Clostridium difficile

• Gram positive bacteria
• Anaerobe
• Was first isolated in 1935 from the stools of healthy neonates
• Is diffiuclt to isolate and culture
• Neonates are the only age group with a high prevalence of C.difficile toxin and no clinical diease
• Is a frequent cause of infectious colitis, usually as a complication of antibiotic therapy in elderly hosipatlised patients.

• In the mid to late 90's C.difficile in actue care hospitals in the US had remained stable at 30-40 cases per 100,000 of population
• In 2001 this rose to almost 50 cases per 100,000 of population
• The cause for concern was an increase in those with severe or fatal infection. E.g. in England C.difficile was listed as the primary cause of death for 499 patients in 1999, but this increased to 11998 patients in 2005 and then 3393 in 2006.
• In 2003 in Quebec all major acute care hospitals in the region was simultaneously affected in the same way - accompanied by an increase in severity and mortality.
• The increase in severity was due to a single strain accounted for at least 1/2 the isolated from 5 facilities in the US. 82% of stool samples from the Quebec outbreak were postivitve for the same strain.
• Global Epidemic Strain - North American Pulsed Field Type 1 (NAP1) - PCR ribtoype 027
• BI/NAP1/027

• Has large toxins A and B - main virulence factors
• Also has an extra toxin called the binary toxin
• Binary toxin was first discovered in 1988
• Until recently was only found in <6% of isolated from hospitalised patients
• Has othe rmutation in tcdC
• However increased virulence alone does not explain why a previously uncommon strain has become an epidemic.

Mutations in tcdC alleles

• In 027 strains there is a non-specific in-frame 18bp deletion and a point deletion at position 117, which causes a frameshift mutation and introduces a stop codon.
• Therefore a truncated, inactive TcdC protein, caused by the deletion at 117 is produced. TcdC normally regulated toxin production. The truncation results in increased toxin production.
• The 18bp deletion does seem to have an affect also, although it does appear with the deletion.

Increased resistance to fluoroquinolones

• Quinolones inhibit bacterial DNA snythesis.
• E.g. Ciprofloxacin, nalidixic acid
• The current NAP1 isolates are more resistant than previous isolates of the same strain --> selective advantage
• Antimicrobial therapy is the most widely recognised risk factor for C.difficile associated disease (CDAD)
• Drugs disrupt the normal gut flora, and allows for a niche for C.difficile multiplication
• All antibiotics could cause this, but it is mostly associated with fluoroquinolones
• Increased duration of antimicrobial therapy, use of broad-spectrum antimicrobials and the use of multiple agents all contribute to this
• Resistance is associated with mutations in the gyrA and gryB genes.

Commensal bacteria

• In healthy adults, the colon has ~10^12 bacteria per gram of contents.
• Commensal bacteria provide an important host defence by preventing  colonisation and overgrowth of C. diff and other pathogens
• Antimicrobial therapy can disrupt this host defence

Clindamycin

• Clindamycin therapy is commmonly associated with  C. diff infection
• Clindamycin-resistant strains have now been associated with large outbreaks
• Clindamycin-resistant strains of C.diff can thrive in environments where other commensal flora are suppressed
• Also seen with cephalosporin and fluoroquinolones, with resistant strains of C.diffcile

• However, not all patients who receive antimicrobials and are exposed to C.difficile will develop CDAD.
• Antibodies to C. difficile toxin can be made by the host immune system.  
• Patients who did not develop antibodies to toxin A in response to first exposure to C. difficile were 48x more likely to develop recurrent infection than patients who did mount an adequate immune response
• C. difficile infection is also increasing in communities without the traditional risk factors for C. diff associated disease. Although they have not received antibiotics they have received proton-pump inhibitors (used to decrease stomach acid production. E.g. peptic ulcer treatment).

All 027 strains are sensitive to metronidazole and vancomycin.

