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CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester MODULE 1 Polynomial - strings of Latin words and phrases Parts of Bacterial Cell and Techniques in Bacterial...

CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester MODULE 1 Polynomial - strings of Latin words and phrases Parts of Bacterial Cell and Techniques in Bacterial containing up to 12 words. Identification Binomial - two-part name for each species. Microbiology Classification by Phenotypic and Genotypic Microbiology is the study of very small living Characteristics organisms called microorganisms or microbes Subdivision of subspecies based on: (ubiquitous-virtually everywhere). ○ phenotypic differences (abbreviated “subsp.”) Advanced course of Biology (study of living Serovarieties based on: organisms). ○ serologic differences (abbreviated “serovar.”) Micro - anything so small that it must be viewed with Biovarieties, based on: a microscope. ○ Biochemical test result differences (abbreviated The various categories of microorganisms include “biovar”) viruses, bacteria, archaeans, certain algae, protozoa, and certain fungi. Schemes of Classification Basis of cell organization, cells are classify into: Importance of Medical Microbiology Five Kingdom - monera, protista, fungi, plantae, animalia Cultivation/ Propagation of organisms from patient Three Kingdom - plantae, animalia, protista specimen Two Kingdom - prokaryotes and eukaryotes Study of technique for isolation, cultivation and identification of pathogens Most biologists use a six-kingdom system: Study of microbial physiology, growth of 1. Animalia requirements, morphology, biochemical, molecular 2. Plantae characteristics for diagnosis and treatment of 3. Fungi diseases (Antimicrobial susceptibility test). 4. Protista 5. Archaebacteria Microbial Taxonomy 6. Bacteria 3 distinct disciplines: 1. Classification - organization of microorganisms that Whittaker’s Classification (1969) was devised by share similar morphologic, physiologic, and genetic Robert H. Whittaker based on the nutritional pattern of traits into specific groups or taxa. microorganisms: 2. Nomenclature - naming of organisms according to established rules and guidelines for universal Kingdom Mode of Procuring recognition , with a binomial system ( genus and Food/Nourishment species). 3. Identification - process by which a microorganism’s Animalia Ingestive key features are delineated, which can be compared Mycetae Absorptive with other characterized microorganisms. Plantae Photosynthetic The Classification of Organisms Classification - multi level grouping of individuals. Protista Unicellular, plant-like, animal-like Organisms first classified by Aristotle over 2,000 and fungal-like organism years ago. Classification scheme of the Middle Ages Monera Bacteria and Cyanobacteria (polynomial system) was replaced with a binomial system by Linnaeus about 250 years ago.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Nomenclature The family name is capitalized and has an –aceae ending. e.g., Micrococcaceae Marguelles et. al (1978) came up with the modified The genus is capitalized and followed by the species classification scheme based on the type of cells: epithet, which begins with a small letter; both genus and species should be italicized in print but Eucaryotae Plantae underlined when written in script. e.g., Mycetae Staphylococcus aureus or Staphylococcus aureus Animalia Some the genus name is abbreviated by using the Protista first letter of the genus followed by a period and the Procaryotae Monera species of epithet. e.g., S. aureus The abbreviation “sp.” is used to designate single species. e.g., Penicillium sp. Groups of Microorganisms The abbreviation “spp.” is used to designate more 1. Algae - simple organisms, unicellular, others are than one species. e.g., Staphylococcus spp. aggregation of similar cells with little or no differentiation in complex structure or function. Nicknames and slang terms frequently used within 2. Viruses - very small non-cellular parasites or hospitals are GC and gonococci (for Neisseria pathogens of plants, animals and bacteria, even gonorrhoeae), meningococci (for Neisseria meningitidis). protists. Visualized only with the use of an electron microscope. Can be cultivated only in living cells. By convention: 3. Bacteria - minute, unicellular prokaryotic organism, ➔ first word of binomial name is genus and is multiply by binary fission, plant-like, microscopic always capitalized organism which lacks chlorophyll. ➔ second word refers to specific epithet and is not 4. Protozoa - unicellular eukaryotic organism, capitalized differentiated on the basis of morphology, physiology ◆ together form a scientific name, written and nutrition. in italics 5. Fungi - eukaryotic lower plants devoid of chlorophyll, ➔ a complete scientific name may include the usually multicellular but not differentiated into roots. author’s name. Ex. Bacillus anthracis stems, and leaves, unicellular single-celled (yeast), composed of mycelium (filaments and masses of Prokaryotic and Eukaryotic cells which make up the body, reproduce asexually and or sexually).  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester ❖ Prokaryotic cells have complex cell walls consisting of proteins, lipids, and polysaccharide. ❖ Eukaryotic cells contain membranous structures and many membrane bound organelles. ❖ Prokaryotic cells possess no membrane other than the cell membrane that encloses the cytoplasm. ❖ Eukaryotic ribosomes are larger and more dense than those found in prokaryotes. Comparison of Human and Bacterial Classification Recap of Structural differences between Prokaryotic Important Parts of Bacterial Cell and Eukaryotic Cells Flagella ❖ Eukaryotic cells are divided into plant and animal ○ Atrichous = absence of flagella types. ○ Monotrichous = single flagellum at one end ❖ Animal cells do not have cell walls, whereas plant ○ Amphitrichous = single flagellum at both cells have simple cell walls, usually containing ends cellulose. ○ Lophotrichous = tuft/bundle of flagella at one ❖ Cellulose, a type of polysaccharides, is a rigid or both ends polymer of glucose. ○ Peritrichous = bacterium surrounded with flagella  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Pili/Fimbriae Capsule Dark-field microscope - the light is directed toward the Cell Wall specimen from the side so that the only light to reach the Endospores objective is reflected from the bacteria or object to be Inclusions/Metachromatic granules studied. Thus, the microbes appear as bright objects on Nucleoid a dark background. Plasmid Ribosome Plasma Membrane Microbial Classification and Identification Criteria 1. Phenotypic Criteria - readily observable characteristics (visually present) Macroscopic morphology/Cultural/Colonial Characteristics - culture media characteristics Microscopic morphology - characteristics of microorganisms Staining Characteristics Environmental & Nutritional Requirement Resistance profile/ Antimicrobial Testing Antigenic profile/ Serological Testing - based on the substance that has been produced in the Phase-Contrast Microscope - it is used to observe body (e.g. antibodies) living microorganisms without staining because the light Subcellular or Biochemical properties/test refracted by the living cells is different from the 2. Genotypic Criteria surrounding medium to see the object more easily. DNA base composition Nucleic Acid (DNA and RNA) base sequence Microscopy Bright field microscope - magnification is obtained by a system of optical lenses. Compound Bright-field Microscope - equipped with two lenses and a visible light source that passes through the specimen and lenses to the observer's eye. The eyepiece contains the ocular lens, the next lens is in the objectives near the object to be viewed. Form a dark image against a brighter background (Resolving power - 0.2000 um.) Objectives: Low-power objectives - 100x, High-power objectives - 400X, Oil-immersion objectives - 1000X Fluorescence Microscope - this microscope uses Resolving power -ability of the lens to distinguish ultraviolet (UV) light source that illuminates the object two adjacent points or objects at a particular without passing into the objective of the microscope. The distance apart. It depends on the wavelength of ultraviolet light strikes certain dye (fluorescein the light source and the numerical aperture. The isothiocyanate, rhodamine B) and pigments they emit numerical aperture is proportional to the fluorescent against a dark background. resolving power.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester e.g Enterococcus spp., Neisseria spp., Staphylococcus spp., and Streptococcus spp. B. Bacilli may be short or long, thick or thin, pointed or with curved or blunt ends. From very short coccobacilli to long filamentous rods. Bacilli with tapered, pointed ends are termed fusiform. e.g include the members of the family Electron Microscope - It uses electron beam instead of Enterobacteriaceae (e.g Enterobacter, visible light and magnets instead of lenses to focus the Escherichia, Klebsiella , Proteus, Salmonella, beam. The electron passes through the dry specimen, and Shigella spp.), Haemophilus influenzae, which is mounted in wax or plastic, and the image is Pseudomonas aeruginosa, Bacillus spp., and seen on the fluorescent screen. Clostridium spp. Transmission electron microscope - the object can be viewed on the screen and allows C. Curved and spiral-shaped bacilli are placed into a examination of cellular ultrastructure, as well as third morphological grouping. viruses. Image is two dimensional. Resolving e.g Vibrio spp., such as Vibrio cholerae and power- 0.0005 um. Maximum magnification - Vibrio parahaemolysticus 200,000X Scanning electron microscope - it shows a three dimensional view of the specimen. It is also useful in examining surface structure of cells and viruses. Resolution is limited compared with transmission electron microscopy. Resolving power- 0.0200 um. Maximum magnification- 10,000X. Bacterial Morphology Bacteria vary in size from 0.4 to 2μm. They occur in the three basic shapes. ❖ Cocci (spherical) ❖ Bacilli (rod-shaped) ❖ Spirochetes (spiral) A. Cocci may be seen singly or in pairs (diploccoci), chains (streptococci), clusters (staphylococci), packets of four (tetrads). Or packets of eight (octads), depending on the particular species and the manner in which cells divide.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Types of Bacterial Staining Procedures Some bacteria may lose their characteristic shape because adverse growth conditions prevent the production of normal cells. Such cell wall deficient bacteria are called L-forms. Some L-forms revert to their original shape when placed in favorable growth conditions, whereas others do not. Mycoplasma do not have cell walls; thus , microscopically they appear in various shapes. The ability to exist in a variety of shapes is known as GRAM STAIN pleomorphism. Dr. Hans Christian Gram - developed a staining technique that bears his name Growth Curve Most important staining procedure in the ❑ During an initial LAG phase the rate of growth or cell bacteriology laboratory. division is very slow. Differentiates between “Gram-positive” and ❑ Growth or cell division then starts to accelerate into “Gram-negative” bacteria. the EXPONENTIAL phase. Primary procedure: VIAS ❑ Growth will start to DECELERATE (DECLINE). Crystal Violet (V) - primary stain (1-2 mins.) ❑ This may be followed by a STATIONARY phase, Gram’s Iodine (I) - mordant (1 min.) during which there is no discernible change in cell Alcohol (A) - until no blue color comes -off concentration or biomass. Safranin (S) - counterstain (30 sec.-1min) ❑ Finally, we may observe a phase of CELL DEATH and LYSIS - which results in a decrease in cell number Gram Staining Procedure: and/or biomass. 1. Add the Crystal Violet dye to one edge of the coverslip “tunnel.” Draw the dye through using an absorbent paper towel applied to the opposite edge of the cover slip. Draw the dye through until all the water has been replaced by dye. Stain for 30 seconds.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester 2. In a similar manner, wash the slide by drawing decolorization step appearing as blue-to-purple water under the coverslip with a paper towel bacteria. until no further purple color is removed. Thick layer of peptidoglycan makes it difficult to 3. Add the Gram's Iodine solution by adding remove the crystal violet iodine complex during several drops of the reagent to the same edge of the decolorization step. the coverslip and drawing it through with a paper Gram-negative. The crystal violet was removed towel. Treat for about 1.5 minutes. from the cells during the decolorization step, and the 4. Decolorize the slide by drawing 95% alcohol cells were subsequently stained by the safranin; under the coverslip until no further purple due is they will be pink-to-red. removed. Thin layer of peptidoglycan in the cell walls of 5. Wash the slide as in step b. Gram-negative bacteria make it easier to remove 6. Counter stain the slide by placing Safranin dye the crystal violet. Some stains of bacteria are at the same end of the “tunnel” and drawing it neither consistently blue-to-purple nor pink-to-red; through with a paper towel. Stain for 1/2 minute. they are referred to as Gram-variable bacteria. 