Introduction to Bacteriology to Gram positive PDF

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Intro to MicrobiologyHepatitis B - the only DNA Virus (double stranded). The rest A,C,D- RNA VirusGram positive bacteria – purple color when stained, cocci shapedGram negative bacteria – red color when stained, rod shapedLactobacillus acidophilus – normal flora of the female genital tractFrancesco r...

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Intro to Microbiology

Hepatitis B - the only DNA Virus (double stranded). The rest A,C,D- RNA Virus Gram positive bacteria – purple color when stained, cocci shaped Gram negative bacteria – red color when stained, rod shaped Lactobacillus acidophilus – normal flora of the female genital tract Francesco redi – demonstrated an experiment that maggots cannot arise from decaying meat John Needham – boiled mutton broth; microbes came through the broth Lazzaro Spallanzani – microbes moves through air Louis Pasteur – developed the vaccine against anthrax (bacillus anthracis – bacteria that causes anthrax) and rabies Ferdinand Cohn – bacteria has endospores and can withstand heating and boiling. -

4 group classification of bacterias: cluster, rod, cocci, spiral

John Tyndall – tyndallization Robert Koch – discovered Mycobacterium tuberculosis and Bacillus anthracis -

Koch Postulates – Germ theory of disease

Edward Jenner – Smallpox vaccine; vaccine – Latin word ‘vacca’ for cow Ignaz Semmelweis – routine handwashing Joseph Lister – introduced antiseptic surgery in Britain; phenol – as an antimicrobial agent Alexander Fleming – discovered Penicillin (Penicillium chrysogenum); discovered lysozyme Paul Ehrlich – discovered treatment of syphilis (asphenamine (Salvarsan) – treatment)

Bacterial Cell Structure Prokaryote – one chromosome, not in a membrane, no organelles (lack mitochondria, Golgi apparatus) Peptidoglycan cell walls, multiplies by binary fission, no histones. 1. External structure: A. -

Glycocalyx – surface coating. (Greek word “Kalux” – husk/pod) Also called, Pericellular Matrix – tissues that surrounds a cell; gel-like layer Coating of molecules external to the cell wall Made up of sugar and or proteins

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Two types of glycocalyx: Slime layer – thin, loosely organized and attached. Capsule – highly organized and tightly attached – to the cell wall. (Mucoid)

B. Flagella – bacterial propellers; locomotor organ of the bacteria. - Consist of three parts: Filament – long, thin, helical structure composed of protein (flagellin); tail of the bacteria. Hook – curved sheath Basal body – stack of rings *basal body and hook are embedded in the cell surface; filament is free on the surface of the bacterial cell* Flagellar arrangements: Trichous – having a hair like part of structure -

Atrichous – no flagellum Monotrichous – single polar flagellum Lophotrichous – 2 or more flagella at one pole (Lopho – combinding form indicating a Tuft) Amphitrichous – 2 or more flagella (tuft) on both poles Peritrichous – with flagella all over the surface/ all over

Endoflagella (axial filament) – special kind of flagella found in the periplasmic space of spirochetes. Corkscrew movement of the spirochete. C. D. -

Fimbriae – fine, proteinaceous hairlike bristle from the cell surface. Function is not for motility but for adherence to other cells and surfaces. Protein: Fimbrin Pili – hairlike structure Protein: Pilin Two types: Common Pili – used for adherence to cell surface Sex Pili – transfer of genetic material (DNA) from the donor to the recipient during a process called Conjugation (genetic recombination process).  Comparison between Fimbriae and Pili:  Fimbriae – present in multiple numbers. Its function is to adhere to host tissues  Pili – longer than the fimbriae; present singly or in pairs. Adheres to other bacteria during DNA transfer.  Comparison between Flagella and Pili:  Flagella: size: Large; thickness: thicker; Origin: cell membrane; Organ for locomotion: check  Pili: size: Small; thickness: thin; Origin: Cell wall; Organ for locomotion: none

2. Cell Envelope -

Boundary layer of bacteria; external covering outside the cytoplasm and maintains cell integrity. Two basic layers: Cell wall and Cell membrane

A. Cell wall -

2 major types of cell wall (according to their staining): Gram Positive – Purple stain and Gram Negative – Red stain (Gram + and Gram – is composed of Peptidoglycan) Peptidoglycan - is the primary component of bacterial cell wall. - is a multilayered structure that is composed of N-acetyl Muramic Acid (NAM) and N-acetyl Glucosamine (NAG) backbones cross linked with peptide chain and pentaglycine bridge. - Also called as, Murein layer

Components of cell wall of Gram-negative bacteria 1. 2. 3. 4.

