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A 63-year-old man was seen in the emergency room with the complaints of sudden onset of fever, chills, and abdominal pain, accompanied by mild diarrhea. The blood pressure was 140/84, the pulse rate 82/minute, and the body temperature 39.8C. A blood sample was drawn for a complete blood count, and a blood culture. A second blood culture was drawn from the opposite arm, with 10 mL of blood being placed into each an aerobic and an anaerobic bottle, following customary practice. The complete blood count revealed a hemoglobin of 15.8 mg/dL, a hematocrit of 45%, and a white blood count of 4.2/L. The neutrophils were 39%, lymphocytes 45%, monocytes 10%, eosinophils 4% and basophils 2%. The platelet count was 255/L. The patient was admitted to the hospital for further work-up and empiric antibiotic therapy. Within 24 hours after admission, the body temperature had decreased to 38.2C, although the mild diarrhea persisted. A stool toxin test for Clostridium difficile was negative and neither enteric pathogens nor Campylobacter species were recovered in stool culture after 24 hours incubation. Fecal neutrophils were not seen on direct examination. The anaerobic blood culture became positive 36 hours after inoculation.

The growth observed on the anaerobic blood agar plate after 48 hours incubation (see upper image), revealed a spreading colony. The spreading nature of the colony is better observed in the lower image. No growth was observed on subcultures incubated aerobically indicating that this isolate is truly an anaerobe (although aerotolerance studies would be needed for confirmation). The spreading nature of the colony and the lack of hemolysis are highly suggestive of Clostridium septicum. However, biochemical confirmation is necessary.

Stool culture is very effective in detecting C. difficile. Unfortunately, non-toxigenic strains will also grow, requiring strains to be tested for toxin production. The greatest disadvantage to culture is the length of time that is needed before results are available, which may be up to four days. However, antibiotic sensitivity testing following culture is useful for strain-typing that would provide necessary epidemiological information during nosocomial outbreaks.Colonies of C. difficile will appear white, flat, and spreading on blood agar (see top image on the right). Cycloserin- cefoxitin-fructose agar(CCFA) is a selective media that is used for isolation of C. difficile. There is however, no distinction between pathogenic and commensal strains, which all produce yellow colonies with a characteristic "ground glass" appearance. as shown in the bottom image on the right. The characteristic odor of "horse manure" aids in identification of C. difficile. Stool samples are directly inoculated onto CCFA and incubated in an anaerobic atmosphere at 37°C for 48 hours. Large, thin, gram-positive bacilli with spores will be observed on a Gram stain of a typical colony, as shown below.

Clostridium difficile-associated diarrhea (CDAD) is a unique hospital infection that occurs almost entirely in patients who have received previous antimicrobial treatment. Anaerobic gut flora are crucial to colonization resistance, so any disruption of the normal colonic flora (through illness, therapeutic procedures or, most commonly, antibiotic use) is essential to the pathogenesis of C. difficile infection. The association of CDAD with antibiotic use is significant. Early attention (1970s) focused on clindamycin but later on (1980s,1990s & continuing today) the cephalosporins, especially third generation, and broad spectrum penicillins (e.g., amoxycillin/ampicillin) were also implicated. The risk of CDAD is increased if C. difficile is resistant to the particular antimicrobial. In the case of clindamycin, C. difficile resistance is variable. Risk of infection due to a clindamycin-resistant strain increases with use of the drug. For the third generation cephalosporins, C. difficile is universally resistant; thus, any toxigenic strain is capable of causing CDAD during cephalosporin use. Other less commonly implicated antibiotics are the macrolides, e.g., erythromycin, azithromycin, clarithromycin. However, prolonged courses of any antibiotics will increase the risk of disease. Even those antibiotics used to treat colitis (metronidazole, for example) have sometimes been reported to cause CDAD.The fluoroquinolones have been in use since the 1980s. Ciprofloxacin was approved in 1987, but it is only in recent years with the emergence of the epidemic strain 027/NAP1/BI, which is resistant to the fluoroquinolones, that this class of drugs has been implicated in Clostridium difficile disease. The fluoroquinolones were initially considered to be low risk but their use has been increasing, both with hospital inpatients and in the community, and fluoroquinolones are now implicated as a risk factor for C. difficile infection. The newer fluoroquinolones, e.g., gatifloxacin, moxifloxacin, have better activity against anaerobes, but poor in vitro activity against C. difficile, thus increasing the likelihood of CDAD. The CDC now recommends that all fluoroquinolones, as a class, be used sparingly as each poses an increased risk for CDAD.

Are used to collect sterile blood samples from patients who may be septic (have bacteria or other organisms growing in their bloodstream). Different blood culture bottles are used for aerobic, anaerobic, and pediatric collections.

Toxin Comment Most Likely Means of Dissemination Primary Route of Entry General Signs and Symptoms Laboratory Testing Botulism toxin: Gram stained image of C. botulinum courtesy of CDC Produced by Clostridium botulinum Could be purified and used in a bioterrorist event to contaminate food or aerosolized to cause disease Aerosol Food contamination Inhalation Ingestion Difficulty speaking or swallowing Blurred or double vision Drooping eyelids (ptosis) Dilated pupils Dry mouth, decreased gag reflex Weakening of the reflexes (hyporeflexia) Abnormal sensations such as numbness, tingling, and progressive arm or leg weakness Flaccid paralysis Culture, anaerobic Digoxigen-labeled IgG ELISA to detect A, B, E, and F toxins Mouse Bioassay for all toxin types and to confirm DIG ELISA Ricin toxin: Extracted from Castor beans Inhibits protein synthesis Causes death approximately 72 hours after initial exposure As an aerosol Inhalation Fever Cough Chest tightness Dyspnea Cyanosis Gastroenteritis Necrosis Antibody detection in clinical specimens Clinical testing not performed unless known exposure has occurred

Blood is normally sterile. Any bacteria in the bloodstream is abnormal. A blood culture is collected to detect the presence of bacteria in the bloodstream. Blood is collected into appropriate media to allow for growth and identification of bacteria or other organisms that may be in the patient's bloodstream. A blood culture set usually consists of two bottles: an aerobic bottle and an anaerobic bottle. Blood cultures are usually ordered in multiple sets drawn from separate sites at different times. An improperly collected blood culture can have a serious impact on the care and treatment of a patient. If bacteria enters the culture vial from sources other than the blood, as a result of improper specimen collection, a patient may needlessly be treated for an infection that is not present. On the other hand, some collection errors may cause negative culture results when the patient actually has bacteria in his/her blood. A false-negative culture result could be a life-threatening error.