Integrated Multivariate Analysis with Nondetects for the Development of Human Sewage Source-Tracking Tools Using Bacteriophages of <i>Enterococcus faecalis</i>

We developed sewage-specific microbial source tracking (MST) tools using enterococci bacteriophages and evaluated their performance with univariate and multivariate analyses involving data below detection limits. Newly isolated <i>Enterococci faecalis</i> bacterial strains AIM06 (DSM100702) and SR14 (DSM100701) demonstrated 100% specificity and 90% sensitivity to human sewage without detecting 68 animal manure pooled samples of cats, chickens, cows, dogs, ducks, pigs, and pigeons. AIM06 and SR14 bacteriophages were present in human sewage at 2–4 orders of magnitude. A principal component analysis confirmed the importance of both phages as main water quality parameters. The phages presented only in the polluted water, as classified by a cluster analysis, and at median concentrations of 1.71 × 10² and 4.27 × 10² PFU/100 mL, respectively, higher than nonhost specific RYC2056 phages and sewage-specific KS148 phages (<i>p</i> < 0.05). Interestingly, AIM06 and SR14 phages exhibited significant correlations with each other and with total coliforms, <i>E. coli</i>, enterococci, and biochemical oxygen demand (Kendall’s tau = 0.348 to 0.605, <i>p</i> < 0.05), a result supporting their roles as water quality indicators. This research demonstrates the multiregional applicability of enterococci hosts in MST application and highlights the significance of multivariate analysis with nondetects in evaluating the performance of new MST host strains

List of primers

<p>List of primers</p

Gel mobility shift assays and DNase I footprinting of EstR bound to the <i>estR-estC</i> promoters.

<p>A, Gel mobility shift assays of the [³²P]-labeled 314-bp DNA fragment spanning the <i>estR</i> and <i>estC</i> promoters with purified EstR protein (0–125 nM) were performed. The complexes were separated on native PAGE. The specificity of the binding was validated by adding the cold DNA fragment (CD) to the binding mixture or adding bovine serum albumin (BSA) to the reaction instead of the EstR protein. CHP represents the bound complexes treated with 1 mM CHP. B and F indicate bound and free probes, respectively. B, DNase I protection assays were performed with the [³²P]- labeled <i>estR-estC</i> promoter fragment and purified EstR protein at the indicated concentrations. The digested and protected DNA fragments were separated on 8% denaturing DNA sequencing gels beside the sequence ladders (G, A, T, C) generated from pGEM-3Zf (+) using the pUC/M13 forward primer. The numbers on the left side are the size in base pairs of the bands. C, The depicted sequence shows the <i>estR-estC</i> promoter region. +1 and Met indicate the transcription and translation start sites. The -10 and -35 regions are underlined. RBS represents the ribosome binding site. The sequences corresponding to the sites of OI and OII protection are shaded. Arrows indicate palindromic sequences. Small letters above the sequence line represent the putative EstR binding box derived from site OI protection. Identical nucleotides are marked by asterisks.</p

IscR-regulated <i>nfuA</i> expression and <i>nfuA</i> promoter analysis.

<p>(A) IscR-regulated <i>nfuA</i> expression. RNA samples were isolated from uninduced and 0.5 mM plumbagin (PB)-induced cultures of the indicated <i>P</i>. <i>aeruginosa</i> strains. qRT-PCR using primers BT2841 and BT2860 for monitoring <i>nfuA</i> expression and performed as described in the Materials and Methods. Relative expression (fold) is defined as the changes in the <i>nfuA</i> expression levels across multiple samples relative to the level of the uninduced culture of PAO1. The data are presented as the means ± SD from three independent experiments. (B) The primer extension assay was performed using ³²P-labeled primer BT3577 and RNA extracted from <i>P</i>. <i>aeruginosa</i> PAO1 and Δ<i>iscR</i> grown under uninduced (UN) and 0.25 mM PB-induced conditions. G, A, T, and C represent the DNA ladder sequence prepared using ³²P-labeled primer BT3577 and plasmid pP_<i>nfuA</i> as the template. The arrowhead indicates the transcription start site (+1). The -10 and -35 elements are in italic type. The consensus sequence of the Type-I <i>E</i>. <i>coli</i> IscR-binding site is aligned above the corresponding underlined sequence, and the homologous bases are marked with asterisks. The mutated IscR-binding site on the <i>nfuA</i> promoter was aligned below the underlined sequence line. The putative ribosome-binding site (RBS) is indicated in bold type. (C) The electrophoretic mobility shift assay was performed using ³²P-labeled native or mutagenized <i>nfuA</i> promoter fragments and increasing concentrations of purified IscR. CC and HD represent an addition of 1 μg unlabeled <i>nfuA</i> promoter and 2.5 μg of heterologous DNA (pUC18 plasmid), respectively, to the binding reaction mixtures containing 3.0 μM IscR. F and B indicate free and bound probes, respectively.</p

Determination of the resistance levels against oxidative and metal stresses in <i>P</i>. <i>aeruginosa</i> strains.

