CLINICAL MICROBIOLOGY 1
001 Introduction to Bacterial Identification
Contents
- Bacterial Taxonomy
- Steps in Diagnostic Isolation and Identification of Bacteria
- Isolation and Identification Method ofs
- Isolation and Culturing of Microorganism
- Microscopic Examination
- Biochemical Testing of Microorganism
- Antimicrobial Sensitivity Testing (AST)
1. Introduction
Infectious diseases caused by different bacteria have a variety of clinical courses and consequences
Isolation and identification of bacteria aids treatment
Susceptibility testing of isolates (e.g: establishing Minimum Inhibitory Concentration, MIC) can help in selection of antibiotics for therapy
Recognizing that certain species (or strains) are being isolated atypically may suggest that a disease outbreak has occurred
2. Bacterial Taxonomy
- Taxonomy is the practice and science of classification
- Family is a group of related genera
- Genus is a group of related species
- Species is a group of related strains
- Type means sets of strain within a species (e.g: biotypes, serotypes)
- Strain is one line or a single isolate of a particular species
- Reviewing naming convention of bacterial species: Streptococcus pyogenes (abbreviation: S. Pyogenes)
3. Steps in Diagnostic Isolation and Identification of bacteria
Step 1 Isolate and observe growth in pure culture
- Samples of body fluids (blood, urine, CSF) are streaked on culture plates
- Isolated colonies of bacteria which are visible to the naked eye appear after incubation for one to several days
- Observe colonies for size, texture, color, hemolytic reactions
Step 2 Microscopic examination
- Colonies are Gram stained and individual bacterial cells observed under the microscope
Step 3 Species identification
- The bacteria are speciated using the isolated colonies. This often requires an additional 24 hours of growth. Generally using physiological tests.
Step 4 Antibiotic susceptibility testing
- A test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics
4. Isolation and Culturing of Microorganism
Culturing the organisms in dishes or tubes containing the nutrients required for microbial growth
To identify a pathogen it is necessary to isolate the organism in pure growth from a single colony
A pure growth refers to a culture which contains only a single species of organism
A mixed culture is one which contain more than one species
5. Microscopic Examination
The appearance of bacteria is observed under the microscope
- Gram positive
- Morphology (rod, coccus, spiral, pleomorphic etc)
- Arrangement (single, chains, pairs etc)
- Size
Besides the Gram stain, there are other less commonly employed stains (e.g: spore, capsule stains)
6. Morphology of Bacteria
Most bacteria are classified according to shape
- Bacillus: rod shaped
- Coccus: spherical
- Spirillum: spiral
Some bacteria have quite different shapes
- Coccobacilli: elongated coccal form
- Filamentous: bacilli that occur in long threads
- Vibrios: short, slightly curved rods
- Fusiform: bacilli with tapered ends
7. Arrangement of Bacteria Cells
Bacteria sometimes occur in groups, rather than singly, and the single cell's shape influenced the cell arrangements that they form through binary fission
Bacilli divide along a single axis, so sometimes they are seen in pairs or chains. Since they only divide along one axis, you will not find them in clusters, such as those formed by staphylococcal bacteria
Cocci divide on one or more planes, producing cells in
- Pairs (diplococci)
- Chains (streptococci)
- Packets (sarcinae)
- Clusters (staphylococci)
8. Bacterial Colony Morphology
Different types of bacteria will sometimes produce colonies with a distinctive appearance. The characteristics of a colony (shape, size, color, etc) are termed the colony morphology.
- Shapes: punctiform (under 1 mm diameter), round, filamentous, irregular
- Forms: circular, filamentous
- Elevation: turn the petri dish on end
- Margin: smooth (entire), curled, wavy, lobate, filamentous
- Surface: smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled), concentric, contoured
- Opacity: transparent, opaque, translucent (almost clear but distorted vision, like looking frosted glass), iridescent (changing colors in reflected light)
- Chromogenesis (pigmentation): white, buff, red, purple
9. Biochemical Testing of Microorganism
OF glucose
Used to identify the oxidative and fermentative abilities of microorganisms. Organism to be tested in stabbed into 2 tubes of the semi-soft Agar medium. One tube remains open to air and one tube is covered with oil. Tubes are incubated and results read by noting the color of the bromthymol blue indicator. If glucose is used, acid end-products are generated, the pH is lowered and the bromthymol blue indicator turns yellow.
Open tube turns yellow=oxidative
Bromthymol blue green turns yellow=fermenting glucose
Alcaligenes faecalis: glucose not metabolized (non saccharolytic)
Escherichia coli: fermentative and oxidative metabolism
Pseudomonas aeruginosa: oxidative metabolism
Phenol Red Fermentation Broth
Used to identify acid and gas production from a variety of sugars such as lactose, mannitol, sucrose. Broths are inoculated and incubated. The results are determined by the color of the phenol red indicator. If sugar is fermented, acid end-products are generally and the phenol red indicator turns yellow.
Staphylococcus aureus: alkaline, non glucose and lactose fermenter
Escherichia coli: acid, glucose fermenter and gas bubbles in Durham tube
Shigella sonnei: acid, glucose fermenter and no gas bubbles in Durham tube
Pseudomonas aeruginosa: glucose fermenter but non lactose fermenter, gas bubbles in Durham tube
Simmons Citrate Agar
Used to identify organisms that can utilize citrate as their sole carbon source. A slant is inoculated and incubated. The result is determined by determining the color of the bromthymol blue indicator. Citrate is used and the pH of the medium rises to turn the tube a bright Prussian blue color.
Enterobacter aerogenes, Salmonella enteritidis, Salmonella typhimurium, Proteus vulgaris: positive
Escherichia coli: negative
MRVP (Methyl Red-Vogues Proskauer)
Used to identify organisms that ferment to produce stable acid end-products or neutralized acids into acetoin
MR:
Escherichia coli: red, positive
Klebsiella, Enterobacter aerogenes: yellow/orange, negative
VP:
Escherichia coli: negative
Klebsiella, Enterobacter aerogenes: pink, positive
Urease test
Urease is an enzyme produced by some microorganisms. It attacks the nitrogen and carbon bond in aminde compounds and forms alkaline end product ammonia. The urease test is particularly helpful in identifying Proteus from other species due to the rapid action of the enzyme by Proteus compared to other non lactose fermenting enterics. The pH indicator phenol red changes to a deep pink color in the present of the alkaline ammonia.
Escherichia coli: negative
Proteus vulgaris: pink, positive
Urease positive bacteria include Helicobacter pylori, Klebsiella, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus aureus, Haemophilus influenzae, Mycobacteria except MAC, Cryptococcus, Ureaplasma, Diptheroids
Indole production test
Tryptophan is an essential amino acid that can undergo oxidation by the action of the bacterial enzyme tryptophanase. Not all bacteria possess this enzyme and so this test can be used as a biochemical differentiator. The SIM Agar which contains tryptophan is used to identify the presence of indole. Kovac's reagent is added to the top of an SIM Agar deep that has been inoculated and incubated with a test organism. The formation of a cherry red reagent ring is a positive result.
Escherichia coli, Proteus vulgaris, Vibrio cholerae: positive
Klebsiella pneumoniae, Enterobacter sp, Proteus mirabillis, Staphylococcus aureus, Salmonella arizonae: negative
Hydrogen Sulfide test
Gaseous H2S may be produced in 2 ways by bacteria. SIM Agar may be used to detect this production of gas. SIM Agar contains peptone, sodium thiosulfate, and ferrous ammonium sulfate. When H2S is produced, a precipitation reaction will occur with the ferrous ammonium sulfate. An insoluble black precipitate is seen as a positive result.
Escherichia coli, Staphylococcus aureus: negative
Proteus vulgaris: positive
Triple Sugar Iron
May also used to determine H2S production as well as carbohydrate fermentation.
Pseudomonas, Acinetobacter, Moraxella: K/K, K/NC, non fermenter, uses peptone
Escherichia coli, Klebsiella, Enterobacter, Serratia: A/A, ferments glucose, lactose and sucrose, gas
Proteus: A/A, H2S
Shigella, Morganella, Providencia: ferments glucose, uses peptone, K/A, no gas
Salmonella: ferments glucose, K/A, H2S, gas
Phenylalanine Deaminase test
Used to identify bacteria with the ability to deaminate phenylalanine into phenylpyruvic acid. After incubation, the slant is covered with ferric chloride solution. The ferric chloride will react with any phenylpyruvic acid present and produce a dark green color. A yellow color is a negative result.
