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Abstract This study was conducted to detect E. coli O157:H7, Legionella spp. and H. pylori from groundwater, sea water, River Nile water, El-Rahawy Drain and hospital wastewater in Egypt during the study period extend from June, 2010 to July, 2011. E. coli O157:H7 was detected using culture and multiplex PCR methods. Legionella spp. and H. pylori were detected using culture methods. In addition to this, conventional bacterial indicators (TVBC, TC, FC and FS) were also examined. ● To determine the sensitivity and specificity of multiplex PCR in detection of E. coli O157:H7, E. coli O157:H7 ATCC 35150 was used. It was found that the detection limit of E. coli O157:H7 by multiplex PCR was 100 CFU/ml after enrichment step. Furthermore, the multiplex PCR was specific for E. coli O157:H7, no positive band were detected when E. coli ATCC 25922, Enterobacter cloacae, Klebsiella pneumonia, Proteus mirabilis, Listeria monocytogenes, Salmonella Typhimurium ATCC 14028, Pseudomonas aeruginosa, Staphylococcus aureus and E. coli were used. ● Regarding to the results of groundwater collected from New Valley Governorate. It was found that, 16 out of 40 groundwater samples did not comply with Egyptian Standard (2007) for drinking water due to the high bacterial counts in 16 wells samples and presence of TC in 3 well samples including one sample which containing FC. Summary - 195 - ● All treated groundwater samples which were collected from 10 wells, Qalyubia Governorate complied with the Egyptian Standard (2007). ● 15 sea water samples collected from the Mediterranean Sea, Marsa-Matroha Governorate had relatively high average counts of TVBC at 37 and 22oC which were 9.2x102 and 1.4x103 CFU/ml, respectively. ● Considering the fifty water samples which were collected from Rossita Branch along about one Km, the results of bacterial indicators indicated that, the minimum values were observed before the mixing point with El- Rahawy Drain. While, the maximum values were observed at the mixing point with El- Rahawy Drain. The prevalence of E. coli O157:H7, Legionella spp. and H. pylori were 32 (64%), 18 (36%) and 33 (66%), respectively. E. coli O157:H7 was detected in 35 out of 50 (70%) by multiplex PCR and the presence of six virulence genes (flic, stx1, stx2, eae, rfbE and hly) was 0 (0%), 10 (20%), 25 (50%), 23 (46%), 15 (30%) and 0 (0%), (with fragment size at 949, 655, 477, 375, 296 and 199 bp), respectively. ● Among the recording 20 water samples collected from 4 sites distributed along about 5 km of El-Rahway Drain, the averages counts of TVBC were 9.5x105 and 1.5x106 CFU/ml at 37 and 22oC, respectively. The averages counts of TC, FC and FS were 2.2x105, 1.2x105 and 3.1x105 MPN-index/100ml, respectively. The prevalence of E. coli O157:H7 and H. pylori was 20 out of Summary - 196 - 20 (100%), while the prevalence of Legionella spp. was 16 out of 20 (80%) water samples. E. coli O157:H7 was detected in 20 out of 20 water samples by multiplex PCR with prevalence percentage 100%, and the presence of six virulence genes (flic, stx1, stx2, eae, rfbE and hly) was 1 (5%), 8 (40%), 17 (85%), 19 (95%), 15 (75%) and 1 (5%), respectively. ● 10 separate wastewater samples were collected from El-Kasr El-Aini hospitals. Average values of TVBC at 37 and 22oC were 6.4x104 and 5.9x104 CFU/ml respectively, while the average values of TC, FC and FS were 1.5x104, 8.6x103 and 6.2x102 MPN- Index /100ml respectively. In addition to this the average counts of E. coli O157:H7, Legionella spp. and H. Pylori were 3.6x102, 2.1x103 and 2.2x103 CFU/100ml respectively. E. coli O157:H7 was positive in 5 out of 10 (50%) using both culture and multiplex PCR methods, with only three detected virulence genes (stx2, eae and rfbE). Legionella spp. and H. pylori were present in 10 (100%), 6 (60%) out of 10 hospital wastewater samples respectively. ● The O antigen (rfbE) gene in six local E. coli O157:H7 