GRAM POSITIVE, SPORE-FORMER BACILLI

Prepared by: nahaeminrmt

SPORE FORMING, NON-BRANCHING, CATALASE POS BACILLI

Bacillus spp.
General Characteristics
 It commonly inhabits the soil
 Aerobic and facultative anaerobic (may also form spore under these conditions)
 Causative agent of anthrax
 Considered as a bio terror agent - B. anthracis
 Some species are thermophile (55°C)
 Grow well on SBA and other enriched media but not in Columbia CNA
Bacillus anthracis
 Pathogenic and lethal in human
Virulence Factors  Capsule (polygamma D glutamic acid)
 Protection against phagocytosis
 Polypeptide of D-glutamic acid - resistant to proteolytic enzymes hydrolytic
action
 Anthrax Toxins - act synergistically to produce damaging effects
 Protective Antigen/Protective Capsular Antigen - binding molecule of edema
factor and lethal factor so that they could adhere on the host cell surface
 Edema Factor - Edema (EF + PA); adenylate cyclase that increases the
concentration of cyclic adenosine monophosphate (cAMP) in host cells
 Lethal Factor - death (PA + LF); protease that is capable to kill host cell by
disrupting extracellular signal transduction (so walang APC ang makakaabot
ke macrophage)
Clinical Manifestations MOT: animals - ingestion of spores; human - animal contact/occupational hazard
 Cutaneous Anthrax
 Most common type but less severe
 S/S: wounds contaminated with spores
 “Black Eschar”/Malignant pustule - papule/pimple (2-3 days after exposure) →
vesicle development → erythematous ring → ulcerates and dries up →
depressed black necrotic areas
 Black eschar is painless and non-pyogenic and heals after 1-2 weeks
 If untreated = 20% fatality rate
 Inhalation Anthrax/”woolsorter’s disease”
 It affects the pulmonary parenchyma
 S/S: it resembles upper RTI (colds and flu)
 Complications: bronchopneumonia, mediastinitis, and septicemia
 May be severe and highly fatal = respiratory problems (dyspnea, cyanosis,
pleural effusion) then followed by disorientation, coma, and death
 Moratality rate = almost 100%
 Gastrointestinal Anthrax
 Most fatal among all anthrax
 MOT: ingestion of spores
 S/S: abdominal pain, nausea, anorexia, vomiting, and may cause bloody
diarrhea (prostration that may lead to death)
 Injectional Anthrax
 Soft tissue infections associated with skin popping and heroin used (Scotland
outbreak in 2009-2010)
 Injection of spores into tissues
 Complications: necrotizing fasciitis, shock, coma, organ failure, and meningitis
 Lack of eschar and higher mortality rate compared to cutaneous anthrax
 Complication: septicemia → purulent meningitis
Laboratory Diagnosis Microscopy  Gram Stain: gram pos/gram variable, square ended rod

LOUISSE NICOLE B. MANLICLIC, RMT (2023)

