Molecular enrichment for detection of S. aureus in recreational waters

The identification of rapid methods for the control of recreational water and of aquatic environments with similar characteristics is necessary to provide adequate levels of health safety for users. Molecular techniques have been proposed in recent years as a viable alternative to traditional microbiological methods, as they offer various advantages and are less time consuming than traditional tests. An innovative protocol based on molecular enrichment that allows the identification of low concentrations of Staphylococcus aureus in recreational water has been developed. The method is based on the specific amplification of prokaryotic genomic DNA by the usage of universal primers for 23S rDNA; subsequently, a second amplification step is performed with specific real-time polymerase chain reaction (PCR) primers and probe. This approach shows sensitivity levels similar to those observed with microbiological tests, with the additional benefits of the specificity typical of nucleic acids techniques. This methodology is easily applicable also to other microbiological parameters, representing an important milestone in hygiene monitoring by the detection of specific pollution indicators. doi: 10.2166/wst.2012.435 F. Valeriani S. Giampaoli L. Buggiotti G. Gianfranceschi V. Romano Spica (corresponding author) University of Rome ‘Foro Italico’, P.zza L. De Bosis, 6, 00135 Roma, Italy E-mail: vincenzo.romanospica@uniroma4.it


INTRODUCTION
In the last two decades a strong increase in sport activity has been reported in many western countries and a positive trend was also registered in swimming and physical activity in swimming pools (Vandelanotte et al. ). The increase of users in these recreational environments needs specific procedures for monitoring and maintaining hygiene and water quality (World Health Organization ). Microbial contamination is indeed one of the major health risks for swimming pool users and operators, due to the fact that pathogens can find favorable conditions for proliferation and infection. Main sources of this contamination are environment (especially for outdoor facilities) and swimmers. The bathers are largely responsible for the corruption of the water. It has been estimated that each swimmer can release into the pool a significant quantity of urine and sweat, with large amounts of microorganisms (Sato et al. ; De Laat et al. ). Moreover, the release of bacteria may be from the peeling of the skin, accidental fecal release or residual fecal material on swimmers' bodies (World Health Organization ; Elmir et al. ).
The microbiological monitoring of water quality is an essential tool for maintenance of adequate hygienic conditions. This is done by using indicator microorganisms, which are easy to enumerate and are useful to measure the potential presence of hard-to-detect target pathogenic organisms. Staphylococcus aureus is an indicator of anthropogenic contamination, and in aquatic environments its presence has been related to the number of bathers; it is also considered a risk indicator for skin, eye and ear diseases (Gabutti et al. ). Different studies show that human body continuously releases large quantity of coagulasepositive staphylococci, in particular S. aureus (Alico & Dragonjac ). For these reasons, this microorganism can be suggested as an indicator of overcrowding in swimming pools and of the efficiency of recirculation and disinfection systems (AFSSET ).
Procedures for the detection of Staphylococcus aureus in water samples are based on the membrane filtration (MF) microbiological method. The past editions of Standard Methods for the Examination of Water and Wastewater have included two methods for the enumeration total of staphylococci or S. aureus in water samples. Unfortunately, these methods do not respond simultaneously to the requirements of specificity, selectivity and speed of detection. In particular, the simple identification of S. aureus by cultural methods and coagulase test can lead to incorrect classification, mainly due to the presence of different species of coagulase-positive staphylococci (cf Blaiotta et al. ), but also to the presence of S. aureus coagulase negative (Vandenesch et al. ). These points are well addressed by molecular approaches and in the last few years several authors proposed interesting protocols applied to the detection of indicator microorganisms in environmental water samples (Noble & Weisberg ; Brandi et al. ). These techniques are less time consuming and provide highly specific signals due to the fact that they are based on the identification of species-specific nucleotide sequences (Girones et al. ). In particular, real-time polymerase chain reaction (PCR) protocols allow the detection and quantization of bacterial species in an accurate and fast mode (Guillemet et al. ). A recent study showed that in less than 5 h without the need of culture enrichment, two Gram-negative bacterial species (Escherichia coli and Shigella) can be detected also at very low concentration (1 cell in 100 ml of water) by real-time PCR performed after a molecular enrichment step based on an isothermal procedure that amplifies genomic DNA (Maheux et al. ).
In the present work a different protocol, still built on the principle of a molecular enrichment, but based on an alternative strategy with specificity for prokaryotic DNA sequences, has been utilized in the detection and quantification of S. aureus contamination in recreational waters. The new method has been compared with real-time PCR without molecular enrichment and classical microbiological tests. This is the first time that a PCR approach has been suggested for the identification of S. aureus in recreational waters.

