Chapter 25

The sequence of events in a typical outbreak of staphylococcal food poisoning

1. Food containing protein is cooked (bacteria usually killed). 2. Then food is contaminated by worker with staphylococci on hands (competing bacteria have been eliminated). 3. Food is left at room temperature. Organisms incubate in food (temperature abuse) long enough to form and release toxins. (Reheating will eliminate staphylococci but not the toxins.) 4. Food containing toxins is eaten. 5. In 1-6 hours, staphylococcal intoxication occurs.

The stages of tooth decay

1. Healthy tooth with plaque 2. Decay in enamel 3. Advanced decay 4. Decay in dentin 5. Decay in pulp

Periodontal Disease

An expansion of the biofilm beneath the gingival surface occurs with shift from gram-positive, facultative bacteria to one predominated by gram-negative, anaerobic species occurs The supragingival (above the gingival surface) and subgingival (beneath the gingival surface) habitats differ in terms of pH, redox potential, and nutrient availability facultative anaerobes that colonize first, lower oxygen tension so anaerobic organisms take hold When you begin to get this increasing infection in gingival sulcus get sequential addition of bacteria Get aerobic bacteria- use up all the oxygen Facultative anaerobes can grow and then obligate anaerobes can grow Different Types of bacteria (oxygen-dependence) can grow over time

The Tooth

Enamel Dentin Pulp on inside Blood vessels and nerves Gingiva- gum Gingival sulcus- space between tooth and gum, pretty narrow, more space is bad, want a tight adherence between gum and tooth to have a very narrow gingival sulcus

Salmonellosis

*S. enterica: primates* - diarrhea, fever, vomiting, and abdominal cramps 12 to 72 hours after infection *a large inoculum is necessary to overcome stomach acidity and to compete with normal intestinal flora.* selectively attach to M cells, internalized, transported within phagosomes, and then released in the lamina propria promote an influx of neutrophils Bacteria that is predominantly responsible for diarrhea Takes A LOT of salmonella to create an infection in diarrhea because it is sensitive to acidity of stomach, takes a lot more than Shigella If engulfed in food, they can be protected from the acidity

*Normal Microbiota*

*Saliva* contains millions of bacteria *Stomach and small intestine have few because of acid and rapid movement of food through small intestine* - Heliobacter pylori - Fluid flow through small intestine is very rapid *Large intestine has millions of bacteria: facultative anaerobes* - *Predominantly in gamma proteobacteria, gram-negative* Assist in breakdown of food

*Typhoid Fever*

*Salmonella enterica typhi* *bacteria enter the lymphatic system of small intestine, liver, spleen, and bloodstream* - *cause a systemic form of salmonellosis* *Endotoxins act on the vascular and nervous system* - *increased permeability and decreased tone of the vessels, upset thermal regulation, vomiting/diarrhea.* Taken up by and multiply within phagocytic cells and are disseminated by them - Phagocytes lyse and release the bacteria into the blood stream On test **cause of typhoid fever- systemic form of salmenollosis, slamonella enterica typhi Chickenpox- Varicella-zoster virus (human herpesvirus 3) Virus enters respiratory system and localizes to skin cells causing a vesicular rash Complications of chickenpox include encephalitis and Reye's syndrome. After primary infection, virus enters peripheral nerves and may remain latent in dorsal root ganglia- activation causing shingles - characterized by a vesicular rash along the affected cutaneous sensory nerves - Unaffected by the immune system because antibodies can't get into cells and no viral antigen is expressed on host cell surface (no T-cell response)

Salmonella Mechanism

*Salmonella enterica* *Infection: Endotoxin* Cells enter M cells, multiply, then may pass through to lymphatic nodes and the blood stream - cells replicate in macrophages High incidence in chickens and eggs Salmonella, M cell, Epithelial cell lining intestinal tract Membrane ruffle 1. Salmonella enters an epithelial cell. 2. Salmonella multiplies within vesicle inside the cell. 3. Salmonella multiplies in mucosal cells; an inflammatory response results in diarrhea. The bacteria may cross the epithelial cell membrane and enter the lymphatic system and bloodstream. Lymph node --> blood stream M cells take them and move them to underlying tissue of epithelium- lamina propria- where there are a lot of macrophages Inflammatory response associated with diarrhea

