SCREENING FOR ACTIVITYCOMMERCIAL PRODUCTIONASSAY AND TESTING METHODSANTIBIOTIC PRODUCTION
SCREENING FOR ACTIVITYsimple, rapid methods have been developed forscreening microorganisms for antibiotic-producingabilitysoil samples are commonly employed in thescreen since they are a rich source of antibiotic-producing organismsapproximately 85% are produced byactinomycetes, 11% by fungi and 4.5% by bacteriaantibiotics currently used in therapy are producedby surprisingly few groups of distantly relatedorganisms
SCREENING FOR ACTIVITY Soil samples are treated with cyclohexamide and phenol (1:10dilution) to inhibit the growth of interfering bacteria and fungibut not affect actinomycetes varying dilutions of the treated soil sample are streaked on agarplates containing medium which will support the growth ofactinomycetes incubate for 3 to 7 days at 25° to 30°C then examine the platesfor characteristic colonies of actinomycetes transfer onto fresh medium selected colonies and incubate againto allow growth plugs are cut from the colonies which include not only theorganism, but also the underlying agar (if the organism producesan antibiotic, it should diffuse into the agar medium) the plugs are placed on an agar plate which has been seeded witha test organism that will give an indication of the potentialusefulness of the antibiotic.
SCREENING FOR ACTIVITY example, activity against Gram-positive bacteriacan be determined with Staphylococcus aureus orBacillus subtilis activity against Gram-negative bacteria withEscherichia coli or Salmonella typhiactivity against fungi with Neurospora crassathe test plates are incubated under conditionsappropriate for maximal growth of the testorganism, and if after incubation there is a clearzone around the plug of the actinomycete, it canbe assumed that an antibiotic was present
SCREENING FOR ACTIVITY determine whether the chemical substance whichproduced the inhibition is a new antibiotic or a knowncompound by using bioautographythis assay employs paper chromatography or thin-layerchromatography and a biological assaythe antibiotic is first detected using chromatography bycomparing its Rf value to known substancesthe developed chromatogram is then placed on anagar medium which has been seeded with anappropriate test organismclear zones on the agar due to inhibition of growth ofthe test organism indicate the position of theantibiotics on the chromatogram.
SCREENING FOR ACTIVITYafter it has been established that a microorganism has beenisolated which produces a new antibiotic, quantitativeassays are employed to monitor the antibiotic titer the two most commonly employed assays are theturbidimetric (tube dilution) assay and the plate (agardiffusion) assay In the turbidimetric assay, the test organism is grown in testtubes containing different concentrations of the antibioticClear tubes indicate a higher antibiotic concentration thanturbid tubes, and the lowest concentration of antibiotic thatcompletely prevents the appearance of turbidity is knownas the minimum inhibitory concentration (MIC)
SCREENING FOR ACTIVITYIn the plate assay, filter paper discs areimpregnated with solutions of antibiotic ofvarying concentration, allowed to dry, placed onagar medium seeded with an appropriate testorganism, and incubated.the size of the clear zone of growth inhibitionaround the filter paper disc is related to theconcentration of antibiotic.
PRODUCTIONRaw Materials the fermentation broth is an aqueous solution made up ofall of the ingredients necessary for the proliferation of themicroorganisms.contains a carbon source like molasses, or soy meal, both ofwhich are made up of lactose and glucose sugars as a foodsource for the organismsnitrogen is another necessary compound in the metaboliccycles of the organisms (an ammonia salt is typically used)trace elements are needed for the proper growth of theantibiotic-producing organisms like phosphorus, sulfur,magnesium, zinc, iron, and copper introduced throughwater soluble saltsto prevent foaming during fermentation, anti-foamingagents such as lard oil, octadecanol, and silicones are used.
MANUFACTURE the desired antibiotic-producing organism mustbe isolated and its numbers must be increased bymany timesa starter culture from a sample of previouslyisolated, cold-stored organisms is created in the lab.a sample of the organism is transferred to an agar-containing platethe initial culture is then put into shake flasks alongwith food and other nutrients necessary for growthcreating suspensions, which can be transferred toseed tanks for further growth.
MANUFACTUREthe seed tanks are designed to provide an idealenvironment for growing microorganisms, including warmwater and carbohydrate foods like lactose or glucose sugarsadditionally, they contain other necessary carbon sources,such as acetic acid, alcohols, or hydrocarbons, and nitrogensources like ammonia saltsgrowth factors like vitamins, amino acids, and minornutrients round out the composition of the seed tankcontentsseed tanks are equipped with mixers, which keep thegrowth medium moving, and a pump to deliver sterilized,filtered airafter about 24-28 hours, the material in the seed tanks istransferred to the primary fermentation tanks.
MANUFACTUREthe fermentation is filled with the same growth mediafound in the seed tank and also provides anenvironment inductive to growththe microorganisms are allowed to grow and multiplywhere they excrete large quantities of the desiredantibioticthe tanks are cooled to keep the temperature between73-81° F (23-27.2 ° C)it is constantly agitated, and a continuous stream ofsterilized air is pumped into itsince pH control is vital for optimal growth, acids orbases are added to the tank as necessary.
