Clinical Microbiology Molecular typing and antimicrobial susceptibility of Clostridium perfringens from broiler chickens Saad Gharaibeh a, * , Rami Al Rifai b , Ahmad Al-Majali a a Department of Pathology and Animal Health, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan b Department of Diseases Surveillance, Irbid Health Directorate, Jordanian Ministry of Health, Irbid 21110, Jordan article info Article history: Received 26 May 2010 Received in revised form 9 August 2010 Accepted 12 October 2010 Available online 20 October 2010 Keywords: Clostridium perfringens Chicken Minimum inhibitory concentration Typing abstract Clostridium perfringens (Cp) causes necrotic enteritis disease in commercial poultry. Antimicrobials are used to control and treat this disease and sometimes clinical outbreaks do not respond well to certain treatments. This study was designed to isolate Cp from clinical cases, type these isolates by multiplex PCR, and determine their antimicrobial susceptibility by micro-dilution method. A total of 67 Cp isolates were obtained from 155 broiler chicken flocks. All isolates were classified as type A and non-enterotoxin producers. Lincomycin, erythromycins, and tilmicosin showed very high minimal inhibitory concentra- tion (MIC) 50 of 256 mg/ml. However, tylosin, amoxicillin, ampicillin, penicillin, florfenicol, dano- floxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline had variable MIC 50 of 64, 0.5, 1, 1, 8, 4, 8, 4, 8, 0.5 mg/ml, respectively. It is recommended that Cp infections in Jordan be treated with either penicillins or tetracyclines especially amoxicillin and oxytetracycline. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Necrotic enteritis (NE) is an important enteric disease of chickens caused by Clostridium perfringens (Cp) usually types A or C and rarely type D [1,2]. Cp is also responsible for a wide range of diseases in other animals [2]. Different types of Cp are associated with different clinical infestations in animals and humans. Identifying Cp type by the presence of these certain toxins is important for understanding Cp pathogenesis and epidemiology. Pathogenicity of Cp is associated with the production of four major toxins including alpha (a), beta (b), epsilon (e), and iota (i). Cp strains are classified into five types (AeE) based on their ability to produce the above toxins. Type A and all other Cp types produce a toxin. Additionally, type B produces b and e toxins, type C produces b toxin, type D produces e toxin and type E produces i toxin [3]. In addition to the above major toxins, a minority of Cp strains especially type A, produce an enterotoxin (CpE), which is responsible for the symptoms of common Cp type A food poisoning in humans [4]. The classification of Cp isolates into toxigenic types has been traditionally performed as toxin neutralization bioassay in mice or guineaepigs [5,6]. These time consuming, expensive techniques were replaced by enzyme linked immunosorbent assay (ELISA) [7]. During the last decade these methods have been replaced by the more convenient PCR-based detection of toxin genes [2,8e13]. Usually, outbreaks of NE in chickens can be readily treated by the administration of certain antibiotics in water such as linco- mycin, bacitracin, oxytetracycline, penicillin, and tylosin tartrate. To prevent the disease, antibiotics such as bacitracin, lincomycin, vir- giniamycin, penicillin, avoparcin, or nitrovin have been added to the feed. However, there are reports of resistance of some isolates to certain antimicrobial such as bacitracin and lincomycin [14]. This study was designed to determine the type of Cp isolated from broiler flocks with enteritis, and to determine its sensitivity to commonly used antimicrobials. 2. Materials and methods 2.1. Samples During the period between April and December of 2006, 155 broiler chicken flocks from Jordan with history of enteritis and retarded growth were examined. These flocks were between 3 and 5 weeks of age and had intestinal lesions varying from catarrhal to necrotic enteritis. Jejunal swabs were collected for bacterial culture. 2.2. Bacterial isolation All incubations were performed at 37  C for 18e24 h anaero- bically using plastic pouches and anaerobic gas generating kit * Corresponding author. Tel.: þ962 2 7201000; fax: þ962 2 7201081. E-mail address: saadgh@just.edu.jo (S. Gharaibeh). Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe 1075-9964/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2010.10.004 Anaerobe 16 (2010) 586e589