Institutionen för kvinnors och barns hälsa Biomedicinska analytikerprogrammet
Examensarbete 15 hp
POLYVINYLALCOHOL-CARBAZATE (PVAC) INHIBITS BACTERIA GROWTH Jakob Syk
Handledare: Carl Påhlson
Examinator: Anneli Stavreus-Evers
Adress: Institutionen för Kvinnors och Barns Hälsa, Akademiska sjukhuset, 751 85 Uppsala Telefon: 018-611 28 31
Abstract
Introduction
This study evaluated the effect of the polymer polyvinylalcohol-carbazate (PVAC) on the bacteria Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and
Pseudomonas aeruginosa. PVAC is a polymer with a carbazate moiety that neutralizes free
aldehydes and has shown great promise in stabilizing erythrocytes during long term storage. It has also been shown to reduce intraperitoneal adhesions after trauma. For this study, two Gram positive and two Gram negative bacteria strains were used with PVAC to evaluate its effect.
Materials and methods
PVAC was obtained from the research team at Ångström Laboratory, Uppsala. The bacteria were obtained from Clinical Microbiology and Hospital Hygiene, Academic Hospital, Uppsala. The methods used were spectrophotometric assessment of bacteria growth, use of FITC-conjugated PVAC to study adherence to bacteria, use of FITC-antibodies to study PVAC’s effect on bacterial adherence to erythrocytes and a qPCR for quantification of E.
coli.
Results and discussion
PVAC displayed a clear effect of inhibition of bacteria growth in the study as shown by use of spectrophotometric assessment. Trials with FITC-PVAC showed that the polymer adheres directly to the bacteria, displaying a possible function of its inhibitory properties. The qPCR assay was able to detect the bacteria in all the dilutions used.
Keywords
Introduction
Bacteria are responsible for a multitude of infections of various morbidity and mortality. Therefore, medical science is devoted to developing drugs and equipment to combat and control them. Although many diseases are caused by bacteria, our own microbiome consists of many different commensals that protects us against foreign invading pathogens.
In this study we chose two pathogenic bacteria, the Gram positive S. aureus and the Gram negative E. coli and also two relative commensals, Gram positive S. epidermidis and Gram negative P. aeruginosa. Since the cell walls differ dramatically between Gram positives and Gram negative bacteria, using both major groups of bacteria is vital in evaluating PVACs effect.
specificity, followed by a core polysaccharide which is the same for all members of the same family of Gram negatives. The core polysaccharide is followed by the lipid portion, called lipid A. It consists of a disaccharide phosphate group with fatty acids which tethers the LPS to the outer membrane. Beneath the outer membrane lies the cytoplasmic membrane, and
between the two is a space called the periplasmatic room. Just over the cytoplasmic membrane lies a thin PG-layer [1].
Polyvinylalcohol-carbazate (PVAC) is a plasticizer that contains a carbazate moiety that functions as an aldehyde scavenger and has shown great promise in stabilizing erythrocytes in blood bags by neutralizing the free aldehydes that cause damage to the erythrocytes cell membrane. It also shows some effect in stabilizing the erythrocyte membranes when the erythrocytes were introduced to a hypotonic solution [2]. It has also been demonstrated that sutures impregnated with PVAC reduce intraperitoneal adhesions in a controlled animal study of 110 Sprague-Dawley rats that underwent laparotomy, cecal abrasions and construction of a small bowel anastomosis [3]. This study presents a first attempt at evaluating PVACs effect on various bacteria. PVAC could show a stabilizing effect on the bacteria, as it did with the erythrocytes, making it a useful additive in bacteria storage. In contrast, it could show an inhibitory effect on bacteria, which would only further solidify PVAC as a candidate for addition to blood bags during long-time storage, as it would also inhibit any eventual bacteria in the bag, aside from its effect on the erythrocytes.
concentrations of bacteria yields higher or lower absorbance, respectively. In the study, a customized system with 3D-printed culture tubes and spectrophotometers were used, but the experiment can theoretically be performed by use of any spectrophotometer [4]. Another study showed that a spectrophotometer could also be used to detect the formation of biofilm by staining with a crystal violet solution, and then de-stain with acetic acid. By acquiring absorbance measurements at 550 nm (the color corresponding with the purple hue of crystal violet), the amount of biofilm in the sample can be assessed [5].
