Anaerobic Gram-Positive Spore-Forming Bacilli

Anaerobic Gram-Positive Spore-
Forming Bacilli

Clostridium perfringens (Clostridium welchii)
• Gram reaction & characteristics:
• Gram positive or gram variable bacilli, sore forming, obligate anaerobe, non-motile.
brick-shaped rods/box car. Spores rarely seen. Spores are subterminal but difficult to
induce.
• Habitat:
• Common inhabitant of the colon.
• Virulence factor:
• Produces several exotoxins; alphatoxin, the most important, mediates destruction of host
cell membranes; enterotoxin inserts and disrupts membranes of mucosal cells; beta-toxin
is a cytotoxin. Hemolysin, necrotizing toxin.
• Disease:
• Cellulitis, gas gangrene.
• Alpha toxin (lecithinase) → muscle cell necrosis, degradative enzymes → subcutaneous
gas bubbles → crepitus myonecrosis with crepitus (crackles), gangrenous muscles →
black fluid exudate leaking from skin.
• Post-abortion sepsis, abdominal infections, and enterocolitis, septicemia.

• Major cause of food poisoning (from meats and gravy), resulting in a mild to moderate
diarrhea without vomiting.
• Enteritis necroticans (necrotizing enteritis; NEC): Life threatening infection that causes
ischemic necrosis of the jejunum.
• Diabetics and patients with circulatory disorders are more prone to infection.
• Mode of transmission:
• Bacteria are acquired through puncture wound or by ingestion. C. perfringens is normal
flora of the GI tract and can spread from this site following trauma.
• C. perfringens is also normal flora of the female genital tract and can cause post-abortion
infections.

• Lab diagnosis:
• Samples: wound swabs, necrosed tissues, muscle fragments, exudates, pus, stool, food.
• Culture characteristics:
• Dome shape, glossy, dew drop, produces a double zone of beta-hemolysis on SBA or
BRU/BA incubated anaerobically.
• C. perfringens exhibits a positive (enhanced hemolysis) reverse CAMP test. In this
assay, Streptococcus agalactiae (group B streptococci) is substituted for
Staphylococcus aureus in the standard CAMP test.
• Biochemical tests:
• Positive: Positive for lecithinase and glucose, lactose, maltose, and fructose
fermentation. Catalase negative.
• Treatment:
• Surgical removal of infected areas (amputation).
• Hyperbaric oxygen to kill anaerobic organisms.
• Penicillin, clindamycin (effective only in local, weak infections).

• Nagler test:
• Antilecithinase antibody is swabbed onto half of an egg yolk agar plate. The
isolate is inoculated onto both halves of the plate. C. perfringens produces
lecithinase, which will produce an opaque zone on the half of the plate without
the antibody. The antibody will neutralize lecithinase, preventing the opaque
zone from forming. This test is not performed much today.

Clostridium tetani (drum-stick bacillus)
• Gram-positive bacilli with round/terminal spores that resemble drumsticks or tennis
racket/lollipop obligate anaerobe, motile. Becomes gram neg after 24 hr.
• Habitat:
• Soil, dust, intestinal tract of various animal.
• Produces tetanospasmin (TeNT), a neurotoxic exotoxin that disrupts nerve impulses to
muscles.
• Disease:
• Spastic paralysis, tetanus (lock jaw or trismus), risus sardonicus (sneering):
• C. tetani produces tetanospasmin, a neurotoxin that affects the anterior horn cells of the
spinal cord, resulting in involuntary muscle contractions. Contractions begin with the
neck and jaw ("lock jaw") and progress to a backward arching of the back muscles.
• Bacteria and spores gain entry into the host by puncture wounds contaminated with
soil, or by wounds, which may include gunshots, burns, or animal bites. The bacteria
produce little necrosis. Contamination of existing wound or puncture by objects
contaminated with toxigenic Clostridium spp.
• Drug injections. Human & animal bites.

