Paste This dissertation was to asses if Staphylococcus. epidermidis and strain ATCC 35984 Staphylococcus. epidermidis had a minimum inhibitory concentration (MIC) and if that MIC could be increased to determine the possibility of susceptibility. The main body of this dissertation discusses problems with increasing resistance, how that resistance occurred and incorrect use of disinfectants that consequently lead to susceptibility and resistance. From the experiments completed it was clear that the bacteria had MICs and could be grown in sub lethal was in fact viable. The results also identified that those MICs could be increased when the experiment was completed with bacteria previously grown in those sub lethal concentrations.
INDEX
Abstract 5
1 Introduction 6
1.1 Staphylococcus. epidermidis 6
1.2 Understanding the Potential Problems 7
1.3 Resistance, Acquired or Intrinsic 8
1.4 Disinfectants 9
1.5 Minimum Inhibitory Concentration 11
1.6 Objectives of Project
1.7 Hypothesis 11
2 Method 12
2.1 Nutrient Agar 12
2.2 Nutrient Broth 12
2.3 Serial Dilutions 13
2.4 Mueller Hinton Broth 13
2.5 Saline Solution 13
2.6 MIC Procedure 14
2.7 15
2.8 Exact Range of susceptibility
2.9 15
3. Results 16
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3. Discussion
4.1 Discussion of Results
4.2 Significance
4.3 Limitations
4.4 Future Works
4.5 Conclusion
Acknowledgments
Firstly, I would like to thank my supervisor Dr. Philip Wood for excepting me as one of his dissertation students, for his encouragement as much was needed and his guidance and support and helping me focus when all else failed. I would also like to thank Mary Wood for enduring my constant questions and her help in finding my confidence in the lab, along with her general direction concerning laboratory procedures.
I would also like to thank Dr. Paul Kowabnik of St. Helens college, science section for everything he has done for me over the past 3 years and getting me this far, I am sure without his help and support I would not have completed the foundation degree and would defiantly not be able to have completed this dissertation.
Lastly but definitely not least, I would like to thank every one of my family, as without their help and encouragement this would not have been possible.
Abbreviations
1
Introduction
This dissertation is to determine if Gram Positive Staphylococcus. epidermidis a normal commensal of the natural human flora, and Strain ATCC 35984 Staphylococcus. epidermidis (biofilm forming) both potential pathogens have or can develop a resistance/susceptibility to disinfectant, in particular Dettol surface spray.
1.1
Staphylococcus. epidermidis
The definition of pathogenic is the ability of a microorganism to cause disease in a healthy individual, the virulence of a pathogen is determined by structural features, biochemical processes and the genetics of an organism and how they interact with a host (Thomas & Elkinton, 2004).
Staphylococcus. epidermidis is a common bacteria of the natural human flora (90%) it is a Gram positive non-motile bacteria. Currently it is one of the most common causes of healthcare acquired infections (HCAIs), it also has the ability to form adherent biofilms. Biofilms are the accumulation of a cluster of cells on many types of surface including living tissue and medical devices either inside or outside the body, that cannot be removed by regular mechanical cleaning. This cluster of cells is usually enclosed in a matrix of primarily polysaccharide material (Donlan, 2002).
The virulence of S. epidermidis is increased by the presence of biofilms, the cells in the biofilms are generally more tolerant to antibiotics than single cells leading to a problem in treatment of infections associated with biofilms (Cerca et al., 2012).
The ATCC strain used in this dissertation are specially developed by the company ATCC, they offer a range of isolates that clinically relevant multi-drug resistant strains. That allow works to be carried out in vitro on a variety of factors that involve resistance, with one being the study of disinfectants and new antimicrobial products (Lgcstandards-atcc.org, 2016).
The ability to form biofilms is dependent on a surface and a water based medium, forming an ideal environment for the attachment and growth of the bacteria. The characteristics of the solid surface are important, Characklis et al noted that the extent to which the bacteria colonizes is dependent on the roughness of the surface, as this increases surface area increases. The chemical properties of a surface also influence the attachment, as hydrophobic nonpolar surfaces such as plastics increases chances, hydrophilic surfaces such as metal and glass does not (Fletcher and Loeb, 1979).
