MICROBIOLOGY OF AQUATIC SYSTEMS Water Sources in a Zoological Park Harbor Genetically Diverse Strains of Clostridium Perfringens Type A with Decreased Susceptibility to Metronidazole Sergio Álvarez-Pérez 1 & José L. Blanco 1,2 & Teresa Peláez 3,4 & Eva Martínez-Nevado 5 & Marta E. García 1 Received: 9 February 2016 /Accepted: 13 April 2016 # Springer Science+Business Media New York 2016 Abstract The presence of Clostridium perfringens in water is generally regarded as an indicator of fecal contamination, and exposure to waterborne spores is considered a possible source of infection for animals. We assessed the presence and genetic diversity of C. perfringens in water sources in a zoological park located in Madrid (Spain). A total of 48 water samples from 24 different sources were analyzed, and recovered iso- lates were toxinotyped, genotyped by fluorophore-enhanced repetitive polymerase chain reaction (rep-PCR) fingerprinting and tested for antimicrobial susceptibility. C. perfringens was recovered from 43.8 % of water samples and 50 % of water sources analyzed. All isolates (n = 70) were type A and 42.9 % were β2-toxigenic (i.e., cpb2+), but none contained the enterotoxin-encoding gene (cpe). Isolates belonged to 15 rep-PCR genotypes and most genetic diversity (88 %) was distributed among isolates obtained from the same sample. Most isolates displayed intermediate susceptibility (57.1 %; MIC = 16 μg ml -1 ) or resistance (5.7 %; MIC ≥ 32 μg ml -1 ) to metronidazole. No resistance to other antimicrobials was detected, although some isolates showed elevated MICs to erythromycin and/or linezolid. Finally, a marginally signifi- cant association between absence of cpb2 and decreased sus- ceptibility to metronidazole (MIC ≥ 16 μg ml -1 ) was detected. In conclusion, our results reveal a high prevalence of C. perfringens type A in the studied water reservoirs, which constitutes a health risk for zoo animals. The elevated MICs to metronidazole observed for genetically diverse isolates is a cause of additional concern, but more work is required to clarify the significance of reduced metronidazole susceptibil- ity in environmental strains. Keywords Clostridium perfringens . Metronidazole . Rep-PCR typing . Toxins . Water . Zoological park Introduction Clostridium perfringens is among the most important toxin- producing bacteria currently known [1, 2]. Apart from four major toxins (alpha, α; beta, β; epsilon, ε; and iota, ι), which classify the species into five types (A to E), this Gram-positive anaerobe can produce a varied arsenal of additional toxins with a role in pathogenesis [1–5]. Although C. perfringens has been traditionally implicated in several enteric diseases of pets and farm animals, the path- ogenicity for some species remains to be further elucidated and veterinary clinical diagnosis is still challenging [1, 3–5]. Wild and zoo animals are also susceptible to C. perfringens infection [1, 6–10]. Metronidazole is still considered a first-line drug for the treatment of anaerobe infections in human and animal Electronic supplementary material The online version of this article (doi:10.1007/s00248-016-0772-2) contains supplementary material, which is available to authorized users. * José L. Blanco jlblanco@ucm.es 1 Department of Animal Health, Faculty of Veterinary, Universidad Complutense de Madrid, Madrid, Spain 2 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain 3 Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain 4 Department of Medicine, Faculty of Medicine, Universidad Complutense de Madrid, Madrid, Spain 5 ZOO-AQUARIUM Madrid, Madrid, Spain Microb Ecol DOI 10.1007/s00248-016-0772-2