Novel antibiotic resistance proteins in

Vibrio parahaemolyticus

Francis Higgins: 09542639

Submitted to the National University of Ireland, Galway in partial fulfilment of the requirements for the Bachelors of Science Honours

Degree

Supervisor: Doctor Aoife Boyd

Department of Microbiology

National University of Ireland, Galway

December 2012

Contents

1. Abstract..........................................................................................................................3

2. Introduction....................................................................................................................4

2.1 Vibrio parahaemolyticus.....................................................4

2.2 Efflux pumps…………………...........................................5

2.3 emrA & emrB......................................................................6

2.4 pET101 expression vector...................................................6

2.5 Aims of the project..............................................................7

2.6 Workflow followed throughout the project.........................8

3. Materials.......................................................................................................................9

3.1 Bacterial strains....................................................................9

3.2 Growth Media and antibiotics..............................................9

3.3 Expression plasmids.............................................................9

3.4 Equipment............................................................................9

4. Methods.......................................................................................................................10

4.1 Preparation of E.coli stock in glycerol.................................10

4.2 Preparation of competent cells.............................................10

4.3 Transformation of competent cells.......................................11

4.4 MIC Assay............................................................................11

i. Inoculating E.coli in LB broth.............................11

ii. Induce protein expression.....................................11

iii. Set up micro-titre plate.........................................12

iv. Inoculate micro-titre plate....................................12

v. Growth measurement...........................................12

5. Results.........................................................................................................................13

5.1 Transformation of E.coli strains............................................13

5.2 MIC Assay results..................................................................13

6. Discussion.....................................................................................................................22

6.1 Critical review of methods used............................................22

6.2 Analysis of data generated.....................................................23

6.3 Future Directions....................................................................25

6.4 Conclusion..............................................................................25

7. Bibliography................................................................................................................26

2

1. Abstract

Vibrio parahaemolyticus is a major cause of gastroenteritis in humans, commonly known as

food poisoning, following the consumption of raw, undercooked or contaminated seafood

(Shen et al., 2010). Gastroenteritis is characterised by inflammation of the stomach lining

and small intestine, leading to symptoms such as vomiting and diarrhoea (Singh, 2010).

A number of MIC assays were carried out using four different antibiotics to determine

whether a set of two genes identified in V. parahaemolyticus , called emrA and emrB, are

encoding for an efflux pump. This efflux pump would act as a direct path from the cytoplasm

of the organism to the extracellular space (Nikaido, 1996), allowing for the expulsion of

antibiotics before they can have an effect on the bacteria. The genes were cloned on an

expression vector and introduced into E. coli. The results of the assays were inconclusive but,

following further research and testing, it could be found that the genes do in fact encode for

an efflux pump in V.parahaemolyticus.

3

2. Introduction

2.1 Vibrio parahaemolyticus

Vibrio parahaemolyticus is a member of the genus Vibrio (Joseph et al, 1982). It is a gram-

negative, rod shaped bacterium found mainly in marine environments, and is isolated from a

number of seafood’s such as shrimp, crab, lobster and other shellfish (Su & Liu, 2007). The

organism was first discovered in the 1950’s following a severe outbreak of food poisoning in

Osaka, Japan (Honda et al, 2008). It is the causative agent of gastroenteritis in humans

following the consumption of contaminated, undercooked seafood, and is also known to

cause infections of wounds and septicaemia (Devi et al., 2009). As well as being detrimental

to our health, it also affects the economic state of the marine environment, so it is a

particularly important bacterium to study and understand (WHO, 2011).

The pathogenicity of V. parahaemolyticus has been found to be directly associated with the

production of a thermostable direct haemolysin (TDH) (Zhao et al., 2011). It is known to

induce beta-haemolysis when introduced to Wagatsuma blood agar, a reaction known as the

Kanagawa phenomenon. This response is seen in most clinical isolates of V.

parahaemolyticus (Caburlotto et al, 2008). As such, TDH is recognised as a major virulence

factor of V. parahaemolyticus. It is encoded by the tdh gene, the presence of which is often

used to identify pathogenic strains of V. parahaemolyticus (Martinez-Urtaza et al., 2004).

Similar to many other gram-negative bacteria, V. parahaemolyticus also possess genes

encoding for two distinct sets of type 3 secretion systems (Yeung & Boor, 2004) (Honda et

al, 2008). The type 3 secretion system acts as a virulence mechanism by sending or injecting

effector proteins from the bacteria directly into eukaryotic cells (Hueck, 1998). These

proteins alter cell functions by interfering with host cell signalling proteins and pathways.