Escherichia coli: negative
Providencia stuartii: positive
10. Antimicrobial Sensitivity Testing (AST)
002 Gram Positive Cocci
Contents
- Genus: Staphylococcus
- Species and Pathogenicity
- S. Aureus
- S. Epidermidis
- S. Saprophyticus
- Laboratory Diagnosis
- Microscopy
- Culture
- Biochemical Reaction
- Antimicrobial Sensitivity
1. Introduction
More than 30 species of staphylococci have been recognized
Gram-positive, non-motile, non sporing cocci
Occurs singly, in pairs, and in irregular clusters
Size may be variable
Colonies are opaque and maybe white or cream
Occasionally yellow or orange
Optimum growth temperature 30~37°C
Facultative anaerobes and have a fermentative metabolism
Salt tolerant and highly resistant to drying
Some species are hemolytic
2. Staphylococcus natural habitat
Staphylococcus aureus and Staphylococcus epidermidis are normal flora of the skin
Staphylococcus aureus colonizes moist environments (nasal passages and axillae)
Other species of staphylococci are infrequent human commensals
Staphylococcus epidermidis is rarely pathogenic, Staphylococcus aureus is an opportunistic pathogen
3. Staphylococcus aureus
Staphylococcus aureus can cause many forms of infection
Superficial skin lesions (boils, styles) and localized abscesses
Deep seated infections in debilitated persons from chronic illness, trauma, burns or immunosuppression
Staphylococcus aureus is the major cause of hospital acquired infection of surgical wounds
Food poisoning (release of enterotoxin in food)
Toxic shock syndrome (release of superantigen into blood)
Staphylococcal scalded skin syndrome
4. Enzymes and toxins produced by Staphylococcus aureus
Coagulase
Leucocidin
Lytic toxin
DNAse
Hyaluronidase
Lipase
Staphilokinase
Exfoliatin
Enterotoxin B
Beta lactamase
5. Other species
Staphylococcus epidermidis causes infections associated with inwelling medical devices include artificial valve endocarditis, catheter and shunt infections, and prosthetic infections
Staphylococcus saprophyticus is mainly associated with cystitis, pyelonephritis and acute urethritis in females
Laboratory Diagnosis
6. Identification
7. Microscopy
Direct gram stain of specimen collected from the lesion/wound/pus/sputum
Blood samples require culturing first because of the small numbers of bacteria in blood
8. Culture
Primary isolation media
Material from clinical specimen or blood culture streaked onto media
Blood Agar: 16~48 h incubation in 5~10% CO2 at 35~37°C
Specimens likely to be contaminated with other microbes plated on mannitol salt Agar
Colonial morphology
Varies with species
Usually opaque and maybe white or cream
Sometimes yellow to orange on Blood Agar
Hemolysis may be detected
MSA is both selective and differential
It contains 7.5% NaCl which selects for organisms that are halotolerant
It also contains mannitol and phenol red
Organisms able to ferment mannitol produce acid fermentation products which lowers pH
Phenol red indicator turns yellow
Staphylococcus aureus is able to ferment mannitol
Other staphs grow on MSA but do not ferment mannitol
9. Biochemical Reaction
Catalase test
Catalase is an enzyme produced by most aerobic and facultative anaerobe bacteria. It decomposes hydrogen peroxide into oxygen and water. Some bacteria from hydrogen peroxide as an oxidative end product of aerobic breakdown of sugars. If allowed to accumulate is highly toxic to bacteria. Enzyme present in staphylococci but lacking in streptococci. Bubbling indicates a positive result. Scant or no bubbling indicates a negative result. Catalase test should not be done on Blood Agar (blood itself will produce bubbles).
Coagulase test
Coagulase is an extracellular protein which through the conversion of fibrinogen to fibrin results in the clotting of blood plasma. It exists in 2 forms: bound coagulase (clumping factors) that bound to cell wall and free coagulase that liberated by the cell wall. It present in Staphylococcus aureus but lacking in most other staphylococci. Some isolates of Staphylococcus aureus may be defective in coagulase and give a negative result.
DNAse test (Staphylococcus aureus is DNAse positive)
Modified oxidase test to differentiate micrococci from staph
Lysostaphin test (commercial identification kit)
10. Antimicrobial Sensitivity
Antibiotic against Staphylococcus aureus is penicillin, cephalosporin, erythromycin, lincomycin, Clindamycin, vancomycin
Many strain of Staphylococcus aureus are penicillin-resistant due to production of penicillinase (a beta lactamase)
Novobiocin is a antimicrobial drug
Mode of action is to prevent supercoiling of DNA by DNA gyrase (Inhibitory effect on DNA replication)
Most strains of Staphylococcus saprophyticus are resistant whereas most strains of Staphylococcus epidermidis are sensitive (zone of inhibition >17 mm)
003 Gram Positive Cocci
Contents
- Genus: Streptococci
- Species and Pathogenicity
- Streptococcus pyogenes (Lancefield Group A)
- Streptococcus agalactiae (Lancefield Group B)
- Streptococcus enterococci (Lancefield Group D)
- Streptococcus pneumoniae
- Laboratory Diagnosis
- Microscopy
- Culture
- Biochemical Reaction
- Antimicrobial Sensitivity
1. Introduction
Large group of Gram positive bacteria
Inhabit a wide range of hosts as normal microbial flora including humans, horses, pigs and cows
Can cause diseases from subacute to acute or chronic
Taxonomic note:
Streptococci and related organisms have undergone extensive taxonomic revisions
Phenotypic methods alone may not adequately identify organisms to species level
Further identification (molecular methods) may be necessary where indicated clinically or epidemiologically
2. General characteristics
Gram-positive cocci often occuring in pairs and chains
On Blood Agar, species exhibit various degrees of hemolysis
2 important factors in presumptive identification: hemolytic activity of colonies on Blood Agar and Lancefield serological grouping
Facultatively anaerobic
Catalase negative
Metabolizes carbohydrates fermentatively (major metabolite produced is lactic acid)
3. Lancefield classification
Based on proteins found in cell walls of streptococci
Species classified into groups A through Medically important are Group A, B and D
4. Species of medical importance
Streptococcus pyogenes (Group A)
Streptococcus agalactiae ( Group B)
Enterococcus sp (Group D)
Streptococcus pneumoniae (do not possess Lancefield protein)
Viridans streptococci (Alpha hemolytic streptococci that lack Lancefield protein)
More than 40 species of streptococci and related organisms have been reported to cause human infection
5. Streptococcus pyogenes (Group A)
Pharyngitis and cellulitis
Impetigo, Streptococcal toxic shock syndrome
Rheumatic fever (inflammation of body's organ system)
Glomerulanephritis
Enzymes and toxins produced by Streptococcus pyogenes
Streoptolysin o
Streptolysin S
Streptokinase
Hyaluronidase
DNAse
DPNase
Erythrogenic toxin
6. Streptococcus agalactiae (Group B)
Causes neonatal meningitis and septicemia
After transmission from normal vaginal flora of mother
7. Enterococcus
Found in the gut flora
Infection often follows from fecal contamination
Significant cause of UTI
Opportunistic infection (intraabdominal, septicemia and endocarditis)
8. Streptococcus pneumoniae
Leading cause of pneumonia in all ages often follows viral infection
Causes otitis media
Often spreads causing bacteremia and meningitis
9. Identification
10. Microscopy
Gram stain
Streptococcus and Enterococcus are Gram positive, round or ovoid cells occuring in pairs, short or long chains
Streptococcus pneumoniae is Gram-positive, lanceolate cells occuring in pairs, often with a visible capsule
Non-motile
Some may have capsule
11. Culture
Hemolysis on Blood Agar
Types of hemolytic activity demonstrated by streptococci:
Alpha hemolysis
Partial lysis of the RBCs surrounding a colony, causes a greenish discoloration of the medium
Beta hemolysis
Complete lysis of RBCs surrounding a colony, causes a clearing of the blood from the medium
Non-hemolytic (Gamma hemolysis)
No hemolysis takes place
No change in the media surrounding a colony
Primary isolation media
Blood Agar
Incubated at 5~10% CO2 at 35~37°C for 16~48 h
Throat swabs: anaerobically at 35~37°C for 16~48 h
Staph/Strep Agar
In air at 35~37°C for 16~48 h
CLED Agar
In air at 35~37°C for 16~48 h
Colonial appearance
12. Biochemical Reactions
Catalase test
As described with Staphylococcus, streptococci and morphologically similar organisms are usually catalase negative
Bile Aesculin Hydrolysis test used to differentiate Enterococci from Streptococci. Ability of the bacterium to grow in the presence of 40% bile and produce aesculinase. The aesculetin combines with ferric ions in the medium to form a black complex. 2 methods of examining results: examine for presence of dark brown to black halo around bacterial growth (negative has no color change) and examine under UV light at 360 nm (positive has no fluorescent; negative has fluorescent). Aesculin is a glycoside composed of glucose and a dihydroxycoumarin compound. Certain bacteria such as the Enterococcus are able to grow in the presence of bile and hydrolyze the esculin. The liberated dihydroxycoumarin complexes with ferric citrate present in the media to form a dark brown/black soluble compound. Enterococci, Lancefield Group D Streptococci hydrolyze esculin in 40% bile while others don't.