*Shigellosis*

*Shigella expresses adherence factors for adherence to M cells in small intestine* M cells take up antigen (bacteria) from the lumen of the small intestine by endocytosis or phagocytosis transport antigens from the lumen to cells of the immune system (dendritic cells and T cells) that reside below the M cells, thereby initiating an immune response However, Shigella multiply in M cells, spread to other cells, produce toxin which destroys tissue

*Bacillary Dysentery*

*Shigella* G- (S. sonnei, S. flexneri, S. dysenteriae) *Infection: Endotoxin and Shiga exotoxin* - As few as 10-100 organisms will cause disease - Shiga toxin blocks protein synthesis in the epithelial cells leading to *destruction of the epithelial cells of the intestinal mucosa* - family of related toxins expressed by genes that are part of the genome of lambdoid prophages. Have to ingest the organisms, takes a while for the symptoms to appear Dysentery- blood involved in diarrhea influx Toxin comes from gene from prophage being inserted into host DNA and making the new characteristics of the gene virulent

*Microbial Antagonism Inhibits Tooth Decay*

*Strep. sanguinis, Strep. gordonii, Strep. oralis, Strep. mitis, Strep. sobrinus* - normal inhabitants of the healthy human mouth found in dental plaque - *modify the environment to make it less hospitable for other strains of Streptococcus that cause cavities (Streptococcus mutans)* - may compete with the mutans streptococci for colonization sites on tooth surfaces biochemically or through H2O2 In the mouth have a variety of different kinds of strep in addition to strep mutans

INTERPRETATION: Typical colony morphology on Mitis Salivarius Agar

*Streptococcus salivarius* - Large, pale-blue, sticky, mucoid colonies that are glistening (i.e., "gum-drop") in appearance. *Streptococcus mitis* - Small, flat, hard colonies, blue in color with a domed center *Streptococcus mutans* - Raised,convex, undulate, opaque, pale-blue colonies that are granular (i.e., frosted glass") in appearance. -Colonies may exhibit a glistening bubble on the surface due to excessive synthesis of glucan from sucrose. *Streptococcus sanguis* - Raised, smooth,hard colonies embedded in agar *Enterococci* Blue-black, shiny, and slightly raised colonies The media we were using was selective for strep

Paneth Cells

*To keep pathogenic bacteria out of small intestine, antimicrobial defenses include Paneth cells which line small intestine* - *Phagocytize bacteria and secrete lysozyme and proteins called defensins* - Defensins- peptides that can insert themselves into the membranes of bacterial cells creating pores, disrupting architecture, making cell more permeable Defensins: peptides that have hydrophobic and positively-charged domains - insert into membranes to form pores that disrupt bacterial membrane function Defensins are secreted when bacteria are exposed to lipopolysaccharide, muramyl dipeptide and lipid A.

*Dental Disease: Tooth Decay*

*Tooth Decay* Of the 200 to 300 species isolated from plaque, only *S mutans, and to a lesser extent the lactobacilli,* are primarily associated with dental decay When sucrose is consumed frequently, Strep mutans emerges as the predominant organism *Sucrose is used for energy metabolism; sucrose is catabolized into glucose and fructose molecules, which are then converted to lactic acid by the glycolytic pathway.* One species of bacteria that is the major contributor of tooth decay- Strep mutans When you eat a lot of sugar, creating an environment in which strep mutans can survive well on the surface, uses the sugar for carbon and energy, sucrose --> glucose and fructose, glucose goes to glycolytic pathway and is converted to lactic acid

