PHARMACEUTICAL MICROBIOLOGYPHARMACEUTICAL MICROBIOLOGY

JIGAR SHAHJIGAR SHAH

INSTITUTE OF PHARMACYINSTITUTE OF PHARMACY

NIRMA UNIVERSITYNIRMA UNIVERSITY

TAXONOMYTAXONOMY�� TaxonomyTaxonomy (Greek(Greek taxistaxis –– arrangementarrangement oror order,order, andand nomosnomos –– lawlaw oror

nemein)nemein) isis thethe sciencescience consistsconsists ofof threethree separateseparate butbut interrelatedinterrelated partsparts::

11)) Classification,Classification, 22)) Nomenclature,Nomenclature, 33)) IdentificationIdentification..

11)) ClassificationClassification::ArrangementArrangement ofof organismsorganisms intointo groupsgroups oror taxataxa basedbased ononmutualmutualsimilaritysimilarity ororevolutionaryevolutionaryrelatednessrelatedness..mutualmutualsimilaritysimilarity ororevolutionaryevolutionaryrelatednessrelatedness..

22)) NomenclatureNomenclature::ConcernedConcerned withwith thethe assignmentassignment ofof namesnames toto taxonomictaxonomicgroupsgroups inin agreementagreement withwith publishedpublished rulesrules..

33)) IdentificationIdentification:: ProcessProcess ofof determiningdetermining aa particularparticular isolateisolate whichwhichbelongsbelongs toto aa recognizedrecognized taxontaxon.. ItIt isis thethe practicalpractical approachapproach tototaxonomytaxonomy..

��It’s the scientific study of organisms with the ultimate object of It’s the scientific study of organisms with the ultimate object of characterizing and arranging them in an orderly manner.characterizing and arranging them in an orderly manner.

��Taxonomic Ranks:Taxonomic Ranks:

�� The most commonly used levels or ranks, in ascending order, are The most commonly used levels or ranks, in ascending order, are species, genera, families, orders, classes, phyla or divisions, and species, genera, families, orders, classes, phyla or divisions, and kingdoms.kingdoms.

�� Means places the m.organisms within a small, homogenous group Means places the m.organisms within a small, homogenous group �� Means places the m.organisms within a small, homogenous group Means places the m.organisms within a small, homogenous group which is itself a member of larger groups in a nonwhich is itself a member of larger groups in a non--overlapping overlapping hierarchical arrangement.hierarchical arrangement.

�� At each level or rank, microbial groups name with ending or suffixes At each level or rank, microbial groups name with ending or suffixes characteristic of the level. characteristic of the level.

RANKS EXAMPLESKingdom ProcaryotaeDivision (Phylum) TenericutesClass MollicutesOrder MycoplasmatalesFamily MycoplasmatalesGenus MycoplasmaSpecies M. pneumoniae

Major Characteristics Used in Taxonomy

�� Following characteristics are used in classifying and identifying Following characteristics are used in classifying and identifying m.organisms.m.organisms.

�� Morphological Characteristics:Morphological Characteristics:

�� Morphological features are easy to study and analyze.Morphological features are easy to study and analyze.

�� These are important because structural features depend on the expression These are important because structural features depend on the expression of many genes. of many genes.

�� They are usually genetically stable, and normally do not vary greatly They are usually genetically stable, and normally do not vary greatly with environmental changes.with environmental changes.

FeatureFeature Microbial GroupMicrobial Group

Cell Shape, size, cilia, flagella, color, colony All major groupsCell Shape, size, cilia, flagella, color, colony All major groups

Endospore shape & locationEndospore shape & location Endospore forming bacteriaEndospore forming bacteria

MotilityMotility Gliding bacteria, spirochetesGliding bacteria, spirochetes

Staining behaviorStaining behavior Bacteria, some fungiBacteria, some fungi

Physiological & Metabolic CharacteristicsPhysiological & Metabolic Characteristics

�� Related to the nature and activity of microbial enzymes and transport Related to the nature and activity of microbial enzymes and transport proteins.proteins.

