Family Micrococcaceae y
Includes Genus Staphylococcus and Micrococcus
y
All
are spherical Gram (+) cocci
y
All
are Catalase (+)
y
All
are non-motile
y
All
are strongly pigmented
y
May be aerobic (need O2) or facultativey anaerobic (can use 02 or use fermentation)
y
They
y
Colonies are circular, opaque and smooth
reduce nitrates to nitrites
Staphylococcus y
These
are non-motile cocci
y
Arranged
y
Only some may produce Capsule and Slime Layer which increases its virulence.
y
They
are facultative anaerobes. They produce acid from glucose a naerobically (fermentation)
y
They
produce acid from glycerol in the presence of erythromycin (antibiotic)
y
They
are susceptible to lysis with Lysotaphin (antibiotic against Staphylococcus aureus)
in Grape-Like Clusters (Greek: Staphyle)
Staphylococcus Aureus + + + + Golden Yellow
Coagulase Test Blood Agar Hemolysis Mannitol Salt Agar Trehalose Broth T est Pigment
Staphylococcus epidermidis +/White
* Coagulase Test (+) serum will coagulate.
* Mannitol Salt
* Blood Agar Hemolysis (+) will show clear/faint halo in culture.
* Trehalose Broth Test (+) turns medium bright yellow with gas bubbles
Three
Agar
Staphylococus saprophyticus + + Lemon Yellow
(+) yellow colonies w/ yellow zones.
major par ts of the Cell Wall tha t will distinguish Staphylococcus aureus from Micrococcus
1.
Peptidoglycan
2.
Teichoic
gives the cell wall a tight structure and it will help the c ell survive in host tissue. It is thicker in Gram (+) bacteria.
Acid (specifically: Ribitol Phosphate acid) acid)
y
These
y
It is important in the maintenance of t he normal physiologic function of the cell by regulating the cationic environment controlling the autolytic
are bacterial polysaccharides found in t he genera Staphylococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium and Listeria.
enzymes that function in the growth of the cell wall and the s eparation of daughter cells. It is an essential component of p hage receptor Staphylococcus aureus
y
Protein
3.
A It is a major protein component of the cell wa ll of Staphylococcus aureus. It is group antigen specific for most strains of Staphylococcus a ureus.
Coagulase (+) Staphylococci Staphylococcus
aureus
Location - It is frequently part of the skin flora found in the nose and on skin. Cultural Charac teris tics 1.
It is a facultative Anaerobe. (Grows in the absence of O2)
2.
Growth occurs at a optimum temperature of 37 C but (ideally) for pigment formation at 20 C - 25 C.
3.
Colonies are smooth, round, glistening, low convex with golden yellow colora tion on solid media.
4.
Hemolytic on Blood Agar
5.
Mannitol salt agar is used as selective and differential medium for Staphylococcus aureus from other Staph.
6.
It is Catalase (+) and is used to distinguish staphylococci from enterococci and streptococci.
7.
It is
8.
It is Oxidase (-)
9.
Staphylococcus saprophyticus resembles Staphylococcus aureus particularly on Mannitol Salt Agar.
Determinan ts
I.
Coagulase (+) which makes i t the most dis tinguishing characteris tic of Staphylococcus aureus from o ther Staphylococci .
of Pathogenici ty of Staphylococcus aureus
Surface An tigens
a.
Polysaccharides
possess antiphagocytic activity, it enables the encapsulated staphylococci to spread rapidly through tissue especially through
loose connective tissue. b. II.
Protein
recep tors proves the organism with adhesion mechanisms.
Extracellular Enzymes
a.
Coagulation/Coagulases this will clot the fibrinogen in th e blood.
b.
Lipases will explain the intense colorization of Stap hylococcus aureus in the sebaceous glands of greatest activity.
c.
Hyaluronidase
d.
Staphylokinase
e.
Nuclease
facilitate the spread of i nfection by degrading hyaluronic acid. (other name: Fibrinolysin) Fibrinolysin) converts pro-enzyme plasminogen to plasmin causing dissolution of clots.
responsible for thermostability. (65 C causes structural disruption)
III.
Toxins
a.
Entero toxin
i. b.
c.
6 types (A1 , B1 , C1, D1, D2, E) (ABCDEF in the book)
Type
A is most frequently associated w ith Staphylococcal food poisoning.
Exfoliative toxins
i.
Causes Scalded Skin Syndrome (SSS) in newborns
ii.
It does not illicit inflammatory response.
iii.
It does not primarily cause cell death.
iv.
It causes lysis of the intracellular attachment of cells in the glandular layer of th e epidermis.
Pyrogenic toxins
i.
It is associated with Toxic Shock Syndrome (TSS) severe and often fatal disorder characterized by multiple organ dysfunctions with (-) blood cultures.
d.
Cytolytic toxins i.
Hemolysins 1.
(Alpha) -hemolysin disrupts lysosomes and is cytotoxic for a variety of tissue cells. It usually involves macrophages and platelets but monocytes are immune.
2.
(Beta) -hemolysin has the ability to produce hot-cold lysis. It will enhance hemolysis.
3.
(Gamma) -hemolysin consists of 2 protein components that act synergistically, both being essential for hemolysis and toxicity.
4. ii.
(Delta) -hemolysin responsible for the damaging effect on the membranes.
Leucocidin (Leukocidin) this alters the cation permea bility of the White Blood Cells.
IDENTIFICATION OF ORGANISMS
COAGULASE TEST y
It detects the enzyme coagulase which binds plasma fibrinogen causing the organism to agglutinate or clot.
y
Coagulase has 2 forms o
Bound Coagulase (Clotting fac tor)
It is bound to the Cell Wall.
Bound coagulase reacts directly with fibrinogen from the plasma and alters it so it precipitates on the cell wall, causing the c ell to clump when bacterial suspension is mixed with plasma.
o
It is detected by the Slide Test.
Free Coagulase
It is liberated by the Cell Wall (extracellular protein enzyme)
Causes clot when colonies are incubated with plasma in 1-4hours at 37 C.
Clotting mechanism involves the activation of a plasma coagulas e reacting factor. (PRF)
Plasma with citrate is not suitable for use in this test because citrate utilizing bacteria may yield false (+) results.
A
It is detected by the Tube Test.
(+) coagulase test is us ed in differentiating Staphylococcus aureus from other Staphylococci.
CATALASE TEST y
The
presence of catalase enzyme in the test isolate is detected using hydrogen peroxide. If bubbles or froth forms, the organism is said to be catalase-
positive. o y
y
Staphylococci and Micrococci are catalase-positive.
If not, the organism is catalase-negative. o Streptococci and Enterococci are catalase-negative. Catalase (+) and Coagulase (-) organisms res embling staphylococci by Gram stain and c olonial morphology are Stomatococcus mucilaginosus.