• Lab diagnosis:
• Samples: excised bits of tissue from necrotic depths of wounds. Wounds swabs are not good.
• Colonies on BRU/BA are irregular and rhizoid, gray, matte, translucent, and usually with a
narrow zone of -hemolysis. Often, a C. tetani colony swarms over the surface of a moist agar
plate (proteus of anaerobe world).
• Positive: gelatinase, spot indole and motility positive.
• Negative: catalase, lecithinase and lipase negative.
• Treatment:
• Clean wound
• Antitoxin (human anti-tetanospasmin immunoglobulin)
• DTaP vaccine booster
• Penicillin, metronidazole
• Diazepam (a GABA agonist).
• Prophylaxis:
• DTaP vaccine: tetanus toxoid, boosters required.
• Note:
• DTaP vaccines are administered at 2, 4, 6, and 18 months, again before starting school, and
every 10 years thereafter.
• Spores can affect neonates through cut umbilical cord, especially when the mother is not
immunized.

Body posture known as opisthotonos,
caused by the neurotoxin tetanospasmin.

Clostridium botulinum
• Gram positive or gram variable, bacilli, spore forming, obligate anaerobe, motile. Spores
are oval/subterminal and resemble tennis rackets.
• Habitat:
• Soil, dust, water, vegetables, animals.
• Botulinal toxin (BoNT), A, B, C, D, E, F ,G is a neurotoxin that binds to the synapse of
nerve fibers (blocks acetylcholine release), resulting in acute (flaccid/rag doll) paralysis
and death.
• Disease:
• Botulism. Flaccid paralysis and the four Ds: diplopia (double vision), dysarthria (speech
disturbance), dysphonia (altered voice), and dysphagia (difficulty swallowing).
• Adult botulism (classical food poisoning). Botulism due to ingestion of toxin in
inadequately cooked or improperly canned foods.
• Infant botulism/intestinal botulism (floppy baby syndrome). Infant botulism (due to
ingestion of spores in honey) is the most common type of botulism. Bacteria are ingested
and grow in the infant GI tract and can cause a rapidly fatal infection.
• Wound botulism is the result of contamination of wounds with spores of C. botulinum,
with subsequent germination, multiplication, and production of botulinal toxin in vivo.
• No fever!

• Botulism is usually acquired by ingestions of spoiled, home-canned foods in which the
spores are not destroyed.
• Wound botulism from injection drug use.
• Lab diagnosis:
• Samples: stool, wounds specimen, food.
• Botulinal toxin in serum, feces, gastric contents, vomitus, or the implicated food.
• Toxicity in mice.
• On egg yolk agar (EYA), yellow precipitation, exhibit a pearly surface iridescence as a
result of lipase positivity.
• Positive: Lipase, glucose, and motility.
• Negative: lecithinase, spot indole, catalase.
• Treatment:
• Antitoxin, respiratory support.
• Note:
• Botulinum toxin has been applied successfully to treat a number of conditions,
including strabismus, muscle spasticity, and facial wrinkles (Botox).

Clostridium difficile
• Gram positive bacilli, may arrange in chains (2-6 cells) spore forming (subterminal), obligate
anaerobe, motile. Brick-shaped bacilli
• Habitat:
• C. difficile is normal GI flora in a small percentage of the population, and as many as 30% of
hospitalized patients may carry the bacteria.
• toxin A (TcdA), enterotoxin → alters fluid secretion → watery diarrhea (AAD).
• toxin B (TcdB), cytotoxic to epithelial cells → pseudomembranous colitis.
• Disease:
• Antibiotic treatment disrupts normal flora but C. difficile survives by forming spores.
• Pseudomembranous Colitis (PMC), diarrhea (AAD).
• Person-to-person spread.
• Spores of C. difficile are frequently transmitted among hospitalized patients and are often
present on the hands of hospital personnel caring for patients.