7. Wash the slide as in step b. 8. Observe the slide under high dry or oil Rules in Gram Staining: immersion microscopy. ➔ Gram Positive = Cocci ➔ Gram Negative = Bacilli ➔ All are GRAM POSITIVE COCCI except NVBMAS (Neisseria, Veillonella, Branhamella, Megasphera, Acidaminococcus, Simonsiella) ➔ All are GRAM NEGATIVE BACILLI except BCMCNELL (Bacillus, Clostridium, Mycobacterium, Corynebacterium, Nocardia, Erysipelothrix, Listeria, Lactobacillus) ➔ Higher forms of organisms like Actinomyces, Streptomyces, yeast, and molds are Gram Positive ➔ All spiral organisms are reported as Gram Negative ➔ Not GRAM stained are Rickettsiae; Chlamydia; Mycoplasma; Ureaplasma (wall less) ACID-FAST STAIN Contains wax-like lipoidal material affecting staining quality. Carbolfuchsin is the primary stain. Acid fast organisms resist decolorization with acid alcohol. After decolorization, methylene blue is added to organisms to counterstain any material that is not acid fast. Gram Reactions: Gram positive. If the bacteria (stained with crystal violet) were not decolorized during the  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Methylene Blue ➔ Traditionally used to stain Corynebacterium diphtheriae for observation of metachromatic granules. Primary procedure: CAM ➔ It is also used as counterstain in acid-fast staining Carbolfuchsin (bright red dye) (C) - heat for 5 procedures. mins Acid Alcohol (A) - until red color comes-off Methylene Blue (M) General Rule: All bacteria are none or not acid fast except Mycobacterium and Nocardia-slightly acid fast. Types of Dye Acridine Orange ➔ It is a fluorochrome dye that stains both Gram positive and Gram negative bacteria, living or dead. ➔ Used to locate bacteria in blood cultures and other Lactophenol Cotton Blue specimens. ➔ Used to stain the cell walls of medically important fungi grown in slide culture. Calcofluor White ➔ It is fluorochrome that binds to chitin in fungal cell Endospore Stain walls. ➔ Malachite green, the primary stain, is applied and ➔ It fluoresces as a bright apple-green or blue-white, heated to steaming for about 5 min. allowing visualization of fungal structures with a ➔ Washed for 30 sec. to remove the primary stain fluorescent microscope. ➔ Applied the counterstain safranin to the smear ➔ It appears green, within pink-or-appearing bacterial cells  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester c. Chemoautotroph - Some bacteria use O2 in the air to oxidize inorganic compounds and produce ATP (energy). - The energy is enough to convert CO2 into organic material needed for cell growth. - Ex: Thiobacillus (sulfur S), Nitorsomonas (ammonia), Nitrobacter (nitrite), Various genera (hydrogen etc.) d. Anaerobic Respiration - Final electron acceptor: never be O2 India Ink - Sulfate reducer: final electron acceptor is ➔ It is a negative stain used to visualize capsules sodium sulfate (Na2SO4) surrounding certain yeasts, such as Cryptococcus - Methane reducer: final electron acceptor is CO2 spp. - Nitrate reducer: final electron acceptor is sodium ➔ The fine particles are excluded from the capsule. nitrate (NaNO3) Leaving a dark background and a clear capsule - O2/H2O coupling is the most oxidizing, more surrounding the yeast. energy in aerobic respiration. - Therefore, anaerobic is less energy efficient. Microbial Growth and Nutrition 3 Major Nutritional Needs for Growth Source of carbon (for making cellular constituents) ○ 50% of the dry weight of a bacterium B. Oxygen Requirements Source of nitrogen (for protein synthesis) Major Groups Requiring Oxygen ○ 14% of the dry weight Obligate aerobes - require oxygen for growth Source of energy (ATP, for performing cellular ○ Ex: Mycobacterium, Pseudomonas, and functions) Bacillus ○ 4% are: Phosphate for nucleic acids and Facultative aerobes - either with or without oxygen phospholipids of cell membranes, and ○ Ex: Staphylococcus and Streptococcus, E. sulfur for protein synthesis coli, Salmonella Microaerophilic - bacteria grow in a reduced level of A. Nutritional Requirement oxygen a. Autotrophic or lithotrophic ○ Ex: Helicobacter and Campylobacter - carbon dioxide as the sole source of carbon, Aerotolerant aerobes - can grow in the presence of with only H2O and inorganic salts required. oxygen, but grow best in an anaerobic environment b. Heterotrophic or organotrophic ○ Ex: Lactobacillus - requires more complex substances for growth. - require an organic source of carbon.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Obligate anaerobes - does not require Nutrients are used up oxygen/cannot grow in the presence of oxygen Toxic products accumulate ○ Ex: Bacteroides and Clostridium Sporulation begins to occur New cells equal the number of dying cells C. pH Requirements Neutrophile - most bacteria grow well at pH 7.0 to Death phase/Stage of Decline 7.5 Death of cells Acidophile - grows at an acidic pH = 3.0 (Lactobacillus) Alkalophile - grows at an alkaline pH = 10.5 (Vibrio) D. Temperature Psychrophilic - 0-20 degrees Celsius Mesophilic - 20-45 degrees Celsius Thermophilic - 50-125 degrees Celsius Thermoduric - not growing well in the presence at high temperatures but can withstand exposure to high temperatures. E. Salt, Carbon Dioxide Requirements Types of Medium Based on Microbial Requirement Capnophilic - require concentrations of 5% to 10% Minimal medium - laboratory growth medium CO2 for optimal growth whose contents are simple. ○ Ex: Streptococcus and Neisseria Nutrient media - more complex and made of Halophilic - High salt concentrations extracts of meat or soybeans. ○ Vibrio Selective media - media containing additives that inhibit the growth of some bacteria but allows others Bacterial Growth to grow. Bacteria replicate by binary fission. Differential media - ingredients that allow Generation time or doubling time time required for visualization of metabolic differences between one cell to divide into cells: groups or species of bacteria. ○ 20 minutes for the bacteria in the culture to Transport medium - when a delay between generate for fast growing organism collection of the specimen and culturing the e.g. E. coli specimen is necessary. ○ 24 hours for a slowing bacterium e.g. Mycobacterium tuberculosis Bacterial Biochemistry and Metabolism Metabolism Stages of Bacterial Growth breakdown organic compounds to synthesize Lag Phase/Adjustment Phase new bacterial parts from the resulting carbon Initial phase skeleton. Detoxifying medium Turning on enzymes to utilize medium Fermentation and Respiration Number of cells at beginning equal number of cells Fermentation Respiration at the end of lag phase obligate and facultative Molecular oxygen is the Log Phase anaerobes final receptor Rapid exponential growth less efficient in energy Certain anaerobes can generation than carry out anaerobic Stationary phase/Plateau  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Carbohydrate Utilization respiration respiration such as Nitrate Bacteria has a greatly diversity in the ability to Sulfate use Carbohydrates Lactose Utilization - the most important 2 important diagnostic Final electron receptor carbohydrate determination test test used in identification Lactose of the - A disaccharide consisting of glucose Enterobacteriaceae: and galactose connected by a galactose Voges-Proskauer (VP) Methyl red tests bond. Lactose Degradation - used to differentiate bacterial species Chemoautotroph ○ Lactose Fermenters Some bacteria use O2 in the air to oxidize Able to ferment lactose inorganic compounds and produce ATP ○ Non-lactose Fermenters (energy). Unable to ferment lactose The energy is enough to convert CO2 into Enzymes necessary for bacterium take up organic material needed for cell growth lactose Thiobacillus (Sulfur, S) 1. β-galactoside permease - serves as a Nitrosomonas (Ammonia) transport enzyme that facilitates entry of Nitrobacter (Nitrite) lactose molecule across plasma Various genera (hydrogen etc.) membrane. 