Recently, two genes were discovered in strains of V. parahaemolyticus, called emrA and

emrB due to the fact they somewhat resemble genes of the same name found in other bacteria

such as Escherichia coli (Lomovskaya & Lewis, 1992). These genes are believed to form an

efflux pump which would remove antibiotics from the bacterium (Nikaido, 1996). A better

understanding of these genes could ameliorate the treatment of infections of V.

parahaemolyticus, in the hope that the genes could be targeted and supressed in order to stop

them from functioning.

4

2.2 Efflux pumps

Resistance to antibiotics is a threat that grows with each passing day, having been identified

as a serious public health concern by WHO (Noorlis et al., 2011). Resistant strains are

becoming more and more apparent due to the constant overuse of antibiotics. This includes

antibiotic treatment of bacterial infections and, in the case V. parahaemolyticus, seafood

farms and marine environments (de Melo et al., 2011)(Noorlis et al., 2011).

Multidrug-resistance efflux pumps are one mechanism of antibiotic resistance that many

gram-negative bacteria have developed. These structures actively recognise and expel a range

of antibiotics (Fernandez-Recio et al., 2004), and are composed of three proteins. An

example of an efflux pump found in E. coli consists of an inner membrane transporter protein

AcrB, the outer membrane protein, and a membrane fusion protein AcrA that is located in the

periplasm. (Drew et al., 2008). The pump utilises proton electrochemical force to displace

antibiotics from the organism. The third component of the AcrAB pump that is also present in

many other efflux pumps (including the one we are studying in this project) is the outer

membrane protein, TolC. TolC represents a large family of proteins found in a wide variety

of gram–negative bacteria (Sharff et al, 2001).

The study of efflux pumps is imperative in the battle against antibiotic resistant bacteria.

Through identification of the molecular and genetic bases that comprise multi-drug resistant

efflux pumps, steps can be taken to repress their functioning (Pagès & Amaral, 2009).

Specifically, a number of chemical compounds known as efflux pump inhibitors (EPI) have

been designed that restrict the activity of efflux pumps. They reduce the resistance of bacteria

to antibiotics that are normally expelled by their respective efflux pumps. For example, a

number of efflux pump inhibitor compounds have been developed to reduce the activity of

the AcrAB- TolC and MexAB- OprM efflux pumps, which prevents them from filtering out

antibiotic substances. It could thus be said that EPI compounds rejuvenate the antibiotics’

power.

The four antibiotics used in this project to test our efflux pump belong to the quinolone

family, two of which fall into the subset of fluoroquinolones. All quinolones possess a

carboxylic acid molecule present at C-3 (Heeb et al, 2011). Fluoroquinolones differ from

quinolones in that they possess a fluorine atom which is attached to their central ring.

Quinolones and fluoroquinolones inhibit the unwinding and synthesis of DNA by targeting

the enzymes DNA gyrase or topoisomerase IV (Hooper, 2001).

5

The antibiotics used were as follows:

Quinolones -

1. Nalidixic acid

2. Oxolinic acid

Fluoroquinolones –

1. Ciprofloxacin

2. Norfloxacin

2.3 emrA & emrB

The genes in question that have been identified in V. parahaemolyticus have been dubbed

emrA and emrB, owing to the fact that they are somewhat similar to the emr genes found in E.

coli (Lomovskaya & Lewis, 1992). We believe that these genes encode for novel proteins

which may play a part in antibiotic resistance in V. parahaemolyticus through the formation

of an efflux pump.

2.4 pET101 expression vector

In order to test whether our genes are conferring antibiotic resistance to the bacteria, they

have been cloned onto a commercial expression vector pET101 (see Fig.1) to be introduced

into an E. coli strain BL21(DE3) that is hypersensitive to antibiotics . The pET101 vectors

utilise a T7 promoter (taken from T7 bacteriophage) to control expression of our genes [1]. It

also possesses a gene coding for resistance to ampicillin (meaning our cells are grown in the

presence of ampicillin to ensure that no other cells grow). In order for expression to take

place T7 RNA polymerase must be used (supplied in the BL21(DE3) E. coli strain) [2].

Heterologous protein expression is the term used to describe the expression of proteins within

host cells that are not a natural part of the cell (Weng et al, 2006).

The RNA polymerase that is present in E. coli cannot recognise the T7 promoter used in front

of our genes on our expression plasmid. The lac repressor (LacI) binds the lac operon, which

blocks our T7 RNA polymerase from binding our promoter. This prevents transcription of

our genes of interest.

6

The lac repressor protein (LacI) will sense lactose if it is present and bind to it, leaving our

operator sequence to bind the T7 RNA polymerase, allowing for expression of our desired

proteins. Thus, IPTG is introduced to our transformed cells when they are being grown. IPTG

mimics lactose structurally, meaning the lac repressor will fall off the operator DNA

sequence in front of our gene and bind to it. This allows our T7 RNA polymerase to bind the

T7 promoter in front of our genes, which begins transcription of said genes. IPTG is a more

useful inducer than lactose, as it does not belong to any metabolic pathway. This means that

the concentration will stay the same, as it is not broken down or used by the cell [4].

