Tanja Kostić

Evaluation of quantitative PCR combined with PMA treatment for molecular assessment of microbial water quality.

Microbial water quality assessment currently relies on cultivation-based methods. Nucleic acid-based techniques such as quantitative PCR (qPCR) enable more rapid and specific detection of target organisms and propidium monoazide (PMA) treatment facilitates the exclusion of false positive results caused by DNA from dead cells. Established molecular assays (qPCR and PMA-qPCR) for legally defined microbial quality parameters (Escherichia coli, Enterococcus spp. and Pseudomonas aeruginosa) and indicator organism group of coliforms (implemented on the molecular detection of Enterobacteriaceae) were comparatively evaluated to conventional microbiological methods. The evaluation of an extended set of drinking and process water samples showed that PMA-qPCR for E. coli, Enterococcus spp. and P. aeruginosa resulted in higher specificity because substantial or complete reduction of false positive signals in comparison to qPCR were obtained. Complete compliance to reference method was achieved for E. coli PMA-qPCR and 100% specificity for Enterococcus spp. and P. aeruginosa in the evaluation of process water samples. A major challenge remained in sensitivity of the assays, exhibited through false negative results (7-23%), which is presumably due to insufficient sample preparation (i.e. concentration of bacteria and DNA extraction), rather than the qPCR limit of detection. For the detection of the indicator group of coliforms, the evaluation study revealed that the utilization of alternative molecular assays based on the taxonomic group of Enterobacteriaceae was not adequate. Given the careful optimization of the sensitivity, the highly specific PMA-qPCR could be a valuable tool for rapid detection of hygienic parameters such as E. coli, Enterococcus spp. and P. aeruginosa.

Microbial diagnostic microarray for food- and water-borne pathogens.

A microbial diagnostic microarray for the detection of the most relevant bacterial food‐ and water‐borne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence‐specific end labelling of oligonucleotides and the pyhylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus/species level) and sensitive (0.1% relative and 104 cfu absolute detection sensitivity) detection of the target pathogens. Validation was performed using a set of reference strains and a set of spiked environmental samples. Reliability of the obtained data was additionally verified by independent analysis of the samples via fluorescence in situ hybridization (FISH) and conventional microbiological reference methods. The applicability of this diagnostic system for food analysis was demonstrated through extensive validation using artificially and naturally contaminated spiked food samples. The microarray‐based pathogen detection was compared with the corresponding microbiological reference methods (performed according to the ISO norm). Microarray results revealed high consistency with the reference microbiological data.

Multi-laboratory evaluation of the rapid genoserotyping array (SGSA) for the identification of Salmonella serovars

Salmonella serotyping is an essential first step for identification of isolates associated with disease outbreaks. The Salmonella genoserotyping array (SGSA) is a microarray-based alternative to standard serotyping designed to rapidly identify 57 of the most commonly reported serovars through detection of the genes encoding surface O and H antigens and reporting the corresponding serovar in accordance with the existing White-Kaufmann-Le Minor serotyping scheme. In this study, we evaluated the SGSA at 4 laboratories in 3 countries by testing 1874 isolates from human and non-human sources. The SGSA correctly identified 96.7% of isolates from the target 57 serovars. For the prevalent and clinically important Salmonella serovars Enteritidis and Typhimurium, test specificity and sensitivity were greater than 98% and 99%, respectively. Due to its high-throughput nature, the SGSA is a rapid and cost-effective alternative to standard serotyping for identifying the most prevalent serovars of Salmonella.

Propidium monoazide–quantitative polymerase chain reaction for viable Escherichia coli and Pseudomonas aeruginosa detection from abundant background microflora

Nucleic acid-based techniques represent a promising alternative to cultivation-based microbial water quality assessment methods. However, their application is hampered by their innate inability to differentiate between living and dead organisms. Propidium monoazide (PMA) treatment was proposed as an efficient approach for alleviating this limitation. In this study, we demonstrate the performance of PMA-quantitative polymerase chain reaction (qPCR) for the detection of indicator organisms (Escherichia coli and Pseudomonas aeruginosa) in a background of a highly abundant and complex microflora. Treatment with 10 μM PMA resulted in the complete or significant reduction of the false positive signal arising from the amplification of DNA from dead cells.

Oligonucleotide and DNA Microarrays: Versatile Tools for Rapid Bacterial Diagnostics

The rapid and unambiguous detection and identification of microorganisms, historically a major challenge of clinical microbiology, gained additional importance in the fields of public health and biodefence. These requirements cannot be well addressed by classical culture-based approaches. Therefore, a wide range of molecular approaches has been suggested. Microarrays are molecular tools that can be used for simultaneous identification of microorganisms in clinical and environmental samples. Main advantages of microarrays are high throughput, parallelism and miniaturization of the detection system. Furthermore, they allow for both high specificity and high sensitivity of the detection. Microarrays consist of set of probes immobilized on a solid surface. Even though the first application of the microarrays can be seen as relatively recent (Schena et al. 1995), the technology developed rapidly reaching the milestone of 5,000 published papers in 2004 (Holzman and Kolker 2004). This development encompasses both the successful transfer of various technological aspects as well as the expansion of the application scope. The most important technological elements of custom-made platforms as well as the characteristics of the commercially available formats are reviewed in this chapter. Furthermore, application potential is presented together with considerations about quality control.