MATERIALS AND METHOD
Bacterial strains, culture and DNA preparation The S. aureus strain used in the study was purchased from the American Type Culture Collection (ATCC). S. aureus was grown at 37 W C in TSB (Tryptic Soy Broth, Oxoid, Germany) for 12 h before DNA extraction (GenElute™ Bacterial Genomic DNA Kit, Sigma Aldrich, USA). DNA from other bacteria species (E. coli, Enterococcus faecalis, Pseudomonas aeruginosa, Lactobacillus acidophilus, Streptococcus agalactiae, Streptococcus salivarius, Legionella pneumophila serogroup 1, Legionella pneumophila serogroup 10, Lactobacillus crispatus, Lactobacillus gasseri) were similarly extracted.

DNA sequence analysis and design of the universal and specific primers and probe
Sequence alignments of 23S rDNA of several Eubacteria were performed by using MEGA software, version 5.0 (Tamura et al. ). In particular the following species were analyzed: Staphyloccocus aureus, Bacillus cereus, Listeria grayi, Listeria monocytogenes, Enterococcus faecalis, Streptococcus parasanguinis, Streptococcus downei, Lactobacillus crispatus, Clostridium difficile, Escherichia coli, Salmonella enterica, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella longbeachae. Primer pairs and probes were designed using web-based Primer3 (v. 0.4.0) program and blasted against the other available sequences in GenBank by using BLAST network service. Moreover, gradient PCR of the universal primers was performed to determine the optimum annealing temperature (range between 40 and 68 W C).

Detection of S. aureus in water samples
Several water samples with S. aureus titers ranging from 10 1 to 10 3 cfu/ml were prepared for tests. Aliquots of 5 ml were analyzed in duplicate by using both microbiological and molecular methods. The microbiological analysis was conducted by membrane filtration, using nitrocellulose filters (0.45 μm pore size) according to the Standard Methods (APAT-IRSA CNR ). The membrane was transferred aseptically in Baird Parker agar (tryptone 10.0 g/l, 'Lab-Lemco' powder 5.0 g/l, yeast extract 1.0 g/l, sodium pyruvate 10.0 g/l, glycine 12.0 g/l, lithium chloride 5.0 g/l, agar 20.0 g/l, pH 6.8 ± 0.2 at 25 W C, completed with 50 ml egg yolk tellurite emulsion equivalent to 3 ml of 3.5% potassium tellurite, Oxoid) and incubated at 37 ± 1 W C for 40 to 48 h.
Gray-black shiny convex colonies with narrow white entire margin surrounded by zone of clearing (coagulase reaction) were identified as S. aureus. In the molecular analysis, the water aliquots were centrifuged at 4,000 × g for 10 min, bacterial pellet was resuspended in 200 μl of lysis solution buffer for Gram-positive bacteria supplemented with lysozyme (Sigma Aldrich) and transferred in 1.5 ml vial. Glass beads were then added, the pellet was homogenized by pestle and samples were incubated at 37 W C for 30 min. The extraction was then carried out according to the manual of the GenElute™ Bacterial Genomic DNA Kit (Sigma Aldrich). Final elution was performed in 60 μl. Four microliters were analyzed according the procedure described in the following section.

PCR and real-time conditions
PCR was carried out in 25 μl reaction mixture consisting of 1 × Taq master mix (Promega, USA), 1 μM of forward and reverse universal primers (

Standard curve for real-time quantification
Serial 10-fold dilutions were performed after photometric determination of the DNA concentration of S. aureus. Four microliter aliquots of each dilution were used for PCR and real-time PCR (in triplicate) to generate the standard curve.