2. β-galactosidase - hydrolyzes lactose Anaerobic Respiration into glucose and galactose Final electron acceptor: never be O2 ➔ After hydrolyzed, glucose is Sulfate reducer: final electron acceptor in available for metabolism sodium sulfate (Na2SO4) through the pathways: Methane reducer: final electron acceptor is CO2 ◆ Embden-Meyerhof Nitrate reducer: final electron acceptor is pathway (glycolysis) sodium nitrate (NaNO3) ◆ Entner-Duodoroff O2/H2O coupling is the most oxidizing, more pathway energy in aerobic respiration. Therefore, LFs possess both enzyme unlike the NLFs anaerobic is less energy efficient. which do not possess either Late/Delayed Lactose Fermenters (dLFs) - MODULE 1 bacteria that have β-galactoside permease but Biochemical Tests possess β-galactosidase. Principle of Biochemical Reaction Sugar Fermentation Test Based on enzymatic reactions Synthesis of enzymes produce by the organisms Triple Sugar Iron Agar (TSI) or utilization of substrate Contains glucose, lactose, sucrose containing Available substrate in the culture medium ammonium sulfate Formation of the end-product after Red colored medium alkaline enzyme-substrate reaction Yellow colored medium acid Addition of the indicator/s. The color change, initial to the final color reaction TSI Reactions Group 1 Ac slant & butt w/ gas, no H2S  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester bacteria utilized the peptones producing Group 2 Ac slant & butt w/ gas & H2S an alkaline reaction. Group 3 Alk slant/ac butt w/ gas 3. Lactose Fermentation A/A - acid/acid Group 4 Ac slant/ac butt w/o gas Glucose fermenter attacked the glucose then the lactose Group 5 Alk slant & butt w/o gas Acid production from the fermentation of the additional sugar is suffering to keep Red colored medium (alkaline) change to yellow both the slant and the butt acid (yellow) - acid 4. Hydrogen Sulfide Production Red colored medium - no change (red) - alkaline K/A, H2S - alkaline slant/acid butt, H2S Initial pH indicator used - phenol red (7.3-7.6) in butt TSI and KIA are useful in the presumptive A/A, H2S - acid slant/acid butt, H2S in identification of gram-negative bacteria butt pH indicator: Phenol Red 2 indicators (to detect H2S): Sodium Ferrous sulfate and sodium thiosulfate is added thiosulfate and Ferrous sulfate to detect production of H2S 2 step process: Useful in detecting the ability of the ○ Bacterium (acid environment) + microorganisms to ferment carbohydrate, sodium thiosulfate -> H2S gas glucose, and lactose ○ H2S + Ferric ion —> ferrous To produce gas from fermentation of sugars sulfide (black precipitate) To detect the production of H2S 5. Gas Production (Aerogenic) or no gas Both put on slant (aerobic) or butt (anaerobic) production (Nonaerogenic) Reactions must be read within 18-24 hours Formation of bubbles poor splitting of otherwise it will be erroneous. the media in the butt or complete Reactions on TSI Agar or KIA displacement 1. No Fermentation - K/A - glucose is fermented ALK/ALK or K/K - alkaline slant/alkaline - A/A - two or more sugars fermented butt - K/K - no sugar fermented ALK/no change or K/NC - alkaline - Space or crack - gas formation slant/no change - Blackening - H2S production Unable to ferment either lactose or glucose Krigler Iron Agar (KIA) Organisms can degrade the peptones Glucose and Lactose with ferrous ammonium present in the medium aerobically or sulfate anaerobically. For hydrogen sulfide production Resulting in production of alkaline pH indicator: phenol red by-products in slant/deep. Indicator: deep color red Russel Double Sugar Agar (RDS) 2. Glucose Fermentation only, no Lactose Contains glucose, lactose, no ferrous Fermentation ammonium sulfate K/A - alkaline slant/acid butt pH indicator: Andrade's indicator TSI agar and KIA contains glucose Dissolve the fuchsin in distilled water and add concentration 0.1% the sodium hydroxide. If the fuchsin is not Yellow indicator: Because of acid sufficiently decolorized after several hours, an produced in concentration of glucose. additional 1 or 2 ml sodium hydroxide is added. Deep color red indicator: because after pH = 7.2 - colorless 12 hours, glucose will be consumed and ○ Pale yellow or colorless - alkaline  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester ○ Pink or red - acid C. Bacteria can utilize carbohydrates by: All are distributed or dispense at the butt/slant 1. Oxidation - aerobically Ferrous ammonium sulfate - H2S indicator 2. Fermentative - anaerobically ○ Black - (+) reaction 3. Or both - Facultative anaerobes D. Asaccharolytic - they do not use any Glucose Metabolism and Its Metabolic Products carbohydrate instead they use other organic molecules for energy and carbon sources. A. Process of Metabolism Oxidation-Fermentation Tests (O/F) Important test in the identification of bacteria Carbohydrate Fermentation Test ○ determine the ability of a microorganism to ferment a specific carbohydrate incorporated into a basal medium. Glucose enters the glycolysis pathway Formation of pyruvic acid End product is an acid or acid with gas ○ Acid Formation - detected with oh indicators added to medium ○ Homolactic Acid Fermenters - bacteria that produce primarily a single acid Example: streptococci ○ Mixed Acid Fermenters - bacteria that produce several acids like lactic acid, propionic acid, and succinic acid. Methyl Red (MR) and Voges Proskauer Oxidation Test (VP) Test Glucose enter the glycolysis pathway Detect the end products of glucose Pyruvic formed oxidized to CO2 fermentation It requires: Part of the IMViC Reactions: ○ Oxygen (aerobic respiration) ○ Insole ○ Inorganic Molecule (anaerobic ○ Methyl Red respiration) ○ Voges-proskauer Example: Nitrate (NO3) as a ○ Citrate terminal electron acceptor Bacteria can be positive for either of two Higher acidity is formed during fermentation than but not for both. in oxidation. Bacteria can be negative for both. The same medium is for oxidation and fermentation test B. Other Carbohydrates Oxidizer and fastidious fermenter often produce Monosaccharides, Disaccharides, either weak or small amounts of acids from Polysaccharides carbohydrates. ○ Examples: maltose, rhamnose, sucrose, raffinose, arabinose Fermentation/Oxidation Reaction Sugars - collective polyhydric alcohols Energy production by breakdown of sugars ○ Examples: adonitol, dulcitol, through a degradation process coupled with mannitol, sorbitol oxidation-reduction reaction.