1.2
Understanding the Potential Problems
Antibiotic resistance is no longer a problem for the future it is occurring now, and having a very negative affect on the ability to treat even the simplest of infections. The extensive and excessive misuse of antibiotics in hospitals and community settings has aided in this crisis (Neu, 1992). With antibiotic resistance being a major problem, the use of other antimicrobials has increased significantly. Disinfectant and antiseptics are now continually being used not only in hospital setting but in the home and in the work place as well as personal use via hand gels (Levy, 2002). Disinfectants and antiseptics or at least the active ingredients in them have been used for hundreds of years. Russel et al, (1998) suggest that a low level resistance to biocides has been recognised in antibiotic resistant strains of Staphylococcus. aureus and Staphylococcus. epidermidis.
There is a possibility that the continued extensive use of disinfectants in particular may lead to a bacterial resistance or susceptibility. Bacteria is termed resistant when it remains viable or continues to grow when subjected to conditions that would normally effect it, either by killing(bactericidal) it or just allowing it to be inhibited (bacteriostatic) (Cloete, 2003). A study by Saperkin, Kovalishena and Blagonravova, (2013) stated that in 2011, 82% of bacteria tested from 40 hospitals showed a susceptibility to quaternary ammonium compounds (QACs), that are found in disinfectant’s used in hospitals and the home.
The word resistance when used with disinfectants would be deemed incorrect, as it is the dilutions of disinfectants that cause a change in MIC the word susceptibility would be better used.
1.3
Resistance, Acquired or Intrinsic
The resistant properties of bacteria can be divided into intrinsic where the bacteria have a natural ability to become resistant, and acquired which is either via mutation or acquisition of plasmids or transposons. Although most bacteria with intrinsic resistance are Gram negative, mycobacteria or spore forming. There are some staphylococci spp. that depending on conditions can also possess intrinsic resistance, over recent years, acquired resistance to certain types of biocides have been detected, mainly in staphylococci (McDonnell and Russell, 1999).
Acquired resistance in bacteria can occur via a mutation or the acquisition of genetic material in the form of plasmids or transposons, possibly the bacteria most commonly known for this is Staphylococcus. aureus. It was originally thought that plasmids played no role in the acquired resistance of disinfectants (Chopra ,1982) but there are reports linking the presence of plasmids in bacteria with increased tolerance to chlorhexidine and QACs.
Sidhu et al, (2002) suggested that widespread use of QACs has been shown to increase selective pressure that contributes to disinfectant resistance. Known QAC resistant genes also display resistance to the aminoglycoside antibiotic gentamicin, particularly in and are plasmid borne. It is thought that it is the qacA gene that shows resistance to benzalkonium and chlorhexidine and are located on plasmids, the qacC gene which also shows resistance to QACs and is also located on plasmids. (Mayer, 2001).
Zmantar et al (2011) conducted a study, and the MIC results showed that 74% of the isolates of staphylococci showed a resistant to quaternary ammonium compound in particular benzalkonium chloride, 56% of these BC-resistant staphylococcus isolates have at least one of the three resistant disinfectants genes (qac A, qac B and qac C).
Rouch et al, (1990) state that these genes qacA -qacG encode for efflux pumps, efflux pumps are transporter proteins that are involved in the transportation or removal of toxic substances and specifically with the qac gene QACs out of a cell membrane, they can be present in Gram positive and Gram negative bacteria. They can be responsible for both intrinsic and acquired resistance to antibiotics and as mentioned above QACs (Webber, 2002).
The implications of the continued use of disinfectants along with resistance is the continual removal of resident bacteria on the body, this is the first line of defence and if continually disrupted along with the resistance of bacteria to antimicrobials in general would have a huge impact on the fight against infectious disease.
1.4
Disinfectants
With the rapid increase of antibiotic resistance, the use of disinfectants and other antimicrobial products have become more widespread, saying this the knowledge people have of the mode of action of these products is very little in comparison with antibiotics. This along with increased use has led to speculation and concerns of the resistance in potentially pathogenic bacteria (McDonnell and Russel, 1999). Russell (1996) reported that Gram-negative bacteria are generally more resistant to antiseptics and disinfectants than Gram-positive bacteria.
Disinfection is usually a chemical agent used to control the growth of vegetative microorganism on living and inanimate objects (Angelillo et al, 1998). It is the active component in the disinfectant that determines the effectiveness of the product; Chloroxylenol and benzalkonium chloride (BC) perform this role in Dettol the UPAC name is Dimethylphenol (Pubchem.ncbi.nlm.nih.gov, 2015).
