Bacillus, Clostridium

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Bacillaceae

The family BacillaceaeThe feature they all share is formationof endospores2 clinically important genera:Bacillus(aerobic spore-formers)Clostridium(anaerobic spore-formers)

BacillusMore than 70 speciesB anthracis: -the most important

-the most feared agent of biological warfare-Anthrax (cutaneous,

gastrointestinal, inhalation)B. cereus: -gastroenteritis, ocular

infection,catheter-relatedsepsis, opportunistic infections

Bacillus anthracisGreek: anthrakis : coal –the blacklessions of the infectionLarge gram-positiveArranged as single or paired rods or as long, serpentine chainsFacultative anaerobeSpores (in 2-3-day-old cultures, are not seen in clinical specimen): in the center

Bacillus anthracis’s virulence

Capsule: coded by a plasmidPolypeptide capsule (consisting of poly-D-glutamic acid) in clinical specimenThis capsule is not produced in vitrounless special growth conditions areused.

Bacillus anthracis’s virulence

Three exotoxins:a second plasmid-Protective antigen (PA)-Edema factor (EF)-Lethal factor (LF)

PA + EF = Edema toxin (EdTx)PA + LF = Lethal toxin (LeTx)

Bacillus anthracis

Edema toxin: protectiveantigen(responsible for binding to thehost cell)+edema factorLethal toxin: protective antigen+lethalfactor

Bacillus anthracis - Epidemiology

Anthrax is primarily a disease of herbivores.Rarely isolated in developed countriesIs prevalent in impoverished areaswhere vaccination of animals is not practiced

Bacillus anthracis

The spores of the organismcontaminate soil or animal products andremain infectious for many yearsIndividuals at risk include people in contact with infected animals orcontaminated soil.Anthrax as a threat of biological warfare

Bacillus anthracis

acquired by-inoculation(cutaneous anthrax-mostcommon)-ingestion-rare-inhalation-the most deadly form

B.anthracis: an agent of bioterrorism

1979 in Sverdlovsk in the former SovietUnion: 79 cases of anthrax with 68 deathsContamination of employees of US Postal Service with letters containingB.anthracis (22 cases)

B.anthracis-Lab. diagnosis

Microscopy: long, thin, gram-positiverods, arranged singly or in long chainsSpores are not observed in clinicalspecimens but only in culturesincubated in a low CO2 atmosphere(special spore stain: malachite greenstain)In the center of nonmotile bacilli

B.anthracis-Lab. diagnosis

The capsule is produced in vivo but is not typically observed in cultureContrasting stain such as India ink (theink particles are excluded by thecapsule so that the background but not the area around bacteria appears black)Direkt fluorescent antibody (DFA)

B.anthracis-Lab. diagnosisCan grow on most non-selective mediaGrow rapidly and firmly adherent to theagarColonies not hemolytic, nonmotile in motility testsHave a dry "ground glass“ surface andirregular edges with projection alongthe lines (medusa head)

Bacillus cereusTwo enterotoxins:Heat-stable: emetic formHeat-labile: diarrheal form. Is similar tothe enterotoxins produced byEscherichia coli and Vibrio cholerae; each stimulates the adenilate cyclase-cyclic adenosine monophosphatesystem

The pathogenesis of B.cereusocular infections

Three toxins:Necrotic toxin (a heat-labileenterotoxin)Cereolysin (a hemolysin)Phospholipase C (a lecithinase)

Bacillus cereus- Diseases

Food poisoning (rice, meat, vegetables)Ocular infectionCatheter-related sepsis

Bacillus cereus

Are ubiquitous organisms, present in virtually all environmentIs responsible for two forms of foodpoisoning:Vomiting disease (emetic form)Diarrheal disease (diarrheal form)

The emetic form (vomitingdisease)

results from the consumption of contaminated riceMost bakteria are killed during the initialcooking of rice, but spores survive.The spores germinate and the heat-stable enterotoxin is released1-6 hour incubation period