Fluorescence microscopy is an effective method to study the effect of different adhering substances, as only the fluorescent molecules will be visible in the microscope. One substance that can be used to stain bacteria for fluorescence microscopy is
4',6'-diamidino-2-phenylindole, also known by its abbreviation DAPI. DAPI is a substance that binds to DNA and fluoresces with a blue color. It's an effective method for studying bacteria, as it outlines the shape of the bacteria well. In a study, the DAPI staining was used to detect E. coli. in water samples. It has previously been shown that DAPI clearly stains the bacteria and was proved to be an effective method of observing bacteria in a fluorescence microscope [6, 7]. Another common fluorophore that is widely used, is fluorescein isothiocyanate (FITC), which fluoresces with a green color when viewed with a FITC filter in a fluorescence microscope. Antibodies directed against specific structures on the bacterial surface can be conjugated with FITC-molecules, and thus be used to specifically detect certain bacteria. The use of FITC-conjugated antibodies is an easy method to study adherence to cell structures, as the fluorescence makes it easy to see where the antibodies have bound [8].
complementary to the nucleotide sequence of the target just ahead of the primer binding sites. They are also conjugated with a reporter and a quencher. The reporter fluoresces faintly, but it is absorbed by the quencher molecule entirely while the probe is intact. When the DNA polymerase in the PCR reaction elongates the strand from the primers, the probes are
hydrolyzed, and the reporter released from the quencher. When released into the solution, the reporter emits fluorescence that is measured by the real-time PCR machine. It provides the simple formula that 1 hydrolyzed probe equals 1 amplified copy of the target section of the subject DNA. PCR can be used to identify small quantities of bacteria in samples, and many assays have been developed for E. coli, with the importance mostly focused on detecting the bacterium in food and fecal samples. [9].
The aim of this study was to evaluate PVACs interaction with the bacteria Escherichia coli,
Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa by,
through a range of different methods, assessing PVACs adherence to and effect on bacterial samples.
Methods
Bacteria
Bacteria used in this study were obtained from KMB (Clinical Microbiology and Hospital Hygiene, Uppsala Academic Hospital, Uppsala, Sweden). The bacteria used were Escherichia
coli (strain ARU 638), Staphylococcus aureus (strain CCUG 15915), Staphylococcus
epidermidis (strain CCUG 18000A) and Pseudomonas aeruginosa (strain CCUG 17619). The
PVAC
Freeze-dried PVAC was obtained from the research team at Ångström Laboratory (Uppsala, Sweden). The PVAC was cut into pieces and then weighed to the desired amount. It was then dissolved in PBS to two stock solutions, 10 mg/ml and 20 mg/ml. The solutions were sterile filtered before use. FITC-conjugated PVAC used in experiments was freeze-dried and suspended in 600 µl of PBS to a stock solution of 5 mg/ml. The FITC-PVAC was not sterile filtered before use.
PVACs effect on bacteria growth rate
A single colony from each bacterial strain were added to 10 ml of 1x LB (Luria-Bertani growth medium, Spectrum) in 50 ml Falcon tubes (Sarstedt) and were incubated overnight in 37°C on a rotating plate at 200 rpm. The suspensions were then diluted with LB to a turbidity of 0.5 McFarland, and then diluted 1000 times with LB. The diluted suspensions were then added to each of the wells of a 96-well ELISA plate, 20 µl each, along with PVAC (10 mg/ml stock solution, and a 20 mg/ml solution for the 5 mg/ml wells) and LB, to reach PVAC concentrations of; 0 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2.5 mg/ml and 5 mg/ml. Total well volume was 250 µl. The plate was incubated for 18 h in an ELISA plate reader
(Molecular Devices VERSAmax™ microplate reader, USA) in 37°C, with absorbance readings acquired every 30 minutes at 600 nm, to assess PVACs effect on bacterial growth rate.
