Removal, Inhibition and Destruction of Microorganisms

In this article, we will discuss about the removal, inhibition and destruction of microorganisms.

History:

When it was assumed that the com­municable diseases were caused by microorganisms, the methods of sterilisation, disinfection and sanita­tion were adopted in daily practice. In early nineteenth century, the hospitals were dirty buildings accommo­dating the patients on mats spread on the floor, over­crowded together, dead and dying in the same beds.

Surgeons wore black coat with linen thread wound around the button of his coat, which was used as suture material. His scalpel, kept attached to his breast pocket, was used periodically for the incision and was sharpened on the heel of his boot. The doctors washed their hands when they were soiled with pus and blood. The sleeve of the coat was rolled back to pre­vent from getting it spoiled.

Such unhygienic condi­tions prevailed in those days in the hospital. Then, during this 19th century came, Louis Pas­teur (1863) who proved that the microbes caused the diseases. Robert Koch (1865) described many mi­crobial diseases and isolated the causative microbes in pure culture.

Later, Lister (1867) advocated in sur­gery the use of dilute carbolic acid as disinfectant. In obstetric and gynecology practice, the hand washing before delivery and operation was made compulsory. The practice of antisepsis and disinfection started from 1867.

About 200 years ago Napoleon Bonaparte or­dered to serve better food for his armies. In order to obey his order, a French scientist, Nicholas Appert, developed a process which was called an “Appetizing” which involved steam, heat, and pres­sure — as in autoclaving.

This principle is even now adopted in the modern method of sterilisation in every hospital. Later Pasteur advocated a method to check spoilage of beer by subjecting it to a temperature of 50° – 60°C for a few minutes and named it “Pasteurization” which is used in our day-to-day practice, i.e., Pasteurization of milk. Koch sterilised the culture media by a method of intermittent heat at 100°C.Tyndall modified and perfected the method and called it as “Tyndallisation”.

In 1880, Pasteur constructed a miniature early autoclave similar to the modern pressure cooker. In 1890, for the first time in the world, an autoclave was installed in Rochester City Hospital, New York. It is well established that the microorganisms (both harmful and harmless) are ubiquitous in nature i.e., they exist everywhere.

The pathogenic (harmful) bacteria should be killed or eliminated completely — particularly in surgery, surgical environment, hos­pital and public health. This procedure can be accom­plished by the professional trained nurse by her skill­ful intelligent application of her nursing microbiol­ogy knowledge in the medical and nursing practice.

In bacteriological laboratory, mostly pure cultures grown on nutrient media are dealt with. There- fore, the disinfection and sterilisation methods advo­cated for pure cultures were followed in the diag­nostic laboratory, whereas, in nursing practice, the situ­ations are different unlike laboratory.

So the ingen­ious professional nurse has to deal with mixed cul­tures, e.g., resistant forms and vegetative forms of microorganisms living in tissues, exudates, discharges, faeces mucus, pus, blood, Therefore, the intelligent well-trained nurse must exercise her own judgement after evaluating each situation to adopt the best meth­ods to remove, disinfect, inhibit or destroy completely the pathogenic microorganisms.

The following are the best possible procedures:

1. Removal by:

(a) Passing the fluids through very fine filter which retains bacteria;

(b) High speed centrifugation.

2. Inhibition by:

(a) Low temperature (dry ice);

(b) Desiccation (drying process);

(c) Combination of low temperature and des­iccation;

(d) High osmotic pressure (brine);

(e) Microbiostatic chemicals and drugs

(i) Dyes — eosin, methylene blue, crys­tal violet;

(ii) Chemical agents — sulphonamide, antiseptics.

3. Destruction by:

(a) Heat — dry (hot air oven); moist (auto­clave);

(b) Chemical agents (disinfectants);

(c) Radiation (X-rays, ultraviolet);

(d) Mechanical agents (crushing).

Inhibition of Microorganisms: Antimicrobial Agents:

Antimicrobial agent is a chemical which inhib­its the growth and causes the death of organisms.

