Clostridium difficile is an obligate anaerobic, spore forming, Gram-positive bacillus, and is

identified as the leading cause of nosocomial diarrhea worldwide. The clinical presentation of
Clostridium difficile infection (CDI) can range from mild diarrhea to more serious disease
such as toxic megacolon and pseudomembranous colitis. The severity of hospital acquired
CDI is determined by many factors, including the age and immune status of the patient,
duration of hospitalization as well as the receipt of antimicrobial therapy.
Increase in incidence and severity of CDI in recent years, is partly linked to the emergence of
the endemic C. difficile strain PCR ribotype 027. PCR RT 027 is hypervirulent and is one of
the ten most common ribotypes in Europe. Ribotype 014 has been demonstrated as the
predominant PCR type in many European countries, and has recently found to be more
prevalent than RT 027 in Europe. Common ribotypes have been associated with certain
phenotypic characteristics including increased toxin production, better growth rates, and
higher antimicrobial resistance. Other common ribotypes in Europe include ribotypes 078,
015, and 005.
Currently, antimicrobials used for the treatment of CDI include metronidazole, vancomycin
and fidaxomicin. Metronidazole is currently used as first line therapy for treatment of mild to
moderate CDI. It has a lower propensity for selection of vancomycin-resistant enterococci
and is more economical. Severe or more complicated CDI is typically treated with
vancomycin.
Exposure to almost all classes of antimicrobials has been associated with the induction of
CDI. The gut microbiota plays an important role in the protection of the host against
pathogens, including C. difficile, through colonization resistance. Disruptions in the gut
microbiota as a result of broad-spectrum antimicrobial use, can have deleterious effects. C.
difficile is an opportunistic pathogen which may subsequently thrive due to perturbation of
the gut microbiota.
Antimicrobial therapy, typically given for treatment of a different condition, can make the
patient more susceptible to CDI if the patient is exposed to a toxigenic strain of the organism.
Fluoroquinolones are the most recent class of antimicrobial drugs which have strongly been
associated with CDI and to severe epidemics. CDI that is resistant to fluoroquinolones such
as moxifloxacin, is typically caused by the more virulent strain) ribotype 027.
Target mutations in genes gyrA and gyrB, which encode subunits A and B of DNA gyrase
mediate resistance to fluoroquinolones. Resistance is also conferred by mutations in gene
parC and parE, encoding the respective subunits of A and B of topoisomerase IV, as well as
mutations which result in the reduction of the accumulation of the drug in the cell. In C.
difficile resistance is determined by alterations in the amino acid sequence in the QRDR of
either gyrA or gyrB. Recent data on fluoroquinolone resistance of C. difficile isolates for the
UK are limited.
Reduced susceptibility as well as resistance to treatment agent metronidazole has also been
reported, therefore periodic monitoring of antimicrobial resistance in C. difficile isolates is
essential. Furthermore, Studies testing metronidazole resistance in the common ribotypes in
Europe, using the agar incorporation method are limited. Barbanti et. al (2012) report
differences in the classification of strains as susceptible or having reduced susceptibility due
to low categorical agreement between the E-test and agar dilution. The MIC value of certain
strains acquired using E-test were lower than those tested by agar incorporation. Agar
incorporation is recommended as the gold standard for antimicrobial susceptibility testing. It
delivers accurate results and it has a superior ability to detect strains with reduced
susceptibility over agar dilution.
In this study, the antimicrobial susceptibilities of common European ribotypes 027, 024, 015
and 005 to metronidazole, moxifloxacin, linezolid and valnemulin were determined using a
wilkin chagrens agar incorporation method.