RESULTS AND DISCUSSION
DNA sequence analysis of 15 Eubacteria allowed the identification of two primers (forward and reverse) annealing on all the genomes considered, generating an amplicon of approximately 1.5 kb (Table 1). In parallel, a set of oligonucleotides for real-time PCR (forward and reverse primers, FAM/quencher probe) was identified annealing specifically on the S. aureus sequence (Figure 1). The specificity of these oligonucleotides was also investigated with an alignment of nucleotide sequences of other species of the genus Staphylococcus (S. intermedius, S. pseudointermedius, S. haemolyticus, S. epidermidis, S. capitis): the consensus sequence obtained showed several points of mismatch, in particular in the annealing regions of the forward primer and of the labeled probe, underlining a theoretical specificity of these oligonucleotides for S. aureus genome (data not shown). The universal primers were used in a PCR (molecular enrichment step) of only 12 cycles, in order to avoid to reaching the reaction plateau. In fact, an important consequence of reaching plateau is the increase of nonspecific products (due to mispriming events), and amplification bias that can strongly affect the representativeness of each molecule originally present in the template sample ( Jung et al. ; Hawkins et al. ; Crotty et al. ).
Part of the molecular enrichment product (20% of the reaction) was then amplified in the real-time step. The choice to not use all the product in the next step of the assay, is mainly due to possible interferences due to different buffer compositions. The specificity of forward/reverse  primers and FAM/quencher probe for S. aureus was previously empirically verified on genomes from different bacterial species: a nonspecific signal was detectable at 36 C T only with L. gasseri and E. faecalis genomic DNA at a concentration of >100 ng per reaction. The nonspecific amplification can be explained by the elevated concentration of template DNA: in fact, the same genomic samples were negative when amplified at lower concentrations (<10 ng per reaction).
The identification of 23S gene as a target for this approach is based on the consideration that rRNA genes are highly conserved, allowing the design of universal primers annealing on genomes of bacteria belonging to different phyla. At the same time it is possible to identify more variable regions for the discrimination between close species, as well documented for lactic acid bacteria (Mohania et al. ).
In order to test sensitivity and linearity limits of the proposed approach, serial 10-fold dilutions of S. aureus genomic DNA were analyzed in triplicate, both with and without the molecular enrichment step. The results showed a constant gain of sensitivity in the molecular enrichment protocol, quantified in approximately 7 C T , for reaction starting from 10 fg-10 pg of template DNA (Figure 2). The two sets of data were characterized by a correlation coefficient close to 1 (R 2 ¼ 0.99). This positive observation sustains the usage of this innovative protocol for the quantization of S. aureus in water samples, as observed in other studies utilizing real-time PCR for detection of bacteria (Shannon et al. ).
Moreover, the lower limit of detection was of 10 fg of DNA, equivalent to the genomic contents of 2-3 cells of S. aureus (Francois et al. ). This level of sensitivity is in line with several national laws that require detection of less than 10 cells per liter (cf. Italian Republic ).
In order to verify the reliability of the method on environmental samples, six water samples with S. aureus titers ranging from 10 1 to 10 3 cfu/ml were assayed. Each sample was microbiologically tested for the accurate detection of the number of S. aureus cells presents in 5 ml. Subsequently another aliquot of 5 ml has been processed for DNA extraction and PCR real-time analysis (with or without molecular enrichment). The results reported in Table 2 confirm the increment of sensitivity after molecular enrichment. In addition, considering the data reported in the plot in Figure 2 as standard curves, it is possible to estimate the number of genomes detected by the amplification reaction: the data obtained are comparable with the microbiological quantification.

CONCLUSION
In conclusion, the method outlined here can be adopted for the estimation of S. aureus cells in water samples, with significantly reduced time to results when compared with standard microbiological tests. The technical approach proposed is not a conclusive validated method but a research output that clearly needs additional developments and comparative tests. The choice of universal oligonucleotides for the molecular enrichment step, is a key element for future expansion of the assay. An appropriate selection of primers/probes combination can lead to the assembling of multiplex real-time reactions that, starting from the same molecularly enriched samples, easily detect more bacterial species. This possible improvement clearly reduces the cost/benefits ratio.
On the other hand, molecular tests maintain a well known limit, due to the fact that they cannot discriminate between cultivable and noncultivable cells (including injured or dead bacteria). In this situation, it is possible to have an overestimation when compared with classical methods. For this reason many concerns have been raised for the application of nucleic acid techniques to drinking water analysis. While further comparison studies will be useful in determining this limit, in the case of S. aureus as indicator of overcrowding in swimming pools, the method outlined in this paper could be important, because it is able to correlate the total number of cells with the amount of bathers who access the facility.
Table 2 | Analysis of six environmental water samples characterized by different titer of S. aureus. 'MF': S. aureus microbiological titer determined by membrane filtration method. 'No. of genomes': estimated number of genomes present in 4 μl of extracted DNA and amplified by PCR. 'C T ': cycle threshold values with or without molecular enrichment. 'Genomes detected by amplification': extrapolation of the number of genomes detected by real-time PCR after molecular enrichment, considering the data reported in the plot in Figure 2