isolates was chosen for characterization, because it was the most frequent gene in isolated local E. coli O157, also this gene represent O antigen gene which synthesis O157 LPS. It was found that, sequence analyses of positive PCR products of E. coli O157:H7 revealed high homology with two E. coli O157:H7 strains, it Summary - 197 - deposed in database namely (EC 4024) and (09BKT078844) with Blast identity ranging from 94% to 100%. ● Regarding the statistical analyses. The statistical analyses were carried out in order to find any possible correlation between tested pathogenic bacteria and bacterial indicators; It was found that, there were differences in correlations either by positive or negative with different variety of significance in different water tested. ●Regarding to antibiotic sensitivity test of E. coli O157:H7 and Legionella spp. isolates. It was found that, E. coli O157:H7 isolates were resistant to amoxicillin (100%), clarithomycin (77%), streptomycin (11%) and tetracycline (7%). While E. coli O157:H7 ATCC 35150 (strain 1) was resistant to amoxicillin and clarithomycin. On the other hand, L. pneumophila ATCC 33152 was resistant to cefaxime and clarithromycin. While confirmed Legionella isolates were resistant to tetracycline (100%), clarithromycin (100%), ciprofloxacin (80%), amoxicillin (70%), cefaxime (40%), and streptomycin (30%). ●In survival experiment, E. coli O157:H7 survived in all sterilized tested water types longer than non sterilized water. It was found that the E. coli O157:H7 isolated from wastewater sample had the longest survival time (98 days) in sterilized wastewater compared to the other strain (reference strain and isolate of River Nile). Summary - 198 - ● In chlorine experiment, in order to reach 99.9% inactivation of E. coli O157:H7 by chlorine. It was found that, both E. coli O157:H7 environmental strains form River Nile and wastewater required higher dose of that than required to inactive the reference strain E. coli O157:H7 ATCC 35150. Comparing four log reduction of E. coli O157:H7 ATCC 35150 and L. pneumophila ATCC 33152 numbers, it was found that L. pneumophila ATCC 33152 (2.6 mg/l) required higher chlorine dose than E. coli O157:H7 ATCC 35150 (1.4 mg/l). Conclusions and Recommendations ● El-Rahawy Drain is the main source of the deterioration of River Nile (Rossita Branch), thus, it is recommended that El- Rahawy Drain should be treated before discharging to Rossita Branch. ● Hospital wastewater should be treated onsite before mixing with sewerage system. ● The cultivation of E. coli O157:H7 on HiCrome EC O157:H7 selective agar media is considered inexpensive and reliable detection methods in concentrated water sample; samples with low counts were easily detected. ● The Multiplex PCR is sensitive, specific, for detection of E. coli O157:H7; and also, provides a clue for the degree of virulence; can be used to detect at least one or more of the Summary - 199 - virulence genes which culture, biochemical and serological tests cannot. ● Combining both culture and PCR methods should be recommended for the detection of E. coli O157:H7. ● Culture methods for detection of Legionella spp. and H. pylori were reliable and gave consistent results, but they were time consuming (4-10 days). It is recommended faster methods to detect Legionella spp. and H. pylori. ● The absence of consistent, clear and significant correlation between the presences of bacterial indicators and tested pathogenic bacteria, it is recommended that monitoring of bacterial pathogens from time to time a long routine bacterial indicators examination. ● Due to abuse and misuse of antibiotics, environmental pathogenic bacteria develop strategies for resistance. Restriction of antibiotics used should be enforced. ● It is recommended that, test for disinfections control in Egypt, should be carried out on local pathogenic bacterial isolates because they showed higher resistance than reference strains. |