Clostridium perfringens, Pseudomonas aeruginosa, and Bacillus anthracis

Dr. Shler Ghafour RaheemBSc., MSc., PhD Medical Microbiology

shler.ghafour@tis.edu.iq

GENUS: CLOSTRIDIUM

General features of clostridia

• Large, gram-positive blunt-ended rods.

• Anaerobic

• They form endospores; the position of the developing spore within the vegetative cell is useful in identifying the species

• Most species are motile.

Species of Medical Importance

Clostridium tetani tetanus (“lockjaw”)

Clostridium botulinum botulism

Clostridium perfringens Gas gangrene & food poisoning

Clostridium difficile pseudomembranous colitis

Gas Gangrene

• When blood supply to a tissue isinterrupted, for example by a wound, acondition known as ischemia develops. Thetissue becomes anaerobic, and necrosissets in.

• If the wound is infected withendospores of anaerobic Clostridiumthen gas gangrene develops in thedead tissue.

Clostridium perfringens

Distinguishing Features

• Large gram-positive, spore-forming rods (spores rare in tissue), nonmotile

• Anaerobic: “stormy fermentation” in milk media

• Double zone of hemolysis

Reservoir: Soil and human colon

Transmission: Traumatic implantation

https://en.wikipedia.org/wiki/Clostridium_perfringens

Virulence factors

A- Exotoxins: Elaborates at least twelve exotoxins. The most important of these, is alpha toxin.

C. perfringens strains are grouped, A through E on the basis of their spectrum of exotoxins. Type A strains, which produce both alpha toxin and enterotoxin, are responsible for most human clostridial infections

B- Hydrolytic enzymes: C. perfringens is a metabolically vigorous organism that produces a variety of hydrolytic enzymes, including proteases, DNases, hyaluronidase, and collagenases, which liquefy tissue and promote the spread of infection.

.

Pathogenesis

Spores germinate under anaerobic conditions in tissue.

Vegetative cells produce:

– Alpha toxin (phospholipase C) is a lecithinase. It disrupts membranes, damaging RBCs, platelets, WBCs, endothelial cells → massive hemolysis, tissue destruction, hepatic toxicity.

Identified by Nagler reaction: egg yolk agar plate—one side with anti- α-toxin; lecithinase activity is detected on side with no antitoxin.

Eleven other toxins damage tissues.

Gas gangrene (myonecrosis)

Clostridial spores are introduced into tissue, - Contamination with infected soil

- Endogenous transfer from the intestinal tract.

Severe and open wounds, such as compound fractures and other ischemia-producing injuries, are a prime predisposing condition.

Alpha toxin and other exotoxins are secreted and extensive cell killing ensues.

Production of enzymes that break down ground substance

facilitates the spread of infection.

Fermentation of tissue carbohydrates yields gas, and an accumulation of gas bubbles in the subcutaneous spaces produces a

crinkling sensation on palpation (crepitation); hence, the name gas

gangrene.

Untreated clostridial myonecrosis is uniformly fatal within

days of the initiation of gangrene.

Laboratory identification

Diagnosis of clostridial myonecrosis or cellulitis rests largely on clinical impression.

Specimens from diseased tissue usually show vegetative clostridialforms (large, gram-positive rods. It contains spores with central or sub-terminal spores) accompanied by other bacteria and cellular debris.

http://bacteriologynotes.com/morphology-of-clostridium-perfringens

Laboratory Identification

When cultured anaerobically on blood agar, C. perfringensgrows rapidly, producing colonies with a unique double zone of hemolysis

http://atlas.sund.ku.dk/microatlas/food/bacteria/Clostridium_perfringens

Treatment and prevention

• Immediate removal of foreign material and devitalized tissue.

• Exposure of the wound to O2. Hyperbaric oxygen chambers increase the tissue O2 tension in the affected part.

• Amputation, when anatomically possible, is still mandatory in gangrene.