Streptococci Grouping (commercial kit)
PYR uses L-pyrrolidonyl-Beta-naphthylamide to detect the presence of an enzyme (L-pyrrolidonylarylamidase). Product of hydrolysis reacts with a reagent (N, N-methyl-aminocinnamaldehyde) to produce a red color. Enterococci and Streptococcus pyogenes are positive. A small amount of colony is rubbed on the surface of a PYR disc (available commercially). A drop of detector reagent is added. Observed for red color within 5 minutes. (Negative is orange/yellow color, no color change).
Bile solubility (optional) is used to differentiate between Streptococcus pneumoniae and other Alpha hemolytic streptococci. It determines ability of bacterial cells to lyse in presence of bile salts within a specific time. Streptococcus pneumoniae possess an autolytic enzyme which lyses the cell's own wall during division. Addition of bile salts (sodium deoxycholate) activate the autolytic enzyme and the organism rapidly autolyse. Other Alpha hemolytic streptococci do not possess this system and do not dissolve in bile. Rapid lysis of the pneumococcus in the presence bile cause by the activation of cell wall autolytic enzyme as a consequence of the detergent action of bile.
CAMP test (Christie, Atkins, Munch, Peterson) to identify Group B Streptococci. The hemolytic activity of streptococcal-hemolysin on RBC is enhanced by an extracellular factor produced by Group B Streptococci called the CAMP factor. Therefore, wherever the 2 reactants overlap in sheep BAP, an accentuation of the Beta-hemolytic reaction will be noted.
Single streak of the streptococcus perpendicular to a strain of Staphylococcus aureus that is known to produce beta hemolysin is made. The 2 streak lines must not touch one another. Plate incubated with an ambient atmosphere (room temperature). The plate should not be incubated in an anaerobic environment because many Group A Streptococci are positive in the absence of oxygen. Group B Streptococcus are positive.
13. Antimicrobial Sensitivity Testing (AST)
Optochin Sensitivity Test detects the organisms susceptibility to the chemical optochin. The chemical tests fragility of the bacterial cell membrane. It differentiates Streptococcus pneumoniae from other alpha hemolytic strep. Optochin disc placed at edge of primary inoculum on blood plate of specimen. Incubate as for Blood Agar culture. Positive result: zone of inhibition of ≥5 mm radius from edge of disc. Organism is Streptococcus pneumoniae. Negative result: no zone or zone <5 mm radius. Organism is not Streptococcus pneumoniae. Optochin causes Streptococcus pneumoniae to lyse due to changes in surface tension.
Bacitracin Sensitivity Testing differentiates beta hemolytic group A Streptococci from other beta hemolytic streptococci. Bacitracin is an antibiotic produced by Bacillus subtilis. It is used as topical antibiotic. It inhibits cell wall synthesis. Growth of Group A Streptococcus pyogenes is inhibited by low concentrations of bacitracin on Blood Agar. Most other staphylococci are not inhibited. Diameter of zone of inhibition (>10 mm = sensitive). Streptococcus pyogenes is sensitive to bacitracin and will not grow around the antibiotic disc. Other beta hemolytic streptococci are not sensitive to bacitracin.
004 Gram Negative Rods
Contents
- Species and Pathogenicity
- E. Coli
- Enterobacter
- Proteus spp.
- Klebsiella spp.
- Serratia
- Laboratory Diagnosis
- Microscopy
- Culture
- Biochemical Reaction
- Antimicrobial Sensitivity
1. Species and Pathogenicity
Enterobactericae is a large family of Gram negative rod. It is found in colon as part of normal flora. It can cause variety of diseases with different pathogenic mechanisms.
2. General Features
The family enterobactericae is facultative anaerobes.
All ferment glucose.
None have cytochrome oxidase.
All contain endotoxin in their cell walls.
Non spore forming.
All members are similar in morphology and cultural characters.
All can be differentiated by biochemical reaction.
Gram-negative bacilli (rods)
Mobile with peritrichate flagella
Some are non motile (Klebsiella, Shigella)
3. Escherichia Coli
E. Coli is the majority of GI normal flora (T/F). It is one of the opportunistic pathogens. It can cause extraintestinal infection include UTI, septicemia and neonatal meningitis.
Extraintestinal E.Coli infections are the common causes of septicemia and meningitis in neonates. 40% of gram negative meningitis cases is caused by E. Coli.
Newborn acquires infection in birth canal just before or during delivery when mother's vagina is heavily colonized.
4. Klebsiella
Gram-negative bacilli, short, non-motile, capsulate
Commensal in intestine
Klebsiella pneumoniae causes Fried-Lander pneumonia (severe fatal bronchopneumonia), neonatal meningitis, lung abscess, wound infection and UTI.
5, Enterobacter
Motile
Colony morphology resembles Klebsiella on MacConkey
Most commonly isolated species include E. Cloacae, E. Aerogenes. Both have been isolated from wounds, urine, blood, and CSF.
6. Serratia
Opportunistic pathogens associated with nosocomial outbreaks
Ferments lactose slowly
Resistant to multiple antibiotics
S. Marcescens
- Species that is clinically important
- Produces characteristic pink to red pigment (prodigiosin)
- Cause hospital acquired infections of urinary or respiratory tract
- Bacteremic outbreaks in nurseries and cardiac surgery and burn units
7. Proteus
Normal intestinal flora
Opportunistic pathogens
Cannot ferment lactose
4 species
2 species human pathogens include P. Mirabilis and P. Vulgaris. Both have been isolated from urine, wounds and ear and bacterimic infections.
8. Laboratory Diagnosis: E. Coli Diarrhea
Specimen: stool
Microscopic examination of fresh preparation
Isolation on MacConkey Agar plates
Identification by sugar fermentation
9. Laboratory Diagnosis: Klebsiella
Specimen: sputum, urine, pus
Microscopy: Gram stain shows short Gram-negative rods with unstained halo around them due to the presence of the capsule
Isolation: on Blood Agar and MacConkey Agar media, it shows mucoid lactose fermenting colonies
10. Laboratory Diagnosis: Proteus
Specimen: urine, pus
Microscopy: Gram-negative rods
Culture:
swarming growth occurs on Blood Agar
Non-lactose fermenting colonies occurs on MacConkey Agar
Identification: sugar fermentation (rapid urease activity)
11. Double Sugar Iron Agar Reaction
Differential Agar slants contain more than one sugar, and possess other indicators. In these slants, both respiration (aerobic) and fermentation (anaerobic) growth can take place. It is a very useful media for distinguishing among this group of organisms.
This media contains lactose and glucose, in a ratio of 10 lactose molecules to 1 glucose molecule, a ferric salt, and phenol red as indicator (yellow below pH 7, red above).
It is inoculated by stabbing into the butt of the tube with a needle, withdrawing the needle and streaking the surface of the slant.