*Cholera*

*Vibrio cholerae:* Requires ingestion of about 100 million bacteria to cause cholera in a normal healthy adult found in two animal populations: shellfish and plankton *Grow in small intestine, secrete exotoxin (cholera toxin) encoded by prophage* *Toxin causes host cells to secrete electrolytes (Na+, K+, Cl−, and HCO3− ) and water* Loss of fluids causes shock, collapse, and death Exceptionally sensitive to stomach acids Rehydration therapy is highly effective Very sensitive to acid Infection, grow in small intestine Toxin interacts with the epithelium, causing cells to secrete electrolytes and water

Type three secretion system

*promote the uptake of the bacterium by the host cell.* Effectors injected into the host cell induce the host to engulf the bacterium and to practically "eat" it. Bacterial effectors manipulate the cytoskeleton machinery of the host cell. Inject molecules which will promote the uptake of the bacteria by the host Take over cytoskeleton Bacterial pathogen (Chlamydia or Salmonella) Enters eukaryotic host cell plasma membrane Pathogen-containing vacuole- interact with different host cell pathways to mediate intracellular proliferation (and *virulence*) of the bacteria Use type III secretion systems to inject host cells effector proteins across the plasma membrane (e.g. for cell invasion) and across the vacuolar membrane Once they get in (like legionella) once in vacuole, can inject effectors into the cytoplasm to protect the bacteria from lysosomal components Molecules direct the host cell to do the bidding of the bacteria to protect them from the host cell

Can modify the environment so that strep mutans growth is inhibited

Hydrogen peroxide is used by bacteria to defend themselves or inhibit growth that they have to compete with, can produce and secrete it into environment, have enzymes on themselves to protect themselves from toxic oxygen of hydrogen peroxide radicals, strep mutans cannot protect itself, this allows the other bacteria to compete successfully

*Inhibition ability of S. sanguinis, S. gordonii, and S. mutans*

Inhibition ability of S. sanguinis, S. gordonii, and S. mutans. S. gordonii or S. sanguinis was inoculated first and grown for 16 h at 37°C S. mutans was then inoculated next to the pioneer colonizer, and the plates incubated overnight. Take strep sanguinis and try to grow strep mutants with it, kill the strep mutans, same with strep gordonii Have lots of bacteria producing H2O2 and killing strep mutans, less likely to get cavities slide 18

Peyer's Patches

Intestinal wall lined with scattered Peyer's Patches M cell surrounded by epithelial cells - Th cells - B cells - Phagocytic cells - IgA

*Dental Disease*

These glucans and especially mutan enables S. mutans to strongly adhere to and accumulate on *smooth* tooth surfaces causing decay *S. mutans* is also responsible for tooth decay in pits and fissures where glucans are not as important - Bacteria can colonize in pits and fissures Other aciduric bacteria (produce acid and can survice in low pH) such as Lactobacilli and Strep. faecalis may also be involved in the pits and fissures All the glucans + mutan allows the bacteria to adhere to the tooth surface causing decay Most bacteria can't adhere

Periodontal Disease- amount of bacteria a person can have in their mouth

Total bacteria count in plaque at gingival sulcus is 10^8 micro/mg plaque. Amount of plaque in periodontal disease is 200 mg plaque 200x10^8 = 20 billion bacteria

Oral microbial system: oral biofilm

most prevalent oral biofilm, dental plaque - attached to the tooth surface. - Gram-positive, gram-negative, aerobic, facultative, and anaerobic microorganisms deposited on the tooth surface in a sequential fashion begins with the adherence of early colonizers, streptococci and actinomycetes to glycoproteins, mucins, and other proteins coating the tooth surface - pili and outer membrane proteins mediate this initial attachment. biofilm continues to develop as late colonizers, such as veillonellae, prevotellae, propionibacteria, and certain streptococci, begin to colonize the tooth surface Dental plaque- oral biofilm where bacteria attach to surface of our tooth and start to grow When you brush your teeth- get rid of biofilm, as soon as saliva comes into mouth again, contains proteins, proteins deposit onto surface of teeth and bacteria can adhere to proteins, always ongoing

