ENTEROPATHIC E. COLI (EPEC)

• Watery diarrhea and vomiting.
• Occurs most often in toddlers and infants in developing countries.

EPEC gastroenteritis

• Adhesion and effacing lesions formed on the epithelial cells of the small intestine.
  – The lesions destroy the microvillus brush border, which is where the final stages of carbohydrate and protein digestion and absorption take place.
• Virulence factors and gene locations:
  – Bundle-forming pili (BFP)* mediate initial attachment to host and bacterial cells and form microcolonies.
  BFP genes are found on the EPEC adherence factor plasmid in typical strains.
  – Interactions between intimin and the translocated intimin receptor (Tir) mediate more intimate attachment; their genes are found on the Locus of Enterocyte Effacement (LEE) pathogenicity island.
  – The Type 3 Secretion System (T3SS) translocates Tir and various cytotoxic effectors into the host cell cytoplasm; EPEC cytotoxic effectors are encoded by pathogenicity islands and phages.

ENTEROTOXIGENIC E. COLI (ETEC)

• Watery diarrhea with vomiting, fever, and abdominal cramps.
• Travelers and infants in developing countries.

ETEC gastrotenteritis

• Attaches to epithelial cells of the small intestine via colonization factor antigens I-III; the genes for CFAs are carried on plasmids.
• Flagellar adhesions and outer membrane proteins Tia and TibA also promote adherence; these traits are carried on pathogenicity islands.
• Heat-stable and heat-labile enterotoxins cause water and ion loss by altering activity in the cells of the small intestine; as a result, water and ions are flushed out of the body in diarrhea. The genes for both enterotoxins are on plasmids.
  – Heat-stable enterotoxin (STa) increases cGMP in host cells; As a result, sodium absorption decreases and water secretion increases.
  – Heat-labile enterotoxin increases cAMP; as a result, chloride secretion increases and sodium and chloride absorption decreases.

ENTEROAGGREGATIVE E. COLI (EAEC)

• Watery diarrhea with inflammatory cell infiltration, vomiting, and dehydration.
• Causes gastroenteritis in travelers and infants; in some children, it can lead to chronic diarrhea and growth retardation.

EAEC gastroenteritis

• Characterized by aggregative adherence to each other and the epithelial cells of the small and large intestines; this results in a “stacked brick” aggregation pattern
  – Attachment is mediated via aggregative adherence fimbriae; the genes for AAF are carried on a plasmid.
• AAF also trigger mononuclear cell infiltration and inflammation.
• Cytotoxins damage the intestinal mucosa.
  – The specific cytotoxins vary by strain.
  – Plasma-encoded toxin (Pet), which is coded for by plasmid genes, and induces exfoliation.
  – Be aware that some isolates produce Shigella enterotoxin 1 and EAST1.

SHIGA TOXIN-PRODUCING E. COLI (STEC)

• Watery diarrhea that can progress to hemorrhagic colitis with severe abdominal cramping; some patients develop hemolytic uremic syndrome.
• Children younger than 5 years old are most commonly infected by STEC.
• Enterohemorragic E. coli (EHEC) is generally considered to be a subset of STEC, and there is phenotypic variation even within EHEC isolates.
• Animals, particularly cattle, are key reservoirs for STEC.
• Humans are infected upon ingestion of foods, especially undercooked meat and raw fruits and vegetables; water is also a source of infection.
• Undercooked hamburger was once the main cause of STEC infection in the U.S.; increased awareness of proper handling has reduced the rate of hamburger-associated infections.
• Because STEC has a low infectious dose, person-to-person transmission is also possible.
• Antibiotics increase the risk of HUS complications, and should NOT be given to patients with STEC infections; it is thought that antibiotics cause phage lysis and increase toxin release.

STEC gastroenteritis

• Some strains produce adhesion and effacing lesions that damage microvilli brush border; as in EPEC strains, this is mediated by intimin and Tir;
• Hemorrhagic coli pilus is a type IV pilus.
• Cytotoxins cause necrosis and cell death.
  – More specifically Shiga toxins damage the microvasculature; the genes for these toxins are on phages.
• In some cases of severe infection, Shiga toxins destroy red blood cells resulting in hemolytic uremia syndrome, aka, HUS.
  – HUS is characterized by microvascular thrombi, thrombocytopenia, hemolytic anemia and renal damage that leads to renal failure.

ENTEROINVASIVE E. COLI (EIEC)

• Watery, then bloody, diarrhea and abdominal cramps.
• Fever helps to distinguish EIEC from STEC.
• EIEC infection is rare, and the bacteria are closely related to Shigella. In fact, many authors consider them to be members of the same species, and much of what we know about EIEC comes from research on Shigella.
• Unlike most other strains of E. coli, EIEC are non-motile, and do not ferment lactose.

EIEC gastroenteritis

• EIEC bacteria are intracellular pathogens that invade cells of the colon.
• Genes for the following invasion virulence factors are on the Invasion plasmid (InvP):
  – The type 3 secretion system and IpaC effector protein promote bacterial entry into cells
  – IpgD, IpaA, and VirA effectors facilitate bacterial invasion of phagosomes
  – IpaB, IpaC, and IpaD effectors facilitate phagosome escape
  – OpsB, OpsF, and OpsG effectors inhibit host immune responses
• Intra- and intercellular movement is facilitated by VirG, which promotes actin polymerization; these actin tails push the bacteria within and between host cells.
• Severe infection can lead to Shigellosis, aka, bacillary dysentery, which is characterized by colon epithelium destruction and inflammation. However, the EIEC-associated form is rare.