PVACs adherence to bacteria using FITC-conjugated PVAC
Centrifugation method
mixed with FITC-PVAC to desired concentrations; 0 mg/ml, 0.5 mg/ml, 1 mg/ml and 2.5 mg/ml. The solutions were then incubated in room temperature for 1 h. After incubation, the tubes were centrifuged, and the supernatant was extracted. The pellet was resuspended in 500 µl PBS and incubated in RT for 15 min. From this suspension, 100 µl was extracted and added to another tube. The suspension was then centrifuged, the supernatant extracted, and the pellet was dissolved another time. Another sample was taken from the new suspension to another tube. This was repeated 4 times to obtain several levels of specific conjugated FITC-PVAC to the bacteria. A single drop from the 4 suspensions were added to a microscope slide with wells. The slide was then dried in room temperature for 1 h. When dry, the slide was mounted with Vectashield Mounting Medium with DAPI (Vector, California, USA) and analysed in a fluorescence microscope.
Smear method
A drop of PBS was added at the end of a microscope slide (n=6 slides), and a single colony of
S. aureus and P. aeruginosa was suspended in the drop, and then smeared using the standard
blood smear method. The slides were air-dried. When dry, 50 µl of FITC-PVAC was added to the slides in the concentrations; 0 mg/ml, 1 mg/ml and 2.5 mg/ml, and smeared across the slide with a pipette tip. The slides were incubated in a humidity chamber for 30 min in the dark. After incubation, the slides were air-dried and mounted with Vectashield DAPI and analysed.
Bacterial adherence to erythrocytes in solution containing PVAC
concentrations; 0 mg/ml, 1 mg/ml och 2.5 mg/ml. A 0.5 McF suspension of S. aureus was prepared and then added to the tubes, 50 µl to each. Total tube volume was 300 µl, with varying volumes of PVAC stock solution (10 mg/ml), and the rest PBS. Viable count (VC) of the spiked blood samples was prepared by adding 1 µl from each spiked blood sample to a blood agar plate (total 3 plates). The tubes were then incubated overnight (t=20 h) in 4°C. The next day, the tubes were vortexed for a few seconds (due to layering of the blood). Then 1 µl from each tube was added to three blood agar plates for comparison of the VC. A single drop (10 µl) from each tube was then added to a microscope slide (in duplicates, n=6 slides) and smeared. The slides were then fixated in methanol for 5 min. An anti-human IgG polyclonal rabbit antibody (Dako, Denmark), conjugated with FITC, was diluted 1:20 and 50 µl of the dilution was added to each of the slides. They were then incubated for 30 min in the dark in a humidity chamber. After incubation, the slides were washed twice for 5 min in PBS and then air-dried.
After drying, 3 of the slides (1 slide per concentration of PVAC) were stained with a few drops of a 0.4% trypan blue solution (Sigma) and mounted with glycerol-PBS (Euroimmun, Germany). The remaining 3 slides were mounted with Vectashield DAPI (without staining). The slides were then analyzed in a fluorescence microscope.
Quantification of E. coli using real-time PCR
In this study, a real-time PCR assay was developed to acquire another method of quantifying
E. coli (strain ARU 638).
Master Mix
A PCR master mix was prepared for the ARU 638 strain just before each run in the optimization trials, where reagents were added in the following order; nuclease-free H2O,
Taq-polymerase and MgCl2 solution. All master mix materials were obtained from Qiagen, except
for the nuclease-free H2O (Roche). The primers were obtained from Invitrogen. For the
real-time PCR, reagents were added in the following order; nuclease-free H2O, TaqMan Master
Mix (Qiagen), TaqMan-probes, primers and MgCl2. The probes were acquired from Tib
Molbiol, Germany. Total reaction volume for all PCR runs was 25 µl, with 23 µl master mix and 2 µl of the samples.
