Bacillus, Clostridium
Doç Dr Nevriye Gönüllü
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