GENERAL PRINCIPLES

• Gram-negative Rods
• Colon microbiome
  – E. coli are common members of the colon microbiome.
  – Opportunistic pathogens: upon transfer to new anatomical niches, these strains can cause endogenous infections.
• Found in soil and water
  – As a result of animal or human fecal contamination.
  – Upon ingestion, pathogenic strains cause exogenous infections in humans.
• Because they reside in the intestines of animals and humans, E. coli are “enteric” bacteria.
• E. coli common pilus
  – Aids in adhesion to host surface and biofilm formation.
• Flagellar motility facilitates movement.
• Rapid growth
  – Simple nutritional requirements
• Facultative anaerobe
• Catalase-positive
• Oxidase-negative
• Reduce nitrate
• Ferments glucose
• Ferments lactose
  – Coliforms: Gram-negative enteric lactose fermenters.
  – Also includes strains from Klebisella, Serratia, Klebsiella, and Citrobacter.
  – Eosin methylene blue agar and MacConkey’s agar are two types of selective media used to identify coliforms; both inhibit Gram-positive bacterial growth and signify acid production from lactose fermentation.
  EMB agar: lactose fermentation and strong acid production yield a characteristic greenish hue.
  MacConkey’s agar: E. coli lactose fermentation produces a pinkish-purple color.

COMMON VIRULENCE FACTORS

Many virulence factors are acquired via horizontal gene transfer of plasmids, bacteriophages, and pathogenicity islands.

• E. coli endotoxin comprises heat-stable lipopolysaccharide:
  – Its A-antigen has classic endotoxin activity: it triggers the host inflammatory responses, disseminated intravascular coagulation, and hypovolemic shock.
  – The core polysaccharide is common to all Enterobacteriaceae.
  – The O-antigen is used to classify E. coli serotypes.
• Exotoxins promote cell death or fluid loss.
• Adhesins are present on pili, fimbriae, or strain-specific surface antigens.
• Type III secretion systems inject virulence effector proteins into host cells.
  – Virulence effectors promote attachment, invasion, and cell destruction.
• Phase variation alters the expression of O, K, and flagellar H antigens and pili.
  – Alteration of surface antigens allows E. coli to avoid host immune system and, possibly, to adjust energy expenditure according to environmental factors.
• Antibiotic resistance
  – Resistant phenotypes are rapidly spread via HGT, which makes antibiotic treatment of E. coli infections particularly difficult.
• Siderophores, special receptors, and transporters
  – Acquire iron from the host; recall that iron is necessary for bacterial growth.
• Polysaccharide capsule lends repels phagocytes and avoids serum killing.

E. COLI INFECTIONS

Extraintestinal Infections

• Causative strains are called Extraintestinal pathogenic E. coli (ExPEC).
• ExPEC are often commensals that act as opportunistic pathogens when exposed to new anatomical sites.

• Urinary Tract Infections
  – Uropathic E. coli (UPEC) causes most urinary tract infections.
  – Adhesins: Type 1 and P pili and Dr fimbriae attach to urothelium.
  – Hemolysin A causes cell damage and facilitates bacterial movement through superficial cell layers.
  – Strains lacking these virulence factors are easily removed from the body in the urine.

• Neonatal Meningitis
  – E. coli is the second most common cause of bacterial neonatal meningitis.
  – Neonatal Meningitis-causing E. coli (NMEC) have special virulence factors that enable translocation across the blood-brain barrier:
  Type I pili and Outer Membrane Protein A (OmpA) attach to brain microvascular endothelial cells.
  Ibe (invasion of brain endothelial cells) proteins, CNF1 (cytotoxic necrotizing factor 1) and other mediators facilitate invasion of the blood brain barrier.
  K1 capsular antigens are thought to prevent lysosome fusion to allow successful movement of live bacteria across the barrier.

• Septicemia
  – Septicemia can occur when urinary or gastrointestinal tract infections spread to the blood; this can happen, for example, upon trauma to the abdomen.

Gastroenteritis

• E. coli pathotypes responsible for gastroenteritis are collectively referred to as “Intestinal Pathogenic E. coli” (IPEC).
  Infection typically follows ingestion of contaminated food or water; human or animal feces is often the source of the bacteria.
• IPEC can affect the small and/or large intestine:
  – Small intestine: Enteropathogenic E. coli and Enterotoxigenic E. coli
  – Small and Large intestines: Enteroaggregative E. coli
  – Large intestine: Enteroinvasive E. coli and Shiga toxin-producing E. coli
  Virulence factors specific to each pathotype determine the pathogenesis of infection.

PATHOGENIC ENTEROBACTERIACEAE

Be aware that we will not include all pathogenic members, and that details can be found elsewhere.

Common features of enterobacteriaceae:

• They are ubiquitous in the environment, and some are commensals.
• They are a significant cause of disease, particularly urinary tract and gastrointestinal infections.
  The following characteristics are helpful in identification:
• The bacteria in this family share a common surface antigen.
• They are facultative anaerobes.
• The ferment glucose and reduce nitrate
• They are catalase positive and oxidase negative
• They have common virulence factors, including endotoxin, antibiotic resistance, and others.

Ferments lactose: Escherichia coli

• When grown in MacConkey’s agar, lactose-fermenters, including most strains of E. coli, produce bright pinkish-purple colonies; in Eosin methylene blue agar, lactose-fermenters produce colonies with a bright green sheen.
  Escherichia coli produce extra-intestinal and gastrointestinal infections, depending on the bacterial strain.

• Extra-intestinal infections are typically caused by opportunistic pathogens, such as commensals that migrate to new anatomical niches.
  – Primary cause of urinary tract infections.
  – Significant cause of neonatal meningitis.

• Gastrointestinal infections are caused by pathogenic strains in the GI tract; they are usually introduced via contaminated food or water.
• Pathogenesis and symptoms vary by pathotype
• Infants and children are most likely to suffer long-term consequences of dehydration due to severe diarrhea and/or vomiting.
• Pathotypes that tend to produce their effects in the small intestine and cause watery diarrhea and vomiting (illness is often referred to as “traveler’s diarrhea):
  – Enteropathogenic, Enterotoxigenic, and Enteroaggregative; Enteroaggregative E. coli also affects the large intestine.
• Pathotypes that primarily affect the large intestine and produce watery, then bloody diarrhea:
  – Shiga toxin-producing Escherichia coli, as its name suggests, produces Shiga-like toxin.
  Enterohemorrhagic Escherichia coli (EHEC) is usually considered a subset of Shiga toxin-producing Escherichia coli.
  In severe infection, a condition called hemolytic uremic syndrome (HUS) can develop when red blood cells and platelets are destroyed; the damaged cells can obstruct the renal filtration system and cause renal failure.
  Antibiotics increase the risk of HUS in patients with Shiga toxin-producing Escherichia coli infection.
  – Enteroinvasive Escherichia coli is similar to Shigella, which produces inflammatory lesions and ulcers in the colon.

