PathogenicGram-Negative

Bacteria

PathogenicGram-Negative

Bacteria

Baldwin Toye M.D., FRCPCDivision of Microbiology

Division of Infectious Diseases Children’s Hospital of Eastern Ontario

The Ottawa HospitalUniversity of Ottawa

Baldwin Toye M.D., FRCPCDivision of Microbiology

Division of Infectious Diseases Children’s Hospital of Eastern Ontario

The Ottawa HospitalUniversity of Ottawa

Objectives• Give a general classification for medically important

Gram negative bacteria. • List which gram negative organisms are part of the

normal human microbiome (colonization, carrier, etc.).

• Recognize medically important Gram negative pathogens in common infectious diseases.

• Identify important virulence factors among Gram negative bacteria (using Escherichia coli and Pseudomonas aeruginosa as examples).

Nature 511, 108–111 (03 July 2014) doi:10.1038/nature13484

O antigens

3-deoxy-D-mannooctulosonic Acid (KDO, keto-deoxyoctulosonate)

Clinically Important Gram Negative Bacteria

Gram-Negative Bacilli Gram-Negative Cocci

H. influenzae

Enterobacteriaceae(glucose fermenters)

Non-glucose Fermenters

Haemophilus

• Pseudomonas• Acinetobacter etc.

Neisseria

N. meningitidisN. gonorrhoeae

Enterobacteriaceae on MacConkey Agar

ProteusMorganellaSalmonellaShigella

Escherichia coli EnterobacterSerratiaCitrobacter

Klebsiella

Natural Habitat - Enterobacteriaceae• GI tract (“coliforms”)

– Normal human microbiota– e.g., E. coli, Klebsiella, Citrobacter, etc.

• Hospital environment• Water, sewage, soil, plants, animals

Case

24 yr old, otherwise healthy, woman1-2 day history of urinary frequency, urgency, dysuriaNo feverUrine culture grew E. coli

Dx of urinary tract infection

What can Enterobacteriaceae cause?

• Urinary tract infection• Bacteremia (bloodstream infection)• Pneumonia (usually hospital-acquired)• Skin & soft tissue infection

Virulence Factors

• Adherence factors (e.g., fimbriae)• Capsule (antiphagocytic)

e.g., Klebsiella, Enterobacter• LPS (endotoxin)• Exotoxins

What can Enterobacteriaceae cause?• Urinary tract infection• Bacteremia (bloodstream infection)• Pneumonia (usually hospital-acquired)• Skin & soft tissue infection• Gastroenteritis

E. coli Salmonella Shigella Yersinia

ingestion of contaminated food or water

Case35 yr old male vacationing in Mexico5 days into vacation, developed watery non-bloody diarrhea (5-6 x/day)Associated with abdominal cramps & nausea but no fever

What is the diagnosis?

Traveler’s diarrhea likely due to enterotoxigenic E. coli

Case11 yr old girl, 2 day history of watery diarrhea that became increasingly bloodyAssociated with fever, headache, lower abdominal painNo history of travelHad eaten a hamburger at a picnic 3 days prior1 week after onset of diarrhea, developed reduced urine output and had elevated serum creatinine

What is the diagnosis?

E. coli Diarrhea - Mechanisms

Pathotype Adhesin Toxin Other Virulence Factors

Enterotoxigenic (ETEC)

CFA (colonization factor antigens)

LT (heat labile enterotoxin)ST (heat stable enterotoxin)

Enteropathogenic (EPEC) Bfp (bundle forming pili)

Intimin

Shiga toxin producing E. coli (STEC) Enterohemorrhagic (EHEC)

Lpf(long polar fimbria)

STx (shiga toxin) Intimin

Enteroinvasive (EIEC) none invasiveness

Enteroaggregative (EAEC) AAF(aggregative adherence fimbriae)

EAST (enteroaggregative ST)

Diffusely adherent E. coli (DAEC)

Afa/Dr adhesins(afimbrial & fimbrial)

E. coli Diarrhea - Mechanisms

Pathotype Adhesin Toxin Other Virulence Factors

Enterotoxigenic (ETEC)

CFA (colonization factor antigens)

LT (heat labile enterotoxin)ST (heat stable enterotoxin)

Enteropathogenic (EPEC) Bfp (bundle forming pili)

Intimin

Shiga toxin producing E. coli (STEC) Enterohemorrhagic (EHEC)

Lpf(long polar fimbria)

STx (shiga toxin) Intimin

Enteroinvasive (EIEC) none invasiveness

Enteroaggregative (EAEC) AAF(aggregative adherence fimbriae)

EAST (enteroaggregative ST)

