Novel antibiotic resistance proteins in V parahaemolyticus
1. 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
2. 2
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
3. 3
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.
4. 4
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.
5. 5
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).
6. 6
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.
7. 7
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
Fig.1: pET101 expression vector
including our genes of interest, the
T7 promoter and a gene encoding
for ampicillin resistance [3].
8. 8
out work and research independently in order to gain a real understanding of the topic in
question.
2.6 Workflow followed throughout the project
Preparation of materials
-Media
-Frozen stocks of bacteria
Transformation of competent cells
to take up our expression plasmids
MIC Assays carried out for a range of
antimicrobials on our transformed
cells
Analysis of results and comparison
of MIC values for transformed E. coli
cells containing our genes and the
cells containing the empty vector
10. 10
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
11. 11
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.
12. 12
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.
Fig. 2: 96 well plate used for MIC
assay (obtained from Paul McCay
lecture slides).
E= cells with empty vector
AB = cells with vectorcontaining
emrA+B
13. 13
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.
14. 14
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.
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+)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
OpticalDensityat595nm
Ciprofloxacin (ng ml -1)
15. 15
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.
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 conferring antibiotic
resistance to the cells containing them, as the MIC for AB+ and E+ are the same (19.53 ng
0.00
0.10
0.20
0.30
0.40
0.50
0.60
OpticalDensityat595nm
Ciprofloxacin (ng ml -1)
16. 16
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.
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 ).
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
50 25 12.5 6.25 3.13 1.56 0.78 0.39 0.20 0.10 Pos
Cont.
OpticalDensityat595nm
Nalidixic acid (µg ml -1)
17. 17
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.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
50 25 12.5 6.25 3.125 1.56 0.78 0.39 0.20 0.10 Pos
cont.
OpticalDensityat595nm
Nalidixic acid (µg ml -1)
18. 18
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.
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70OpticalDensityat595nm
Norfloxacin (ng ml -1)
19. 19
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)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
OpticalDensityat595nm
Norfloxacin (ng ml -1)
20. 20
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).
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
1000 500 250 125 62.5 31.25 15.63 7.81 3.91 1.95 Pos
cont.
OpticalDensityat595nm
Oxolinic acid (ng ml -1)
21. 21
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.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
1000 500 250 125 62.5 31.25 15.63 7.81 3.91 1.95 Pos
cont.
OpticalDensityat595nm
Oxolinic acid (ng ml -1)
22. 22
6. Discussion
6.1 Critical review ofmethods 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.
23. 23
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
24. 24
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
25. 25
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.
26. 26
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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-
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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;
