Escherichia Coli Is More Resistant Towards Antimicrobial Agents Biology Essay

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Antimicrobial agents which include antiseptics, disinfectants and antibiotics are used to kill or inhibit the growth of bacteria. It has been acknowledged that gram negative bacteria Escherichia coli is more resistant towards antimicrobial agents compared to gram positive bacteria Staphylococcus aurous. This was assessed through two experiments which involved looking at the effectiveness of antiseptics and disinfectants on Staphylococcus aurous and Escherichia coli. The second experiment involved gram positive and gram negative antibiotic multidisc system M14 and M43 with Staphylococcus aurous and Escherichia coli. The zone of inhibition of the Staphylococcus aurous and Escherichia coli was measured for the different antimicrobials.

In the 19th century, surgery was unsafe and dangerous, patients were at high risk of infection, due to surgery not being carried out under aseptic conditions. Aseptic condition is referred to the absence of microorganisms. Louis Pasteur a French scientist demonstrated that diseases were caused by invisible microbes.

It was acknowledged during the 19th century that sepsis infection was the reason behind the high mortality rate during surgery as almost half of the patients died even though the operation had been successful. Sepsis can be referred to as a systemic inflammatory response syndrome (SIRS) where the body is responding to an infection. It is a condition where the body is fighting against a severe infection which has been spread by the means of the bloodstream. The condition can cause wide spread inflammation as well as blood clotting.

Joseph Lister an English surgeon was inspired by Pasteur's work. Lister used Pasteur's germ theory of disease and applied it to surgery which was the start of modern antiseptic surgery. A solution called carbolic acid (phenol) was used by Lister to disinfect . The carbolic acid resulted in a 45% decrease in mortality of those patients being operated upon. The solution was sprayed around using a handheld sprayer in the operating room.

Microbial growth can be defined as the increase in the number of cells being produced rather than the size of the cell increasing, vast amount of microorganism growth occurs by binary fission.

Microorganisms can be divided into five groups: bacteria, fungi, helminths, protozoa, viruses. Bacteria are prokaryotes single celled organisms which contain both Deoxyribonucleic acid (DNA) and Ribonucleic acid RNA, although do not have a defined nucleus. Bacteria can be classified as cocci (spherical), bacilli (straight rod), spiral or curved rod. Gram stain is a procedure which is used widely in medical bacteriology, due to bacteria being colourless and very difficult to be seen by a light microscopy.

Sterilisation is the technique used to destroy and eliminate bacteria, an object or substance can either be sterile or non sterile. There are various ways in which sterilisation can take place which includes using chemicals, heat, radiation or physically removing the cells.

Gram stain allows you to determine whether bacteria is gram negative (Escherichia coli) or gram positive (Staphylococcus aurous), the procedure was established by a Danish Dr Hans Christian Gram. It also allows you to determine whether the bacterium is spherical or rod shaped. The cell wall of bacteria is made up of peptidoglycan, gram positive bacteria have thick cell walls whereas gram negative bacteria have thin cell walls with an outer phospholipid bilayer membrane. A crystal violet stain is used, if a blue colour appears it indicates that the bacterium is gram positive and has a thick cell wall as the crystal violet has been retained by the cells. Whereas, if a red colour appears it indicates that the bacterium is gram negative.

Antimicrobial agents are used to control microbial growths which include chemical preservatives, antiseptics and disinfectants as well as those drugs used in the treat infectious diseases from plants and animals. They are chemicals which inhibit or kill the growth of microorganisms. The antimicrobial agents that kill cells are called cidal agents whereas those that inhibit the growth of cells are called static agents.

Antiseptic is a chemical agent that is applied to living tissues to prevent infection, antiseptics destroy or inhibit the pathogens. Disinfectant is an agent which is normally a chemical that kills microorganisms but is unsuitable to be applied on living tissues. Disinfectants and antiseptics can be distinguished by assessing whether it is safe to be applied on mucous membranes, which can be influenced on how concentrated the compound is

An antibiotic is a drug which is used to slow down the growth of a bacteria or if not kill the bacteria. Antibiotics are classified as antimicrobials that are used to treat infection caused by microorganisms. Severe infections caused by bacteria have become a global problem in the 21st century as the infections have become resistance to the common antibiotics prescribed.

The first aim of the investigation is to determine how effective disinfectants or antiseptics with bacteria and whether there is any difference when using gram negative or gram positive bacterium. The second aim of the investigation is to determine the most effective antibiotic in gram negative and gram positive.

A culture of Staphylococcus aurous and Escherichia coli was grown in a nutrient broth. An inoculum of each culture was prepared aseptically by diluting 0.1 ml of culture into 9 ml of fresh nutrient broth, which was then shaken to ensure that the culture had thoroughly mixed.

