The Colonisation of Calves in Czech Large-Scale Dairy Farms by Clonally-Related Clostridioides difficile of the Sequence Type 11 Represented by Ribotypes 033 and 126

To investigate a possible Clostridioides difficile reservoir in the Czech Republic, we performed a study in 297 calves from 29 large-scale dairy farms. After enrichment, faecal samples were inoculated onto selective agar for C. difficile. From the 297 samples, 44 C. difficile isolates were cultured (prevalence of 14.8%, 10 farms). The Holstein breed and use of digestate were associated with C. difficile colonisation (p ˂ 0.05). C. difficile isolates belonged to the ribotype/sequence type: RT033/ST11 (n = 37), RT126/ST11 (n = 6) and RT046/ST35 (n = 1). A multiple-locus variable-number tandem-repeat analysis revealed four clonal complexes of RT033 isolates and one clonal complex of RT126 isolates. All isolates were sensitive to amoxicillin, metronidazole and vancomycin. Forty isolates were resistant to ciprofloxacin, twenty-one to clindamycin, seven to erythromycin, seven to tetracycline and six to moxifloxacin. Moxifloxacin resistant isolates revealed an amino-acid substitution Thr82Ile in the GyrA. In conclusion, the calves of Holstein breed from farms using digestate as a product of bio-gas plants are more likely to be colonised by clonally-related C. difficile of ST 11 represented by ribotypes 033 and 126. The identified resistance to moxifloxacin with a Thr82Ile substitution in the GyrA highlights the need for further monitoring by the “One health approach”

Low Rates of Antimicrobial-Resistant Enterobacteriaceae in Wildlife in Taï National Park, Côte d’Ivoire, Surrounded by Villages with High Prevalence of Multiresistant ESBL-Producing Escherichia coli in People and Domestic Animals

Antimicrobial resistance genes can be found in all ecosystems, including those where antibiotic selective pressure has never been exerted. We investigated resistance genes in a collection of faecal samples of wildlife (non-human primates, mice), people and domestic animals (dogs, cats) in Côte d’Ivoire; in the chimpanzee research area of Taï National Park (TNP) and adjacent villages. Single bacteria isolates were collected from antibiotic-containing agar plates and subjected to molecular analysis to detect Enterobacteriaceae isolates with plasmid-mediated genes of extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR). While the prevalence of ESBL-producing E. coli in the villages was 27% in people (n = 77) and 32% in dogs (n = 38), no ESBL-producer was found in wildlife of TNP (n = 75). PMQR genes, mainly represented by qnrS1, were also present in human- and dog-originating isolates from the villages (36% and 42% in people and dogs, respectively), but no qnrS has been found in the park. In TNP, different variants of qnrB were detected in Citrobacter freundii isolates originating non-human primates and mice. In conclusion, ESBL and PMQR genes frequently found in humans and domestic animals in the villages were rather exceptional in wildlife living in the protected area. Although people enter the park, the strict biosecurity levels they are obliged to follow probably impede transmission of bacteria between them and wildlife

Characteristics of isolates with PMQR genes.

<p>Isolates were obtained on MCA-ciprofloxacin.</p><p>For legend see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113548#pone-0113548-t001" target="_blank">table 1</a>.</p><p>Characteristics of isolates with PMQR genes.</p

Characteristics of plasmids obtained by transformation.

<p>“NT”–not typable. Note: transconjugants obtained in this study contained more than one plasmid and therefore were not used for plasmid characterization.</p><p>Characteristics of plasmids obtained by transformation.</p

Dendrogram of resistant <i>E. coli</i> isolates’ PFGE profiles.

<p>Generated by cluster analysis of the Dice similarity indices in the BioNumerics fingerprinting software (optimization 1%, band matching tolerance 1%, tolerance change 1%). Isolates marked with “cip” were obtained by cultivation on ciprofloxacin and harbored PMQR genes, isolates with “ctx” represent the CTX-M-15 producing <i>E. coli</i>. “H” isolate from human, “D” isolate from dog, “C” isolate from cat.</p

Characteristics of ESBL-producing isolates from villages.

<p>All isolates were obtained on MCA-cefotaxime.</p><p>“D” – isolate from dog, “H” – isolates from human, “C” – isolate from cat. “T” in brackets means that the resistance gene was successfully transformed into competent cells, “C” in brackets means that the gene was conjugated. Plasmids were isolated and characterized from isolates in bold font. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113548#pone-0113548-t002" target="_blank">Table 2</a> for plasmid characteristics.</p><p>PG = phylogroup, Amp = ampicillin, Cef = cephalotin, Caz = ceftazidime, Amc = amoxycilin-clavulanate, Nal = nalidixic acid, Cip = ciprofloxacin, Tet = tetracycline, Sxt = trimethoprim-sulfamethoxazole, Spt = streptomycin, Sul = sulfonamides compounds, Gen = gentamicin, Chl = chloramphenicol, Cpd = cefpodoxime.</p><p>Characteristics of ESBL-producing isolates from villages.</p