Media may be dispensed in bottles with rubber-lined metal screw caps, polypropylene caps or tubes plugged with non-absorbent cotton wool. Small bottles may be sterilized with their caps screwed down firmly, but not packed tightly in the baskets. The larger bottles should have their caps loosened before heating and subsequently tightened for storage. A useful method of distinguishing between types of media is the use of colored non-absorbent cotton wool plugs in tubes or, in the case of bottles, colored caps or beads may be used.
Cotton wool plugs must not be so tight that air is excluded. On the other hand, they should be firm enough to allow one to raise each tube by its plug. Media for current use should always be stored in a dust-free cupboard or in a cool, moist atmosphere. For longer periods store at 4-6 degree Celsius. Each batch should be tested before use and labeled with a batch number. The shelf-life of media varies considerably. Poured plates will be used within a few days of preparation but agar slopes should be checked to ensure that moisture is still present. Supplies of the basal media should be such that each batch is renewed within 3 months of manufacture.
In normal bacteriology, the most important requirement of a culture medium is the ability to allow detectable growth from a minute inoculum, possibly a single organism, within the shortest period of incubation. The medium which forms the basis of the majority of culture media is referred to as nutrient broth. It is designed to support the growth of a wide range of bacteria and consists in the main of meat extracts, peptone and mineral salts in clear solution at a pH of approximately 7.4.
Meat extracts supply a wide range of growth factors including mineral salts and amino acids. Peptone is a source of nitrogen obtained by the peptic digestion of protein to give a heat-stable mixture of proteoses, peptones, polypeptides and amino acids. Several varieties of peptone are commercially available, two of the most widely used types being bacteriological and proteose peptones. The latter is especially rich in amino acids such as tryptophan, which is necessary for satisfactory indole production. The mineral salts essential to growth consist of sulphates, chlorides and phosphates of the acid radicals and calcium, phosphate and sodium among the bases.
Resistance to one or more antibiotics can be transmitted from one bacterial strain to a related but previously sensitive strain by bacteriophage transduction. It is not clear yet whether this mechanism is an important source of therapeutic difficulties. Transferable or infective resistance presents a threat to antibiotic control of all infectious diseases. It is important for the following reasons:
patients who are distributing these strains into their environments; always giving anti-bacterial drugs in adequate doses; and using drug combinations when appropriate. Another approach to the problem that has had some successes is to introduce an antibiotic policy for a hospital or area. This usually involves designating certain antibiotics as available for general use but withdrawing others from circulation or permitting their use only on rare and special occasions. If all goes well (and in particular, if all relevant clinicians abide by the policy), the incidence of strains resistant to the reserved antibiotics falls considerably in the months following institution of the policy, and in due course it may be judged right to reintroduce these drugs for general use and to withdraw others.
An antibacterial drug kills or inhibits a bacterium by interfering with some vital process. Any one drug is effective against only some bacterial groups and species, and usually against only some strains of any given species. The remaining bacteria owe their resistance to:
(1) Impenetrability of their outer layers to the drug.
(2) Lack of any important metabolic process to which the drug has any relevance for example sulphonamide resistance due to possession of a dihydrofolic acid reductase which does not 'confuse' sulphonamides with PABA .
(3) Ability to destroy the drug or convert it into an inactive form for example penicillinases (β-lactamases), which open up the β-lactam rings of penicillins and to variable extents also of cephalosporins; or the acetyltransferases by means of which some bacteria can acetylate chloramphenicol. Since the bacteria that produce such enzymes are as a rule intrinsically sensitive to the drug, their resistance may not be manifested when small numbers of them are exposed to an adequate concentration of the drug without being given time to produce enzyme; this is particularly the case when the enzyme is inducible rather than constitutive.
Even when an antibacterial drug is first introduced, the existence of naturally resistant bacterial species and strains places limits on its usefulness. However, there can have been few pathogenic bacteria in circulation 30 years ago which could have survived the onslaught of our present-day armoury of drugs if these had all become available at once. Many of our modern therapeutic problems are due to the fact that in most cases the introduction of a new drug has been followed by proliferation of bacterial strains resistant to it. Staphylococcus aureus has been notably successful in keeping pace with new discoveries. In relation to penicillin it is thought that evolution of new strains has played a relatively small part, the increase in frequency of penicillin-resistant Staphylococcus aureus and other species is largely due to existing resistant strains as their more sensitive colleagues were eliminated. But with most other drugs sensitive strains give rise to rare resistant mutants; these normally have no particular survival value, but in the presence of an appropriate concentration of the drug in question they alone are able to multiply, giving rise to a new strain with increased drug resistance.