2.5 Aims of the project

The aim of this project was to investigate the proteins produced by the genes emrA and emrB.

Specifically, we wanted to examine their role in the antibiotic resistance of Vibrio

parahaemolyticus, i.e. whether they form an efflux pump that would transport antibiotics

from the bacteria before they could have an effect.

In order to do this, the genes have been cloned on to an expression vector and introduced to a

strain of E. coli, BL21(DE3). The antibiotic resistance profile of the transformed E.coli was

determined by finding the Minimum Inhibitory Concentrations (MIC) for a number of

antibiotics, which were then compared to an E. coli strain containing an empty vector.

The personal aims of working on this project were to gain experience of work in a research

lab and working as part of a team to carry out the project in the most efficient and

comprehensive way. Undertaking this project also allowed me to take the initiative to carry

7

Fig.1: pET101 expression vector

including our genes of interest, the

T7 promoter and a gene encoding for

ampicillin resistance [3].

out work and research independently in order to gain a real understanding of the topic in

question.

2.6 Workflow followed throughout the project

8

3. Materials

1.1 Bacterial strains

Escherichia coli BL21(DE3) (Invitrogen)

E.coli BL21(DE3)ΔacrB (Boyd lab)

E.coli TOP10 (Invitrogen)

1.2 Growth media and antibiotics

LB Broth, Miller (Luria – Bertani broth) (Difco ™ ref no. 244620)

LB Agar, Miller (Luria – Bertani broth) (Difco ™ ref no. 244520)

SOC medium (Sigma- Aldrich) filter sterilized using 0.2 μm pore filter and syringe

Ampicillin sodium salts (Sigma) (stock concentration – 12,000 µg ml -1)

Ciprofloxacin (Fluka) (stock conc. – 5000 µg ml -1)

Nalidixic acid (Sigma) (stock conc. – 10,000 µg ml -1)

Oxolinic acid (Sigma) (stock conc. – 1,000 µg ml -1)

Norfloxacin (Fluka) (stock conc. – 10 µg ml -1)

1.3 Expression plasmids

Invitrogen Champion™ pET101 vector (cat no. K101-01)

Invitrogen Champion™ pET101emrA+B vector.

1.4 Equipment

Autoclave: HiClave HV-85L Autoclave

Centrifuge: eppendorf Centrifuge 5415 D

Microfuge: Beckman Coulter Microfuge 22R Centrifuge

Spectrophotometer: Spectronic 20 GENESYS

Electronic Scales: Sartorius TE212

Sartorius TE64

MIC Assay plate: Greiner bio-one PS- Microplates

Plate reader: Tecan Sunrise plate reader

9

4. Methods

2.1 Preparation of E.coli in glycerol for freezing

A loop full of bacteria was taken from the plate containing BL21(DE3) colonies and

suspended in 1 ml LB Broth (25 g l-1 ). The tubes were centrifuged twice for 1 min at 13,000

rpm with the pellet being re-suspended in 1 ml LB Broth after first spin and the supernatant

being removed after each spin. The pellet was re-suspended in 0.7 ml LB Broth. 0.7 ml of a

20% glycerol solution (Sigma) was added. The solution was transferred into 2 cryotubes and

frozen at -80°C.

2.2 Preparation of competent cells

Day 1- Cells must be competent to accept transformed plasmid.

A single bacterial colony (2-3 mm in diameter) of BL21 (DE3) was selected using an

inoculating loop and transferred into 25 ml LB broth in a 250 ml flask. The flask was then

incubated for 6-8 h at 37°C with vigorous shaking (250 – 300 rpm). This starter culture was

then used to inoculate three 250ml flasks (each containing 40 ml LB Broth) as follows:

1 ml starter culture was pipetted into first flask.

0.33 ml starter culture was pipetted into second flask.

0.1 ml starter culture was pipetted into third flask.

All three flasks were incubated at 30°C with moderate shaking overnight.

Day 2- The three 250 ml flasks containing our cultures were removed from incubation and

stored on ice. The OD600 of the three cultures were measured using a spectrophotometer and

culture closest to 0.55 Absorbance units (0.1 ml culture; 1.456 Absorbance units) was

selected. 1.5 ml of this culture was pipetted into 8 eppendorf (eppi) tubes and centrifuged at

3000 rpm for 20 min. The supernatant was removed using a pipette and the open eppis were

tapped on tissue paper to remove excess medium. The cells were re-suspended in 500 µl ice-

cold Inoue transformation buffer (MnCl2●4H20 0.55g/l Sigma, CaCl2●2H20 0.11g/l AnalaR,

KCl 0.94g/l Sigma) and centrifuged at 2000g in microfuge at 4°C for a duration of 10 min.