�� Since proteins are gene products, analysis of these characteristics Since proteins are gene products, analysis of these characteristics provides an indirect comparison of microbial genomes.provides an indirect comparison of microbial genomes.

Carbon & Carbon & Nitrogen sourcesNitrogen sources

Cell wall Cell wall constituentsconstituents

Energy sourcesEnergy sources Fermentation Fermentation productsproductsNitrogen sourcesNitrogen sourcesconstituentsconstituents productsproducts

General General nutritional typenutritional type

Growth Growth temperature temperature

Oxygen Oxygen relationshipsrelationships

Mech. Of energy Mech. Of energy conversionconversion

pH optimum & pH optimum & growth rangegrowth range

Photosynthetic Photosynthetic pigmentspigments

Salt Salt requirements requirements

Osmotic Osmotic ToleranceTolerance

Storage Storage inclusionsinclusions

Sensitivity to Sensitivity to antibioticsantibiotics

Sensitivity to Sensitivity to metabolic metabolic inhibitorsinhibitors

Secondary Secondary metabolites metabolites formedformed

Ecological CharacteristicsEcological Characteristics�� MM..organismsorganisms livingliving inin variousvarious partsparts ofof humanhuman bodybody markedlymarkedly differdiffer

fromfrom oneone anotheranother andand fromfrom thosethose growinggrowing inin freshwater,freshwater, terrestrialterrestrial andandmarinemarine environmentsenvironments..

�� LifeLife cyclecycle patterns,patterns, naturenature ofof symbioticsymbiotic relationships,relationships, abilityability toto causecausediseasedisease inin particularparticular host,host, andand habitathabitat preferencespreferences (such(such asas requirementrequirementforfor temperature,temperature, pH,pH, oxygen,oxygen, andand osmoticosmotic concentration)concentration) areare thetheexamplesexamples ofof taxonomicallytaxonomically importantimportant ecologicalecological propertiesproperties..

Genetic AnalysisGenetic Analysis�� MostMost ofof thethe eucaryoteseucaryotes areare ableable toto reproducereproduce sexually,sexually, so,so, geneticgenetic

analysisanalysis ofof mm..organismsorganisms isis considerableconsiderable importantimportant inin classifyingclassifying themthem..

�� ProcaryotesProcaryotes dodo notnot reproducereproduce sexually,sexually, so,so, thethe studystudy ofof chromosomalchromosomalgenegene exchangeexchange transformationtransformation andand conjugationconjugation isis oftenoften importantimportantduringduring theirtheir classificationclassification..

Molecular CharacteristicsMolecular Characteristics�� StudiesStudies ofof proteinsproteins andand nucleicnucleic acidsacids havehave recentlyrecently beenbeen introducedintroduced asas

powerfulpowerful approachesapproaches toto taxonomytaxonomy.. TheThe comparisonscomparisons ofof proteinsproteins andandnucleicnucleic acidsacids alsoalso yieldyield usefuluseful informationinformation aboutabout truetrue relatednessrelatedness..

�� ComparisonComparison ofof ProteinsProteins::��TheThe aminoamino acidacid sequencessequences ofof proteinsproteins havehave directdirect reflectionsreflections ofof mRNAmRNA

sequences,sequences, and,and, therefore,therefore, closelyclosely relatedrelated toto thethe structuresstructures ofof thethe genesgenescodingcoding andand theirtheir synthesissynthesis.. TheThe comparisonscomparisons ofof proteinsproteins fromfrom differentdifferentmicroorganismsmicroorganismsareareveryveryusefulusefultaxonomicallytaxonomically..microorganismsmicroorganismsareareveryveryusefulusefultaxonomicallytaxonomically..

��TheThe sequencessequences ofof cytochromes,cytochromes, otherother electronelectron transporttransport proteins,proteins,histones,histones, varietyvariety ofof enzymes,enzymes, andand electrophoreticelectrophoretic mobilitymobility ofof proteinsproteinsareare alsoalso importantimportant inin studyingstudying relationshipsrelationships atat thethe speciesspecies andandsubspeciessubspecies levelslevels..

