Anaerobic bacteria
lect:3
Dr. Khalid Waleed
M.B.ch.B., Msc., PhD. Immunology
Objectives
The objectives of this lecture are to know
1. Diseases related to Clostridium perfringens infections.
2. Diseases related to Clostridium difficile infections.
3. Other clinically important species of anaerobic organisms.
Clostridium perfringens
It is an anaerobic, spore-forming Gram-positive rod, produces
several exotoxins; Alphatoxin, the most important, mediates destruction
of host cell membranes. Beta-toxin is a cytotoxin. Enterotoxin inserts
and disrupts membranes of mucosal cells.
Diseases and pathogenesis
Gas gangrene, or clostridial myonecrosis, is an invasive, anaerobic
infection of muscle and is characterized by extensive tissue necrosis and
the production of gas. The infection begins with the entry of C.
perfringens from contaminated soil or clothing, into a traumatic wound,
followed by the multiplication and spread of the organism. The
incubation period of gas gangrene is usually short (6-8 hours). There is
reduction of the local oxidation-reduction potential, which creates the
anaerobic environment that is required for the growth of C.
perfringens. The growing C. perfringens vegetative cells produce
alpha toxin and perfringolysin O, which cause local and regional
necrosis in muscle, allowing rapid and progressive spread of the
infection. In addition, these toxins can enter the systemic circulation to
induce organ damage, circulatory problems and death.
Other diseases
1. C. perfringens food poisoning. C. perfringens is associated with two
types of food poisoning—type A, a relatively mild and self-limited GI
illness, and type C, a more serious but rarely seen disease (C. perfringens
type C food poisoning (enteritis necroticans). C. perfringens foodborne
disease usually follows the ingestion of enterotoxin-producing strains in
contaminated food Usually self-limiting and benign; manifested by
abdominal cramps, diarrhea, and vomiting.
2. Necrotizing enteritis (NEC): Life threatening infection that causes
ischemic
necrosis
of
the
jejunum.
Often
associated
with
immunocompromised patients (e.g., those with diabetes, alcohol-induced
liver disease, or neutropenia).
Laboratory Diagnosis
Smears of tissue and exudate samples show large gram-positive rods.
Spores are not usually seen because they are formed primarily under
nutritionally deficient conditions.
The organisms are cultured anaerobically and then identified by sugar
fermentation reactions and organic acid production. C. perfringens
colonies exhibit a double zone of hemolysis on blood agar (alpha
surrounding beta).
Nagler’s reaction (Lecithinase test): is a biochemical test used to
identify organisms which liberate phospholipases (lecithinases) e.g.
Clostridium perfringens. The M.O. is inoculated on medium
containing egg yolk. The alpa (α) toxin of C. perfringens has
phospholipase activity and hence, helps in differentiation of C.
perfringens from other Clostridium spp.
Clostridium difficile
It causes life-threatening diarrhea. It is a leading cause of hospital
associated gastrointestinal illness. This infections mostly occur in people
who have had recent medical care (hospitalized or recently hospitalized
patients), or recent antibiotic use, or recent chemotherapy. This
organism is part of the GI biota in about 5% of individuals, although the
colonization rate in patients associated with long-term care facilities, such
as nursing homes and rehabilitation facilities, can reach 20% of the
population.
Clostridium difficile characteristics
Clostiridum difficle is a major nosocomial enteric pathogen in
hospitals.
It is an anaerobic, Gram positive rod.
Transmission
The hands of health-care workers.
Direct exposure to contaminated patient-care items.
High-touch surfaces in patients’ bathrooms.
Toxins
Produces toxin A (TcdA), an enterotoxin, and toxin B (TcdB), a
cytotoxin.
Both toxin A and toxin B are classified as large clostridial cytotoxins.
The toxins glycosylate guanosine triphosphate (GTP) signaling
proteins, leading to a breakdown of the cellular cytotoxin and cell
death.
Pathogenesis
Following antimicrobial therapy, many bowel biota organisms other
than C. difficile are killed. Thus allowing C. difficile to multiply with less
competition and produce two toxins: toxin A and B. Bloody diarrhea
with associated necrosis of colonic mucosa is seen in patients with
pseudomembranous colitis. C. difficile is a common cause of health care–
associated (nosocomial) infection. The organism is frequently
transmitted among hospitalized patients and is present occasionally on the
hands of hospital personnel. Diarrhea caused by C. difficile is being seen
with increasing frequency in outpatients who have received antimicrobial
therapy. Fatal cases of pseudomembranous colitis leading to bowel
perforation and sepsis.