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Substrate is oxidized as it donates electrons to ○ Utilized by the organism producing e’-acceptor molecules, w/c then reduced. acidity and can be observed upon the Mediated by carrier molecules such as NAD and addition of methyl red (indicator). NADP If the pH of the media reaches 4.5 or less the Which involved fermentative metabolism positive reaction appears with a red color, but (anaerobic) producing alcohols, acids, CO2 and if the pH is less acidic (6.2), it displays a H2. negative reaction with a yellow color (4 days Oxidative phosphorylation involve e’ transport incubation at 37 C) system in which O2 is the final e’ acceptor Indicator: Methyl Red (4.4-6.4, acid) - red to (aerobic respiration) yellow Anaerobic respiration - O2 is not the final e’ ○ Positive Result - red (acid) acceptor. ○ Negative result - yellow (alkaline) Methyl Red (MR) Test Indole Production Bacteria are incubated in a broth medium Tryptone broth is rich in amino acid tryptophan containing glucose (3-5 days) which can be used by some bacteria as a Glucose is metabolized by the mixed acid source of carbon and nitrogen. The organism fermentation pathway, stable acid, and end hydrolyzed tryptophan producing indole, pyruvic products are produced, low pH. acid (alanine), and ammonia. MR (+) develops a red color upon addition of Plus ether or xylene indicator methyl red. Ehrlich and Kovack’s reagent react to MR (-) remains yellow. methylindole producing red colored reaction. Indole - one of the degradation products of the amino acid tryptophan organism that possess the enzyme tryptophan are capable of deaminating tryptophan, with formation of Voges-Proskauer Test degradation products of indole, pyruvic acid, and Production of acetyl methyl carbinol from ammonia. glucose after 48hrs. Ehrlich Indole Test By adding 15 drops of 5% alphanapthol in indole is extracted from the broth culture by absolute ethanol (Barritt’s rgg A) and 10 does of addition of xylene and Ehrlich 0.3% creatinine in 40% KOH (Barritt rgt B) reagent(withPDAB) Producing positive reaction upon the Positive for indole: red color appearance of pink red color in 15-30 minutes. More sensitive than Kovac reagent Media: Clark Lub’s broth, glucose broth, MR-VP Kovac Reagent Test broth. Kovac reagent use as an alternative because it Positive result - red from oxidation of contains PDAB (-dimethylaminobenzaldehyde) acetylmethylcarbinol in the presence of Xylene is not used in this method potassium hydroxide alkaline & atmospheric air. Positive for indole: red color Negative result - colorless broth Voges Proskauer (VP) Test Methyl Red Acids formed during fermentation , metabolized pH Indicator to 2, 3 butanediol through acetoin. Clark-Lub’s broth or Glucose broth, MR-VP α-nalthol is added as a catalyst ○ Contains peptone, dextrose or glucose, 40% KOH or NaOH is added VP (+), presence and dipotassium phosphate. of potassium forms red complex, pH is neutral Dextrose  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Negative Result ○ Streptococci and Pneumococci No evolution of gas Reagent: 3% H2O2 Citrate Utilization Organism enzyme (Catalase) + H2O2 — H2O + Utilization citrate as the only source of carbon in O2 gas a medium containing inorganic nitrogen giving a ○ False (+) result obtain due to Prussian blue color or blue which is a positive contamination of RBC result in 24-48 hours of incubation (using Do not use blood agar: Use tryptic soy agar Simmon Citrate Agar) (TSA); blood has catalase and will give a false positive test Simon Citrate Agar Bromthymol Blue (pH 6.9) Oxidase Test Positive result - blue color medium Colonies producing enzymes indophenol Negative result - green color medium oxidase become pink, changing to red, and finally black or dark purple upon addition of Koser Citrate Broth oxidase reagent (1% Result - presence of growth of organism tetramethylparaphenylene-diamine-dihydrochlori (turbidity) is the positive result de or Disadvantage: turbidity of the medium when paraamino-dimethyl-aniline-monohydrochloride) large inoculum is used. Media Use: Chocolate Agar for Neisserria Nutrient Agar for pseudomonas, moraxella, and Citrate Utilization aeromonas, campylobacter fetus. Determines whether an organism can see ○ Positive result: Black or dark purple sodium citrate as a sole carbon source. Neisseria, Pseudomonas, Moracella, Simmons citrate (medium) Aeromonas, Campylobacter, Vibrio ○ Contains ammonium salt as the sole ○ Negative Result: no change in color nitrogen source. Not satisfactory for colonies ○ Thereby releasing ammonia. growing on selective media or ○ Changes the pH indicator (bromocresol media containing glucose blue) from green to blue. Fermentation inhibits enzyme Light inoculum is important to use because dead oxidase activity and leads to a organisms can be a source of carbon, producing false negative result for a false-positive reaction. indophenol oxidase. Christensen citrate (medium) Use platinum loop or wooden ○ Incorporates phenol red (pH indicator) applicator, iron in the nichrome and organic nitrogen loop can give a false-positive ○ At alkaline pH, indicator turns from reaction. yellow to pink Oxidase Test Determines the presence of the cytochrome Catalase Test oxidase system that oxidizes cytochrome with Decomposition of H2O2 into free O2 and H2O by molecular oxygen. enzyme catalase from the organism resulting to Oxidase negative: enterobacteriaceae a rapid evolution of oxygen gas which is the Oxidase positive: Neisseria, Pseudomonas, positive reaction. Campylobacter, Vibrio Positive Result ○ Micrococci, Staphylococci, Bacillus Other Oxidase Test species are all Gram (+) organisms. Modified Oxidase Test  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester ○ To distinguish Staphylococcus from Initial pH of the medium 6.0 (purple) Micrococcus Kovac Oxidase Test ○ Lavender color within 10 to 15 seconds is a positive reaction. Urease Test Some organisms can hydrolyze urea producing urease and ammonia, thus making the medium alkaline and change color from salmon ti cerise or cherry color. ○ Proteus hydrolyze urea rapidly (2-4hrs) Christensen’s Urea Agar Phenol red - indicator UREA SERVE AS SUBSTRATE: Positive result: ○ pink → red color → Proteus (+) Negative result: salmon color, no change Results Urease Test ○ First few hours of incubation,the Determines whether a microorganism can organism attacks glucose first, changing hydrolyze urea pH to Acid (yellow, negative result) Urease hydrolyze urea to form ammonia, water, ○ If organism produce decarboxylase and and CO2 hydrolyzes the amino acid in the Christensen's urea broth or agarB medium, the enzyme attacks the amino ○ Phenol red is the indicator acid and release amine product, ○ Alkaline pH is bright pink color changing pH to ALKALINE (purple - + result) Decarboxylation/Dihydrolase Test Determines whether the bacterial species Lysine Decarboxylase possess enzymes capable of decarboxylating Some organisms can decarboxylate lysine (removing the carboxyl group) the specific amino converting it to cadaverine, producing a purple acids. color as a positive result. Some organisms Degradation of Amino Acids and their deaminate lysine producing red slant and yellow Specific End-Products: butt due to fermentation of glucose. ○ Lysine (amino acid) + Lysine For differentiation of member of decarboxylase → Cadaverine (amine) + Enterobacteriaceae CO2 MEDIA USED: LYSINE - IRON AGAR ○ Ornithine + ornithine decarboxylase → Positive reaction: yellow color will be produced putrescine due to alkalization of medium which is the sign ○ Arginine + arginine dihydrolase → of initial fermentation of glucose citrulline → ornithine