MOLECULAR IDENTIFICATION OF EXTENDED SPECTRUM Β-LACTAMASE (ESΒL)
PRODUCING KLEBSIELLA SPECIES RETRIEVED FROM DISEASED CAGE-CULTURED SEABREAM AND SEABASS

Riad H. Khalil1,*; Hany M.R. Abdel-Latif1 and Amany M. Diab2

1Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University.

2Aquatic Microbiology, Faculty of Aquatic and Fisheries Sciences, Kafr El-Sheikh University

* Corresponding author e-mail: – ryadvet_2013@yahoo.com

Received 6/ 11/ 2016

Accepted 22/ 12/ 2016

Abstract

Extended spectrum β-lactamase (ESβL) producing Klebsiella spp have the opportunistic ability to the aquatic biota, and they can cause disease problems of the infected fish under stressful culture conditions. This study was mainly focused on the confirmatory molecular identification of the ESβL producing Klebsiella spp isolated from diseased cage-cultured marine fishes, using a multiplex polymerase chain reaction (mPCR). Significant mortalities with noticeable septicemic signs of cage-cultured seabass and seabream had occurred during summer 2016 in two localities in Egypt [Borg-El Arab, Alexandria, and Eldeepa, Damietta]. Moribund and dying fishes were sampled, clinically examined, and bacteriologically demonstrated (phenotypic characterization, and API 20E testing). Additionally, antimicrobial susceptibility testing was done using 10 antibiotic discs, followed by a confirmatory mPCR for identification of bla TEM, bla CTX-M, and bla SHV genes that are responsible for the detection of the EsβL production. Diseased fish showed septicemic signs with significant mortalities. From 250 retrieved isolates, results demonstrated the occurrence of Klebsiella
spp, Escherichia coli, Citrobacter freundii, Proteus vulgaris, and Enterobacter cloacae with a prevalence of 60, 8, 5, 12, and 7 % respectively. Bacteriologically, there was the predominant existence of Klebsiella spp, about 150 Klebsiella isolates were determined, of which 85 (57%) were recognized as K. pneumoniae and 65 (43%) as K. oxytoca depending on phenotypic characterization. Additionally, the antibiogram testing showed that the strongest resistance to Cefotaxime, Ceftriaxone, Nalidixic acid and Trimethoprim-sulfamethoxazole, intermediate susceptibility to Cefepime, while some of the isolates were susceptible to Ciprofloxacin, Ofloxacin, Gentamycin, Amoxycillin-clavulanic acid, and Amikacin. Furthermore, the position of bands on an agarose gel, 964 bp for bla TEM, 593 bp for bla CTX-M, and 854 bp for bla SHV confirmed the EsβL production. In this work, it was demonstrated that ESβL producing K. pneumoniae and K. oxytoca have encountered in mortalities of marine fish, and the multiplex PCR is considered a more confirmatory, reliable, relatively rapid, effective, and possibly a potential tool for the routine identification of ESβL producing Klebsiella species.

Keywords: Extended-spectrum β-lactamase (ESβL) – Klebsiella species – Seabass – Seabream.