• Administration of antibiotics in high dose. C. perfringens is sensitive to penicillin

Pseudomonas aeruginosa

• Patients whose skin has beenburned away by a fire or exposureto steam. In such patients,opportunistic pathogens gain access tothe moist, nutrient-rich environment of

the fascia and deeper tissues.

• The most common microorganismseen in burn victims is Pseudomonasaeruginosa.

Pseudomonas aeruginosa

Distinguishing Features

• Oxidase-positive, Gram-negative rods, nonfermenting

• Strict aerobes

• Pigments: pyocyanin (blue-green) and fluorescein

• Grape-like odor

• Capsule

• Non–lactose fermenting colonies on EMB or MacConkey

• Biofilm

Reservoir—ubiquitous in water

Transmission—raw vegetables, flowers

Pathogen and Virulence Factors

• Pseudomonas aeruginosa is a Gram-negative, aerobicbacillus that metabolizes a wide range of organic carbon and nitrogensources.

• It is almost everywhere in soil, in decaying organic matter,and in almost every moist environment, including swimmingpools, hot tubs, sponges, washcloths, and contact lens solutions.

• In hospitals, it grows in sinks, moist foods, vases of cutflowers, sponges, toilets, floor mops, dialysis machinesrespirators, and humidifiers.

P. aeruginosa has numerous virulence factors:

Fimbriae and adhesins attach to host cells and enable biofilmformation.

Its capsule plays role in bacterial attachment and biofilmformation, and it shields the bacterium from phagocytosis.

Neuraminidase modifies host cell receptor molecules to makebacterial attachment to the cells more likely.

Elastase breaks down elastic fiber, degrades complementcomponents, and cleaves immunoglobulins A and G (IgA and IgG).

• Endotoxin (lipid A) can trigger fever, blood clotting,inflammation, or possibly shock.

• Exotoxin A and exoenzyme S, which inhibit eukaryoticprotein synthesis, lead to host cell death.

• Pyocyanin, the blue-green pigment of Pseudomonas,triggers the formation of reactive forms of oxygen (superoxideradical and peroxide anion) that damage host cells.

Signs and Symptoms

• When Pseudomonas aeruginosa invades thebloodstream, it causes fever, chills, and shock.

• Massive infections are often readily diagnosedbecause the bacterium typically produces ablue-green pigment, pyocyanin, that colorssuch infections

Laboratory Diagnosis

•Pseudomonads are gram-negative rods

that resemble the members of the

Enterobacteriaceae but differ in that they

are strict aerobes (i.e., they derive their energyonly by oxidation of sugars rather than by

fermentation).

•Because they do not ferment glucose, they

are called nonfermenters, in contrast to themembers of the Enterobacteriaceae, which do

ferment glucose.

Gram stain of Pseudomonas aeruginosa

•Grows as non–lactose-fermenting

(colorless) colonies on MacConkey’s or

EMB agar.

•It is oxidase-positive.

•Blue-green pigment on ordinary nutrient

agar and a fruity aroma are sufficient to

make a presumptive diagnosis.

•The diagnosis is confirmed by

biochemical reactions.Blue-green pigment (pyocyanin) produced by

P. aeruginosa diffuses into the agar

Treatment

• P. aeruginosa is resistant to many antibiotics, treatment must be tailoredto the sensitivity of each isolate

• The treatment of choice is an antipseudomonal penicillin (e.g.,piperacillin/tazobactam) plus an aminoglycoside (e.g., gentamicin oramikacin).

• For infections caused by highly resistant strains, colistin (polymyxin E) isuseful.

Prevention

• Pasteurization or disinfection of water-related equipment, hand washing; promptremoval of catheters.

• No flowers or raw vegetables in burn units

GENUS: BACILLUS

Genus Features• Gram-positive rods

• Spore forming

• Aerobic

• Species of Medical Importance

• Bacillus anthracis anthrax

• Bacillus cereus food poisoning

Note/ Anthrax has three distinct clinical manifestations:

1. Inhalation

2. cutaneous.

