Pollutant profile complexity governs wastewater removal of recalcitrant pharmaceuticals.

Organic pollutants are an increasing threat for wildlife and humans. Managing their removal is however complicated by the difficulties in predicting degradation rates. In this work, we demonstrate that the complexity of the pollutant profile, the set of co-existing contaminants, is a major driver of biodegradation in wastewater. We built representative assemblages out of one to five common pharmaceuticals (caffeine, atenolol, paracetamol, ibuprofen, and enalapril) selected along a gradient of biodegradability. We followed their individual removal by wastewater microbial communities. The presence of multichemical background pollution was essential for the removal of recalcitrant molecules such as ibuprofen. High-order interactions between multiple pollutants drove removal efficiency. We explain these interactions by shifts in the microbiome, with degradable molecules such as paracetamol enriching species and pathways involved in the removal of several organic pollutants. We conclude that pollutants should be treated as part of a complex system, with emerging pollutants potentially showing cascading effects and offering leverage to promote bioremediation.

Living with sulfonamides: a diverse range of mechanisms observed in bacteria.

Sulfonamides are the oldest class of synthetic antibiotics still in use in clinical and veterinary settings. The intensive utilization of sulfonamides has been leading to the widespread contamination of the environment with these xenobiotic compounds. Consequently, in addition to pathogens and commensals, also bacteria inhabiting a wide diversity of environmental compartments have been in contact with sulfonamides for almost 90 years. This review aims at giving an overview of the effect of sulfonamides on bacterial cells, including the strategies used by bacteria to cope with these bacteriostatic agents. These include mechanisms of antibiotic resistance, co-metabolic transformation, and partial or total mineralization of sulfonamides. Possible implications of these mechanisms on the ecosystems and dissemination of antibiotic resistance are also discussed. KEY POINTS: • Sulfonamides are widespread xenobiotic pollutants; • Target alteration is the main sulfonamide resistance mechanism observed in bacteria; • Sulfonamides can be modified, degraded, or used as nutrients by some bacteria.

Subsistence and complexity of antimicrobial resistance on a community-wide level.

There are a multitude of resistance strategies that microbes can apply to avoid inhibition by antimicrobials. One of these strategies is the enzymatic modification of the antibiotic, in a process generally termed inactivation. Furthermore, some microorganisms may not be limited to the mere inactivation of the antimicrobial compounds. They can continue by further enzymatic degradation of the compounds' carbon backbone, taking nutritional and energetic advantage of the former antibiotic. This driving force to harness an additional food source in a complex environment adds another level of complexity to the reasonably well-understood process of antibiotic resistance proliferation on a single cell level: It brings bioprotection into play at the level of microbial community. Despite the possible implications of a resistant community in a host and a lurking antibiotic failure, knowledge of degradation pathways of antibiotics and their connections is scarce. Currently, it is limited to only a few families of antibiotics (e.g. ß-lactams and sulfonamides). In this article, we discuss the fluctuating nature of the relationship between antibiotic resistance and the biodegradation of antibiotics. This distinction mainly depends on the genetic background of the microbe, as general resistance genes can be recruited to function in a biodegradation pathway.

Biodegradation of antibiotics: The new resistance determinants - part I.

History shows that the discovery of, and the resistance to, antibiotics go hand in hand. While knowledge of resistance mechanisms, their impact and distribution is vast, over the years, the topic of antibiotic degradation has often been overlooked and regarded as being discrete from the research on resistance. As a result, understanding of the degradation of antibiotics and the impact of antibiotic degraders on the environment and human health are, for most classes, neither thoroughly documented nor understood. Current information on the biodegradation of antibiotics is described in two review articles. This first part focuses on sulfonamides, trimethoprim, aminoglycosides, amphenicols and tetracyclines. Detailed metabolic and molecular aspects as well as the role of the degraders in natural microbial communities are discussed. An integrated analysis of the accumulated data indicates that appreciation of the interplay between resistance and degradation is quite fragmented, and closing this gap will require novel experimental approaches.

Biodegradation of antibiotics: The new resistance determinants - part II.

Antibiotic residues are widespread in the environment and their presence is known to contribute to the propagation of antibiotic resistance. Nevertheless, knowledge on processes involved in their degradation is scattered. This second part of a two part review aims at compiling knowledge on the (bio-) degradation of antibiotics, focusing on ß-lactams, macrolides, quinolones and ionophores, as well as some less common classes. Detailed metabolic and molecular aspects are discussed, as well as the role of antibiotic degraders in natural microbial communities. This exercise led to the conclusion that among the classes analyzed, the majority of antibiotics are prone to microbial cleavage or transformation.

Comparative genomics reveals a novel genetic organization of the sad cluster in the sulfonamide-degrader 'Candidatus Leucobacter sulfamidivorax' strain GP.

