The drinking water industry needs to be able to screen microorganisms rapidly to better protect public health. Polymerase chain reaction (PCR) allows us to detect DNA of the indicator bacteria E. coli and enterococci within hours. However, PCR does not reveal whether the pathogens are still viable. In addition, humic acids or metal particles can interfere with the analysis and lead to false-negative results: although pathogenic bacteria are present, the sample produces a negative PCR result.
Such interfering substances makes it difficult to detect pathogenic bacteria at low concentrations using PCR. For this reason, methods such as pre-culture PCR have been developed in food diagnostics, where products are checked on a daily basis. Pre-culture PCR methods have a lower detection limit and limit the effects of humic acids, particles or other substances in the matrix. They also reduce the probability of false-positive detection (caused by dead cells).
Vitens Water Expertise Centre (WEC) has studied the use of pre-culture PCR for screening for faecal contamination in drinking water. A pre-culture PCR procedure for E. coli and enterococci was developed and validated according to the NEN-EN-ISO 16140 standard for this purpose. This included assessing whether the method was sensitive to interfering substances in drinking water. Standardised culture methods for the detection of E. coli and enterococci in drinking water were used as reference. These standards are based on NEN-EN-ISO guidelines for the detection of E. coli and enterococci in drinking water testing. The relevant test for E. coli takes 24 hours and the test for enterococci as long as 48 hours. The government has incorporated these tests into drinking water regulations and they are currently the standard for monitoring E. coli and enterococci.
Pre-culture PCR
In pre-culture PCR, DNA from a target organism is multiplied and examined in two steps. In the first step, target organisms are cultivated in the sample under controlled conditions (pre-cultivation). Subsequently, detection by PCR is relatively easy.
The process for pre-cultivation is as follows: we filter 200 millilitres of drinking water. The filter is then incubated for nine hours in a generic liquid medium at 37 °C to propagate E. coli and enterococci. S. epidermidis is added as a control. After incubation, DNA from the pre-cultivation medium is isolated followed by PCR to multiply and detect DNA from faecal indicator organisms and S. epidermidis. The method is qualitative, which means that it detects culturable E. coli, enterococci and S. epidermidis, but does not provide information on the number of colony-forming units (cfu) or DNA copies. Figure 1 shows an example of a pre-culture PCR result.
Figure 1. A pre-culture PCR result for E. coli from three samples. In PCR, a fluorescence signal is measured in a cyclic process. E. coli shows an increase in this fluorescence signal. Purple with crosses = two samples positive for E. coli; purple with circles = one sample negative for E. coli; green = S. epidermidis (control). If this green line does not show a smooth s-curve, there was interference in the PCR.
Validation of the pre-culture PCR
For the purpose of validation, the selectivity, sensitivity, accuracy and detection limit of the pre-culture PCR were determined using drinking water as the matrix. We compared the pre-culture PCR results with those of standard culture methods.
The pre-culture PCR used was found to be selective: 89 reference strains of enterococci and E. coli gave positive results, while 34 other strains (not enterococci or E. coli) gave negative results. The comparison to the standard methods (sensitivity and accuracy) involved testing 134 samples, 64 of which were intentionally contaminated with E. coli and/or enterococci. Of the samples, 27% contained E. coli and 37% contained enterococci. The results of the validation are shown in Table 1.
Table 1. Comparison of pre-culture PCR to standard culture methods in terms of detection of E. coli and enterococci. ND = negative deviation; PD = positive deviation.
International standards have been developed for reliable validation. The NEN-EN-ISO 16140 standard is often used for this purpose. According to this standard, the difference (acceptable limit) between the pre-culture PCR and the standard culture method must not exceed 4. The result for the limit in our comparison was one for both organisms, meaning that the results of the pre-culture PCR are consistent with those of conventional culture methods and therefore meet the requirement which are stated in the standard.
For the detection limit of the pre-culture PCR, we examined low levels of contamination using both the pre-culture PCR and standard culture methods (see Table 2).
Table 2. Determination of the detection limit of the pre-culture PCR used. Positive means the sample is contaminated, and negative means the sample is free of E. coli and enterococci.
The experiment shows that low amounts of E. coli and enterococci species (0.7 cfu and 1.5 cfu/100 ml) are more frequently detected by the pre-culture PCR than by conventional culture methods.
To determine the sensitivity of the pre-culture PCR to PCR inhibitors, a further 28 drinking water samples were tested. Most samples were contaminated with low concentrations of E. coli and enterococci. The results of the pre-culture PCR and standard culture methods were broadly in line in these 28 samples tested. One sample tested negative with the pre-culture PCR and positive with the standard method (showing 1 cfu/100ml of E. coli). This difference can potentially be explained as a result of detection probability of culturable organisms in the sampled water. Nevertheless, the conclusion of this experiment is that the pre-culture PCR is not sensitive to PCR inhibitors, and the probability of a false negative result is low.
Use with other bacteria
After examining faecal indicator organisms, we also explored the suitability of the pre-culture PCR for detecting Clostridium perfringens and coliform bacteria. A small study by Vitens WEC showed that pre-culture PCR was able to detect thermotolerant coliforms Klebsiella, Enterobacter and Citrobacter. Although pre-culture PCR still requires improvement, the prospects are encouraging: a single pre-culture medium has the potential to detect thermotolerant coliforms, enterococci, E. coli and C. perfringens. This reduces costs, the use of plastics and the need for culture media, making the pre-culture PCR more sustainable than conventional standardised culture methods. Finally, Vitens WEC is also working on a pre-culture PCR method that can detect viable L. pneumophila within 48 hours. This can achieve a time saving of as much as five days compared to the conventional method. The initial results are promising.
Scope for using pre-culture PCR for drinking water research
In this study, we investigated whether a pre-culture PCR method from food research is suitable for screening drinking water for E. coli and enterococci. The validation showed that the pre-culture PCR performs similarly to standard culture methods and is not sensitive to interfering substances in drinking water. The method is easy to automate, which is of interest to Vitens WEC, which tests more than 200 samples daily. The pre-culture PCR can provide results within 10 to 12 hours: a significant time saving compared to standardised culture methods, which take one to two days. The method is effective in detecting low amounts of faecal indicator organisms. Pre-culture PCR offers a fast and accurate alternative to routine drinking water testing.
In conclusion, the pre-culture PCR can be a rapid, simple and sustainable alternative to current culture methods for screening for faecal indicator and other organisms. The aim is to deploy pre-culture PCR methods further and share knowledge about their capabilities. We also aim to standardise these methods further to ensure that they are available to other interested companies, as has happened in the food industry.
Pre-culture polymerase chain reaction (PCR) is a technique from food diagnostics for the rapid screening of viable pathogenic bacteria. Research by Vitens Water Expertise Centre (WEC) has shown that this method is also suitable for screening water, including drinking water. In the study, drinking water is filtered and the filter is then incubated in a growth medium, followed by DNA analysis using PCR. Compared to standard culture methods, pre-culture PCR offers savings in terms of time, culture media, costs and the use of plastics.