crwad petruzzi presentation
TRANSCRIPT
Characterization of Biofilm Formation
in Pasteurella multocida
Briana L. PetruzziInzana Lab
Virginia Maryland College of Veterinary Medicine
Respiratory Diseases due to Pasteurella multocida
http://npaper-wehaa.com/pubfiles/13287235204f32b64017593/pub/Hartford-Magazine-November2012/lib/1350059946507847aa59b16.jpg
• Bovine Respiratory Disease (BRD)
• Avian Cholera
• Atrophic Rhinitis
• Porcine Respiratory Disease Complex
(PRDC)
Outbreak at the Great Salt Lake: Loss of 15,000 Eared GrebesUtah Division of Wildlife 2007
http://thecelestialconvergence.blogspot.com/2013/04/mass-bird-die-off-over-12000-birds-drop.html
Focus on BRD and Avian Cholera
Primary cause of Morbidity and Mortality in the US Beef Industry
Zoetis
http://dap-steenbergen.nl/studiostone01/wpcontent/uploads/2012/11/pinkengriep1-300x252.jpg
Avian Cholera
• Mucous discharge from the mouth• Nasal Discharge• Soiling or matting of the feathers around the vent, eyes, and bill• Pasty, yellow or blood-stained droppings• Bloody lesions around heart, lungs, intestines• Highly contagious, spread rapidly• Can be acute or chronic
Clinical Signs Include:
Bovine Respiratory Disease Complex
• Polymicrobial: bacterial, viral, parasitic • Onset occurs after stress:
• Transportation, viral infection, poor ventilation, handling, weaning• Lack of appetite• Fever, one of the earliest signs of disease• Nasal discharge and salivation• Coughing
Rockefellar Institute, Circa 1930• Irridescent Strains
• Highly Virulent (50% mortality)
• Blue Strains• Avirulent (0% mortality)• All infected birds became carriers• Survived in the nasal clefts of birds for up to 65 days
• Intermediate Strains• Virulence ranging from 0 – 60% mortality
(Hughes et al., 1930 – 1931)
Mucoid / Encapsulated Isolates
Non-Mucoid / Acapsular Isolates
Hypothesis1. Pasteurella multocida is capable of making a biofilm
2. Biofilm formation is essential for chronic avian cholera, and is Probably associated with the carrier state seen in birds
3. Biofilm formation by P. multocida contributes to polymicrobial biofilms in disease complexes such as BRD, PRDC
Biofilm Formation was not Uniform Among P. multocida Clinical Isolates &
StrainsA56
2C
ryst
al V
iole
t
Blue(Non-M
ucoid)
Intermed
iate
Irides
cent (M
ucoid)
0
1
2
3
4****
Isolate Capsule Designation
Mean Biofilm A562
Serotype Isolated From
9877 + Cap 0.316 A Bovine Lung
276 + Cap 0.212 A Bovine Lung
0202 - Cap 2.7195 A Bovine Lung
538 + Cap 0.297 A Bovine Nasal
232 + Cap 0.223 A Bovine Lung
1904 ++ Cap 0.29 A Bovine Lung
134 - Cap 2.5 A Bovine Lung
378 ++ Cap 0.243 A Bovine Nasal
One But Not Both: Capsule vs. Biofilm
Congo Red Uptake
P1059
WT
P1059
-8
P1059
ACAP
P1062
WT
P1062
ACAP
X73 W
T
X73 ACAP
0
1
2
3
4
A56
2 Cry
stal
Vio
let
*********
***
**** ****
A490 / P
elle
t Wei
ght i
n G
ram
s
P105
9 W
T
P105
9 A
CA
P
P105
9-8
P106
2 W
T
P106
2 A
CA
P
X73
WT
X73
AC
AP
0
5
10
15
20***
**** * ***
Biofilm Formation between Acapsular & Encapsulated Isolates
P1059 ACAP P1059 WT
Average Thickness: 38.57 µm3/µm2
Average Thickness: 52.06 µm3/µm2
Average Biomass: 30.35 µm3/µm2
Average Biomass: 37.27 µm3/µm2
Hyaluronidase Treatment Increases Biofilm Forming Potential in Encapsulated Isolates
010
030
0 010
030
0 010
030
0 010
030
0 010
030
0 010
030
0 010
030
0
0
1
2
3
4
5P1059 WTP1059 ACAPP1059-8P1062 WTP1062 ACAPX73 WTX73 ACAP
**
Units Hyaluronidase
A562 C
ryst
al V
iole
t
** **
010
030
0 010
030
0 010
030
0 010
030
0 010
030
0 010
030
0 010
030
0
0
1
2
3
4P1059 ACAPP1059-8P1062 WTP1062 ACAPX73 WT
Units Hyaluronidase
Mili
gram
s Pr
otei
n
P1059 WT
X73 ACAP* **
****
Quantitative Real Time-PCR to Identify Genes Significantly Upregulated During
Biofilm FormationGene Function
csrA Carbon Storage RegulatorpldB Lysophospholipase sgbU L-xylulose 5-phosphate 3-epimerasexylB carbohydrate kinase FGGY
Pasteurella multocida Biofilm Formation Correlates with Chronic Infection
P. multocida was isolated from all birds at euthanasia
- All 10 Birds Survived until Euthanasia- Displayed signs of Chronic Pneumonia
- 14/15 Birds Survived until Euthanasia- Displayed signs of Chronic Pneumonia
- 3/15 Birds Survived until Euthanasia- 12 Birds Succumbed to Acute
Infection- 3 Birds showed signs of Chronic
Pneumonia
Pasteurella multocida Biofilm Forming Potential Correlates with Virulence
Non-Infected Control 756 Proficient Biofilm FormerX73 Deficient Biofilm Former 775 Proficient Biofilm Former
Polymicrobial Relationships Between P. multocida and Histophilus somni
H. somni P. multocida H. somni + P. multocida
Polymicrobial Biofilm Observations• When H. somni is grown as a biofilm, very few cells are present, but a large amount of extracellular matrix material is made
• When P. multocida, including highly mucoid poor biofilm forming strains, are added to an H. somni biofilm, P. multocida becomes well established in the biofilm.
• However, when H. somni is added to a P. multocida biofilm, H. somni is unable to become established.
• We believe P. multocida takes advantage on the H. somni biofilm to infect and persist in the lower respiratory tract.
• Assays with differentially labelled nucleic acid probes (FISH) are being used to clarify the interactions between H. somni and P. multocida together in a biofilm.
Conclusion and Summary• Pasteurella multocida makes a single species and polymicrobial
biofilm
• Biofilm formation is inhibited by capsule production
• Proficient Biofilm Formers are less Virulent than Biofilm Deficient Isolates
• Proficient Biofilm Formation is Associated with Chronic Pneumonia, and responsible for the spread of disease
Acknowledgements & Questions
Funding Provided by:USDA-NIFA #2013-67015-21314VA-MD College of Veterinary MedicineVirginia Tech College of Agriculture and Life Sciences
Dr. Thomas Inzana & Lab:Dr. Abey BandaraAnna ChampionAnne C. M. HymanKelly FreudenbergerYu PanIndra Sandal
BMVS Department: Dr. Avery, Becky Jones
Dr. Bob Briggs; USDA National Animal Diseases CenterW. E. Swords, Wake Forest University