Genetic Relatedness, Antibiotic Resistance, and Effect of Silver Nanoparticle on Biofilm Formation by Clostridium perfringens Isolated from Chickens, Pigeons, Camels, and Human Consumers

Abstract

In this study, we determined the prevalence and toxin types of antibiotic-resistant Clostridium perfringens in chicken, pigeons, camels, and humans. We investigated the inhibitory effects of AgNPs on biofilm formation ability of the isolates and the genetic relatedness of the isolates from various sources determined using RAPD-PCR. Fifty isolates were identified using PCR, and all the isolates were of type A. The cpe and cpb2 genes were detected in 12% and 56% of the isolates, respectively. The effect of AgNPs on biofilm production of six representative isolates indicated that at the highest concentration of AgNPs (100 µg/mL), the inhibition percentages were 80.8–82.8%. The RAPD-PCR patterns of the 50 C. perfringens isolates from various sources revealed 33 profiles and four clusters, and the discriminatory power of RAPD-PCR was high. Multidrug-resistant C. perfringens isolates are predominant in the study area. The inhibition of biofilm formation by C. perfringens isolates was dose-dependent, and RAPD-PCR is a promising method for studying the genetic relatedness between the isolates from various sources. This is the first report of AgNPs’ anti-biofilm activity against C. perfringens from chickens, pigeons, camels, and humans, to the best of our knowledge.

Antibiogram pattern and molecular characterization of Clostridium perfringens isolated from different species.

“…Within sterile freshly prepared Cooked Meat broth (CMB) (Oxoid), each sample was introduced into tubes and incubated anaerobically in gas pack anaerobic jars using anaerobic kits (anaerogen, Oxoid ltd, England) at 37°C for 24 hrs. A loop-full from each incubated broth was streaked in 10% defibrinated sheep blood agar plates containing neomycin sulphate 200 μg/ml and incubated for 48 hr at 37˚C under anaerobic conditions (Carter and Cole, 1990).All samples were inoculated on reinforced Clostridial agar (RCA; Oxoid, CM0151) and anaerobically incubated for 24-48hr at 37˚C (Ahmed et al, 2022).The suspected colonies were kept on CMB for further confirmation on Egg Yolk agar (oxoid, SR00885) and Tryptose Sulphate Cycloserine agar (TSC; oxoid CM 0587B). Then the isolated colonies were identified by the cultural, morphological, and biochemical characteristics: (Lecithinase, Catalase, Indol, Gelatin liquefaction, Litmus milk and Sugar fermentation test) (Macfaddin, 2002).…”

“…Typing of C. perfringens isolates was carried out by PCR amplification of cpa, cpb, etx and itx genes using the specific primers for each gene according to (Yoo et al, 1997) and following the thermal cycling conditions of primary annealing at 94 ˚C for 5 minutes, then amplification for 35 cycles of denaturation at 94˚C for 30 sec followed by annealing at 55˚C for 40 sec and extension at 72˚C for 45 sec ended with a final extension step at 72˚C for 10 minutes. PCR program was done using the primers shown in table (1).…”