putrescine ○ Yellow color will be turned into a violet Moeller Decarboxylase Base Medium color due to decarboxylation of the Test to detect decarboxylation amino acid. A broth containing glucose; peptones ○ Yellow color after several days of 2 indicators: incubation is (-) or absence of enzymes ○ Bromocresol purple decarboxylase or dihydrolase ○ Cresol red Specific amino acid concentration: 0.1%  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester ○ All positive tests should be compared Deaminase Test with the control tube, which remains Removed an amine group yellow. Phenylalanine deaminase (PAD) test Lysine Iron Agar Slant (LIA) Determines whether an organism possess the It is the most useful in conjunction with TSI in enzyme that deaminates phenylalanine to screening stool specimens for the presence of phenylpyruvic acid pathogens (agar slant) Medium: Agar Slant (with 0.2% conc. of Contains the amino acid lysine, glucose ferric phenylalanine) ammonium citrate, and sodium thiosulfate pH indicator: bromocresol purple It is primarily to determine bacteria decarboxylate or deamine lysine H2S production is detected Ortho-Nitrophenyl-β-D-galactopyranoside Test (+) result for lysine decarboxylation: dark ONPG and PNPG determines whether the purple or black precipitate due to the H2S organism is a dLF or NLF (+) result for lysine deamination: light purple ONPG is similar to lactose but easily transported color to plum or reddish-purple color. through the bacterial plasma membrane and ○ Example: Salmonella (lysine+) and does not require β-galactoside permease Citrobacter (lysine-) Test can be performed by making a heavy suspension of bacteria in sterile saline and Motility Indole-Ornithine Agar adding commercially prepared ONPG disks or A semisolid agar used to detect motility and tablets. indole and ornithine decarboxylase production. The suspension is incubated at 37◦C and Useful in differentiating Klebsiella spp. From positive results can be seen within 6 hours Enterobacter and Serratia spp. ➔ ONPG - Motility is shown by a clouding of the medium or o-nitrophenyl-β-d-galactopyranoside spreading growth from the line of inoculation test Ornithine decarboxylation is indicated by a ➔ PNPG – purple color throughout the medium p-nitrophenyl-β-d-galactopyranoside It does not overlayed with mineral oil to provide test anaerobic condition. ➔ NLF - non - lactose fermenters It is detected by adding Kovac’s reagent ➔ DLFs - late/delayed lactose Positive result: pink to red color fermenters Motility is read before addition of Kovac reagent DNase Phenylalanine Deaminase Test DNA - a polynucleotide composed of repeating Some organisms can deaminate phenylalanine purine and pyrimidine mononucleotide converting it to phenyl acid by phenylalanine monomeric units. deaminase produced by bacteria. Then later Bacterial DNases – are undonuclease cleaving reacts with ferric ions to produce a green color. internal phosphodiester bonds resulting in ○ MEDIA: Phenylalanine Agar smaller subunits. ○ (+) result - rxn appear after addition of DNase test medium usually contains 0.2% DNA ferric chloride Inoculum of bacteria is streak in a straight line ○ Positive result: dark green - Proteus, Plate is incubated at 35 ̊ C for 18-24 hours Klebsiella, and Yersinia 1N HCl is added (unhydrolyzed DNA is insoluble ○ Negative result: yellow - other in HCl and form precipitate) Enterobacteriaceae Oligonucleotide formed and dissolved in acid forming clear zone around inoculum  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester DNase Test Hydrolysis/depolymerization of FNA by enzyme DNase which produces a clear zone in a DNA-methyl green medium. Positive: Clear Zone Negative: Pale Green Gelatin Liquefaction Gelatin – A protein derived from animal collagen Gelatinases – Proteolytic enzymes that break down gelatin into amino acids produced by bacteria. Gelatinase activity – detected by loss of gelling (liquefaction) of gelatin affected by some factors including size of inoculum and incubation at room temperature. Gelatinase Test Motility Hydrolysis of gelatine by enzyme gelatinase that causes liquefaction. Motility Test Media Positive: liquid medium Have agar concentration of 0.4% Negative: solid medium after refrigeration ○ To allow the free spread microorganisms A single stab into the center of the medium is Malonate Utilization made ○ Best result if it is made straight Malonate Test After incubation: Determines whether the organism is capable of ○ Movement away from the stub or hazy using sodium malonate as its sole carbon appearance indicates a motile organism. source Incubation temperature is important because Malonate broth contains bromothymol blue as a some bacteria are motile only at room pH indicator temperature. Positive Test: Increase alkalinity, changing indicator from green to blue Nitrate Test Reduction of nitrate to nitrite which can be determined after the addition of sulfanilic acid  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester and alpha-naphthylamine, nitrite react with detected by oxidation with ninhydrin reagent sulfanilic acid to form diazonium salt w/c couples resulting in the production of purple color. with alpha- naphthylamine to form red, water Negative: Yellow or Pink soluble azo dye. Positive: Red or Gas PYR Test Negative: Yellow, no gas L-pyrroglutamyl aminopeptidase hydrolyzes the Nitrate Reduction substrate L-pyrrolidonyl-B-naphthylamide (PYR) Determines the ability of the organism to reduce to produce a B-naphthylamine that combines nitrate to nitrite and further reduce nitrite to with cinnamaldehyde reagent producing bright nitrogen gas red color. ❖ N,N dimethyl-α-naphthylamine and Negative: orange (other streptococci) sulfanilic acid is added (indicator) Positive: red for S.pyogenes,Enterococci ❖ Positive for the presence of nitrite: red Acetamide Utilization ❖ No color develops indicate that nitrate Acetamide is the source of carbon, which has not been reduced or nitrate is deaminate acetamide to form ammonia resulting reduced to N2, nitric oxide, nitrous oxide. in an increase in pH that changes the medium ❖ Zinc dust - helps to detect the result if it from green to royal blue. is a true negative result or due to the reduction beyond nitrate. Bile Esculin Agar Bile inhibition of organisms other that streptococci and enterococci which grow on 40% bile and hydrolyze esculin forming esculetin which turn the indicator ( ferric ammonium citrate) a dark brown color due to combination of esculetin with ferric ions to form a phenolic iron complex. Bile Solubility Test Differentiates S. pneumoniae from alpha hemolytic streptococci (negative). Sodium deoxycholate lysis the cell due to the presence of intracellular autolytic enzymes. Bile salt lower Sulfide-indole-motility agar the surface tension between the cell membrane A semisolid agar helpful in differentiating and the medium gram-negative bacteria in the family Enterobacteriaceae CAMP Test Positive for motility: cloudiness spreading from Group B streptococci produce a diffusible the inoculation extracellular protein (CAMP