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Agnoletti F.*, Ferro T., Guolo A., Cocchi M., Drigo I., Bacchin C., Marcon B., Bano L.
Istituto Zooprofilattico Sperimentale delle Venezie, Viale Brigata Treviso 13/a, 31100 Treviso, Italy *Corresponding author: [email protected] ABSTRACT
Clostridium spiroforme causes severe rabbit gastroenteritis with subsequently important production
losses. Outbreaks are controlled with therapy but field data report frequent failures and suggest C.
being of high drug resistance. In order to establish whether therapy failure is due to
intrinsic or acquired drug resistance, 30 field strains of C. spiroforme were tested against spiramycin,
amoxicillin, doxycycline, sulfadimethoxine, norfloxacin, and tiamulin, respectively representative of
the following classes of antimicrobials: macrolides, β-lactams antibiotics, second-generation
tetracyclines, sulfonamides, quinolones and diterpenes. The minimum inhibitory concentration was
determined with the agar diffusion method (NCCLS M11-A6 manual, 2004). Resulted MICs were:
sulfadimethoxine MIC50: 256 µg/ml and MIC90: 256 µg/ml; spiramycin MIC50: 256 µg/ml and MIC90:
256 µg/ml; tiamulin MIC50: 64 µg/ml and MIC90: 128 µg/ml; norfloxacin MIC50: 32 µg/ml and MIC90:
32 µg/ml; amoxicillin MIC50: 0.063 µg/ml and MIC90: 0.063 µg/ml; doxycycline MIC50: 8 µg/ml and
MIC90: 16 µg/ml. Among all antimicrobials tested, only doxycycline showed MICs supposed to be of
therapeutic efficacy. Results support the field hypothesis of an extensive acquired resistance of C.
to antimicrobials and the presence of intrinsic resistances of importance. Both findings
suggest the necessity to give greater priority to prophylaxis of enteritis caused by C. spiroforme by
attempting to reduce risk factors rather than controlling outbreaks by therapy. The ambitious and
complex objective can be pursued by optimising technical management and by a prudent use of
Key words: Clostridium spiroforme, Minimal Inhibitory Concentration (MIC), Drug susceptibility,
Clostridium spiroforme is one of the main pathogens of the rabbit’s gastroenteric system and can lead to serious haemorrhagic typhlitis with rapid onset and usually unfortunate ending (Boucher and Nouaille, 2002; Marlier et al., 2003; Songer, 1996). It usually affects rabbits during weaning and can be related to production of a binary toxin similar to the iota toxin of Type E C. perfringens. The toxin is composed of independent sub-units “Sa” and “Sb” that are respectively coded by the genes “sas” and “sbs” (Perelle et al., 1993). In the past, the antigenic resemblance between C. spiroforme toxin and C. perfringens iota toxin attributed to Type E C. perfringens caused the enteric pathology likely to be attributed to C. spiroforme (Borriello and Carman, 1983). The pathogenicity of C. spiroforme in rabbit became clear in 1982 (Carman and Boriello, 1982); in the same period it was demonstrated the possibility to experimentally reproduce the disease by oral administration of lincomycin (Yonushonis et al., 1987). The antibiotic-associated origin of mostly of C. spiroforme outbreaks can have an indirect evidence in sporadic outbreaks due to accidental rabbit feeding with feed cross contaminated with small amounts of amoxicillin: rabbits developed symptoms of serious intoxication associated with imbalance in caecal microbiota and abnormal proliferation of C. spiroforme thanks to destruction of antagonist bacteria due to the β-lactam unintentionally swallowed. A similar mechanism is known in the “antibiotic associated diarrhoea” due to C. difficile and affecting several animal species and in humans (Voth and Ballard, 2005). In addition to sporadic and accidentally intoxication, the increased use of antimicrobial agents to control the enzootic rabbit 9th World Rabbit Congress – June 10-13, 2008 – Verona – Italy enteropathy (ERE, a disease causing important losses in rabbit breeding during the last ten years) is for sure a factor increasing C. spiroforme clostridiosis that affect nowadays industrial farms. Other trigger factors may include hyper-protein diets that induce an increase trypsin secretion which through the enzymatic scission of the sub-units “Sa” and “Sb” is a potent activator of C. spiroforme binary toxin (Ellis et al., 1991). High carbohydrate and low fibre diets can also induce clostridial overload (Percy et al., 1993). The