12: OUTBREAK

According to the Centers for Disease Control and Prevention (CDC), about 48 million Americans become sick from foodborne illnesses each year. About 128,000 people are hospitalized, and 3,000 die. In 2000, 406 cases of the same foodborne illness were reported across ten states, mainly on the West Coast. Epidemiologists found that the pathogen spread through the fecal–oral route (the spread of a pathogen when fecal material enters a person’s mouth, often through contaminated food, water, hands, or surfaces). The same type of bacterium was found in stool samples from 98% of people who had similar signs and symptoms.

Several bacteria in the Enterobacteriaceae family (a group of Gram-negative, non-endospore-forming, facultative anaerobic bacteria) could have caused the outbreak. Common foodborne pathogens in this family include Shigella, Salmonella, and Escherichia coli. These bacteria can cause similar signs and symptoms. However, treatment may differ depending on the species or strain causing the infection.

Illnesses caused by these pathogens can range from mild diarrhea to severe dysentery. The severity of the illness depends partly on the toxins produced by the bacteria. Some strains of Shigella produce Shiga toxin (a powerful toxin that damages human cells). This toxin can cause severe illness and may lead to death. Shigellosis is often self-limiting, which means the infection may go away without medication. However, the person should be monitored and kept hydrated.

Enteroinvasive E. coli (EIEC) (a type of E. coli that enters and damages cells in the intestine) is closely related to Shigella and can cause similar signs and symptoms. Infections caused by E. coli O157:H7 may also begin with symptoms that resemble shigellosis. However, the infection can lead to serious complications if it is not identified and managed properly.

Salmonella bacteria cause an illness called salmonellosis. Treatment usually focuses on replacing fluids and electrolytes to prevent dehydration. Antibiotics are generally used only for severe infections or for people who have a greater risk of complications.

Identifying the exact organism causing an illness is important because different pathogens can cause similar signs and symptoms but may require different treatments. Some infections require only fluids, rest, and monitoring, while others may require antibiotics. In some cases, using the wrong antibiotic could make the illness worse. Antibiotics are generally avoided in infections caused by Shiga toxin-producing E. coli, such as E. coli O157:H7, because they may increase the risk of serious kidney damage. Quickly identifying the pathogen helps healthcare providers choose the safest treatment, watch for possible complications, and prevent the pathogen from spreading to others.

The toxins and other disease-causing features of a bacterium are determined by its genome (all the genetic material in a cell, including its chromosome and plasmids). In the past, laboratories mainly used bacterial cultures and metabolic tests to identify pathogens. These methods may take several days. Today, clinical laboratories can examine bacterial DNA to identify pathogens more quickly. Two methods used for this purpose are polymerase chain reaction and gel electrophoresis.

Polymerase chain reaction (PCR) (a laboratory method used to make millions of copies of a selected DNA region) uses repeated cycles of heating and cooling. Heating separates the two strands of DNA. Cooling allows primers to attach to matching DNA sequences. DNA polymerase then builds new DNA strands and copies the selected region.

Primers (short, single-stranded pieces of DNA that mark the beginning and end of the region to be copied) give PCR its specificity. PCR uses a set of two primers instead of only one because DNA polymerase can build DNA in only one direction. Each primer set contains a forward primer and a reverse primer. These primers attach to opposite DNA strands on either side of the target region. This marks the exact section of DNA that will be copied and allows both strands to be copied during each PCR cycle. As the cycles are repeated, the number of copies increases quickly.

Each primer set is designed to attach to DNA sequences from a particular pathogen. If both matching sequences are present, the primers attach to the DNA, and DNA polymerase copies the region between them. If the matching sequences are not present, the primers do not attach correctly, and the expected DNA fragment is not made. Different pathogen-specific primer sets can therefore be used to tell pathogens apart, even when they cause similar signs and symptoms.

During this lab, PCR will be used to examine an unknown bacterial sample for DNA from possible foodborne pathogens, such as Shigella, Salmonella, or a disease-causing strain of E. coli. A different pathogen-specific primer set will be added to each PCR reaction. Each primer set will search for a DNA region linked to one of the possible pathogens.

During PCR, the reaction mixture will go through repeated cycles of heating and cooling. Heating will separate the two strands of bacterial DNA. As the mixture cools, the forward and reverse primers will attach to matching sequences on opposite strands. DNA polymerase will then extend from each primer and make new copies of the target region. Repeating these steps will produce millions of copies of the target DNA.

If the unknown sample contains the sequences recognized by a primer set, PCR will produce a DNA fragment with an expected number of base pairs. If the target sequences are not present, the expected DNA fragment will not be produced.

The PCR products will be examined using gel electrophoresis (a laboratory method that separates DNA fragments by size). DNA has a negative charge. When an electric current is applied, the DNA fragments move through the gel toward the positive electrode. The agarose gel contains tiny pores that act like a filter. Smaller DNA fragments move through these pores more easily and travel farther than larger fragments.

The DNA bands from the unknown sample will be compared with a DNA ladder (a mixture of DNA fragments of known sizes), a positive control, and a negative control. The DNA ladder will help determine the size of each PCR product. The positive control will show what a successful PCR result should look like. The negative control will help reveal contamination or other errors.

A visible band at the expected location will show that the unknown sample contains the DNA sequence targeted by that primer set. The pathogen will be identified by determining which pathogen-specific primer set produces a band of the expected size. This information can help healthcare providers select the safest treatment and respond quickly to the foodborne outbreak.

During this lab, you will use pathogen-specific primer sets and PCR to copy selected DNA regions from an unknown bacterial sample. You will then use gel electrophoresis to separate the PCR products by size. By comparing the DNA bands with a DNA ladder and positive and negative controls, you will determine whether the unknown sample contains DNA from Shigella, Salmonella, or a disease-causing strain of E. coli. The results will be used to identify the pathogen and explain why accurate identification is important when choosing a safe and effective treatment.