Fundamentals Block 5: Gram Negative Bacteria 1 and 2

go back over the tests and add those to the other lecture, go over ETEC

What is a Capsule? (4)

A shared structure found in both gram negative and gram positive bacteria. An important structure for survival
→ poorly antigenic
→ antiphagocytic
→ a major virulence factor acting as a barrier to detergents and promoting binding to other bacteria
→ produce biofilm

What is the Gram Negative Wall?

The Gram Negative Cell Wall is more complex than gram positive, it comes in two layers:
1) Outer membrane: unique to GN, bilayered permeability barrier and maintains protection from the outside
2) in between is the periplasmic space containing transport systems for macromolecules and enzymes
3) Thin peptidoglycan layer
4) Cytoplasmic Membrane

What are Lipopolysaccharides and Lipooligosaccharides?

Endotoxins that are only found in gram-negative bacteria’s outer membrane
1) Lipopolysaccharides are made of three things
→ Lipid A, Core Polysaccharide, and O-side chain (antigenic)

2) Lipooligosaccharides are made of the same things but lack the O-side chain

How do you select for gram negative bacteria on media? how does it work?

You would initially streak all unknown bacteria onto Sheep Blood Agar before growing it on MacConkey agar which grows and selects for gram negative bacteria
→ bile salts will prevent the growth of gram positive bacteria

What are the two aerobic gram negative bacteria?

Neisseria. gonorrhoeae and Neisseria. meningitidis

What are Neisseria?

A family of bacteria that are gram negative diplococci
→ there are two relevant ones: N. gonorrhoeae and N. meningitidis which are differentiated based on their growth on blood agar and nutrient agar (chocolate agar)
→ N/ meningitidis grows on blood while N. gonorrhoeae only grows on chocolate
→ all are oxidase positive with most being catalase positive

What is N. gonorrhoeae structure?

A gram negative diplococci
→ pili on the bacteria allow for attachment to the host epithelial cells
→ use Lipooligosaccharide and IgA protease

What is N. Meningitidis structure

Gram negative diplococci
→ polysaccharide capsule is the main virulence factor
→ Lipooligosaccharide and IgA Protease

What does N. gonorrhoeae cause in men and women

The bacteria is spread via sexual contacts, and is expressed differently in men and women. The disease shows up more in men

Men
→ Urethritis - results in urethral discharge and painful urination

Woman
→ Cervicitis - cervical discharge and bleeding

In both men and women
→ Pharyngitis
→ Rectal Infection
→ Conjunctivitis

How is N. gonorrhoeae diagnosed?

Urethral gram stain in men observing for Gram-negative diplococci
→ diagnosis of choice clinically is Nucleic Acid Amplification PCR
→ Culture with specialized media - chocolate-agar (Thayer-Martin agar)

How is N. meningitidis spread?

spread via respiratory droplets or oral secretions
→ spread most easily via household contact or crowding in small spaces
→ you have a much greater risk for meningitidis infection if you have a deficiency in proteins responsible for MAC formation

How does N. meningitidis present? (3)

Diverse spectrum of disease
→ respiratory tract infection
→ bacteremia
→ Meningococcemia (bacteremia with septic complications)
→ Meningitis-classic triad of fever, neck stiffness, and photophobia

How is N. meningitidis diagnosed?

1) Gram stain of gram negative diplococci in the CSF
2) Growth on chocolate and sheep blood agar and chocolate agar

How would you differentiate between N. gonorrhoeae and N. meningitidis in culture?

N. gonorrhoeae
→ needs chocolate agar in order to grow
→ oxidase and catalase positive
→ needs glucose

N. meningitidis
→ can grow on both blood and chocolate agar
→ oxidase and catalase positive
→ needs both glucose and maltose

What is Enterobacterales

Large order of bacteria with some shared key features
→ Non-spore forming gram negative rods
→ share a common antigen
→ facultative anaerobes

extremely simple growth requirements
→ reduce nitrate
→ oxidase negative
→ ferment glucose
→ catalase positive

What are the three key antigenic structures of enterobacterales

There are three different antigens found on enterobacterales that allows for us to classify them

O antigens
→ Lipopolysaccharide

K antigens
→ found in the capsule

H antigens
→ found on the flagella

What are Escherichia?

The most common Enterobacterales in the gut
→ E. coli is the most relevant one, and can cause GI and extra-intestinal disease if they have acquired virulence factors from plasmids/bacteriophages
→ a common index of fecal pollution in food and water
→ Lactose positive, Indole positive

What are the clinical syndromes of Escherichia?