Enterobacteriaceae that do not ferment lactose (or do so very slowly)

• They produce colorless colonies on MacConkey’s agar, as opposed to the bright pink colonies of E. coli.

Salmonella

• Transmitted via the fecal-oral route, usually via contaminated foods.
• Causes typhoid and paratyphoid fevers, which are characterized by flu-like, nonspecific and gastrointestinal symptoms.
  – More specific signs of typhoid and paratyphoid fever include *gallbladder infection due to bacterial replication, liver and spleen enlargement, and the development of so-called rose spots over the skin of the torso.
• Also causes gastroenteritis without typhoid fever; this is the most common form of infection in the United States.
• Can cause extra-intestinal problems, such as osteomyelitis and septicemia; recurrent septicemia is an AIDS-defining condition.

Shigella

• Transmitted via the fecal-oral route, and show that it is an intracellular pathogen.
• Shigellosis is most common in children, and is easily transmitted due to its low infectious dose.
• Associated with watery, then bloody diarrhea.
• Shigella strains that produce the Shiga toxin can cause Hemolytic uremia syndrome (HUS), often in children.

Yersinia

• Zoonotic infections.
• Fleas transmit the strain of Yersinia that causes the plague, which is characterized by fever, chills, and weakness.
  Three key forms of the plague: bubonic, system, and pneumonic.
• Bubonic plague is characterized by infection of the lymphatic system, and that bacterial replication in the lymph nodesproduces buboes.
  – Localized lymph node destruction produces these inflamed, raised areas that are tender or even painful.
• Septicemic plague occurs when the bacteria enter the bloodstream. Patients experience extreme weakness, as well as pain in the abdominal area.
  – Disseminated intravascular coagulation occurs, producing necrosis and even organ failure without treatment.
• Pneumonic plague can occur if bubonic plague spreads to the lungs, or, in other cases, as the result of human-human transmission via respiratory droplets.
  – Inflammation and necrosis in the lungs can rapidly lead to respiratory failure.

• Entercolitis is caused by other strains of Yersinia
• Transmitted via consumption of infected animals, especially pork products.
  – Whereas younger children are likely to have bloody diarrhea, older children tend to experience pain the right abdomen that may be mistaken for appendicitis.

ACTINOMYCES ISRAELII

• A. israelii is anerobic, slow-growing, and has low virulence.
• Opportunistic pathogen:
  – It is a common colonizer of the oral cavity and upper respiratory tract, and is sometimes present in the gastrointestinal and urogenital tracts.
• Branching, fungus-like morphology.
• Colonies, aka, sulfur granules
  – Granules comprise bacteria and calcium phosphate, and have a dimpled, molar-like appearance.
  – They do not contain sulfur.
• Most A. israelii infections occur when the bacteria invade deeper tissues of the oral cavity after trauma or surgery. From there, infections can spread.

Actinomyces:

• Chronic, slow forming granulomatous lesions that become abscesses that drain pus with sulfur granules.
• Infection most commonly occurs in the cervicofacial region following dental trauma.
  – Produces localized swelling, often in the mandibular region: “lumpy jaw.”
  – The abscess may form sinus tracts that erupt on the face.
• When infection occurs in other sites, more serious complications can arise.
  – Central nervous system infection can manifest as a single abscess with headache and focal neurological signs. In the image of an abscess removed from the brain, we see examples of “dust bunny” formation; this reflects aggregation of the filamentous bacteria.
  – Thoracic infection typically produces nonspecific symptoms such as fever and non-productive cough, and lung abscesses may form.
  – Abdominal cavity infection can affect any organ, and can produce fever and fatigue. Be aware that digestion problems and inflammation may be mistaken for signs of Crohn’s diseases, and masses have been mistaken for tuberculosis and cancerous tumors.
  Abdominal cavity infection can affect any organ, and can produce fever and fatigue.
  – Pelvic actinomycosis has been associated with long-term use of intra-uterine devices; masses can easily be mistaken for tumors.

• Prevention of actinomyces includes good oral hygiene, and, in the case of dental procedures, prophylactic antibiotics.
• Treatment includes drainage or surgical debridement when necessary and administration of penicillin.

Be aware that other species of Actinomcyes are also associated with actinomcyosis.

NOCARDIA

Includes several species that can infect and cause nocardiosis.

Be aware that Nocardia nomenclature and classification has changed dramatically over the years. As a result, some isolates that were once commonly associated with nocardiosis no longer are. For example, several isolates formerly identified as Nocaria asteroides have been reclassified.

• Nocardia are weakly acid-fast, with a delicate “beaded”appearance.
• Aerobic and catalase-positive, with slow growth.
• Unique aerial hyphae, with filaments that grow upward from the colony.
• Nocardia are not considered part of the normal human microflora.
• Found in soil.
• Gain entry to human hosts via inhalation and aspiration.
• Individuals with defective cellular immunity are particularly susceptible to infection.
• Pathogenic Nocarida have multiple ways to avoid phagocytic destruction, which, as you may recall, is key to innate immunity.
• Enzymes catalase and superoxide dismustase protect them from the harmful effects of phagocytic reactive oxygen species.
• Cord factor: When phagocytosed by macrophages, Nocarida cord factor (aka, trehalose dimycolate) prevents phagosome-lysosome fusion, which means the bacteria avoid bactericidal molecules.
  – Nocardia can survive and replicate within macrophages, which travel throughout the body.

Nocardiosis

Overall, Nocardiosis is rare, and manifests as non-specific symptoms.
However, it should be ruled out early to avoid delayed diagnosis and treatment. Fortunately, the bacteria’s weakly acid-fast nature and aerial hyphae make it easy to identify.