THERMOSTABLE DEOXYRIBONUCLEASE TEST y
Colony in 5% blood agar is plac ed into the center of a microcapillary tube with the ends sealed. Then the tube is heated in a boiling water bath for 15 minutes. After it cools, break open the tube and inoculate onto DNA agar (toluidine blue DNA agar). Incubate at 37 C for 1-4hours and observe for a pink halo surrounding the innoculum. A (+) halo (zone of inhibition) has thermostable deoxyribonuclease and is identified as Staphylococcus aureus. A
y
LYSOSTAPHIN TEST Involves observing the smear for lysis. Staphylococcus aureus is ( +). Add 0.5ml of blood culture to 2 t ubes with Brain heart infusion agar. A dd 0.05ml lysostaphin to one tube. Incubate at 37 C with agitation on the shaker for 30 minutes then centrifuge for 10 minutes. Discard Supernatant fluid and place sediment on a slide, air dry, heat fi x, then Gram stain. There should be no cells present in the smear with lysostaphin because Staphylococcus aureus is susceptible to lysostaphin and there should be 90% of cells present in the control. y y y y y
MODIFIED OXIDASE TEST The oxidase test determines whether a microbe can oxidize certain aromatic amines to form colored end products. A colony from blood agar is placed on a filter paper and a drop of oxidase reagent (tetramethyl phenylene diamine dihydrochloride) in methyl sulfate is added. (+) test is indicated by a dark blue color in 2 minutes. Staphylococcus aureus and Enterobacteriaceae are Oxidase (-) Pseudomonas and Neisseria (Moraxella) are Oxidase (+) y y
y y y
BACITRACIN SUSCEPTIBILITY TEST Colony from Tryptic (Trypticase) Soy Agar (TSA) or broth is transferred t o Mueller-Hinton agar (MHA) A 0.04 bacitracin disc (antibiotic) is placed in the aga r, incubate at 37 C for 18 hours. No Growth = Micrococcus | Has Growth Staphylococcus y
y y
POLYMYXIN SUSCEPTIB ILITY TEST y y
Same procedure with Bacitracin but different antibiotic used. Opposite Results No Growth = Staphylococcus aureus
PARTICLE A GGLUTINATION TEST y y
It is used for rapid differentiation of Staphylococcus a ureus. It uses latex particles.
PHAGE TYPING y y
It is used in epidemiologic study It is time consuming and is not done routinely
PENICILLIN SUSCEPTIBILITY y
85-90%
of Staphylococcus aureus, even those acquired in the community ar e Penicillin (PCN) resistant; resistance is due to beta-lactamase production and
are affected by extrachromosomal plasmids. NONCULTURE ME THODS FOR IDENTIFICATION OF STAPHY LOCOCCUS AUREUS y
Antibody
Detection Radioimmunoassay(RIA), Enzyme-linked immunosorbent assay (ELISA), crossed immunoelectrophoresis, agar gel diffusion and
immunoprecipitation method Micrococcus Benzidine (+), Catalase (+) and Lysostaphin negative (not lysed) Does not produce acid in glycerol in th e presence of erythromycin. Does not produce acid from glucose anaerobically. y y y
Growth in 10% NaCl Nitrate Reduction Acid from glucose anaerobically Oxidase Activity Pigment
Micrococcus Varians + + + Yellow
Micrococcus Luteus + Yellow
Micrococcus Roseus + or + Pink
Coagulase (-) Staphylococci Staphylococcus
epidermidis
y
It normally resides in the skin and mucous membrane of humans and animals.
y
Has low virulence but life threatening if immune system is compromised.
y
Colonies are smooth circular and usually pale-transparent white.
y
Has high salt tolerance like Staphylococcus aureus b ut differ in being o
Coagulase, Thermonuclease, Mannitol (-)
y
Basically saprophytic (grows on and derives its nourishment from d ead or decaying organic matter) rather tha n pathogenic.
y
Infection is associated with cardiac valves, CNS shunt insertion and total hip replacement.
y
Some strains produce a viscous extracellular substance t hat facilitate colonization on plastic or metal.
Staphylococcus y
saprophyticus
Closely resembles to epidermidis but is said to be distinct by being o
Resistant to novobiocin
o
Resistant to Nalidixic acid
o
It does not grow on MacConkey agar
y
It produces an intense yellow pigment
y
It has a predilection to the urinary tract in sexually active women. It is t he common cause of Urinary Tract Infection (UTI); occurs in normal skin and in the peri-urethral and urethral flora but transiently in small numbers.
y
It does not ferment glucose ana erobically.
y
It is non-hemolytic and it does not contain protein A.
Stomatococcus
Mucilaginosus
y
Normal flora from clinically important sites.
y
It has been recovered from c ompromised patients, particularly drug users, as an agent of endocarditis and septicemia.
y
Colony and microscopic appearance is similar to Staphylococci but ad heres to the agar because of their capsule.
y
They
y
Inablility to grow in media with 5% NaCl distinguishes them from all other Micrococcaceae
y
They
are Catalase (-) and are vancomycin resistant. are susceptible to Penicillin drug of choice for initial therapy.
Strep tococci y
They
y
Oxidase (-)
are facultative anaerobes
y
Catalase (-)
y
Gram (+) cocci that grow in chains in liquid media.
y
Non-motile
y
Non-sporeforming
y
It will form large quantities of lactic acid as t he end product of carbohydrate metab olism.
Hemolysis Beta Beta Beta Alpha or none Alpha Alpha
Group A Group B Group D enterococci Group D non-enterococci Viridans Pneumococci There
Bacitracin Susceptibility + + + or -
Camp Test + -
Bile Esculin Medium + + -
Growth in NaCl + + -
Optochin and Bile +
are two classifications based on hemolysis on blood agar plate and the major cell wall carbohydrate present.
1.
Browns Classfication makes use of the Greek alphabet
2.
Lancefeild Classification makes use of the English alphabet. It is the most reliable classification. The basis for classification is on the antigenic group specific C substance which is expressed in t he cell wall of the organism. a.
Hemoly tic
i.
ii.
iii. b.
Beta Hemolytic will produce clear zone around colony as a result of complete lysis of RBC. 1.
Group A Streptococci
2.
Group B Streptococci
3.
Group C and G Streptococci
4.
Minute colonies intermediate, has alpha and beta hemolysis
Alpha Hemolytic will produce a zone of partial hemolysis with a greenish discoloration of the medium. 1.
Group D
2.
Streptococcus Pneumoniae
3.
Streptococcus Viridans
Gamma Hemolysis this group are non-hemolytic, will produce no hemolysis on blood agar.
Non-hemoly tic
Group A Streptococci ( Strep tococcus Pyogenes) y
One of the most frequent pathogen of humans.
y
Normal individuals will harbor the organism without symptoms in the d ormant state.
y
Infection will only occur in 3 conditions o
When the host is immunocompromised.
o
When the organism is able to exert its virulence. (If it is in abundance)
o
If it is introduced in vulnerable tissue or host.
Morphology y
Grows in short or moderately long chains
y
When grown in liquid medium, some strains will produce very long chains.
y
Has rigid cell wall where fimbriae (finger-like) appendages ar e found on its external surface .
Cultural Charac teris tics y
Mostly Beta-hemolytic in sheep blood agar.
y
Humans have inhibitory substances for streptococci, should not be used for culture.
y
Optimum pH = 7.4 7.6 at 37 C.
y
Enhanced growth in culture can be obtained in reduced O2 and increased CO2.
Laboratory Identification 1.
Bacitracin Tes t distinguishes between Beta Hemolytic Streptococci, it is no longer recommended because some strains of Streptococci are also sensitive to bacitracin.
2.
Pyrolydonyl
beta Napthylamine test (PYR) hydrolysis of this substrate via e nzyme to produce Beta Napthylamine.
Determinan ts
1.
a.
Positive result will produce a bright red color in 5 minutes.
b.
Negative Result will produce no color or light orange color.
of Pathogenici ty
Cellular Components a.