One of the active ingredients in Dettol is Chloroxylenol this is a low level disinfectant with a bactericidal effect, is a chlorinated phenolic compound that displays broad spectrum antimicrobial activity in Gram-positive and gram negative bacteria, according to Goddard and McCue (2001) this activity may be dependent on the formula used.
Phenol based disinfectants or their derivatives, work by injuring plasma membranes and inactivating cytoplasmic enzymes by forming unstable complexes. If the concentration of the disinfectant is high, it inhibits enzymes resulting in denaturation and resulting in a progressive leaking of the intracellular contents (Bruch, 1996).
A significant problem with disinfectants is that no single active ingredient will affect all microorganisms (Prescott, Harley & Klein, 2005).
Benzalkonium chloride (BC) (also known as alkyldimethylbenzalammonium chloride) is also an active ingredient in Dettol and is classified as a quaternary ammonium compound (QAC). QACs display a broad spectrum of antimicrobial activity and are amphoteric surfactants and are widely used in clinical and industrial setting. QACs, during the last decade have also become increasingly popular in domestic setting and the products that are used there (MacBain et al, 2004). Benzalkonium chloride in particular due to its broad-spectrum antimicrobial activity and its surfactant properties have made it the chosen ingredient in disinfectant cleansing formulations (Bloomfield, 2002).
Each QAC exhibits its own antimicrobial characteristics or mode of action. This can be displayed in five main ways. The main target in most QACs is the cytoplasmic membrane. Salton, (1968) originally hypothesised that a sequence of events would occur on exposure to the QACs.
1. Adsorption and penetration
2. Membrane disorganization via reaction of cytoplasmic membrane to QACs
3. Leakage of intracellular content
4. Degradation of proteins and nucleic acid
5. Cell wall lysis
The cell membrane has the ability to control which substances enter or leave the cell, therefore attacking the cell membrane disrupts the interactions that occur within the cell, which can lead to various causes of death such as loss of structure and/or function of the cell which will then result in the leakage/loss of material that is essential to the survival of the cell (CDC, 2008).
A study carried out by (Reichel et al., 2014), stated that there are discrepancies between studies with some stating that disinfectants particularly containing BC are generally effective against both Gram negative and Gram positive bacteria, and that others suggested that they are not effective against Gram negative bacteria.
Another identified problem is the contaminated disinfectant solutions, some strains of bacteria particularly biofilm forming have the ability to survive and grow in disinfectant solutions in particular benzalkonium chloride (Weber, Rutala and Sickbert-Bennett, 2007).
1.5
Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration (MIC) is the test that is widely used to determine the resistance of a bacterial strain to an antibiotic and other antimicrobial, there are two methods of MIC, the agar method sometimes known as the Kirby Bauer method and the broth method. The MIC is defined as the lowest concentration of antimicrobial were no growth occurs after overnight incubation (Andrews, 2001).
MIC results are used to determine susceptibilities of bacteria and also to measure the activity and susceptibility of new antimicrobial agents. Agar dilution or Kirby Bauer method involves the addition of different concentrations of the antimicrobial into a nutrient agar followed a bacterial suspension to the surface of the agar plate. For broth dilution, often completed in 96-well microtiter plate, bacteria are inoculated into a liquid growth medium along with different concentrations of an antimicrobial (Wiegand, Hilpert and Hancock, 2008).
1.6
Objectives of project
The aim of the dissertation was to ascertain if the Bacteria Staphylococcus. epidermidis and strain ATCC 35984 Staphylococcus. epidermidis.
1. Does use of antimicrobial cleaning products increase susceptibility in bacteria.?
2. Are colonies left behind after antimicrobial treatment viable, and can they be cultured and grown?
3. Does the ability to form biofilms change the effectiveness of the products used?
1.7
Hypothesis
Each strain of S. epidermidis will have an MIC at sub lethal concentrations to Dettol surface spray, and on the assumption that the bacteria will be viable when removed from those concentrations, the MIC will be increased.
2
Method
Every step of the method was completed following aseptic techniques. A range of disinfectants along with two strains of Staphylococcus. epidermidis were chosen and a serial dilution was completed following on each to identify the most effective product. Dettol surface spray, Dettol, and Zoflora was used.