1. Increased production of toxins A and B

2. Fluoroquinolone resistance

3. Production of the binary toxin

4. Hypersporulation

• Major virulence determinants of C.difficile
• Toxin negative strains are non-pathogenic
• 19.6kb pathogenicity locus
• The locus has 5 genes - two toxin genes (tcdB and tcdA), and three regulatory genes
• The gene tcdE has an uncertain role, but is thought to facilitate toxin release.
• tcdC appears to inhibit toxin transcription in early exponential period of growth, however nn  NAP1/027 strains there is the deletion mutation in tcdC. This leads to increased toxin production  mediating colonic tissue injury and inflammation in C difficile infection
• Toxins A and B show sequence and structural homology
• In 027 strains there is a different 3 end to the TcdB sequence which causes different C-terminal binding properties. This may increase toxic activity.
• The binding domain binds to a cell surface receptor --> receptor mediated endocytosis occurs. This is followed by pore formation by the hydrophobic domain - DXD glucosyltransferase enters the cytoplasm of the host cell.
• The target of the toxin gluocosylate is Rho GTPases, which leads to the disintegration of the cytoskeleton, and cell death

• Some strains have a binary toxin - CDT
• Is associated with hypervirulence
• It consists of a binding component and an enzymatic component --> actin-specific ribosyl transferase, which produces cytoskeletal disorganisation (increaes microtubule protusions which increases C.difficile adherence)
• CdtR is the regulatory protein
• The toxin may potentiate the toxicity of TcdA and TcdB and cause more severe disease
• Unrelated to locus encoding A and B.
• C.difficile produces the binary toxin in the absence of A and B --> non-pathogenic
• However is thought to be synergistic with A and B in causing severe colitis

• Midl diarrhoea with normal colonic mucosa to pseudomembranoue colitis (PMC)
• PMC - fibrin, mucin, sloughed mucosal epithelia, and acute inflammatory cells.
• Characteristic summit lesions

• Predominantly affects the elderly and frail hospital and nursing home patietns.
• Also at risk are the young, and previously healthy who have been in close contact with patients with C.difficile
• Direct person-to-person transfer
• Most commonly identified as the cause of antibiotic-associated diarrhoea --> 15-25% of cases

• Since 1970's C diff infection treated with either metronidazole or oral vancomycin
• Metronidazole has been the initial treatment since the 1990's
• Despite increase in incidence and severity in the last decade both remain the treatment of choice
• Metronidazole is used first for mild infection (cheaper, concern with vancomycin-resistance) 
• Vancomycin is more effective in severe infections
• Intravenous antibodies to C.difficile have shown some benefit
• Experimental use of toxin-binding polymers
• Firstly, discontinue treatment with the anitmicrobial which has resulted in C.difficile infection
• Previously effective in 25% of patients in 48-72hrs, however there is now an increase in fulminant infection (ie. rapid onset & pseudomembraneous colitis), so delayed treatment is no longer recommended

1st and 2nd episodes

• Metronidazole - longest history and cheaper
• Also Vancomycin - both are equally effective
• If a 2nd episode develops, use the same treament

Recurrent (3 or more)

•  Most recur within  7-14 days of treatment completion - relapse rather than reinfection
• Can recur for years - due to poor host immune response --> inability to mount effective anti-toxin A IgM or IgG
• Age is a factor, as immune response becomes less effective
• Antibiotics may prevent recolonisation with normal colonic flora – makes patient more vulnerable. The favoured treatment is to start with a high dose of vancomycin then taper off the dose. This allows recolonisation with normal flora but still suppresses C. difficile

• Disease can recur after successful initial treatment
• Recurrence is similar with either antibiotic therapy or re-infection with a different strain or persistence of the original strain
• There is a risk of recurrence is increased in patients who have already had one recurrence
• There has been an escalation in recurrence (60% risk after two or more recurrences)
• This increase risk probably caused by selection of patients without protective immunity – there are vulnerable to repeated attacks
• C difficile infection develops in only half of patients colonised by toxigenic C difficile as a complication of antimicrobial therapy – the remainder are symptomless carriers

Sympyomless carriers

• Have an increase in serum IgG against toxin A
• In patients in whic infection develops, there is no increase in serum IgG