 Lipid – present Peptidoglycan – thin layer (1-2 sheets) Lipoprotein Phospholipids Lipopolysaccharide – it contains 3 regions: Antigenic O, Core polysaccharide, and inner Lipid A (endotoxin) – the outer membrane of the gram negative cell wall  Lipid A – responsible for much of the toxicity of the gram negative bacteria.

Components of cell wall of Gram-positive bacteria  Lipid – absent or minimal (scant) 1. Peptidoglycan – thick layer (40-65 sheets) 2. Teichoic acid – anchored/linked to the peptidoglycan 3. Lipoteichoic acid – anchored to the cell membrane  Teichoic and Lipoteichoic acid – unique to the gram + cell wall. Its function is to provide rigidity to the cell wall. Penicillin – an antibiotic that prevents the synthesis of peptidoglycan  Gram staining – invented by Hans Christian Gram  Fixation – heat fixed  Crystal Violet – put crystal violet; it is the primary stain; basic dye (PURPLE STAIN!!! bacterial cell wall has a negative charge in G+ bacteria due to the presence of Teichoic acid; negative charge in cell wall of Gbacteria due to the presence of phospholipids and LPS) 30 seconds babad, wash in running water then iodine treatment.  Iodine Treatment – mordant; binder. STILL PURPLE STAIN, 60 seconds babad in Iodine wash in running water. If not added, color of Gram Positive will be pinkish. Iodine acts as a mordant in the process.  Decolorization – using Alcohol (decolorizer; can interact with lipids in the cell membrane) or acetone. (Crystal violet will be dissolved in the Gram negative since it contains lipids that is insoluble to alcohol. While Gram positive will remain as is). 10 seconds  Safranin – counter stain; red dye; wait for 30 secs; wash off with water. (Gram Positive will remain purple since it didn’t got decolorized like

Gram Negative, only the Gram Negative will turn into color Red as an end product since it absorbed the red dye or safranin) Acid Fast Cell Wall -

(found in Mycobacterium, Nocardia and Corynebacterium spp.) Composed of Mycolic Acid layer Cell wall is fast acid resistant; cell wall is rich in lipids 2 acid fat stain that uses Carbolfuchsin as their primary stain: Kinyoun (cold method) and Ziehl Neelsen (hot method) – dormant method Primary Stain - Carbol fuchsin (red);  Only binds to the bacteria that has a waxy cell wall Decolorizer – acid alcohol Counter stain – Methylene blue

No cell wall -

Found in Mycoplasma spp. Pleomorphic (filamentous to cocci or doughnut shape)

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B. Cell Membrane 30% of the dry weight of bacterial cell Composed of 60% protein, 20-30% lipids, and 10-20% carbohydrate Fluid Mosaic Model – phospholipid bilayer with embedded proteins

3. Internal Structures - Chromosomes – single, circular, double stranded DNA molecule that contains all genetic info required by a cell.  Nucleoid – dense area where the DNA is tightly coiled. - Plasmids – Extra chromosomal genetic material; free into the chromosome and duplicated and passed on to offspring. (not essential to bacterial growth and metabolism)

100,000 CFU – UTI Septicemia – Blood infection Urine – 50 mL Yellow top for blood culture Chocolate Agar Plate – CAP (Brown color media) Blood Agar Plate – BAP

BAP -> CAP – heat; due to heat hemoglobin turned brown Mycobacterium gordonae – tap water bacillus 1st morning urine – most acidic urine sample; not ideal Cary-blair medium – transport medium for rectal swab or feces Selenite F. – transport medium for Salmonella Alkaline Peptone Water – transport medium for Vibrio organisms Culture and isolation – 24 hours

Staining I.

Simple Stain – consist of the addition of 1 dye

Crystal violet – primary stain Safranin – counter stain Two types of Simple Stain: Direct Stain – stains the bacteria; Negative Stain – stains only the background. II.

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 1. 2. 3. 4.

Differential Stain – consist of the addition of more than 1 dye; results to differences in color and shade  Primary stain  Mordant (Iodine)  Decolorizer  Counter Stain A. Gram Stain & Acid Fast Stain Gram stain - to differentiate gram + and gram – microorganism Acid Fast Stain – if the patient is infected with Mycobacteria B. Gram Stain General rule:  All cocci are Gram + except: Neisseria (G- bacteria)  All bacilli are Gram – except: D.A.T.T.TA (Diphtheria, Actinomyces, Tetani, Tuberculosis, Anthrax)  Spiral forms are hard to stain; once stained they are Gram negative. Factors causing False Gram - reaction: Removal of MgRNA++ by precipitation with bile salts Using an acidic Gram’s Iodine Technical error – over decolonization Aging/dying cell

 Factors causing False Gram + reaction: 1. Too thick smear

2. Incomplete decolorization

Acid Fast Stain -

Note: AFBs are difficult to stain; but once stained, they resist decolorization by mineral acids.

