الفهرس 
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Abstract
The present study aimed to investigate the prevalence of Cronobacter sakazakii in commercial powdered infant formula milk and powdered infant foods available in the Egyptian market. Also, the study aimed to determine factors that affect survival and growth of C. sakazakii in powdered infant formula milk in order to control the spread of the organism.
The selective media (Brilliance Enterobacter sakazakii isolation agar) was used to isolate C. sakazakii. The organism was isolated from 9 out of 173 samples of powdered infant formula milk and one out of 61 powdered infant foods making a total of 10 with a prevalence rate of 4.27%. Powdered infant formula milk exhibited a higher frequency of isolation of the organism (5.2%) compared with powdered infant food (1.6%).
Among the 7 clinical specimens, only one Cronobacter sakazakii isolate was recovered, while no detection of organism was found in environmental samples.
Isolates identification was based on the cultural, morphological and biochemical characteristics after isolation on the selective Brilliance Enterobacter sakazakii agar media for Cronobacter sakazakii. Presumptively positive isolates producing blue green colonies were further identified using RapiD API 20E kit.
The identity of C. sakazakii was further confirmed by PCR amplification of 1680 bp fragment of the gluA gene that encodes α- glucosidase enzyme, where all isolates were found to have gluA gene.
The 10 brands of powdered infant products that gave positive isolation for C. sakazakii were subjected to microbiological investigation to examine the bacterial load and results revealed aerobic plate counts ranging from 0.6xto 4.9 xcfu/gm. Two samples exhibited high viable counts ranging from 7xto 8.3xcfu/gm. All samples failed to comply with microbiological criteria for aerobic plate count of C. sakazakii according to Codex Alimentarius Commission standards (2008).
The presence of Omp A gene as a virulence factor was examined in all isolates by PCR amplification of 469 bp fragment for all isolates of Cronobacter sakazakii, where all isolates were found to harbor OmpA.
All C. sakazakii isolates were tested in vitro for their susceptibility to 16 antimicrobial chemotherapeutic agents by disc diffusion method. All isolates demonstrated resistance to rifampicin (100%) and ampicillin (100%). All isolates were sensitive to levofloxacin (100%), norfloxacin (100%) and ofloxacin (100%). High susceptibility was observed to ciprofloxacin, naldixic acid, gentamicin, imipenem, ceftazidime, sulphmethoxazole/Trimethoprime (90.9% each), aztronam (81.8%), and streptomycin (72.7 %). A lower rate of sensitivity was observed to cefotaxime (54.5%), amoxicillin /clavulanic acid (27.3 %) and cephalexin (9.09 %).
The clinical Cronobacter sakazakii isolate showed higher resistance to most of the tested antimicrobial chemotherapeutic agent compared to isolates from powdered infant products.
The Minimum Inhibitory Concentrations (MICs) of some selected chemical antibacterial agents used as a disinfectant and preservative were determined for the isolates of C. sakazakii by broth dilution method. Lactic acid at concentration 0.2% inhibited all isolates of C. sakazakii except of the clinical isolate that was inhibited by 0.4% all other isolates. MICs ranged from 0.0125 to 0.05% and 0.05 to 0.2% for potassium sorbate and methyl paraben (methyl p-hydroxy benzoate), respectively. MICs ranged from 0.0005 to 0.002%, 0.0025 to 0.01% and 0.0025 to 0.02% for thiomersal, Benzalkonium chloride and cetrimide, respectively.
One isolate of C. sakazakii was selected to test the effect of temperature on survival of the organism in reconstituted infant formula milk (e.g. complete balanced, lactose-free and soy protein infant formula) at different temperatures (55, 60, 65, 70, 75, 80, 85 and 90˚C).
For complete balanced and lactose free infant formula milk, the numbers of the organism decreased with time at all temperatures used. At 70˚C, 7 reductions in cfu were obtained after 15 minutes with D-values of 2.5 minutes, while no viable organism was detected after 20 minutes. The increase in temperature from 55˚C to 70˚C reduced D-values by about three folds.