Isolation and characterization of toxins produced by Cronobacter species and the subsequent virulence characterization of the toxin producing strains

Global Medical Microbiology Summit & Expo

November 28-29, 2016 San Francisco, USA

Ziad W Jaradat, Waseem Al Mousa, Ahmed El Beteiha, Qutaibah Ababneh and Anas Al Nabulsi

Jordan University of Science and Technology, Jordan

Posters & Accepted Abstracts: J Med Microb Diagn

Abstract :

Cronobacter spp. is a group of Gram negative pathogens that have been implicated as the causative agents of necrotizing enterocolitis and meningitis in infants. Progress in understanding the virulence mechanism of Cronobacter has been poor to date and not much is known about the factors involved in its pathogenicity. The phylogenetic analysis based on 16S rRNA gene sequences of the 47 Cronobacter isolates used in this study identified four main clusters with C. sakazakii was the most represented species (78.72%) followed by C. muytjensii (14.89%), C. turicensis (4.26%) and C. dublinensis (2.13%). The antibiotic resistance profile of the isolates revealed a low resistance profiles with only 10.64%, 8.51% and 4.26% were resistant to ampicillin, cefoxitin and amoxicillin-clavulanic acid, respectively. Most of the isolates (76.6%) were susceptible to all the antibiotics used. PCR screening of putative virulence genes showed that C. sakazakii isolates harbor virulence genes more frequently than other Cronobacter species with siderophore interacting protein gene (sip) and iron acquisition gene clusters (eitCBAD and iucABCD/iutA) were the most detected genes. To address potential virulence factors and toxins produced by Cronobacter, the filtrates of the isolates were tested on cell culture. The results of Vero cells assay varied between the 47 isolates, whereas most isolates exhibited a weak cytotoxicity. Nine isolates were selected subsequently to be evaluated for enterotoxin production in suckling mice. The filtrates tested were consistently negative, even when two types of media (BHI and TSB) were used to culture the isolates. Concentrating the culture filtrates at least 20-fold using freeze drying showed a positive enterotoxin production for C. sakazakii 12C and C. turicensis Jor170. The toxin was purified from these strains by stepwise ammonium sulfate precipitation followed by Sepharose 6-B gel filtration chromatography. The molecular mass of the enterotoxin purified was determined to be around 40-50 kDa. Suckling mice were also challenged both intraperitoneally and orally to determine the minimum lethal dose for the nine isolates that were selected for enterotoxin assay. All the isolates caused death at 107 CFU per mouse when injected intraperitoneally, while seven isolates were lethal at the same dose by the peroral route. However, an obvious difference in infectivity between the strains was observed. Results obtained in this study might provide more answers about the virulence characteristics of Cronobacter species.

Biography :

Email: jaradatz@just.edu.jo