factor) that acts Production of H2S: formation of black synergistically with the beta-lysin of S. aureus to precipitate cause enhanced lysis of red blood cells forming Positive result: colorless to red color after an arrowhead zone of beta hemolysis e.g. addition of Kovac’s reagent or Erlich’s reagent + S.agalactiae. chloroform or ether Cetrimide Test Hippurate Hydrolysis Determine the ability of the organism to grow on Hydrolysis of hippuric acid by enzyme cetrimide which is toxic to many organisms. hippuricase that produces glycine. Glycine is (+) growth/green  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester reaction endpoints in minutes to hours. A LAP (Leucine Aminopeptidase) Test positive reaction is measured by enzymatic Used to identify catalase negative gram positive activity and color change. cocci. Leucine B-naphthylamide impregnated on a disk is hydrolyzed by LAP forming a red Test Bacterial Mode of Applications B-naphthylamine upon the addition of Enzyme Action cinnamaldehyde reagent. ONPG Β-galactosi An ester Determine de linkage of lactose Rapid and Automated Identification Systems ortho-nitrophen fermentation yl moieties to in slow Rapid Methods various lactose Conventional methods carbohydrates fermenters Hydrolysis Miniaturized biochemical test using small results in amount of substrates and inoculum release of Principles of Identification yellow ortho-nitrophen Categories: ol ○ pH-based reactions ○ Enzyme-based reactions Oxidase Cytochrom A blue Differentiation ○ Utilization of carbon sources eC compound is of non- oxidase produce when fermenters; ○ Visual detection of bacterial growth tetramethyl-phe aids in ○ Detection of volatile or non-volatile fatty nlenediamine identification acids by gas chromatography reacts with Neisseria Numeric code: Cytochrome C sp., Aeromonas ○ Generated based on the metabolic sp. profiles of each organisms Metabolic reaction translated into 2 LAP Leucine LAP hydrolyzes Presumptive responses: aminopepti the substrate to identification dase leucine of catalase ○ Plus (+) for positive and negative, ○ Minus (-) for negative α-naphthylamin gram-positive Binary code: e which reacts cocci ○ re computer converted into code profile with DMAC to form a red color numbers that represent the identifying phenotype of specific organism Catalase Catalase Breakdown of Differentiation Clinical outcome of rapid and accurate reporting hydrogen of peroxide into staphylococci of results should directly affect the patient care oxygen and from in 2 ways: water resulting streptococci 1. Early diagnosis in rapid 2. Subsequent selection of appropriate production of bubbles antimicrobial therapy Rapid method – encompasses a variety of procedures and techniques and has been loosely applied to any procedure affording Rapid Methods results faster than conventional method. ○ ex: common stains and fluorescent antibody to detect specific organism Rapid test for detection of end products resulting from carbohydrate metabolism or enzymatic tests using chromogenic substrate produce CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Enterotube ID System - one shot innoculation Other rapid methods include ○ Micro-ID ○ Rap ID ○ Enteric-Tek ○ Quantum II - multipurpose instrumental system using plastic cartridge Pie-shaped wedges in a circular plastic tray Automated Identification Systems These systems uses turbidity, colorimetric, fluorescent assay principles Lyophilized reagents are provided in micro titer trays. Advantages ➔ Interface to laboratory information systems leading to decreased turn around times for results ➔ Statistical prediction of correct identification ➔ Increase data acquisition and epidemiologic analysis ➔ Automated standardization of identification profiles that can reduce analytical errors  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Micro Scan System Takes a totally different approach; identification Consist of plastic standard-sized 96-well is based on the 9 to 20 carbon fatty acid microtiter trays in which as many as 32 reagent composition of microorganisms substrates are included for the identification of Fatty acids are located in the plasma membrane bacteria and yeasts of bacteria and are modified by the bacteria 3 Systems: depending on the environmental condition. a. Touch scan - Reagents are added to This system compares the fatty acid profile of some wells, and the panels are the unknown with the database and determines interpreted visually. Biochemical results a similarity index are recorded with a computer b. Auto scan - Can be used to detect bacterial growth or color changes by differences in light transmission. c. Walk away - Fully automated with capabilities to incubate more than one panel; (add reagent, interpret panel results, and print results), all without operator intervention Sensititre System Offers two automated systems: a. Sensititre Autoreader b. Full Automated Sensitire Evaluation of Identification Systems It use the same gram-negative intracellular ○ To provide results with greater speed diplococcic (GNID) for determination for gram and precision than traditional method negative bacteria ○ Highly accurate and can provide results It use fluorescent technology to detect bacterial in as soon as 2 to 4 hours growth and enzyme activity ○ New system for accuracy Comprises 32 biochemical test ○ Cost effectiveness ○ Effect on work flow Vitek System ○ Provide decrease sensitivity First introduced in 1980’s EBC provide automatic identification of Molecular Method Enterobacteriaceae within 8 hours of incubation. Nucleic Acid-Based Methods The computer software collates the reading and a. Hybridization matches them to the automated database for b. Amplification & Sequencing final identification. c. Enzymatic Digestion of Nucleic Acid NonNucleic Acid- based Analytic Methods BD Phoenix 100 System a. Chromatography Released in 2003, it is the latest automated b. High-Performance Liquid system Chromatography Panels are read every 20 minutes for up to 16 c. Electrophoretic Protein Analysis hours. Molecular ID Results are generally available in 8 to 12 hours Sherlock Microbial Identification System  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Serological ID MODULE 2 Introduction Staphylococci: Electrophoretic Analysis Catalase positive gram positive cocci. Which of the bacterial isolates are of the same Can cause infections in humans. species? Colonize various skin and mucosal surfaces. Which of the isolates are related to one another? Transmitted from person to person. Which of the isolates is not totally related to May become established as part of the other? recipient's normal flora. Can be introduced to sterile sites by trauma or Immunochemical ID invasive procedures. Production of Antibodies S. aureus is the most virulent species of Precipitin test Staphylococci encountered. Particle Agglutination Catalase negative, gram positive cocci: Immunofluorescent Includes Streptococcus and Enterococcus. Enzyme Immunoassays Commonly found as part of normal human flora. Encountered in clinical specimens as contaminants or components of mixed cultures with minimal or unknown clinical significance. Can cause life-threatening infections when they gain access to normally sterile sites. Streptococcus pneumoniae: Found as part of the normal upper respiratory flora. Leading cause of bacterial pneumonia and meningitis. Streptococcus pyogenes: Rarely considered normal flora. Clinically important whenever encountered. Species of the genus Neisseria and Moraxella catarrhalis: Oxidase positive, gram negative diplococci.