E. coli patients will present with:
→ urinary tract infections
→ common cause of gastroenteritis
→ neonatal meningitis

What are the three potential pathogenic mechanisms of infectious diarrhea

1) Organisms that make toxins before a food is consumed
→ rapid onset of symptoms, causing diarrhea

2) Organisms that make toxins once they have been ingested
→ symptoms take longer to develop and can cause watery or bloody diarrhea

3) Microbes that invade by damaging epithelial cell surface or invading across the intestinal barrier
→ cause wide range of symptoms and different kinds of diarrhea

What is Enterotoxigenic E. coli?

Enterotoxigenic E.coli also known as ETEC is the most common cause of bacterial diarrheal disease in developing countries
→ leading cause of Traveler’s Diarrhea (30%) and is acquired from consumption of fecally contaminated food and water
→ secretory diarrhea developing after 1-2 days

What is the Pathogenesis of Enterotoxigenic E. coli?

Requires bacterial attachment to the small bowel epithelium by bacterial surface protein where it then produces two kinds of enterotoxins
1) Heat stable toxins (STa and STb)
→ binds to transmembrane guanylate cyclase C receptor increasing cGMP leading to a hypersecretion of fluids resulting in a loss of electrolytes and water

2) Heat labile toxins
→ increase cAMP levels leading to secretion of chloride and sodium

What is the structure of bacterial toxins?

Contain a A-B structure
→ A domain is effector function - protein-protein interactions within cell
→ B (binding) domain is receptor binding function, allowing for A-domain to get through lipid bilayer

What is Enterohemorrhagic E. coli or Shiga toxin producing E. coli

EHEC/STEC
→ caused by undercooked contaminated food or contaminated water
→ person to person spread can occur and can cause symptoms like mild diarrhea to severe abdominal pain and bloody diarrhea
→ can cause hemorrhagic colitis and in young children can cause hemolytic uremic syndrome

What is the toxin produced by Enterohemorrhagic E. coli or Shiga toxin producing E. coli

EHEC and STEC produce Shiga toxin 1 or 2
→ A-B subunit, binds to glycolipids on the host cells such as intestinal villi and renal endothelial cells
→ after the A subunit is internalized it is cleaved into A1 and A2, with A1 binding and causing cessation of protein synthesis

How can Shiga toxin producing E. coli be diagnosed?

Can be diagnosed by shiga toxin-based assays trying to detect E.coli 0157:H7 which is classically associated with shiga-toxin
→ most labs will test for 0157:H7 using special sorbitol MacConkey (SMAC) plates
→ 0157:H7 will ferment sorbitol slower compared to other E. coli and produce clear colonies on sorbitol MacConkey plates

What is Salmonella (disease, spread, secretion)

Enterobacteriaceae that can colonize all animals and infects us from ingestion of contaminated food products or direct fecal-oral spread
→ the bacteria will bind to the small intestine and invade into the microfold cells in the Peyer patches of the small intestines
→ utilizes the type III secretion system that injects virulence factors into host cells
→ Salmonella is H2S positive

What are the two major clinical syndromes that Salmonella can cause?

1) Enteric Fever: illness with little to no diarrhea
→ two kinds: S. typhi (typhoid fever) or S. paratyphi (paratyphoid fever)
→ bacteria are engulfed by macrophages where they can replicate in the liver, spleen and bone marrow

2) Gastroenteritis: ingestion of contaminated food products - also known as food poisoning
→ non bloody diarrhea, vomiting, and nausea

What is Nontyphoidal Salmonella?

S. enterica
→ spread via contaminated food and water and has a wide variety of reservoirs
→ Poultry and eggs and some exotic pets such as reptiles

What is the Xylose Lysine Decarboxylase test used for?

The Xylose Lysine Decarboxylase Test is used to test between Salmonella and Shigella based on the presence of H2S
→ Salmonella will ferment sugar and lower the pH by metabolizing thiosulfate to hydrogen sulfide producing a black medium which is positive result
→ Shigella will not do this

What can Shigella cause clinically?

Can cause two different clinical diseases
→ Shigellosis: Abdominal cramps, diarrhea, fever, and bloody stool 1-3 days after ingesting
→ Bacteremia

What is the pathogenesis of Shigella?

Shigella invades the colon and replicates there
→ utilizes type III secretion system secreting proteins into epithelial cells and macrophages, leading to their engulfment into macrophages where they can replicate
→ produce exotoxin Shiga toxin similar to STEC, which disrupts protein synthesis leading to intestinal epithelial damage

How would you lab diagnose Shigella?

