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• Lecture #63 is on Microbiology: Bacterial G.I. Infections, specifically Normal Flora and Pathogenic E. coli
• The lecture is by Charles Pavia, Ph.D., Professor; Dept. of Biomedical Sciences

Session Objectives

• Epidemiology, microbial virulence factors, normal microbial flora (microbiome), and host-defense mechanisms play key roles in preventing or contributing to gastrointestinal tract infections caused by pathogenic bacteria.
• Normal GI bacteria and pathogenic E. coli are associated with causing gastrointestinal infections, based on unique microbiologic features.
• Clinical microbiology labs use various test procedures to identify pathogenic E. coli bacteria, which aids clinicians in making correct diagnoses.

Outline of the lecture

• An overview of the bacteria (normal flora?) residing in the G.I. tract (microbiome)
• The key microbiologically-related properties of the enteric bacteria are discussed.
• A review of the epidemiology of the G.I. tract pathogens is provided.
• Microbiologic and clinical aspects of the pathogenic E. coli are covered.

Microbiologic Overview of the G.I. tract: Special Defenses

• Saliva and drinks are part of the flow of liquids and contain lysozyme and normal flora.
• Peristalsis is a flow of liquids.
• Low pH environments.
• Peristalsis is a flow of liquids that aids in the shedding of epithelium.
• Peyer's patches IgA, and mucus also play roles in this shedding.
• Normal flora, peristalsis, and shedding of epithelium all contribute defensively.

Microbiologic Overview of the G.I. tract: Microbial Flora (examples)

• Normal: predominantly anaerobes (lots of species), alpha-streptococci, Neisseria, diphtheroids, Lactobacilli, spirochetes, mycoplasma, and other bacteria
• Sparse flora are normally present.
• Lactobacilli and Fusobacterium constitute sparse flora.
• Helicobacter pylori is a sparse flora.
• An increasing number of bacteria with distance are normally found from the stomach
• Anaerobes (lots of species), coliforms, streptococci (enterococci), Lactobacilli, and spirochetes are predominantly normal
• Abnormal: Beta-streptococci, staphylococci, Fusobacterium, many other bacteria, Herpesvirus, Coxsackievirus, other viruses, and Candida can be abnormal
• Candida and Herpesvirus are abnormal.
• E. coli (ETEC, EPEC strains) Vibrio, Yersinia, Aeromonas, rotavirus, other viruses, Giardia, Cryptosporidium, Strongyloides, Ascaris, Diphyllobothrium, and other worms are abnormal.
• Shigella, E. coli (EHEC strains), Campylobacter, Entamoeba histolytica, Trichuris, and other worms are abnormal

GI Tract and typical Numbers of Bacteria at the Main Sites

• Gram-positive flora, coliforms, and anaerobes are present at different levels in Stomach, Duodenum, Jejunum, Ileum, and Colon.

Composition of the Intestinal Flora of Adult Humans

• Stomach: total viable count of 0-103
• Jejunum: total viable count of 0-105
• Ileum: total viable count of 102-107
• Colon: total viable count of 1010-1012

Aerobes or facultative anaerobes

• Enterobacteria: levels ranging from 0-102 in the stomach to 104-1010 in the colon
• Streptococci: levels ranging from 0-102 in the stomach to 105-1010 in the colon.
• Staphylococci: levels ranging from 0-102 in the stomach to 104-109 in the colon.
• Lactobacilli: Levels range from 0-103 in the stomach to 104-1010 in the colon.
• Fungi levels range from 0-102 in the stomach to 104-106 in the colon.

Anaerobes

• Bacteroides are rare in the stomach and increase to colon levels of 1010-1012.
• Bifidobacteria are rare in the stomach and increase to colon levels of 108-1012.
• Streptococci are rare in the stomach and increase to colon levels of 1010-1012.
• Clostridia are rare in the stomach and increase to colon levels of 106-1011.
• Eubacteria are rare in the stomach and increase to colon levels of 109-1012

Normal flora of the upper GI tract

• Esophagus and stomach bacteria have low numbers of 10^4/ml, surviving from the upper respiratory tract and food, which includes Mycobacterium spp.
• Small intestine bacteria include Lactobacillus spp., Mycobacterium spp., Enterobacteriaceae, S. aureus, Enterococcus, Gram-negative anaerobic spp., Bacteroides spp., and Clostridium spp.