Slant color:
Red does not ferment either lactose or sucrose
Yellow ferments lactose and/or sucrose
Butt color:
Red does not ferment glucose
Yellow some fermentation of glucose has occurred, acid has been produced
Gas formed seen as cracks in the Agar, bubbles or the entire slant may be pushed out of the tube
Black precipitate means H2S has been produced
Results:
E. Coli
- Facultative anaerobe
- Ferments glucose and lactose as a carbon source
- Produce gas
- Does not produce H2S
Klebsiella pneumoniae
- Facultative anaerobe
- Ferments glucose and lactose as a carbon source
- Produces gas
- Does not produce H2S
Enterobacter aerogenes
- Facultative anaerobe
- Ferments glucose and lactose as a carbon source
- Produces gas
- Does not produce H2S
Proteus vulgaris
- Ferment glucose but not lactose as carbon source
- Produces H2S
12. Enterotube Reactions
Yet, another set of media to differentiate among these genera is the commercially available Enterotube II (Roche Diagnostics, Montclair, N.J), which contains biochemical tests in miniature form.
Note: the percentage shown above a test in the images below indicates the likelihood (as percent) that a particular colony /clone of that specific organism (genus, species) would yield a positive test.
005 Gram Negative Rods
Contents
- Species and Pathogenicity
- Salmonella spp.
- Shigella spp.
- Laboratory Diagnosis
- Microscopy
- Culture
- Biochemical Reaction
- Antimicrobial Sensitivity
1. Species and Pathogenicity
a) Salmonella
Gram-negative, facultative anaerobic rods
Clear, colorless colonies
Non-lactose fermenters
Most produce H2S
2 species include S. Enterica and S. Bongori
Antigenic structures
- O antigen
- LPS on outer membrane
- heat stable
- Flagellar H antigen
- Vi antigen capsular antigen
- Surface polysaccharide
- prevents phagocytosis
- found in serotype Typhi (used in identification)
Humans are infected by ingesting organisms in food, water and milk contaminated with human or animal excreta.
Salmonella ser. Typhi and ser. Paratyphi have no known animal reservoir. Infections only occur in human.
Other Salmonella infect various animals become reservoirs and sources for human infection.
Enterocolitis: S. Enteritidis, S. Typhimurium, S. Choleraesuis
• Defined as an infection in the lining of the small intestine.
• It occurs when you swallow food or water that is contaminated with the Salmonella bacteria.
– Source primarily poultry, milk, eggs, egg products
• Insufficiently cooked
– Handling infected pets
• Transmission
– Cooking utensils to prepare contaminated meat can spread contamination to other food (e.g. chicken to salad)
– Direct person-to-person reported in institutions.
• Dehydration from diarrhea, especially in young children and infants, is a dangerous complication
Septicemia: S. Choleraesuis, S. Typhimurium, S. Enteritidis, S. Hirschfeldii
• Accounts only about 5-10 % of Salmonella infections
• Occurs in one of two settings
– A patient with chronic disease such as sickle cell anemia / cancer. Example of type of infection is osteomyelitis in a child with sickle cell anemia.
– Child with enterocolitis
Enteric fever: S. Typhi, S. Paratyphi A, S. Schottmuelleri, S. Hirschfeldii
• Typhoid fever
• Caused by Salmonella ser. Typhi
• Infection begins in small intestine, multiply
• Originate from infected individuals or carriers
• No known animal reservo
• Occurs most often in tropic & subtropic countries
• Improper sewage disposal, poor sanitation, lack of modern water systems assoc with outbreaks when organism reaches water source
• Carriers (esp. food handlers) are important source of infection
– Paratyphoid fever
• Similar to typhoid fever but less severe & fatality rate lower
b) Shigella
Not normal gastrointestinal flora
All species cause biliary dysentery (bloody diarrhea)
Presence in blood, mucus and pus in stool
Non-motile
Generally form clear, non-lactose fermenting colonies
Not produce H2S
Shigella are fragile organisms
Susceptible to physical and chemical agents (disinfectants, high concentration of acids or bile)
4 biochemically similar species
Divided into 4 major O antigen groups
- Shigella dysentery (group A)
- Shigella flexneri (group B)
- Shigella boydii (group C)
- Shigella sonnei (group D)
All have O antigen
Some strains have K antigen
Clinical infections
All species cause dysentery
Vary in epidemiology, mortality, severity of disease
Most common in developing countries
Shigella dysenteriae is the most virulent species with significant morbidity and mortality
Shigella boydii
Baciliary dysentery (enterocolitis)
Penetration of intestinal epithelial cells
Attachment of organism on mucosa
Local inflammation
Shedding of intestinal lining
Formation of ulcers following epithelial penetration
Symptoms vary from asymptomatic to severe
High fever, chills, abdominal cramps
Pain
Symptoms appear 24~48 hrs after ingestion of organism
Multiple in small intestine and move to colon
Watery diarrhea
Followed by bloody stools containing mucus and leukocytes
Patients suffer from extremely painful bowel movements
Some cases are life-threatening especially if extra intestinal complications develop
2. Specimen collection and transport
In enterocolitis, the organism is most likely easily isolated from a stool sample
In enteric fever, a blood culture is done for 2 weeks of illness
Bone marrow cultures are often positive
Sputum from hospitalized patients containing enteric bacteria need complete identification
3. Culture
MacConkey
- 16~24 hrs incubation in air 35~37°C
- Pink (lactose fermenting) or colorless (non-lactose fermenting)
- Size and shape vary
CLED
- 16~24 hrs incubation in air 35~37°C
- Yellow (lactose fermenting) or blue (non-lactose fermenting)
Xylose-Lysine-Deoxycholate Agar (XLD)
- Stool sample or black halo
4. Identification
Conventional biochemical tests
Commercial identification systems
5. Growth on MacConkey Agar
6. Double Sugar Iron Agar Reaction
Differential agar slants contain more than one sugar, and possess other indicators.
In these slants both respiration (aerobic) and fermentation (anerobic) growth can take place.
It is a very useful media for distinguishing among this group of organisms.
Double Sugar Iron Agar slant contains lactose and glucose, in a ratio of 10 lactose molecules to 1 glucose molecule, a ferric salt, and phenol red as indicator (yellow below pH7, red above).
It is inoculated by stabbing into the butt of the tube with a needle, withdrawing the needle and streaking the surface of the slant.
Results:
Salmonella enteritidis ser Typhimurium
- Ferments glucose but not lactose as a carbon source
- Produces gas and H2S
Salmonella Typhi
- Ferments glucose but not lactose as carbon source
- Produces H2S
- Unlike S. Enteritidis ser Typhimurium, does not produce gas
Shigella boydii
- Ferments glucose but not lactose as carbon source
- Does not produce gas or H2S
7. Enterotube Reactions
Yet, another set of media to differentiate among these genera is the commercially available Enterotube II (Roche Diagnostics, Montclair, N.J), which contains biochemical tests in miniature form.
Note: the percentage shown above a test in the images below indicates the likelihood (as percent) that a particular colony /clone of that specific organism (genus, species) would yield a positive test.