The primer sequences for ARU 638 were; 5’-CTG CTG TCG ATA AAG CGC AC-3’ (forward) and 5’-GAA AGC TAT CTG CAC GCT GC-3’ (reverse). The probe sequence was 6’-FAM-TGC TTC CGC CAG TTC CAC--DB.
Optimization of PCR
The first step for the E. coli PCR was to optimize it for use. To do this, a temperature gradient was used for the annealing; 6 temperatures, 55-65°C, with 2° increments. A 10 min lysis step at 95°C was added at the start of the PCR to lyse whole bacteria and to activate the hot start Taq polymerase. After PCR, the samples were pipetted to a 2% agarose gel (E-Gel,
Invitrogen) together with a DNA size ruler (O’GeneRuler, Thermo Scientific). The gel ran for 15 min and was then evaluated under UV illumination.
A specificity test for the primers was also performed. This was done by testing other Gram negative bacteria with the primers for ARU 638. The other bacteria used were Klebsiella
pneumoniae (strain CCUG 225), Klebsiella oxytoca (strain CCUG 42935), Enterobacter cloacae (strain CCUG 59627) and Proteus mirabilis (strain CCUG 26767). The PCR products
were analyzed by gel electrophoresis as described above.
Real-time PCR
E. coli strain ARU 638 was taken from a blood agar plate with a loop and added to PBS to a
concentration of 0.5 McF. It was then serial diluted to the concentrations; 105, 104, 103, 102,
101 and 100 cfu/µl. A PCR master mix was prepared as described above, this time also
containing probes. The probes were acquired from Tib Molbiol, Germany, and had the nucleotide sequence 6’-FAM-TGC TTC CGC CAG TTC CAC-DB-3’. The PCR protocol used was; lysing of the bacteria at 95°C for 10 min and cycling at (95°C 15 s, 59°C 30 s, 72°C 30 s)x45. The data was compiled after analysis and evaluated.
Viable count was performed on 6 blood agar plates (1 plate per bacteria concentration), where 1 µl of each suspension was spread on each plate with a sterile loop, and the plates were then incubated in 37°C overnight. The colonies were counted and evaluated the following day.
Ethics considerations
The blood trial was viewed as a development project to increase the long-term survival of erythrocytes in storage and did not require an approval from the ethical committee. For reference, the ID-number for the development project is KITM17001, in use at Uppsala Academic Hospital Blood Central.
Results
The polymer polvinylalcohol-carbazate (PVAC) was used with different bacteria in a range of experiments to evaluate its effect on them. These experiments include spectrophotometric assessment of PVACs effect on bacteria growth, PVACs adherence to bacteria using
FITC-conjugated antibodies directed to S. aureus surface protein A. A real-time PCR assay was also developed for detection of E. coli, strain ARU 638.
PVACs effect on bacteria growth rate
PVAC was incubated with various bacteria in a 96-well ELISA plate, with different
concentrations of PVAC, to evaluate its effect on the bacteria growth. The figures below show the acquired absorbance from the 96-well plate with mounting concentrations of PVAC. Figure 1 shows the absorbance for E. coli, figure 2 shows S. aureus, figure 3 shows S.
epidermidis and figure 4 shows P. aeruginosa.
E. coli showed a marked response to the polymer even at lower concentrations of PVAC, with a less steep log phase present in the read with 1 mg/ml PVAC compared to the control with no PVAC added. This trend is continued at higher concentrations, as shown by the gradually diminishing growth curves. At 2.5 mg/ml PVAC, E. coli displays only a weak log phase before entering the stationary phase. At this concentration, the absorbance also dips after the log phase. At 5 mg/ml PVAC, the growth curve of E. coli has flatlined, with a slightly visible log phase followed by a dip to a value corresponding with the absorbance acquired at the start of the read (Figure 1 a-d).
