12: OUTBREAK

According to the Centers for Disease Control and Prevention (CDC), about 48 million Americans become ill from foodborne diseases each year. About 128,000 are hospitalized, and 3,000 die. In 2000, the CDC received reports of 406 cases of the same foodborne illness across ten states, primarily on the West Coast. Epidemiologists investigating the outbreak determined 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 isolated from stool samples collected from 98% of people with similar signs and symptoms.

Several members of the Enterobacteriaceae family (a group of Gram-negative, non-endospore-forming, facultative anaerobic bacteria) could have caused the outbreak. Foodborne pathogens in this family include Shigella, Salmonella, and Escherichia coli. These bacteria can cause similar signs and symptoms, but the safest and most effective 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 usually self-limiting (an illness that generally resolves without medication), but the affected person should be monitored and kept hydrated.

Enteroinvasive E. coli (EIEC) (a type of E. coli that enters and damages cells lining 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, these infections can progress to serious complications if they are not identified and managed correctly.

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

The toxins and other disease-causing traits produced by a bacterium depend on its genome (all the genetic material within a cell, including its chromosome and plasmids). For diagnosis and treatment, it is often critical to quickly identify the species and strain causing an infection.

Identifying the exact organism 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, 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. Rapid identification helps healthcare providers choose the safest treatment, monitor for possible complications, and limit the spread of the pathogen.

In the past, laboratories relied mainly on signs and symptoms, bacterial cultures, and metabolic tests to identify pathogens. Most metabolic tests require 48–72 hours of incubation before results are available, which can delay diagnosis and treatment. In addition, some metabolic tests cannot distinguish between closely related bacterial strains. Hospitals and clinical laboratories now commonly use polymerase chain reaction and gel electrophoresis to analyze pathogen DNA and identify infectious organisms more quickly.

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 DNA strands. Cooling allows primers to attach to matching sequences on the separated strands. DNA polymerase then builds new DNA strands, copying 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 because DNA polymerase can build DNA in only one direction. Each set contains a forward primer and a reverse primer. The primers attach to opposite DNA strands on either side of the target region. Together, they mark the exact DNA section that will be copied and allow both strands to be copied during each PCR cycle.

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 either matching sequence is absent, the primers do not attach correctly, and the expected DNA fragment is not produced. Different pathogen-specific primer sets can therefore be used to distinguish among pathogens that cause similar signs and symptoms.

During PCR, the reaction mixture passes through repeated cycles of heating and cooling. Heating separates the two strands of bacterial DNA. As the mixture cools, the forward and reverse primers attach to matching sequences on opposite strands. DNA polymerase then extends from each primer and makes new copies of the target region. Repeating these cycles produces 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 a predictable number of base pairs. If the target sequences are absent, the expected 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 pass through the pores more easily and travel farther than larger fragments.

The bands produced by the unknown sample will be compared with a DNA ladder (a mixture of DNA fragments of known sizes) and known positive and negative controls. The DNA ladder will help determine the size of each PCR product. The positive controls will show the expected results when the target DNA is present. The negative control will help detect contamination or other errors.

A 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 choose the safest treatment and respond quickly to a 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 bands from the unknown sample with a DNA ladder and known positive and negative controls, you will determine whether the sample contains DNA from Shigella, Salmonella, or a disease-causing strain of E. coli.