Arranged by Sarah Mohssen

Section I– Microbiology By Dr. Mohammed Ayad

Lecture Three

Gram-positive bacilli

Aerobic non-spore forming bacilli

Usual Corynebacterium

Unusual Arcanobacterium, Rothia

Acid-fast Rhodococcus, Nocardia, Gordonia

Aerotolerant anaerobes non-spore forming bacilli

Actinomyces, Propionibacterium

Corynebacterium

Usual C diphtheriae

Less C amycolatum, C minutissimum, C jeikeium, C pseudodiphtheriticum, C striatum, C urealyticum,

C xerosis

Corynebacterium species and Propionibacterium species are normal flora in skin and mucus membrane, so they

frequently contaminated our specimens; although the Corynebacterium diphtheriae is a pathogenic microbe that

produces a powerful exotoxin which causes diphtheria disease.

Corynebacterium bacteria tend to be clubbed and irregular shaped, the coryneform or diphtheroid bacteria is a

convenient name for this group. Corynebacterium has high concentration of guanosine and cytosine, its 0.5-1

µm in diameter and 3-4 µm in length; it posses irregular unipolar swelling (club shaped) and it composed of

granules deeply stained by aniline dye known as metachromatic granules.

Figure shows Corynebacterium tends to be found in irregular distribution or parallel or on acute angles

(Chinese letters)

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Section I– Microbiology By Dr. Mohammed Ayad

Figure shows Corynebacterium diphtheriae measurement

It colonies onto BA are small granular gray in color with irregular edges and have small hemolysis zone

around, if the plate containing potassium tellurite so the colonies become black brown with a black brown halo

as it reduced intracellular.

Figure shows Corynebacterium diphtheriae growth onto potassium tellurite agar plate

Corynebacterium have 4 biotypes or serovars; gravis, mitis, intermedius, belfanti, they are classified

depending on colony morphology, biochemical reactions, and disease severity. C diphtheriae grow on most

culture media, on loeffler serum medium it grows readily on it more than other respiratory microbes.

C diphtheriae found in 2 strains either toxigenic by lysogenic conversion or non toxigenic strains and if it

infected by bacteriophages so the offspring will be toxigenic also; so bacterial toxins are under phage gene

control while bacterial invasiveness is under bacterial gene control.

C diphtheriae cause respiratory and cutaneous infections (skin and soft tissues), so it spread from person to

other by droplets or direct contact; as it habitat the area it will start toxin production. Bacterial exotoxin

elaboration depend on media nitrogen or carbon or pH or amino acids or osmotic pressure or iron contents

C diphtheriae exotoxin is heat labile polypeptides and it composed of 2 fragments; fragment B act as a receptor

of the toxin to special epithelial cell receptors and facilitate the fragment A entrance to the human cell which

act upon inhibition of polypeptides chain elongation through inactivation of elongation factor-2 (EF-2), so

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Section I– Microbiology By Dr. Mohammed Ayad

arrest cell protein production and this step is responsible for tissue necrosis, this action is similar to the action

of P aeruginosa exotoxin.

As the bacterial exotoxin is released it enters the cell and destructs them with embedding of fibrin, RBCs,

WBCs exudate yielding grayish pseudomembrane onto pharynx, tonsils, larynx, any attempt to remove it will

lead to bleeding, and the bacteria inside the membrane will continuo exotoxin production which will be

absorbed to the blood to reach liver, heart, kidneys, adrenals, nerves and produce tissue necrosis among them

i.e. the disease is not due to the bacterial cell itself but it due to the effect of its exotoxin.

Regarding the cutaneous infection there is the same membrane on wound failed to be healed, but here the

exotoxin absorption is slight so the systemic picture is negligible, as this small quantity will elicit the

production of antitoxin antibodies that neutralize it.

In diphtheria the clinical presentation is due to suffocation caused by membrane obstruction to the airways, the

patient is feverish, irregular heartbeats, blurred vision or speech difficulties or swallowing, and weakness in

arms or legs.

In general serovar gravis produce the sever presentation more than the serovar mitis.

C diphtheriae laboratory diagnosis

It is significant in order to improve the clinical impression plus for epidemiological purposes, it is very

important not to delay the proper treatment.