006 Gram Negative Rods
Genus
- Species and Pathogenicity
- Campylobacter
- Helicobacter
- Vibrio
- Laboratory Diagnosis
- Microscopy
- Culture
- Biochemical Reaction
- Antimicrobial Sensitivity
1. Species and Pathogenicity
a) Campylobacter: Campylobacter jejuni and Campylobacter coli
Important properties:
Gram-negative bacilli
Actively motile
Small, slender, spirally shaped
Microaerophilic (5%)
Grow best at 42~43°C
b) Helicobacter
Morphology and characteristic:
Gram negative
Spiral
Biochemically inactive
Motile, unipolar
Noncapsulated
Produces a very powerful urease, catalase and oxidase
Strictly microaerophilic
c) Vibrio
Found as natural resident of aquatic environments
Most important pathogens of human are
- Vibrio cholera
- Vibrio parahaemolyticus
- Vibrio vulnificus
Morphology:
Gram negative
Single polar flagellum
Curved or rod shaped
Noncapsulated
Non sporing
2. Laboratory Diagnosis: Microscopy, Culture, Biochemical reaction, Antimicrobial sensitivity
a) Campylobacter
Laboratory diagnosis:
- Diarrhea-stool sample culture on Skirrow's medium (blood agar containing antibiotics inhibit other fecal flora)
- Plate incubate at 42°C, in microaerophilic (5%)
- Identified by failure to grow at 25°C
- Oxidase positive
Microscopy:
pleomorphic (many kinds of shapes)
Close up view of Campylobacter jejuni on Blood Agar illustrating raised, gray-white and somewhat mucoid colonies
Gram stain of Campylobacter jejuni illustrating pleomorphic gram-negative bacilli, some short and curved, others forming spirals
b) Helicobacter
Direct microscopy biopsies
- Warthin-starry silver staining
- Haematoxylin-eosin staining
- Gram staining: short curved, spiral gram negative bacteria
Culture
- Inoculated on chocolate agar and campylobacter agar (Skirrow's)
Non-cultural diagnosis
Biopsy urease test
- Biopsy tissue in urea solution containing a pH indicator
- Positive within 2 min to 2 hrs if H. pylorus is present
Urea breath test
- Urea C¹³ or C ¹⁴
- If positive the isotope gives high reading in the CO2 emitted in the breath
c) Vibrio
Culture
Specimen: watery stool, rectal swab
Nutrient Agar: moist, round disc colonies
MacConkey Agar: colorless then pink
Blood Agar: do not produce hemolysis
TCBS media: V. Cholerae produces yellow colonies (sucrose), V. Parahaemolyticus and V. Vulnificus produce green colonies (non-sucrose fermenting)
Transport or holding media
- Venkataraman-Ramakrishnan (VR) medium
- Contains 20gm salt and 5g peptone in 1L dH2O
- Vibrios not multiply, remain viable for several weeks
- Cary-Blair medium
- Suitable transport medium for Salmonella, Shigella and Vibrio
- Enrichment medium
- Alkaline peptone water
Biochemical reaction
- Ferment glucose, mannitol, sucrose, maltose with acid production
- Non-lactose fermenting
- Catalase and oxidase positive
Other tests: growth of V. Parahaemolyticus in 8% NaCl
007 Gram Negative Cocci and Rods
1. Gram Negative Cocci
Medically relevant organisms
Neisseria gonorrhoeae: Gonorrhoeae
Neisseria meningitidis: Meningitis
Moraxella catarrhalis: Respiratory illness
Acinetobacter baumanii
Coccobacilli (rod in exponential phase, spherical in stationary phase)
Associated with nosocomial infections
Bacteremia, secondary meningitis, ventilator associated pneumonia
2. Lab Diagnostic Identification
Most Neisseria, Moraxella and Acinetobacter can grow on simple media, under varying temperatures
N. Meningitidis and N. Gonorrhoeae (fresh isolates) are sensitive to fatty acids and trace metals present in media
Require an enriched medium containing blood or blood products
CO2 atm and the incubation at 37°C required for growth
3. Culture
4. Microscopic Appearance
Neisseria gonorrhoeae
Gonococcus
Diplococci
Coffee-bean shape
Fragile, do not survive long outside human body
5. Identification Scheme for Gm Neg Aerobic Cocci
Positive Oxidase Test
Dip a sterile cotton-tipped wooden stick into a fresh dye solution
Press the cotton tip against the colony to be tested
A colour change within 10 seconds indicated a positive result
Detects presence of cytochrome oxidase (enzyme) using a dye
The dye is colorless in the reduced state. Purple in oxidized state.
Oxidase Test
Positive test commonly used for presumptive identification of Neisseria sp.
Organisms contain large amounts of the enzyme (cyt. oxidase)
M. Catarrhalis and some Gram negative bacilli (Pseudomonas) also give positive test
Ability to grow on NYC media
NYC media is an enriched media containing antibiotics (Vancomycin, colistin, nystatin and trimethoprim)
NYC media permits the growth of the pathogenic N. Meningitidis and N. Gonorrhoeae but not the nonpathogenic Neisseria, Moraxella catarrhalis and other gram negative organisms
Carbohydrate degradation
Oxidative breakdown of the sugars glucose, maltose and sucrose can help to distinguish among Neisseria meningitidis, Neisseria gonorrhoeae and Moraxella catarrhalis
Haemophilus
6. Important properties
Catalase positive, oxidase positive, and ferments glucose
Small, gram-negative rod
Growth factors
7. Morphology
Pleomorphic Gram-negative rods
In clinical specimens, haemophilus is small, uniform coccobacillus
In cultures, haemophilus is longer, filamentous rods
8. Growth factors
All require blood for growth
Requirement based on X and V factors
X factor is haemin
V factor is nicotinamide adenine dinucleotide (NAD) or phosphate (NADP)
9. Growth on laboratory media
Chocolate Agar with X and V factors
Horse or Rabbit Blood Agar with X and V factors
Ordinary Blood Agar contains low level of V factor
S. Aureus excrete V factor
Growth around S. Aureus colonies is called satellitism
10. Diseases
Meningitis
Epiglottis
Bacteremia
Septic arthritis
Pneumonia
Otitis media, sinusitis
Cellulitis
Occur in children 6 months to 6 years
Polysaccharide capsule is the major virulence factor
11. Specimens
CSF
Blood
Throat swab
Sputum, pus
Collected in sterile containers, should never be refrigerated
12. Culture
CSF is plated promptly on Blood Agar or Chocolate Agar
Blood culture is usually positive in case of epiglottitis and pneumonia
Sputum culture is homogenized by shaking with sterile water and glass beads for 15~30 mins
H. Influenzae requires X and V factors
Grows on Chocolate Agar
Satellitism around S. Aureus on Blood Agar
13. Satellitism
H. Influenzae streak on Blood Agar
S. Aureus streak across the same Blood Agar plate
Incubate
The colonies of H. Influenzae will be large alongside of S. Aureus
X factor is available in high concentration near streptococci growth
Identified by its colony morphology, satellitism, Gram-staining
14. X and V requirement of Haemophilus sp
15. Direct microscopy
Colonies of H. Influenzae appear as convex, smooth, pale, gray or transparent colonies
Gram-negative, coccobacilli, with no specific arrangement
Catalase and oxidase positive
16. Important properties of Brucella
Small, Gram-negative rods
Without capsule
3 animal reservoirs are B. Melitensis (goats and sheeps), B. Abortus (cattle), B. Suis (pigs)
Brucellosis is also considered an occupational disease because of a higher incidence in people working with animals (slaughterhouse cases)
17. Laboratory Diagnosis
Requirements of CO2 for growth
Production of urease and H2S
Use of specific antiserum
Grow slowly on Blood and Chocolate Agar
Fail to grow on MacConkey, EMB or other enteric media
Brucella sp are not included in the databases of commercially available kits for the identification of gram-negative organisms
18. Moraxella
Gram-negative, aerobic, oxidase positive
Diplococcus
Rarely caused infections in humans
008 Gram Positive Bacilli
Contents
- Species and Pathogenicity
- Bacillus
- Cornyebacterium
- Listeria
- Laboratory Diagnosis
- Microscopy
- Culture
- Biochemical Reaction
- Antimicrobial Sensitivity
Species and Pathogenicity
1. Bacillus anthracis
Disease
Cause anthrax, common in animal, rare in human
Human disease occur in 3 main form: cutaneous, pulmonary and gastrointestinal
2001 outbreak 18 cases, 5 died
2. Important properties
Large Gram-positive rod with square end
Found in chains
Non motile
Its antiphagocytic capsule is composed of D-glutamate
3. Transmission
Spores of organism persist in soil for years
Cutaneous: Human most often infected (trauma to the skin). Allow the spores on animal products (wool, bristles) enter the skin
Gastrointestinal: Contaminated milk are ingested
4. Clinical findings
Cutaneous anthrax
95% of human cases of anthrax
Route of entry: skin
Sites involved: face, neck, hands, arms and back
Malignant Pustules: satellite lesions filled with serum or yellow fluid arranged around a central necrotic lesion which is covered by a black eschar.