• Lung infection is most common.
  – Illness onset is associated with nonspecific symptoms.
  – Lung abscesses and necrosis can develop, and dissemination to other organs can occur.
• CNS infection is the most serious form of nocardiosis, and results in abscesses with non-specific symptoms (such as fever and headache); meningitis is possible but infrequent.
• Cutaneous infections manifest as granulomas, ulcers, or cellulitis, and may involve nearby lymphatics. Infection can be primary or secondary.

• Treatment
• Nocardia response to antibiotics varies, so testing for antimicrobial susceptibility is crucial.
• In general, pulmonary infections are treated with trimethoprim-sulfamethoxazole (TMP-SMX) and amikacin;
• CNS infections are treated with trimethoprim-sulfamethoxazole and imipenem or cephalosporin.
• Prolonged antibiotic treatment is recommended to avoid relapse.

CORYNEBACTERIUM DIPHTHERIAE

Causative agent of diphtheria.

• Club-shaped, and often arranged in “L” or “V”- shaped formations.
• Pathogenic C. diphtheriae contain granules with volutin, aka, polyphosphate, which provide intracellular energy storage and stain metachromatically.
• Non-motile
• Airborne transmission, human-human is typical.
• Bacteria colonize the oropharynx and skin of asymptomatic carriers, which maintains their presence within populations.
• Four subtypes of C. diphtheriae; the mitis biotype is most often responsible for human disease.

Diptheria Toxin

• Lysogenic bacteriophages introduce the Diphtheria toxin gene into the bacteria.
• The A subunit acts via ADP-ribosylation of elongation-factor 2to inactivate host cell protein synthesis.
• The B subunit has two regions:
  – Binding region binds heparin-binding EGF-like growth factor, which is anchored to the membranes of many host cells, particularly heart and nerve cells.
• Translocation region facilitates movement of diphtheria toxin into the host cell.

Respiratory diphtheria

• Sudden onset of fever, sore throat, and adenopathy.
• Pharyngeal exudate forms a pseudomembrane that can extend to the larynx.
  – Comprises immune cells, bacteria, and fibrin, and, unique to diphtheria.
  – It is firmly adhered to the underlying tissue.
• Complications:
  – Obstructed airways.
  – Neurotoxicity, which tends to manifest as cranial nerve weakness beginning in the pharynx.
  – Myocarditis is common in diphtheria, and tends to appear a week or two after illness onset.
  – Edema with inflammation in the myocardium.
  – Arrhythmia, heart failure, and death.

Cutaneous diphtheria

• Occurs after skin contact with an infected person.
• Characterized by chronic ulcers, which may be covered by gray membranes.

Prevention with DPT Vaccination:

• Typically, in the U.S, children are given a series of injections with a combined preparation of diphtheria, pertussis, and tetanus antigens.
• Booster immunizations should be given every 10 years after the last childhood injection.

Treatment:

• Antitoxin should be given immediately because the toxins bind irreversibly and cause cell death. – Test for hypersensitivity because serum sickness can occur.
• Antibiotics such as Penicillin G or erythromycin should be prescribed.
• Vaccinate after recovery.
  – Many people do not develop protective antibodies in response to natural diphtheria infections.

LISTERIA MONOCYTOGENES

Causes febrile gastroenteritis and meningitis.

• Animals, plants, and soil.
• Also grows readily in cold temperatures.
• In the U.S., infectious outbreaks are associated with contaminated unpasteurized dairy products and deli meats.
• Short rods; some authors categorize them as coccobacilli.
• Gray, weakly beta-hemolytic colonies on blood agar plates.
• Unique tumbling motility, which makes it appear as if it’s doing summersaults in broth media.
• Facultative intracellular anaerobes.

Virulence factors

Adhesion and invasion, vacuole escape, and movement are regulated by positive regulatory factor (PrfA), a transcription factor that is activated upon host cell infection.
These virulence factors allow L. monocytogenes to move from the lumen of the gut to infect the meninges and other body tissues.

• Internalins A & B facilitate attachment and entry into host cells:
  – Internalin A recognizes receptors on host enterocytes.
  – Internalin B interacts with a wider range of cells, including endothelial cells, fibroblasts, and enterocytes.
• Listeriolysin O and Phospholipase C
  – Upon entry into the host cell, bacteria release listeriolysin O and phospholipase C to escape from vacuoles.
  – This promotes microbial evasion of phagolysosome destruction and access to the cytosol for reproduction.
• Actin assembly-inducing protein (ActA) facilitates intra- and intercellular movement via “comet tails”.
  – ActA is also associated with aggregation and biofilm formation, and avoidance of autophagy.

L. monocytogenes meningitis pathogenesis:

1. Ingestion of L. monocytogenes-contaminated foods.
2. Interalins facilitate entry to enterocytes from the lumen of the GI tract.
3. Actin tails push the bacteria out of enterocytes and to macrophages, where replication occurs.
4. The parasitized macrophages disseminate the bacteria throughout the body.
5. Ultimately, L. monocytogenes can cross the blood-brain barrier and cause meningitis.

Listeriosis

Infection with L. monocytogenes can induce a range of dysfunction: some individuals are asymptomatic carriers, while others, particularly those with defective cellular immunity, experience disease.

• Healthy adults: flu-like symptoms and gastroenteritis with watery diarrhea, fever, aches, and abdominal cramps.
• Immunocompromised adults, including pregnant women, the elderly, and transplant recipients: bacteremia and meningitiscan develop.
  – L. monocytogenes meningitis is associated with high mortality.
• Neonate: Early and late onset diseases:
  – Early onset infection occurs when the bacteria cross the placenta; this can result in spontaneous abortion, pre-term birth, or granulomatosis infantiseptica, which is characterized by rash, abscesses and granulomas in the liver, lungs, spleen, and other organs.
  – Late-onset infection is acquired during or soon after birth, and can result in meningitis that appears up to a month after birth.

Prevention

• Because L. monocytogenes is ubiquitous in the environment, and there is no vaccine, prevention is difficult.
• At-risk populations, including pregnant women, are advised to avoid unpasteurized dairy foods and cold deli foods.

Treatment

• Invasive infections can be treated with ampicillin and gentamicin.

