Characterization of Clostridium Perfringens Isolates Collected from Three Agricultural Biogas Plants over a One-Year Period

Abstract

Digestate produced by agricultural biogas plants (BGPs) may contain pathogenic bacteria. Among them, <i>Clostridium perfringens</i> deserves particular attention due to its ability to grow under anaerobic conditions and persist in amended soil. The aim of this study was to examine the potential pathogenicity and the antimicrobial resistance of <i>C. perfringens</i> in manure and digestate collected from three agricultural biogas plants (BGPs). A total of 157 isolates (92 from manure, 65 from digestate) were screened for genes encoding seven toxins (<i>cpa</i>, <i>cpb</i>, <i>etx</i>, <i>iap</i><i>cpe</i>, <i>netB</i>, and <i>cpb</i>2). The 138 <i>cpa</i> positive isolates were then screened for <i>tetA</i>(P), <i>tetB</i>(P), <i>tet</i>(M), and <i>erm</i>(Q) genes and tested for antimicrobial susceptibility. The toxinotypes identified in both manure and digestate were type A (78.3% of the isolates), type G (16.7%), type C (3.6%), and type D (1.4%), whereas none of the isolates were type F. Moreover, half of the isolates carried the <i>cpb2</i> gene. The overall prevalence of <i>tetA</i>(P) gene alone, <i>tetA</i>(P)-<i>tetB</i>(P) genes, and <i>erm</i>(Q) gene was 31.9, 34.8, and 6.5%, respectively. None of the isolates harbored the <i>tet</i>(M) gene. Multiple antimicrobial resistant isolates were found in samples that were collected from all the manure and digestates. Among them, 12.3% were highly resistant to some of the antibiotics tested, especially to clindamycin (MIC ≥ 16 µg/mL) and tilmicosin (MIC > 64 µg/mL). Some isolates were highly resistant to antibiotics used in human medicine, including vancomycin (MIC > 8 µg/mL) and imipenem (MIC > 64 µg/mL). These results suggest that digestate may be a carrier of the virulent and multidrug resistant <i>C. perfringens</i>.