�� AntibodyAntibody –– antigenantigen reactionsreactions inin vitrovitro::�� AntibodiesAntibodies cancan discriminatediscriminate betweenbetween veryvery similarsimilar proteinsproteins..

ImmunologicImmunologic techniquestechniques areare usedused toto comparecompare proteinsproteins fromfrom differentdifferentmm..organismsorganisms..

�� Nucleic Acid Base Composition:Nucleic Acid Base Composition:

�� Here, determination of DNA base composition is carried out.Here, determination of DNA base composition is carried out.

�� DNA contains 4 purine & pyrimidine bases: adenine (A), guanine (G), DNA contains 4 purine & pyrimidine bases: adenine (A), guanine (G), cytosine (C), and thymine (T). In doublecytosine (C), and thymine (T). In double--stranded DNA, A pairs with stranded DNA, A pairs with T, and G pairs with C.T, and G pairs with C.

�� The (G+C) / (A+T) ratio or G+C content, the percent of G + C in The (G+C) / (A+T) ratio or G+C content, the percent of G + C in DNA, reflects the base sequence and varies with sequences changes as DNA, reflects the base sequence and varies with sequences changes as follows:follows:

Mol. % G + C = G + C Mol. % G + C = G + C X 100X 100

G + C + A + TG + C + A + T

�� The G + C content of DNA from animals and higher plants averages The G + C content of DNA from animals and higher plants averages around 40% and ranges between 30 and 50%.around 40% and ranges between 30 and 50%.

�� Procaryotic G+C content is the most variable, ranging from around 25 Procaryotic G+C content is the most variable, ranging from around 25 to almost 80%. to almost 80%.

�� Despite such wide variation, G+C content of strains within a Despite such wide variation, G+C content of strains within a particular species is constant.particular species is constant.

�� If two organisms, differ in their G+C content by more than about 10%, If two organisms, differ in their G+C content by more than about 10%, their genomes have quite different base sequences. their genomes have quite different base sequences.

�� Its not mean that with very similar G+C contents also have similar Its not mean that with very similar G+C contents also have similar DNA base sequences because two very different base sequences can DNA base sequences because two very different base sequences can be constructed from the same proportions of AT & GC base pairs.be constructed from the same proportions of AT & GC base pairs.

�� If organism in the same taxon are too dissimilar in G+C content, the If organism in the same taxon are too dissimilar in G+C content, the taxon probably should be divided.taxon probably should be divided.

�� Useful in characterizing procaryotic genera since the variation within Useful in characterizing procaryotic genera since the variation within a genus is usually less than 10% even though the content may very a genus is usually less than 10% even though the content may very a genus is usually less than 10% even though the content may very a genus is usually less than 10% even though the content may very greatly between genera.greatly between genera.

�� For example, staphylococcus has G+C content of 30 to 38%, whereas For example, staphylococcus has G+C content of 30 to 38%, whereas Micrococcus DNA has 64Micrococcus DNA has 64--75%G+C; yet these genera of gram75%G+C; yet these genera of gram--positive cocci have many other features in common. positive cocci have many other features in common.

�� Nucleic Acid Hybridization:Nucleic Acid Hybridization:�� These studies are used to compare similar genomes more directly.These studies are used to compare similar genomes more directly.�� DNA DNA ––DNA hybridization is only used to study closely related DNA hybridization is only used to study closely related microorganism. DNA molecules are very different in sequence, will not microorganism. DNA molecules are very different in sequence, will not form a stable, detectable hybrid.form a stable, detectable hybrid.�� More distantly related organisms are compared by carrying out More distantly related organisms are compared by carrying out DNADNA--RNA hybridization experiments using radioactive ribosomal or RNA hybridization experiments using radioactive ribosomal or transfer RNA.transfer RNA.

�������� Nucleic acid Sequencing:Nucleic acid Sequencing:�� Genome structure can be directly compared by sequencing DNA and Genome structure can be directly compared by sequencing DNA and

RNA. RNA.

�� RNA sequencing has been used more extensively in microbial RNA sequencing has been used more extensively in microbial taxonomy.taxonomy.

