Listeria monocytogenes - jifsan [PDF]

Listeria m onocytogenes

a Model System for Studying Persister Cells

Stephen Knabel Professor of Microbiology Dept. of Food Science, UP

What are Persister Cells? 

Historical Definition 



A small subpopulation of bacteria that are tolerant to (not resistant to) antibiotics Formed by all free-living bacteria tested to date

Bigger, 1944

mRNAs lacking cut sites are not degraded

Survival & Death Proteins! Toxin: Sequence-specific Endoribonuclease

Growth Arrest and Persister Cell Formation

Toxin-Antitoxins Play a Key Role in Persister Cell Formation

Listeria monocytogenes

A Unique Model System for Understanding Long-term Survival  



Foodborne bacterial pathogen “Rod-shaped”

“Non-spore-forming” 



Forms persister cells 



Like most bacteria

Like all free-living bacteria

Possess a highly repeatable Life Cycle 

Including Death, Long-Term Survival (LTS) and Germination Phases

CFU/ml TSBYE

Log

Stationary

Early-death

Late-death

LTS

Long-Term-Survival (LTS) Phase

1.0E+10 1.0E+09 1.0E+08 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00

0

100

200

300

400

500

600

700

800

Growth time (h) at 35°C in TSBYE Listeria enters a coccoid long-term-survival phase upon extended incubation

Time Rods

0h

Cocci

Germination

1.5 h Outgrowth

2.5 h

3.5 h 5h Replication

Cocci

Wen et al., 2009

15 h

Rods

CFU/ml TSBYE

Log

Stationary

Early-death

Late-death

LTS

Long-Term-Survival (LTS) Phase

1.0E+10 1.0E+09 1.0E+08 1.0E+07 1.0E+06 1.0E+05 1.0E+04 1.0E+03 1.0E+02 1.0E+01 1.0E+00

Growth Curve Life Cycle! 0

100

200

300

400

Germination

500

600

700

800

Growth time (h) at 35°C in TSBYE

Proposed Model

Persister Cells are Common in Biofilms 



Persister cells form within biofilms due to a decrease in nutrients Allow survival and transmission of Lm to foods

Kim Lewis

Are LTS phase cells persister cells? If so, what causes their formation?

9

90

8

80

7

70

6

60

5

50

4

40

3

30

2

20

1

10

0

0

0

24

48 72 Incubation time (h) @ 35C

96

TSBYE

T = Tryptone S = Soytone YE = Yeast Extract Frequency Persister Cell (%)

100

Log CFU/mL

10

Persister frequency TSBYE

10

9

90

8

80

7

70

6

60

5

50

4

40

3

30

2

20

1

10

0

0

0

24

48 72 Incubation time (h) @ 35C

96

-S (TBYE) T (Tryptone) = Pancreatic Digest of Casein Frequency Persister cell (%)

100

Log CFU/mL

10

Persister frequency TBYE

11

Log CFU/mL

9

100 90

8

80

7

70

6

60

5

50

4

40

3

30

2

20

1

10

0

0

0

24

48 72 Incubation time (h) @ 35c

96

-T (SBYE) S (Soytone) = Papaic Digest of Soy Protein Frequency Persister Cell (%)

If use Pancreatic Digest of Soy (Peptone S) rather than Papaic Digest of Soy (Soytone) get same results as with TSBYE and TBYE

10

Persister frequency

SBYE

12

Acknowledgements 

PSU     



University of Turin  





IIT  



Dr. Sara Lomonaco Virginia Filipello

University of Milan 



Dr. Jia Wen Dr. R. Anantheswaran Matthew Doan Dr. Edward Dudley Dr. Tom Wood, Dept. CE & BMB

ISU  

Dr. Mirella Pontello Ettore Amato Dr. Wei Zhang Joelle Salazar



Materials Characterization Lab - PSU  



Flow Cytometry Lab - PSU 



Ruth Nissly

Undergraduates:    

Dr. Aubrey Mendonca Fei Wang

Tim Tighe Trevor Clark



Adrian Paskey Gregory Park Spencer Lovrinic Sin Yen Tan Azree Zaffran