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Integrated multivariate analysis with nondetects for the development of human sewage source-tracking tools using bacteriophages of Enterococcus faecalis Bencharong Wangkahad, Skorn Mongkolsuk, and Kwanrawee Sirikanchana Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04714 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 17, 2016
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Environmental Science & Technology
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Integrated multivariate analysis with nondetects for the
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development of human sewage source-tracking tools
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using bacteriophages of Enterococcus faecalis
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Bencharong Wangkahad†, Skorn Mongkolsuk‡,ǁ,§,and Kwanrawee Sirikanchanaǁ,§,*
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†
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for Emerging Bacterial Infections, Faculty of Science, Mahidol University, Bangkok, Thailand 10400;
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ǁ
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Chulabhorn Graduate Institute, Bangkok, Thailand 10210; ‡Department of Biotechnology and Center
Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand 10210; §Center of
Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Thailand 10400.
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ABSTRACT We developed sewage-specific microbial source tracking (MST) tools using enterococci
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bacteriophages and evaluated their performance with univariate and multivariate analyses involving
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data below detection limits. Newly isolated Enterococci faecalis bacterial strains AIM06
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(DSM100702) and SR14 (DSM100701) demonstrated 100% specificity and 90% sensitivity to human
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sewage without detecting 68 animal manure pooled samples of cats, chickens, cows, dogs, ducks, pigs,
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and pigeons. AIM06 and SR14 bacteriophages were present in human sewage at 2–4 orders of
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magnitude. A principal component analysis confirmed the importance of both phages as main water
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quality parameters. The phages presented only in the polluted water, as classified by a cluster analysis,
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and at median concentrations of 1.71 × 102 and 4.27 × 102 PFU/100 ml, respectively, higher than non-
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host specific RYC2056 phages and sewage-specific KS148 phages (p 0.05). The two sewage-specific enterococci strains included
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strain AIM06 from the wastewater of a 100-unit residential building and strain SR14 from a 164-
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inpatient bed hospital. Both AIM06 and SR14 host strains showed clear plaques of approximately 3
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mm in size when incubated under aerobic conditions for 24 h (Figure S2). Both host strains presented
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similar 817 bp DNA sequences in the 16s rRNA gene region. The results from the BLAST program
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showed a 99% identity with E. faecalis at 99% coverage. Biochemical identification confirmed both
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isolates as E. faecalis species. The DNA sequences of strains SR14 and AIM06 were submitted to
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GenBank with accession numbers KX670425 and KX670426, respectively. Enterococcus strains SR14
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and AIM06 were also deposited in the Leibniz Institute DSMZ–German Collection of Microorganisms
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and Cell Cultures with numbers DSM100701 and DSM100702, respectively.
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Bacteriophages of AIM06 and SR14 in human sewage. The detected concentrations of the
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bacteriophages of the AIM06 and SR14 host strains, together with FIB and the phages of RYC2056
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and KS148, in randomly assigned groups of the 10 human sewage samples (i.e., human sewage sets 1
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and 2) are presented as box plots in Figure 1. In both data sets, TC showed the highest concentrations,
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followed by E. coli and intestinal enterococci. The median concentrations of both AIM06 and SR14
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phages were 5.12 × 102 and 3.11 × 103 PFU/100 ml, respectively. The AIM06 host could detect phages
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in almost all 10 water samples, except one septic tank sample that contained 3.40 x 106 CFU/100 ml,
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1.83 × 101 PFU/100 ml, and 0.05). Accordingly, these results highlighted that the new enterococci
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phages can be potentially used to indicate sewage contamination in environmental water.
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Principal component analysis (PCA) of observations in environmental water. PCA was performed
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to extract more information on the major parameters that govern the observed data in environmental
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water samples with potential contamination of sole human sewage source. The first three PCs showed
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eigenvalues of 6.1129, 1.2869, and 1.0550, respectively. These PCs presented significant governance
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of the data because their eigenvalues were greater than 1, which is an average of eigenvalues for a
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correlation matrix.55 The proportions of variance from PC1, 2, and 3 were 0.509, 0.107, and 0.088,
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respectively. The first PC, which constituted the highest variance of 50.9%, was mainly influenced by
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AIM06 phages and SR14 phages, as well as FIBs and BOD, with loadings ranging from 0.309 to 0.379
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(Table S2). Moreover, the small angles among these main parameters, as shown in the biplot (Figure
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2), revealed that the parameters are associated with one another. No outlier was observed, as calculated
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by the Mahalanobis distance (data not shown). This finding suggested that AIM06 and SR14 phages
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tended to be found at high levels in sites where TC, E. coli, and enterococci were high in
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concentrations. This result further reiterated the applicability of both phages to assist in identifying
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human sewage source in contaminated sites where subsequent action would be required to improve the
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current water quality parameters and thus meet water quality standards.
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Cluster analysis for grouping polluted water versus non-polluted water. With the purpose of
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evaluating the new enterococci phages as indicators of sewage-polluted water, an approach to
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categorize the polluted versus non-polluted sample groups needed closer attention. The similarity
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among the physicochemical and microbiological parameters of the water samples was examined with
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cluster analysis (Q mode). All 41 water samples were grouped into two clusters (Figure 3). Cluster 1
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represented water samples mainly from WWTPinf (n=8), canals (n=11), and lakes (n=2), whereas
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cluster 2 represented samples from WWTPeff (n=8), canals (n=2), lakes (n=4), and rivers (n=6). The
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cluster centroids, which were a measure of cluster location, were calculated for all parameters used to
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group the samples into these clusters (Table S3). The results showed that cluster 1 provided higher
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loadings of TC, E. coli, enterococci, RYC2056 phages, KS148 phages, AIM06 phages, SR14 phages,
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BOD, and SS, but lower loadings of pH, DO, and temperature. Therefore, cluster 1 was used to
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represent the relatively polluted water samples, whereas cluster 2 was used to represent the relatively
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non-polluted water samples. Interestingly, the cluster centroids of AIM06 and SR14 phages were
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relatively similar to those of FIBs, BOD, and SS, which indicated that regardless of the use of phage
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levels or FIBs, BOD, and SS parameters, similar classifications between the polluted versus non-
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polluted groups would result. The cluster analysis results again reinforced PCA results on the
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applicability of both phages in helping identify sources in hot-spot sites where water quality
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parameters tend to fail standards.
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Bacteriophages of AIM06 and SR14 and other water quality parameters in polluted and non-
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polluted water. The summary statistics of microbial parameters are presented as box plots (Figure 4)
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and physicochemical parameters as tabulations (Table S1). None of the phages were found in any of
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the non-polluted water samples. In the polluted samples, the distributions of AIM06 phages and SR14
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phages were not significantly different (p>0.05), and their median concentrations were 1.71 × 102 and
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4.27 × 102 PFU/100 ml, respectively. The co-presence of AIM06 phages and SR14 phages was
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observed in 12 out of 14 positive samples. Both phages showed significantly higher concentrations
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than RYC2056 phages and KS148 phages (p
