6.4.1 E. coli enumeration (I, II) and confirmation (I)
E. coli was enumerated with the Colilert-18 method (I-II) and the MMPN method (I). A volume of 100 ml sample was filled on 51-well Quanti-tray (IDEXX) and the tray was incubated at (36 ± 2) °C for 18-21 hours. Then, following the manufactur-ers' guidelines, the E. coli MPN count was enumerated based on yellow wells of the tray that also exhibited fluorescence under the UV-light (ISO 9308-2 2012). In the MMPN method, a volume of a 19.2 ml (96 × 200 µl) water sample was pipetted into the 96-well microtiter plate containing Merck product Chromogenic Coliform agar (Bio-Rad Laboratories Inc., France) and the plate was incubated at (44 ± 0.5) °C first for 48 - 51 hours and then for 69–72 hours. The fluorescent wells recorded after the
44
incubation were counted as E. coli and used in generating the MPN count estimate (ISO 9308-3 1998).
In the study I, the false positive rate of Colilert-18 and MMPN methods was in-vestigated by conducting additional confirmation tests. The false-negative rate was only investigated for the Colilert-18 method, which includes the determination of coliform bacteria, other than E. coli (yellow wells that do not exhibit fluorescence).
All E. coli positive (fluorescent) wells on the Colilert tray and MMPN plate and five without fluorescence per the Colilert tray were sub-cultured on the Chromocult Coliform Agar medium (CCA; Merck, Germany) at 36±2 °C overnight for confirma-tion tests.
Again, the isolates were sub-cultured from CCA to tryptone soya agar (TSA, Oxoid Ltd, UK) at 36 ± 2˚C overnight. The oxidase test was conducted for all the colonies. Then, oxidase negative colonies were passed to Fluorocult Lauryl Sulfate Broth (Merck, Germany) tubes containing durham-tube for gas collection at 44 ± 0.5
˚C for 21 ± 3 h. The fluorescence of the Fluorocult tubes was recorded under UV light, the gas formation was studied. The indole test was carried out by using the Kovac’s reagent. The oxidase negative strains that were positive for indole and fluorescence tests were confirmed as E. coli bacteria.
6.4.2 Enterococci enumeration and confirmation (II & III)
ENT were enumerated with the ISO 7899-2 (2000) method (II-III). The volume of water samples filtered for analysis varied from 1 ml to 1000 ml for obtaining around 10-100 presumptive colonies per membrane, according to the ISO 8199 (2005) principle. After filtration, the membrane (GN6, Pall Life Sciences, Michigan, USA) was incubated on the S&B medium (Oxoid Ltd. Basingstoke, Hampshire, England) at 36±2 °C for 44±4 h. All colonies with red, maroon, or pink in colour were considered as presumptive ENT. Then, after counting the presumptive ENT, the membrane was transferred on bile aesculin azide agar medium (BEA, Scharlau, Barcelona, Spain) for incubation at 44±0.5 °C for 2 h. All the presumptive colonies, which changed into black or brown colour on the BEA medium, were confirmed as iENT.
6.4.3 Secondary confirmation of enterococci isolates (II & III)
All, or at least 10, confirmed and 10 unconfirmed colonies per analyzed sample were sub-cultured on non-selective TSA medium (TSA, Oxoid Ltd, UK) and incu-bated at 36±2 °C for two days. The isolates were stored at -75 °C in nutrient broth containing 15 % glycerol. Then, bacterial biomass was transferred into 0.1 ml sterile deionized water and stored at -18 °C for preparing the total genomic DNA. Then, the bacterial suspension was heat-treated at 95 °C for 10 minutes. The partial 16S ribosomal RNA (rRNA) gene sequences were obtained by using universal bacterial primers (Table 6, Ryu et al. 2013). Further, isolates were identified with the
Entero-45 coccus spp. specific qPCR assay targeting the 23S rRNA gene (Ludwig and Schleifer, 2000).
Unique phylogenetic contigs were selected, based on sequence homology with bio-informatics software CD-hits (Li and Godzik, 2006). The representative contigs were aligned with reference sequences collected from the National Center for Bio-technology Information (NCBI) GenBank. Then, the phylogenetic tree was made on Molecular Evolutionary Genetics Analysis software version 6 with 1000 bootstrap values with a muscle sequence aligner (Tamura et al. 2013). The isolates were identi-fied, based on phylogenetic clades and additional in silico confirmation with a clus-ter sequence aligner in MEGA-6 with Enclus-terococcus genus-specific primer Ent1 tar-geting to 16S rRNA sequence. Species-specific primers Faecium1 (E. faecium), Fae-calis1 (E. faecalis) and Casseli1 (E. casseliflavus) were tested in silico (Ryu et al. 2013) (for primer sequences, see Table 6).