CLOSTRIDIUM OVERVIEW

• Rapid-growing
• Thrive in anaerobic conditions, such as the intestinal tract, sewage, water, and soil.
• Pathogenic strains produce histolytic toxins, enterotoxins, and neurotoxins.
• Four strains that cause infection in humans:
  – Clostridium difficile causes diarrhea and colitis.
  – Clostridium perfringens causes gas gangrene and food poisoning.
  – Clostridium tetani causes tetanus.
  – Clostridium botulinum causes botulism.

CLOSTRIDIUM DETAILS

Clostridium difficile

• Transmitted via the fecal-oral route
• Common colonizer of the human colon.
• Historically associated with hospitalized patients, we’ve seen a rise in community-acquired cases.
• A major cause of C. difficile infection is antibiotics that suppress the non-pathogenic colonic flora, which allows the opportunistic C. difficile to flourish.

Virulence factors include potent toxins:

• Toxin A is an enterotoxin that attracts neutrophils, which release cytokines.
  – Toxin A also increases intestinal wall permeability by attacking colonic epithelial tight junctions.
• Toxin B is a cytotoxin that acts on enterocyte actin to destroy cytoskeletal integrity.
• Binary toxin: Some strains of C. difficile produce Binary toxin, aka, C. difficile transferase (CDT).
  – The role of Binary toxin in C. difficile infection is uncertain: Some studies suggest that Binary toxin increases bacterial adherence to host cells and promotes cell death; others report that the toxin suppresses eosinophil activity. It is possible that the toxin has multiple virulence effects.

Infection:

• C. difficile infection acts on the colon, where it induces a range of gastrointestinal issues, from mild diarrhea to severe colitis.
  – Pseudomembranous colitis is the most serious form of C. difficile infection; characterized by yellowish-white exudate on the mucosal surface of the colon. The pseudomembrane comprises fibrin and inflammatory cells in mucus.

Treatment & Prevention:

• Withdrawal of the associated antibiotic often suffices to treat diarrheal symptoms.
• Metronidazole or vancomycin can be administered in more serious cases.
• Colitis relapse is common. Patients with multiple relapses may benefit from fecal microbiota transfer from a healthy donor, which replenishes the healthy bacteria in the colon.

Clostridium perfringens

• Spores are rarely seen clinically, and, that colonies are flat and irregular; it is beta-hemolytic.
• C. perfringens can be divided into subtypes based on toxin production.

Virulence factors:

• Alpha toxins cause hemolysis, vascular leakage, liver toxicity, and cardiac dysfunction.
  – Present in all C. perfringens subtypes.
• Several other toxins form pores and/or induce necrosis.
• Food poisoning enterotoxins alter the intestinal membrane permeability, leading to fluid and ion loss; like some other bacterial toxins, this enterotoxin is a superantigen.

Infection:

• Range of soft tissue infections, including cellulitis, fasciitis, myositis, and myonecrosis.
  – Myonecrosis, aka, gas gangrene, which is a life-threatening disease that destroys muscle tissues. Bacterial metabolic activity produces characteristic gas bubbles, which appear as purlplish-black bulges under the skin. Tissue necrosis and other complications can lead to death.
• C. perfringens food poisoning typically results from consumption of contaminated meat products; the enterotoxin acts on the small intestine to produce abdominal cramps and watery diarrhea without fever or vomiting.

Treatment & Prevention:

• Soft tissue infections:
  – Treatment for C. perfringens soft-tissue infections includes antibiotics and surgical debridement of necrotic tissues.
  – Proper wound care is essential for prevention of infection.

• Food poisoning:
  – Treatment for food poisoning includes rehydration; antibiotics are not recommended because this type of food poisoning is self-limiting.
  – Prevention is achieved by refrigerating and thorough reheating of foods to at least 74 degrees Celsius.

Clostridium tetani

• Distinctive tennis-racket shape during spore formation.
• It is extremely oxygen sensitive, and spores can survive for extended periods of time in the soil.
• Infection typically occurs when wounds encounter contaminated soil.
• Wounds provide ideal necrotic and anaerobic environments for C. tetani growth.

Virulence factors:

• Tetanospasmin, is a heat-labile neurotoxin that blocks the release of inhibitory neurotransmitters, such as GABA and glycine
  – The neurotoxin is endocytosed, then transported along axons to reach neuronal somas in the spinal cord.
  – Because it blocks inhibitory neurotransmitter release, neuronal excitatory activity is unregulated.
• Tetanolysin is another toxin produced by C. tetani.
  – It is an oxygen-labile hemolysin thought to promote tissue necrosis.

Infection:

• Tetanus is characterized by spastic paralysis:
  – This can manifest systemically or locally as lockjaw, grimace (aka, risus sardonicus), and opisthotonos, in which spasms of the extensors of the head, neck, and back produce extreme back extension. Tetanus can also cause fever and sweating.
  – Cephalic tetanus involves the cranial nerves.
  – Maternal tetanus is associated with pregnancy, specifically, contamination during medical or spontaneous abortion and delivery.
  – Neonatal tetanus occurs when infection spreads from the umbilical stump.

Treatment & Prevention:

• Vaccination effectively prevents tetanus.
• Treatment for infection involves wound debridement and administration of metronidazole and antitoxins are necessary to prevent death.

Clostridium botulinum

• Foodborne disease: the spores tend to contaminate vegetables and meat.
• Toxins are protected from degradation within the gastrointestinal tract.

Virulence factors:

• C. botulinum produces exotoxins A-G.
• Types A, B, and E are responsible for most human infections.
• Toxins are pre-formed, particularly in canned goods, which provide ideal alkaline and anaerobic environments.
• From the gut, the neurotoxins are absorbed and delivered to motor neurons; upon endocytosis, neurotoxins block acetylcholine release at peripheral motor junctions.

Infection:

• Botulism
  – Descending flaccid paralysis, blurred vision with dilated pupils, dry mouth, and constipation.
  – Respiratory failure leads to death.
  – Wound infection produces similar symptoms; in the US, this form is associated with injection drug use.

Treatment & Prevention:

• Treatment includes metronidazole or penicillin and antitoxins.
  – Respiratory support may be necessary, and gastric irrigation can aid in toxin removal.
• Prevention of botulism involves boiling of home-canned goods and discarding damaged cans
• Honey can contain C. botulinum organisms, so it should not be given to infants because they have not yet acquired the competitive microflora that prevents C. botulinum survival in adults.