Lipotechoic acid is present in the cell wall of Group A streptococci. It is highly cytotoxic for a variety o f host cells.
b.
M protein - is the major virulence factor, it is found in the fimbriae of the cell membrane projecting on the bacterial cell wall of the organism. Mechanism of Pathogenici ty will bind to host fibrinogen and block the binding of complement t o the underlying peptidoglycan. It
i.
will allow survival of the organism by inhibiting phagocytosis. 2.
Extracellular Produc ts
a.
Hemolysin
i.
Strep tolysin O (SLO)
is a leukocidin that is toxic to neutrophils and induces a strong a ntibody response. The strong antibody
response is used as an in dicator for streptococcal infection. It is toxic to WBC and RBC. ii.
Strep tolysin S (SLS)
is a leukocidin which is t oxic to polymorphonuclear leukocyte. Is responsible for RBC lysis observed on Sheep
Blood Agar. b.
Pyrogenic Exotoxin
(A, B, C) its primary effect is Fever. Is also referred to as super antigen because of their a bility to stimulate T-cells to
proliferate even at a very low concentration. i.
Type
C Increases permeability of the blood-brain barrier to endotoxins and bacteria. It directly exerts its pyr ogenic effect on the
hypothalamus. c.
Enzyme
i.
Strep tokinase
enzyme that causes lysis of fibrin by activating plasminogen to plasmin. It is also antigenic and will produce
antistreptokinase. ii.
Hyaluronidase
it will lyse the gel-like consistency of hyaluronic acid.
Clinical Infection y
Impetigo
y
Streptococcal sore throat
y
Pharyngitis with scarlet fever
y
Post-Sterptococcal complication rheumatic fever and acute glomerulonephritis.
Group B Streptococci Streptococcus Agalac tiae y
They
colonize the Gastrointestinal Tract and genitourinary tract.
y
Often asymptomatic but can cause infection in infected host.
y
There
y
They
are destroyed by Pasteurization.
y
They
are the only streptococci with a specific morphological criterion.
are more in the low er GI tract especially in the rectal sid e than in then genitourinary tract.
Morphology y
In liquid media o
Appears
o
They
diplococcic or in short chains
have a longer chain in type A, C and G
y
There
is a double zone of hemolysis in rabbit blood agar.
y
Colonies are large, translucent to opaque and mucoid with a relatively small zone of hemolysis (1-2mm)
Presumptive Identification y
The
ability of the organism to hy drolyze sodium hippurate.
y
(+) Camp tes t is an extracellular infusible protein called Camp Factor w hich will interact with streptococcal beta hemolysin in sheep bl ood agar.
y
A
positive test is indicated by the presence of an arrow-shaped zone of hemolysis perpendicular to the organism.
Group C Streptococci y
Streptococci equi causes disease in horses.
y
Streptococci equisimilis will produce streptolysin O and streptokinase and other extracellular products. It is the source of Streptokinase used in thrombolytic therapy in humans.
y
Streptococci zooepidemicus is a co mplication associated with poststreptococcal Glormerulonephritis acquired after drinking milk. Trehalose
Streptococci equisimilis Streptococci equi and zooepidemicus Streptococci dysgalactitiae
+
Sorbitol + -
Disease Respiratory tract infection in humans and animals Post-Streptococal sequelae do not occur Pharyngitis, puerperal sepsis and endocarditis
Group D Streptococci They
are divided into 2 categories: Streptococci faecalis Streptococci faecium Streptococci durans
Enterococci
Streptococci Bovis
Non-en terococci
Streptococci Equinus
Grow in the presence of 6.5% NaCl and are not susceptible to penicillin. Increasingly seen in cases of Subacute bacterial endocarditis (SBE) and other infections. ---------------------
Enterococci
y
o
Are
the normal flora of the colon
o
They
o
Can grow in the presence of 6.5% NaCl and in the presence of bile salts.
are causes of opportunistic Urinary Tract Infection and some wound infections.
Non-en terococci
y
o
Can grow in the presence of bile salts but not with NaCl
o
They
may cause endocarditis.
Biochemical and Grow th Characteris tics
y
o
They
o
Some have distinct buttery odor in medium.
may be alpha-hemolyric or non-hemolytic.
o
NaCl plate medium will yield false negative results due to the presence of paraffin.
o
They
have the capacity to grow at 45 C and withstand temperatures above 60 C
o
They
can grow in the presence of 40% bile and hydrolyze esculin (selective and differential medium which is used to presumptively identify enterococci)
o
They
o
fail to grow in Mueller Hinton Agar (MH medium) but grow very well on Blood Agar. They can be differentiated from streptococci by their ability to grow at 45 C and by being thermostable at 60 C for 30 minutes.
o
Another
o
They
o
Serologic test Coagglutination Test and Rapid Latex test system
o
Treatment:
o
The
useful biochemical test is with the use of Brain Heart Infusion Br oth (BHIB) containing 6.5% NaCl in 18-24hours.
can be differentiated from streptococci by reduction of litmus milk in 4 h ours. Penicillin and Ampicillin
most accurate presumptive test for Group D is with the use of bile esculin medium (BEM) by forming a dark brown or black compound. Bile Esculin Medium (BEM) + + -
Strep tococcus
Salt Tolerance + -
Viridans
o
Normal flora of the oral, respiratory and gastrointestinal mucosa.
o
Oppurtunistic pathogen with low virulence
o
Generally Facultative, some are obligatory anaerobes.
o
Will grow well in most enriched media.
Pathogenici ty o
Ability
to adhere to epithelial and endothelial cells. Its adherence is the key factor in their ability to cause disease.
Cultural Charac teris tics o
They
o
Cannot grow in broth containing 6.5% NaCl
are alpha-hemolytic and produce a green discoloration on blood agar.
o
There
is a variation in colony formation.
o
If the organism has large amounts of M protein colony is large mucoid
o
If the organism has small amounts of M protein colony is smooth and glossy
Strep tococcus Pneumoniae o
Inhabits the nasopharyngeal or oropharyngeal area
o
Transmission
o
Bile sensitive
o
Alpha-hemolytic
is from person to person via r espiratory secretions and aerosols.
Morphology o
Encapsulated gram (+) lanceolate shape as observed under sputum smear
o
Occurs singly, in pairs or in short chains.
o
Fresh specimens are surrounded by a capsule.
Enterococci Non-enterococci Non-Group D Streptococci
Determinan ts o
o
of Pathogenici ty
Cell wall o
Peptidoglycan
o
Techoic
acid determinant for C p olysaccharide antigenic activity
Plasma membrane contains choline containing techoic acid
Cultural Charac teris tics o
It requires an enriched medium for primary isolation a nd routine culture.
o
For those strains that require an increased CO2 c oncentration, use the candle jar or CO2 i ncubator for primary isolation on solid media.
o
Young culture of encapsulated of pneumococci on blood agar plates will produce circular, glistening, dome-shaped c olonies.
o
Colonies of type 3 pneumococci: the colonies ar e larger and more mucoid t han those in the other types.
o
Trypticase
or brain heart infusion agar enriched with 5% defibrinated blood (sheep, rabbit or horse)
Laboratory Identification 1.
2.
Optochin Suscept ibili ty Tes t
a.
It is a quinine derivative that has like a detergent-like action.
b.
It causes selective lysis of pneumococci.
c.