�� The rRNAs are almost ideal for studies of microbial evolution and The rRNAs are almost ideal for studies of microbial evolution and relatedness since they are essential to a critical organelle found in all relatedness since they are essential to a critical organelle found in all microorganisms.microorganisms.

NUTRITION OF BACTERIANUTRITION OF BACTERIA

�� The nutrition requirements for microbial growth can be divided into The nutrition requirements for microbial growth can be divided into two main categories: PHYSICAL & CHEMICAL.two main categories: PHYSICAL & CHEMICAL.

�� Physical aspects include temperature, pH & osmotic pressure.Physical aspects include temperature, pH & osmotic pressure.

�� Chemical requirements include sources of carbon, nitrogen, sulfur, Chemical requirements include sources of carbon, nitrogen, sulfur, �� Chemical requirements include sources of carbon, nitrogen, sulfur, Chemical requirements include sources of carbon, nitrogen, sulfur, phosphorus, oxygen, trace elements, and organic growth factors.phosphorus, oxygen, trace elements, and organic growth factors.

�� Bacteria require following elements:Bacteria require following elements:

In large amounts In large amounts -- Carbon, Nitrogen, Oxygen, Phosphorus, Carbon, Nitrogen, Oxygen, Phosphorus, SulphurSulphur..

In smaller quantities In smaller quantities -- Iron, Calcium, Magnesium, Potassium, SodiumIron, Calcium, Magnesium, Potassium, Sodium

In trace quantities In trace quantities -- ManganezeManganeze, Zinc, Copper, Silica, Bromine, Iodine., Zinc, Copper, Silica, Bromine, Iodine.

Nutrition Of Bacteria…Nutrition Of Bacteria…

�� OneOne systemsystem ofof classifyingclassifying bacteriabacteria isis toto groupgroup themthem accordingaccording toto thethewayway theythey useuse certaincertain elementselements andand toto theirtheir sourcessources ofof energyenergy..

�� AutotrophicAutotrophic bacteriabacteria:: CanCan synthesizesynthesize carbohydratescarbohydrates andand obtainobtainenergyenergy fromfrom simplesimple inorganicinorganic sourcessources ofof carboncarbon dioxidedioxide oror carbonatescarbonateswithwith oror withoutwithout usingusing lightlight..

�� ThoseThose thatthat requirerequire lightlight areare calledcalled photosyntheticphotosynthetic bacteriabacteria andand havehavepigmentspigments eithereither inin plastidsplastids oror inin solutionsolution inin thethe cytoplasmcytoplasm..

�� FewFew bacteriabacteria cancan utilizeutilize nitratesnitrates andand ammoniaammonia asas solesole sourcessources ofofnitrogennitrogen byby completecomplete setset ofof enzymesenzymes theythey havehave..

��AutotrophsAutotrophs areare ofof interestinterest inin pharmacypharmacy becausebecause theythey causecausedeteriorationdeterioration ofof productsproducts andand packagingpackaging materialsmaterials..

�� HeterotrophicHeterotrophic bacteriabacteria:: CanCan growgrow withwith elaboratedelaborated sourcessources ofof carboncarbonandand manymany requirerequire complexcomplex sourcessources ofof nitrogennitrogen..

�� SomeSome organismsorganisms cancan bebe utilizedutilized aminoamino acidsacids forfor energyenergy productionproduction..

�� TheyThey areare oftenoften subdividedsubdivided inin toto twotwo classesclasses::

11)) ParasitesParasites:: RequireRequire environmentenvironment ofof livingliving cellscells..

�� CanCan bebe growngrown underunder artificialartificial conditions,conditions, usingusing highlyhighly specializedspecializedmediamedia..

22)) SaprophytesSaprophytes:: CanCan growgrow onon organicorganic mattermatter fromfrom deaddead plantsplants ororanimalsanimals..

�� TheyThey areare scavengersscavengers ofof bacterialbacterial worldworld becausebecause theythey areare responsibleresponsibleforfor thethe decaydecay ofof deaddead materialmaterial..

