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

List of primers used in this study.

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

Expression levels of <i>soxR</i>, <i>iscR</i> and their targeted genes in <i>P</i>. <i>aeruginosa</i> strains.

<p>Analysis of the expression levels of genes encoding [2Fe-2S]-containing transcriptional regulators, namely, SoxR and IscR, and their targeted genes, <i>soxR</i>, <i>PA2274</i>, <i>iscR</i> and <i>fdx2</i>, in <i>P</i>. <i>aeruginosa</i> wild type (PAO1::Tn7T), <b>Δ</b><i>nfuA</i> mutant (<b>Δ</b><i>nfuA</i>::Tn7T) and the complemented strain (<b>Δ</b><i>nfuA</i>::NfuA) grown in uninduced and 0.5 mM Paraquat-induced conditions. qRT-PCR was performed as described in the Materials and Methods, and the data are shown as the fold expression relative to the level of the uninduced PAO1 (PAO1::Tn7T).</p

Iron-sulfur cluster-containing protein activities.

<p>(A) Aconitase and succinate dehydrogenase (Sdh) activities in <i>P</i>. <i>aeruginosa</i> PAO1 and <i>fprB</i> mutant harboring empty vector (pBBR), pFprB, or pISC strains under normal growth condition were measured. Cell culture conditions, protein preparation and enzymatic activity assays were performed as described in the experimental procedures. The data shown are means and SD of triple biologically independent replications. The asterisk indicates a statistically significant difference (P < 0.01) compared with the PAO1/pBBR strain and pBBR referred as an empty vector control. (B) Real-time RT-PCR analysis of <i>anr</i>, <i>narG</i>, <i>soxR</i> and PA2274 in indicated <i>P</i>. <i>aeruginosa</i> strains cultivated under normal growth condition. RNA isolation and real-time RT-PCR were performed as described in the experimental procedures. The data shown are means and SD of three independent experiments. The asterisk indicates a statistically significant difference (P < 0.01) compared to those of the PAO1/pBBR strain.</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