For a superficial infection of the skin or an accessible mucous surface it may be possible to apply the drug in high concentration directly to the lesion; but unfortunately such topical application, notably of the penicillins, is particularly liable to provoke a hypersensitivity reaction and so to deprive the patient of subsequent systemic use of the group of antibiotics in question. A different form of local application that gives less trouble and is sometimes indicated is direct injection into a body cavity:
Oral administration is possible only if the drug can be produced in a palatable form; if it survives the action of the gastric secretions or it can be protected from it in capsules that dissolve in the small intestine; if it does not provoke significant gastro-intestinal upset; and above all if it is reliably absorbed into the blood (unless its site of action is to be the bowel lumen) and passes through the liver without being inactivated. Sometimes an ester or other derivative, itself not an effective antibacterial drug, meets all of these requirements and is converted into the active form after absorption. Oral preparations have, in addition to their unsuitability for patients who are vomiting or cannot swallow, the disadvantage that their absorption may be unreliable in very ill patients, in whom it is particularly important to achieve good levels rapidly and consistently.
Some antibacterials, notably metronidazole, are well absorbed when given as rectal suppositories, provided that the patient does not have diarrhoea.
Parenteral that is non-alimentary administration is usually intramuscular or intravenous. For intramuscular injection it is necessary to produce a strong solution (so that the volume is tolerable) which is of physiological pH and which does not cause excessive pain, or damage the injected muscle; this is not possible for some antibiotics. Also, having been injected, the drug must be rapidly and reliably absorbed into the blood (unless it is deliberately given as a slow-release depot preparation, as is sometimes done with penicillin). Intravenous injection or infusion is in some ways the ideal way of getting a drug into the blood, but it is seldom the most practicable or convenient. Furthermore, some drugs are difficult to give repeatedly by this route because they cause local phlebitis and thrombosis and a consequent shortage of accessible veins.
Once in the blood-stream, drugs vary in their distribution to tissues and body fluids and in their renal handling, and therefore a high blood level is no guarantee of good tissue levels. Indeed, a high and sustained blood level could well be due to the drug's inability to get out of the blood! Part of this variability in tissue penetration is related to differences in binding to plasma proteins. Virtually all antibacterials are bound to some degree, but some very much more than others— even others in the same group. Bound drug is in general no longer active against bacteria, so two drugs may give comparable total blood levels but with one mostly bound and inactive and the other mostly free and active. There is a reversible equilibrium between bound and unbound drug, but it may be only the unbound portion that is free to diffuse out of the circulation—and even then its troubles are by no means over, as it may bind to tissue proteins.
Rapid excretion of an antibacterial drug by the kidneys may make it highly suitable for dealing with urinary tract infections, provided that it is not, like chloramphenicol, excreted mainly in an inactive form. Such rapid excretion also necessarily means that blood and tissue levels are not well sustained—which may be an advantage, as we shall see below, under. Conversely, persistent high levels will be achieved if a drug that is not excreted in bile or metabolized in the liver or elsewhere is only slowly excreted by the kidneys— either because of its nature or because of renal failure.
Antibacterial drugs also vary widely in their ability to pass into body fluids other than urine. For example, sulphadiazine or chloramphenicol levels in cerebrospinal fluid are usually 40-80% of the prevailing blood levels. On the other hand, only traces of the penicillins or of streptomycin reach the cerebrospinal fluid from the blood if the meninges are healthy, though much larger amounts go through and much higher cerebrospinal fluid levels are achieved when the meninges are inflamed. Similarly, when ampicillin is used in treatment of suppurative chronic bronchitis it may pass fairly readily into the sputum at first, but as the infection is brought under control and the sputum ceases to be purulent its ampicillin content falls sharply. However, penetration of the closely related drug amoxycillin into sputum is far less dependent upon local inflammation. Penicillin given to pregnant women may reach much higher concentrations in the liquor amnii than in the maternal blood; yet streptomycin, the tetracyclines and chloramphenicol hardly penetrate to the liquor at all. These examples indicate something of the complexity of a subject which is of great clinical importance but is far from being thoroughly understood.