The supernatant was again discarded and the open eppis were tapped on tissue paper, then the

pellet was re-suspended in 125 µl ice cold Inoue buffer. All suspensions were then combined

into 1 eppendorf tube and 76 µl DMSO (Sigma) was added. The eppi was mixed by swirling

10

and stored on ice for 10 min.50 µl aliquots of the suspension were then pipetted into each of

20 chilled eppendorf tubes.

The tubes were stored at -80°C.

2.3 Transformation of competent cells

2 tubes containing competent cells were removed from the freezer and thawed by holding in

the palm of your hand before being stored on ice for 10 min. Two plasmids were added to the

competent cells:

1) pET101 (empty vector)

2) pET101 + emr A+B

1 μl of the plasmids was added to each of the two tubes and stored on ice for 30 min.

The cells were then heat shocked by transferring to 42°C for exactly 90 sec, and were then

put back on ice for 1 – 2 min. 800 μl SOC medium was added to each tube and the tubes were

then incubated for 45 min at 37°C. 100 μl bacteria was then taken from each tube and spread

onto LB + Amp (0.12g/10ml) plates. The plates were inverted and left to incubate at 37°C

overnight.

It was first attempted to transform BL21(DE3)ΔacrB cells to take up the plasmids. Following

several failed attempts at this, BL21(DE3) cells were used and plasmids were successfully

transferred to cells.

2.4 MIC Assay

Day 1- i. Inoculating E.coli in LB broth:

A single colony was selected from the BL21(DE3) plates (1 from plate containing empty

vector, and 1 from emrA+B vector) and inoculated into tubes containing 2ml LB + Amp

(0.12g/10ml). The tubes were then incubated while being shaken overnight at 37°C.

Day 2 - ii. Induce protein expression:

60 µl of each culture was added to two tubes containing 2 ml LB + Amp. The tubes were then

incubated with shaking at 37°C for 90 min. IPTG was added (to a final concentration of

50µM) to one tube for each culture. Tubes were incubated again at 37°C for 90 min.

11

iii. Set up micro-titre plate: (2 plates set up, 1 for incubation at 30°C, and 1 for 37°C)

The plates were set up as follows:

100 µl LB + Amp into all wells except columns 1 and 12 (using multi-channel pipette)

200 µl LB + Amp into column 12 wells

200 µl LB + Amp + Test antibiotic added to each well of column 1

Highest concentration of antibiotics on plate:

Ciprofloxacin - 5000 ng ml-1,

Nalidixic acid- 50 μg ml-1

Norfloxacin- 500 ng ml-1

Oxolinic acid – 1 μg ml-1

Starting at column 1, 100 µl transferred from column to column (pippeted up and down

several times to ensure mixing) up to column 10.

100 µl then discarded from column 10.

iv. Inoculate micro-titre plate:

Cultures were diluted 1:2 in LB + Amp or LB + Amp + 50µM IPTG where applicable

100 µl diluted cultures were added to relevant rows in columns 1 – 11 using a pipette.

Plates were incubated at 30°C or 37°C overnight.

Day 3- v. Growth measurement

Plates were examined for growth and the optical density of the wells was measured at 595nm

using Tecan Sunrise machine. Results were then plotted in tables using Excel.

12

Fig. 2: 96 well plate used for MIC

assay (obtained from Paul McCay

lecture slides).

E= cells with empty vector

AB = cells with vector containing emrA+B

5. Results

3.1. Transformation of E.coli strains

In order to test whether our emrA+B genes confer antibiotic resistance through the formation

of an efflux pump, the genes were inserted into pET101 expression vectors and transformed

into an E.coli strain that is hypersensitive to antibiotics (BL21(DE3)) by means of a modified

Inoue method for transformation. This involved the heat shocking of the bacterial cells to

create pores in the membrane to allow uptake of the plasmids containing our genes.

Transformation of the BL21(DE3)ΔacrB to take up our two plasmids were first attempted,

but proved unsuccessful. Some colonies of the cells containing our empty vector formed

initially but the vector containing emrA+B did not grow. Subsequent attempts to grow

colonies of our two plasmids all failed. After successful uptake of our plasmids in E. coli

TOP10 cells and formation of colonies we concluded that our competent BL21(DE3)ΔacrB

were the problem, and proceeded to use BL21(DE3) cells instead of BL21(DE3)ΔacrB for

the remainder of the project.

3.2 MIC Assay Results

Bacteria were incubated in LB Broth at 37°C overnight prior to testing with MIC Assays.