Gastrointestinal anthrax: vomiting, abdominal pain and bloody diarrhea
Pulmonary anthrax: non specific respiratory tract symptoms (influenza)
Dry cough and substernal pressure
Death
5. Lab Diagnosis
Microscopy morphology
Gram stain, large square ended Gran positive rods, may appear end to end giving a bamboo appearance
Colonical morphology
Non hemolytic on 5% Blood Agar, raised large, grayish white
6. Cornyebacterium sp.
Found as free living saprophyte
Members of usual flora of humans and animals
Cornyebacterium diphtheriae is the most significant pathogen
Other species may cause infections in immunocompromised host
7. Important properties
Gram-positive rods
Has a characteristic"Chinese letter" arrangement, often with club-shaped swellings at the poles with irregularly staining segments or granules
8. Transmission
Human is the only natural host
Toxigenic and non toxigenic organism reside in the upper respiratory tract
Transmitted by airborne droplet
Also infect skin at the site of preexisting skin lesion
Occur in tropic
9. Pathogenesis
Organism establish and maintain itself in the throat
Exotoxin production
- Diphtheria toxin
- Inhibit protein synthesis
- Affect all eukaryotic cell
10. Clinical findings
Thick gray, adherent pseudo membrane over the tonsils and throat
Fever, sore throat, malaise
Toxin absorbed to bloodstream carried systemically
11. Growth of diphtheria bacilli
12. Identification
Microscopy
Gram stain: Gram-positive bacilli, Chinese letter pattern
Albert's stain for metachromatic granules
13. Culture
Isolation of bacilli requires media enriched with blood, serum or egg
Blood Agar
Loeffler's serum slope
Rapid growth, 6~8 hrs
Tellurite Blood Agar
Tellurite is reduced to tellurium, gives gray or black color to the colonies
14. Listeria monocytogens
General characteristics
Gram positive
Widespread in nature
Known to infect a wide variety of animals
Able to grows well at cold temperatures
15. Disease
Immuno suppressed adults
- Febrile gastroenteritis
- Meningitis and sepsis
- Renal transplant patients
Newborns
- Meningitis and sepsis
- Transmission across the placenta or during delivery
16. Clinical findings
Newborn is infected at the time delivery can have acute meningitis 1~4 weeks later
The infected mother either is asymptomatic or has an influenza like illness
Watery diarrhea, fever, headache, abdominal cramps
Little vomiting
17. Laboratory Diagnosis
Gram staining
- Appearance of gram positive rods
- Resembling diphteroids
Culture
- Small, gray colonies with narrow zone of beta hemolysis on Agar plate
- Confirmed with the presence of motile organism
009 Mycobacterias and Spirochetes
Contents
- Species and Pathogenicity
- Mycobacterium
- Sphirochete
- Laboratory Diagnosis
- Microscopy
- Culture
- Biochemical Reaction
- Antimicrobial Sensitivity
1. Mycobacterias
Fungus-like bacteria, Acid-fast bacteria
Slender bacilli
Show branching filamentous forms resembling fungal mycelium
Difficult to stain, but resist decolorization
Aerobic, non motile, non capsulated, non sporing
2. Mycobacterium tuberculosis (Koch's bacteria)
• Important properties
Grows slowly (doubling time 18 h)
Clinical specimen must be held for 6~8 weeks before being record as negative
Obligate aerobe
Resistant to acids and alkalis (NaOH concentrate specimens)
Resistant to dehydration
• Disease
Tuberculosis
Cause more death than any other single microbial agent
Humans are the natural reservoir
Transmitted from person to person via aerial route
The droplet are expelled during coughing, sneezing and even talking
• Laboratory Diagnosis
Sputum is collected in clean wide mouthed container
Morning specimen collected 3 consecutive days
Children-gastric washing (tend to swallow sputum)
• Culture
Acid fast staining of sputum-initial test
Culture on Lowenstein-Jensen medium for 8 weeks
Will not grow on Blood Agar plate
BACTEC medium
Production of radioactive carbon dioxide in 2 weeks
Liquid medium
• Biochemical Reaction
Niacin test
Metabolic by product all mycobacteria
Canary yellow color shows positive
Human tubercle-positive test
Catalase-peroxidase test is positive
• Treatment
Multidrug therapy
Isoniazid (INH), rifampin (6 months)+pyrazinamide (2 months)
3. Mycobacterium leprae
Slender, curved bacilli
Acid fast
Seen singly and in groups
• Important properties
Has not been grow in laboratory/artificial media
Grown in mouse footpad
Human are the natural host
OT is 30 C, grows preferentially in the skin and superficial nerve
Grows very slowly
Antibiotic therapy
• Disease
Tuberculoid Leprosy
- Cell mediated immune (CMI) response to the organism limit it's growth
- Very few acid fast bacilli seen
Leprotamous leprosy
- CMI is poor
- Skin and mucous membrane
- Contain large numbers of organism
• Laboratory Diagnosis
Acid fast stain of skin lesion or nasal scraping
• Treatment
Dapsone
Combination therapy
Dapsone+rifampin+clofazimine for leprotamous leprosy)
Dapsone+rifampin for Tuberculoid form
2 years or until the lesion free of organism
4. Spirochetes
• General characteristics
Elongated, motile (endoflagella-axial filament), spiral bacteria, Gram-negative
Structurally complex
Central protoplasmic cylinder bounded by a cytoplasmic membrane and cell wall of similar structure to that of Gram-negative bacteria
Larger spirochetes are gram-negative, others are too thin to be seen with light microscope
Seen under Dark Ground Microscope
• Classification
Human pathogens belong to following 3 genera
- Treponema
- Leptospira
- Borrelia
Others (saprophytes) are found in water, sewage and in mouth and genital tracts of humans
5. Treponema pallidum
• Important properties
Thin, delicate with tapering ends
Have about 10 regular spirals
They are seen only by dark field microscopy, immunofluorescence
Have not been grow on bacteriologic media or cell culture
• Characteristics
Morphology
Thin, 10u with tapering ends
Culture
Do not grow on artificial media
Antigens induce specific antibodies which can be detected by immunofluorescence
• Transmission
From spirochetes-containing lesions of skin or mucous membranes (genitalia, mouth, rectum) of infected person
By intimate contact
Can also transmitted from pregnant woman to their fetus
• Pathogenesis
Produce no toxin or enzyme
Infect endothelium of small blood vessel
Causes Syphilis which can be
- Venereal
- Congenital
- Non Venereal
Primary lesions
Laboratory Diagnosis
• Microscopy
Dark field microscopy
Direct fluorescent antibody (DFA) test
Not seen on a Gram stain smear
• Non specific serology test
Wasserman complement fixation test
Kahn flocculation test
VDRL (Venereal Disease Research Laboratory)
RPR (Rapid Plasma Reagin)
• Specific serology test
Using Nichol's strain
T. Pallidum immobilisation (TPI) test
T. Pallidum hemagglutination test (TPHA)
Fluorescent treponemal Ab absorption test (FTA-ABS)
Very specific, standard reference test
• Treatment
Penicillin
2nd line
Erythromycin, Tetra/Doxycycline
Neurosyphilis
Ceftriaxone
010 Actinomycetes & Propionibacterium
1. Actinomycetes
Classification
- Order: Actinomycetales
- Shows fungus-like characteristics such as branching in tissues or in culture (look like mycelia).
- The filaments frequently segment during growth to produce pleomorphic, diphtheroidal, or club shaped cells.
- The cell wall and the internal structures are typical of bacteria rather than fungi.
- Some are aerobic and others are anaerobic
- All are slow growing
The anaerobic genera: Actinomyces, Arachnia, Bifidobacterium
Morphology and cultural characteristics
- G+ branching, or diphtheroid-like bacilli
- Anaerobic and require CO2 for growth
- Non-sporing
- Will grow on anaerobic BA or PEA.
- A. israelii, the most commonly isolated species, produces rough, granular colonies that resemble molars.
Biochemistry
- ID by gas liquid chromatography (GLC) of metabolic by products or fluorescent antibody studies
Clinical significance
- Are part of the NF found in the cavities of humans and other animals.
- All may cause actinomycosis or “lumpy jaw” which is a cervicofacial infection that used to occur following tooth extractions or dental surgery which provided traumatized tissue for growth of the microorganism which may also invade the bone.
- This is rare today because of prophylactic antibiotic therapy.
- May cause thoracic or abdominal infections
- May cause meningitis, endocarditis or genital infections
Granules
Actinomyces from Greek "actino" that means mucus and fungus, is a genus of the actinobacteria class of bacteria. They are all Gram-positive and are characterized by contiguous spread, suppurative and granulomatous inflammation, and formation of multiple abscesses and sinus tracts that may discharge sulfur granules. They can be either anaerobic or facultatively anaerobic . Actinomyces species do not form endospores, and, while individual bacteria are rod-shaped, morphologically Actinomyces colonies form fungus-like branched networks of hyphae.
Biochemistry
- The organisms are identified based on sugar fermentations and hydrolysis reactions (caseine, tyrosine, etc)
2. Propionibacterium
Classification
- Two species P. Acnes and P. Granulosome
- Are described as anaerobic diphtheroids, though some can grow in CO2.