Type three secretion system (Injectisome)

protein appendage (needle-like structure) found in several Gram-negative bacteria. In pathogenic bacteria, used as a sensory probe to detect the presence of eukaryotic organisms and secrete proteins (effectors) that help the bacteria infect them. essential for the pathogenicity of many pathogenic bacteria Shigella (bacillary dysentery), Salmonella (typhoid fever), Escherichia coli (food poisoning), Vibrio (gastroenteritis and diarrhea), Burkholderia (glanders), Yersinia (plague), Chlamydia (sexually transmitted disease), Pseudomonas Secretion system- bacteria inject molecules into a cell that will help them get in or survive in that cell- Legionella's disease Secretion systems involve a needle-like structure that can detect a sensory probe, presence of eukaryotic organisms, probe to determine presence of epithelial cell in your gut When they inject, effector molecules direct the host cell to do something that will help the bacteria in one way or another Don't need all the details

Plate for oral bacteria*

Mitis salviarius agar is used to differentiate among species of Streptococcus and the very close genus Enterococcus that are flora in the mouth. These organisms have been associated with dental caries. The sugars in this medium are sacharose and glucose, as well as the dyes trypan blue and crystal violet. Trypan blue causes the colonies to become blue. The crystal violet, and 1% tellurite inhibit gram - bacteria as well as many other gram + bacteria.  Differential because it can differentiate between the different colonies slide 10

Oral microbial system

Oral cavity contains over 500 different microorganisms The sites colonized by microorganisms range from the tooth surface to the epithelium - Predominant groups include Streptococcus, Neisseria, Veillonella, Actinomyces and other obligate anaerobes - maintain a mutualistic relationship with the host by preventing pathogenic species from adhering to the mucosal surface microorganisms colonizing these surfaces are present as a biofilm The mouth is perpetually populated by pathogenic microorganisms Mutualistic relationship between us and bacteria in our mouth, normal microbiota inhibits pathogens adhering to mouth Biofilm- can be made up of multiple bacteria which have secreted polysaccharides, other carbs, proteins, etc. can feel it on surface of teeth- slimy stuff

The human digestive system

Parotid (salivary) gland Oral cavity Tongue Teeth Pharynx Esophagus Liver Gallbladder Stomach Duodenum Pancreas Small intestine Large intestine Rectum Anus

*Shigella will kill the macrophages that engulf them*

Shigella, M cell, Epithelial cell lining intestinal tract Membrane ruffle 1. Shigella enters an epithelial cell 2. Shigella multiplies inside the cell 3. Shigella invades neighboring epithelial cells, thus avoiding immune defenses 4. An abscess forms as epithelial cells are killed by the infection. The bacteria rarely spread in the blood stream Mucosal abscess Shigella will kill the macrophages that engulf them M cell- no cilia, takes bacteria, moves it to under side where there are macrophages and components that can break it down and kill it Membranous cellular ruffles surrounding the cell take the bacterium into the cell, bacteria multiply and spread to neighboring cells producing shiga toxin that destroys the tissue- dysentery is the result of damage to the intestinal tract Can move to other cells, M cell dies and other cells die How they can produce dysentery or diarrhea- destruction of the cells 714