Three MIC assays were carried out for each antibiotic at 30°C and at 37°C by inoculating 96–

well plates with our bacteria in growth medium and varying concentrations of antibiotics, as

well as one column containing bacteria and medium only (Positive control) and one column

containing only LB Broth (Blank). This was carried out to demonstrate whether the cells

containing the emrA+B genes would have increased resistance to the antibiotics, measured by

obtaining and comparing the Minimum Inhibitory Concentrations of our 4 samples to 4

different antibiotics

The 4 samples analysed were E. coli BL21( DE3) containing: Empty pET101 vector without

IPTG (E-) , Empty vector with IPTG (E+), emrA+B vector without IPTG (AB-) and emrA+B

vector with IPTG (AB+). IPTG is added to 2 of the samples to induce protein expression and

left out of the other 2 to see how much of an effect it would have on the growth of our

bacteria. All our MIC assays included a positive control column containing bacteria and

medium only, to see how each of our four strains would grow in these conditions without

antibiotics. Data shown in our tables are the average of 2 replicates ± Standard deviation of

one experiment. Each experiment was carried out three times.

13

4 MIC assays of Ciprofloxacin were carried out. The first was discarded due to an error in

the dilution of the antibiotic, resulting in it being 10 times more concentrated then it should

have been (highest concentration on the plate was 50,000ng ml -1 ) and as such there was no

growth in the wells except at the lower concentrations.

It can be observed in Fig. 3, and also in the rest of our tables, that there is more growth from

the cells containing the empty vector that are grown in the absence of IPTG (black bars) then

all the other samples. This is especially noticeable when looking at the positive control. The

OD values for our positive control in Fig. 3 demonstrates this and are as follows: E- 0.41 A,

E+ 0.24 A, AB- 0.32 A and AB+ 0.33 A. We believe that our E- cells are growing much

better than our E+ because the E+ in the presence of IPTG are seeing increased expression of

the T7 polymerase. This overexpression could be taking up too much energy for the cells to

survive and as such we see less growth. This may also be the case for our cells containing

emrA+B in the presence of IPTG and, even in the absence of IPTG, the AB- cells can still see

some expression of the genes.

5000

2500

1250 62

531

2.5

156.

2578

.13

39.0

619

.53

9.77

Pos co

nt.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Ciprofloxacin (ng ml -1)

Op

tic

al D

en

sit

y a

t 5

95

nm

Fig. 3: Optical density of our bacterial cells in Ciprofloxacin at 30°C

Growth of E.coli BL21(DE3)containing pET101 vectors:

Black bars – Empty vector without IPTG (E-)

White bars – Empty vector with IPTG (E+)

Dark grey bars – emrA+B vector without IPTG (AB-)

Light grey bars – emrA+B vector with IPTG (AB+)

14

Positive controls contain our 4 bacterial samples and growth medium only (no antibiotics)

It can be seen from Fig. 3 that there was very minimal growth of bacteria in most of the wells

for the MIC assay for ciprofloxacin at 30° .This may be due to too high a concentration of

ciprofloxacin or errors made when inoculating the wells. This lack of growth is very clear

when compared to the positive control (+bacteria –antibiotic) where growth of both vectors

of E and AB were much higher (0.24-0.41 A). The MIC values were E - 9.77 ng ml -1, E +

9.77 ng ml -1, AB- 9.77 ng ml -1 and AB+ 9.77 ng ml -1. From these results there is no

indication that the emrA+B genes are increasing antibiotic resistance of our cells to

ciprofloxacin at 30°C as the MIC of our four samples are all 9.77 ng ml -1.

5000

2500

1250 62

531

2.5

156.

2578

.13

39.0

619

.53

9.77

Pos C

ont.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Ciprofloxacin (ng ml -1)

Op

tic

al D

en

sit

y a

t 5

95

nm

Fig. 4: Optical density of our bacterial cells in ciprofloxacin at 37°C (Legend as in Fig. 3)

Our wells in Fig. 4 of the MIC assay of ciprofloxacin at 37°C showed

irregular growth when compared to the Positive Control. This could be due

to improper inoculation of our wells, considering more growth is seen

from concentrations 19.53 ng ml -1 – 312.5 ng ml -1 than is seen at the lowest

measured concentration 9.77 ng/ml ( it appears that only our E + cells

grew properly at this concentration). The MIC values were E – 9.77 ng ml -1, E +

19.53 ng ml -1, AB- 19.53 ng ml -1 and AB+ 19.53 ng ml -1. Our genes do not seem to be

15

conferring antibiotic resistance to the cells containing them, as the MIC for AB+ and E+ are

the same (19.53 ng ml-1). From our results at 30°C and 37°C we can conclude that if emrA+B

do encode for an efflux pump, ciprofloxacin is not a substrate of it.

