This document discusses Clostridium difficile, a gram-positive anaerobic bacterium that is the most common cause of hospital-acquired diarrhea. It was first identified in 1935 but was misclassified as a commensal organism until the 1970s when it was recognized as the causative agent of pseudomembranous colitis. C. difficile infection is associated with prior antibiotic use and spreads through spores that can persist in the hospital environment. The document outlines the risk factors, mechanisms of pathogenesis, clinical manifestations, diagnostic tests, and treatment approaches for C. difficile infection.

1. Dr. Meenakshi Sharma
Assistant Professor
Department of Microbiology
Mayo Institute Of Medical Sciences

2.  C difficle first described in1935 gram-positive
anaerobic bacillus
 “difficult clostridium”-difficult to grow in
culture
 Found in stool specimens from healthy neonates
leading to misclassification as a commensal
organism
 1970s:“clindamycin colitis” pseudomembranous
colitis in hospitalized patients
 1978: C diffficle recognized as causative
organism

3.  Most common cause of nosocomial diarrhea.
 Rate and severity of C. difficile-associated
diarrhea (CDAD) increasing.
 New strain of C.difficile with increased
resistance and virulence identified.

4.  Present in environment.
 Hospital is major reservoir. Spores can be
recovered from surfaces for months.
 Spread primarily on hands of HCW.
 Fecal-oral transmission.
 Transmission may occur from asymptomatic
colonized persons.
 Colonizes the colon of up to 3% of healthy adults.
 5 – 25% of debilitated and antibiotic-treated
hospitalized adults colonized.
 Implicated in approx. 25% of cases of antibiotic-
associated diarrhea

5.  Cephalosporins
 Clindamycin
 Erythromycin
 Penicillins, such as amoxicillin and ampicillin
 Quinolones, such as ciprofloxacin and
levofloxacin
 Tetracyclines, such as doxycycline and
minocycline

6.  Previously experienced antibiotic-associated
diarrhea while taking an antibiotic medication
 Are age 65 or older
 Have had surgery on your intestinal tract
 Have recently stayed in a hospital or nursing
home
 Have a serious underlying illness affecting
intestines, such as colon cancer or
inflammatory bowel disease

7. Disruption of protective colonic flora
(AB)
Colonization with toxigenic C. difficle
by fecal-oral transmission
Toxin A and B production
A/B: Cytoskeletal damage, loss of tight
junctions.
A: Mucosal injury, inflammation, fluid
secretion. Colitis and Diarrhea

8.  Pseudomembranous colitis
 Toxic mega colon
 Perforation of the colon
 Sepsis
 Death

9.  Proper selection , collection and transport of
clinical specimens are extremely important.
 Suspected gas gangrene or Necrotizing fascitis
 Requires rapid clinical diagnosis
 Multiple tissue specimens should be sampled from
the active sites of infection
 Films from the muscles at the edge of the affected
area, from the tissues in the necrotic area and
from the exudate in the deeper parts of the wound
 Necrotic tissue and muscle fragments

10.  Suspected C.perfringens foodborne illness
 Freshly passed fecal specimen and the suspected food
are the preferred specimens
 Suspected Enteritis Necroticans (C.perfringens type
C)
 Three different blood cultures from three different
venipuncture sites
 Stool (at least 25g or 25ml if liquid)
 Bowel luminal contents or tissue from the involved
bowel
 Suspected CDI
 A single freshly passed fecal specimen (ideally 10-20 ml
of watery stool; minimum of 5ml or 5g ) is ideal for
culture and toxin assays

11.  Suspected Neutropenic enterocolitis involving
C.septicum
 Three blood cultures collected from three different
venipuncture sites
 Stool
 Luminal contents or tissue from the involved
ileocecal area
 Suspected Botulism or Tetanus
 Feces ,enema fluid, gastric aspirates or vomitus
 Tissue or exudates
 And post mortem specimens
 Serum specimen as soon as possible after the onset
of symptoms
 Inform the higher centers

12.  Diagnosis is made primarily on clinical grounds
 Function of the lab is to provide confirmation
 Gram stained films provide presumptive
information about the species involved
 Gram positive bacilli without spores-C.perfringens
 Citron bodies and boat or leaf shaped pleomorphic
bacilli with irregular staining – C.septicum
 Large bacilli with oval, subterminal spores –C.novyi

13. Boat or leaf shaped C.septicum

14.  Isolation and appearance on plated media
 Aerobic and anaerobic cultures are made on fresh
and heated blood agar, preferably on 5-6% agar to
prevent swarming
 Robertson’s cooked meat broth inoculated with
mixtures of bacteria incubated at 45˚C for 4-6 hrs
serves as enrichment media
 In RCM meat turns pink but is not digested with a
sour odour –C.perfringens
 Stormy fermentation seen in litmus milk
 Target hemolysis seen on blood agar
 Lecithinase production is seen as opalescence in
serum or egg yolk media

17. Nagler’s reaction

19.  Microscopy is unreliable, demonstration of
typical drumstick gram positive bacilli in wound
is not diagnostic
 Culture is more dependable
 Fildes’ technique- Water of condensation at the
bottom of NA slant is inoculated with mixed cultures
and incubated anaerobically. Sub culture from the
top of the tube will yield pure growth of tetanus
 Granular or ground glass appearance on NA
 Egg yolk agar No opalescence or pearly layers

20.  Growth in RCM
 Turbidity
 Some gas formation
 Meat is turned black on prolonged incubation
 Meat is reduced in volume Foul smelling end products
 Mixed cell cultures are inoculated laterally on BA
Tetanus bacilli spread over the entire plate as a thin
film with dentate spreading edge

21.  Gelatin stab culture: C. tetani liquefies gelatin
anaerobi-cally. Hence, the bacillus produces a fir-
tree type of growth in gelatin stab culture under
anaerobic incubation.

22.  Inoculation on RCM
 3 tubes are used
First-80˚C for 15 mnts
Second-80˚C for 5 mnts
Third – left unheated
 Subculture on one half of BA pates daily for up to 4
days
 Subculture from the swarming edge of the growth
 Selective media
 Incorporation of polymyxin B

23.  BA with 4% agar having tetanus antitoxin
spread over one half
 Strains are stab inoculated to each half of
plate
 Hemolysis around the colonies on the half
without antitoxin
 Indicates the production of tetanolysin
 unreliable

24.  Guinea pigs Or mice
 0.2ml of 5- 10 day old cooked meat broth
culture
 To the right of the base of tail of mice
 Stiffness & paralysis of the tail & hind limbs
which progress further
 Animal ties in 2 days
 Spine of the animal tend to curve towards right
 Control animal Protected with 500 - 1500 units
0f anti toxin 1 hour before the test
Intracutaneously /intra peritoneally

26.  Endoscopy (pseudomembranous colitis)
 Culture
 Cell culture cytotoxins test
 ELISA toxin test
 PCR toxin gene detection

27.  Enhanced infection control measures
 Targeted antibiotic restriction
 Appropriate antibiotic therapy
 Adjunctive therapy –probiotics