S. aureus acquired a less steep growth curve at 0 mg/ml PVAC compared to E. coli. By 1
mg/ml PVAC, S. aureus displays a markedly lower log phase, followed by a stationary phase which displays a dip in absorbance. At 2.5 mg/ml PVAC, S. aureus displays a long log phase, with a visible stationary phase following. The stationary phase is similar to 1 mg/ml PVAC, but shorter due to the timeframe of the experiment. At 5 mg/ml PVAC, S. aureus displays what would be best described as a long log phase, one that does not appear to end by the end of the read (Figure 2 a-d).
defined lag, log and stationary phases. At 1 mg/ml PVAC, the bacteria also showed a slightly decreased log phase, followed by a dip in absorbance in the stationary phase. At 2.5 mg/ml, the growth curve is similar to S. aureus curve at 5 mg/ml, with a log phase that does not end before the end of the read. At 5 mg/ml, the growth curve is very similar to 2.5 mg/ml, with a faintly lowered stationary phase (Figure 3 a-d).
P. aeruginosa displayed the highest absorbance readings of all bacteria used in the experiment at 0 mg/ml PVAC. At 1 mg/ml PVAC, the log phase is elongated and not very steep, followed by a clearly visible stationary phase. At 2.5 mg/ml PVAC, P. aeruginosa displays a late onset of the log phase, one that does not end before the end of the read. By 5 mg/ml, no visible increase in absorbance could be observed (Figure 4 a-d).
Figure 2. Absorbance readings for the 96-well plate for S. aureus, with LB as medium. Graph a) shows the growth curve with no PVAC added, b) 1 mg/ml PVAC, c) 2.5 mg/ml PVAC and d) 5 mg/ml PVAC.
Figure 4. Absorbance readings for the 96-well plate for P. aeruginosa, with LB as medium. Graph a) shows the growth curve with no PVAC added, b) 1 mg/ml PVAC, c) 2.5 mg/ml PVAC and d) 5 mg/ml PVAC.
PVACs adherence to bacteria using FITC-conjugated PVAC
E. coli, S. aureus and P. aeruginosa were stained with FITC-conjugated PVAC and then
observed in a fluorescence microscope to evaluate if PVAC adheres to bacteria, seen as bright spots in the images (Figures 5-7).
The smear method was also used for E. coli (Figure 6). In the DAPI image (right), the bacteria are outlined well, while in the FITC image (left), a few green spots could be observed. Only the smear method was used for E. coli.
In the final trial, P. aeruginosa was used with the smear method (Figure 7). In the DAPI image (right), the bacteria are not as clearly defined as the other bacteria used. Only a few clear blue spots can be seen, with less stained spots around them. The FITC image (right) is similar to the smear method image for S. aureus, with green fluorescence concentrated around clusters of bacteria. The dark lines in the images were due to scraping when applying the bacterial suspension to the microscope slide.
Figure 6. E. coli, smear method. Fluorescence microscopy image at 630x magnification of E. coli with DAPI filter (left) and FITC filter (right). The PVAC concentration for this stain was 2.5 mg/ml.
Bacterial adherence to erythrocytes in solution containing PVAC
Erythrocytes were incubated with S. aureus, and then with FITC-conjugated antibodies complementary to S. aureus' surface protein A, to assess how PVAC affects bacterial
adherence to erythrocytes. The trypan blue negated most of the background fluorescence from FITC in all images, evident by the reduced green hue of the trypan blue images (right in all figures). It also improved the red colour of the erythrocytes as compared to the samples unstained with trypan blue (left in all figures).
The negative control (Figure 8) showed clearly defined erythrocytes in both images. Both the stained (right) and unstained (left) images displayed the round outline of the erythrocytes well. The trypan blue image (right) had red cells, with a green hue outlining them.
The stain with 1 mg/ml added PVAC (Figure 9) showed a high fluorescence intensity in the unstained image (left), while the stained image showed an intense green colour. The round outline of the cells was not clearly visible.