BACILLUS ANTHRACIS

• “Anthrax” means “charcoal,” which describes appearance lesions it produces on the skin.
• Is the causative agent of anthrax.
• Resides in the soil, where its spores can persist for years; spores are also found on animals, especially on hides and wool.
• Non-motile
• In clinical specimens, spores are rarely seen; colonies are non-hemolytic.
• Current treatment recommendations for B. anthracis infection include ciprofloxacin or doxycycline with antitoxins
  – Amoxicillin is often used to treat cutaneous anthrax.
• In areas where B. anthracis is endemic, vaccination of humans and animals can help control disease.
• Infected animals should be incinerated, as spores can remain in the soil for many years.

Virulence factors:

• Unique polypeptide capsule comprising D-glutamic acid, which enables host immune evasion (recall most bacterial capsules comprise polysaccharides).
• Exotoxins: Edema toxin and Lethal toxin.
  – Toxins comprise A and B subunits:
  B subunit is Protective antigen (PA); this is the region of the toxin that binds with host cells.
  – A subunits are factors that combine with Protective antigen to form active toxins:
  Edema factor (EF) is an adenylate cyclase that increases intracellular cyclic adenosine monophosphate (cAMP), resulting in edema.
  Lethal factor (LF) is a protease that inactivates mitogen-activated protein kinase (MAPK) pathways, resulting in cell death.

Be aware that these factors are nontoxic on their own; they must combine with protective antigen to enter host cells and cause damage.

Infections:

• B. anthracis primarily infects non-human animals.
• Interaction with contaminated animal products can lead to three types of human infections:
  • Cutaneous, Inhalation, Gastrointestinal.

• Cutaneous anthrax
  – Most common form.
  – Presents as skin lesions with central necrotic eschar surrounded by edema. The pustule is initially painless, but infection can progress to produce systemic signs of edema, and bacteremia can be fatal.
  – Typically, cutaneous anthrax is due to exposure to contaminated soil or animal hides, hair, or wool; however, outbreaks have also been reported among injection drug users.

• Inhalation anthrax
  – Initially presents with nonspecific symptoms, including fever, non-productive cough, and myalgias.
  – However, as the spores travel from the lungs to the nearby lymph nodes, edema and mediastinal lymph node enlargement occurs; in chest X-rays, mediastinal widening is an important diagnostic cue. Respiratory failure can ensue, and, in about half the cases, meningeal symptoms occur.
  – Historically, inhalation anthrax in humans was associated with spore inhalation while working with animal products. However, weaponized anthrax has been used in bioterrorism; person-to-person transmission does not occur, because bacterial replication occurs in the lymph nodes.

• Gastrointestinal anthrax
  – Aka, ingestion anthrax, occurs upon consumption of contaminated meat.
  – Manifests in the upper and lower gastrointestinal tract:
  In the oral cavity, pharynx, and esophagus, anthrax produces lymphadenopathy, edema, sore throat, and can lead to sepsis. In some patients, pseudeomembranes form; these are grayish coverings that comprise fibrin, leukocytes, and other exudates.
  In the lower gastrointestinal tract, particularly the ileum and cecum, infection causes ulcerative lesions and edema, with nausea, vomiting, and bloody diarrhea.

BACILLUS CEREUS

• “Cereus” means “wax-like”; these bacteria produce flat, grayish white colonies on blood agar plates.
• Environmentally ubiquitous, motile, and beta-hemolytic.
• It causes two forms gastroenteritis, aka, food poisoning, upon consumption; symptoms depend on the type of toxin ingested.
  – Symptomatic treatment is given, infection resolves itself.

Emetic food poisoning

• An intoxication caused by pre-formed cereulide, which is heat stable.
• Spores survive initial cooking, and germinate if the food is not refrigerated; importantly, reheating does NOT kill the enterotoxin.
• It is commonly associated with rice and other starchy foods left at room-temperature for long periods of time.
• Toxin produces nausea, abdominal cramps, and vomiting; in rare complications, liver failure occurs when large quantities impair mitochondrial fatty acid metabolism.
• Quick: the incubation period < 6 hours after consumption, and illness duration < 24 hours.

Diarrheal food poisoning

• Infection by vegetative cells that produce heat labile enterotoxin in the intestine.
• Within the intestinal epithelial cells, the toxin increase the concentration of cyclic AMP.
• The bacteria tend to reside on meats and vegetables.
• They multiply in the gastrointestinal tract, where heat labile enterotoxin produces nausea, abdominal cramps, and watery diarrhea.
• Long duration: incubation period is > 6 hours; duration > 24 hours.

Other B. cereus infections:

• Ocular infections associated with trauma, surgery, or bacteremia.
  – At least three toxins are associated with ocular infection: necrotic toxin, cereolysin, and phospholipase C. Interactions of these toxins, and perhaps factors, leads to rapid infection progression and eye loss.
  – Clindamycin or vancomycin is urgent.
• Severe pneumonia* that mimicks inhalation anthrax.
• Intravenous catheter and CNS shunt infections, endocarditis, bacteremia, and meningitis.
  – Immunocompromised patients.

Spore-Producing Rods
Bacillus (aerobic) and Clostridium (anaerobic)

• Bacillus anthracis is the causative agent of anthrax, which takes on cutaneous, respiratory, and gastrointestinal forms.
  – Cutaneous anthrax is the most common and least dangerous form.
  Contracted from handling animal hides or other products that carry anthrax spores; intravenous drug use can also spread cutaneous anthrax.
  Lesions tend to be on exposed areas of the body, including the face, neck, forearms, and hands. Indicate that lesions comprise a red area of edema with a central necrotic eschar.
  – Inhalation anthrax is the most lethal form of anthrax.
  Upon inhalation of the spores, the mediastinal lymph nodes enlarge; pulmonary edema occurs (which produces a bat-wing appearance on x-ray), and, respiratory failure from collapsed lung can result without effective treatment.
  – Gastrointestinal anthrax is the result of consumption of contaminated meats.
  The effects can occur throughout the GI tract, and include lesions and ulcers; patients can experience vomiting and diarrhea.