2.1
Nutrient agar
28 g of powder were weighed and placed in a Duran bottle containing 1 litre of deionised water (to the shoulder) allowed to soak for 10 minutes and was swirled to dissolve. It was then sterilised in the autoclave for 15 mins at 121 ̊ C then cooled in a water bath to 47 ̊ C then poured into plates.
When correct temperature 84 plates of nutrient agar was poured and left overnight to cool and set.
2.2
Nutrient Broth
25g of powder were weighed and dispersed into Duran bottles containing 1 litre of deionised water, it was soaked for 10 minutes and swirled to mix. This was then dispensed into appropriate tubes or bottles and sterilised for 15 minutes at 121 ̊ C.
A broth of bacteria was made using 1-2 single colonies from a previously streaked plate labelled and incubated overnight at 37 ̊ C.
2.3 Serial dilutions
Using 7 bottles as per the number of serial dilutions, 8ml of distilled water was placed into each with 2 ml of a disinfectant in the first bottle, 2 ml was then removed from the first bottle and placed into the second bottle this was repeated up to the seventh bottle.
Plates were labelled accordingly and a pipette was used to place 0.1 μl of bacterial culture on each plate and spread to create a lawn of bacteria over the agar, a well was then made using a metal borer in the centre of each plate 7.5mm in size. 0 .1 μl of each dilution was placed in the well in each of seven plates and repeated twice, this was done for each disinfectant sealed then incubated overnight at 37 ̊ C. Results were then recorded by measuring the zones of inhibition using digital callipers around each well.
Minimum Inhibitory Concentration (MIC)
These were prepared 24hours before for sterilization:
2.4
Mueller Hinton Broth
A 1 litre batch of Mueller Hinton (MH) was prepared by weighing 21 g and placing into a Duran bottle with 1 litre of deionised water, it was allowed to soak for 10 minutes. Swirled and gently warmed to mix. The broth was then divided into 10 flats each containing 99ml, and then was sterilised in the autoclave for 15 minutes at 121 ̊ C.
2.5
Saline Solution
The saline solution was also prepared by dissolving 0.8g of powder into 60 ml of water to dissolve the powder then topped up to 100ml and sterilised.
Plates were streaked to single colonies of the two bacteria on nutrient agar and incubated overnight.
24 hours later, 5ml of the saline solution was removed and placed in sterilised bottles, the solution was prepared equal to a McFarland standard, (1-3 colony forming units were removed and placed in Saline)
1 ml was then removed from the solution and placed in to a flats bottle containing 99 ml of Mueller Hinton suspension, once prepared this has to be used within 15 minutes.
The appropriate microtiter plate was selected and labelled accordingly, 12rows down 8 across.
Fig 1(Microplate – 96 Well U Bottom, 2016)
2.6
MIC procedure
100 μl of neat disinfectant were placed using a pipette in the 1st row, then 50 μl of MH broth were placed in every well except the last row (12), 100 μl of MH broth were placed into each well on this row.
50 μl were removed from the first row and all wells containing the neat disinfectant and was placed in the row below containing 50 μl of MH broth and mixed. This happened for each subsequent row until the last which contained 100ml of MH broth, 50 μl of this was removed and discarded so that every row and well contains only 50 μl of solution.
1 ml was then removed from the saline solution and placed in to a flats bottle containing 99 ml of Mueller Hinton suspension, once prepared this has to be used within 15 minutes.
Each well was then inoculated with 50 μl of MH solution containing bacteria, the plate was then sealed with tape and incubated at 37 ̊c overnight. Results were observed and recorded and showed that there was a susceptibility to the concentrations of the disinfectant and growth occurred.
This procedure was completed for both bacteria.
2.7
Viability
Previously prepared MH broth (see section 2.4) and saline solution (see section 2.5) were used for the next experiment, after 1st experiment, using an inoculating ring, bacteria was removed from the MIC were growth occurred and streaked to single colonies on nutrient agar and incubated overnight, to ensure viable colonies.