3. Gastrointestinal. Gastrointestinal anthrax is rare in humans

Bacillus anthracis

Distinguishing Features

• large gram-positive rod with square ends, frequently found in chains

• spore-forming rods

• Capsule is composed of D-glutamate (This is unique—capsulesof other bacteria are polysaccharides.)

• Potential biowarfare agent

• Reservoir―animals, skins, soils

• Transmission―contact with infected animals

Cutaneous Anthrax

• Cause Bacillus anthracis

• Virulence factors Endospore, capsule, threeanthrax toxins.

• Portal of entry Direct contact of endosporeswith wounded skin, infected animals, or

contaminated animal products. (Two other forms ofanthrax—gastrointestinal and inhalation—have routes of entry via

the gastrointestinal tract and respiratory tract, respectively.

Pathogenesis

Capsule―polypeptide, antiphagocytic, immunogenic

Anthrax toxin includes 3 protein components:

• Protective antigen (B component)—mediates entry of lethal factor oredema factor into eukaryotic cells.

• Lethal factor—kills cells. A protease that cleaves the phosphokinase thatactivates the mitogen-activated protein kinase (MAPK) signal transductionpathway

• Edema factor is an adenylate cyclase that causes an increase in theintracellular concentration of AMP. This causes an outpouring of fluid fromthe cell into the extracellular space, which manifests as edema

• Signs and symptoms

Localized itching followed by a raised lesion that eventually forms apainless, black eschar within 7 to 10 days.

• Incubation period

Immediate response, within one day.

• Susceptibility

The most commonly infected individuals are animalhandlers.

Laboratory Diagnosis

• Smears show large, gram-positive rods in chains. Spores are usually not seen in smears of exudate because spores form when nutrients are insufficient, and

nutrients are plentiful in infected tissue.

• Nonhemolytic colonies form on blood agar aerobically. Colonies on blood agar typically have a characteristic flared “comet’s tail” appearance.

In case of a bioterror attack, rapid diagnosis can be performed inspecial laboratories using”

• Polymerase Chain Reaction (PCR)-based assays.

• Direct fluorescent antibody test that detects antigens of theorganism in the lesion.

• Serologic tests, such as an enzyme-linked immunosorbent assay(ELISA) test for antibodies.

• Treatment Doxycycline or ciprofloxacin are the preferredantimicrobials. There is no treatment for the toxin.

• Prevention Vaccinate livestock. The vaccine, consideredsafe for humans but recommended only for high-riskpersonnel, requires six doses over an 18-month period andannual boosters.

References

• Warren E. Levinson, Peter Chin-Hong, Elizabeth Joyce, Jesse Nussbaum, BrianSchwart. 2018. Review of Medical Microbiology & Immunology, 15th edition.McGraw-Hill Education.

• Kaplan. 2018. USMLE™ Step 1 Lecture Note. Immunology and Microbiology

• Robert W. Bauman, Todd P. Primm. Microbiology with Diseases by BodySystem. 2018. Fifth edition, Pearson.

• Sarah Friebe, F. Gisou van der Goot, and Jérôme Bürgi. The Ins and Outs ofAnthrax Toxin. Toxins (Basel). 2016 Mar; 8(3): 69. doi: 10.3390/toxins8030069.

• Dennis L. Stevens Dennis L. Stevens. The Role of Clostridial Toxins in the Pathogenesisof Gas Gangrene. Clinical Infectious Diseases, Volume 35, Issue Supplement_1, 1September 2002, Pages S93–S100, https://doi.org/10.1086/341928.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Friebe S[Author]&cauthor=true&cauthor_uid=26978402https://www.ncbi.nlm.nih.gov/pubmed/?term=van der Goot FG[Author]&cauthor=true&cauthor_uid=26978402https://www.ncbi.nlm.nih.gov/pubmed/?term=Bürgi J[Author]&cauthor=true&cauthor_uid=26978402https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810214/https://dx.doi.org/10.3390/toxins8030069https://doi.org/10.1086/341928