*Campylobacter Gastroenteritis*

*Campylobacter jejuni:* *Caused by Infection* *Leading cause of food borne illness in the US* infective dose is 1000-10000 organisms Sensitive to stomach acid but antacids can reduce infective dose adhere to the gut enterocytes and can then induce diarrhea by toxin release (enterotoxin and cytotoxin) 2,000,000 cases in US/year Self-limiting High optimum growth temperature matches that in poultry (42 degrees C) Clostridium difficile *growing problem in healthcare facilities* - kills 14,000 people a year in America - *outbreaks occur when humans accidentally ingest spores in a medical facility.* *flourishes when other bacteria in the gut are killed during antibiotic therapy* - leads to colitis (antibiotic-associated diarrhea) rate of C. difficile acquisition estimated to be 13% with hospital stays of up to two weeks, and 50% with stays longer than four weeks Go to hospital and have an infection of another bacteria- give you broad spectrum antibiotics to kill a variety of bacteria- problem, it kills everything in your gut including your microbiota which protects you from pathogens Clostridium makes endospores, those endospores are all over the place, common for patients without a normal microbiota to ingest endospore- very difficult to get rid of Ingest campylobacter and taking antacids, antacids reduce acidity in stomach and can make the environment better for campylobacter survival Self-limiting- get sick then get better, drink water and Gatorade Grow best in chickens at a temp that is higher than our body temp

*Staphylococcal Food Poisoning*

*Caused by Staphylococcus aureus* - *Caused by intoxication of Enterotoxin* Heat stable to 30 min if boiling Will last through 30 min of boiling, would have to boil for a very long period of time *Enterotoxin: protein exotoxin that targets the intestines (pore-forming toxins)* - Cytotoxic, kill cells by increasing the membrane permeability of the mucosal epithelial cells of the intestinal wall - Increased permeability leads to chloride leakage into the lumen followed by sodium and water movement. - causes vomiting, diarrhea, and abdominal pain Caused disease by intoxication- ingesting an enterotoxin Cytotoxic- disrupts membrane permeability Diarrhea can be caused by- increasing membrane permeability, Cl- leaks out so Na+ leaks out also to neutralize charge in intestine and water comes out with it

The Effects of H2O2 Production on Oral Biofilm Development

*Competitors are eliminated;* the integration of H2O2 compatible species into the developing biofilm is promoted. *H2O2 production causes the release of DNA into the environment;* extracellular DNA promotes biofilm formation and cell-cell aggregation. *H2O2 causes DNA damage;* can lead to beneficial mutations Other oral streptococci can take up the changed extracellular DNA (transformation; horizontal gene transfer)

*Diseases of Lower Digestive System: Gastroenteritis*

*Gastroenteritis: inflammation of stomach and intestinal mucosa: diarrhea, dysentery* - Blood in diarrhea *Treatment: oral rehydration therapy* Few bacteria in the small intestine, however, there is a disease- small intestinal bacterial overgrowth, same symptoms and restrictions as someone who has celiac disease, no leafy green vegetables Diarrheal Cholera- lots of people pressed closely together in an area with a lack of clean water

Gum Disease

*Gingivitis: mild inflammation of the gums* - *No loss of bone and tissue that hold teeth in place.* *Periodontitis: more severe inflammation* - infectious disease caused with multiple types of bacteria being the primary etiologic factor plus yeast and protozoa - *gums pull away from the teeth and form spaces (pockets) that become infected* - *bacterial toxins and immune response to infection break down the bone and connective tissue that hold teeth in place leading to tooth loss* - Bacteria use gingival sulcus- becomes deeper and wider and more and more infected, if not taken care of with hygienist, start getting degradation and dissolving of the bone Can be a chronic disease, hereditary

*Diseases of Lower Digestive System: Infection*

*Infection: growth of a pathogen in intestines* - *Organisms penetrate and destroy intestinal mucosa* - Incubation is from 12 hours to 2 weeks - *Fever* - Need to ingest the bacteria itself pathogen enters GI tract and multiplies microorganisms can penetrate into intestinal mucosa and grow there or can pass through to other systemic organs delay in appearance of GI disturbance and a fever

*Diseases of Lower Digestive System: Intoxication*

*Intoxication: ingestion of a preformed toxin* - *Symptoms appear quickly after ingestion* - *No fever* caused by ingestion of some pathogens that cause disease by forming toxin that affects the GI tract characterized by very sudden appearance- usually a few hours