Spread plates were prepared by spread plating 0.1 ml of inoculums over the surface of the nutrient agar. Two spread plates were prepared in this manner for each bacterium.

The bottom of each spread plate was labelled with the name of the bacterium. Eight products of antiseptics or disinfectants were obtained and labelled from 1-8.

The first spread plate of the bacterium was subdivided into four sections and labelled from 1 - 4 with a marker pen. The second spread plate was again subdivided into four sections and labelled 5 - 8 with a marker pen. In total four spread plates, two for each bacterium labelled from 1 - 8. The sections 1-8 refer to the 1-8 labelled bottles of antiseptics or disinfectants.

The tip of the forceps were sterilised by passing the tip through the flame of the Bunsen burner two to three times. The sterile filter paper disc was picked up in an aseptic technique with the sterile forceps and then dipped into the chosen antiseptic or disinfectant labelled 1. It was important that the excess antiseptic or disinfectant was drained off.

The sterile filter paper disc was placed into the centre of the section labelled 1 on the Staphylococcus aurous inoculated plate. The procedure was repeated for the same antiseptic or disinfectant labelled 1 for section 1 on the Escherichia coli inoculated plate.

The forceps were washed thoroughly with alcohol prior to the next step of the procedure.

Steps 5 and 6 were repeated for the seven antiseptics or disinfectants for the remaining 2 - 8 sections on both the Staphylococcus aurous inoculated plate and Escherichia coli inoculated plate.

Ensuring before a new antiseptic or disinfectant is used the tip of the forceps is washed thoroughly with alcohol to make it sterile, preventing contamination between the antiseptic or disinfectant.

The tip of the forceps was flamed so that it was sterile and then used to press down gently the sterile filter paper discs to ensure that there was contact with the nutrient agar.

When all four plates contained the eight soaked sterile filter paper discs of the antiseptics or disinfectants. Four soaked sterile filter paper discs on each plate of the bacterium.

The plates were sealed with parafilm, followed by the plate being inverted and then finally incubated at 37°C for 24 hours.

The plates were recovered after 24 hours and zones of inhibition of the bacterial growth were measured in millimetres (mm) by measuring the diameter.

As described in experiment 1 spread plates for Staphylococcus aurous and Escherichia coli were prepared.

Using aseptic technique the tip of the forceps were sterilised by passing the tip through the flame of the Bunsen burner two to three times.

The multidisc was carefully placed onto the lawn of Staphylococcus aurous and Escherichia coli on each of the bacterium plate.

There were two multidisc systems M14 and M43. The M14 is a gram negative multidisc system this was placed in the Escherichia coli plate.

The M43 is a gram positive multidisc system which was placed in the Staphylococcus aurous plate.

To ensure the tip of the forceps was sterile it was flamed two to three times. The forceps were then used to press down gently the M14 and M43 multidisc to ensure that there was contact with the nutrient agar.

The plates were sealed with parafilm, followed by the plate being inverted and then finally incubated at 37°C for 24 hours.

The plates were recovered after 24 hours and zones of inhibition of the bacterial growth were measured in millimetres (mm) by measuring the diameter.

Staphylococcus aurous and Escherichia coli are different types of microorganisms which is why the response to antiseptics, disinfectants and antibiotics varies. This is the result of the microorganism's cellular structure, composition and physiology being different. Intrinsic resistance can be demonstrated by gram negative bacteria Escherichia coli. (McDonnell, G Russell, A D, 1999)

Staphylococcus aurous produces invasive and toxin diseases, which are thought to cause disease. Escherichia coli assists in the adherence to mucosal cells, invasion into underling tissues or it can alter mucosal function. (Ingles, TJJ, 2007)

From experiment 1 (see table 1 & graph 1), the most effective disinfectant was the walk pine with a 34mm zone of inhibition of the Staphylococcus aurous bacterium. The most effective antiseptic for Staphylococcus aurous bacterium with a 24mm zone of inhibition was germolene. For the Escherichia coli bacterium, walk pine was the most effective disinfectant with a 22mm zone of inhibition. Savlon was the most effective antiseptic with an 18mm zone of inhibition for the Escherichia coli bacterium. Savlon's active ingredient is Chlorhexidine gluconate, its mechanism of action involves membrane disruption. (McDonnell, G Russell, A D, 1999)

The least effective disinfectant and antiseptic for Staphylococcus aurous were Mr Muscle and TCP. Dettol surface cleaner, Mr Muscle and TCP were the least effective disinfectants and antiseptic for Escherichia coli. From the eight disinfectants and antiseptics walk pine was the most effective towards Staphylococcus aurous and Escherichia coli bacteria. Therefore, disinfectants are the most effective at killing or inhibiting the growth of bacteria, due to the high concentrations of active ingredients used and toxicity compared to antiseptics.