The 2.5 mg/ml stain (Figure 10) showed a relatively low fluorescence intensity for the unstained image (left). The stained image (right) displayed clearly round red cells, with faint green fluorescence surrounding them.
Figure 8. Fluorescence microscopy image at 400x magnification of red blood cells with a fluorescent antibody against S. aureus protein A. Unstained image with FITC filter (left) and trypan blue stained image with FITC filter (right). The PVAC concentration for this trial was 0 mg/ml (control).
Figure 10. Fluorescence microscopy image at 400x magnification of red blood cells with a fluorescent antibody against S. aureus protein A. Unstained image with FITC filter (left) and trypan blue stained image with FITC filter (right). The PVAC concentration for this trial was 2.5 mg/ml.
Optimisation of PCR
A real-time PCR assay was optimized for use in quantification of E. coli, strain ARU 638, to acquire an additional method of quantifying the bacteria. A series of optimizations were performed to maximize the performance of the assay.
The optimisation of the annealing temperature resulted in 59°C providing the best bands on the agarose gel (Figure 11a, indicated with an orange arrow).
The specificity test of the primers (Figure 11b) showed that the primers for the ARU 638 strain of E. coli also amplified the CCUG 17620 strain. None of the other Gram negative bacteria used in the trial were amplified.
By adding 2 mM of MgCl2 to the PCR mix, visible product was acquired down to a bacteria concentration of 101 cfu/µl (Figure 11d, indicated with a blue arrow).
Figure 11. Compound image of agarose gels used in the PCR optimisation.
a) The picture shows a 2% agarose gel under UV illumination for the annealing temperature
optimisation, with fluorescent PCR products. Wells 2-7 shows the PCR product for E. coli strain ARU 638, 103 cfu/µl, for the annealing temperatures 55-65°C, with 2°C increments
(well 2 shows 55°C, well 3 57°C and so forth). Well 1 contained a DNA size ruler.
b) The picture shows a 2% agarose gel under UV illumination for the primer specificity
c) The picture shows a 2% agarose gel under UV illumination for the primer sensitivity
optimisation. All samples are E. coli, strain ARU 638. Well 1; 105 cfu/µl. Well 3; 104 cfu/µl.
Well 5; 103 cfu/µl. Well 7; 102 cfu/µl. Well 9; 101 cfu/µl. Well 11; 100 cfu/µl. Well 13 contains
PCR product from E. coli, strain CCUG 17620, 105 cfu/µl. The wells with even numbers
contains negative controls (water). Well 15 contains a DNA size ruler.
d) The picture shows a 2% agarose gel under UV illumination for the MgCl2-concentration
optimisation, with 2 mM MgCl2 added. All samples are E. coli, strain ARU 638. Well 1
contains a DNA size ruler. Well 2; 105 cfu/µl. Well 3; 104 cfu/µl. Well 4; 103 cfu/µl. Well 5; 102
cfu/µl. Well 6; 101 cfu/µl. Well 7; 100 cfu/µl. Well 8 contains a negative control. Well 9
contains PCR product from E. coli, strain CCUG 17620, 105 cfu/µl.
Quantification of E. coli using real-time PCR
A single run was performed with real-time PCR on ARU 638. The results from the varying concentration of E. coli are displayed in the figure below. Ct; cycle threshold, the amount of cycles required for the sample fluorescence to overcome the background fluorescence. R^2; correlation coefficient, e.g. how well the result corresponds with the specified concentration of the sample. M; slope of the standard curve. B; intercept with the ordinate (a theoretical value of the nucleic acid concentration of a sample before the first cycle of PCR). The 105
cfu/µl sample was redacted from this figure due to an error with the PCR machine. The PCR reaction provided amplification curves above the cycle threshold for all the concentrations used in the trial (100-104 cfu/µl). The colours in the table correspond to the colours of the
Table 1. Results from Viable Count from the E. coli-suspensions used in the real-time PCR. Confluent; the colony count was too high to visually count.