<p>The resistance levels of PAO1::Tn7T, Δ<i>nfuA</i>::Tn7T and the Δ<i>nfuA</i>::NfuA mutant strains expressing transposed wild-type NfuA or mutated NfuA (NfuA_C152S, NfuA_C155S, and NfuA_C43,47S) against substances were determined using plate sensitivity assays on plates containing oxidants (A) i.e., 250 μM Paraquat, 1 mM Plumbagin, 0.5 mM H₂O₂, 1.6 mM CuOOH, and 0.045% NaOCl, and metals (B) i.e., 1.2 mM 2,2’-dipyridyl, 4.2 mM CuCl₂, 0.8 mM CdCl₂, 0.5 mM CoCl₂, and 5 mM MgCl₂. Survival (%) was defined as the percentage of colony-forming units (CFU) on plates containing oxidants over the number of CFU on plates without oxidants. The data shown are the means ± SD from three independent experiments. The asterisk indicates statistical significance (paired <i>t</i>-test, <i>p</i> < 0.05) compared with PAO1 treated with the same condition.</p

Multiple alignments of Atu5211 (EstR) and Atu5212 (EstC).

<p>A, The deduced amino acid sequence of Atu5211 (EstR) was aligned with OhrR from <i>Xanthomonas campestris</i> pv. phaseoli (OhrR_Xcp), <i>A</i>. <i>tumefaciens</i> (OhrR_Atu) and <i>Bacillus subtilis</i> (OhrR_Bsu). Cysteine residues are marked by arrow heads. B, A phylogenetic tree constructed from the amino acid sequences of transcriptional regulators belonging to MarR super family. EstR_Atu, <i>A</i>. <i>tumefaciens</i> EstR; OhrR_Xcp, <i>Xanthomonas campestris</i> pv. phaseoli OhrR (AAK62673); OhrR_Bsu, <i>Bacillus subtilis</i> OhrR (NP_389198); OhrR_Sco, <i>Streptomyces coelicolor</i> OhrR (CAB87337); OhrR_Atu, <i>Agrobacterium tumefaciens</i> OhrR (AAK86653); ExpG_Sme, <i>Sinorhizobium meliloti</i> ExpG (CAB01941); MepR_Sau, <i>Staphylococcus aureus</i> MepR (AAU95767), GraR_Rhi, <i>Rhizobium sp</i> GraR (BAF44528); TcaR_Sau, <i>Staphylococcus aureus</i> TcaR (AAG23887); ScoC_Bsu, <i>Bacillus subtilis</i> ScoC (NP_388880); PecS_Ech, <i>Erwinia chrysanthemi</i> PecS (CAA52427); HucR_Dra, <i>Deinococcus radiodurans</i> HucR (NP_294883); MexR_Pae, <i>Pseudomonas aeruginosa</i> MexR (AAO40258); MarR_Eco, <i>Escherichia coli</i> MarR (ABE11597); SlyA_Sty, <i>Salmonella typhimurium</i> SlyA (NP_460407); SlyA_Efa, <i>Enterococcus faecalis</i> SlyA (NP_816617); MobR_Cte, <i>Comamonas testosterone</i> MobR (BAF34929); BadR_Rpa, <i>Rhodopseudomonas palustris</i> BadR (NP_946008); MoaI_Kae, <i>Klebsiella aerogenes</i> MoaI (BAA09790); and HpcR_Eco, <i>Escherichia coli</i> HpcR (AAB25801). C, The deduced amino acid sequence of Atu5212 (EstC) was aligned with EstC from <i>Burkholderia gladioli</i> EstC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168791#pone.0168791.ref038" target="_blank">38</a>]. The Ser-His-Asp catalytic triad residues are indicated by asterisks. The bar above the sequences represents the esterase-lipase active domain. Alignment was performed using ClustalW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168791#pone.0168791.ref032" target="_blank">32</a>].</p

List of primers used in this study.

<p>List of primers used in this study.</p

Gene organization of the <i>estC</i> locus in various bacteria.

<p>The arrow indicates the orientation of the transcription. The number in the arrows represents percentage of identity of each sequence to the corresponding <i>A</i>. <i>tumefaciens</i> genes. The asterisk indicates a truncated gene. <i>scd</i>, short chain dehydrogenase; <i>zdh</i>, zinc dehydrogenase; <i>cdh</i>, choline dehydrogenase; and <i>gst</i>, glutathione S-transferase.</p

Expression analysis of <i>estC</i>.

<p>A, Northern analysis of <i>estC</i> in <i>A</i>. <i>tumefaciens</i> NTL4 wild-type and the <i>estR</i> mutant was performed using the [³²P]-labeled <i>estC</i> probe. Equal amounts of RNA (10 μg) prepared from bacterial cultures grown under uninduced (UN) conditions or induced with 1 mM CHP, 250 μM H₂O₂, 200 μM MD or 250 μM plumbagin (PB) were used in the experiments. The 23S rRNA used as the amount control is shown below the hybridized autoradiograph. B, Esterase activity in <i>A</i>. <i>tumefaciens</i> NTL4 and the <i>estR</i> mutant strains was assayed in crude lysates prepared from cultures cultivated as described in A. BHP, culture induced with 500 μM <i>t</i>-butyl hydroperoxide. C and D show the <i>estC</i> and <i>estR</i> promoter activities <i>in vivo</i>, respectively, in NTL4, the <i>estR</i> mutant, and the complemented strain (<i>estR</i>/pEstR) carrying the plasmid either pP_estC that contains the <i>estC</i> promoter-<i>lacZ</i> fusion (C) or pP_estR that contains the <i>estR</i> promoter-<i>lacZ</i> fusion (D). The activities were monitored in samples of lysates prepared from uninduced (UN) and oxidant-induced cultures (1 mM CHP, 25 μM LHP, 500 μM BHP, 250 μM H₂O₂, and 250 μM MD). The β-galactosidase activity is expressed in international units. pEstR means harboring the <i>estR</i> expression plasmid. Asterisks indicate a significant difference (<i>P</i> < 0.05) from the uninduced control.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p