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

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Nalidixic acid (µg ml -1)

Op

tic

al

De

ns

ity

at

59

5n

m

Fig. 5: Optical density of our bacterial cells in nalidixic acid at 30°C (Legend as in Fig. 3)

Growth is seen in Fig. 5 of the MIC assay of nalidixic acid at 30°C at lower concentrations

similar to that seen in the positive controls and a clear MIC can be identified for each of our 4

samples. The MIC values were E - 3.13 µg ml -1, E + 3.13 µg ml -1, AB- 3.13 µg ml -1, and

AB+ 3.13 µg ml -1. Growth of our cells containing the empty vector in the absence of IPTG

(white bars) at the 25 µg ml -1 concentration is due to suspected contamination. There is no

obvious indication that emrA+B is allowing the cells containing said genes more resistance to

nalidixic acid at 30°C then those not possessing the genes, as the MIC of all four samples is

the same (3.13 µg ml -1 ).

16

50 25 12.5 6.25 3.125 1.56 0.78 0.39 0.20 0.10 Pos cont.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Nalidixic acid (µg ml -1)

Op

tic

al D

en

sit

y a

t 5

95

nm

Fig. 6: Optical density of our bacterial cells in nalidixic acid at 37°C (Legend as in Fig. 3)

Fig. 6 of the MIC assay of nalidixic acid at 37°C shows that the MIC for our emrA+B

containing cells in the presence of IPTG is higher than that of the empty vector in IPTG. The

MIC values were E - 6.25 µg ml -1, E + 3.13 µg ml -1, AB- 6.25 µg ml -1 and AB+ 6.25 µg ml -

1. From this we could say that the genes do have an effect on our cells antibiotic resistance to

nalidixic acid at 37°C, but since this effect was not seen for nalidixic acid at 30°C we cannot

say for certain whether nalidixic acid is a substrate for the efflux pump if emrA+B do indeed

encode for one.

17

500

250

125

62.5

31.2

515

.63

7.81

3.91

1.95

0.98

Pos co

nt0.00

0.10

0.20

0.30

0.40

0.50

0.60

Norfloxacin (ng ml -1)

Op

tic

al D

en

sit

y a

t 5

95

nm

Fig. 7: Optical density of our bacterial cells in norfloxacin at 30°C (Legend as in Fig. 3)

The MIC assays for norfloxacin at 30°C and 37°C were initially tested with

the highest concentration on the plate at 5000 ng ml -1, but was diluted

down 10 x after little or no growth was seen on the plate. The MIC values for

the MIC assay of norfloxacin at 30°C seen in Fig. 7 were E - 15.63 ng ml -1, E + 15.63 ng ml -

1, AB- 15.63 ng ml -1 and AB+ 15.63 ng ml -1. We believe growth of the AB – cells after its

MIC (15.63 ng ml -1) is due to suspected contamination. There is no indication from

Fig. 6 that emrA+B are allowing more antibiotic resistance to norfloxacin

at 30°C to the cells containing the genes then those without the genes.

We know this as the MIC’s of the 4 samples are all 15.63 ng ml -1.

18

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Norfloxacin (ng ml -1)

Op

tic

al D

en

sit

y a

t 5

95

nm

Fig. 8: Optical density of our bacterial cells in norfloxacin at 37°C (Legend as in Fig. 3)

The MIC values of the MIC assay for norfloxacin at 37°C as seen in Fig. 8 were E - 31.25 ng

ml -1, E + 15.63 ng ml -1, AB- 62.5 ng ml -1 (growth at 125 ng/ml concentration due to

suspected contamination), and AB+ 31.25 ng ml -1 (no growth seen at 3.91 ng ml -1

concentration due to accidental omission of bacterial culture from wells). It appears as

though the genes are having a direct effect on the antibiotic resistance of

our cells to norfloxacin at 37°C. The MIC for our cells containing emrA+B

in the presence of IPTG (31.25 ng ml -1) is twice that of the MIC of the empty

vector cells in IPTG (15.63 ng ml -1)

19

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

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Oxolinic acid (ng ml -1)

Op

tic

al D

en

sit

y a

t 5

95

nm

Fig. 9: Optical density of our bacterial cells in oxolinic acid at 30°C (Legend as in Fig. 3)

The MIC values for the MIC assay of oxolinic acid at 30°C as seen in Fig. 9 were E - 125 ng

ml -1, E + 62.5 ng ml -1, AB- 125 ng ml -1 and AB+ 62.5 ng ml -1. We can conclude from our

results that our genes do not confer our bacterial cells resistance to oxolinic acid at 30°C. We

know this as the MIC values for our empty vector cells in the presence of IPTG and the cells

containing the emrA+B in the presence of IPTG are the same (62.5 ng ml -1).