*Dental Disease: Lactic acid*

*Lactic acid fermentation leads to a rapid drop in the pH, to 5.0 or lower leading to dissolving of enamel.* - Normally, saliva buffers the acid during meals but not between meals - S. mutans tolerates low pH Plaque organisms contain enzymes that split sucrose and attach the glucose to glucose to form polymers known as a glucans. - There are several different types and different bacteria vary in the types they make However, S. murtans synthesizes all of them including one that is unique, mutan Lactic acid produced by metabolism of the sugar, lowers pH, leads to the dissolving of your enamel Saliva has buffering capacity Begin eating sugar between meals, amount of saliva produced doesn't compensate for lactic acid being produced Strep mutans can survive and grow under conditions of low pH, can split sucrose and use the glucose for energy, carbon, and to form glucose polymers- glucans which help the bacteria adhere to surface of tooth

Bacterial Endocarditis

*Oral strep bacteria (viridans; α-hemolytic) may gain entrance to the bloodstream (dental cleanings and surgeries) and colonize the mitral and aortic heart valves: it is the most common cause of subacute bacterial endocarditis* *Subacute endocarditis* Predisposing factor is preexisting heart abnormalities e.g., malformed stenotic valves, - In the company of bacteremia, valves become infected, via adhesion and colonization of the surface area. - This causes an inflammatory response leading to destruction of collagen Standard treatment is a minimum of four weeks of high-dose intravenous penicillin with an aminoglycoside such as gentamicin.

*Enterohemorrhagic EC*

*Primary virulence is from Shiga toxin.* *Toxin is released upon lysis of cell* - *Antibiotic treatment makes it worse cause of cell lysis* * Responsible for outbreaks in US due to serotype O157:H7 Cattle are the main reservoir - Runoff from feedlots contaminates veggies - ID50=< 100 bacteria May lead to hemolytic uremic syndrome - Often leads to kidney failure in children Primary virulence factor- shiga toxin ***Give a person antibiotics (especially with gram-negative bacteria), person will get worse before they get better because antibiotics are killing bacteria, releasing endotoxin into the system, get sicker and then when body begins to clear everything out, will get better* Serotype (strain)

Peptostreptococcus

Gram-negative cocci in our gram-stain may be this peptostreptococcus

Causative Organism and Clinical Features of IE (infective endocarditis)

Streptococcus viridans - This organism accounts for approximately 50-60% of cases of subacute disease. - Most clinical signs and symptoms are mediated immunologically. Streptococcus intermedius group - These infections may be acute or subacute. - S intermedius infection accounts for 15% of streptococcal IE cases. - Members of the S intermedius group, especially S anginosus, are unique among the streptococci in that they can actively invade tissue and form abscesses, often in the CNS. Group D streptococci - Most cases are subacute. - The source is the gastrointestinal or genitourinary tract. - It is the third most common cause of IE. - They pose major resistance problems for antibiotics. Group B streptococci - Acute disease develops in pregnant patients and older patients with underlying diseases (eg, cancer, diabetes, alcoholism). - The mortality rate is 40%. - Complications include metastatic infection, arterial thrombi, and congestive heart failure. - It often requires valve replacement for cure. Group A, C, and G streptococci - Acute disease resembles that of S aureus IE (30-70% mortality rate), with suppurative complications. - Group A organisms respond to penicillin alone. - Group C and G organisms require a combination of synergistic antibiotics (as with enterococci). Strep can cause infective endocarditis

*Shigellosis (Bacillary Dysentery)*

Symptoms: mild abdominal discomfort to full-blown dysentery - characterized by cramps, diarrhea, fever, blood, pus, or mucus in stools. - Onset time = 12 to 96 hours, recovery = 5 to 7 days without antibiotics Infections are associated with mucosal ulceration, rectal bleeding, and drastic dehydration transmitted through food - Food contamination through the fecal-oral route approximately 90 million cases of severe dysentery annually, 100,000 deaths, mostly children in the developing world - 18,000 cases in the United States annually