• Lab diagnosis:
• Stool specimen: stool, toxin B present in feces.
• Colonoscopy: pseudomembranes (yellow-white plaques) present.
• Because C. difficile can be normal flora, stool cultures can sometimes be too sensitive.
Cultures for C. difficile should only be performed on watery or unformed stools.
• It is also important to test isolates for toxin production.
• Samples:
• Non-hemolytic on BRU/BA.
• Cycloserine-cefoxitin-fructose agar (CCFA) is used for isolating C. difficile from stool
specimens, fructose positive, yellow flattened colonies. The product will also
fluoresce yellow-green.
• Positive: glucose and fructose fermentation.
• Negative: Lecithinase, lipase, catalase and spot indole.
• Treatment:
• Oral metronidazole or oral vancomycin. Withdraw causative antibiotic (often
clindamycin).
• Note:
• Major cause of hospital-acquired diarrhea, often transmitted by the hands of hospital
personnel. Inflammation by toxin A can sometimes cause hypoalbuminemia.

Treponema pallidum
• Characteristics:
• Spirochetes, long, slender, helically curved bacilli, endoflagella, cannot usually be seen on
Gram stain, microaerophilic, sensitive to high temperature. Special stains such as silver and
Giemsa will stain spirochetes, silver for all spirochetes and Giemsa only for Borrelia.
• Habitat:
• Coat itself with host proteins.
• Tropism to arterioles; infection ultimately leads to endarteritis (inflammation of the lining
of arteries) and subsequent progressive tissue destruction.
• Disease:
• Venereal syphilis, 3 stages:
• Primary: Chancre (painless ulcer) at the site of inoculation (genitalia). The primary stage
is extremely infectious, because the lesion contains a large number of organisms.
• Secondary: Skin rash and lesions on oral mucosa (fever, weight loss, malaise, and loss of
appetite are present). Common symptoms are flulike (fever, sore throat, lymphadenopathy,
Patchy hair loss may occur, such as loss of eyebrows (“moth-eaten” area). highly infectious
state. Aseptic meningitis,
• Latent: Absence of clinical symptoms (subclinical).

• Tertiary: CNS disorders (neurosyphilis), aneurysms, and skin, liver, and bone disorders,
cardiovascular abnormalities, eye disease, and granuloma-like lesions (gummas) that are
soft, painless, and noninfectious and found on the skin or in the bones or visceral organs.
• Congenital syphilis is transmitted from a mother to an unborn fetus during any stage of
infection but is most often associated with early syphilis. The unborn fetus may develop
an asymptomatic infection or symptomatic infection.
• Symptoms known as Hutchinson triad (deafness, blindness, notched peg-shaped teeth)
may occur. Additionally, poor bone formation may result, such as “saber shin” bowing of
the tibia and the “bull dog” appearance of a deformed maxilla. Finally, neurosyphilis or
neonatal death can occur.
• Other clinically important species include T. pallidum subsp. pertenue (yaws), T.
pallidum subsp. endemicum (endemic syphilis), and T. carateum (pinta).
• Transmitted by sexual contact, direct blood transmission, or transplacentally (congenital
syphilis).

• Lab diagnosis:
• Spirochetes can be observed by darkfield or phase-contrast microscopy.
• Samples: from ulcers, lesions, CSF, umbilical cord section, placenta.
• chancer (containing live spirochetes), for primary syphilis diagnosis.
• Serology:
• Nontreponemal antigen tests include the Venereal Disease Research Laboratory
(VDRL) and rapid plasma reagin (RPR) tests, which detect antibodies to cardiolipin-
lecithin-cholesterol and are nonspecific. These antibodies are sometimes referred to
as reagin. Biologic false positives are caused by Lyme disease, various viruses,
autoimmune disorders (e.g., systemic lupus erythematosus), IM and pregnancy.
• Treponemal antigen tests include the fluorescent treponemal antibody absorption
(FT A-ABS) test and the Treponema pallidum particulate antigen (TP-PA) test,
which are specific and confirmatory.
• Direct fluorescent antibody tests can be used to detect T. pallidum in tissues and
material obtained from primary or secondary lesions.
• T. pallidum cannot be cultured in the laboratory. The bacteria exhibit corkscrew
motility seen by darkfield microscopy on material taken from lesions.
• T. pallidum subsp. pallidum can be propagated in the testicles of live rabbits.