- Most clinical isolates are P. acnes which is part of the NF of skin.
Morphology and cultural characteristics
- Pleomorphic, small G+B, may have Chinese letter configurations or may be branching.
- Grow well on CBA, producing tiny translucent to opaque and white to gray colonies.
- Growth may be slow.
- Anaerobic, though occasional strains of P. granulosum grow in CO2
Biochemistry
- Catalase positive
- Indole +/-
- Ferment glucose
- Produce caseinase
Virulence factors
- Protease
Clinical significance
- Is part of skin NF
- Has been implicated in causing acne
- During adolescence more sebum is produced, and P. acnes metabolizes it to produce fatty acids.
- These may contribute to the inflammatory response seen in acne.
- Has also been isolated from joint infections
011 Chlamydia Mycoplasma
1. Chlamydia
Are obligate intracellular parasites
Cell walls are similar to the cell walls of G-B, but lack muramic acid
Have a complex developmental cycle
The infectious form is called an elementary body (EB) which is circular in form and is taken into the cell by induced phagocytosis.
Inside the phagocytic vesicle replication takes place
• Identification
Direct methods – stain tissues with Giemsa or use a direct fluorescent antibody technique.
The most sensitive method is to culture the organisms in tissue cultures and then stain the infected tissue culture cells
A complement fixation serological test is available as are DNA based tests.
• Virulence factors
Toxicity from attachment and penetration
• Clinical significance
Chlamydia trachomatis – serotypes A-K and L1,2,3; the serotype determines the clinical manifestation.
Genital tract infection (serotypes D-K) – is the major cause of non gonococcal urethritis; is sexually transmitted and frequently found concomitantly with N. gonorrhoeae
In males symptoms include urethritis, dysuria and it sometimes progresses to epididymitis
In females symptoms include mucopurulent cervical inflammation which can progress to salpingitis and PID.
Inclusion conjunctivitis – this occurs in both newborns and adults and a genital tract infection is the source of the infection (serotypes D-K); is a benign, self-limited conjunctivitis which heals with no scarring
• Clinical Syndromes
1. Trachoma
Chronic infection or repeated infection
inflammation and follicle formation involving the entire conjunctiva
Lymphogranuloma venereum (serotypes L1, 2, 3) is a venereal disease that occurs in poor, tropical areas.
Upon infection, widespread dissemination takes place and a primary, painless lesion (either a vesicle or an ulcer) occurs at the site of entry within a few days.
This heals with no scarring.
A secondary stage occurs 2-6 weeks later with symptoms of regional suppurative lymphadenopathy (buboes) that may drain for a long time and be accompanied by fever and chills.
Arthritis, conjunctival, and CNS symptoms may also occur.
A tertiary stage may occur and is called the urethrogenital perineal syndrome.
This is characterized by structural changes such as non-destructive elephantiasis of the genitals and rectal stenosis.
• Laboratory diagnosis
1. Cytology
Examination of stained cell scrapings for the presence of inclusion bodies
2. Culture
the most specific method for diagnosis
cultures of susceptible cells
iodine-staining inclusion bodies
3. Serology
Detection of high titer IgM antibodies is indicative of a recent infection
2. Chlamydia a life cycle
3. Mycoplasma
2 medically important genera:
Mycoplasma
Ureoplasma
Three common clinical isolates – M. pneumoniae, M. hominis, and U. urealyticum
• Morphology and cultural characteristics
Do not possess the distinctive cell wall of bacteria
Plasma membrane is the outermost part of the organism and is unique in bacteria in that it has a high content of sterols that act to prevent osmotic lysis
Very small in size (too small to be seen with an ordinary light microscope) and highly pleomorphic
Don’t stain with a Gram’s stain
Non-motile
May possess a capsule
Although some are free living, most are closely adapted parasites
Grow on media enriched with serum (need cholesterol)
Grow beat at 35-370 C either aerobically or anaerobically
M. pneumoniae grows in 5-14 days, M. hominis in 2-4 days, and U. urealyticum in 24-28 hours.
M. pneumoniae colonies resemble fried eggs and can be stained with Dienes stain (they stain blue)
• Identification
M. pneumoniae
Isolation in culture
Ability of colonies to hemolyze guinea pig RBCs
Rise in specific antibody titer
Cold agglutinin test – a nonspecific test in which the patient produces cold reacting antibodies that agglutinate type O human RBCs at 40 C, but not at 370 C
A single titer of 1:128 is significant and occurs in 7 days and disappears in 6 weeks.
• M. hominis
Isolation in culture
No hemolysis of guinea pig RBCs
• U. urealyticum
Urease production
• Virulence factors
Not invasive and simply colonize cell surfaces through specific binding
Damage to host tissues may be due to toxic metabolic products
• Clinical significance
M. pneumonia – the major cause of primary, atypical pneumonia (walking pneumonia)
Transmitted by droplet infection
Genital tract infections - caused by M. hominis and U. ureolyticum which may also be found as part of the NF in the genital tract
May cause nongonococcal urethritis, PID, post-partum fever, infertility, stillbirth, spontaneous abortion, and acute urethral syndrome
Quiz 1 notes
Bacterial Taxonomy
Gram staining
Biochemical Testing
Microscopy
Catalase
Coagulase
AST (novobiocin) Staphylococcus saprophyticus resistant, Staphylococcus epidermidis sensitive
Lancefield Group & Hemolysis Streptococcus
Biochemical Reaction
Quiz 2 notes
Shigella boydii is a glucose fermenting org
Skirrow's campylobacter
Gastric ulcers helicobacter short curved spiral gram -ve Choco Agar
Vibrio culture TCBS not Loeffler's serum transport medium Venkataraman Ramakrishnan
Swarming Proteus glucose fermentor
Enterobacteriacaea not oxidase positive
Shigella boydii cause bacillary dysentery
Salmonella Mac
Klebsiella gram -ve short non motile capsulated TSI glucose lactose
Salmonella enterica produce H2S
Oxidase positive Pseudomonas
Non motile clear non lactose fermenting not produce H2S Shigella
Quiz 3 notes
Mycobacterium tuberculosis, acid-fast staining prepare smear of sputum suspected, Direct smear: ZN, droplet, × Blood Agar, sputum sample
Mycobacterium leprae, Leprotamous leprosy, Tuberculoid leprosy
Mycobacterium, × facultative anaerobe, ✓ obligate aerobe
VDRL, syphilis, Treponema pallidum, RPR, Dark field microscopy, transmission: intimate contact
Neisseria gonorrhoeae grow on NYC, glucose
Dark ground microscopy, Leptospira interrogans, spiral, × Gram-positive
Haemophilus, haemin and nicotinamide adenine dinucleotide (Choco Agar),
Haemophilus ducreyi, requires X factor
Bacillus anthracis, bamboo appearance, non hemolytic on 5% Blood Agar, gram-positive square end, non motile, antiphagocytic capsule D-glutamate
Actinomycetes, hyphae, endospores anaerobic, diphteroid-like bacilli
Mycoplasma pneumoniae, droplet
Cornyebacterium diphtheriae, Chinese pattern, Albert's stain for metachromatic granules, Tellurite Blood Agar, transmitted by airborne droplet
SUMMARY
Chapter 1
Taxonomy is the practice and science of classification.
Family: (a group of related) genera
Genus: species
Species: strains
Type: a set of strain within a species
Strain: single isolate of a particular species
Decolorization fail, retain purple.