Bacteria that had grown in the dilutions of disinfectant were removed and streaked onto a plate and incubated overnight to determine viability. The MIC procedure above (see section 2.6) was performed again,
2.8
Exact Range of susceptibility
Once MIC had been established a similar procedure was carried out to observe the range, previously prepared solutions and broths (see sections 2.3 and 2.4) were used and a microtiter plate (see fig 1) labelled accordingly
100 μl of neat disinfectant placed in each well on the first row, then 50 μl 0f MH broth was placed in each row and well below the first until row 6 (3.125 % dilution) when using strain ATCC35984 of Staphylococcus. Epidermidis. In the next 3 rows 45 μl of MH broth was placed in each well. The procedure (see section 2.5) from above was then followed until row 6 were only 5 μl was removed and placed into the 45 μl of MH broth until row 9, 50 μl of the MH suspension containing the bacteria was then added to each well and row.
The same was repeated up to row 7 (1.5626 %) when using normal strain of Staphylococcus. Epidermidis. The next 2 rows only contained 45 μl of MH broth then 50 μl of MH suspension containing the bacteria was placed in each row and well. Each plate was then taped securely and incubated overnight at 37 ̊c.
2.9
Section 2.7 was repeated according to section 2.6
3 Results
3.1
Table 1 The level of susceptibility of Staphylococcus. epidermidis to Dettol surface spray
Disinfectant
% A B C D E F G H
1
100% No No No No No No No No
2
50% No No No No No No No No
3
25% No No No No No No No No
4
12.5% No No No No No No No No
5
6.25% No No No No No No No No
6
3.125% No No No No No No No No
7
1.562 % No No No No No No No No
8
0.781% Growth No Growth Growth No Growth No No
9
0.390% Growth Growth Growth Growth Growth Growth Growth Growth
10
0.195% Growth Growth Growth Growth Growth Growth Growth Growth
11
10.097 % Growth Growth Growth Growth Growth Growth Growth Growth
12
0% Growth Growth Growth Growth Growth Growth Growth Growth
These results were obtained by carrying out the procedure described in section 2.6, they show the findings obtained from a single observation.
Table 1 above showed 12 wells down and 8 wells across, the 12 wells down contain the dilutions of the disinfectants. Row 1 being neat disinfectant and bacteria, no MH broth and row 12 having no disinfectant only MH broth and bacteria.
The experiment was to determine if the bacteria Staphylococcus. epidermidis possessed any susceptibility, to the disinfectant Dettol surface spray.
The lowest concentration that no growth occurred was row 7 (shown in red) so the MIC of Staphylococcus. epidermidis was 1.562% OR 1.562μg/cm3
Table 2 below was also to determine if the strain ATCC35984 Staphylococcus. epidermidis displayed any susceptibility to the Dettol surface spray and its active ingredients, and also to determine if the was a comparison between the two strains.
The lowest concentration that no growth occurred was row 6 (shown in red) so the MIC of strain ATCC35984 Staphylococcus. epidermidis is 3.125% or 3.125μg/cm3.
3.2 Table 2 The level of susceptibility of strain ATCC35984 Staphylococcus. epidermidis to Dettol surface spray.
Disinfectant
% A B C D E F G H
1
100% No No No No No No No No
2
50% No No No No No No No No
3
25% No No No No No No No No
4
12.5% No No No No No No No No
5
6.25% No No No No No No No No
6
3.125% No No No No No No No No
7
1.562 % No Growth No Growth Growth Growth Growth Growth
8
0.781% Growth Growth Growth Growth Growth Growth Growth Growth
9
0.390% Growth Growth Growth Growth Growth Growth Growth Growth
10
0.195% Growth Growth Growth Growth Growth Growth Growth Growth
11
0.097 % Growth Growth Growth Growth Growth Growth Growth Growth
12
0% Growth Growth Growth Growth Growth Growth Growth Growth
These results were obtained by carrying out the procedure described in section 2.6, they show the findings obtained from a single observation.
3.3
A sample was then removed from each microtiter plate. (Table 1 row 8 well A) and (Table 2 row 7 well B) were growth occurred, and streaked onto a plate and incubated overnight at 37 ̊ c, to determine viability of bacteria removed from the dilutions.
Fig 2 The colonies were viable as shown below and used to make the next set of solutions for the MIC.
The MIC for experiment 2 remained the same for each of the bacteria:
Staphylococcus. epidermidis was 1.562% OR 1.562μg/cm3
ATCC35984 Staphylococcus. epidermidis is 3.125% or 3.125μg/cm3.
3.4
Table 3 Displays a more focused Range at which susceptibility occurred Staphylococcus. epidermidis.
