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Detection of diarrhoeagenic Escherichia coli in clinical and environmental water sources in South Africa using single-step 11-gene m-PCR.

Omar KB, Barnard TG - World J. Microbiol. Biotechnol. (2014)

Bottom Line: Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested.In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types.The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Water and Health Research Centre, University of Johannesburg, Doornfontein, PO Box 17011, Johannesburg, 2028, South Africa, kousaro@uj.ac.za.

ABSTRACT
Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

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Agarose gel of the PCR products obtained from samples (lane 4–11, 13–18). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1 and 12). The positive reference control is shown in (lane3)
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Fig2: Agarose gel of the PCR products obtained from samples (lane 4–11, 13–18). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1 and 12). The positive reference control is shown in (lane3)

Mentions: A total of 701 samples were analysed, samples composed of 239 clinical isolates, 171 environmental water isolates and 291 samples from the Colilert® Quanti-Tray®/2000 (Fig. 2); these samples were obtained from various provinces in South Africa. Isolates and water samples were subjected to the protocols described in the methodology, with 100 % (171/171) of environmental water isolates, 85 % (202/239) of the clinical isolates and 100 % (291/291) of the water samples testing positive for the mdh house-keeping gene (Fig. 2). For the 15 % (37/239) of clinical isolates in which the mdh gene was not detected, it is possible that these do not contain the malate dehydrogenase but the malic acid dehydrogenase gene, which is also a housekeeping enzyme of the citric acid cycle (Hsu and Tsen 2001). When the study was initiated Tarr et al. (2002) article was used, who included the malate dehydrogenase gene and indicated in their tests positive results for all the E. coli strains tested. Based on this the mdh gene was used as a control to confirm the microbiology results in case no pathogenic genes tested for were detected. It is only later that for a separate study the malic acid dehydrogenase gene was tested (also referred to as mdh by Hsu and Tsen 2001) that not all the E. coli strains present in the samples tested positive. The reason could be that the original work by the authors were done on strains that could not be present in South Africa or that we have strains that have different genetic characteristics. No false positives and no PCR inhibition were indicated in the m-PCR as the external control gene (gapdh) was detected in 100 % (701/701) of the samples. A supposedly negative test result for an infectious agent can influence therapeutic decisions, such as withholding antibiotic and antiviral drugs (Cone et al. 1992; Hartman et al. 2005). Therefore, the additions of the internal and external controls are important to ensure that there are no PCR inhibitors in the reaction as well as to validate the accuracy of the PCR in distinguishing false negative from true negative PCR results. Fig. 2


Detection of diarrhoeagenic Escherichia coli in clinical and environmental water sources in South Africa using single-step 11-gene m-PCR.

Omar KB, Barnard TG - World J. Microbiol. Biotechnol. (2014)

Agarose gel of the PCR products obtained from samples (lane 4–11, 13–18). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1 and 12). The positive reference control is shown in (lane3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4150989&req=5

Fig2: Agarose gel of the PCR products obtained from samples (lane 4–11, 13–18). No template control (NTC) in (lane 2). The molecular weight marker is shown in (lane 1 and 12). The positive reference control is shown in (lane3)
Mentions: A total of 701 samples were analysed, samples composed of 239 clinical isolates, 171 environmental water isolates and 291 samples from the Colilert® Quanti-Tray®/2000 (Fig. 2); these samples were obtained from various provinces in South Africa. Isolates and water samples were subjected to the protocols described in the methodology, with 100 % (171/171) of environmental water isolates, 85 % (202/239) of the clinical isolates and 100 % (291/291) of the water samples testing positive for the mdh house-keeping gene (Fig. 2). For the 15 % (37/239) of clinical isolates in which the mdh gene was not detected, it is possible that these do not contain the malate dehydrogenase but the malic acid dehydrogenase gene, which is also a housekeeping enzyme of the citric acid cycle (Hsu and Tsen 2001). When the study was initiated Tarr et al. (2002) article was used, who included the malate dehydrogenase gene and indicated in their tests positive results for all the E. coli strains tested. Based on this the mdh gene was used as a control to confirm the microbiology results in case no pathogenic genes tested for were detected. It is only later that for a separate study the malic acid dehydrogenase gene was tested (also referred to as mdh by Hsu and Tsen 2001) that not all the E. coli strains present in the samples tested positive. The reason could be that the original work by the authors were done on strains that could not be present in South Africa or that we have strains that have different genetic characteristics. No false positives and no PCR inhibition were indicated in the m-PCR as the external control gene (gapdh) was detected in 100 % (701/701) of the samples. A supposedly negative test result for an infectious agent can influence therapeutic decisions, such as withholding antibiotic and antiviral drugs (Cone et al. 1992; Hartman et al. 2005). Therefore, the additions of the internal and external controls are important to ensure that there are no PCR inhibitors in the reaction as well as to validate the accuracy of the PCR in distinguishing false negative from true negative PCR results. Fig. 2

Bottom Line: Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested.In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types.The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Health Sciences, Water and Health Research Centre, University of Johannesburg, Doornfontein, PO Box 17011, Johannesburg, 2028, South Africa, kousaro@uj.ac.za.

ABSTRACT
Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

Show MeSH
Related in: MedlinePlus