Wilko pine disinfectant's active ingredient is non - ionic surfactant Quaternary ammonium compound (QAC). Surfactants are surface active agents which two regions hydrophilic (water loving) and hydrophobic (water hating). QAC's are used in disinfectants, antiseptics as well as hard surfaces and deodorisation, they are membrane active agents. (Hugo, W. B and M. Frier, 1969) QAC's have the ability to damage the outer membrane of gram negative bacteria, enabling them to promote their own uptake. (McDonnell, G Russell, A D, 1999)

QAC cetrimide demonstrated to have an effect on the proton motive force (PMF) in Staphylococcus aurous. QAC's are sporostatic as they inhibit the spores out growth however not the actual germination processes. (McDonnell, G Russell, A D, 1999)

A QAC product can induce disintegration as well as morphology changes of human hepatitis B virus (HBV), which results in the loss of infection. (Prince D L et al, 1993)

Mr Muscle contains additives that cause the disinfectant to become a dilute concentration of the active ingredient therefore less effective as a disinfectant, compared to the walk pine which it is more concentrated disinfectant.

Phenol is the active ingredient in germolene, phenols have membrane active properties which help contribute to their activity as a whole. Phenol induces leakage of intracellular components. It was demonstrated by Pulvertaft and Lumb that when low concentrations of phenols are used there is rapid lysis of growing cultures of Escherichia coli. (McDonnell, G Russell, A D, 1999)

Experiment 2 demonstrated that the most effective antibiotic on the M43 - Systemic Gram Positive Ring (see table 2 & graph 2) was Penicillin G (1 unit) with the largest zone of inhibition of 32mm of the gram positive Staphylococcus aurous bacterium. However, Trimethoprim (1.25mg) and Sulphamethoxazole (25mg) demonstrated resistance towards gram positive bacterium, another possibility for this outcome could be that there was insufficient concentration for the antibiotic to have an effect.

Trimethoprim works by blocking am important stage in the bacteria's system which involves new DNA being produced. As antibiotic individually Trimethoprim can kill bacteria but it is very slow process. This is the reason why it is combined with another antibiotic Sulphamethoxazole which kills bacteria much more efficient and rapid. The combination of the two antibiotics is also able to kill bacteria which are partly resistant to the antibiotics alone. (Vinay N. Reddy, M.D, 2008).

Within some zones of inhibition small colonies were present, where the bacterium is resistant towards the antibiotic.

For Penicillin G to be administered to a patient it is important to establish the toxicity levels of the antibiotic, to prevent a hypersensitive reaction and to ensure it is not harmful to the patient. Trimethoprim and Sulphamethoxazole have demonstrated to work synergistically against a number of bacterium. (Rein M F et al, 1980) Antibiotic synergism occurs when combined antibiotics have a greater effect that an antibiotic by itself. (Mayer G, 2010)

Cotrimoxazole is the most effective antibiotic amongst the M14 systemic gram negative ring (see table 3 & graph 3) with a 28mm zone of inhibition of the Escherichia coli gram negative bacterium. Cotrimoxazole is the combination of two antibiotics Trimethoprim and Sulphamethoxazole. Resistance towards the gram negative bacterium was observed by Ampicillin. Antibiotic resistant to the bacteria for various reasons which include the synthesis of enzymes resulting in the inactivation of the drug, inactivation of the drug or that the target site has been modified. (Medoff G et al, 1999)

Penicillin G and Ampicillin resistance mechanism involves the hydrolysis of β - lactam ring by the β - lactamase. (Medoff G et al, 1999)

The values for Staphylococcus aurous are much higher for zone of inhibition when compared to Escherichia coli through out the two experiments. This is due to the cell wall of bacteria is made up of peptidoglycan, gram positive bacteria have thick cell walls whereas gram negative bacteria have thin cell walls with an outer phospholipid bilayer membrane. The antimicrobials are more effective towards Staphylococcus aurous as there is less penetration required compared to Escherichia coli, as Escherichia coli has a cytoplasmic membrane which the antimicrobial has to cross therefore zone of inhibition much smaller. (Gregersen T, 2004)

From the above data collected it is clear that disinfectants are more effective than antiseptics towards Staphylococcus aurous and Escherichia coli. There are number of factors that should be taken into account which may influence the zone of inhibition for the antimicrobials. The factors include temperature of incubation, the mechanism by which the antimicrobial will use to kill or inhibit the bacteria. The concentration of the antimicrobial being administered. The molecular weight will influence how effectively the antimicrobial crosses the membrane alongside the thickness of the membrane

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