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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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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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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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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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).

3- Staphylococci aureus considered susceptible to vancomycin if its MIC (minimum inhibitory concentration) is ≤ 2

µg/ml, intermediate if MIC 4-8 µg/ml which known as (VISA) and resist if MIC ≥ 16 µg/ml (VRSA). Staphylococci resistance

is due to bacteria cell wall changes and not due vancomycin resistance gene (van gene) which occur in Enterococci.

4- Tolerance; it is usually due to failure of the drug to induce the cell wall autolytic enzymes.

Staphylococci certain colonies sometimes differ from other population in shape, color, hemolysis, enzymes production,

and pathogenicity, in nafcillin resistance as 107 will be resist at 37 ºC but in 30 ºC incubation only 103 will be resist.

Staphylococci antigen

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Its cell wall peptidoglycan is important in production of endogenous pyrogen (interlukin-1) plus opsonic antibodies by

monocytes beside it act as chemotactant for the polymorphonuclear leucocytes (PMNs), also it has endotoxin like

activity to enhance the complements.

In addition the teichonic acid in the cell wall (composed of glycerol or ribitol phosphate polymers) which connects to

the peptidoglycan can enhance antibodies detected by gel diffusion in patients with Staph endocarditis.

Another antigen is the cell wall protein A act as antigen and adhesin of bacteria to the target cell wall and it attach to Fc

portion in the IgG molecules to enhance the agglutination process to specific antigen (coagglutination).

Some strains gain a capsule that inhibits bacteria phagocytosis by PMNs cells.

Coagulase (aggregation or clumping factor) also found in the cell wall, that connect enzymatically to fibrinogen

Staphylococci extracellular enzymes & toxins

The main pathogenesis is by invasion + multiplication + enzymes & toxins production

The bacterial enzymes are under plasmids or chromosomal control and they are:

1- Catalase: that convert H2O2 to H2O and O2 in Staph but not in Strep

2- Coagulase & clumping factor: it coagulate oxalated or citrated plasma; as it bind to prothrombin to be activated and

initiate fibrin polymerization, there after the clumping factor is responsible for bacteria adherence to the fibrinogen.

3- Hyalorunidase, staphylokinase that produce fibrinolysis, proteinase, lipase.

The toxins are:

1- α-toxins (hemolysin) act upon leucocytes cell wall

2- β-toxins act upon RBCs act upon leucocytes wall

3- γ-toxins (hemolysin) act upon leucocytes wall

4- δ-toxins act upon human epithelial cells

5- Exfoliative toxins (A+B) they are epidermolytic toxins, cause skin desquamation (scalded skin), type A is under

chromosomal control and it heat stable while type B is under plasmid control and it heat labile.

6- Toxic shock syndrome toxin (TSST-1) it bind to major histocompatability complex class II (MHC-II) molecules and this

will lead to T-lymphocytes stimulation.

7- Enterotoxins (emetic effect) and it types are (A-E, G-J, K-R, and U, V); it preformed as Staph grow in carbohydrate or

protein diets and the personnel ingest the already produced toxins not the microbe itself.

Pathogenesis

S epidermidis usually found normally onto skin, respiratory, GIT. 20-50% of people normally harboring S aureus

nasally (this is important in nursery), also found into fomites, clothing and lines.

Staphylococci infections

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Local infection or abscess with painful inflammatory reaction that goes to central suppuration and heals as soon the pus

is drained. The fibrin and immune cells around the lesion try to prevent infection spread, so must prevent its

manipulation or trauma rupture.

Staphylococci occur also directly through wound contamination (surgical or traumatic) as meningitis after skull fracture

or osteomyelitis after open fracture surgery. If the bacteria transmitted by blood stream we get bacteremia, pulmonary,

osteomyelitis, meningitis, arthritis, etc.

In food poisoning the incubation period is short (1-8 hours); with vomiting and diarrhea & NO FEVER

Staphylococci laboratory diagnosis

The specimens are pus swab, blood, tracheal or abscess aspirates, urine, CSF

1-Gram stained smears

2-Culture onto 5% blood agar plate (BAP), Mannitol salt plate as S aureus ferment it to gain yellow colonies, in

suspected mixed growth use CAN (Columbia agar plate) which contain colistin + nalidixic acid in order to suppress the

G- bacteria; in addition we can use chromogenic culture media especially for nasal carriers detection.

3-Catalase test to detect cytochrome oxidase enzyme as using 3% hydrogen peroxide solution

4-Hemolysis in BAP

5-Coagulase test which is carried out in 2 ways (slide); we use particles covered with fibrinogen and IgG antibodies

that bind coagulase to gain clumping within 20 seconds if the IgG is present in low concentration we turn onto tube

method as we mix equal volumes from bacterial broth and citrated plasma then incubate it for 1-4 hours at 37 ºC to gain

the clot in positive reaction (Coagulase positive Staph) (CPS); S intermedius usually give positive test although it is not

regarded as human pathogen.

((Most of the CPS is pathogenic to human))

1-Susceptibility testing done by broth microdilution or disk diffusion and as known 90% of S aureus is producing β –

lactamase.

Resistance to nafcillin, oxacillin, and methicillin occur in 65% of S aureus and in 75% of S epidermidis isolates. As

commercially methicillin disk production is ceased, so can be replaced with oxacillin disk; if the bacteria resist it that

mean it resist methicillin i.e., it is MRSA.

2-Detection of mec gene through PCR, most laboratories use Mueller-Hinton agar (MHA) contain 4% NaCl + 6 µg/ml

oxacillin in order to inoculate S aureus on it, if the bacteria grow that’s mean it resist nafcillin and gain mec gene.

3-Rapid staph detection through quantitative PCR (qPCR) also called real-time (RT-PCR) as in the ordinary culture we

need few days to gain the bacteria while by PCR few copies of bacterial DNA enough to detect its presence within few

hours; in it we duplicate DNA using thermus aquaticus (Taq polymerase) and to start duplication need free nucleotides

(primers).

4-Serology by agglutination to the isolates of little values nowadays.