Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

Abstract

Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex 100 system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli and Shigella spp., Bacteroides distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.

Fig. 1

Median fluorescence intensity (MFI) of Luminex probes hybridized against DNA from a variety of bacterial cultures. Results are shown for beads in multiplex format for A) DNA amplified by singleplex or multiplex PCR and hybridized at 55 °C and B) DNA amplified by multiplex PCR containing a single DNA template or a pool of template “positive” and hybridized at 53 °C. “thetamic.” is thetaiotaomicron; “Positive” was a mixture of E. coli, B. distasonis, B. vulgatus, and Ent. Faecalis DNA; “Negative” was a mixture of Enterobacter aerogenes and K. pneumoniae DNA.

Fig. 2

Corrected fluorescence intensity (cMFI) of Luminex probes versus concentration of genomic DNA used in the PCR reaction for sensitivity analysis of A) E. coli B) Ent. faecalis C) B. distasonis and D) B. vulgatus. Column height depicts the mean and bars the range for replicate analysis, which included separate PCR and detection reactions. A positive response is a cMFI value >2.

Fig. 3

The average number of cells of various bacterial types filtered for DNA extraction for river water (n=15), wet sand (n=6), dry sand (n=6), and beach water (n=11) for samples collected between February 2004 and April 2005. Values of %CV ranged from 52% for Bacteroides spp. in creek water to 240% for E. coli in river water.

Fig. 4

Luminex response to DNA extracted from various types of environmental samples. The positive controls are E. coli, B. distasonis, and B. vulgatus; the negative is a no template control. Background fluorescence (F_o) from TE controls containing no PCR product was removed to yield values of median fluorescence intensity (MFI). Column height depicts the mean and bars the range for replicate analysis, which included separate multiplex PCR and detection reactions.

Fig. 5

A) Sensitivity of the Luminex assay designed to target the 23S rRNA gene region of Enterococcus (Entero23S) versus the assay designed for the 16S rRNA gene region of Ent. faecalis (Efaec2) for a serial dilution of Ent. faecalis DNA. Results are reported as cMFI to aid interpretation of the detection limit; positive is cMFI >2. B) Comparison of probe sensitivity for samples of raw sewage (#31) and for seawater collected from Hobie Beach (#59, #73). Column height depicts the mean and bars the range for 2 separate detection reactions from a single multiplex PCR. Results are reported as MFI (F-F_o) to show the intensity of the fluorescence obtained.

Fig. 6

Phylogram of PAUP* neighbor joining tree analysis of 16S rRNA gene sequences (498 positions) obtained by Bacterfor/Unirev 800 amplification of river water followed by shot-gun cloning. The sequence labeled G7 contained a perfect match to the Bfra3 probe. Bootstrap values are shown on internal nodes for values greater than 50, as space allowed; “*” denotes visible nodes with bootstrap values <50.

Fig. 7

A) Phylogram of PAUP* neighbor joining tree analysis of 16S rRNA sequences (444 positions) obtained by Unifor/Unirev800 amplification of bacterial colonies isolated from BBE or BVSA media used to isolate Bacteroides spp. from river and beach water. Bootstrap values are shown on internal nodes for values greater than 50, as space allowed. B) Luminex response for samples included in the phylogram, except for samples 184, 186, and 193, which were not tested. Column height depicts the mean and bars the range for replicate analysis, which included separate multiplex PCR and detection reactions. Groups labeled with the probe names Colinsitu2, Bfra3, and Bdis3 (see Table 2) contained sequence that matched the probe within 0–1 bp. The group labeled Bfra3 also contained sequences with 2 bp mismatches to the Bdis3 probe.