The diarrheal disease (diarrhealform)

Consumption of contaminated meat, vegetables or saucesA longer incubation period, duringwhich the organism multiplies in thepatient’s intestinal tract and produceheat-labile enterotoxin

Ocular infections

Occur after traumatic, penetratinginjuries of the eye with soil-contaminated object

Laboratory diagnosisThe implicated food (e.g., rice, meat, vegetables) should be culturedFecal colonization is common: isolationof the organism from the patient shouldnot be attemptedspecimens collected from infected eyes, intravenous culture sites: Gram stainand culture

Clostridium

All anaerobic: Most strictanaerobesSome aerotolerant

Gram-positiveForms endospores(except C. perfringens-rarely)

The traditional method forclassifying an isolate in Clostridium

-demonstration of spores-growth in anaerobic conditions-a complex set of biochemicaltests-gas chromatography analysis

more than 177 species

ClostridiumUbiquitous (everywhere): soil, water, sewage,

gastrointestinal flora of animalsand humans

Most are harmless saprophytesSome are well known pathogens:

-tetanus(C. tetani)-botulism(C. botulinum,..)-gas gangrene(C. perfringens,..)

Clostridia-Diseases

Skin and soft tissue infectionFood poisoningAntibiotic associated diarrhae and colitis

The Clostridia

Ability to survive adverse environmentalconditions through spore formation

Rapid growth in a nutritionally enrichedoxygen deprived environment

Production of numerous histolytic toxins, enterotoxins and neurotoxins.

Pathogenic Clostridia: siximportant human pathogens

C.difficile-commonC.perfringens-commonC.septicum-uncommonC.tertium-uncommonC.botulinum-uncommonC.tetani-uncommon

Rare clostridia

C.baratii, C.butyricum, C.histolyticum, C.novyi,C.sordellii

Clostridium perfringens

Gram-positive large rod shaped bacteriaSpores are rarely observed after in vivoor in vitro cultivationNonmotile, but rapidly spreadinggrowth on laboratory media

Clostridium perfringens-culture

Rapidly grows in tissue and in cultureHemolyticMetabolically activeLethal toxins (α, β, ε, ι toxins)5 types (A to E)

C.perfringens - Pathogenesis

Can cause severe life-threateningdiseaseSelf-limited gastroenteritisSoft tissue infections(cellulitis, myositis, myonecrosis or gas gangrene)

C.perfringens - Toxins

12 toxins and enzymesα toxin: the most important toxinA lecithinase (phospholipase C)Lyses erythrocytes, platelets, leukocytes andendothelial cellsIt increases vascular permeability, resulting in massive hemolysis, bleeding, tissuedestruction, hepatic toxicity and myocardialdysfunction

C.perfringens - Toxins

β toxin: responsible for the necrotic lesions in necrotizing enteritisΕ toxin: a protoxin activated by tripsin andincreases the vascular permeability of thegastrointestinal wallι toxins: the fourth major lethal toxin, has necrotic activity and increases vascularpermeability

Clostridium perfringens

Enterotoxin (cytotoxic, enterotoxic): Heat-labile;Leads to altered membrane permeability in ileum and loss of fluid and ions;Neurominidase –promotes capillary

thrombosis

C.perfringens-EpidemiologyType A commonly inhabits the intestinaltract of humans and animalsWidely distributed in nature, particularlysoil and water contaminated with fecesType A is responsible for most humaninfectionsType C is responsible for one importantinfection in humans: enteris necroticans

C.perfringens- Diseases

Soft-tissue infections :CellulitisFasciitis, myositisMyonecrosis or gas gangrene (gas in tissue)

C.perfringens- Diseases

Clostridial food poisoning: shortincubation period (8-24 hours)Necrotizing enteritis (enteritisnecroticans or pig-bel)Septicemia

C.perfringens-Lab.diagnosisPerforms a confirmatory diagnosis of soft-tissue diseases because therapymust be initiated immediatelyThe microscopic detection of gram-positive rods in clinical specimens, usually in the absence of leukocytes; characteristic morphologyCan be detected on simple media