Treatment:
Penicillin G.
Note:
Organisms that cross placenta and
therefore allow infection to pass from
pregnant mother to fetus (TORCHES):
TOxoplasma gondii
Rubella
Cytomegalovirus
HErpes, HIV
Syphilis

Mycobacterium tuberculosis
• Characteristics:
• Gram positive, acid-fast bacilli, thin rods, non-motile, obligate aerobe. Mycolic acid cell
wall: acid-fast staining, renders cell resistant to phagocytosis, germicides, and dryness.
• Habitat:
• Sulfatides in cell wall inhibit phagosome from fusing to lysosome, facultative I/C.
• Disease:
• Primary tuberculosis:
• Infection begins in the middle or lower areas of the lungs.
• The bacteria can spread (extrapulmonary tubercles) to the lymphatic system
(scrofula), CNS, and heart (pericarditis), vertebral bodies (Pott’s disease), kidneys,
GI.
• Macrophages phagocytize the bacteria and form multinucleated cells, which are
eventually surrounded by fibroid cells. Together the cells form granulomatous lesions
called tubercles, which can be seen on chest X-rays. The lesions can calcify, at which
point they are called "ghon complexes." While the bacteria are contained within the
granulomas, the patient is typically asymptomatic. This stage of the disease is called
a latent infection.
• Primary TB may not lead to active TB in people with healthy immune systems.
• Bloody sputum.

• A mycolic acid in the cell walls of M. tuberculosis, called cord factor, is associated with
several biologic activities, including host cell membrane cytotoxicity and inhibition of
white blood cell migration.
Serpentine cording
Middlebrook 7H10

• Reactivation or secondary tuberculosis:
• Occurs in people who have had latent TB.
• Dormant phase (macrophages) → mycolic acid cell wall allows intracellular survival
and proliferation. Reactivation of pulmonary tubercles, may disseminate to other
sites through lymphatics and blood (miliary TB),
• Reactivation, because of alteration in the cell-mediated immune response, can be
triggered by poor nutrition, alcoholism, or hormonal factors associated with
pregnancy and diabetes.
• Treatment requires long-term combination therapy, which can last up to 24 months.
First-line drugs include isoniazid, rifampin, ethambutol, and pyrazinamide.
• Spread by person-to-person contact via infected droplets.

special safety precautions must be practiced when working
with mycobacteria
• Negative-pressure room with air exiting through HEPA filters;
• Biosafety cabinet.
• Special centrifuges to contain aerosol production.
• Protective clothing, such as masks, disposable gowns, gloves, caps, shoe protection, and
sometimes, respirators.
• Personnel should be monitored annually with the Mantoux tuberculin test (PPD), in
which a mycobacterial antigen is injected into the skin.

• Lab diagnosis:
• Specimens:
• Lower respiratory tract: Sputum and bronchial washings, usually 3 to 5 samples, are
collected early in the morning on different days.
• Urine: 3 to 5 different morning voids
• Blood and bone marrow.
• Tissue and body fluids.
• It is necessary to decontaminate samples containing normal flora before culturing, and
sputum must also be digested.
• Digestion and decontamination of sputum samples:
• The mycobacteria are slightly more resistant to acids and alkalis than contaminating
bacteria making up the normal flora. Therefore, mild treatments, such as 2% NaOH
with N-acetyl-L-cysteine (NALC), benzalkonium chloride, oxalic acid are effective.
Only specimens containing normal flora, such as sputum that contains bacteria from
the oral cavity or feces, urine, gastric lavage, need to be decontaminated. Specimens
from normally sterile sites (e.g., CSF, blood, etc.) do not require decontamination.
• NALC is a mucolytic agent that liquefies mucus in respiratory specimens,
releasing mycobacteria.
• NaOH is digestant and decontaminating agent, increases the pH to a level that is
antibacterial.