 found in single or in chains with unstained central pore
(bamboo fishing rods)
 Gram pos - young age; gram variable - aged and
under nutritional stress
 Clear zone = capsule; stimulation of capsular
formation (increase CO2)
 Spore stain: spores (green); vegetatitive cells (red)
Culture  Spx: blood, lung tissue, and CSF
Characteristics  BAP - non-hemolytic, gray, and flat with an irregular
margin due to outgrowth of long filamentous
projections (“Medusa Head”) and have a tenacious
consistency (“beaten egg whites”)
 Prolonged incubation of heavy growth = may
appear weakly hemolytic
 PEA - growth is usually weak; presumptive identification
of B. anthracis in stool (CDC)
 Other culture media - see reviewer
Biochem Tests Catalase pos
Non-motile
Ferments glucose but not mannitol, arabinose, or xylose
Growth on EYA
Growth in 7% salt and acidic pH (<6)
Susceptible to penicillin
String of Pearl MHA with penicillin (0.05 units)
Susceptibility Penicillin (10 units)
Test
Capsule - India Ink; M’Fadeyen; direct fluorescent antibody
assays for a cell wall polysaccharide and a capsule antigen
NAAT
Bioterror compenent -SPORE
-Handled by reference laboratory
-BSL II (processing); BSL III (culture)
Treatment Penicillin; tretracycline, fluoroquinolones, and chloramphenicol
Initial IV therapy: ciprofloxacin or doxycycline
Injectional Anthrax - metronidazole
To inhibit exotoxin production - clindamycin
Bacillus cereus
 Motile, salicin fermenter, growth on EYA
 Contaminated fried rice; food poisoning (nausea, abdominal pain, vomiting, occasional diarrhea and NO FEVER
 Diarrheal or Emetic in form
 Culture of food is preferred: 100000 organism/gram of food (same value is also applied in stool); it is compared to stool
colony as B. cereus may also present in small amount in health people
 BAP: beta-hemolytic with frosted glass appearance (37degC)
 Other culture media: reviewer
 Eye Infection - most common type of non-GI infection
 Endolphthalmitis, panophthalmitis, and keratitis with abscess formation
 Non-GI Infections - Commonly observed in drug abusers, immunocompromised, and post-surgical patients
 Meningitis, septicemia, and endocarditis, pneumonia, and ear infection
 Treatment/Drug of Choice - Penicllin (R); Vancomycin/Clindamycin with or without aminoglycoside (S)
Diarrheal/Bacillus spp. (Henry’s) Emetic/TYPE 1/B.cereus (Henry’s)
Incubation period 8-16 hrs 1-5 hrs(Mahon)/1-6 hrs (Henry’s)
Diarrhea Very common Fairly common
Vomiting Occasional Very common
Duration of Illness 12-24 hours 6-24 hrs
Food implicated Meat products, poultry, soups, vegetables, Fried or boiled rice
puddings, sauces
LOUISSE NICOLE B. MANLICLIC, RMT (2023)
 Stability to heat - +
Bacillus thuringiensis
 Larvicidal/insect pathogen
 Parasporal crystals
Other Bacillus: B. subtilis (common lab contaminant), B. licheniformis, B. circulans (vancomycin resistant), B. pumilus, and B.
sphaericus
- usually non pathogenic; contaminants
- food poisoning, bacteremia, meningitis, pneumonania, and other infections
- drug of choice: fluoroquinolines, tetracyclines, aminoglycosides, chloramphenicol, vancomycin
Other gram positive aerobic bacilli, spore producer
 Paenibacillus macerans - contamination of culture bottles in NICU
 Paenibacillus pasadenensis - isolated in spacecraft facilities; mediastinitis following heart surgery

GRAM , SPORE-FORMER BACILLI

Prepared by: Louisse Nicole B. Manliclic

Clostridium spp.

General Characteristics
 Predisposing factors: human or animal bite wounds; aspiration of oral contents after vomiting, periodontal procedures;
GI tract surgeries/traumatic puncture of the bowl; genital tract surgery or traumatic puncture of genital tract; soil
contamination of wound
 Indication of probable involvement of anaerobes: infection in close proximity to a mucosal surface; fould odor; large
quantity of gas; black color or brick red fluorescence; presence of sulfur granules
VIrulence Factors of Clostridium spp.
Virulence Factor Function Clostridium spp.
Collagenase Catalyze the degradation of collagen Certain clostridium spp
Cytotoxins Toxic to specific types of cells Clostridium difficile
DNAses Destroys DNA Certain clostridium spp
Enterotoxins Toxic to cells of the intestinal mucosa Clostridium difficile
Hemolysins Catalyzes the hydrolysis or hyaluronic acid, the cementing Certain clostridium spp
substance of tissues
Lipases Catalyze the hydrolysis of ester linkages between fatty acids Certain clostridium spp
and glycerol of triglycerides and phospholipids
Neurotoxins (e.g., Destroy or disrupt nerve tissue Clostridium botulinum, C.
botulinum, toxin, tetani
tetanospasmin)
Phospholipases Catalyze the splitting of host phospholipids (lecinthinase) Certain clostridium spp
Proteases Split host proteins by hydrolysis of peptide bonds Certain clostridium spp
Clinical Infections
Clostridium tetani  Tetanus
 MOT: inoculation through wounds
 Neurotoxin tetanospasmin - it prevents the release of neurotransmitters
(spastic paralysis)
 Toxin → gangliosides of CNS, lymph nodes, and bloodstream →
inhibition of modulator (gangliosides) → painful spastic paralysis
 Ganglisodes - modulator of cell-to-cell interaction, membrane proteins,
and ion channels
 Trismus (lock jaw), risus sardonicus (distorted grin), difficulty in breathing
 Symptoms onset: 7 days after injury; incubation period (distance from the
site of injury to the CNS): 3-21 days
 S/S: muscular rigidity (muscular spasms in the pharyngeal area); rigidity of
the abdomen, chest, back, and limbs
 Diagnosis (Henry’s) - patient symptoms and clinical manifestations
 Preventive measures: diphtheria-tetanus-acellular pertussis vaccine
LOUISSE NICOLE B. MANLICLIC, RMT (2023)