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

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Oxolinic acid (ng ml -1)

Op

tic

al D

en

sit

y a

t 5

95

nm

Fig. 10: Optical density of our bacterial cells in oxolinic acid at 37°C (Legend as in Fig. 3)

The MIC values for the MIC assay of oxolinic acid at 37°C as seen in Fig. 10 were E - 250

ng ml -1, E + 125 ng ml -1, AB- 125 ng ml -1 and AB+ 125 ng ml -1. Again no indication of the

genes affecting resistance to oxolinic acid as the MIC values of both the cells possessing the

empty vector and those containing the emrA+B vector, in the presence of IPTG, are the same

(125 ng ml -1) We can conclude that emrA+B are not conferring resistance to oxolinic acid to

our cells at either 30°C or 37°C.

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6. Discussion

6.1 Critical review of methods used

Transformation of competent cells

In our project we initially intended to introduce the vectors containing our genes into the E.

coli strain BL21(DE3)ΔacrB. The ΔacrB gene encodes for a protein that gives resistance to

kanamycin, so the medium we used to grow our transformed cells contained ampicillin and

kanamycin). However, only a few colonies of the cells containing the empty vector grew, and

no colonies formed for our transformed cells that contained our genes. The protocol was

repeated, with the amount of each plasmid added to the competent cells increased from 1μl to

2μl. This was an acceptable increase as long as the volume of the plasmid added didn’t

exceed 5% of the 50μl concentration of competent cells, i.e. 2.5μl (Sambrook & Russell,

2006)). Once again no growth was seen, so the protocol was repeated with new plasmids.

This also failed, meaning we had to reconsider our protocol and figure out why our cells were

not growing. We came to two possible hypotheses:

1. Expression of our genes was proving to be toxic to the BL21(DE3)ΔacrB strain

(which are a strain that are hypersensitive to certain antibiotics and other

substances). To test this we transformed our cells as normal but added 1% glucose

(Sigma) to our growth medium. Glucose represses transcription of the lac promoter

which would stop our genes from being expressed and promoting their possibly

toxic proteins in the cells, allowing the cells to grow. However no growth was seen

at all.

2. No colonies were forming due to improper preparation on our behalf. To test this

we transformed E. coli TOP10 cells to take up our plasmids and plated them onto

LB + Ampicillin plates (no kanamycin as the cells did not contain the ΔacrB gene).

Growth was seen for cells containing each of our two plasmids.

Therefore, since our competent BL21(DE3)ΔacrB did not grow even when expression of the

genes was repressed by the glucose, and we saw exponential growth of the E. coli TOP10

cells containing our plasmids, we could conclude that our BL21(DE3)ΔacrB cells were the

problem. We carried out another transformation, this time using BL21(DE3) cells, (these did

not possess the ΔacrB and thus were grown in the presence of ampicillin without kanamycin).

Colonies grew for both of our plasmids. We then proceeded to use the BL21(DE3) strain

containing our plasmids to carry out our MIC assays.

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MIC Assays

The first antibiotic we carried out our MIC assays on was ciprofloxacin. After incubating our

MIC assay plates overnight at 30°C and 37°C and reading them using the Tecan Sunrise

plate, no growth was seen. This should not have been the case. After reviewing the method

by which we set up the plates, we realised there was an error in the dilution of the antibiotic.

The stock concentration was 50 mg ml-1, as opposed to 5 mg ml-1 which is what we believed

to concentration to be. This means that when we diluted it down the highest concentration on

our plates was 50,000 ng ml-1, 10 times more concentrated then it should have been (the

concentration in column 1 should have been 5,000 ng ml-1).

When inoculating the plates to be incubated overnight, it is very important that it is carried

out very slowly and carefully. Often times one or more of the wells in column 12 (the

negative controls containing only growth medium) would have seen exponential growth

higher than any of the other wells in columns 1 to 11, due to accidental contamination. While

this did not affect the results too drastically (the averages of all the wells in column 12 not

contaminated was obtained, any contaminated wells were omitted), it illustrates how precise

one must be when preparing the assay plates to ensure a fair and correct reading. Evidence

that contamination may also have occurred after the MIC of some of our antibiotics can be

seen in our results. Examples include growth in Fig. 5 of E- (white bar) at 25 µg ml -1 and

growth in Fig. 7 of AB- (dark grey bar) at 125 ng ml-1.

6.2 Analysis of data generated

Ciprofloxacin

The results seen for ciprofloxacin do not indicate that there is any increased resistance of the

cells containing the EmrAB efflux pump to the antibiotic. However with the low levels of

growth seen in the assays (particularly in Fig. 3) it is difficult to discern whether the cells

containing our genes are growing considerably better than the cells with the empty vector. I

would suggest that a lower concentration of ciprofloxacin be used when carrying out the MIC

assays, as there is minimal growth even at the lowest concentration (9.77 ng ml -1 ). A higher

volume of bacteria could also be added before incubating the plates overnight which, in

combination with a lower concentration of antibiotics, should produce more growth in the

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wells which would allow a definitive MIC for each of the four samples to be identified and

compared.