Centers for Disease Control and Prevention acid-fast smear evaluation and reporting criteria

• Solid media:
• Lowenstein-Jensen (LJ) contains egg components for growth and malachite green to
inhibit growth of normal flora (nonpigmented (tan or buff, rough & tough), dry, heaped,
and granular in 14-21 days at 37° C).
• Lowenstein-Jensen-Gruft is made selective by the addition of penicillin and naladixic
acid. It is also supplemented with RNA.
• Middlebrook medium is agar based and contains 2% glycerol to support the growth of M.
aviwn complex (MAC). These media generally exhibit growth several days before egg-
based media. Antimicrobials can be added to make the media selective for the
mycobacteria.
• Liquid media:
• Middlebrook 7H9 broth is often used to maintain stock cultures and prepare isolates for
biochemical tests.
• Mycobacterium growth index tube (MGIT®) by Becton Dickinson (Franklin Lakes, NJ)
contains a modified 7H9 broth. The large amount of oxygen in the broth quenches the
fluorescence of a fluorochrome. As mycobacteria grow, they consume the oxygen, and the
fluorochrome will fluoresce when exposed to ultraviolet light.
• BACTEC 460TB system contains a radioactively labeled substrate that can be metabolized by
mycobacteria. Growth of mycobacteria releases radioactive CO2 that is detected by the
instrument.

+ve niacin accumulation test
(right)

• Acid-fast stain of sputum, PCR.
• Purified protein derivative (PPD) test: DTH reaction in active or previous infection
• Ghon complex on CXR.
• Measure IFN-gamma released by lymphocytes exposed to M. tuberculosis antigens (e. g.,
Quantiferon Gold assay/IGRA).
• Mycobacteria resist Gram staining because of lipids in their cell wall that prevent
penetration of crystal violet and safranin.
• Mycobacteria are acid-fast and are referred to as acid-fast bacilli (AFB). The primary
stain in the acid-fast stain is carbol fuchsin. The Ziehl-Neelsen stain requires heating
during the staining step, whereas the Kinyoun's stain does not.
• M. tuberculosis is niacin positive, nitrate reduction positive, and 68°C catalase test
negative.
• Treatment:
• Multidrug-resistant M. tuberculosis (MDR-TB), defined as simultaneous resistance to
isoniazid and rifampin, was first discovered in 1991, and, if present, indicates a poor
prognosis for recovery.
• Extremely-drug-resistant M. tuberculosis (XDR-TB) is defined as resistance to any
fluoroquinolone, and at least one of three injectable second-line drugs (capreomycin,
kanamycin, and amikacin), in addition to isoniazid and rifampicin.
• Treat with RIPES (multiple drugs to avoid resistance): rifampin, isoniazid, pyrazinamide,
ethambutol, streptomycin prophylaxis (for +ve PPD).
• BCG (Bacille Calmette-Guerin) vaccine: live attenuated vaccine for cell-mediated
immunity, renders patient PPD +ve.

• Mycobacterium avium complex (MAC) is comprised of two species that are difficult to
distinguish, M. avium and M. intracellulare, collectively called Mycobacterium avium-
intracellulare (MAI).
• MAI infections are clinically indistinguishable from pulmonary and systemic tuberculosis
infections. They are a major opportunistic pathogen in AIDS patients with low CD4 counts
and are resistant to multiple drugs.
• Treatment is with a macrolide ( azithromycin or clarithromycin) + ethambutol + rifamycin
(rifampin or rifabutin).
• M. bovis is found in unpasteurized milk and can lead to GI tuberculosis. Alternatively, it can
lead to pulmonary tuberculosis if inhaled.
• +ve PPD test: 10 mm induration (or 5 mm induration if immunocompromised, in AIDS often
false-negative).

• Runyon groups:
• Photoreactivity:
• 1) Photochromogens produce yellow to orange pigment only when exposed to light.
• 2) Scotochromogens produce yellow to orange pigment in the light and in the dark.
• 3) Nonchromogens (nonpigmented) do not produce pigment.
• Growth rate:
• Rapid growers produce colonies on solid media within 1 week. Most common pathogens
are slow growers, and weakly pathogenic species are rapid growers.
• Members of Runyon group 1 are slow growers and photochromogens.
• Group 2 members are slow growers and scotochromogens.
• Mycobacteria that are slow growers and nonchromogens belong to group 3. Group 4
contains the rapidly growing mycobacteria. Because of variation within the species,
Runyon groups are no longer commonly used.