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Differential stain to distinguish Acid Fast organisms from Non-Acid Fast (found in Mycobacterium, Nocardia, and Corynebacteria)

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Primary Stain: Carbol fuchsin (Red in color) Mycolic Acid (Hydroxymethoxy Acid) – long chain of fatty acids (high lipids content ang acid fast bacilli) Corynebacteria Nocardia – slightly Acid Fast Bacilli

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Tergitol – wetting agent Red – Carbolfuchin color HCl, Alcohol – Decolorizer Methylene blue – counterstain Acid fast – positive bacilli – Red color Acid Fast – negative bacilli – Blue color

Gram + Bacteria Aerobic Gram Positive bacteria 

Staphylococcus aureus – 30% of the population carries S. aureus as resident flora - Isolated from the abscess, wound infection and carbuncles (boils) - causes food poisoning, pneumonia, endocarditis, wounds, SSS (Staphylococcus scalded skin syndrome) - produces six types of enterotoxin F and TSST-1 (Toxic Shock Syndrome Toxin-1) - Positive in DNAse test!!!

Panton – vanlentine Leucocidin – destruction of neutrophils and macrophage. Staphylokinase – Dissolves the fibrin clot Hyaluronidase – “Duran-Reynal Factor”

- Enzyme that catalyzes the breakdown of hyaluronic acid in the body. Also called “speading factor”. Staphylococcal scalded skin syndrome – infection causes peeling skin over large parts of the body. Carbuncles – painful, pus-filled bumps  Identifying Characteristics of S. aureus  Gram + cocci  Opaque and smooth colonies  Beta hemolytic (full zone of hemolysis) in BAP  Catalase +  Coagulase + (to differentiate the Staphylococcus spp.)  PYR (-); pyrrolidonyl a-naphthylamide  Ornithine (-)  Can tolerate 7.5% to 10% NaCl (high salt content) in Mannitol Salt Agar (MSA)  Ferments Mannitol and produces yellow colonies in MSA; lowers the pH, so the culture media becomes acidic. * Red – Original color of Mannitol Coagulase negative Staphylococci – very common skin flora and are mostly non-pathogenic.   

Colonies appear white to gray appearance (BAP); non-hemolytic or gamma-hemolytic Catalase (+) Coagulase (-)

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Staphylococcus Epidermidis – most common species of coagulase negative staphylococci (can be seen in the normal flora of skin. * Novobiocin (Susceptible) * Causes: Prosthetic Valve Endocarditis

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Staphylococcus saprophyticus – cause UTI among sexually active women. * Novobiocin (Resistant) * Novobiocin testing – what test to differentiate S. epidermidis (S) and S. saprophyticus (R) S. lugdunensis – frequent cause of endocarditis * Ferments Mannitol * PYR test – test used to differentiate S. aureus (PYR -) and S. lugdunensis (PYR +).

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 Micrococcus - normal flora of the skin and mucous membrane - arranged in “tetrads” and appear large than Staphylococcus spp. on Gram stain.

- Yellow colonies and non-hemolytic on BAP

o To differentiate Micrococcus and Staphylococcus - Micrococcus – Positive on Modified oxidase test - Bacitracin (0.04 unit disk) – Susceptible - Staphylococcus – Negative on Modified oxidase test - Bacitracin (0.04 unit disk) – Resistant

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Streptococcus spp.

General characteristic:  Catalase (-)  Gram-positive cocci arranged in pairs and chains.  Could be Alpha or Beta Hemolytic or Non-Hemolytic on BAP.  Lancefield grouping is based on a cell wall antigen.  Streptococcus pyogenes (group A streptococci) - infections caused; Strep throat, Impetigo, cellulitis, “Scarlet Fever”, pneumonia, “otitis media” (middle ear infection) and Necrotizing fasciitis - Rheumatic fever and post – streptococcal glomerulonephritis - Bacitracin (Susceptible) - PYR (+) !!! - Pinpoint colonies (1mm, flat, creamy, and show small zones of beta hemolysis.  CAMP test (+) – test for S. agalactiae !!!  Hippurate hydrolysis (+)  PYR (-); unlike S. pyogenes (+)  Bacitracin (resistant); S. pyogenes (Susceptible)  Group D Streptococcus – Normal fecal and oral flora  Group D strep in blood culture means the patient has “colon cancer”  Colonies: gray to white, translucent, round and convex  Bile-esculin (+)  Growth in 6.5% NaCl (-) !!!  PYR (-)

