20: CYA Later
- Page ID
- 106481
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- Describe the structure and function of the GI tract.
- Identify normal flora of the GI tract.
- Identify common causes of diarrheal diseases.
- Describe pathways to diarrheal disease and prevention.
- Utilize and interpret different types of selective and/or differential medias.
Introduction to CYA: Culturing Your Anus
Gastrointestinal diseases are a leading cause of morbidity and mortality worldwide. The CDC reports that diarrhea kills 2,195 children every day worldwide; that's 801,000 child deaths a year! Often times, the cause of such gastrointestinal (GI) diseases is never determined. This is due to the inconvenient and sensitive nature of diagnostic testing. In most diagnostic situations, a fecal sample is used to inoculate culture medium to determine the causative agent of a given diarrheal disease. However, collection of fecal specimens can be difficult, especially when you consider the specimen must be collected in a small sterile cup, which poses a significant biohazard risk to the patient, especially in the case of diarrhea. Fecal samples must also be fresh in order to accurately test the specimen. Consequently, there has been a shift in diagnostic assays towards rectal swabs, which may be collect in transport medium that preserves the sample. Rectal swab collection also mediates the difficulty of safely procuring a fecal sample. Today, we will be culturing the microbes from the rectum using the rectal swab method of collection. Before we begin, let's discuss the GI tract as a microbial environment.
The GI Tract as a Microbial Niche
The GI tract is made up of the mouth, tongue, teeth, pharynx, esophagus, stomach, small intestines, and large intestines. Some accessory structures include the salivary glands, liver, gall bladder, and pancreas. It functions in the mechanical and chemical digestion of food. Nearly 70% of the immune system is located in the intestinal tract. Loosely organized lymphoid tissue innervate the gut, including Peyer's patches, which are masses of lymphatic tissue, and M cells, which translocate antigens and microbes to Peyer's patches to allow for immune surveillance of the gut. The GI tract is quite an inhospitable place for microbes, starting in the mouth which contains lysozyme and the flow of saliva which dilutes bacteria and keeps them moving in the direction of the stomach with every swallow. In the stomach, the low pH and digestive enzymes prevent many microbes from growing. Stomach contents move through the small and large intestine where most nutrients are absorbed by the host. Here we see a plethora of microbiota which play a pivotal role in GI immunity.
Microbiota of the Intestines
The most studied population of normal flora in the microbes living in the intestines. This population of microbes is commonly referred to as the gut microbiota or gut microbiome. Although the bacterial species most commonly associated with the intestines is Escherichia coli, it is actually not the most numerous in the intestine. Bacteria in the phylum Bacteroidetes form a large proportion of bacteria in the gut. The Gram-positive Firmicutes (such as Lactobacillus and Clostridium) and Actinobacteria (including Bifidobacterium) can be equally numerous. In healthy individuals, Proteobacteria (including E. coli and other Enterobacteriaceae) are the least abundant of the major bacterial groups in the intestines. There are many other groups of microbes found in the intestines, including fungi such as Candida. It is shifts in the proportions of these groups of microbes that are typically studied when investigating the role of normal flora on human health.
Pathways to Diarrheal Illness
Rotavirus is the most common cause of childhood diarrhea, however, Campylobacter, E. coli O157H7, Salmonella, and Shigella also contribute to diarrheal diseases. Most often, diarrheal illness is spread via the fecal-oral route. Roughly 88% of diarrhea related deaths are caused by unsafe water, poor sanitation, and poor hygiene. In the absence of proper sanitation facilities, sick people or animals may defecate in or near a water source that is used to irrigate crops. Those crops then make it to the kitchen table as they are used to prepare meals. Alternatively, if a drinking water source is contaminated, people may drink contaminated water leading to disease. Contamination can also come from contaminated hands after a trip to the bathroom. There are numerous events of contamination worldwide, even in more developed nations, again underscoring the pervasiveness of GI diseases.
Preventing Diarrhea
Certain causes of diarrheal disease, like rotavirus, can be prevented with vaccination. Other infectious agents require that safe water, adequate sanitation, and proper human waste disposal are provided by governments and ministries of health. Proper handwashing can also significantly reduce risk of contracting diarrheal disease. Treatment can limit spread of disease and help to replenish lost water that occurs as a result of diarrhea. Rehydration therapy and antibiotics can significantly help to limit infection and reduce dehydration and death. Healthcare providers and food handlers should also be provided with sufficient training on proper handwashing.
Laboratory Instructions
In this experiment, a rectal swab will be collected and cultured on blood agar, MacConkey agar, EMB agar, and TSA.
Prepare Petri Plates and Collect Rectal Swabs
- Prepare 1 plate of each media type per student.
- Label the plates with your name, date, and inoculum source (rectal swab).
- Using proper aseptic technique, insert the swab 1-1.5 inches into the anus and gently rotate for 10 seconds. Fecal material must be visible on the tip of the swab.
- Immerse the swab in 1mL of TSB in a 1.5mL microcentrifuge tube and let it sit for 5 minutes before gently mixing the swab to resuspend the specimen in the media.
- Close the microcentrifuge tube and mix by inverting up and down 10 times or by flicking 10 times.
- Using a micro-pipette, plate 200uL of the resuspended specimen on each type of media (4 plates total)
- Invert the petri plates and incubate for 24 – 48 hours at 37 degrees Celsius.
Results & Questions
Report your results using the table below. Identify the different colony types for each type of media, then speculate on what you think might be growing on your plates.
| TSA | EMB Agar | MacConkey Agar | Blood Agar | |
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- How many distinct types of microbes can you identify on the petri plates (each distinct type would have a different colony morphology)?
2. Which type of media showed the most diversity of microbes (or the most different colony morphologies)?
3. True or false. The microbes growing in your gut under normal circumstances are dangerous to your health.
4. What is the most common method of transmission for most diarrheal diseases?
5. How do food and water sources become contaminated?
6. What is the cause of death in severe cases of diarrheal disease?
7. What can be done to prevent contamination of food and water sources?
8. What is the role of the GI microbiome in preventing diarrheal diseases?
Attributions
- CDC. “Diarrhea: Common Illness, Global Killer.” Centers for Disease Control, Https://Www.cdc.gov/Healthywater/Pdf...arrhea508c.Pdf.
- Jean, Sophonie, et al. “Culture of Rectal Swab Specimens for Enteric Bacterial Pathogens Decreases Time to Test Result While Preserving Assay Sensitivity Compared to Bulk Fecal Specimens.” Journal of Clinical Microbiology, vol. 57, no. 6, June 2019, https://doi.org/10.1128/jcm.02077-18.
- Tortora, Gerard J., et al. Microbiology: An Introduction. 13th ed., Pearson, 2019.