46 Table 6. The characteristics of primers and probes. Assay name (Study) Target speciesSequence 5′to 3′Length (bp)Reference Universal bacterial primers used for endpoint PCR Universalbacteria (II, III)Bacteria 8F: AGAGTTTGATCCTGGCTCAG 787R: CGACTACCAGGGTATCTAAT 770Ryuet al. (2013) General faecal indicator markers used for qPCR and RT-qPCR Entero1 (II &III)Enterococcus spp.ECST748F: AGAAATTCCAAACGAACTTG; ENC854R: CAGTGCTCTAC- CTCCATCATT; GPL813TQ: 6FAM- TGGTTCTCTCCGAAATAGCTTTAGGGCTA-TAMRA
92Ludwig & Schleifer (2000) EC23S857 (II)E. coli F: GGTAGAGCACTGTTTtGGCA; R: TGTCTCCCGTGATAACtTTCTC; P: 6FAM-TCATCCCGACTTACCAACCCG-TAMRA 88Chern et al. (2011) GenBac 3 (II)Bacteroidetes spp.GenBactF3: GGGGTTCTGAGAGGAAGGT; GenBactR4: CCGTCATCCTTCACGCTACT; GenBactP2: 6FAM- CAATATTCCTCACTGCTGCCTCCCGTA-TAMRA
129Siefringet al. (2008) Host-specific markers used for MST with qPCR and RT-qPCR HF183 (II)Human- specific Bac- teroidales
HF183-1: ATCATGAGTTCACATGTCCG; BthetR1: CGTAGGAGTTT- GGACCGTGT; BthetP1: 6FAM-CTGAGAGGAAGGTCCCCCACATTGGA- TAMRA 167Haugland et al. (2010) Pig-2-Bac (II)Pig-specific BacteroidalesPig-2-Bac41F: GCATGAATTTAGCTTGCTAAATTTGAT; Pig-2-Bac163Rm: ACCTCATACGGTATTAATCCGC; Pig-2Bac113MGB: 6FAM-TCCACGGGATAGCC-BHQ1
117Mieszkin et al. (2009) Rum-2-Bac (II)Ruminant- specific Bac- teroidales
BacB2-590F: ACAGCCCGCGATTGATACTGGTAA; Bac708Rm: CAATCGGAGTTCTTCGTGAT; BacB2-626P: 6FAM- ATGAGGTGGATGGAATTCGTGGTGT-BHQ1
99Mieszkin et al. (2010) Gull4 (II)Gull-specific Catellicoccus marimammali- um
qGull7F: CTTGCATCGACCTAAAGTTTTGAG; qGull8R: GGT TCT CTG TAT GCG GTA TTA GCA; qGull7P: FAM-ACACGTGGGTAACCTGCCCATCAGA-TAMRA
116Ryuet al. (2012)
47
Table 6 continue... Assay name Target speciesSequence 5′to 3′Length (bp) Reference Viral markers used as an enumeration of human virus Adenoviruses (II)Human ade- novirusesJTVXF: GGACGCCTCGGAGTACCTGAG; JTVXR: ACIGTGGGGTTTCTGAACTTGTT; JTVXP: 6FAM- CTGGTGCAGTTCGCCCGTGCCA-BHQ1 96Jothikumaret al. (2005) Noroviruses (II)GI norovirusesNVGIF: GCYATGTTCCGCTGGATG; NVGIR: CCTTAGACGCCATCATCATT; NVGIP-MGB: VIC- TGGACAGGAGAYCGC-MGBNFQ
95Kauppinen et al. (2014) Noroviruses (II)GII norovirus- esQNIF2d: ATGTTCAGRTGGATGAGRTTCTCWGA; COG2R: TCGAC- GCCATCTTCATTCACA; RING2-TP: 6FAM- TGGGAGGGCGATCGCAATCT-BHQ1
88Loisyet al. (2005), Kageyama et al. (2003) Vibrio markers used for qPCR and RT-qPCR Vcho (III)V. choleraeompW-F: TCAATGATAGCTGGTTCCTCAAC ompW-R: CGATGATAAATACCCAAGGATTGA ompW-Probe:TGGTATGCCAATATTGAAACAACG
126Garrido-Maestu et al., (2014), Garrido- Maestu et al., (2016) Vibrio (III)Vibrio genusF: (567F) GGCGTAAAGCGCATGCAGGT R: (680R) GAAATTCTACCCCCCTCTACAG114Thompson etal. (2004) Markers used forin silico test Ent1 (II)Enterococcus spp.Ent151F: ACACTTGGAAACAGGTGC; Ent376R: TCGGTCAGACTTKCGTCC243Ryuet al. (2013) Faecalis1 (II)Enterococcus faecalisFaecalF: CGCTTCTTTCCTCCCGAGT; FaecalR: GCCATGCGGCATAAACTG; FaecalP: 6FAM-CAATTGGAAAGAGGAGTGGCGGACG-TAMRA 143Ryuet al. (2013) Casseli1 (II)Enterococcus casseliflavusCasselF: GGAGCTTGCTCCACCGAA; CasselR: TTTCTTCCATGCGGAAAATAGT; CasselP: 6FAM- CGAACGGGTGAGTAACACGTGGGTAA-TAMRA
132Ryuet al. (2013) Faecium1 (II)Enterococcus faeciumCiumF: TTCTTTTTCCACCGGAGCTT; CiumR: AACCATGCGGTTTYGATTG; CiumP: 6FAM-AGTAACACGTGGGTAACCTGCCCATCAGA-TAMRA
141Ryuet al. (2013)
48
6.4.4 MS2 coliphage enumeration (III)
The MS2 coliphage was enumerated by using a culture-based method with a dou-ble agar layer technique from 0.5 mL samples (USEPA 2001), using E. coli (ATCC 15597) as a host and was presented as PFU/100 ml from water and as PFU/100 mg (wet weight) from sediment and vegetation samples.