1. Isolation and observe growth in pure culture
- streaking
- colonies visible to naked eye appear after incubation
- size, texture, color, hemolysis
2. Microscopic examination
- gram stained
3. Species identification
- bacteria are speciated using isolated colonies-add 24 hrs
- generally use physiological tests
4. AST
- susceptible to specific antibodies
Gram positive purple/blue, Gram negative pink
Lab temperature 37
OF glucose, oxidative & fermentative, bromthymol blue green turns yellow fermentative
Simmon citrate, utilize citrate as carbon source, bromthymol blue indicator green turns Prussian blue
Phenol red fermentation broth, acid and gas production from variety of sugars, phenol red indicator turns yellow (sugar fermented, acid)
Urease, identify Proteus from non-lactose fermenting enterics, phenol red turns deep pink (alkaline ammonia)
H2S, SIM agar is used, insoluble black precipitate is positive (ferrous ammonium sulfate)
Phenylaline deaminase, deaminate phenylaline into phenylpyruvic acid, dark green (ferric chloride react with phenylpyruvic acid)
TSI, H2S production as well as carbohydrate fermentation
Indole, SIM agar with tryptophan, cherry red reagent ring is positive
MRVP, produce acid or neuralize into acetoin, MR red positive, VP pink positive
Chapter 2
Gram+, non sporing, non motile, occurs in singly, pairs and in irregular clusters, opaque colonies, facultative anaerobes, salt tolerant, highly resistant to drying, hemolytic
Staphylococcus aureus, coagulase/DNAse +, opportunistic, NF of skin, food poisoning, scalded skin, toxic shock syndrome
Staphylococcus epidermidis, sensitive to novobiocin, NF of skin, endocarditis, catheter
Staphylococcus saprophyticus, resistant to novobiocin, cyctitis, pyelonephritis, urethritis
Micrococci, gram +
Specimen collection: wound, lesion, pus, sputum
Culture: Blood Agar, MSA (selective & differential)
Chapter 3
Gram+, pairs/chains
Streptococcus pyogenes, group A, beta hemolysis, bacitracin +, PYR + (red)
Streptococcus agalactiae, group B, beta hemolysis, bacitracin -, CAMP + for group B
Streptococcus enterococci, group D, gamma hemolysis, 40% bile esculin + (black halo, fluorescence UV), PYR +
Streptococcus pneumoniae, alpha hemolysis, bile solubility & optochin +
Streptococcus viridans, alpha hemolysis
Lancefeld, proteins on cell wall
Alpha, partial lysis of RBC, greenish discoloration
Beta, complete lysis, clearing of blood, grey
Gamma, no hemolysis
Catalase, staphy +, strep -
Chapter 4
opportunistic=cause disease under certain condition/in certain hosts
primary=in anyone
facultative anaerobes, ferment glucose, non spore forming, none have cytochrome oxidase, contain endotoxin in cell wall
E. coli, primary & opportunistic, NF of GI, UTI, septicemia, neonatal meningitis, MacConkey red (lactose+), no H2S
Enterobacter, opportunistic MacConkey red, no H2S, motile
Proteus, opportuinistic swarming growth, gram -, non lactose, nosocomial UTI, oxidase -
Klebsiella, primary & opportunistic, gram -, non motile, capsulate, MacConkey red, no H2S, Fried-Lander pneumonia, neonatal meningitis, lung abscess, wound infection, UTI
Serratia, opportunistic hospital acquired infections, nosocomial & bacteremic outbreaks, TSA pink to red, prodigiosin, ferments lactose slowly, resistant to multiple antibiotics
Chapter 5 Primary pathogen
Salmonella spp, gram -, facultative anaerobes, non lactose, most produce H2S, enterocolitis, septicemia, enteric fever
Salmonella enteritidis ser typhi, produce H2S and gas
Salmonalle typhi, no gas
Salmonella bongori
Shigella, dysentery, non motile, no H2S, fragile, non lactose, O antigen
Shigella dysenteriae, group A
Shigella flexneri, group B
Shigella boydii, group C, no H2S and gas
Shigella sonnei, group D
Mac, CLED, XLD
Chapter 6
Campylobacter jejuni/coli, gram -, motile, slender spiral, microaerophilic (5%), grow at 42~43, oxidase+, Blood Agar, mucoid colonies, pleomorphic, Skirrow's, stool, diarrhea
Helicobacter pylori, gram -, spiral, biochemically inactive, motile unipolar, non capsulated, produces urease, catalase and oxidase, microaerophilic, needs CO2, Warthin-starry silver stain, H&E, choco & Skirrow's, Biopsy Urease -placed in urea contain pH indicator -positve within 2m~2h, Urea Breath (urea C13/14) positive if isotope gives high reading in CO2
Vibrio, gram -, unipolar, curved/rod, non sporing & capsulated, Mac (colorless then pink), Blood (no hemolysis), Nutrient (moist, round discs), ferment glucose, mannitol, sucrose, maltose with acid production, non lactose, catalase & oxidase +
TCBS: cholera yellow (sucrose), parahaemolyticus (8% NaCl) & vulnificus green
Transport medium: Venkatraman-Ramakrishnan, Cary-Blair (shigella & salmonella), Alkaline peptone water
Chapter 7
Neisseria, gram -, oxidase +, sensitive to fatty acids & trace metals, growth on Choco Agar 37
N. gonnorrhoeae, diplococci, fragile, gonococcus, NYC +, glucose
N. meningitidis, NYC+, glucose, maltose
Moraxella catarrhalis, oxidase +, NYC -, x sugar fermentation
Actinetobacter, oxidase-, catalase+
Haemophilus, small, gram-, rod or coccobacilli, catalase&oxidase+, glucose+, convex, smooth, pale, grey, transparent colonies
growth factor VX
X haemin , ducreyi
V nicotiamide adenine dinucleotide, parainfluenzae
XV, influenzae, aegyptius
Specime collection: CSF, blood, throat swab, sputum, pus, never be refrigerated
Brucella, non capsulated, B. melitensis (goats and sheeps), B. abortus (cattle), B. suis (pigs), Brucellosis, slaughterhouse case, grow slow on Blood Agar, Choco Agar, produce urease, H2S, fail to grow on EMB, MacConkey, need CO2
Moraxella, aerobic, oxidase +, diplococcus, rarely caused infections in humans
Chapter 8
Bacillus anthracis, anthrax, gram + rod with square end, chains, bamboo appearance, antiphagocytic capsule is composed of D glutamate, cutaneous, GI, pulmonary, non hemolytic on Blood Agar (greyish white)
Cornyebacterium, free living saprophyte, gram + rod, Chinese letter, airborne droplet, tropic, Blood Agar (rapid growth 6~8 hrs), Tellurite Blood Agar (grey/black colonies), Loeffler's serum slope, Albert's stain for metachromatic granules.
Listeria monocytogens, gram+. cold temp, meningitis, sepsis, resembling diphteroids, motile, small grey colonies (beta-hemolysis)
Chapter 9
Mycobacterium TB, Koch's Bacillus, acid-fast, fungus-like, obligate aerobe, resistant to acids, alkalis, dehydration, discoloration, non motile, non capsulated, non sporing, slow growing, airborne droplet, sputum, Lowenstein-Jensen medium (8 w), no growth on Blood Agar, BACTEC (2 w, produce radioactive CO2), liquid medium, catalase/oxidase +, Niacin (canary yellow)
Mycobaterium laprea, not grown in artificial media, grow in mouse footpad, slow growing, Tuberculoid leprosy, Leprotamous leprosy
Spirochetes, elongated, motile (endoflagella), spiral, gram -, dark ground microscopy
Treponema pallidum, immunofluorescence, dark field, tapering ends, intimate contact, syphilis (nevereal, nonvenereal, congenital), Direct Fluorescent Antibody, Wasserman complement fixation test, Kahn flocculation, VDRL, RPR, Nichol's strain, TPImmobilasation, TPHaemaAgglutination, FluoresecentTAntibody-ABSorption, penicillin, ceftriaxone, erythromycin
Chapter 10
Actinomycetes, gram+, fungus-like (mycelia) (hyphae), pleomorphic, diphteroid, sluc shaped, some aerobic others anaerobic, slow growing, non sporing, NF in cavities, actinomycosis (lumpy jaw), thoracic, abdominal, meningitis, endocarditis, genital infections, granulomatous inflammation, no endospores
Actinomyces, Arachnia, Bifidobacterium, A. israelii
Propionibacterium, gram+, anaerobic diphteroids, pleomorphic, Chinese letter, grow well on CBA (translucent to opaque/white to grey colonies), slow growing, catalase+, indole+/-, ferment glucose, produce caseinase, protease as virulence factor, isolated from joint infections
P. acnes NF of skin, P. granulosum
Chapter 11
Chlamydia, obligate intracellular parasite, cell wall lack muramic acid, Giemsa stain, DFA, trachomatis, genital tract infection, inclusion conjunctivitis (eye), lymphogranuloma venereum
Mycoplasma, small pleomorphic, non motile, may posses a capsule, free living/adapted parasites, Diene's stain (fried egg), hemolyze guinea pig RBC, Cold agglutinin test
M. pneumoniae (droplet), M. hominis (no hemolysis, NF GI), Ureoplasma urelyticum (produce urease, NF GI)
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