تأثير الحرارة وتراكيز الملح على نمو وبقاء الإشيرشا القولونية O157: H7 المعزولة من الحليب الخام ومنتجات الألبان للأبقار والجاموس المباعة في محافظة كربلاء

The effects of temperature and salt concentrations on survival and growth of E. coli O157:H7 isolated from Cattle’s and Buffalo’s Raw Milk

Abstract
The current study which aims to isolate, diagnose the spread of E. coli O157:H7 and study the effect of temperature and salt concentrations in milk products for cows and buffaloes. where 240 samples were collected, from cow and buffalo milk and their milk products, raw milk, cheese and yogurt, These products were divided in two parts 60 samples from street vendors (20 samples for each) and 60 samples from shops and stores (20 samples for each )This study included three aspects :
The first aspect includes confirmation of E. coli O157:H7 by conventional bacterial methods (culture media for growth and differentiation), biochemical examinations (indole, motility, oxidase, urease, simmon citrate and kligler iron tests) and diagnosis by using the vitek -2 device. The bacteria also were confirmed by applying PCR, to detect rfb (O157) encoding the O. antigen this specific for E.Coli O157:H7 and fliCH7, E. coli O157:H7 isolates were serotypped for detection of the O157 and H7 antigens., non-sorbitol fermenting (NSF) isolates. Additionally, the virulentor for detection the serotype (rfbO157and fliCH7) genes by PCR procedures too. Total of 18 isolates from 120 E. coli O157:H7 in cows (15%) and 14 from 120 (11%) buffalo. All fourteen samples isolates from cows milk were reported as positive (rfbO157and fliCH7) (100%) genes, however, only 13 from 18 isolates from buffalo were positive.
E. coli O157:H7 which were isolated from 27 (11.25%) of 240 sample isolates were biochemically typical for E. coli O157:H7 (sorbitol non-fermenting and confirmation with Vitek-2 system) and motile expressing the H7 antigen according to culture methods, biochemical tests and PCR using rfbO157gene and fliCH7 that amplified from raw milk, cheese and yogurt isolates via using conventional PCR a product size was approximately 497 bp. using fliCH7 gene that amplified a product size was approximately 625.
In the second aspect, of study the rate of contamination in raw milk, yogurt and cheese of cow collected from street venders were 60%, 50%, and 70%, respectively. while, the contamination rate in raw milk, yoghurt and cheese of buffalo drawn from street venders were 40%, 35% and 35% respectively.
The study had a significant increase effect (p<0.05) than those contaminated samples from shops and stores (50%, 35% and 55%) (55%, 40% and 45%) in raw milk, yogurt, and cheese of cow and buffalo respectively . The study also showed that there are significant differences in contamination rates in cow’s milk compared to buffalo milk and in all products (60% ,50%, 70% for cow street venders ) (50%, 35 and 55% for buffalo street venders ), follows ( 55% , 40% , 45% for cow stores origin ) ( 40% ,35 and 35% for buffalo stores origin ) respectively .
The results were shown in the third aspect the experimental work, which illustrate the effect of temperature on E.coli O157:H7 which found a significant increase in bacterial growth when observed in all dilutions (treatment (3) at 20 °C for (24) hours) (80 , 80 ,3 , 3 and 2)CUF/ml (Treatment (4) at 30 °C for (12) hours) (uncountable , uncountable , uncountable, 70 , 5 and 4)CUF/ml and also in (Treatment (5) at 37°C for (12) hours ) (uncountable , uncountable , uncountable , 95 , 16 and 3 ) CUF/ml the last treatment (6 )at 43 c for 12 hours ) (uncountable , uncountable , 133 , 17 and 8) CUF/ml in the dilution ( 10-1 , 10-2 , 10-3 , 10-4 ,10-5 and 10-6 ) respectively and comparing all treatments vertically and each for dilutions. There were high significant differences in the growth of numbers of bacteria E.coli O157H7 in a (treatment 3 , treatment 4, treatment 5 , treatment 6 ).
The result of the study also showed that using concentration of (1 %) NaCl had significant growth in treatment ( 2) at 20°C for (24) hours (uncountable , uncountable, 11 , 7, 1 and 1 ) CUF/ml in dilution (10-1 , 10-2 , 10-3 , 10-4 , 10-5 and 10-6 ) respectively, while showing the treatment concentration of 5% NaCl at (20 °C for 24 hours) high growth of E.coli o157:H7 (uncountable , uncountable , 51 , 6 and 1) CUF/ml in dilution (10-1 , 10-2 , 10-3 , 10-4 and 10-5), At salt levels in 9% NaCl at (37°C for 12 hours) Bacterial growth was observed in the dilutions (uncountable, uncountable , 100 ,7 , and 5 ) CUF/ml in dilution (10-1 , 10-2 , 10-3 , 10-4 and 10-5) , whil taking a concentration 10 % NaCl , no bacterial growth was observed in all treatments .the findings of the current study concluded that treatment with a temperature (4ᵒC, 7ᵒC, 46ᵒC, 53ᵒC and 72ᵒC) was efficient to kill E.coli O157:H7 bacterial cells in milk , while adopting a concentration of ( 10% NaCl) was adequate to destroy all living E.coli O157:H7 .