Streptococcus pneumoniae exhibit zones of inhibition of at least 14mm or more (14-16mm) in diameter around the disc.
Bile Solubility Test a. b.
3.
Neufeld
It will selective lyse the colonies of Streptococcus pneumoniae while the other strains are i mmune to bile. Result is (+) if it appears clear, (-) if it is turbid. Quellung Reaction
a.
It is the most accurate and most reliable method for identification of pneumococci and other types.
b.
A
c.
Result: Positive reaction is when the capsule of pneumococci becomes more refractile and greatly swollen in appearance.
loopful of emulsified sputum with a loopful of antipneumococcal serum and methylene blue
Laboratory Diagnosis 1.
Direct Sputum Examination
2.
Culture
3.
Flexibility to antimicrobial agents.
Family Neisseriaceae Four Genera of Neisseriaceae Morphology Gram (-) cocci Gram (-) cocci Gram (-) rods Gram (-) rods Gram (-) rods
Neisseria Moraxella - Branhamella Moraxella Acinetobacter Kingella
Oxidase Test + + + -
Glucose + -
Penicillin (PCN) Sensitive Sensitive Sensitive Sensitive Sensitive
Genus: Neisseria Neisseria Neisseria gonorrhoeae Neisseria meningitidis Neisseria sicca Neisseria lactamica Neisseria mucosa Neisseria subflava Neisseria flavescens Branhamella Catarrhalis
Glucose + + + + + + -
Sucrose + + -
Maltose + + + + + -
Only two are common infecting humans: Neiss eria gonorrhoeae and Neisseria meningitides General Morphology for Neisseria o
Gram (-) cocci usually seen in pairs (diplococci) with one side flattened. (coffee-bean shaped)
o
Cell envelope is composed of 3 major elements
o
o
Cytoplasmic membrane
o
Rigid peptidoglycan layer
o
Outer layer contains glycopolysaccharide, phospholipids and proteins that ar e immunologically significant.
In smears, they are often seen in Polymorphonuclear leukocytes. They are referred to as Gram (-) intracellular diplococcic.
Lactose + (very slow) (like N.sicca) (like N.meningitidis) -
Cultural Charac teris tics of Neisseria o
It is a facultative anaerobic organism.
o
They
can produce catalase and cytochrome oxidase. Catalase (+) and Oxidase (+)
o
They
are highly susceptible to adverse c onditions. ( drying, chilling, exposure to sunlight and unfavorable pH)
o
It is a fastidious organism with complex n utritional requirements o
Needs Iron for growth
o
Incorporate in the culture media to n eutralize the inhibitory effect of fatty acid. Starch, cholesterol and albumin
Laboratory Identification Thayer-Mar tin
1.
or Martin Lewis test contains chocolate agar modified by the addition of starch, cholesterol a nd albumin.
a.
If you add vancomycin to inhibit Gram (+) bacteria.
b.
If you add colistin to kill Gram (-) enteric flora
c.
If you add nystatin to i nhibit yeast cells
Oxidase Tes t
2.
a.
It uses an indicator dye, whe n the microorganism is exposed to t he dye, the colonies will turn dark purple within sec onds.
Metabolism of Carbohydrate is used to distinguish the two important Neisseria - gonorrhea and meningitidis.
3.
Neisseria
a.
Neisseria meningitidis will produce acid from both glucose and maltose.
b.
Neisseria gonorrhea will produce acid from glucose o nly.
gonorrhea (gonococcus)
Morphology - Gram (-) intracellular diplococci (inside P olymorphonuclear Leukocyte) Cultural Charac teris tics o
It is more fastidious in its growth requirement than Neisseria meningitidis.
o
When grown in solid medium, they will exhibit several col onies: o
T1, T2
o
T3, T4 colonies
Determinan ts 1.
Pili
2.
Outer
colonies are produced in primary culture, they are s mall and dome shaped and pili a re present.
of Pathogenici ty
projecting from the process of the cell membrane. Adherence of piliated gonococci to a variety of epithelial cells is associated with its virulence. membrane componen ts
a.
Protein II it will promote adherence
b.
Peptidoglycan possess intrinsic toxicity for human fallopian tube mucosa. There will be sloughing off of ciliated c ells from the mucosa.
Immunoglobulin
3.
colonies are larger and flatter and pili are absent.
A Protease (IgA) it will inactivate immunoglobulin A .
Laboratory Diagnosis 1.
Microscopic examination of the organism Gram (-) diplococcic found within Polymorphonuclear Leukocytes.
2.
Culture blood culture (+) in disseminated disease .
3.
Serology Radioimmunoassay (RI A) for pilus antibody and Enzyme-linked i mmunosorbent assay (ELISA) for antipilus antibody.
Presumptive Identification Oxidase test
y
o
All
members of the Genus Neisseria as well as other genera are Oxidase (+)
o
The
o
Freshly prepared 1% solution of tetramethyl-p-phenylenediamine dihydrochloride is used.
o
Platinum loop is used instead of nichrome wir e (may give false (-)
o
Colony is placed on a filter paper strip and a drop of reagent is added.
o
Color change to pink, violet then black is observed within 10 seconds.
o
Slow reactions are not typical of Neisseria.
o
Test
enzyme indophenol oxidase oxidizes a redox dye which results in a change in color of the bacterial colony.
should be repeated on an 18-hour culture on blood agar or other medium not containing carbohydrates. If no colonies are observed, the medium
may be flooded with oxidase reagent to detect inapparent colonies, o
If material is obtained from the G enitourinary Tract, it is inoculated into Modified Thayer-Martin medium. It will show typical Oxidase (+) colonies consisting of Gram (-) diplococcic which is sufficient f or presumptive identification.
Confirmatory Identification y
Carbohydrate Degrada tion there result wherein there is glucose degradation without gas o
Recommended base medium cystine trypticase agar (CTA)
o
pH = 7.6 readily supports the gr owth of gonococci and meningococci.
o
The
cystine trypticase is heavily inoculated with the organism from a prepared subculture from ch ocolate agar plate 18-24 hours old in about 0.5ml of
saline or typtic soy broth. rd
o
Deposit 2-3 drops of the suspension on the surface of the medium and then stab the upper 3 of its depth using sterile cotton plugged capillary pipet.
o
Incubate with added CO2 at 35 C and production of acid is indicated by turbidity and yellow color of the upper layer of the medium.
Clinical Infection 1.
Mode of Transmission is by sexual contact.
2.
Incubation period is 2-8days (average 4 days)
3.
First symptom is painful urination.
4.
Most cases cause purulent urethritis in men a nd purulent cervicitis in women.
5.
Urethritis, prostatitis, epididymitis, cervicitis, salpingitis, proctitis, pharyngitis, perihepatitis and septic arthritis.
6.
Vulvovaginitis in children; opthalmia neonatorum in infants. (Credes prophylaxis, during delivery medicine is dropped in t he eye of the n ewborn)
Treatmen t 1.
Penicillin
2.
If allergic to penicillin, use tetracycline and spectinomycin.
Control/Preven tion report to city health Neisseria
meningitidis
Determinan ts
of Pathogenici ty
1.
Capsular Polysaccharide this property is an invasive property by inhibiting phagocytosis.
2.
Endo toxin
3.
Immunoglobulin
it is implicated in vascular damage ( necrosis and inflammatory response) A Protease (IgA)
Laboratory Diagnosis 1.
Identification of organism by gram staining.
2.