Diffusely adherent E. coli (DAEC)

Afa/Dr adhesins(afimbrial & fimbrial)

Nature Reviews Microbiology 2010; 8:26-38

Enterotoxigenic E. coli (ETEC)

• Most common cause of Travelers’ diarrhea

• Usually self-limited• CFA (fimbriae)• LT binds to GM1

– ADP ribosyl transferase, cleaves NAD

– Activates adenyl cyclase → ↑cAMP → phosphorylates CFTR → ↑ Cl- secretion

• ST binds to guanylin receptor → ↑cGMP → phosphorylates CFTR → ↑ Cl- secretion

Prevention of Travelers’ Diarrhea• Hand hygiene• Avoid undercooked or raw meats/seafood,

unpasteurized eggs/dairy products.• Avoid foods cooked earlier in day and not sufficiently

reheated• Avoid vegetables difficult to clean• Avoid fruits you have not peeled yourself• Drink bottled water with intact seals or boiled water• Antibiotics only in select patients• Vaccine (Dukoral®)?

Shiga Toxin Producing E. coli (STEC)• Reservoir is GI tract of cattle and other

animals• E. coli O157:H7 most common cause• Linked to consumption of undercooked

ground beef & other contaminated food• Ability to induce attaching and effacing effect

(similar to EPEC) via multiple genes including intimin

Attaching & Effacing

Shiga Toxin Producing E. coli (STEC)• Production of shiga toxins (Stx)

– Binds to GB3 (globotriaosylceramide) • Intestinal mucosa• Kidney epithelial cells• Endothelial cells

– Cleaves adenine from 28S rRNA → inhibits protein synthesis, cell death

• Complications include hemolytic uremic syndrome (HUS)

Virulence Factors

• Adherence factors (e.g., fimbriae)• Capsule (antiphagocytic)• LPS (endotoxin) • Exotoxins

Case• 16 yr old male, previously healthy• Felt unwell & lethargic yesterday, didn’t go to

school• Over next few hours, fever, headache, nausea• Unresponsive, brought to ED• On exam: T-39.5oC, BP 80/50, P 125/min not rousable skin rash

Lancet Infect Dis 2003;3:565. Lancet 2007;2196-2210

Neisseria meningitidis• Gram-negative diplococci• Natural habitat: nasopharynx (3-25%)• Transmission via droplets, requires close contact• Virulence factors

– Pili (adherence)– Capsule (13 serogroups)– LOS (lipooligosaccaride)

• Invasive Disease– Bacteremia (meningococcemia)– Meningitis

Effect of Endotoxin on Macrophage Release of Inflammatory Mediators

Inflammatory Responses to Sepsis

NEJM 2006; 355:16

Bacterial Pathogenesis 3rd Ed 2011

Pseudomonas aeruginosa• Strict obligate aerobic Gram-negative bacillus• Ubiquitous – soil, water, plants, etc.• Colonies have grape-like odor• Green blue color

– pyoverdin (green)– Pyocyanin (blue)

Pseudomonas aeruginosa

Natural Habitat• Environmental (high moisture, high humidity)

– Hospital (sinks, taps, shower heads, mops, flower vases, etc.)

– Community (whirlpools, hot tubs, spas, swimming pools, humidifiers, etc.)

• Colonizes humans– Hospital acquired– Selected with broad spectrum antibiotic usage

Opportunistic hospital-associated pathogene.g., UTI, pneumonia, bacteremia, etc.

Virulence Factors – P. aeruginosa• Alginate

– Copolymer of mannuronic and gluronic acids– Adherence, antiphagocytic, protection of biofilm from

host immune system and antibiotics, resists opsonic killing– High level production (mutations in regulatory genes)

results in mucoid colonies, seen in CF• Exotoxin A (ExoA)

– ADP-ribosylation of EF-2– Inhibits host cell protein synthesis, results in cell death

• Elastase – elastin, human Ig, complement, some collagens• Quorum-sensing – gene regulating system• Many many others!!!

Cystic Fibrosis

• Genetic disease affecting chloride channels• Thicker mucus in the lungs and airways• Obstruction of airways, interfere with ciliated

cell host-defense• At risk for chronic and recurrent pulmonary

infections

Alginate Biofilm Production

Sherris Medical Microbiology 6th Edition 2014

P. aeruginosa – Antimicrobial Resistance

• Intrinsically resistant to many antibiotics• Many mechanisms of resistance• Often multi-drug resistant• Challenging to treat

Bacterial Pathogenesis1. Adherence2. Entry into body3. Spread4. Cell or tissue damage

direct toxins and other products indirect (inflammation or immune

response) 5. Strategies to counter host defense