• Niacin
• Niacin (nicotinic acid) is a precursor in the synthesis of NAD. Although all mycobacteria
produce niacin, some species produce an excess amount that is excreted from the cell.
Niacin accumulates in the medium and is detected by reacting with a cyanogen halide.
• M. tuberculosis is one of the few species positive for the accumulation of niacin.
• Growth on MacConkey agar
• MacConkey agar without crystal violet is inoculated with a 7-day broth culture of the
test organism. This is not the same formulation used for gram-negative bacilli.
• M. fortuitum and M. chelonei are the only mycobacteria able to grow on MacConkey agar
in 5 days.
• Susceptibility to thiophene-2-carboxylic acid hydrazide (T2H): differentiates M. bovis
(susceptible) from most other species (resistant).

Antimicrobials
• An antibiotic is a molecule produced by microorganisms that inhibits the growth of
other microorganisms. Antibiotics can also be synthetic.
• Cidal: Kills microorganisms (e.g., bactericidal compound kills bacteria).
• Static: Inhibits the growth of microorganisms (e.g., bacteristatic compound inhibits
bacterial growth)
• Synergy: When two or more antimicrobials are used, and the combined effect is greater
than what would be expected for the simple additive effect of the agents.
• Spectrum of Action:
1. Narrow-spectrum antimicrobial agent: Limited range of action
2. Broad-spectrum antimicrobial agent: Active against a wide range of bacteria.

Classes of Antimicrobial Agents and Their Mode of Action
• Beta-lactam antibiotics:
• Inhibit cell wall synthesis.
• (e.g., penicillins, cephalosporins, monobactams, and carbapenems).
• Cephalosporin:
• Narrow spectrum (first generation),
• Expanded spectrum (second generation),
• Broad spectrum (third generation),
• Extended spectrum (fourth generation).
• Beta-lactamase inhibitors:
• Bacteria can exhibit resistance to the beta-lactam antibiotics by producing an enzyme
(beta-lactamase) that cleaves the beta-lactam ring, inactivating the antibiotic.
• Beta-lactamase inhibitors can be given with a beta-lactam antibiotic to provide effective
treatment. Clavulanic acid, sulbactam, and tazobactam are examples of beta-lactamase
inhibitors.

• Aminoglycosides:
• Inhibit protein synthesis at the 30S ribosomal subunit and are
• Active against gram-negative and gram-positive bacteria
• (e.g., gentamicin, tobramycin and netilmicin). Tobramycin is bactericidal whereas the
others are bacteriostatic.
• They have no activity against obligate anaerobes.
• Because of potential toxicity, dosage should be monitored.
• Tetracyclines:
• Inhibit protein synthesis at the 30S ribosomal subunit.
• (e.g., doxycycline and minocycline).
• They are active against gram-positive and gram-negative bacteria and Mycoplasma and
Chlamydia.
• Increased resistance has limited their use.

• Chloramphenicol inhibits protein synthesis by binding to the 30S ribosomal subunit. It
is broad spectrum and used to treat serious gram-negative infections such as meningitis.
Risk of bone marrow toxicity, aplastic anemia (bone marrow suppression), limits use to
serious infections.
• Macrolides inhibit protein synthesis (e.g. erythromycin and clarithromycin).
• Sulfonamides inhibit folic acid synthesis.
• Glycopeptides inhibit cell wall formation by inhibiting peptidoglycan synthesis;
vancomycin is the only glycopeptide approved for use in the U.S. Vancomycin-resistant
enterococci, vancomycin-intermediate S. aureus, and vancomycin-resistant S. aureus
have been isolated.
• Quinolones inhibit DNA activity by inactivating DNA gyrase. Newer agents are known as
fluoroquinolones (e.g., ciprofloxacin and levofloxacin).
• Polymyxins disrupt plasma membranes; they are used to treat infections caused by
gram-negative bacteria (e.g., polymyxin B and polymyxin E).
• Nitrofurantoin inhibits bacterial enzymes; nitrofurantoin is used to treat UTIs.

Alyazeed Hussein, BSc, SUST
This has been a presentation of Alyazeed Hussein
Thanks for your attention and kind patience
@elyazeed7
@Alyazeed7ussein

Anaerobic Gram-Positive Spore-Forming Bacilli

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