Browsing by Author "Adegun, Busayo Roseline"

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    Phenotypic Identification and molecular Characterization of citrobacter sp isolated from commercial fruits and Vegetables in Ile-Ife,Nigeria
    (Microbiology,Obafemi Awolowo University, 2015) Adegun, Busayo Roseline
    ABSTRACT This study investigated antibiotic susceptibility profile, resistance plasmid DNA as well as resistance and virulence genes of Citrobacter species isolated from fruits and vegetables. This was with a view to determining the safety of fruits and vegetables sold commercially in Ile-Ife market. One hundred and fifty (150) fruits and 90 vegetables samples were purchased at Iso-Obi in Sabo, Ile Ife. Each sample was washed with sterile distilled water and serially diluted. The pre-cut of the fruits was homogenized using a sterile mortar and pestle, while fruits with juice were squeezed mechanically to extract the juice. One millilitre of the serially diluted resultant homogenate was plated onto MacConkey, Salmonella-Shigella and Eosin Methylene blue agar plates using spread plate method, and incubated at 37 ºC for 24 h. Preliminary identification of the isolates was based on their cultural and morphological characteristics. Identities of Citrobacter isolates were confirmed using conventional biochemical tests and analytical profile index (API) kits. The antibiotic susceptibility testing of the isolates was carried out using Kirby-Bauer’s disk diffusion techniques and the zones of inhibition were interpreted based on the Clinical and Laboratory Standard Institute (CLSI) guidelines. Isolation of plasmid DNA was done using TENS (Tris, EDTA, NaOH and SDS) buffer and plasmid curing was carried out using ethidium bromide. Molecular detection of resistance (blaCTX, blaSHV and aac(6´)) and virulence (eae) genes was done by Polymerase Chain Reaction (PCR) using appropriate primers. Forty bacterial isolates were recovered from fruits and vegetables comprising 25 (62.5 %) Citrobacter, 7 (17.5 %) Escherichia coli, 5 (12.5 %) Proteus sp and 3 (7.5 %) Klebsiella sp. Antibiotic susceptibility tests showed that all the Citrobacter species were resistant to ceftriazone and amoxicillin, while most were resistant to piperacillin, gentamycin, cefotaxime, tetracycline, cotrimoxazole, nitrofurantoin and augmentin. There was significant difference (p<0.05) in the percentage resistance of the Citrobacter isolates. Twenty two (88 %) of twenty five Citrobacter isolates obtained in this study, Citrobacter freundii (60 %), Citrobacter braakii (20 %) and Citrobacter youngae (8 %) exhibited multiple antibiotic resistances to between two and four different classes. All the twelve selected multiple antibiotic resistant Citrobacter isolates harboured single plasmid with molecular weight of 1710 bp. The plasmid curing experiment revealed that ceftriazone resistance was plasmid-borne. Five of the 12 representative isolates namely Citrobacter freundii (3), Citrobacter braakii (1) and Citrobacter youngae (1) harboured blaCTX gene with molecular weight of 585 bp. Citrobacter freundii (2) and Citrobacter braakii (1) had blaSHV with molecular weight of 392 bp each. None of the isolates harboured aac6’ resistant gene. Only Citrobacter braakii (1) harboured eae (1917 bp) virulence gene. The study concluded that fruits and vegetables sold in the study area were contaminated with potential Gram negative bacterial pathogens capable of causing food-borne disease in consumers.