C.perfringens-Lab.diagnosis

Forms spores but rarely in clinical specimen orcultureReplicates rapidlyLarge spreading colonies within the first day of cultureDouble zone of hemolysis (complete hemolysisand partial hemolysis by different toxins)

Clostridium perfringens

Produces many toxins and hemolyticenzymesWhite blood cells absent in gram-stainedclinical specimen

Clostridium tetaniLarge, motileSpore-formingGram-positive bacilliTerminal spores (drumstickappearence)Strict anaerobe (very sensitive tooxygen)Difficult to isolate from clinical specimen

Pathogenesis - Virulence

The vegetative cells die rapidly whenexposed to oxygen, but (!!)Spore formation allows the organism tosurvive in the most adverse conditionsProduces two toxins:Tetanolysin: an oxygen-labile hemolysinTetanospasmin: heat-labile neurotoxin

Pathogenesis - Virulence

Tetanospasmin:heat-labile neurotoxin, blocks the release of neurotransmitters: causes spastic paralysisPlasmid-encoded: nonconjugativeReleased when the cell is lysedResponsible for the clinicalmanifestations of tetanus

Virulence - TetanospasminIs synthesized as a single peptid that is cleaved into:

light (A-chain) subunit andheavy (B-chain) subunit by an endogenous

proteaseInactivates proteins that regulate release of the inhibitory neurotransmitters glycine andgamma-aminobutryic (GABA)

C.tetani - EpidemiologyUbiquitousSpores in soilsTransiently colonizes the gastrointestinaltracts of humans and animalsExposure to spores is frequentlyDisease is uncommon (increased risk wherethere is a poor access to vaccine and medicalcare)

Clostridium tetani’s diseases

Tetanus: -Generalized-Localized

cephalic tetanus-Neonatal

C.tetani-Laboratory diagnosis

The diagnosis of tetanus is made on thebasis of the clinical presentationThe microscopic detection or culture is is useful but frequently unsuccessfulCulture are positive in only 30% of patients with tetanus

Clostidium botulinum

The etiologic agent of botulismLarge, spore-formingAnaerobic bacilliSeven toxins (A to G): A,B,E,F the mostcommon: Neurotoxin subunit+nontoxic subunitswhich protect them from stomach acids

Clostidium botulinum

Similar to tetanus toxinThe target neural cells are differentBlocks neurotransmission at peripheralcholinergic synapsesPrevents release of neurotransmitteracetylcholineFlaccid paralysis

Clostidium botulinum

Foodborn botulismInfant botulismWound botulismInhalation botulism (a major concern in this era of bioterrorism)

C.botulinum - Lab. diagnosis

Isolation from specimens contaminatedwith other organisms:Heating the specimen for 10 min at 80°C to kill all non-clostridial cellsCulture on anaerobic media allows theheat-resistant C.botulinum to germinate

C.botulinum - Lab. diagnosis

Lipase production: egg-yolk agar is used and iridescent film on coloniesDigests milk proteinsHydolyzes gelatinFerment glucose

Clostidium botulinum

Demonstration of toxin: mouse bioassayTwo aliquots: one with antitoxin andintraperitoneal incubationIf the antitoxin treatment protects themice, toxin activity is confirmedSample of implicated food, stoolspecimen

Clostridium difficile

Until the mid-1970s the clinicalimportance of C.difficile was not appreciatedToxin A: enterotoxinToxin B: cytotoxinA part of normal flora

Clostridium difficileTaking antibiotics alter the floraThe disease develops in people takingantibiotics, because the drugs alter thenormal enteric floraOcurs if the organisms proliferate in colon and produce their toxinsLife-threatening pseudomembranouscolitis

Clostridium difficile

The organism isolated on highlyselective mediaCytotoxin detectionEnterotoxin detectionIn a stool specimen from a patient withcompatible clinical symptomsCommercial immunoassays