BACKGROUND: Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified. RESULTS: Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%). In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp. ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species. The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively). Phylogenetic analysis of core genes between strain GP and Leucobacter spp. corroborated these findings. Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth. However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype. The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase. Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements. CONCLUSIONS: Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP. Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further. This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP. Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, 'Candidatus Leucobacter sulfamidivorax'.

Biodegradation of sulfamethoxazole by a bacterial consortium of Achromobacter denitrificans PR1 and Leucobacter sp. GP.

In the last decade, biological degradation and mineralization of antibiotics have been increasingly reported feats of environmental bacteria. The most extensively described example is that of sulfonamides that can be degraded by several members of Actinobacteria and Proteobacteria. Previously, we reported sulfamethoxazole (SMX) degradation and partial mineralization by Achromobacter denitrificans strain PR1, isolated from activated sludge. However, further studies revealed an apparent instability of this metabolic trait in this strain. Here, we investigated this instability and describe the finding of a low-abundance and slow-growing actinobacterium, thriving only in co-culture with strain PR1. This organism, named GP, shared highest 16S rRNA gene sequence similarity (94.6-96.9%) with the type strains of validly described species of the genus Leucobacter. This microbial consortium was found to harbor a homolog to the sulfonamide monooxygenase gene (sadA) also found in other sulfonamide-degrading bacteria. This gene is overexpressed in the presence of the antibiotic, and evidence suggests that it codes for a group D flavin monooxygenase responsible for the ipso-hydroxylation of SMX. Additional side reactions were also detected comprising an NIH shift and a Baeyer-Villiger rearrangement, which indicate an inefficient biological transformation of these antibiotics in the environment. This work contributes to further our knowledge in the degradation of this ubiquitous micropollutant by environmental bacteria.

Biotransformation of Sulfonamide Antibiotics in Activated Sludge: The Formation of Pterin-Conjugates Leads to Sustained Risk.

The presence of antibiotics in treated wastewater and consequently in surface and groundwater resources raises concerns about the formation and spread of antibiotic resistance. Improving the removal of antibiotics during wastewater treatment therefore is a prime objective of environmental engineering. Here we obtained a detailed picture of the fate of sulfonamide antibiotics during activated sludge treatment using a combination of analytical methods. We show that pterin-sulfonamide conjugates, which are formed when sulfonamides interact with their target enzyme to inhibit folic acid synthesis, represent a major biotransformation route for sulfonamides in laboratory batch experiments with activated sludge. The same major conjugates were also present in the effluents of nine Swiss wastewater treatment plants. The demonstration of this biotransformation route, which is related to bacterial growth, helps explain seemingly contradictory views on optimal conditions for sulfonamide removal. More importantly, since pterin-sulfonamide conjugates show retained antibiotic activity, our findings suggest that risk from exposure to sulfonamide antibiotics may be less reduced during wastewater treatment than previously assumed. Our results thus further emphasize the inadequacy of focusing on parent compound removal and the importance of investigating biotransformation pathways and removal of bioactivity to properly assess contaminant removal in both engineered and natural systems.

FMNH2-dependent monooxygenases initiate catabolism of sulfonamides in Microbacterium sp. strain BR1 subsisting on sulfonamide antibiotics.

We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.

Complete Genome Sequence of Achromobacter denitrificans PR1.

Achromobacter denitrificans strain PR1 was isolated from an enrichment culture able to use sulfamethoxazole as an energy source. Here, we describe the complete genome of this strain sequenced by Illumina MiSeq and Oxford Nanopore MinION.

The crystal structures of native hydroquinone 1,2-dioxygenase from Sphingomonas sp. TTNP3 and of substrate and inhibitor complexes.

The crystal structure of hydroquinone 1,2-dioxygenase, a Fe(II) ring cleaving dioxygenase from Sphingomonas sp. strain TTNP3, which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been solved by Molecular Replacement, using the coordinates of PnpCD from Pseudomonas sp. strain WBC-3. The enzyme is a heterotetramer, constituted of two subunits α and two ß of 19 and 38kDa, respectively. Both the two subunits fold as a cupin, but that of the small α subunit lacks a competent metal binding pocket. Two tetramers are present in the asymmetric unit. Each of the four ß subunits in the asymmetric unit binds one Fe(II) ion. The iron ion in each ß subunit is coordinated to three protein residues, His258, Glu264, and His305 and a water molecule. The crystal structures of the complexes with the substrate methylhydroquinone, obtained under anaerobic conditions, and with the inhibitors 4-hydroxybenzoate and 4-nitrophenol were also solved. The structures of the native enzyme and of the complexes present significant differences in the active site region compared to PnpCD, the other hydroquinone 1,2-dioxygenase of known structure, and in particular they show a different coordination at the metal center.

Arsenic Mobilization from Historically Contaminated Mining Soils in a Continuously Operated Bioreactor: Implications for Risk Assessment.