Ampicillin given parenterally may be preferred because of its low toxicity although this hazard is very rare with chloramphenicol given for 4 to 5 days only. In cases of haemophilus meningitis that do not respond quickly to chemotherapy with a single drug, streptomycin or sulphadiazine (or sulphadimidine) may be additionally prescribed. The chemotherapy of chronic bronchitis is concerned with:-
(a) Control of the chronic infection.
(b) Treatment of acute exacerbations.
For the former, only cases with purulent sputum will respond and for them, ampicillin or trimethoprim— sulphamethoxazole (cotrimoxazole) may be preferred to tetracycline because of their two-step bactericidal activity. Opinions differ as to whether one of these drugs should be given continuously through the winter months or intermittently, as indicated by sputum purulence or clinical relapse.
There is evidence of an increasing incidence of tetracycline-resistant pneumococci from cases of acute and chronic respiratory infections.For the acute exacerbation, in which haemophili or pneumococci, or both, may be implicated, treatment is urgent and parenteral injections of combined penicillin and streptomycin or of ampicillin for 5 to 7 days are indicated.
The skin may be infected by mammalian tubercle bacilli as in the lesions acquired by pathologists and butchers, or the ulceration of lips, external genitalia, or anus from tuberculosis of the associated organs ; by attenuated mammalian tubercle bacilli in lupus vulgaris ; by the artificially attenuated Mycobacteria boris of Calmette and Guerin from the site of a BCG vaccination; by Mycobacteria leprae; and by Mycobacteria fortuitum to produce a small abscess.
Moreover, chronic skin ulcers may be colonized by commensal mycobacteria which can be isolated when appropriate methods are used. There are two species of mycobacteria — Mycobacteria ulcerans and Mycobacteria marinum—which are exclusively skin pathogens. They remain strictly localized, multiplying only in the cool, super-ficial tissues, and they give rise to chronic skin ulcers. The regional lymph nodes are not enlarged and there is no systemic disturbance.
Infection with Mycobacteria ulcerans occurs in Victoria and Queensland, Australia, in Uganda (Buruli ulcer), the Congo, Nigeria, Malaya and Mexico; and foci of infection, generally near rivers and lakes, are probably widely distributed through-out the tropics. Although it is apparently a strictly human parasite, there is as yet no evidence of its spread from man to man ; nor of carriage by insects although biting flies are prevalent in areas where infection with Mycobacteria Ulcerans is found. The organism may be introduced into the skin of an exposed part, usually an arm or leg, by some slight injury such as the prick of a thorn or an insect bite. In the course of a few weeks an area of induration develops which breaks down, and the ulceration spreads slowly under the skin and into the deeper tissues.
Some of which may be opportunistic pathogens. are usually called diphtheroids. These include:-
Some of these diphtheroids are short and ovoid in shape with a tendency to show polar staining with methylene blue but devoid of volutin granules as demonstrated by selective stains; other diphtheroids which may be present in respiratory secretions or purulent discharges from septic wounds, middle ear infections, etc., resemble Corynebacterium diphtheriae in their morphology and staining reactions, but usually ferment sucrose, and are non-toxigenic.
The corynebacteria are sensitive to penicillin (MIC. 0.004 to 0.02 units/m1) and to other antibiotics active against Gram-positive bacteria. The most useful of these is erythromycin, which may be preferred to penicillin for eliminating diphtheria bacilli from the throat, particularly in treatment of persistent carriers.
Antibiotic therapy can be a useful adjunct to, but not a substitute for, antitoxin in the treatment of clinical diphtheria, since it helps to eliminate the organism but has no effect on preformed toxin which rapidly diffuses from the local lesion and, unless neutralized by antitoxin, soon becomes irreversibly bound to vulnerable tissue cells.
This is why antitoxin must be given as early as possible to any patient with suspected diphtheria as the fatality rate is directly related to the period of delay before giving antitoxin, rising from nil to 20 per cent between the onset and day 5 of the infection: the average case-fatality rate is 5 to 7 per cent.
Has a vast experience in medical bacteriology