Novel antibiotic resistance proteins in Vibrio parahaemolyticus

Frank Higgins

Supervisor: Dr. Aoife Boyd

Introduction• Vibrio parahaemolyticus

• Gram –ve, bacillus, facultative anaerobe

• Found mainly in coastal waters, oceans, estuaries

Fig. 1 & 2 SEM image and gram stain of Vibrio parahaemolyticus

V. parahaemolyticus

Pathogenicity• V. parahaemolyticus pathogenicity linked to production of TDH

(thermostable direct haemolysin)

• Exotoxin that causes lysis of red blood cells

• Induces beta – haemolysis when plated on Wagatsuma blood agar. Known as the Kanagawa phenomenon

• Encoded by the tdh gene, used to identify pathogenic strains of the organism.

Pathogenicity

• Causes gastroenteritis following consumption of contaminated shellfish

• Two distinct sets of Type III Secretion systems in V. parahaemolyticus

• Inject effector proteins into target cells

• Interfere with cell signalling proteins

Fig. 3 Illustration of type III secretion system

Antibiotic resistance:Efflux pumps

• Mechanism of antibiotic resistance in bacteria

• Removes antibiotics from bacteria before they can inhibit or kill the organism.

• Composed of three proteins; An inner membrane transporter protein, an outer membrane protein, and a membrane fusion protein.

Antibiotic resistance: Efflux pumps

• Two genes have been identified within V. parahaemolyticus, emrA & emrB

• Believed to encode for efflux pump

• Direct path from cytoplasm to extracellular space

Fig. 4 emr efflux pump in cell membrane

Goals of project

1. To examine whether the proteins produced by these genes confer resistance to antibiotics via an efflux pump

2. To show which of our antibiotics are removed by the efflux pump

pET101 plasmid• In order to achieve these goals, emrA + emrB transformed into

E.coli [ BL21( DE3) ] strain using pET101 expression vector• 2 different plasmids : 1. pET101 ( empty vector )

2. pET101 + emr A+B

Fig. 5 pET101 expression vector including our genes of interest, the T7 promoter and a gene encoding for ampicillin resistance

IPTG• Expression of our genes cannot take place, while the Lac

repressor (LacI) binds to the Lac operon.

• If lactose is present, LacI repressor senses and binds to it instead.

• IPTG is introduced, as it mimics lactose structurally, so LacI binds to it and our genes can be freely expressed.

Materials and methods

Hypotheses: emrA and emrB form an efflux pump in

V.parahaemolyticus which allows for the expulsion of some antibiotics before they can have an effect.

MIC Assay

• MIC of bacteria with empty vector (E) and bacteria with vector containing emrA + emrB (AB)

• Each bacteria also grown with IPTG and without

• Serial (two fold) dilutions of antibiotics in growth medium

Fig.6 96-well plate used for MIC assays

Results• Results indicate that there is no increased resistance to

Ciprofloxacin or Oxolinic acid

• However, increased resistance is seen to Nalidixic acid and Norfloxacin at 37°C

• Suggests there is potential that our genes are encoding for an efflux pump

Results

MIC values:• E - 9.77 ng ml -1 • E + 9.77 ng ml -1 • AB- 9.77 ng ml -1 • AB+ 9.77 ng ml -1.

Ciprofloxacin

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Fig. 7: Optical density of our bacterial cells in Ciprofloxacin at 30°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

Results

MIC values:• E - 9.77 ng ml -1 • E + 19.53 ng ml -1• AB- 19.53 ng ml -1• AB+ 19.53 ng ml -1

Ciprofloxacin

Fig. 8: Optical density of our bacterial cells in Ciprofloxacin at 37°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

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Results

MIC values:• E - 3.13 µg ml -1• E + 3.13 µg ml -1• AB- 3.13 µg ml -1• AB+ 3.13 µg ml -1.

Nalidixic acid

Fig. 9: Optical density of our bacterial cells in Nalidixic acid at 30°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

50 25 12.5 6.25 3.13 1.56 0.78 0.39 0.20 0.10 Pos Cont.

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Results

MIC values:• E - 6.25 µg ml -1• E + 3.13 µg ml -1• AB- 6.25 µg ml -1• AB+ 6.25 µg ml -1

Nalidixic acid

Fig. 10: Optical density of our bacterial cells in Nalidixic acid at 37°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

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1.56 0.78 0.39 0.20 0.10 Pos cont.

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Results

MIC values:• E - 15.63 ng ml -1• E + 15.63 ng ml -1• AB- 15.63 ng ml -1• AB+ 15.63 ng ml -1

Norfloxacin

Fig. 11: Optical density of our bacterial cells in Norfloxacin at 30°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

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Norfloxacin (ng ml -1)

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Results

MIC values:• E - 31.25 ng ml -1 • E + 9.77 ng ml -1 • AB- 62.5 ng ml -1 • AB+ 31.25 ng ml -1

Norfloxacin

Fig. 12: Optical density of our bacterial cells in Norfloxacin at 37°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

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Results

MIC values:• E - 125 ng ml -1• E + 62.5 ng ml -1• AB- 125 ng ml -1• AB+ 62.5 ng ml -1

Oxolinic acid

Fig. 13: Optical density of our bacterial cells in Oxolinic acid at 30°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

1000 500 250 125 62.5 31.25 15.63 7.81 3.91 1.95 Pos cont.

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Results

MIC values:• E - 250 ng ml -1• E + 125 ng ml -1• AB- 125 ng ml -1• AB+ 125 ng ml -1

Oxolinic acid

Fig. 14: Optical density of our bacterial cells in Oxolinic acid at 37°CGrowth of E.coli BL21(DE3)containing pET101 vectors:Black bars – Empty vector without IPTG (E-)White bars – Empty vector with IPTG (E+)Grey bars – emrA+B vector without IPTG (AB-)Blue bars – emrA+B vector with IPTG (AB+)Positive controls contain our bacterial sample and growth medium only (no antibiotics)

1000 500 250 125 62.5 31.25 15.63 7.81 3.91 1.95 Pos cont.

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Conclusions• No increased resistance to Ciprofloxacin or Oxolinic acid from

our genes.

• Indication that genes conferring resistance to Nalidixic acid and Norfloxacin at 37°C

• Suggests that emrA+B do form an efflux pump, of which the latter two antibiotics are a substrate

• More testing would be required for a conclusive result

Future directions• Testing a wider range of antibiotics to determine whether they

are substrates for our efflux pump

• Mapping the molecular and genetic makeup of the efflux pump

• Developing efflux pump inhibitors specific to our efflux pump to cease its functioning

AcknowledgementsI would like to thank my supervisor Dr. Aoife Boyd, my lab partner Katie Donohue, the Microbiology technical staff, and the 4th year class.

References• Figure 1: http://en.wikipedia.org/wiki/Vibrio_parahaemolyticus • Figure 2: http://pathmicro.med.sc.edu/fox/enterobact.htm• Figure 3: http://

www.rsc.org/Publishing/Journals/cb/Volume/2008/11/bacterial_nanoinjectors.asp

• Figure 4: Piddock, L. (2006). Multidrug-resistance efflux pumps – not just for resistance. Nat Rev Microbiol 4, 629–636.

• Figure 5: http://tools.invitrogen.com/content/sfs/manuals/pettopo_man.pdf (pg. 4)

• Figure 6: Taken from Paul McCay lecture notes