manipulate materials inside the Class III cabinet by using long rubber gloves sealed to the cabinet.

### Filtration in Hospitals

HEPA filters are also commonly used in hospitals and surgical suites to prevent contamination and the spread of airborne microbes through ventilation systems. HEPA filtration systems may be designed for entire buildings or for individual rooms. For example, burn units, operating rooms, or isolation units may require special HEPA-filtration systems to remove opportunistic pathogens from the environment because patients in these rooms are particularly vulnerable to infection.

### Membrane Filters

Filtration can also be used to remove microbes from liquid samples using membrane filtration. Membrane filters for liquids function similarly to HEPA filters for air. Typically, membrane filters that are used to remove bacteria have an effective pore size of 0.2 µm, smaller than the average size of a bacterium (1 µm), but filters with smaller pore sizes are available for more specific needs. Membrane filtration is useful for removing bacteria from various types of heat-sensitive solutions used in the laboratory, such as antibiotic solutions and vitamin solutions. Large volumes of culture media may also be filter sterilized rather than autoclaved to protect heat-sensitive components. Often when filtering small volumes, syringe filters are used, but vacuum filters are typically used for filtering larger volumes ().
 and  summarize the physical methods of control discussed in this section.

### Key Concepts and Summary

1. Heat is a widely used and highly effective method for controlling microbial growth.
2. Dry-heat sterilization protocols are used commonly in aseptic techniques in the laboratory. However, moist-heat sterilization is typically the more effective protocol because it penetrates cells better than dry heat does.
3. Pasteurization is used to kill pathogens and reduce the number of microbes that cause food spoilage. High-temperature, short-time pasteurization is commonly used to pasteurize milk that will be refrigerated; ultra-high temperature pasteurization can be used to pasteurize milk for long-term storage without refrigeration.
4. Refrigeration slows microbial growth; freezing stops growth, killing some organisms. Laboratory and medical specimens may be frozen on dry ice or at ultra-low temperatures for storage and transport.
5. High-pressure processing can be used to kill microbes in food. Hyperbaric oxygen therapy to increase oxygen saturation has also been used to treat certain infections.
6. Desiccation has long been used to preserve foods and is accelerated through the addition of salt or sugar, which decrease water activity in foods.
7. Lyophilization combines cold exposure and desiccation for the long-term storage of foods and laboratory materials, but microbes remain and can be rehydrated.
8. Ionizing radiation, including gamma irradiation, is an effective way to sterilize heat-sensitive and packaged materials. Nonionizing radiation, like ultraviolet light, is unable to penetrate surfaces but is useful for surface sterilization.
9. HEPA filtration is commonly used in hospital ventilation systems and biological safety cabinets in laboratories to prevent transmission of airborne microbes. Membrane filtration is commonly used to remove bacteria from heat-sensitive solutions.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer

### Critical Thinking