Normal flora of the lower GI tract

• Large intestine contains 95% or more species of obligate anaerobes
• Gram-negative anaerobes include Bacteroidaceae spp. and Fusobacterium spp.
• Gram-positive anaerobes include Peptococcus spp. and Peptostreptococcus spp.
• Enterobacteriaceae include E. coli, Klebsiella spp., and Proteus spp.
• Other bacteria are Enterococcus, Lactobacillus, Clostridium spp. (including C. perfringens, C. welchii,and C. difficile), Streptococcus spp., Group B streptococci, and Pseudomonas spp.

Indigenous Anaerobic Flora: Bacteroides fragilis

• It is an aerotolerant anaerobe, but not a strict one located in the colon.
• It is a Gram-negative rod (or bacillus) having a capsule, which can lead to intra-abdominal infections
• It occurs after internal surgery or a traumatic accident
• It has LPS, but lacks endotoxin-like activity due to the lack of a key phosphate group

Indigenous Anaerobic Flora: Clostridium difficile

• An anaerobic, spore-forming, toxigenic gram-positive bacillus.
• It causes antibiotic-associated diarrhea and colitis.
• Patients may develop pseudomembranous colitis following the use of antibiotics like cephalosporins and fluoroquinolones given during and after surgery.
• Clostridium difficile produces two toxins, A and B, that are potent cytotoxins.
• It's recently been implicated as a serious nosocomial pathogen, and can cause spore release, which may colonize and infect other patients.

C. diff.-Associated pseudomembranous colitis

• Endoscopically, it is an appearance of the sigmoid colon showing diffuse pseudomembranes with some hemorrhaging.
• Gram stain of C. difficille shows pale-staining endospores.

Host defenses in the GI tract

• Environment is highly acidic with a range of pH 1-3 in the stomach
• Microbes get smothered in mucous and intestinal fluids to be propelled out during defecation
• The presence of inhibitory agents and secretions includes: lysozyme, proteases, lipases, bile salts, and IgA and host cells (includes breast milk).
• Colon contains indigenous normal flora can create competition (probiotics)

Key Properties of Enteric Bacteria

• Separation of enteric bacteria is based on Serologic properties (surface antigens), Pathogenic features, Biochemical / metabolic tests
• Serologic properties (surface antigens) include the cell wall, capsule, and flagella
• Pathogenic features include toxin production (exotoxin vs. endotoxin)
• Biochemical / metabolic tests include carbohydrate fermentation, substrate utilization / inhibition, enzyme production, and motility

Key Properties of Enteric Bacteria: Lactose Fermentation

• Most E. coli strains ferment this sugar, which helps in their identification from a patient sample.
• MacConkey agar is frequently used for measuring sugar (lactose) fermentation patterns of E. coli and other gram-negative enteric bacteria.
• Not all gram-negatives grow on MacConkey agar, e.g., Haemophilus and Neisseria species; also, all gram-positive bacteria will not grow on MacConkey agar due to inhibitors.
• Red or purple-colored colonies indicate lactose-fermentation; amber or colorless colonies indicate non-lactose fermenters when using a MacConkey agar.

Genetic Properties

• Bacteria can exchange or transfer DNA through conjugation, transduction, and transformation
• There is chromosomal versus plasmid DNA with no nucleus
• Conjugation involves mating via pili
• Transduction is phage (virus)-mediated
• Transformation involves "free" DNA
• Phage typing is unique for a particular species or type of bacterium, where a specific virus recognizes and infects only one type of bacterium.