Discussion
The aim of the study was to study the effect of the polymer polyvinylalcohol-carbazate (PVAC) on various bacteria. The methods used were turbidimetric monitoring of bacteria growth, fluorescence microscopy with FITC-conjugated PVAC and antibodies, and also real-time PCR for quantitative measuring of E. coli.
The turbidimetric analysis displayed clear growth curves for the various bacteria over time, and how their growth was affected by the PVAC. E. coli displayed a clearly defined growth curve at 0 mg/ml PVAC, indicating that the bacteria could grow normally. By 1 mg/ml, the curves log and stationary phase are slightly lowered, indicating an inhibitory effect on growth. At 2.5 mg/ml, E. coli displayed a clearly lower log phase compared to previous concentrations of PVAC, indicating further inhibition. The dip in the stationary phase could also indicate a bactericidal effect on the bacterium by PVAC, which could be cause for further studies. At 5 mg/ml, the absence of clear log and stationary phases further solidifies that growth is
inhibited. The dip in the stationary phase also displays the possible bactericidal effect.
S. aureus acquired a less steep growth curve at 0 mg/ml PVAC compared to E. coli. This
phase could indicate a possible bactericidal effect of PVAC on S. aureus, similar to E. coli. At 2.5 mg/ml PVAC, S. aureus displays a long log phase, indicating an interference of PVAC. The stationary phase is similar to 1 mg/ml PVAC, but shorter due to the timeframe of the experiment. At 5 mg/ml PVAC, S. aureus displayed a long log phase. The results acquired from 2.5 mg/ml and 5 mg/ml PVAC would indicate a certain tolerance to PVAC in S. aureus, with the log phase being elongated instead of terminated as shown in E. coli.
S. epidermidis’ displayed similar growth curves for all concentrations of PVAC as
compared to S. aureus. This is likely because of the two bacteriums many shared properties as members of the same family (Staphylococcus). S. epidermidis displayed a similarly decreased curve at 1 mg/ml, indicating the bactericidal effect of PVAC. At 2.5 mg/ml, the growth curve is similar to S. aureus curve at 5 mg/ml, with a log phase that does not end before the end of the read. This could indicate that S. epidermidis is more sensitive to the polymer compared to
S. aureus. At 5 mg/ml, the growth curve is very similar to 2.5 mg/ml, indicating a tolerance to
the polymer at higher concentrations.
P. aeruginosa’s higher absorbance readings at 0 mg/ml PVAC indicates a more aggressive
formation by clumping bacteria together. At higher concentrations, PVAC likely binds to the bacterium faster than at lower concentrations, and then blocks cellular pathways before any alginate of any consequence can be formed. The bacterium also possesses a range of specific membrane proteins instead of general ones (which is the case for many other bacteria), which could evade clusters of PVAC blocking them. At higher concentrations, PVAC likely blocks most of the membrane surface and then these proteins are also blocked [10].
Spectrophotometric assessment of bacterial growth proved to be an effective method, and has been used in several studies [11, 12], where this method has provided easily calculable results. Further studies on PVAC’s effect on bacteria (or other microbiota) might consider using spectrophotometric methods to assess its effect. A likely explanation for PVACs inhibitory effect is that the carbazate group in PVAC binds to the negatively charged D-Ala groups on LTA and TA on the bacterium’s surface. While bound, PVAC blocks membrane transport proteins, disrupting cellular respiration and disabling cell division.
aureus, where a trial with this bacterium would most likely provide similar results. This could
indicate that Gram positives bind better to PVAC than the Gram negatives. The images for S.