Nalidixic acid

The growth of the cells in the MIC assay for nalidixic acid grew much better than the

ciprofloxacin assays, which allowed us to identify a clear MIC for each of the four samples.

No increased resistance to nalidixic acid was seen in Fig. 5 at 30°C, but there appeared to be

a slight increase to the resistance of the cells grown in Fig. 6 at 37°C. With further testing at

37°C again with increased volume of bacteria, some confident results could be produced

suggesting that nalidixic acid resistance is increased by the presence of our genes. It is

interesting to note that the emr genes found in some E. coli strains are known to provide

resistance to nalidixic acid (Lomovskaya & Lewis, 1992). Both the EmrAB proteins in E. coli

(Piddock, 2006) and our novel proteins discovered in V. parahaemolyticus belong to the

Major Facilitator Superfamily (MFS) of efflux pumps, which is one of the largest families of

membrane transport systems found in bacteria and eukaryotes (Pao et al, 1998).

Norfloxacin

The MIC assays for norfloxacin showed resistance to the antibiotic to our cells containing

emrA+B at 37°C (Fig. 8) but not at 30°C (Fig. 7). Previous studies have shown that the

emrAB genes present in E. coli do not confer resistance to norfloxacin (Nishino &

Yamaguchi, 2001), which is a contrast to the results obtained for our genes. It is important to

note that the assays at the two different temperatures will not have the same level of growth

on the plates. 37°C is the optimum temperature of growth for E. coli (Zhang et al, 1998), so it

is expected that more growth will be seen for the assay plates incubated at this temperature. I

believe that the samples grown at 37°C would give a more accurate result. The more growth

present on the plates, the easier it is to identify the Minimum Inhibitory Concentration of

each sample.

Oxolinic acid

From the results obtained for the MIC assays of oxolinic acid we can deduce that emrA+B do

not confer resistance to our bacterial samples at either 30°C or 37°C. We know this because

the MIC’s for the cells containing our genes grown in the presence of IPTG and the cells

containing the empty vector are the same (62.5 ng ml -1 at 30°C seen in Fig. 9, 125 ng ml -1 at

24

37°C as seen in Fig. 10). These results indicate that V. parahaemolyticus are sensitive to

oxolinic acid, which could be used as an effective therapeutic treatment against infections

from the organism. This finding has been mirrored in previous studies (Ottaviani et al, 2001)

(Liu et al, 2000).

6.3 Future Directions

Future experiments could include MIC assays being carried out for a wider variety of

antibiotics. Strains containing the genes could initially be streaked on plates containing

growth medium + test antibiotics to see whether they are able to grow on the plates or not,

along with strains not containing the genes also being plated to compare colony formation.

This could be used as the basis for selecting what antibiotics for the future MIC assays. From

the results obtained in this project I would recommend carrying out all further assays at 37°C.

More growth was seen all round at 37°C than 30°C, which makes it much easier to identify

the MIC values for each sample.

Following confirmation that the genes emrA+B do indeed encode for an efflux pump, the

next goal would be to map the molecular and genetic makeup of the efflux pump. By doing

so steps can be taken to combat the resistance that depends on the mechanism of the efflux

pump through development of an efflux pump inhibitor specific to the pump produced by the

genes in V. parahaemolyticus. This would be highly beneficial for treatment of infections

from V. parahaemolyticus strains whose resistance to antibiotics can be attributed to our

newly found efflux pump.

6.4 Conclusions

The aim of this project was to investigate whether the proteins produced by the genes emrA

and emrB in Vibrio parahaemolyticus form an efflux pump that would transport antibiotics

from the bacteria before they could have an effect. A number of MIC assays were carried out

to test this hypothesis, but it cannot be said definitively that the genes do confer resistance to

antibiotics as the results were inconclusive. However there is some evidence suggesting that

nalidixic acid and norfloxacin may be substrates for the efflux pump. I believe this warrants

further testing of the genes with these two antibiotics in order to prove this.

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[1] https://products.invitrogen.com/ivgn/product/K10101

[2] http://products.invitrogen.com/ivgn/product/C600003

[3] http://tools.invitrogen.com/content/sfs/manuals/pettopo_man.pdf (pg. 4)

[4]http://www.quora.com/How-does-IPTG-induced-gene-expression-work-at-a-molecular-

level

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This report is an accurate and faithful representation of the work

performed by us between (10/09/12) and (9/11/12). The authorship of any

data included in this report, which was not generated by our own work, is

clearly indicated. .

I have read, understood and have accepted the plagiarism policy of the

Department. To the best of my ability, I have complied with its

requirements.

Francis Higgins

Signed;

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