Refresher on Epidemiologic Terms and Concepts

• A localized epidemic affects a small group of people (usually fewer than 200) within a confined area or space.
• Examples: workplace, restaurant, cruise ship, social event like picnic or weddings, nursing home, or day-care centers
• A community-wide epidemic affects a somewhat larger number of people typically living in a village, town, city or county.
• A nationwide epidemic occurs when a disease outbreak affects a sizeable number of people living in several different cities or regions of a country due to the same pathogen..
• Cholera in Haiti in 2010 following an earthquake and a current cholera outbreak in Yemen are examples of a nation-wide epidemic
• Cholera may be spread from susceptible regions by infected tourists or travelers, imported food or water.
• The 2010 Haitian cholera outbreak is believed to have traveled from Nepal with United Nations peacekeepers headed to the Annapurna military camp in Mirebalais
• Nepal has had Cholera endemic, and a recent Cholera outbreak occurred in 2010 in Kathmandu

Pandemic

• A pandemic is a disease outbreak that affects several different countries worldwide within a relatively short span of time and is often severe with a significant death toll.
• COVID-19 and the 1918 flu pandemic are examples.
• "Endemic" means an infectious agent, illness, or disease has become well established within a community, location, or defined region, with an expected incidence/prevalence rate.
• An organism may become endemic after an epidemic/pandemic caused by a pathogen comes under control and periodic cases are managed.

Bacterial agents of diarrhea

• Classic agents are E. coli, Salmonella, Cholera, and Shigella
• Staph. aureus produces a toxin in food prior to being ingested.
• Botulism is caused by Clostridium botulinum and Bacillus cereus is another agent

Microbial agents of dysentery/bloody diarrhea

• Shigella is a classic bacillary agent.
• Some E. coli, Campylobacter (parasitic), and Entamoeba histolytica.

Escherichia coli pathogens

• Enteropathogenic E. coli (EPEC) causes infantile diarrhea and is a lactose-fermenter, making it, one of the first disease-causing bacterial agents to be isolated and identified.
• Enterotoxigenic E. coli (ETEC) is the common cause of traveler's diarrhea, producing two types of toxins: a labile toxin (LT) and a stable toxin (ST); it is a lactose fermenter.
• Enteroinvasive E. coli (EIEC) causes dysentery similar to Shigella, and is a non-lactose fermenter.
• It is also non-motile which is similar to Shigella and it invades intestinal mucosal cells.

Pathogenic E. coli continued

• Enterohemorrhagic E. coli (EHEC) is also known as Shiga-Toxin Producing E. coli, it causes hemorrhagic colitis with bloody diarrhea.
• It causes hemolytic uremic syndrome (HUS), which can lead to renal failure.
• It produces 2 cytotoxins: one attacks cells in the colon, the other attacks the kidney.
• Enteroaggregative E. coli (EAEC) is rare causes chronic diarrhea and growth retardation in children, mostly in developing countries.
• E. coli virulence factors are Shiga-type toxins which inhibit protein synthesis and cause cytotoxic effects.
• Enterohemorrhagic E. coli (EHEC) is non-motile and it ferments lactose, but doesn't ferment sorbitol.

Enterohemorrhagic E. coli continued

• Serotype H7:0157 is the most common, recent outbreaks associated with eating undercooked hamburgers & raw spinach.
• When viewed under the microscope it appears as pink rods.
• In 2011, Europe dealt with an outbreak of E. coli 0104:H4.
• The outbreak spread to neighboring countries.
• There were 4,000 illnesses, 800 cases of uremic syndrome
• There were 50 deaths in Germany and 15 in countries
• It was identified in contaminated sprouts derived from fecally contaminated fenugreek seeds that were imported from Egypt.
• E. coli strain was a unique version of enterohemorrhagic E. coli, classified as 0104:H4.
• The atypical features are rare, but can still be the cause.
• Food-borne primary transmission also had a secondary unusual transmission
• There was a long incubation period of 8 day

Summary

• The GI microbiome consists of a wide variety of bacteria, most of which are non-pathogenic
• EPEC, ETEC, EIEC, EHEC and EAEC
• Enterotoxigenic E. coli (ETEC is the most common) is associated being with "Travelers Diarrhea", but isn't considered serious.
• EHEC (E. coli 0157) associated with ingestion of uncooked foods especially in young children.