• Bacillus cereus causes food poisoning, both emetic and diarrheal forms.
  – Infection is especially associated with contaminated rice, meats, and vegetables.

• Clostridium difficile causes colitis, which is characterized by inflammation of the colon and diarrhea.
  – A pseudomembranous covering can develop on the wall of the colon. The covering, which comprises inflamed mucosa with raised yellow plaques, looks like a furry rug in gross images.
  – Colitis is associated with antibiotic use, which can kill off the normal, harmless gastrointestinal bacteria and allow for Clostridium difficile overgrowth.

• Clostridium perfringens can cause soft tissue infections, including myocystis, cellulitis, and myonecrosis.
  – Myonecrosis, which is also called gas gangrene, is characterized by areas of necrosis that produce gaseous bubbles in the tissues.
  – Clostridium perfringens can also cause food poisoning.

• Clostridium tetani, which can be found in the soil, causes tetanus.
  – Tetanus is characterized by spastic paralysis; show that this can produce the characteristic extension of the head, neck, and spine, called opistotonos.

• Clostridium botulinum causes botulism; the bacteria can be found in canned goods, and is the reason that infants under one year of age should not be given honey (by childhood, most individuals are resistant to the pathogen in honey).
  – Botulism is characterized by descending flaccid paralysis and respiratory failure.

Non-Spore-Producing Rods

• Corynebacterium diphtheriae
  – The Diphtheria toxin gene is introduced to the bacteria via a bacteriophage.
  – Respiratory diphtheria is characterized by a sore throat with a firmly adhered pseudomembranous covering.
  Serious complications can arise, including airway obstruction, myocarditis, and cranial neuropathies.

• Listeria monocytogenes causes listeriosis.
  – Individuals become infected when they ingest contaminated foods, most often deli meats or cheeses (this is why pregnant women are advised to avoid these foods).
  – The effects of listeriosis depend on the health and age of the individual:
  In healthy, non-pregnant adults, infection can cause flu-like symptoms with watery diarrhea.
  Immunocompromised adults are at increased risk of meningitis.
  Neonates fare the worst, with the possibility of death, organ damage, or meningitis.

OVERVIEW

• Gram-negative cocci
• Neisseria are paired; they tend to look like coffee beans.
• Catalase and oxidase positive
  – Catalase is an enzyme that protects bacteria from oxidative damage.
• Pathogenic Neisseria with Type IV pili have twitching motility (be aware of intertextual variation regarding the motility of Neisseria).
• Invade host cells; in the image of a fluid sample, we see the tiny Neisseria gonorrhoeae inside the larger neutrophils.

Neisseria meningitidis

• Aka, meningococcus.
• Pathogenic serogroups include A, B, C, Y, and W-135.
  – Different serotypes are responsible for geographically distinct epidemics.
• Neisseria meningitidis can be grown on both chocolate agar and blood agar; under some conditions, it can be grown on nutrient agar.

Neisseria gonorrhoeae

• Aka, gonococcus.
• Require highly specific growth conditions, and can only be grown on chocolate agar.
  – Selective Thayer-Martin chocolate agar has antibiotics to exclude other bacterial types present in the sample.

Neisseria Virulence Factors

• Many virulence factors are part of the cell wall.
• Neisseria endotoxin comprises Lipooligosaccharide (LOS), with toxic lipid A region.
  – During infection, Neisseria can release “blebs” of their cell membranes with the endotoxin.
• Pili have multiple effects:
  – Attach to host cells, which is a key step in colonization.
  – Resist neutrophil bactericidal activities.
  – Type IV pili facilitate twitching motility.
• IgA1 protease degrades Immunoglobulin A, which enables the bacteria to reach the mucous membranes of the respiratory and genital tracts.
• Neisseria have special receptors that bind to host transferrin, lactoferrin, and hemoglobin to acquire iron, which is crucial for bacterial metabolism.
  – Notice that this mechanism is different from the siderophores of other bacteria.
• Opacity proteins (aka, Opa), which bind epithelial and phagocytic cells; they also engage in cell to cell signaling.
  – Strains with these proteins appear opaque in culture, hence their name.
• Reduction-modifiable proteins (Rmp) protect porin and lipooligosaccharide from bactericidal antibodies.
• Porin proteins (aka, Por) insert pores into the bacterial cells to allow for movement of nutrients and wastes; they also prevent phagolysosome fusion within neutrophils, which allows the bacteria to survive intracellularly and aid in the invasion of epithelial cells.
  – There are two porin genes:
  PorA is only active in Neisseria meningitidis
  PorB is active in both N. meningitidis and N. gonorrhoeae.

Opa, Por, and Rmp proteins are also referred to as outer membrane protein classes I-V, particularly in Neisseria meningitidis.

• Virulence factors associated with specific species.
  – Neisseria meningitidis:
  Outer polysaccharide capsule that allows it to resist phagocytosis. Several vaccines use the capsule as immunogen, however, this is not effective for Group B meningococcus because its capsule is not immunogenic.
  Factor H binding protein (FHBP) binds Factor H to inhibit complement factor C3b; thus, this protein inhibits opsonization and membrane attack complex formation. And, because it is present on Group B meningococci, it is used as a vaccine immunogen.
  – Neisseria gonorrhoeae:
  Some strains produce beta-lactamase (aka, penicillinase), which promotes penicillin resistance.

NEISSERIA MENINGITIDIS

• Transmitted in airborne respiratory droplets.
• Within the host, pili and other adhesins promote adherence to the pharynx; from here, N. meningitidis can enter the bloodstream.
• Individuals with complement deficiencies are especially at risk for meningococcal disease.
• Some people are chronic carriers that can spread infection to others.
• Meningococcal diseases are largely preventable with vaccines.
• Infections can be treated with Cephalosporin or Penicillin G.

Infections
Meningitis:
– Inflammation of the meninges that cover the brain and/or spinal cord.
– Neisseria meningitidis is the most common cause of meningitis in individuals ages 2-18; Streptococci pneumoniaeis also major cause of bacterial meningitis in this age group.
– Patients have fever, stick neck, headache, and increased neutrophils in their cerebrospinal fluid.