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Except for N. gonorrhoeae, all are normal On 5% sheep blood agar or chocolate agar inhabitants of the upper respiratory tract of the colonies appearance are medium to large; humans. smooth entire, slight raised, translucent; most N. gonorrhoeae: colonies pigmented creamy yellow; most Primarily a pathogen found in the urogenital colonies beta hemolytic; 24 hours of incubation tract. On MSA S. aureus can grow in the presence of Never considered a normal flora; always salt and ferment mannitol produce colonies considered clinically significant. surrounded by yellow halo; for at least 48 to 72 Transmission: sexually transmitted. hours before growth is detected N. meningitidis: Tests for Identification for S. aureus: Spread by contaminated respiratory droplets. M. catarrhalis: TEST RESULTS Infections usually involve a patient's endogenous strain. Catalase POSITIVE Infections are usually localized to the respiratory tract and rarely disseminate. Coagulase (slide and POSITIVE tube method) CATALASE (+) GRAM POSITIVE COCCI Voges - Proskauer Test POSITIVE STAPHYLOCOCCUS, MICROCOCCUS PYR NEGATIVE Staphylococcus aureus Most virulent species of staphylococci Vancomycin RESISTANT encountered Normal flora of human anterior nares, Penicillin and Ampicillin RESISTANT nasopharynx, perineal area and skin Various skin, wound and tissue infections Staphylococcus epidermidis commonly caused by S. aureus - this infections Microscopic Characteristics can rapidly become life threatening if not treated Small to medium; translucent, gray-white and manage appropriately colonies S. aureus produce toxin-mediated diseases, Non-hemolytic such as scalded skin syndrome and toxic Slime-producing and adhere to the agar surface shock syndrome Clinical Significance: Virulence Factors Normal skin flora and mucous membranes alpha, beta, gamma and delta - toxins act on Causes UTI host membrane and mediates destruction Stitch abscess Leucocidin (PVL) - mediates destruction of Prosthetic heart valve infection phagocytes Test for Identification: Clumping factor, coagulase and hyaluronidase - Tube coagulase (-) enhance invasion and survival in tissue 2hr PYR broth hydrolysis (-) exofoliatins, toxic shock syndrome toxin Novobiocin Susceptibility (sensitive) (TSST-1), enterotoxins - potent exotoxins Alkaline Phosphatase (+) Characteristics of Staphylococcus aureus: Polymyxin B (resistant) Gram positive cocci usually in clusters Voges-Proskauer (+) S. aureus will grow on 5% sheep blood and chocolate agar but not on macConkey agar. On selective media Mannitol Salt Agar (MSA) is commonly used.  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Organism Urease Oxidase Catalase Coagulase Staphylococcus saprophyticus S. + - + - saprophyticus In humans, S. saprophyticus is found in the normal flora of the female genital tract and perineum. Culture in Blood Agar Plate (BAP) Staphylococcus saprophyticus is a Non-pigmented, non-hemolytic colonies Gram-positive coccus responsible for Mechanism uncomplicated lower urinary tract infections Bacterial resistance to methicillin and related (UTIs), mainly in young women penicillin and cephalosporin is mediated by S. saprophyticus is second only to E. coli as the acquisition of gene (mecA) that codes for a most frequent causative organism of urinary novel penicillin-binding protein, PBP2a that has tract infection in women. a low affinity for methicillin and related penicillin It was found to contaminate 16·4% of the and cephalosporin. various food samples with a high prevalence of 34 % in raw beef and pork. It was common in Staphylococcus lugdunensis both domestic and imported raw meat products. Normal flora of human skin present in fewer Introduction of endogenous flora into sterile numbers urinary tract, notably in young, sexually active Spread of patient’s endogenous strain to females. A community acquired infection, not normally sterile site, usually as a result of considered an agent of nosocomial infections. implantation of medical devices. Gram (+) cocci in clusters Medium to large, smooth, glossy, entire edge w/ slightly domed center Unpigmented or cream to yellow-orange Staphylococcus haemolyticus Normal flora of human skin present in fewer numbers Spread of patient’s endogenous strain to normally sterile site, usually as a result of implantation of medical devices. Medium, smooth, butyrous and opaque Staphylococcus lugdunensis Can cause endocarditis and bacteremia Coagulase: Slide coagulase (+) PYR: (+) Haemolytic Pattern: Narrow beta hemolytic Vogues Proskauer:  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester Mannitol: (+) Staphylococcus intermedius Coagulase: Slide coagulase (+) PYR: (+) Haemolytic Pattern: Vogues Proskauer: (-) Mannitol: Biochemical tests Staphylococcus haemolyticus Test Results Coagulase: Tube coagulase (+) PYR: Urease - Haemolytic Pattern: Beta hemolytic Vogues Proskauer: Coagulase + Mannitol: Vogues-Proskauer + Staphylococcus schleiferi S. schleiferi is a coagulase-negative organism Micrococcus spp. which has been described as a pathogen Normal flora of human skin, mucosa, and responsible for various nosocomial infections oropharynx including bacteremia, brain abscess, and Mode of transmission is uncertain infection of intravenous pacemakers. Usually considered contaminants of clinical This bacteria has been described to be found specimens; rarely implicated as cause of typically on skin and mucosal surfaces. infections in humans Microscopically, it appears medium to large; Test for identification: smooth, glossy, slightly convex with entire edges Catalase (+) on 5% Sheep Blood agar. Modified oxidase (+) It is susceptible to Novobiocin and produces a Strict aerobe except Kocuria (Micrococcus) heat-stable nuclease. It can be differentiated kristinae which is facultatively aerobic from S. aureus by production of different Bacitracin sensitive nuclease with lack of pigmentation Furazolidone resistant Gram (+) cocci in clusters Lysostaphin resistant Small to medium; opaque, convex; nonhemolytic; wide variety of pigments (white, tan, yellow,orange,pink) cocci growth on BAP CATALASE (-) GRAM POSITIVE COCCI STREPTOCOCCI AND ENTEROCOCCI Streptococcus spp. 1. Beta-Hemolytic Streptococci  CLINICAL BACTERIOLOGY Lecture Assoc. Prof. Rogelio Cruz AY 2024-2025 1st Semester S. pyogenes (Group A ) Can Cause S. agalactiae (Group B) Scarlet fever 2. Group C, F and G beta-hemolytic streptococci Streptococcal toxic shock syndrome 3. Streptococcus pneumoniae Acute pharyngitis 4. Viridans/ Alpha-hemolytic Streptococci Appearance on 5% Sheep Blood Agar