Disinfectant
% A B C D E F G H
1
2100% No No No No No No No No
2
50% No No No No No No No No
3
25% No No No No No No No No
4
12.5% No No No No No No No No
5
6.25% No No No No No No No No
6
3.125% No No No No No No No No
7
1.562 % Growth Growth Growth Growth Growth Growth Growth Growth
8
1.062% Growth Growth Growth Growth Growth Growth Growth Growth
9
0.562 % Growth Growth Growth Growth Growth Growth Growth Growth
These results were obtained by carrying out the procedure described in section 2.8, they show the findings obtained from a single observation.
Table 3 showed that from the point that MIC occurred 1.562% OR 1.562μg/cm3 in Table 1. 5μl were remove instead of 50μl, from the dilution 1.562%, 1.062% and then 0.562%. This shows a more concise concentration that susceptibility occurred.
It remained the same, so susceptibility for Staphylococcus. epidermidis occurred at 1.562% OR 1.562μg/cm3
3.3
Table 4 Displays a more focused range at which susceptibility occurred of strain ATCC35984 Staphylococcus. epidermidis.
Disinfectant
% A B C D E F G H
1
100% No No No No No No No No
2
50% No No No No No No No No
3
25% No No No No No No No No
4
12.5% No No No No No No No No
5
6.25% No No No No No No No No
6
3.125% No No No No No No No No
7
2.625% Growth Growth Growth Growth Growth Growth Growth Growth
8
2.125% Growth Growth Growth Growth Growth Growth Growth Growth
9
1.625% Growth Growth Growth Growth Growth Growth Growth Growth
These results were obtained by carrying out the procedure described in section 2.8, they show the findings obtained from a single observation.
Table 4 showed that from the point that MIC occurred 3.125% OR 3.125μg/cm3 in Table 2. 5μl were remove instead of 50μl, from the dilution 3.125%, 2.125% and then 1.625%. This shows a more concise concentration that susceptibility occurred.
Precise susceptibility occurred at 2.625% or 2.625μl/cm3 instead of the previously determined 3.125% or 3.125μg/cm3.
3.4 Table 5 Displays the Increase in the level of susceptibility of Staphylococcus. epidermidis to Dettol surface spray.
Disinfectant
% A B C D E F G H
1
2100% No No No No No No No No
2
50% No No No No No No No No
3
25% No No No No No No No No
4
12.5% No No No No No No No No
5
6.25% No No No No No No No No
6
3.125% No No No No No No No No
7
1.562 % Growth Growth Growth Growth Growth Growth Growth Growth
8
0.781% Growth Growth Growth Growth Growth Growth Growth Growth
9
0.390% Growth Growth Growth Growth Growth Growth Growth Growth
10
0.195% Growth Growth Growth Growth Growth Growth Growth Growth
11
10.097 % Growth Growth Growth Growth Growth Growth Growth Growth
12
0% Growth Growth Growth Growth Growth Growth Growth Growth
These results were obtained by carrying out the procedure described in section 2.7, they show the findings obtained from a single observation.
As shown in experiment 2, samples were then removed from each microtiter plate on the row where growth occurred, and streaked onto a plate and incubated overnight at 37 ̊ c, to determine viability of bacteria removed from the dilutions. (See Fig 2)
Experiment 2 was repeated again to try and increase the susceptibility of Staphylococcus. epidermidis.
Row 7 in red shows the MIC from experiment 1 Table 1.
The lowest concentration where no growth occurred was row 6 (shown in blue) Table 5 above, so the MIC was 3.125% or 3.125μl/cm3. Showing that an increase in susceptibility occurred.
Table 6 below, showed the Mic from experiment 1 Table 1 (in red) below
Lowest concentration where occurred no growth was row 4, Table 6 Below (in blue). The MIC was 12.5% or 12.5 μl/cm3. Again showing that there was an increase in susceptibility.