Clostridium botulism
BIOTERORROR AGENT - botulism
toxin and C. botulinum
Must know!
Clostridium tetani - spastic paralysis
Clostridium botulinum - flaccid paralysis
Anaerobic cellulitis - no invasion of muscle
Myonecrosis - Anerobic cellulitis with muscle invasions; foul odor
Clostridium perfringens
Clostridium difficile
LOUISSE NICOLE B. MANLICLIC, RMT (2023)
(booster after 10 years)
 Botulism
 MOT: ingestion of toxin
 Botulinum toxin - antigens A to G (A,B, E = associated with human
disease); potent neurotoxin; it prevents the release of acetylcholine in the
neuromuscular junction; most potent toxin in human
 Acetylcholine - neurotransmitter; when inhibited, it causes flaccid paralysis
and death
 Botulinum toxin Type A (botox) - to treat strabismus (wandering eye);
beauty enhancer
 Foodborne Botulism
 Consumption of home-made canned vegetables, home-cured meat,
fermented fish, and other preserved foods
 Toxin → absorbed in small intestine → CNS
 S/S: weakness and paralysis, double or blurred vision, impaired speech,
difficulty in swallowing
 Treatment - antitoxin
 Infant Botulism
 Honey contaminated with C. botulinum
 Floppy baby
 Wound Botulism - contamination of wounds with C. botulinum
 Diagnosis: toxin serum, wound, or food, gastric contents, stool; mouse assay
(exotoxin - heat labile; study of choice)
 Food Poisoning
Characteristic Type A Type C - Enteritis
necroticans
Toxin Enterotoxin Beta-toxin (less
commonly, alpha-toxin)
Incubation 8-30 hrs 5-6 hrs
Food contaminated Meats and gravies
Abdominal pain and Present
diarrhea
Bloody Diarrhea Absent Present
Type C - necrotic inflammation of the small intestine → bowel perforation
 Myonecrosis/Gas Gangrene
 It could also be caused by other clostridium: C. histolyticium, C. septicum,
C. novyi, and C. bifermentans
 Most common causative agent: C. perfringens
 Toxin: alpha-toxin (phospolipase C) - lecithinase (since sinisira niya si
phospholipid, sinisira niya yung cell layer ng skin)
 S/S: pain and swelling in infected area; presence of bullae, serous
discharge, discoloration, and tissue necrosis
 Gram stain (myonecrosis) - large, box-car formation with absence of
neutrophils
 MOT: person-to-person contact (fecal-oral route); exposure to to an
environment contaminated with spores
 Frequent cause of antibiotic associated diarrhea
 Clindamycin, broad-spectrum cephalosporins, carbapenems, and
fluoroquinolones

Other Clostridium spp.
 Pseudomembranous colitis and toxic megacolon
 Enterotoxin tcdA and Cytotocix tcdB - works together to enter and
damage the cell that will lead to apoptosis
 tcdB - major virulent factor
 PCR - to diagnose tcdA and tcdB
 Spx - stool
 Other tests
 Traditional tests - culture and cytoxocity netralization assay
 Rapid antigen test and molecular methods are now used
- ELISA: Glutamate dehydrogenase detection
- NAAT: detection of tcdA and tcdB
 C. Septicum and C. sordelii - gastrointestinal pathology or following abortions,
vaginal deliveries, or ceserean section

Definitive ID for anaerobes

1. Enzyme Based Systems
2. Pre-reduced anaerobically sterilized (PRAS) tube media
3. GLC
4. 16s rRNA detection
5. MALDI-TOF

LOUISSE NICOLE B. MANLICLIC, RMT (2023)