Use Theyer-Martin Selective medium
3.
Detection of meningococcal polysaccharide by coagglutination employing staphylococci with protein
4.
Brudzinskis sign stiffness of neck and involuntary lifting of the legs, positive for meningitis.
A
or countercurrent immunoelectrophoresis.
MacConkey (+) Oxidase (-) Gram (-) rods Family Enterobac teriaceae y
MacConkey (+)
y
Oxidase (-)
y
Gram (-) Rods
y
Most are free-living in nature (soil, water and plants)
y
Some are found in the intestines of man and animals. (Enteric or Enteric Bacilli)
y
Pseudomonas and Vibrio are also enteric but are not Enterobacteriaceae.
They
are divided in to two clinically relevan t members 1.
Opportunistic Pathogens
are part of the n ormal flora of the gastrointestinal tract of man.
a.
Escherichia, Klebsiella, Enterobacter, Serratia, Citrobacter, Proteus Providencia and Morganella
b.
They
normally dont cause disease in the GIT of man but if given the change to invade other body sites, they are the most common cause of
pneumonia, meningitis, septicemia, and urinary tract infection and wound i nfections. 2.
Over t Pathogens
these are the notorious pathogens of the GIT of man. They evade and endure th e gastrointestinal tract.
a.
Salmonella, Shigella and Yersinia.
b.
These
c.
The
pathogens are found only in man during infection. They are not part of the normal flora.
mode of transmission is the Fecal-Oral route except Yersinia Pestis (Plague: through bite of inf ected rat flea)
Biochemical and Morphologic Proper ties of the Enterics y
All
enterics are Gram (-) coccobacilli
y
Oxidase (-) do not have oxidase
y
They
y
All
enterics will ferment glucose. Utilization of glucose may produce acid only (anaerogenic) or acid gas (aerogenic)
y
All
enterics are facultatively anaerobic.
y
They
are motile (peritrichous flagella except Tatumella ptyseos polar flagellated) or n on-motile enteric genus Klebsiella and Shigella
y
They
grow well on routine media blood agar, chocolate agar, brain heart infusion broth and thioglycolate broth.
y
The
y
Large colonies enteric bacillus
y
They
do not form spores. (Asporogenous)
y
They
can reduce nitrate to nitrite except some strains of Enterobacter and Yersinia
y
They
are Catalase (+) except Shigella dysenteriae type I and a species of Xenorhabdus.
grow well on MacConkey Agar and on Eosin Methylene Bl ue (EMB)
eneterics produce large colonies (1-2mm in diameter after 24hours) on blood agar. |
Medium colonies Staphylococci
|
Small colonies Streptococci
Antigenic Structures 1.
2.
3.
Somatic
Antigen - All enterics have this, all Gram (-) bacteria have this. antigen found in the Lipopolysaccharide (endotoxins)
a.
O
b.
This
is heat-stable; it cannot be destroyed by heating.
Capsular An tigen antigen but among the salmonella s pecies it is called the Vi antigen
a.
K
b.
Is found in the capsule that covers the O antigen and this will hinder the reaction between the O antigen and O antibody.
c.
It is heat labile. Boiling 10-60 minutes destroys the K antigen.
Flagellar Antigen a.
Designated as the H an tigen
b.
It is found only in the motile enterics.
c.
It is heat labile. It can be destroyed by ethanol.
Media for isolation 1.
Differential Mildly Selec tive
Media contain carbohydrates and pH indicators with other chemicals that are inhibitory to Gram (+ ) bacteria. It is
differential because it can differentiate lactose ferme nters from nonlactose fermenters. It is mildly selective because it can inhibit Gram (+) bacteria. a.
MacConkey Agar contains lactose
b.
Eosin
c.
Leifson Deoxychola te Agar contains lactose
Methylene Blue agar (EMB) contains lactose and sucrose i.
The
incorporation of lactose will divide the enterics i nto 2 groups Lactose Fermen ters - will produce acid in the medium, reacts with the pH i ndicator and produces colored colonies. Most
1.
of the coliforms are lactose fermenters. Nonlactose
2. 2.
Differential
Fermen ters will produce colorless colonies. Most overt patho gens are nonlactose fermenters.
Moderately Selective designed for the isolation of salmonella and shigella which are overt pathogens. This media is called moderately
selective because it contains carbohydrates and pH indicators with other chemicals that are in hibitory to Gram (+) bacteria and m ost of the coliforms
3.
a.
Salmonella Shigella
b.
Xylose
c.
Deoxycholate
d.
Hektoen Enteric
Highly Selec tive
a.
Lysine Deoxycholate Agar (XLD) Salmonella and Shigella will produce colorless c olonies. Agar (HE) Salmonella and Shigella will produce bluish-green colonies.
Bismuth Sulfite Agar (B SA) Designed for the isolation of Salmonella Typhi will produce jet-black colonies.
Brilliant Green Agar (BGA) i.
c.
Citra te Agar (DCA) Salmonella and Shigella will produce c olorless colonies.
Media designed for isolation of specific bacteria or specific group. i.
b.
Agar (SSA) Salmonella and Shigella will produce colorless colonies.
Designed for the other Salmonella species.
Cefsulodin Irgasan Novobiocin Agar (CIN) i.
Designed for the isolation of Yersinia Enterolitica will produce bulls-eye colonies (dark red or burgundy ce nters with translucent borders)
d.
MacConkey Sorbitol Agar (sMac) i.
It differentiates Escherichia coli O157:H7 which is a notorious overt pathogen - It does not ferment sorbitol as compared from other strains of Escherichia coli.
4.
ii.
Positive = Colorless colonies
iii.
Escherichia coli O157:H7 (O antigen 157 and Flagellar antigen H7)
Enrichmen t Media
|
Negative = Pink Colonies
and Technic this is designed for st ool culture. These suppress the growth of coliforms, supports the growth of enteropathogens
(Shigella, Salmonella and Yersinia) a.
Selenite
F Broth for isolation of Salmonella typhi an d Shigella species
b.
Tetra thionate
c.
Gram Nega tive broth (GN ) for isolation of Salmonella and Shigella from t he stool
d.
Cold Enrichmen t Technic this is done for the isolation of Yersinia enterolitica. i.
Broth for isolation of Salmonella other t han Salmonella typhi
Inoculate a swab or 1gram of the specimen into 5ml phosphate buffer and refrigerate for 3 weeks. Do subculture on Cefsulodin Irgasan Novobiocin Agar (CIN) every 4 hours until you can isolate the bulls-eye colonies.
Biochemical Tes ts 1.
TSI
reaction detects 3 primary characteristics of the enterics.
a.
The
ability to ferment glucose, lactose and sucrose - acid
b.
The
ability to produce gas - H2 a nd CO2)
c.
The
ability to produce H2S cys teinase
i.
Medium Triple Sugar Iron Agar (TSI) components of TSI agar - 1% lactose, 1% sucrose, 0 .1% glucose and peptone.
1.
Active
2.
pH indicator Phenol Red (acidic-yellow|alkaline-pink)
3.
H2S
indicator ferrous sulfate and sodium thiosulfa te
4.
It is prepared in a test tube with a slant and a butt.
5.
Method of inoculation: a.
Wire needle get the in oculum
b.
Make a line on the slant, stab the butt until a few millimeters from the bottom, and then streak across the line on the slant. This
d.
procedure will produce precise results
c.
