Detection of waterborne pathogens by polymerase chain reaction-reverse blot hybridization assay

Abstract

Traditionally, detection and enumeration of waterborne pathogens have focused on the use of selective culture and standard biochemical tests. This classical microbiological methodology relies on the cultivation of indicator bacteria, for example plate counts of total coliforms or Escherichia coli. Such methods have a variety of serious drawbacks, like no correlation to many waterborne pathogens and no valid identification of the pathogen. For example, opportunistic pathogen which normally occur at low levels in the environment tend to incur large errors in sampling and enumeration. Also, some waterborne pathogens (e.g., Shigella spp. and Mycobacterium spp.) are documented to remain viable for extended periods of time, but are unrecoverable by otherwise successful culture protocols. However, recent epidemiological evidence has shown that there is no correlation between E. coli/coliform count and pathological incidences. Hence, conventional water testing methods are clearly outdated and do not adequately safeguard public health. Culture-based methods are time consuming, high cost, and labor intensive. Due to these limitations, examination of water samples for these opportunistic pathogen is usually not performed in routine assessment of water quality. In this regard, there exist absolute needs for developing more comprehensive system for assessing the safety of water. A sensitive, specific, and affordable test for rapid identification of waterborne pathogens is necessary to determine the impact of bacterial pathogens in water samples. The present study developed comprehensive system for assessing the safety of water using polymerase chain reaction-reverse blot hybridization assay (PCR-REBA). The aim of this study was to analyze the 16S rRNA gene diversity of Bacteria, as a supplementary tool to standard practices of microbiological quality in the whole water sample.

This study described the development of waterborne pathogens monitoring system PCR-REBA. The PCR-REBA is capable of simultaneously detecting nineteen different types of waterborne pathogens in complex bacterial mixtures that are normally found in waters. PCR-REBA was developed to detect nineteen waterborne pathogens, using two primer pairs for nested PCR and species-specific probes for each. The method was validated using a reference strain and established both sensitivity and specificity. To assess the ability of PCR-REBA to detect waterborne pathogens, diverse water samples were collected from eight wastewater treatment plants from November 2006 to July 2008. For detection to low population of target bacteria, membrane filtration method was conducted and amplified using the nested PCR for increase of sensitivity. Most of samples collected were positive for indicator bacteria and opportunistic pathogen. This study identified Pseudomonas spp. and Yersinia spp. which are the gram negative bacteria known to cause enteritis, and Enterococcus spp., Listeria spp. and Staphylococcus spp., the gram positive bacteria, in addition to the E. coli that are used as the indicator bacteria in the discharged water quality standard. In addition, as a result of PCR-REBA and sequence analysis after using cetylpyridinium chloride (CPC) and membrane filtration method, contamination of Mycobacterium spp. were confirmed in water samples. Isolated six strains of Mycobacterium spp. were potentially pathogenic mycobacterial species. Since the identified bacteria were continuously detected at all the sampling sites regardless of the seasonal effect and the difference in the disinfection methods, these bacteria suggest to the indicator bacteria for water quality standard. This study shows that even though many innovative bacterial detection methods have been developed, PCR-REBA has the potential for becoming a standardized method for the detection of opportunistic pathogen in diverse water samples. Features of PCR-REBA include high overall rapidity, sensitivity and specificity for identifying waterborne pathogens.