aureus were also acquired at a lower magnification than the other bacteria (400x instead of
630x), because its clear visibility removed the need for larger enhancement. P. aeruginosa shows a similar lack of staining compared to E. coli, with less clear DAPI and FITC stain. The concentrations of the bacteria visible in the image was accepted as proof that the bacteria bind to PVAC, albeit not as well as S. aureus. This could indicate that Gram negative rods bind less well to PVAC. The trial put all together shows that PVAC adheres to bacteria, and this is most likely its function in the inhibition process. PVAC could envelop the bacteria, preventing mitosis through mechanical blocking, or it could interact directly with the cellular chemistry of the bacteria to prevent mitosis. Molecules conjugated with FITC are widely used for easy assessment of adherence. Several studies have used FITC molecules, and its
applications include studying paracellular transport of dextran, as presented by a study [13] where the FITC fluorescence was acquired through SDS-PAGE electrophoresis instead of fluorescence microscopy. It has also been used for studies evaluating intracellular transport of pharmaceuticals in cancer cells, where the fluorescence was observed through
cytofluorometric analysis of the cells [14].
glass where there were more blood cells for the antibody to adhere to. The highest concentration of PVAC was 2.5 mg/ml, where the red blood cells round shape are not as clearly defined by the fluorescence, in both images. It is unknown why the antibody adhered to the erythrocyte cell walls. A possible explanation is that it was mechanically stuck to the cells (without binding to them), and thus avoided being washed away during washing of the objective glasses. Further studies on studying PVAC’s effect on bacterial adherence to erythrocytes might consider using higher concentrations of bacteria to provide results. FITC-conjugated antibodies are widely used in studies on cell cultures, where an antibody can be created that is directed at a membrane protein of ones choosing. One example is a study where FITC-antibodies were used on HeLa cells, where antibodies directed at HeLa-specific
structures were used to simply illuminate the cells and contrast with the other fluorescent substance used[15]. Further studies concerning PVAC’s effect on bacterial interaction with other cells might consider using FITC-antibodies for evaluation of the effect.
The real-time PCR assay was performed only use, and produced curves for all the samples, including the 100 cfu/µl concentration. The machine used was also not functioning properly,
reporting a Ct value for the 105 cfu/µl concentration as 37.22, comparable to 32.81 for the 101
cfu/µl concentration. A higher Ct-value would indicate a lower concentration in the sample, meaning this value was obviously in error. For this reason, 105 was redacted from the results.
The standard curve shows that the remaining samples (104-100) provided good linearity and
were accepted as accurate results. The real-time PCR was originally conceptualized as another method for quantification of E. coli strain ARU 638 (besides spectrophotometry), but due to a lack of time was never used in the experiments. The viable count (VC) performed on the dilutions presented results comparable to the PCR results for all the dilutions, with the exception of 103 cfu/ml, where the colony count was 192, and should have been around 1000
them from view. It might also be because of a pipetting error, where the VC sample was extracted from a portion of the suspension where the bacteria count was low.
An additional experiment was performed to assess PVAC’s effect on biofilm formation, by adding bacteria in LB and PBS to a 96-well plate and incubating the plate in 37°C overnight, and then staining the wells with a crystal violet solution. The stain was then dissolved in acetic acid, and absorbance was acquired from the ELISA plate reader at 550 nm. This experiment was redacted, as wells with higher concentrations of PVAC were stained more than wells with lower concentrations. A likely explanation for this is that PVAC adheres directly to the PVC plastic in the well walls, which could be a subject for further studies on PVAC.
In conclusion, E. coli shows a marked response to PVAC, with growth being gradually more inhibited with mounting concentrations of the polymer. The polymer also displays a possible bactericidal effect on E. coli. PVAC is effective in inhibiting the growth of all the bacteria used in the experiments and adheres directly to them. PVAC displays a clear inhibitory effect on S. aureus’ growth, with lower absorbance readings acquired with mounting concentrations of the polymer. S. aureus also displays a possible tolerance to the polymer. S. epidermidis shows a marked response to PVAC, with growth being inhibited by mounting concentrations of the polymer. It also shows a similar trend response compared to S.
aureus, which is most likely explained by the two bacterium’s many shared properties. P. aeruginosa displays a more aggressive growth at lower concentrations of PVAC compared to
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