seriousness of C. spiroforme pathology is heightened by difficulties in therapeutic control. Field evidences suggest C. spiroforme being of high drug resistance, differently to C. perfringens which diplays good susceptibility to several antimicrobials (Dubreuil and Neut, 2004; Bouvier et al., 2005; Agnoletti et al., 2007) and is involved in rabbit “enteritis complex” (Peeters et al., 1986). C. spiroforme is a fastidious micro-organism and requires enriched selective media and strict anaerobiosis growing. Furthermore the biochemical identification and the interpretative procedure proposed by Kaneuchi et al. (1979) is difficult to apply. Lastly, the Clinical and Laboratory Standard Institute (CLSI, previously named NCCLS) indicates the determination of the Minimum Inhibitory Concentration (MIC) through diffusion in agar as the reference method for the determination of the drug resistance of anaerobic micro-organisms (NCCLS, 2004); the method is complex and time consuming. So many difficulties may be responsible for the lack of data on drug resistance of field strains of C. spiroforme. At the present one study conducted on a limited number of bacterial strains and with antimicrobials that are mostly of no longer nowadays used in farms is available (Carman and Wilkins, 1991). Our study aims to update the knowledge on drug susceptibility of C. spiroforme, both reference and field rabbits isolates, by determining MICs of antimicrobials used in rabbit nowadays therapy. MATERIALS AND METHODS
We examined 30 field strains of Clostridium spiroforme taken from rabbits affected by enteric pathologies in 30 different Italian farms in 2007. Using the selective medium described by Agnoletti et al. (2004) the strains isolated were stored in cryogenic vials (Nalgene) at –80°C until the moment of test execution; the bacterial film was collected in a Reinforced Clostridial Medium (Oxoid) diluted 1:2 in sterile glycerol. Each strain, appropriately cloned and preliminarily identified on the basis of morphology of the colonies and the characteristic microscopic aspect (Borriello et al., 1986), was identified by PCR (Drigo et al., 2007). The strains selected were then tested positive for the Sbs and Sba genes that code for the two sub-units of the binary toxin by PCR multiplex (Drigo et al., 2007) for a further confirmation of their pathogenicity. The following antimicrobial agents were used: spiramycin (Spiramycin from Streptomyces sp., Sigma), sulfadimethoxine (Sulfadimethoxine, Sigma), norfloxacin (Norfloxacin, Sigma), tiamulin (Tiamulin fumarate, Sigma), respectively members of the following classes of antimicrobial: macrolides, β-lactams antibiotics, second-generation tetracyclines, sulfonamides, quinolones and diterpenes. The instructions provided in the respective analysis certificates were followed to solubilize the standard powders, whereas the agar diffusion method provided in the NCCLS/CLSI M11-A6 (2004) manual was adopted for MIC determination. This was followed by the calculation of the MIC50, MIC90, the geometrical mean of the MICs and the MIC range. All MIC values were expressed in µg/ml, when necessary the international units commonly adopted were converted (Sweetman, 2007). Each test batch featured the inclusion of 3 reference strains (C. spiroforme ATCC 2290, C. perfringens ATCC 13124 and Bacteroides fragilis ATCC 25285), even if the Clinical and Laboratory Standard Institute does not indicate acceptable MIC ranges for the active principals and reference strains used in the study. RESULTS AND DISCUSSION

The genus Clostridium is fairly heterogeneous in terms of drug resistance and for this reason permits
the identification of three different groups; C. perfringens, which is usually susceptible to the
antimicrobials, C. difficile, considered to be highly resistant, and the other species of the genus that
present intermediate degrees of susceptibility. The susceptibility of C. spiroforme has only been
scarcely documented and for such reason it has not yet been positioned in any of the three groups
The results of the MIC determination for the 6 antimicrobial products tested against both the field
strains of C. spiroforme and the reference strains are respectively provided in Tables 1 and 2, whereas
Table 3 provides summarized MIC value data, indicating MIC50, MIC90, and the geometrical mean
MIC values and the MIC range.