3.5
Table 6 Displays the increase in the level of susceptibility of strain ATCC35984 Staphylococcus. epidermidis to Dettol surface spray,
Disinfectant
% A B C D E F G H
1
100% No No No No No No No No
2
50% No No No No No No No No
3
25% No No No No No No No No
4
12.5% No No No No No No No No
5
6.25% No No Growth No Growth No No No
6
3.125% Growth Growth Growth Growth Growth Growth Growth Growth
7
1.562 % Growth Growth Growth Growth Growth Growth Growth Growth
8
0.781% Growth Growth Growth Growth Growth Growth Growth Growth
9
0.390% Growth Growth Growth Growth Growth Growth Growth Growth
10
0.195% Growth Growth Growth Growth Growth Growth Growth Growth
11
0.097 % Growth Growth Growth Growth Growth Growth Growth Growth
12
0% Growth Growth Growth Growth Growth Growth Growth Growth
These results were obtained by carrying out the procedure described in section 2.7, they show the findings obtained from a single observation.
3.5
Minimum inhibitory concentrations (MICs) are the lowest concentration of an antimicrobial that will inhibit the visible growth of a bacterium after overnight incubation (Andrews, 2001).
Table 7 Displays a summary of the MICs
Bacteria MIC
Experiment 1
Staphylococcus. Epidermidis Row 7
1.562 %
ATCC35984
Staphylococcus. Epidermidis
Row 6
3.125%
Experiment 2
Staphylococcus. Epidermidis Row 7
1.562 %
ATCC35984
Staphylococcus. Epidermidis Row 6
3.125%
Experiment 3
Staphylococcus. Epidermidis Row 7
1.562%
ATCC35984
Staphylococcus. Epidermidis Row 7
2.625%
Experiment 4
Staphylococcus. Epidermidis Row 6
3.125%
ATCC35984
Staphylococcus. Epidermidis Row 4
12.5%
The Table above displays the results of each experiment undertaken and displays that changes in the MIC were achieved under the conditions provided. Experiment 3 gives a more precise figure at which point growth occurred than just a 2-fold dilution.
3 Discussion
4.1 Discussion of Results
The aim of this dissertation was to determine if Staphylococcus. epidermidis, had a susceptibility to Dettol surface spray and the active ingredients, and if a susceptibility did occur was the bacteria that had grown in the concentrations viable, and also if the presence of biofilms in the strain ATCC 35984 Staphylococcus. epidermidis affected the susceptibility therefore the MIC.
Section 3 Table 1 showed that Staphylococcus. epidermidis MIC was 1.562 μl/mg3, when subjected to different concentrations of the disinfectant it displayed a susceptibility, growth occurred in dilutions containing 0.781% of disinfectant.
Table 2 showed that the strain ATCC 35984 Staphylococcus. epidermidis which has the ability to form biofilms, MIC was 3. 125 μl/mg3, when subjected to concentrations of the disinfectant and it also displayed a susceptibility, growth occurred in dilutions containing 1.562 % of disinfectant.
What Table 1 and 2 indicate and also stated by Raichel et al, (2014) is that incorrect use of the disinfectant, may lead to sub lethal conditions and the ability of that bacteria to grow in those conditions. This is demonstrated in Table 5 and 6 of section 3.
Section 3 Table 5 displayed that Staphylococcus. epidermidis MIC can be increased, when using bacteria previously exposed to sub lethal concentrations of the disinfectant. The MIC increased from 1.562μl/mg3 to 3.125 μl/mg3. Growth occurred in dilutions containing 1.562% of disinfectant.
Section 3 Table 6 displayed that strain ATCC 35984 Staphylococcus. epidermidis MIC can be increased, when using bacteria previously exposed to sub lethal concentrations of the disinfectant. The MIC increased from 3.125μl/mg3 to 12.5μl/mg3. Growth occurred in dilutions containing 6.25% of disinfectant.
MacBain et al, (2004) suggested along with results on Table 5 and Table 6 that incorrect use and sub lethal concentrations of disinfectant, directly affect the susceptibility of the bacteria, with strain ATCC35984 Staphylococcus. epidermidis displaying the most increase. The ability of that strain to form Biofilms affects the outcome of the susceptibility.
4.2 Significance of the study
This study was undertaken because as Neu, (1992) (see section 1.2) suggested the extensive use and misuse of antibiotic and disinfectants along with the increase in resistant strains of bacteria is a major worldwide problem. Levy, (2002) also determined that use of disinfectants previously only used in hospital settings are now being used regularly in the home and sometimes incorrectly.