Meningococcemia:
– Can occur with or without meningitis.
– Characterized by fever, headache, nausea, vomiting, and chills.
– Skin lesions typically begin as small petechiae-like spots that can coalesce into larger hemorrhagic areas; vascular damage can lead to gangrene.
– Musculoskeletal pain, particularly of the joints, is common.
– In severe cases, adrenal gland hemorrhage (aka, Waterhouse-Friderichsen syndrome) and thrombosis with vessel and organ damage can be fatal.

Pneumonia, particularly in older adults.

NEISSERIA GONORRHOEAE

• No vaccine.
• Sexually transmitted
• Can cause local infections of the genitals, rectum, and pharynx; within the blood, it causes disseminated infection.
• Asymptomatic carriers are key reservoirs

Infections

Localized

• In males, the most common site of infection is the urethra; purulent discharge is common.
  – Complications include epididymitis, prostatitis, and development of periurethral abscesses.

• In females, the cervix is the primary site of infection.
  – Many women are asymptomatic; others experience cervical discharge, abdominopelvic pain, and bleeding.
  – Complications occur when infection ascends to the uterus and uterine tubes, resulting in pelvic inflammatory disease.
  – As a result of scarring, ectopic pregnancies and infertility can occur.

• Opthalmia neonatorum
  – Mothers can pass gonorrheal infection to offspring during passage through the birth canal. Affected neonates acquire opthalmia neonatorum, a form of conjunctivitis. Prophylactic use of erythromycin ointment shortly after birth can prevent this.

Systemic

• Gonococcemia
  – Produces fever, chills, and skin pustules on the extremities; septic arthritis can also occur.

OVERVIEW

• Streptococcal agalactiae is beta-hemolytic; it is also referred to as Group B streptococcus.
• Enterococci faecalis and Enterococci faecium, which were formerly classified as members of Group D streptococcus; they are alpha and gamma hemolytic.
• Viridans group streptococci, include several alpha-hemolytic subgroups.
• Streptococcus pneumoniae, aka, pneumococcus, which some authors categorize as members of the Viridans group.

DETAILS

Group B streptococcus

• Virulence factors that contribute to its pathogenicity:
  – Polysaccharide capsule inhibits phagocytosis.
  – Sialic acids in the capsule inhibit activation of the alternative complement pathway and neutrophilic activities, thus facilitating host immune evasion.

• Colonization and Infection:
  – Group B streptococcus commonly colonizes the colon and genitourinary tracts of women.
  – Group B streptococcus can be passed to the neonate, particularly if labor is prolonged; administration of antibiotics to infected mothers can help prevent bacterial transmission.
  – Early-onset diseases occur within the first 7 days after birth, and include bacteremia, pneumonia, and meningitis. This is typically the result of transmission during labor and birth.
  – Late-onset diseases occur between 1 week and 3 months of age, and include bacteremia with meningitis.
  – In women, Group B streptococcus is associated with post-partum endometritis, which is characterized by infiltration of plasma cells and inflammatory cells to the endometrium; it also causes wound and urinary tract infections.
  – In both men and women, Group B streptococcus is associated with bacteremia leading to pneumonia and infections of the bones, joints, skin, and soft tissues; immunocompromised, diabetic, and alcoholic patients are at higher risk of Group B streptococcal infections.
  – Penicillin G is the preferred antibiotic to treat Group B streptococcal infections.

Enterococci faecalis and Enterococci faecium

• Virulence factors:
  – Easily adhere to host surfaces via surface proteins and pili, which promote biofilm formation.
  – Are resistant to most antibiotics; thus, although not as virulent as some other Gram-positive cocci, established infections are difficult to eradicate.

• Infection:
  – Enterococci are particularly problematic in hospital settings, where they are associated with:
  Urinary tract infections, Bacteremia, Endocarditis, and Peritonitis (inflammation of the peritoneum).

Viridans group streptococci

Subgroups: S. mutans, S. salivarius, S. angionosus, S. mitis, and S. sanguinis.
– Fortunately, this group is mostly vulnerable to antibiotic treatments, although S. mitis is an important exception.
– Viridans group streptococci are associated with: Deep tissue abscesses (S. sanguinis); Bacteremia (S. mitis infection in cancer patients with neutropenia); Subacute endocarditis (S. mitis and S. salivarius); and, Dental caries (S. mutans).

Streptococcus pneumoniae

• Virulence factors:
  – Its polysaccharide capsule inhibits phagocytosis, and recombination of capsular genes facilitates antibiotic resistance.
  – Surface proteins bind respiratory tract epithelia, which facilitates respiratory infections.
  – IgA protease keeps the bacteria trapped within the mucin and inhibits clearance.
  – Pneumolysin lyses phagocytic cells and ciliated respiratory epithelium, which also prevents bacterial clearance.
  – Complement activation produces inflammation, and is triggered by pneumolysin, teichoic acid, and peptidoglycan fragments.
• Hydrogen peroxide damages host tissues.
• Phosphorylcholine facilitates entry into host cells, which protects bacteria from removal and promotes migration to deeper tissues.

• Infections:
  – Pneumonia, particularly in the lower lobes: In the sample, we can see that bacteria, red blood cells, and white blood cells infiltrate the alveolar tissue. Symptoms include chest pain (aka, pleurisy) with fever and chills with coughing that produces rust-colored sputum. Be aware that patients with dysfunctional or absent spleens can have severe forms of pneumococcal pneumonia.
  – Otitis media and sinusitis can occur after viral infections that obstruct sinus drainage and allow bacteria to proliferate.
  – Pneumococcal meningitis can occur when Streptococcus pneumoniae spreads to the central nervous system. This can happen as a result of bacteremia or when trauma produces a connection between the nasopharynx and subarachnoid space.

• Vaccine:
  Given the severity of Streptococcus pneumoniae infections, the CDC recommends a pneumococcal vaccine for all children younger than 2 years old, all adults 65 years or older, and for anyone at increased risk of infection.