Microarray Analysis Reveals the Actual Specificity of Enrichment Media Used for Food Safety Assessment

Microbial diagnostic microarrays are tools for simultaneous detection and identification of microorganisms in food, clinical, and environmental samples. In comparison to classic methods, microarray-based systems have the potential for high throughput, parallelism, and miniaturization. High specificity and high sensitivity of detection have been demonstrated. A microbial diagnostic microarray for the detection of the most relevant bacterial food- and waterborne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labeling of oligonucleotides and the phylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus and/or species level) and sensitive (0.1% relative and 10(4) CFU absolute sensitivity) detection of the target pathogens. In initial challenge studies of the applicability of microarray-based food analysis, we obtained results demonstrating the questionable specificity of standardized culture-dependent microbiological detection methods. Taking into consideration the importance of reliable food safety assessment methods, comprehensive performance assessment is essential. Results demonstrate the potential of this new pathogen diagnostic microarray to evaluate culture-based standard methods in microbiological food analysis.

Effect of different heterotrophic plate count methods on the estimation of the composition of the culturable microbial community

Heterotrophic plate counts (HPC) are routinely determined within the scope of water quality assessment. However, variable HPC methods with different cultivation parameters (i.e., temperature and media type) are applied, which could lead to significant effects in the outcome of the analysis. Therefore the effect of different HPC methods, according to DIN EN ISO 6222 and EPA, on the culturable microbial community composition was investigated by 16S rRNA gene sequence analysis and statistical evaluation was performed. The culturable community composition revealed significant effects assigned to temperature (p < 0.01), while for media type no statistical significance was observed. However, the abundance of certain detected bacteria was affected. Lower temperature (22 °C) showed the abundance of naturally occurring Pseudomonadaceae and Aeromonadaceae, whereas at high temperature (37 °C) numerous Enterobacteriaceae, Citrobacter spp. and Bacilli were identified. The highest biodiversity was detected at lower temperature, especially on R2A medium. These results indicate that different temperatures (low and high) should be included into HPC measurement and selection of media should, ideally, be adjusted to the monitored water source. Accordingly, it can be inferred that the HPC method is more suitable for continuous monitoring of the same water source than for single assessments of a water sample.

Novel tools for environmental virology

Routine monitoring of relevant environmental viruses is of great importance for public health and quality assessment. Even though cell culture (i.e., viral infectivity assay) is still regarded as the golden standard, use of new strategies based on the molecular techniques significantly increased in the past years. Specific and rapid detection are main advantages of this alternative approach. Furthermore, integration of cell culture or propidium monoazide treatment with nucleic acid amplification allows for the differentiation of infectious particles. Additional recently reported approaches for the detection of viruses include, among others, whole transcriptome amplification and cell culture combined with Fourier transform infrared spectroscopy. Noteworthy is also the fact, that regardless of the selected detection method, sample preparation still remains the major bottleneck.

Microbial Diagnostic Microarrays for the Detection and Typing of Food- and Water-Borne (Bacterial) Pathogens

Reliable and sensitive pathogen detection in clinical and environmental (including food and water) samples is of greatest importance for public health. Standard microbiological methods have several limitations and improved alternatives are needed. Most important requirements for reliable analysis include: (i) specificity; (ii) sensitivity; (iii) multiplexing potential; (iv) robustness; (v) speed; (vi) automation potential; and (vii) low cost. Microarray technology can, through its very nature, fulfill many of these requirements directly and the remaining challenges have been tackled. In this review, we attempt to compare performance characteristics of the microbial diagnostic microarrays developed for the detection and typing of food and water pathogens, and discuss limitations, points still to be addressed and issues specific for the analysis of food, water and environmental samples.

Verocytotoxin-producing Escherichia coli in chamois (Rupicapra rupicapra) and cattle in Austria.

We assessed the prevalence of verotoxigenic Escherichia coli (VTEC) in chamois (Rupicapra rupicapra) and livestock grazing on a mountain pasture in Austria during June–August 2009. We detected VTEC throughout the sampling period in high numbers in cattle as well as in chamois, leading to the assumption that the degree of contamination of the environment is high. This is the first report of pathogenic E. coli identified in chamois, implicating chamois as a new potential reservoir of these zoonotic pathogens. Because the study area also serves recreational purposes, there is a risk of humans acquiring infection via direct or indirect contact.