 Viridans Streptococci – normal flora of the oral cavity, respiratory tract and GI tract muscosa.  Major cause of “bacterial endocarditis” in ppl with damage heart valves  Alpha hemolytic – partial zone  Optochin (R)  Bile (Insoluble)  Does not grow on “bile-esculin medium”

 Streptococcus pneumoniae – normal upper respiratory tract flora but can cause:  Lobar pneumonia!!!  Otitis media!!!  Meningitis (>29 years old)  Sputum (rust-colored)!!! o Meningitis  Streptococcus agalactiae (birth)  Haemophilus influenza (1-5 months)  Neisseria meningitidis (5-29 years old)  Streptococcus pnuemoniae (>29 years old)  General Characteristics of S. pneumoniae  G+ diplococci

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Lancet/bullet shaped Alpha hemolytic – on Blood Agar Plate Grows on SBA (Sheep Blood Agar) with 5-10% CO2 at 48 hours Colony: Mucoid strains, umbilicated Optochin (will inhibit growth) Bile 10% sodium deoxycholate (soluble)

 Enterococcus – most encountered spp. are E. feacalis and E. faecium  Bile-esculin (+)  Growth on 6.5% NaCl (+)  PYR (+)  Non-hemolytic -

Vancomycin-resistant enterococci (VRE) – resistance due to altered peptidoglycan cross-link target.

 Gemella   

PYR (+) LAP (+); Leucine aminopeptidase Bile-esculin (-)

 Leuconostoc - Linked to osteomyelitis, ventriculitis, postsurgical endophthalmitis, and bacteremia in neonates.  Vancomycin (R)  PYR (-)  LAP (-)  Catalase (-)  Abiotrophia and Granulicatella  Referred as nutritionally variant streptococci.  Required Vitamin B6 (Pyridoxal/ Pyridoxamine) for growth.  Normal flora of the oral cavity.

Aerobic non-spore forming gram-positive bacilli  Listeria monocytogenes – causes spontaneous abortion  Causes Meningitis in animals specifically sheep  Normal flora of the vagina and intestines in humans.

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Identifying Characteristics:  Colonies: small, white with narrow zone of beta-hemolysis

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Closely resembles group B streptococci in SBA Umbrella-like motility (semi-solid media) at room temp. Tumbling motility (wet mount) Hippurate hydrolysis (+) CAMP (+) Esculin (+) Catalase (+) Note: biochemical test is mostly positive; if tests seen all positive, it’s possible that it is Listeria monocytogenes Inverted Christmas tree appearance CAMP reaction (+) -> rectangle block type of hemolysis

 Corynebacterium diphtheria – causes diphtheria (is a pseudomembrane formed by dead cells and exudate at the back of the throat. 

Identifying Characteristics:  Picket fences/Chinese letters can be pleomorphic  Staining with “Methylene blue” will reveal “metachromatic granules” treatment: Penicillin G -

Considered as potential bioterrorism agent and was used as such in a series of attacks in the US in 2001

 3 clinically form of anthrax: 

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Cutaneous anthrax: -

Most common form worldwide

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Characterized by necrotic skin lesion called “black eschar”

Pulmonary anthrax: -

“Wool-sorter’s disease” spread by inhalation of spores from sheep’s wool.

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Endospores are ingested by macrophages.

Gastrointestinal anthrax: -

Rarest form

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Follow ingestion of spores.

General characteristics: 

“Medusa-head colonies”

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“Bamboo rod appearance

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Does not grow on PEA agar at 24 hours

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Gram positive bacilli with spores

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Catalase (+)

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Non-motile!!!

Bacillus cereus (fried rice bacillus) 

Important cause of food poisoning.

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B. cereus and B. subtilis are also common laboratory contaminants.

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Morphology: large, flat, beta hemolytic with irregular edges.

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B. cereus (motile) ; B. anthracis (non-motile)

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10ug Penicillin (resistant)

Bactericidal – kills microorganism; treatment for a life-threatening infection Bacteriostatic – inhibit bacterial growth but do not kill the microorganism Broad-spectrum antibiotic – effective against a wide range Narrow-spectrum antibiotic - effective only against specific organism; either gram (+) or gram (-) Minimal-inhibitory concentration – lowest concentration that can still inhibit bacterial growth Minimal lethal concentration – lowest concentration; can still kill bacterial growth; 25% still effective. Therapeutic index – ratio of toxic dose to the therapeutic dose; High TI – more effective agent TD – toxic dose ED – therapeutic/Effective dose Natural drug – from bacteria/fungi Semi-synthetic – modified natural drugs Synthetic drugs – chemically produced- drugs Carbapanems – actively against ESLB production organism ESLB – can be inhibited by Clavulanic acid.

Clavulanic acid – beta-lactamase inhibitor....