Utilization of sugars will occur aerobically (oxidation) in the slant and ana erobically (fermentation) in the butt.
d.
Anaerobic
e.
It is ideal to use cotton plug or a loose test tube cap to maintain the aerobic condition on the slant.
f.
Incubate at 35-37 C in ambient air for 18-24 hours.
utilization will produce more acid.
Reading and Interpretation i.
Alkaline/Acid - If the slant is pink and the butt is yellow. Only glucose is fermented, lactose and sucrose is not fermented. (reaction for Enterics)
ii.
Acid/Acid If both the slant and the butt are yellow. Glucose, lactose and sucrose are fermented. (reaction for Enterics)
iii.
Alkaline/Alkaline If both the slant and the butt are pink. Glucose, lactose and sucrose are not fermented but peptone is utilized aerobically.
iv.
Alkaline/No Change if the slant is pink and the butt is orange. Glucose, lactose and sucrose are not fermented and peptone is utilized aerobically only in the slant.
v. vi.
Gas Production is indicated by bubbles or cracks in the butt. H2S Production 1.
It is indicated by a black precipitate in the butt.
2.
Cysteinase will act on sodium thiosulfate to pr oduce H2S gas, which is colorless.
3. vii. 2.
3.
enzyme involved: cysteinase
Kligler Iron
Indole Produc tion
H2S will react with ferrous sulfate to produce black FeS precipitate. Agar (KIA) only two sugars are used, glucose (dextrose) and lactose.
tryptophanase splits indole from tryptophan.
a.
Medium tryp tone broth
b.
Reagent: Kovacs reagen t (red ring) or Erlichs Reagen t (purple ring)
c.
Add
d.
(+) red or purple ring
e.
(-) yellow ring
five drops of the reagent and then incubate 18 -24 hours at 37 C
Methyl Red Tes t and Voges-Proskauer Test enterics will utilize glucose and produce strong acid such as acetate and formate. Some enterics will metabolize the carbon compounds of glucose a nd produce acetoin or ac etylmethylcarbinol. a.
b.
4.
Methyl Red Tes t i.
Detects acetate and formate
ii.
Medium MRVP broth (Methyl Red-Voges Proskauer broth or b uffered peptone glucose broth)
iii.
Reagent methyl red indicator
iv.
(+) red medium (pH 4.5 or less)
v.
(-) yellow medium (pH>5.6)
Voges-Proskauer Test i.
Detects acetoin or acetylmethylcarbinol.
ii.
Medium - MRVP broth (Methyl Red-Voges Proskauer broth or buffered peptone glucose broth)
iii.
Reagent 6 drops of alpha-napthol and 2 drops of 40% KOH
iv.
(+) red ring (acetoin or acetylemethylcarbinol is produced from glucose)
v.
(-) no red ring produced
Citra te U tilization Test this will detect whether the enteric bacillus can utilize citrate as the only so urce of carbon for metabolism i. ii.
5.
7.
This
is prepared in a tube with a slant only. (A erobic only)
iii.
Streak the inoculum in a zig zag manner on the slant.
iv.
(+) Blue medium (prussian blue) and growth on slant the organism can utilize citrate.
v.
(-) Green medium and no growth on the slant.
Motility Test i.
6.
Medium Simmons Citrate Agar (CSA)
The
fastest test for motility is the Hanging Drop Method.
ii.
Other than Hanging drop method, we can use semi-solid media.
iii.
Medium: SIM (sulfide, indole, motility)
iv.
No slant, only butt. Stab once a nd incubate. (anaerobically only)
v.
Motile hazy medium
vi.
Non-motile clear medium
Urease Tes t i.
Splits Urea into ammonia and carbon dioxide.
ii.
Medium Urea Broth
iii.
This
test requires heavy inoculation. 5 loopfuls if solution. 2-3 colonies if solid. 24 hours incubation.
iv.
Indicator Phenol Red indicator
v.
(+) dark pink medium (NH3 and CO2)
Nitra te Reduction Test
Ni tratase/N itrate Reductase
i.
Medium Nitrate Agar
ii.
Slant only simple streaking
iii.
After
incubation add 2 drops of s ulfanilic acid and 2 drops of alpha- napthylamine
8.
iv.
(+) Red azo compound (parasulfobenzene azo alpha-naphthylamine) on the slant.
v.
End Product is Nitrite.
ONPG test
i.
(ortho-nitro-phenyl--D-galactopyranoside) Will detect if the organism has the enzyme beta-galactosidase which is one of the enzymes responsible for lactose fermentation.
ii.
All
lactose fermenters have beta-galactosidase but not all have permease.
iii.
It is a rapid test to detect lactose fermenters.
iv.
The
v.
test does not detect lactose fermentation but only detects the presence of beta-galactosidase.
There
are 2 enzymes involved in lactose fermentation 1.
Permease
2.
beta-galactosidase is the enzyme that strips lactose into glucose and galactose
is the enzyme that allows lactose into the bacterial cell
vi.
Lactose fermenters that have both permease and beta-galactosidase are fast lactose fermenters.
vii.
24 hours incubation of the lactose sugar is enough to know whether it is a lactose fermenter or not but some lactose fermenters lack permease so 24 hours incubation is not enough. Most of the time, this group of lactose fermenters are misidentified as non-lactose fermenters because they lack permease.
viii.
9.
10.
Two groups 1.
Fast lactose Fermen ters have both permease and beta-galactosidase
2.
Slow
lactose Fermen ters lack permease
ix.
Medium ONPG broth (3-6 hours incubation)
x.
(+) yellow if it has t he enzyme beta-galactosidase because it hydrolyzes ONPG and liberate ortho nitrophenol which is a yellow compound.
xi.
(-) - c olorless medium
Phenylalanine Deamina tion Tes t
a.
It will detect the enzyme phenylalanine deaminase.
b.
Medium phenylalanine agar.
c.
Prepared with a slant only.
d.
After
e.
(+) will show a green slant
f.
The
enzymes will deaminase phenylalanine to Phenylalanine pyruvic acid.
g.
The
procedure in the manual will produce a brown slant because we use plain phenylalanine agar.
h.
It is only produced by Proteus providencia and morganella.
incubation, add 5 drops of 10% ferric chloride
Lysine Decarboxylation and Deamina tion a.
Medium = Lysine Iron Agar i.
Active Components 1.
Glucose
2.
Lysine
3.
Bromcresol purple as the pH indicator purple in alkaline, yellow in acidic medium.
ii.
This
is prepared with a slant an d a butt.
iii.
The
uninoculated medium is colored purple.
iv.
Lysine Decarboxylation occurs in the butt because it requires anaerobic conditions.
v.
Lysine Deamination occurs in the slant b ecause it requires aerobic conditions.
vi.
Method of Inoculation 1.
Get inoculum using wire needle and stab the butt twice
2.
Streak across the slant. T hen incubate.
vii.
If the butt is purple positive lysine decarboxylase
viii.
If the butt is bright yellow n egative lysine decarboxylase
ix.
Principle all enterics ferment glucose and glucose is present in the lysine iron agar. So the organism will produce acid y ellow medium. Those that have lysine decarboxylase will act on lysine i n the butt and produce ca daverine which is an alkaline c ompound so the butt will revert to purple.
x.
Purple butt during inoculation Yellow butt during incubation P urple butt with yellow tinge after incubation
xi.