Table 1: Frequency of MICs of Clostridium spiroforme

Table 2: Distribution of MICs for ATCC reference strains (µg/ml)

Table 3: Summarized MIC values (µg/ml)
With a MIC value geometric mean of 233 and 256 µg/ml respectively, spiramycin and sulfadimethoxine, appear to have no therapeutic value whatsoever. C. spiroforme seems intrinsically resistant to spiramycin and sulfadimethoxine, whereas the gap between the strain ATCC and field strain MIC values measured for spiramycin suggests that the resistance to this macrolide is acquired, not intrinsic. Intermediate susceptibility values were observed for norfloxacin and tiamulin, which showed geometric mean MIC values of respectively 30.5 and 42.2 µg/ml. Fluoroquinolones were shown to exert pharmacological action even in regard to Gram-positive bacteria, such as genus Clostridium. 9th World Rabbit Congress – June 10-13, 2008 – Verona – Italy Samples of C. perfringens isolated from humans provide values of MIC90 <1 when subjected to testing with numerous fluoroquinolones, whereas up to 26% of the strains of C. difficile of human origin proved resistant (Dubreuil and Neut, 2004). In any case however, fluoroquinolones are not usually used to treat enteric pathologies in the rabbit, and are considered drugs of second choice in particularly for serious cases of colibacillosis. Of greater concern, however, is the extent of resistance to tiamulin, semi-synthetic pleuromutilin derivative, which is still one of today’s leading veterinary drugs against clostridia. The distribution of the MICs and the susceptibility of strain ATCC confirm this to be an acquired and not intrinsic form of resistance; tiamulin, in fact, has been used for many years, but progressive increases in dosage in the field have become necessary in order to achieve the therapeutic results desired. The β-lactams represented by amoxicillin are considered the antimicrobials of preference in the treatment of human clostridiosis and show extremely high efficacy in vitro even against C. spiroforme. These molecules cannot be administered orally to rabbits, however, because they are particularly toxic in this form and therefore cannot be used in the control of enteric pathologies. Lastly, despite the facts that tetracycline is commonly administered orally in rabbit farms and that this class of antibiotic is characterized by phenomena of crossed resistance, doxycycline, a second generation semi-synthetic tetracycline, provides the lowest MIC50 and MIC90 values of all the antimicrobial products tested and may be one of the few pharmacological tools available for the control of C. spiroforme. The MIC values observed demonstrate the elevated resistance to drugs vaunted by C. spiroforme, confirming the evidence from the field regarding the difficulty of controlling cases of enteric pathology caused by C. spiroforme through the use of antimicrobials. From this point of view, C. spiroforme differs from other forms of clostridia, particularly C. perfringens, which is considered highly susceptible to pharmaceutical treatment. This difference may be related to the phylogenetic distance of C. spiroforme from C. perfringens. The latter, in fact, is taxonomically positioned in Cluster I that contains the types of clostridia considered to be the “core species” of the genus Clostridium; instead of C. spiroforme, together with Cl. ramosum and Cl. cocleatum, which are classed in Cluster XVIII (Collins et al., 1994) which is phylogenetically so remote from the former as to justify the hypothesis of taxonomic reclassification in a different genus (Euzéby, 2007). CONCLUSIONS
The results obtained demonstrate the range of resistance to antimicrobials of field strains of C. spiroforme. The gap between the MIC values measured for strain ATCC 2290 and field strains suggests – with the exception of sulfadimethoxine – that the resistances observed are acquired, and not intrinsic in nature. Doxycycline is the only drug that presents MIC values compatible with therapeutic use, due to the fact that amoxicillin, extremely efficacious in vitro, is highly toxic when administered to rabbits orally. Although the in vitro susceptibility of C. spiroforme to other active principals must be tested in the future, the results obtained suggest the need to grant greater priority to C. spiroforme prophylaxis than therapeutic control by attempting to reduce the trigger factors primarily through the careful and prudent use of antimicrobials. REFERENCES

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