The agents can be grouped accordingly:

1. If the substance merely causes a cessation of growth of microorganism — which is reversed when the chemical is removed — it is called static agent, if the substance kills the bacteria, it is called cidal agent. A static agent may become cidal if the concentration is increased. The disinfectants have a cidal action, while chemotherapeutic agents are of­ten static at the concentration used.

2. Agents acting on bacteria are bacteriostatic or bactericidal, those acting on fungi are fungi static or fungicidal.

3. In practice, chemotherapeutic agents can be distinguished:

(a) Disinfectants are chemicals used to kill potentially infective organisms normally present on inanimate objects, surface, water etc. They are potentially toxic to man, when they come in direct contact with man.

(b) Antiseptics (mild disinfectants) are rela­tively non-toxic and non-irritant, antimi­crobial agents which may be used for the topical application on the body surface to kill or inhibit the pathogenic microorgan­isms.

(c) Chemotherapeutic agents are chemicals used to inhibit or kill bacteria already es­tablished in the body tissues and are used for the therapeutic purposes in the treat­ment of microbial infections.

Chemotherapeutic agents should act at a con­centration that can be tolerated by the tissues of the host and, therefore, they must have a selective toxic­ity for the microorganisms compared with the host cells. The most widely used chemotherapeutic agents are the antibiotics which are naturally occurring anti­microbial agents produced by the microorganisms. The most common groups of antibiotic producers are actinomycetes and fungi.

Sulphonamides:

The discovery of sulphonamides in 1935 by Domagk, a German chemist, began with the finding that the red dye, Prontosil, was capa­ble of causing inhibiting infection with Streptococ­cus pyogenes, Prontosil itself had no effect on strep­tococci in vitro, but to be effective, it has to be broken down in the animal to sulphonamide. Sulphonamide itself is effective in non-toxic doses. By substituting different organic groups (represented by R groups in the formula below).

For example, the addition of a pyridine group gave sulphapyridine, a thiazole gave sulphathiazole and so on. In this way, thousands of derivatives called sulphonamides were produced and many of them were found to be effective antimicro­bial agents. Because of the development of newer antibiotics, relatively few sulphonamides have found a practical therapeutic use. It was observed that yeast and meat extract had the property of overcoming the inhibitory effect of sulphonamides on bacteria in vitro.

The sulphonamide “antagonist” in the extracts was isolated and was shown to be para (p)-amino benzoic acid (PABA). It was also shown that PABA is an essential metabolite in any organism and was con­verted by them to dihydrofolic acid then to tetrahydrofolic acid, which acts as a co-factor in reac­tion leading to the synthesis of nucleic acids and pro­teins required for cell growth.

It was found that the sulphonamides inhibit the first stage of the synthesis of dihydrofolic acid from para-amino benzoic acid. The drug, trimethoprim, which was later discovered and introduced into the medicine at much later date, inhibits the conversion of dihydrofolic acid into tetrahydrofolic acid (Fig. 14.1).

Summary:

Since p-amino benzoic acid and sulphonamides have similar chemical structures (Fig. 14.2), there is competition between sulphonamide and PABA for the active site on the surface of the enzyme initiating the conversion of PABA to dihydrofolic acid. To inhibit the conversion of one molecule of PABA into folic acid, a large number of sulphonamides molecules are required in the competitive inhibition (Fig. 14.2).  

Antibiotics Inhibiting Cell Wall Synthesis:

Mucopeptide of the bacterial cell walls is re­sponsible for their mechanical strength. If the syn­thesis of mucopeptide is inhibited, while synthesis of other cell components continue, then the cell can be expected to be lysed rapidly in normal osmotic envi­ronment. Penicillin inhibits the conversion “of a com­plex nucleotide (uridine diphosphate UDP-N-acetyl muramic acid pentapeptide) into mucopeptide (Fig. 14.3)

 

Penicillin was discovered by Alexander Fleming in 1922.

Antibiotics Affecting Protein Synthesis:

A number of antibiotics (Streptomycin, Kanamycin, Tetracyclines, Erythromycin and Puromycin) are known to inhibit protein synthesis. Streptomycin, dis­covered by Waksman, was the first antibiotic to be introduced clinically after penicillin.