Clindamycin was the first antibiotic to be recognized as a cause of
pseudomembranous colitis. At present, second- and third-generation
cephalosporins are the most common causes because they are so
frequently used. Ampicillin and fluoroquinolones are also commonly
implicated. In addition to antibiotics, cancer chemotherapy also
predisposes to pseudo-membranous colitis. C. difficile rarely invades the
intestinal mucosa.
Diseases
The clinical presentation ranges from a mild watery diarrhea to a life-
threatening toxic megacolon, which requires surgical intervention.
Patients with severe disease often present with abdominal pain,
leukocytosis, and fever in addition to the diarrhea.
Diagnosis
Visualization of a characteristic pseudomembrane or plaque on
colonoscopy is diagnostic for pseudomembranous colitis and, with the
appropriate history of prior antibiotic use, meets the criteria for diagnosis
of antibiotic-associated pseudomembranous colitis. Additional patient
risk factors should also be considered for potential diagnosis for C.
difficile infection Such as advanced age (>65 years), immunosuppression
or other severe underlying gastrointestinal disease and use of proton
pump inhibitors,
Laboratory diagnosis
Two major types of tests are available for routine use:
1. Culture for direct detection of the organism .
2. Detection of cytotoxin (toxin A, B or both) by cell culture
cytotoxicity. The test is considered positive if a cytopathic effect
(CPE ) is seen in 50% of cells at 48 h, and the effect is inhibited in the
wells containing C. difficile antitoxin.
3. Enzyme immunoassay (EIA) toxin assay: It Can detect toxin A, toxin
B, or both. Detect free toxin in stool samples. Its rapid and specific,
but Less sensitive than cytotoxin test.
Treatment: The causative antibiotic should be withdrawn. Oral metronidazole
or vancomycin should be given and fluids replaced. Metronidazole is preferred
because using vancomycin may select for vancomycin-resistant enterococci.
However, in life threatening cases, vancomycin should be used because it is
more effective than metronidazole.
Other Spp. of anaerobic organisms
A. Lactobacillus spp.
Lactobacillus spp. are gram-positive, highly pleomorphic bacilli, which may
appear on Gram stain as a coccoid or spiral-shaped organism.
There are more than 100 species of Lactobacillus.
Lactobacilli are widely distributed in nature and foods, as well as in normal
biota in the human mouth, GI tract, and female genital tract.
Associated with advanced dental caries. Organism has also been identified in
endocarditis and bacteremia.
No definitive virulence factors known.
Lactobacillus spp. play an important role in the health of the female vaginal
tract in that they help protect the host from urogenital infections.
Bacterial vaginosis (BV)
BV occurs when the delicate balance of normal vaginal microbiota is
disrupted and replaced by an overgrowth of specific organisms.
In healthy women, the vaginal biota is mainly Lactobacillus spp., which
maintain a pH between 3.8 and 4.5 to prevent the overgrowth and
invasion of pathogenic bacteria by competitive exclusion, competition for
nutrients, and release of antimicrobial substances such as hydrogen
peroxide and organic acids.
Thus, when vaginal lactobacilli are depleted, vaginal pH increases,
allowing the overgrowth of various bacterial species including
Gardnerella vaginalis, Mycoplasma hominis, Ureaplasma spp., and
anaerobes such as Prevotella, Porphyromonas, Peptostreptococcus, and
Mobiluncus.
This mix of organisms leads to the development of an abnormal vaginal
discharge with a distinct unpleasant odor, but lacks a true inflammatory
reaction.
Patients with BV demonstrate a discharge that is usually a milky,
homogeneous, thin liquid adhering to the walls of the vagina.
Lactobacilli produce lactic acid from glycogen, which lowers the vaginal pH
and suppresses the overgrowth of organisms such as Mobiluncus, Prevotella,
and G. vaginalis.
B. Anaerobic, gram-negative bacilli (Endogenous strains)
1. Bacteroide s fragilis group,
2. Prevotella spp.,
3. Porphyromonas spp.
4. Fusobacterium nucleatum and other Fusobacterium spp.
Virulence factors:
Produce capsules, endotoxin, and succinic acid, which inhibit
phagocytosis, and various enzymes that mediate tissue damage.
Require some breach of mucosal integrity.
Infections are often mixed (polymicrobial) with (anaerobic and
facultative anaerobic organisms).
Localized or enclosed abscesses, and may involve the cranium,
periodontium, thorax, peritoneum, liver, and female genital tract.
The hallmark of most but not all infections is the production of a foul
odor.
In general, infections caused by B. fragilis group occur below the
diaphragm; pigmented Prevotella spp., Porphyromonas spp., and
F. nucleatum generally are involved in head and neck and
pleuropulmonary infections.