�� TheyThey areare notnot pathogenic,pathogenic, butbut some,some, ifif theythey gaingain accessaccess toto diseaseddiseased orordamageddamaged tissues,tissues, maymay produceproduce harmfulharmful toxinstoxins thatthat areare difficultdifficult tototreattreat..

Nutrition Of Bacteria…Nutrition Of Bacteria…��������Growth & multiplication:Growth & multiplication:�� Heat relationships:Heat relationships:Bacteria may be classified by their heat Bacteria may be classified by their heat

characteristics which are of two kinds characteristics which are of two kinds ––1) 1) Growth characteristics:Growth characteristics:a) a) Psychrophiles (Cold loving): Psychrophiles (Cold loving): Optimum growth temp. 15Optimum growth temp. 15ºC. cause ºC. cause

problems in food preservation.problems in food preservation.b) b) Psychrotrophs (moderate psychrophiles) :Psychrotrophs (moderate psychrophiles) :Organism will grow Organism will grow

between 20between 20--3030º C. Cause low temp food spoilage because grow well º C. Cause low temp food spoilage because grow well at refrigerator temp.at refrigerator temp.at refrigerator temp.at refrigerator temp.

c) c) Mesophils (moderate temperature loving):Mesophils (moderate temperature loving):Can grow in range of 25 Can grow in range of 25 to 45 º C. They are pathogenic.to 45 º C. They are pathogenic.

d) d) Thermophills (heat loving) :Thermophills (heat loving) : Optimum temperature for growth lie Optimum temperature for growth lie between 55 to 65 º C. In tropical climates they cause spoilage in between 55 to 65 º C. In tropical climates they cause spoilage in sealed containers of food.sealed containers of food.

e) Hyperthermophiles (Extreme Thermophiles): Microbes, member of e) Hyperthermophiles (Extreme Thermophiles): Microbes, member of Arachaea have an optimum growth temperature of 80ºC or higher.Arachaea have an optimum growth temperature of 80ºC or higher.

Nutrition Of Bacteria…Nutrition Of Bacteria…2) 2) Tolerance characteristics:Tolerance characteristics:Organisms that are very resistant to heat Organisms that are very resistant to heat

treatments are said to be thermoduric. Two types of resistance:treatments are said to be thermoduric. Two types of resistance:

1) Soil organisms form spores resist heat treatments, even surviving 121 1) Soil organisms form spores resist heat treatments, even surviving 121 º C for 20min.or more in autoclave, 3hrs. at 160 º C in hot air oven.º C for 20min.or more in autoclave, 3hrs. at 160 º C in hot air oven.

2) Vegetative bacteria can survive abnormally long periods at above 602) Vegetative bacteria can survive abnormally long periods at above 60º º C.C.

�������� Effect of pH:Effect of pH:�� Most bacteria grow best in a narrow pH range near neutrality, Most bacteria grow best in a narrow pH range near neutrality, �� Most bacteria grow best in a narrow pH range near neutrality, Most bacteria grow best in a narrow pH range near neutrality,

between pH 6.5 & 7.5. between pH 6.5 & 7.5.

�� Very few bacteria grow at an acidic pH below about pH 4.Very few bacteria grow at an acidic pH below about pH 4.

�� That’s why we preserve pickles, cheeses from spoilage by acids That’s why we preserve pickles, cheeses from spoilage by acids produced by bacterial fermentation. produced by bacterial fermentation.

�� Some bacteria, called acidophiles, are tolerant of acidity.Some bacteria, called acidophiles, are tolerant of acidity.

�� Chemoautotrophic bacteria (found in drainage water), oxidizes sulfur Chemoautotrophic bacteria (found in drainage water), oxidizes sulfur to sulfuric acid, can survive at pH value of 1.to sulfuric acid, can survive at pH value of 1.