If the slant is red after incubation, then the result is (+) it has the enzyme lysine deaminase and the compound formed is alpha-ketocarboxylic acid)
xii.
Purple Slant negative for lysine deaminase.
xiii.
It is purple if its negative because glucose is used up during 24 hour incubation, peptone is utilized a nd the end product is amine w hich is alkaline. Thats why a negative result is a purple slant.
b.
11.
Lysine Iron Agar reactions i.
P/P lysine deaminase (-) / decarboxylase (+)
ii.
P/Y - lysine deaminase (-) / decarboxylase (-)
iii.
R/Y - lysine deaminase (+) / decarboxylase (+) [ is only produced by proteus providencia and morganella]
iv.
No R/P because the enteric bacillus may have either lysine deaminase or decarboxylase, none of the two but never have both.
v.
End product if R/Y = alpha-ketocarboxylic
vi.
End product if P/P = cadaverine
Ornithine Decarbo xyla tion
a.
Medium MIO (motility, indole, ornithine) it is a s emi-solid medium i. ii.
Active
ingredients glucose, ornithine and bromcresol purple
Use deep butt tube
b.
Method of Inoculation
c.
(+) butt is purple with yellow tinge putrescine is produced in the medium
d.
(-) butt is bright yellow/intense yellow with a purple ri m at the top be cause enteric bacillus will ferment glucose and produce acid.
e.
Motile hazy medium
f.
Non-motile clear medium with growth only on the stab line and the top of the medium.
g.
Indole production add Kovacs reagent (+) result is red ring
h.
In the laboratory, MIO is only for motility and ornithine. Tryptone broth is used for indole production.
i.
Simple stabbing, stab once then incubate.
Genus Escherichia y
It is transmitted via fecal-oral route.
y
It is the predominant facultatively anaerobic species in th e large intestine of man.
y
It is used as an in dex of fecal contamination in water bacteriology.
y
Most strains are motile.
y
It is a lactose fermenter
y
It is aerogenic
y
Triple
y
IMViC (indole, methyl red, VogesProskauer and Citrate Test) [+ + - -]
y
Eosin Methylene Blue Agar (EMB) greenish metallic sheen
y
MacConkey Agar flat, dry, pink colonies
y
Blood Agar some are beta-hemolytic
y
It is the most common cause of Urinary Tract Infection, nosocomial infections and Gram(-) bacteremia.
y
Escherichia coli with K1 antigen causes neonatal meningitis.
Strains
Sugar Iron ( TSI) acid/acid gas
which can cause serious Gastrointes tinal Infec tion Entero toxigenic Escherichia
1.
coli (ETEC) - E TEC strains are non-invasive, and they do not leave the intestinal lumen. E TEC is the leading bacterial cause of
diarrhea in children in the developing world, as w ell as the most common c ause of traveler's diarrhea, Montezumas Reve nge and Delhi Belly. It is a causative agent of diarrhea (without fever) Enteroinvasive Escherichia
2. 3.
coli (EIEC) EIEC infection causes a syndrome that is identical to Shigellosis, with profuse diarrhea and high fever. coli (EHEC)/ Verocy totoxin producing E. coli (VTEC) - The most famous member of this virotype is strain O157:H7, which
Enterohemorrhagic Escherichia
causes bloody diarrhea and no fev er. EHEC can cause hemolytic-uremic syndrome and s udden kidney failure. It uses bacterial fimbriae for attac hment. Enteropathogenic Escherichia
4.
coli ( EPEC) is common among children in orphanages. They utilize an adhesin known a s intimin to bind host intestinal
cells. EPEC cells are moderately invasive (they enter host cells) and elicit an inflammatory response. Enteroaggrega tive Escherichia
5.
coli (EAEC) - So named because they have fimbriae which aggregate tissue culture cells, EAEC bind to the intestinal mucosa
to cause watery diarrhea without fever. E AEC are non-invasive. They produce a hemolysin and an ST enterotoxin similar to that of E TEC. Entero toxigenic
Feature
Escherichia
1° Pathogenic Mechanism 1° Site of Infection
coli (ETEC)
Enteroinvasive Escherichia
coli (EIEC)
Enterohemorrhagic Escherichia
Enterotoxin LT and/or ST
Invasion of Erythrocytes
Verotoxin (cytotoxin)
Small Intestine
Large Intestine
Large Intestine Hemorrhagic Colitis, Hemolytic-uremic syndrome (HUS) Absent Copious and bloody Prominent Absent
Travelers
Epidemiology
Fever of Stool Blood Pus (WBC)
Nature
Diarrhea; Childhood Diarrhea
Uncommon Dysenterylike syndrome
Absent Copious and watery Absent Absent
Common Scanty and purulent Common Prominent
Enteropathogenic
coli (EHEC)
Escherichia
coli ( EPEC) Adherence to enterocytes Small Intestine
?
Common Copious and watery Absent Minimal
Occasional Watery Absent Absent
Stool Exam Entero toxigenic Escherichia
a.
ii.
2.
LT Enterotoxin is a heat-labile Enterotoxin and is destroyed at 65°C for 30 minutes. It is antigenic. 1.
Rabbit Ileal Loop Test (+) dilation of loop
2.
Chinese Hamster Ovary Assay (+) cell damage
3.
Reverse Passive Latex Agglutination Test - (+) agglutination, it is the simplest serological test.
4.
DNA -DNA Hybridization
ST Enterotoxin is a heat-sta ble Enterotoxin even at 100°C. It is poorly antigenic.
Enteroinvasive Escherichia
a.
coli (ETEC)
LT and/or ST Enterotoxin i.
1.
Infant Mouse Assay
2.
DNA -DNA Hybridization
coli (EIEC) Escherichia coli O112
Sereny T est use guinea pigs, (+) keratoconjunctivitis - inflammation of the cornea.
coli (EAEC) to enterocytes ?
Adherence
Infantile and Childhood Diarrhea
Laboratory Diagnosis 1.
Enteroaggrega tive Escherichia
Enterohemorrhagic Escherichia
3.
i.
Verotoxin which damages vascular endothelial cells causing hemorrhagic colitis; can also lea d to hemolytic-uremic syndrome (HUS).
ii.
Most frequently associated with Escherichia coli O 157:H7
b.
MacConkey Sorbitol Agar (sMac) (+) colorless (-) pink
c.
Serotyping use anti O 157 and anti H7
d.
Cytotoxin assay use commercially prepared vero cells from African green monkey kidney cells.
Enteropathogenic Escherichia
4.
coli (EHEC)/ Verocy totoxin producing E. coli (VTEC)
a.
coli ( EPEC) - Escherichia coli O111 and O125
Serotyping anti 0111 and anti 0125
Genus Klebsiella Klebsiella
y
pneumoniae
a.
Friedlanders Bacillus
b.
Capsulated, non-motile, urease (+)
c.
Lactose fermenter, aerogenic
d.
Has O and K antigen
e.
Triple
f.
IMViC (indole, methyl red, VogesProskauer and Citrate Test) [ - - + +]
g.
Eosin Methylene Blue Agar (EMB) and MacConkey agar (Mac) large, pink, mucoid colonies (indicates presence of capsule)
h.
It can cause primary pneumonia am ong elderly and alcoholics.
i.
It can cause a more painful Urinary Tract Infection because it contains urease.
