Evaluation of 16S next-generation sequencing of hypervariable region 4 in wastewater samples: An unsuitable approach for bacterial enteric pathogen identification

Recycled wastewater can carry human-infectious microbial pathogens and therefore wastewater treatment strategies must effectively eliminate pathogens before recycled wastewater is used to supplement drinking and agricultural water supplies. This study characterised the bacterial composition of four wastewater treatment plants (WWTPs) (three waste stabilisation ponds and one oxidation ditch WWTP using activated sludge treatment) in Western Australia. The hypervariable region 4 (V4) of the bacterial 16S rRNA (16S) gene was sequenced using next-generation sequencing (NGS) on the Illumina MiSeq platform. Sequences were pre-processed in USEARCH v10.0 and denoised into zero-radius taxonomic units (ZOTUs) with UNOISE3. Taxonomy was assigned to the ZOTUs using QIIME 2 and the Greengenes database and cross-checked with the NCBI nr/nt database. Bacterial composition of all WWTPs and treatment stages (influent, intermediate and effluent) were dominated by Proteobacteria (29.0-87.4%), particularly Betaproteobacteria (9.0-53.5%) and Gammaproteobacteria (8.6-34.6%). Nitrifying bacteria (Nitrospira spp.) were found only in the intermediate and effluent of the oxidation ditch WWTP, and denitrifying and floc-forming bacteria were detected in all WWTPs, particularly from the families Comamonadaceae and Rhodocyclales. Twelve pathogens were assigned taxonomy by the Greengenes database, but comparison of sequences from genera and families known to contain pathogens to the NCBI nr/nt database showed that only three pathogens (Arcobacter venerupis, Laribacter hongkongensis and Neisseria canis) could be identified in the dataset at the V4 region. Importantly, Enterobacteriaceae genera could not be differentiated. Family level taxa assigned by Greengenes database agreed with NCBI nr/nt in most cases, however, BLAST analyses revealed erroneous taxa in Greengenes database. This study highlights the importance of validating taxonomy of NGS sequences with databases such as NCBI nr/nt, and recommends including the V3 region of 16S in future short amplicon NGS studies that aim to identify bacterial enteric pathogens, as this will improve taxonomic resolution of most, but not all, Enterobacteriaceae species

The asymmetric synthesis of 2-benzopyrans and their quinones through intramolecular diastereoselective ring-closure of titanium phenolates of phenolic aldehydes

Treatment of the phenolic aldehyde (α′S,2S )-2-(5′-hydroxy-2′-methoxy-α′-methylbenzyloxy)propanal 15 with titanium tetraisopropoxide followed by ultrasonication led to its completely diastereoselective cyclisation in high yield to afford (1S,3S,4R)-3,4-dihydro-1,3-dimethyl-8-methoxy-2-benzopyran-4,5-diol 18, which was characterised as its more stable 4,5-diacetate 19. The diastereomeric (α′R,2S )-2-(5′-hydroxy-2′-methoxy-α′-methylbenzyloxy)propanal 17 on similar treatment gave rise to a mixture of (1R, 3S, 4R)- and (1R, 3S, 4S )-3,4-dihydro-1,3-dimethyl-8-methoxy-2-benzopyran-4,5-diols 21 and 23, isolated as the 4,5-diacetates 22 and 24, in a ratio of 3∶1. Oxidative dealkylation of the diol 18 with silver(II) oxide afforded (1S,3S,4R)-3,4-dihydro-1,3-dimethyl-4-hydroxy-2-benzopyran-5,8-quinone 2 in high yield, while the epimeric 1R quinone 40 was similarly obtained from diol 21

An asymmetric synthesis of benzo[c]pyrans related to the aphid pigments

The benzo[c]pyran diacetate 5 was prepared in high yield from the phenol 3via an intramolecular cyclization reaction with titanium tetraisopropoxide. Diacetate diastereomers 8 and 9 were also synthesized

Identification of eukaryotic microorganisms with 18S rRNA next-generation sequencing in wastewater treatment plants, with a more targeted NGS approach required for Cryptosporidium detection

While some microbial eukaryotes can improve effluent quality in wastewater treatment plants (WWTPs), eukaryotic waterborne pathogens are a threat to public health. This study aimed to identify Eukarya, particularly faecal pathogens including Cryptosporidium, in different treatment stages (influent, intermediate and effluent) from four WWTPs in Western Australia (WA). Three WWTPs that utilise stabilisation ponds and one WWTP that uses activated sludge (oxidation ditch) treatment technologies were sampled. Eukaryotic 18S rRNA (18S) was targeted in the wastewater samples (n = 26) for next-generation sequencing (NGS), and a mammalian-blocking primer was used to reduce the amplification of mammalian DNA. Overall, bioinformatics analyses revealed 49 eukaryotic phyla in WWTP samples, and three of these phyla contained human intestinal parasites, which were primarily detected in the influent. These human intestinal parasites either had a low percent sequence composition or were not detected in the intermediate and effluent stages and included the amoebozoans Endolimax sp., Entamoeba sp. and Iodamoeba sp., the human pinworm Enterobius vermicularis (Nematoda), and Blastocystis sp. subtypes (Sarcomastigophora). Six Blastocystis subtypes and four Entamoeba species were identified by eukaryotic 18S NGS, however, Cryptosporidium sp. and Giardia sp. were not detected. Real-time polymerase chain reaction (PCR) also failed to detect Giardia, but Cryptosporidium-specific NGS detected Cryptosporidium in all WWTPs, and a total of nine species were identified, including five zoonotic pathogens. Although eukaryotic 18S NGS was able to identify some faecal pathogens, this study has demonstrated that more specific NGS approaches for pathogen detection are more sensitive and should be applied to future wastewater pathogen assessments

Complementary diastereoselectivity in the intermolecular addition of titanium and magnesium naphtholates to asymmetric lactaldehydes

Addition of 7-benzyloxy-4,5-dimethoxy-1-naphthol 3 as its triisopropoxytitanium naphtholate to (2R, 1′R or S )-2-(1′-ethoxyethoxy)propanal 4 afforded solely (1S, 2R, 1″R or S )-1-(7′-benzyloxy-4′,5′-dimethoxy-1′-hydroxy-2′-naphthyl)-2-(1″-ethoxyethoxy)propan-1-ol 5, being the erythro product arising from anti addition. Complementary reaction of the naphthol 3 as its bromomagnesium naphtholate with aldehyde 4 gave rise solely to the alternative (1R, 2R, 1″R or S ) diastereomer 6 . The naphthol 3 was prepared through the completely regioselective addition of 2-methoxyfuran 9 to 5-benzyloxy-3-methoxydehydrobenzene 8. This differentially protected bisalkoxybenzyne was conveniently prepared from vanillin

A metal-mediated diastereoselective synthesis of precursors to the aphid pigment derivatives

Chiral compounds 16 and 17, precursors to the aphid insect pigment derivatives, have been prepared in good to high yield, with complete control of the diastereoselectivity at the newly-created chiral centre C-1, through the use of metal phenolates derived from naphthol 3