Shigella will be tested for on an XLD plate
→ while salmonella would turn black on an XLD plate, Shigella will create red colonies on the surface of the XLD plate
→ H2S negative

How is Yersinia spread and what is its pathogensis

Yersinia are zoonotic bacteria
→ spread to humans via an animal host, and can kill cells via their ability to resist phagocytic killing and their type III secretion system

What is Yersinia Pestis

Yersinia Pestis is spread via urban plague rates and is transmitted via flea bites. Associated with the plague
There are two clinical manifestations
→ Bubonic plague - patient develops acute febrile lymphadenitis and the bubo (swollen lymph node) is tender
→ Pneumonic plague - inhalation of respiratory secretions and is highly infectious

What is Klebsiella?

Bacteria that has a large capsule responsible for the mucoid appearance of isolated colonies and enhanced virulence
→ Lactose positive, Urea positive
→ Nonmotile
→ B-lactamase production leads to resistant infections

What is Klebsiella disease

Klebsiella can cause several clinical manifestations
1) Pneumonia that results in thick non-purulent bloody sputum and necrotic destruction of alveolar space and abscess formation
2) UTI and meningitis as well

How would you test for Klebsiella

In order to test for Klebsiella we would do the String Test
→ mucous produced by Klebsiella is stretched, and if it reaches a certain length it will be considered a positive string test and is Klebsiella

What is Proteus Species? What is its growth pattern?

Proteus produces infections of the urinary tract
→ the most common member is P. mirabilis which produces large quantities of urease which is a enzyme that splits urea into carbon dioxide and ammonia
→ the presence of urease will raise urinary pH which can cause kidney stones

Proteus species are highly motile causing colony growth that looks like dropped a stone int he water (swarm on plate)

What are the shared features of Pseudomonas?

Small gram negative rods that are non-fermentative (OF test)
→ Obligate aerobes
→ lactose negative on MacConkey agar

What is Pseudomonas? What do they cause

An Aerobic Oxidase Positive bacteria that is commonly found in the soil, organic matter, vegetation, and water
→ P. aeruginosa is the most common and produces a grape-like odor and a blue-green pus

What are the Virulence factors of Pseudomonas? (4)

Pseudomonas use four different virulence factors:
1) Adhesins
2) Toxins: particularly Exotoxin A which disrupts protein synthesis by blocking peptide chain elongation in eukaryotic cells
3) Enzymes
4) Antibiotic Resistance

Which four patients are most susceptible to Pseudomonas infection

An opportunistic pathogen that can cause infections in patients that are considered high risk
1) Cystic Fibrosis
2) Immunocompromised patients
3) Burn patients
4) Patients on broad spectrum antibiotics

What are the five clinical diseases associated with Pseudomonas

Pseudomonas can cause several clinical diseases
1) Pulmonary infections (tracheobronchitis to pneumonia)
2) Primary Skin Infections - folliculitis and ecthyma gangrenosum (hot tub)
3) UTI
4) Ear Infections
5) Eye Infections

How would you lab diagnose Pseudomonas?

MacConkey
Glucose Oxidizer Test
Oxidase Positive

What are Vibrio?

2nd major group of gram-negative, facultatively anaerobic fermentative curved rods
→ oxidase positive
→ have a polar flagella
→ found in estuarine and marine environments because they need salt in order to grow

The most important one
→ Vibrio Cholerae

What is Vibrio Cholerae? Where does it get its toxin from?

Vibrio Cholerae causes rapidly dehydrating diarrheal disease
→ it is a highly motile and salt tolerant bacteria
→ gained genes from Vibrio Pathogenicity Island, following infection from a bacteriophage that encodes for the Cholera toxin

How does Vibrio Cholerae spread and what is a important marker of infection?

Vibrio Cholerae is spread via contaminated water and flourishes in water with chitinous shellfish
→ causes watery diarrhea and vomiting where feces will eventually become colorless and odorless known as rice-water stools

What is Campylobacter?

Small, motile, comma-shaped gram negative rods
→ grow in atmospheres of reduced oxygen and increased carbon dioxide
→ grown on Campy Agar

What disease does Campylobacter cause?

causes gastroenteritis and bacteremia
→ zoonotic, animals serve as reservoirs and is spread through humans via contaminated food and milk
→ acute enteritis: diarrhea, fever, abdominal pain as well as extra-intestinal symptoms like reactive arthritis

What is chronic colonization by Salmonella?

For more than 1 year after symptomatic disease by salmonella, patients can develop chronic colonization with their gall bladder being a reservoir
→ they can spread the bacteria through fecal-oral spread

What is Cholera Toxin

Cholera toxin produced by vibrio cholerae works at the CFTR
→ made of A-B subunits
→ B binds to intestinal cells while the A subunit irreversibly activates adenylate cyclase increasing water and electrolyte secretion