Sugar Iron (TSI) acid/acid gas
Genus Enterobac ter y
They
y
Eosin Methylene Blue Agar (EMB) fish-eye colonies
have small capsules therefore they have less mucoid colonies.
y
Motile, Lactose Fermenter, Aerogenic (abundant gas producer)
y
Triple
y
IMViC (indole, methyl red, VogesProskauer and Citrate Test) [ - - + +]
Sugar Iron (TSI) acid/acid gas ( TSI medium is split and cotton plug is ejected due to abundance of gas)
Lysine Iron Agar (LIA) reaction Lysine Decarboxylase + -
Enterobacter cloacae Enterobacter aerogenes Enterobacter agglomerans
Ornithine Decarboxylase + + -
Urease (-) except Enterobacter cloacae and Enterobacter agglomerans.
y
Genus Serratia y
Serratia
marcescens
a.
Nosocomial infections
b.
Nonlactose fermenter / slow lactose fermenter
c.
Motile
d.
Urease
e.
Triple
f.
IMViC (indole, methyl red, VogesProskauer and Citrate Test) [ - - + +]
g.
Prodigiosin
h.
Sugar Iron alkaline/acid
i.
Red pigment especially at 25°C
ii.
Water insoluble pigment does not diffuse in water, it stays in the col onies.
iii.
Pseudohemoptysis in Respiratory tract infection coughing sputum containing red pigment.
3 extracellular enzymes is unusual among enterics i.
Deoxyribonuclease (DNase)
ii.
Gelatinase
iii.
Lipase
Genus Hafnia y
Hafnia
alvei ( En terobacter hafniae)
a.
Slow lactose fermenter
b.
Indole (-), Citrate (-), Urease (-)
c.
Lysine and ornithine decarboxylase (+)
d.
More positive biochemical tests at 25°C rather than 35°C
Genus Edwardsiella y
Edwardsiella tarda
a.
Normal flora of gastrointestinal tract of cold-blooded animals
b.
Nonlactose fermenter
c.
H2S (+), indole (+)
d.
Motile
e.
Triple
f.
Resembles salmonellae in biochemical reactions
Sugar Iron (TSI) alkaline/acid with H2S
Genus Citrobac ter Citrobac ter Freundii
y
a.
Lactose fermenter, some are slow lactose fermenters.
b.
Urease variable
c.
Motile peritrichous flagella
d.
Most are H2S (+)
e.
IMViC (indole, methyl red, VogesProskauer and Citrate Test) [ - + - +]
f.
Eosin Methylene Blue Agar (EMB) greenish metallic sheen
Proteus-Providencia-Morganella
Group
y
Nonlactose fermenter, rapid motility and abundant urease.
y
Deaminate lysine and phenylalanine
Genus Proteus y
Abundant H2S
y
Noted for rapid motilityL swarming growth on plated m edia with characteristic foul odor.
y
Phenylethyl Alcohol agar (PE A) this inhibits swarming growth of c olonies
y
Urinary Tract Infection is associated with formation of renal calculi stone formation magnesium and potassium
y
Proteus Vulgaris (OX2, OX9), Proteus Mirabilis (OXK) antigens from Proteus used in Weil-Felix reaction to diagnose Rickettsial disease.
Proteus
vulgaris Proteus mirabilis Proteus penneri
Indole Produc tion
Ornithine Decarbo xylase
+ -
+ -
Genus Providencia y
Providencia rettgeri (old name: Proteus rettgeri) causes painful urinary tr act infection.
y
Providencia stuartii major pathogen in burn infection
y
H2S (-), citrate (+), Ornithine Decarboxylase (-)
Genus Morganella y
Morganella morganii (old name: Proteus Morganii) causes painful urinary tract infection
y
H2S (-), Citrate (-), Ornithine Decarboxylase (+)
Genus Salmonella y
Pathogenic for man and animals
y
Does not ferment lactose or sucrose
y
H2S (+) except Salmonella paratyphi A and Salmonella choleraesuis
y
Aerogenic
except Salmonella typhi and Salmonella gallinarum
y
Have peritrichous flagella, motile except Salmonella gallinarum and Salmonella pollorum
y
Lysine decarboxylase (+) except Salmonella paratyphi
y
Ornithine Decarboxylase (+) except Salmonella typhi
y
Indole (-), Methyl Red test (+),VogesProskauer test (-)
y
Citrate (-) for Salmonella typhi and Salmonella paratyphy A; variable for Samonella choleraesuis amd (+) for others.
y
TSI
A
reaction for Salmonella typhi - alkaline/acid with H2S
Salmonellosis y
Fecal-oral route
y
Infective dose 10 organisms because they are sensitive to g astric acid.
y
General Categories
5
o
Gastroen teriti tis
Self limiting which lasts for 2-5 days
It is the most common sa lmonellosis in man infecting small and large intestines
It is caused by any sp ecies, most commonly by Salmonella typhimurium and Sal monella Enteritidis (Gartners Bacillus); they remain in the gastrointestinal tract.
o
Symptoms: nausea, vomiting, diarrhea 1-2 days incubation period.
Bacteremia and Extraintes tinal Infec tions (Septicemia)
From GIT, causative agent enters the bloodstream and infects any tissue.
Infection does not involve the GI T, they just pass through.
Caused by any species, most commonly by Salmonella choleraesuis and Salmonella Dublin
Symptoms: fever, chills, anorexia and anemia.
Perform Blood Culture
o
Enteric
Fever
It is the most life-threatening infection characterized by prolonged fever and multi-organ involvement. Causes death in 2 weeks if not treated.
o
o
Duration in blood stream 1 week
Small intestines > lymph nodes > blood > other organs > liver and spleen
Those
They
Typhoid
that are in the gall bladder are protected from antibodies
invade the macrophages so they cannot be treated with antibodies.
Fever
Most severe form of enteric fever
Salmonella Typhi man is the only known host
It is transmitted by diseased persons or carriers.
Convalescent carriers short period only
Chronic carriers longer than 1 year <= rectal swab culture or stool culture
Incubation period 2 weeks
Symptoms headache, anorexia, abdominal pain, weakness and rose spots (Hallmark of Typhoid Fever)
Parathyroid
Fever
Milder manifestations and shorter in duration
Salmonella paratyphi A = Salmonella para A
Salmonella schottmuelleri = Salmonella para B
Salmonella hirschfeldii = Salmonella para C found only in the Middle East.
Mary Mallon - also known as Typhoid Mary was the first person in t he United States to be ide ntified as a healthy carrier of typhoid fever. Over t he course of her career as a cook, she is known to have infected 53 people, three of whom died from the disease.Her notoriety is in part due to her vehement denial of her own role in spreading the disease, together with her refusal to cease working as a cook. She was f orcibly quarantined twice by public health authorities and died in q uarantine. It is possible that she was b orn with the disease, as her mother had typhoid fever during her pregnancy. Mallon spent the rest of her life in quarantine. Six years before her death, she was paralyzed by a stroke. On November 11 , 1938, aged 69, she died of pneumonia. She was still infectious on the day of her death: an autopsy found evidence of live typhoid bacteria in her gal lbladder. Cholera Dolores she was the first known long-term carrier of Cholera, found in the Philippines, who had suffered from El Tor cholera in A ugust 1962, continued intermittently to excrete vibrios of the same characteristics as th e original isolates until the date of reporting (1966). Duodenal intubation proved that the vibrios ar e lodged in her biliary tract. Her serum antibody titre continued t o remain high in the absence of vaccination against cholera.