Cloete (2003) suggested that bacteria may be defined as resistant when they are not susceptible to a concentration of disinfectant. Raichel et al, (2014) stated that as in this investigation, most other studies on resistance are often carried out with concentrations of disinfectants lower than those that are normally used, and to avoid sub lethal concentrations, recommended concentrations and usage must be adhered to, but this is not always the case. Instructions labelled on the bottle of Dettol surface spray states that the spray should be applied directly to surfaces with no need to remove, this would in affect be 100% concentration of that solution. Russell and McDonnell, (2000) stated that changing the concentration of a disinfectant effects the potency and that for the active ingredients found in Dettol surface spray to be effective the length of time of contact should be altered, for example QACs require the contact time to be doubled if the concentration is halved.
As study by Zmantar et al (2011) (see section 1.3) stated that Staphylococci spp. show a resistance to one of the active ingredients in Dettol surface spray, benzalkonium chloride and that many of these also possess 1 or more of the BC resistant genes, this in turn also results in a resistance to gentamicin which is an antibiotic of the aminoglycoside family. This family of bacteria is used to treat mainly Gram negative bacterial infection, but is also used for Staphylococcus infections so is classed as a broad spectrum antibiotic (Kotra et al, 2000).
This clearly shows that incorrect and extensive use of disinfectants has implications that supersede just basic disinfectant susceptibility, it can have direct effect on treatments of patients needing gentamicin. In addition to these problems enters the strains of bacteria that have the ability to form biofilms and the problems that occur directly because of this. Cerca et al., (2012) identifies an increase in virulence is attributed to these biofilms.
Bridier et al, (2011) stated that during a disinfection process, it is the limited penetration of active ingredients into a biofilm that may result in the low levels of exposure to the disinfectant in deeper areas of the biofilm. This then will allow the biofilm cells to develop a resistance to the sub lethal concentrations of the disinfectant.
Bridier et al, (2011) also stated that the adaptation of biofilm cell populations to disinfectants was first reported in Salmonella, it was noted that biofilm cells were better adapted to benzalkonium chloride than their planktonic cells after continuous exposure.
4.3 Limitations
The limitations of this study was that only one type of bacteria was used, most other studies show a comparison between gram positive and gram negative bacteria
4.4 Future Work
To further develop and enhance this work, a range of other factors could be taken into consideration that could potentially effect the outcomes of the results. Certain physical and chemical influences occur that may enhance or decrease the ability of the active ingredients in the disinfectant formulations. Temperature in most disinfectants increases the disinfectant potential but there are some cases where the opposite occurs resulting in serious problems. Ph also has an effect with an increase causing an increase in QACs disinfectant potential among others, but with the opposite occurring for different types. The chances of being able to control these factors in the home would be difficult and further studies would attempt to take this into consideration as well as exert more control over these factors to determine any change in the results
Studies conducted by Mayer, (2001) and Zmantar et al (2011) state that resistance of staphylococci. spp to BC is mediated by the presence of certain qac genes that are located on plasmids and that this acquired mechanism of resistance is responsible for the increase of the multidrug efflux pumps. Rouch et al., (1990) suggest that these efflux pumps have the ability to pump QACs out of that particular cell. Further studies would include determination of the specific genes associated with these studies, the way in which the efflux mechanism occurs and perhaps ways to inhibit the action of the efflux pump.
4.5 Conclusion
With the increasing problems of resistance as discussed in section 1.2 and the extensive and misuse of disinfectant (see section 1.4). The sub lethal dilutions being used can and will continue to create problems with bacterial resistance to not only disinfectants as mainly discussed throughout this dissertation, but also with antibiotic resistant bacteria as discussed in section 1.3 with the reference to BC resistant Bacteria having the efflux qac genes that also encode for gentamicin resistance. The implications of this along with the strains of bacteria that can produce biofilms is troubling, and more extensive research is required to determine specific reasons for disinfectant susceptibility.
National Guidelines should be made regarding the use of disinfectants in the home with the emphasis on incorrect usage being a major factor in resistance and susceptibility.
It is also abundantly clear that the problems created and that arise from all types of resistance are still continuing to be a major problem, as demonstrated by the emergence of CPE. Carbapenemase-producing Enterobacteriaceae (CPE) are a group of bacteria that have develop a resistance to carbapenems which are a group of antibiotics that are usually used as a last resort when all other attempts have failed, these bacteria produce the enzyme Carbapenemase which in effect breaks down the antibiotic rendering it ineffective. The Nhs state that if this became wide spread the problems that would arise would be effect that of pre-antibiotic era (Nhs.uk, 2014).
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