Antibiotics Affecting Nucleic Acid Synthesis:

Few antibiotics combine with and alter the func­tioning of nucleic acids though they are generally toxic for therapeutic use. Ribonucleic acid (RNA) syn­thesis is prevented by Actinomycin, because it forms a complex with double stranded Deoxyribonucleic acid (DNA), but not with single stranded DNA; how­ever, at higher concentrations, DNA synthesis is in­hibited. On the other hand, mitomycin links compli­mentary strands of DNA resulting into blockage in DNA synthesis.

Antibiotics Acting on the Cytoplasmic Membrane

Polymyxin acts on the cytoplasmic membrane as a cationic detergent binding to the membrane, as a result the semipermeable properties are lost and essential low molecular weight intermediates and coenzymes pass into the environment causing the death of bacteria. Polymyxin should be used with caution as it binds also with the membrane of the host cells.

Source of Antibiotics:

Unlike synthetic sulphonamides, most of the antibiotics are produced in the culture media during the growth of certain microorganisms (Streptomy­ces, Bacillus, Penicillium, Aspergillus). New antibiot­ics are found in plants and animals, terresterial and marine (Table 14.1).

Manufacture of Antibiotics:

The desired antibiotic producing organism is cultivated in large tank of suitable liquid medium for a predetermined length of time. The culture is then centrifuged and filtered to remove the microorganisms. The filtered fluid, containing the antibiotic is then subjected to purification and concentration process. These at length yield crystals of the pure antibiotics which are then tested for purity, sterility, then packed and distributed.

Classes of Antibiotics:

Antibiotics may be grouped as follows:

Group I:

(Penicillin, Streptomycin, Bacitracin, Poly­myxin B) can be used together, they are often synergistic; never antagonistic. For example, when penicillin and streptomycin combined they are valu­able in endocarditis. The combination is more effec­tive than the sum of both.

Group II:

(Tetracyclines, Chloramphenicol, Eryth­romycin, Carbomycin, Neomycin, Oleandromycin, Novobiocin) is neither antagonistic nor synergistic. This sometimes works well in combination with anti­biotics of first group. This second group is generally of the “broad-spectrum” type. There are many other broad-spectrum antibiotics. The usual response to antibiotic therapy and antibiotic resistance or drug fastness are two important properties of organisms.

Use of Antibiotics:

Some of the antibiotics can be used only by oral route; some in ointment only; others are suitable for intravenous or subcutaneous injection. Some are ef­fective chiefly against Gram-positive organisms and some against Gram-negative organisms. Others have a wide range of activity. They are known as broad- spectrum antibiotics.

Drug Resistance or Drug-Fastness:

Microorgan­isms often undergo genetic mutations. These muta­tions generally result in alterations in enzymic equip­ment in the mutant cells. If mutation occurs in few cells of a species of microorganisms (e.g., Staphylo­coccus aureus) altering the enzymatic equipment of these cells which are multiplying in a patient treated with penicillin.

These mutated few cells will produce penicillinase or beta lactamase enzyme acting on beta lactam ring of penicillin become resistant to penicil­lin and grow in presence of penicillin. They are called as antibiotic resistant or drug fast. There resistant cells may grow and finally predominate in spite of the drug.

The predomination is very dangerous to the patient and for everyone around him who may acquire the organisms (e.g., sulphonamide-resistant gonococci). The sufficient quantity of the chemotherapeutic drug in the blood stream affecting even the most resistant species of the infecting organism before it can start growing, can be maintained by using adequate doses. This blood level maintenance can stop the devel­opment of such drug-resistant microorganisms.

Blood Levels Maintenance:

The amounts of drug in the blood can be main­tained by repeated doses at proper intervals of time. If delayed, the patient has to combat a drug-fast in­fection and may succumb to the infection. Therefore, the nurse responsible for giving repeated doses of such drugs should be very careful that the patient receives the drug on time so that his blood levels may not decrease to dangerous low levels.