Nutrition Of Bacteria…Nutrition Of Bacteria…�� Optimum pH of molds and yeasts is usually about pH 5Optimum pH of molds and yeasts is usually about pH 5--6.6.�� To neutralize the acids and maintain the proper pH, chemical buffers To neutralize the acids and maintain the proper pH, chemical buffers

are included in the growth medium.are included in the growth medium.�� The peptones and amino acids in some media act as buffers, and many The peptones and amino acids in some media act as buffers, and many

media also contain phosphate salts.media also contain phosphate salts.�� Phosphate salts have the advantage of exhibiting their buffering effect Phosphate salts have the advantage of exhibiting their buffering effect

in the pH growth range of most bacteria.in the pH growth range of most bacteria.�� They are nontoxic, provide phosphorus, an essential nutrient.They are nontoxic, provide phosphorus, an essential nutrient.

�������� Osmotic pressure:Osmotic pressure:�������� Osmotic pressure:Osmotic pressure:�� M.organisms require 80M.organisms require 80--90% water for their growth.90% water for their growth.�� Osmotic pressure is the pressure required to prevent the movement of Osmotic pressure is the pressure required to prevent the movement of

pure water into a solution containing some solutes.pure water into a solution containing some solutes.�� High osmotic pressures have the effect of removing necessary water High osmotic pressures have the effect of removing necessary water

from a cell.from a cell.�� When a microbial cell is in a solution that has a higher concentration When a microbial cell is in a solution that has a higher concentration

of solutes than in the cell (hypertonic), the cellular water passes out of solutes than in the cell (hypertonic), the cellular water passes out through the plasma membrane to the high solute concentration.through the plasma membrane to the high solute concentration.

Nutrition Of Bacteria…Nutrition Of Bacteria…�� This osmotic loss of water causes This osmotic loss of water causes PlasmolysisPlasmolysis, or shrinkage of the , or shrinkage of the

cell’s cytoplasm.cell’s cytoplasm.

�� So, the growth of the cell is inhibited as the plasma membrane pulls So, the growth of the cell is inhibited as the plasma membrane pulls away from the cell wall.away from the cell wall.

�� Thus the addition of salts to a solution, resulting increase in osmotic Thus the addition of salts to a solution, resulting increase in osmotic pressure, can be used to preserve foods.pressure, can be used to preserve foods.

�� Some organisms, called extreme halophiles, actually require salt for Some organisms, called extreme halophiles, actually require salt for growth. Such organisms often require nearly 30% salt, and the growth. Such organisms often require nearly 30% salt, and the growth. Such organisms often require nearly 30% salt, and the growth. Such organisms often require nearly 30% salt, and the inoculating loop used to transfer them must first be dipped into a inoculating loop used to transfer them must first be dipped into a saturated salt solution.saturated salt solution.

�� Facultative halophiles, are able to grow at salt concentrations up to Facultative halophiles, are able to grow at salt concentrations up to 2%, a conc that inhibits growth of many other organisms.2%, a conc that inhibits growth of many other organisms.

�� A few species of facultative halophiles can even tolerate 15% salt.A few species of facultative halophiles can even tolerate 15% salt.

�� Most m.organisms must be grown in a medium containing 1.5% Agar.Most m.organisms must be grown in a medium containing 1.5% Agar.

�� If higher conc used, the increased osmotic pressure can inhibit the If higher conc used, the increased osmotic pressure can inhibit the growth of somr bacteria.growth of somr bacteria.

Nutrition Of Bacteria…Nutrition Of Bacteria…�������� Chemical requirements:Chemical requirements:

�������� Carbon:Carbon:

�������� Oxygen requirements:Oxygen requirements:Bacteria can be classified according to their oxygen requirements Bacteria can be classified according to their oxygen requirements ––1) 1) AerobesAerobes: Must require atmospheric oxygen to grow.: Must require atmospheric oxygen to grow.2) 2) AnaerobesAnaerobes: Do not employ molecular oxygen as a hydrogen acceptor. : Do not employ molecular oxygen as a hydrogen acceptor. 2) 2) AnaerobesAnaerobes: Do not employ molecular oxygen as a hydrogen acceptor. : Do not employ molecular oxygen as a hydrogen acceptor. �� Oxygen presence produces death in obligate anaerobe, and can Oxygen presence produces death in obligate anaerobe, and can

survive only if they can form spores.survive only if they can form spores.