Amie’s swab from nose, throat and it collected from beneath any membrane seen, then smear is made and fix it

to be stained by Gram or methylene blue; then inoculate the specimen onto BA to rule out Streptococci

inoculate onto loeffler slant and Cystine - tellurite plate (known as modified Tinsdale agar), incubate at 37 ºC

for 36-48 hours, if got any suspected colonies then send our specimen to reference laboratory to investigate

microbial toxigenicity through different ways:

a- Elek’s test as a filter paper containing antitoxin (10 IU) placed onto agar then inoculates the bacteria

7-9 mm far from the disk, after 48 hours if the bacterium is toxigenic so the antitoxin will react with

the toxin to gain precipitin line in the mid line.

Figure shows the Elek’s test reaction

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Section I– Microbiology By Dr. Mohammed Ayad

b- PCR to detect bacterium toxin genes (tox) if it positive and the culture is negative so here the

diphtheria is possible and if both are positive so it is a confirmatory situation. PCR could be applied

upon direct swab specimen or upon the resulted culture.

c- Elisa to detect toxin from specimen directly

d- Immunochromatography strips to detect the exotoxin directly after specimen manipulation and it

highly sensitive.

e- In the past they were detect bacterial isolates toxins through injection of guinea pigs with emulsified

isolates, if it immunized so they will be survive.

Diphtheria treatment relay upon rapid elimination of the bacterium by antimicrobial agents (penicillin or

erythromycin) to prevent toxins production; bacterial antitoxins produced by horses, rabbits, sheep, goats

through repeated purified and concentrated toxoid injections, the antitoxin is injected to the patient in 20,000-

100,000 units i.m. or i.v.

Before immunization diphtheria was disease of small children and as most of cases occurred sub clinically so a

continuous antigenic stimulation is found and the population became immuned to the infection; active

immunization in childhood with toxoid yielded antitoxin level enough till adulthood and adults must given

booster doses of toxoid.

Diphtheria patient must be isolated in order to decrease contact with healthy personnel because without therapy

the patient will continuo shedding the bacilli for weeks or months.

A filtrate broth of toxigenic culture is treated with 0.3% formalin and incubated at 37 ºC till its toxigenicity is

disappeared, then it called (fluid toxoid) which later purified and prepared in flocculation unit (Lf); after that

add aluminum hydroxide or aluminum phosphate to be remain longer as a depot in the injection site beside it is

a good antigenic stimulus.

Diphtheria toxoid is combined with tetanus toxoid plus pertussis vaccine (DPT)

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Section I– Microbiology By Dr. Mohammed Ayad

Lecture Four

Gram positive cocci

Genus Staphylococci

It is gram positive spherical cells (cocci) 1 µm in diameter, arranged in grapelike clusters; producing pigments vary

from white-deep yellow, found as single or pairs or tetrads, or chain in liquid broth, the young stained strongly with

gram and by aging it will become colorless, it is non-motile and non-spore-forming. Some are members of human skin

plus mucus membrane and other cause infections.

Micrococci it resembling Staphylococci found in the environment free, appeared in 4 or 8 cocci together and their

colonies pigments yellow-red-orange; its rarely associated with infections.

Staphylococci got 40 species but 3 are significant in human infections; S.aureus (Coagulase positive), S.epidermidis,

and S.saprophyticus (cause UTI in young females only).

The Coagulase negative Staphylococci (CNS) usually are normal flora in human and cause infections if introduced by

intravascular catheterization or joints prostheses, especially in young or elderly immunocompromised patients. Less

infections caused by CNS is reported (S.ludunensis, S.warneri, S.hominis).

Staphylococci grow in aerobic and microaerphilic conditions grow at 37 ºC and produce its pigments at room

temperature (20-25 ºC), it shape onto solid media round smooth glistening raised and S.aureus colonies color vary from

gray-golden yellow and its size about 3-4 mm with hemolysis area 1 cm around it; while S.epidermidis colonies graywhite in color and without hemolysis. Staphylococci are catalase positive to be differentiated from Streptococci.

Staphylococci drugs resistance

1- β-lactamase producers which under plasmids control, transmitted by transduction or conjugation (e.g., ampicillin,

amoxicillin, ticarcillin, piperacillin)

2- Resist to nafcillin, methicillin, and oxacillin is not due to β-lactamase but due to locus on chromosome called

staphylococci cassette chromosome (SCC mec gene) which reduce drug affinity to PBPs; there are 4 classes of mec genes (I,

II, III) responsible for hospital acquired methicillin resist Staphylococci (MRSA), and class IV responsible for community

acquired resistance of (MRSA).