1. Pathogenic Gram-Negative Bacteria Baldwin Toye M.D., FRCPC
Division of Microbiology
Division of Infectious Diseases
Children’s Hospital of Eastern Ontario
The Ottawa Hospital
University of Ottawa

2. 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).

6. Nature 511, 108–111 (03 July 2014) doi:10.1038/nature13484
O antigens
3-deoxy-D-mannooctulosonic Acid
(KDO, keto-deoxyoctulosonate)
Nature 511, 108–111 (03 July 2014) doi: /nature13484

7. Clinically Important Gram Negative Bacteria
Gram-Negative Bacilli
Gram-Negative Cocci
Enterobacteriaceae
(glucose fermenters)
Non-glucose
Fermenters
Neisseria
Haemophilus
N. meningitidis
N. gonorrhoeae
H. influenzae
Pseudomonas
Acinetobacter
etc.

8. Enterobacteriaceae on MacConkey Agar
Proteus
Morganella
Salmonella
Shigella
Escherichia coli
Enterobacter
Serratia
Citrobacter
Klebsiella

9. Natural Habitat - Enterobacteriaceae
GI tract (“coliforms”)
Normal human microbiota
e.g., E. coli, Klebsiella, Citrobacter, etc.
Hospital environment
Water, sewage, soil, plants, animals
E coli comprises 1% of the total microbial biomass, ~ 10^7 CFU/g feces

10. Case Dx of urinary tract infection
24 yr old, otherwise healthy, woman 1-2 day history of urinary frequency, urgency, dysuria No fever Urine culture grew E. coli
Dx of urinary tract infection

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

12. Virulence Factors Adherence factors (e.g., fimbriae)
Capsule (antiphagocytic)
e.g., Klebsiella, Enterobacter
LPS (endotoxin)
Exotoxins
A pilus (Latin for 'hair'; plural : pili) is a hairlike appendage found on the surface of many bacteria.[1] The terms pilus and fimbria (Latin for 'fringe'; plural: fimbriae) can be used interchangeably, although some researchers reserve the term pilus for the appendage required for bacterial conjugation.
An exotoxin is a toxin secreted by bacteria.[1] An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host. Exotoxins may be secreted, or, similar to endotoxins, may be released during lysis of the cell.

13. 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

14. Case
35 yr old male vacationing in Mexico 5 days into vacation, developed watery non-bloody diarrhea (5-6 x/day) Associated with abdominal cramps & nausea but no fever What is the diagnosis?
EAEC or EPEC or DAEC may also be a cause esp in Latin America
Traveler’s diarrhea likely due to enterotoxigenic E. coli

15. Case
11 yr old girl, 2 day history of watery diarrhea that became increasingly bloody Associated with fever, headache, lower abdominal pain No history of travel Had eaten a hamburger at a picnic 3 days prior 1 week after onset of diarrhea, developed reduced urine output and had elevated serum creatinine What is the diagnosis?

16. 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)
Enteroinvasive (EIEC)
none
invasiveness
Enteroaggregative (EAEC)
AAF (aggregative adherence fimbriae)
EAST (enteroaggregative ST)
Diffusely adherent E. coli (DAEC)
Afa/Dr adhesins
(afimbrial & fimbrial)

17. 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)
Enteroinvasive (EIEC)
none
invasiveness
Enteroaggregative (EAEC)
AAF (aggregative adherence fimbriae)
EAST (enteroaggregative ST)
Diffusely adherent E. coli (DAEC)
Afa/Dr adhesins
(afimbrial & fimbrial)

18. 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
Nature Reviews Microbiology 2010; 8:26-38

19. 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®)?

20. 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

21. Attaching & Effacing
E coli use a type 3 secretion system to inject a receptor, Translocation intimin receptor (Tir), into the host cell to which intimin binds. This leads to formation of a pedestal on the surface of the epithelial cell on which the intimin-containing bacterium sits.

22. 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)
GB3 a glycosphingolipid receptor
Cleaves adenine from 28S rRNA of the 60S ribosomal subunit → inhibits protein synthesis, cell death
Stx binds to GB3 and damages endothelial cells in the glomeruli and epithelial cells in the renal tubules. There is desposition of fibrin in the renal microvasculature, leading to hemolysis and renal failure.

23. Virulence Factors Adherence factors (e.g., fimbriae)
Capsule (antiphagocytic)
LPS (endotoxin)
Exotoxins

24. 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

25. Lancet Infect Dis 2003;3:565. Lancet 2007;2196-2210
Petechial purpuric rash with meningococcal septic shock
Lancet Infect Dis 2003;3:565. Lancet 2007;

27. 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

30. Effect of Endotoxin on Macrophage Release of Inflammatory Mediators

31. Inflammatory Responses to Sepsis
NEJM 2006; 355:16

32. Bacterial Pathogenesis 3rd Ed 2011

33. 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)

35. Pseudomonas aeruginosa

36. 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 pathogen
e.g., UTI, pneumonia, bacteremia, etc.

37. 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!!!

38. 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

40. Alginate Biofilm Production
Sherris Medical Microbiology 6th Edition 2014

41. P. aeruginosa – Antimicrobial Resistance
Intrinsically resistant to many antibiotics
Many mechanisms of resistance
Often multi-drug resistant
Challenging to treat

42. Bacterial Pathogenesis
1. Adherence
2. Entry into body
3. Spread
4. Cell or tissue damage
direct
toxins and other products
indirect (inflammation or immune response)
5. Strategies to counter host defense