MANUFACTURE the growth phase of the organism is termed the trophophaseand the antibiotic production phase is termed the idiophase during the growth phase, the culture becomes thick due to theformation of aggregates of fungal cells called mycelium growth lasts from the beginning of the culture period toapproximately 1 day later (0 to 24 hours) during the growth phase, glucose rather than lactose ispreferentially utilized, since it can be used directly as a source ofcarbon in the growth process, ammonia is liberated by deamination ofamino acids which raises the pH of the medium to 7.0, theoptimum pH for stability, and buffers in the medium maintain thepH close to neutrality
MANUFACTURE some additives may increase antibiotic production through anenzyme induction effect example, the addition during the trophophase, of methionine toa cephalosporin C fermentation stimulates the production of theantibiotic lysine in the culture medium will inhibit antibiotic production another important approach for increasing yield of antibiotic isthrough mutation and strain selection. mutation induced by exposing the parent strain to ultravioletlight, x-rays, or various mutagenic chemicals such as nitrogenmustard and analogs of purines and pyrimidines in the case of induced mutations, lethal levels of the mutagen areadjusted so that approximately 90 to 99% of the cells of theorganism are killed mutants which produce a higher yield of antibiotic are selectedfrom the surviving cells (eg. Penicillin)
MANUFACTUREafter three to five days, the maximum amount ofantibiotic will have been produced and the isolationprocess can begin the fermentation broth is processed by variouspurification methods, example, for antibioticcompounds that are water soluble, an ion-exchangemethod may be used for purificationthe dissolved antibiotic is then recovered using variousorganic chemical meansthe manufacturer is typically left with a purifiedpowdered form of the antibiotic, which can be furtherrefined into different product types
MANUFACTUREthe fundamental approaches which are usuallyconsidered are selective precipitation, selectiveadsorption, or selective extraction with animmiscible solventthe chemical characteristics of various antibioticsand their accompanying metabolites govern themanipulations which will be effective in any givensituation
manufactureonce the crude antibiotic has been recovered fromthe nutrient broth, it is subjected tochromatography, recrystallization, or otherstandard manipulations to effect an appropriatedegree of purificationattainment of chemical purity is usually consideredimpractical and unnecessary for therapeuticpurposesextraneous metabolites such as foreign proteinswhich would cause undesirable side effects areroutinely excluded during purification,
QUALITY CONTROLquality control is of utmost importance in the production ofantibioticsduring manufacturing, the quality of all the compounds ischecked on a regular basisof particular importance are frequent checks of thecondition of the microorganism culture during fermentationsuch as physical and chemical properties of the finishedproduct are checked such as pH, melting point, andmoisture content.In the United States, antibiotic production is highlyregulated by the Food and Drug Administration (FDA)the FDA requires that for certain antibiotics each batch mustbe checked by them for effectiveness and purity. Only afterthey have certified the batch can it be sold for generalconsumption.
ASSAY AND TESTINGMETHODSANTIMICROBIAL SUSCEPTIBILITY TEST in microbiological assays, the response of a growingpopulation of microorganisms to the antimicrobial agent ismeasured 1. Urease assay. When Proteus mirabilis grows in a urea-containing medium, it hydrolyzes the urea to ammonia andconsequently raises the pH of the medium. This production ofurease is inhibited by aminoglycoside antibiotics. In practice, itis difficult to obtain reliable results by this method. 2. Luciferase assay. In this technique, firefly luciferase isused to measure small amounts of ATP in bacterial culture,ATP levels being reduced by the inhibitory action ofaminoglycoside antibiotics. This method may find moreapplication in the future as more active and reliable luciferasepreparations become available.
ASSAY AND TESTINGMETHODSRADIOIMMUNOASSAY OF ANTIBIOTICSRIA utilizes that fact that specific antibody will bindwith a specific antigenin the case of RIA procedures for antimicrobial assay, ithas been found preferable to label the antigen, whichis the antimicrobial agent. A variety of substances (14C, 131I, 3H, 125I) can be used tolabel the antigen. 125Iodine, however, is morecommonly used than either 14C or 3H because it is agamma emitter rather than a beta emitter, thuseliminating the need for liquid scillant and ascintillation counter. In addition, 125I is more popular than 131I because of itslonger half-life and greater isotopic abundance.
ASSAY AND TESTINGMETHODSFLUOROIMMUNOASSAY OF ANTIBIOTICSAminoglycosides may be easily labeled withfluorescein by direct reaction with eitherfluorescein isothiocyanate (FTIC) ordichlorotriazinylaminofluorescein (DTAF)the resulting tracers have excellent shelf-lives andhave been used in most FIA methods.
ASSAY AND TESTINGMETHODSHPLC FOR THE QUANTIFICATION OFANTIMICROBIAL AGENTShigh-pressure liquid chromatography (HPLC) is anadvanced type of liquid chromatography, but ithas played an increasing role in quantitating drugsHPLC is able to assay many agents in a mixture ofantimicrobial agents including the presence ofbioactive metabolites, which may emerge and arenot detected by microbiological agar diffusion.
ASSAY AND TESTINGMETHODS Advantages of HPLC:1. Specificity. HPLC can routinely distinguish between antibiotics in amixture.2. Selectivity. HPLC distinguishes between the unchanged parentcompound and biotransformation products. HPLC differentiates, e.g.,between the original agent and derivatives with antibacterial activitywhich would, consequently, interfere with microbial assays. 3. Rapidity. HPLC renders assay results within minutes (15 to 30minutes) after necessary processing4. Reproducibility, precision. HPLC has higher quantitation accuracythan microbial (5-15%) and many chemical techniques.5. Sensitivity. HPLC can detect concentrations down to themilligram levels; i.e., low concentrations of the order obtained bymost routine assay procedures.
ASSAY AND TESTINGMETHODS6. Resolution. HPLC distinguishes between theparent compound and both its chemical similarbiotransformation-products and other, genericallyrelated compounds.7. Durable columns. The stationary phase of HPLClasts longer than, e.g., in GLC, and is more robust.8. Automation can be incorporated in the HPLCmachinery.9. Uniform methodology is applicable to the HPLCassay for several antibiotics.10. Economical. Low operational costs apply toHPLC.