• The ultimate goal of treament is to discontinue all antibiotics and allow the normal microflora to establish
• Persistent diarrhoea which is resistant to vancomycin or metronidazole is caused by something else, as C.difficile is rarely resistant to these antibiotics.
• The inability to mount a protective immune response to C.difficile and it's toxins underlies the susceptibility to recurrent infection
• However, immunotherapy requires further research
• Recurrent C.difficile infection results from the disruption of normal colonic microflora initiated by antibiotic therapy - some treatments have aimed to restore resistance to colonisation, e.g.  the administration of non-toxigenic C.difficile in order to fill the environmental niche required for infection by toxigenic strains. A filtrate of human faeces (usually from a family member) is currently being developed

• The C.difficile infection rate in the US has tripled from 2000 to 2005
• Morbidity and mortality has also increased in the elderly - there is a need for more effective infection control.
• There are two major approaches for this:

1. Preventing the ingestion of the organism and its spore

2. Reducing the chances of developing C.difficile infection following ingestion

Include tradition infection control strategies

• Target the environment
• Personnel hygine (i.e. staff)
• Barrier methods
• Approaches to minimise or even eliminate antimicrobial exposure

Environmental control, personnel hygiene and barrier methods

• The monitoring of C.difficile infection rates allows for the quick detection of increases above an acceptable level
• Two interventions have been shown to be effective in interrupting disease transmission during outbreaks:

1. Disinfection with hypochlorite - to minimose environmental contamination

2. Barrier precautions during patient contact to prevent transmission e.g. gloves

Environmental and Hand Hygiene

• Patients in rooms which have previously been occupied by patients with a drug-resistant organism have been shown to be at risk of acquiring drug-resistant organisms
• This demonstrates that the environment is a critical source of contamination and it enhances the potential for the spread of infection
• The environment is even more important with spore-forming organisms such as C.difficile. Spores can persist on hard surfaces for prolonged periods
• C.difficile is shed in the faeces - a reservoir of spores
• The spores can resits dessication for months and can persist on hard surfaces for as long as 5 months.
• Hospital wards & intensive care units – C.difficile contamination found on 49% of sites in rooms occupied by patients with C.difficile infection, 29% of sites in rooms were occupied by asymptomatic patients.
• The heaviest contamination was on floors and bedrails
• Healthcare workers become an important vector for transmission

• Commonly used hospital cleaners are not sporicidal - they may in fact encourage sporulation
• Epidemic strains of C.diff (BI/NAP1) are known to hypersporulate, unlike non-outbreak strains.
• The faecal soiling of the environment likely with patients with C.diff infection. Spore forms are predominant after contact with air
• Only chlorine-based disinfectants and high concentration vapourised hydrogen peroxide are sporicidal
• Disinfection with sodium hypochlorite solution is effective in reducing environmental contamination
• Cleaning of frequently touched surfaces – bedrails, call buttons, telephones and floors
• Adherence to good hand hygiene practises - alcohol-based hand gells are not effective against C.difficile or its spores. However their use does promote all-round good hygiene practises.
• Handwashing with chlorhexidine is more effective, as is soap and water
• Medical equipment such as endoscopes should be cleaned with ethylene oxide which is effective against spore forming bacteria
• Single-use thermometers should be used as C.difficile is spread via the faeces
• Gloves and gowns used during outbreaks

• The management and control of antimicrobial use
• Especially Clindamycin and cephalosporins
• CDI prophylaxis: Some clinicians administer metronidazole or oral vancomycin to pateints without diarrhoea &/or signs of CDI who are receiving other antimicrobials for an underling infection. The is not logical and can increase the risk of CDI.
• While non-symptomatic carriers are at no greater risk of developing CDI, they have been linked as the source of nosocomial infections
• The treament of non-symptomatic carriers with antimicrobials is not warranted

• Prompt and precise diagnosis is important
• Test stools for presence of toxins – confirms diagnosis
• Enzyme immunoassay, usual method of diagnosis, but relatively insensitive
• Most common presentation of CDI is diarrhoea associated with antimicrobial use
• Mild to moderate diarrhoea usually only symptom
• Diarrhoea lasting more than 3 days after hospital admission is commonly due to C.difficile
• Cell cytotoxicity assay most sensitive – detects toxin B
• Culture of C.diff from stools – slow, but important
• Allows for molecular typing and is essential for monitoring molecular epidemiology & antibiotic sensitivity