Concentrations of soil arsenic (As) in the vicinity of the former Zloty Stok gold mine (Lower Silesia, southwest Poland) exceed 1000 µg g(-1) in the area, posing an inherent threat to neighboring bodies of water. This study investigated continuous As mobilization under reducing conditions for more than 3 months. In particular, the capacity of autochthonic microflora that live on natural organic matter as the sole carbon/electron source for mobilizing As was assessed. A biphasic mobilization of As was observed. In the first two months, As mobilization was mainly conferred by Mn dissolution despite the prevalence of Fe (0.1 wt % vs 5.4 for Mn and Fe, respectively) as indicated by multiple regression analysis. Thereafter, the sudden increase in aqueous As[III] (up to 2400 µg L(-1)) was attributed to an almost quintupling of the autochthonic dissimilatory As-reducing community (quantitative polymerase chain reaction). The aqueous speciation influenced by microbial activity led to a reduction of solid phase As species (X-ray absorption fine structure spectroscopy) and a change in the elemental composition of As hotspots (micro X-ray fluorescence mapping). The depletion of most natural dissolved organic matter and the fact that an extensive mobilization of As[III] occurred after two months raises concerns about the long-term stability of historically As-contaminated sites.

Ipso-substitution--the hidden gate to xenobiotic degradation pathways.

Screening for metabolites of environmental pollutants, the focus is often on transformation products based on well-known pathways. Hydroxylation at unsubstituted positions of the aromatic ring or side chain modifications, followed by meta ring-cleavage pathways are usually considered, whereas less obvious mechanisms are often ignored. Here, a glimpse of the multitude of transformations involving ipso-substitution events, which are often overlooked as such, is presented. These reactions can be catalyzed by a variety of enzymes, proceed via several mechanisms and will often result in metabolites that are not expected to arise from generally known pathways. Hence, there is a future need when looking into transformations of emerging pollutants, to stray from the 'beaten pathways' and explore the possibilities of less obvious mechanisms.

Biodegradation of sulfamethoxazole and other sulfonamides by Achromobacter denitrificans PR1.

This study aimed to isolate and characterize a microbial culture able to degrade sulfonamides. Sulfamethoxazole (SMX)-degrading microorganisms were enriched from activated sludge and wastewater. The resultant mixed culture was composed of four bacterial strains, out of which only Achromobacter denitrificans PR1 could degrade SMX. This sulfonamide was used as sole source of carbon, nitrogen and energy with stoichiometric accumulation of 3-amino-5-methylisoxazole. Strain PR1 was able to remove SMX at a rate of 73.6 ± 9.6 µmol SMX/gcell dryweighth. This rate more than doubled when a supplement of amino acids or the other members of the mixed culture were added. Besides SMX, strain PR1 was able to degrade other sulfonamides with anti-microbial activity. Other environmental Achromobacter spp. could not degrade SMX, suggesting that this property is not broadly distributed in members of this genus. Further studies are needed to shed additional light on the genetics and enzymology of this process.

Emerging chemicals and the evolution of biodegradation capacities and pathways in bacteria.

The number of new chemicals produced is increasing daily by the thousands, and it is inevitable that many of these chemicals will reach the environment. Current research provides an understanding of how the evolution of promiscuous enzymes and the recruitment of enzymes available from the metagenome allows for the assembly of these pathways. Nevertheless, physicochemical constraints including bioavailability, bioaccessibility, and the structural variations of similar chemicals limit the evolution of biodegradation pathways. Similarly, physiological constraints related to kinetics and substrate utilization at low concentrations likewise limit chemical-enzyme interactions and consequently evolution. Considering these new data, the biodegradation decalogue still proves valid while at the same time the underlying mechanisms are better understood.

Ipso-hydroxylation and subsequent fragmentation: a novel microbial strategy to eliminate sulfonamide antibiotics.

Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso-hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino-N-phenylbenzenesulfonamide, and N-(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso-hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para-position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us.

Exploring the potential of applying proteomics for tracking bisphenol A and nonylphenol degradation in activated sludge.

A significant percentage of bisphenol A and nonylphenol removal in municipal wastewater treatment plants relies on biodegradation. Nonetheless, incomplete information is available concerning their degradation pathways performed by microbial communities in activated sludge systems. Hydroquinone dioxygenase (HQDO) is a specific degradation marker enzyme, involved in bisphenol A and nonylphenol biodegradation, and it can be produced by axenic cultures of the bacterium Sphingomonas sp. strain TTNP3. Proteomics, a technique based on the analysis of microbial community proteins, was applied to this strain. The bacterium proteome map was obtained and a HQDO subunit was successfully identified. Additionally, the reliability of the applied proteomics protocol was evaluated in activated sludge samples. Proteins belonging to Sphingomonas were searched at decreasing biomass ratios, i.e. serially diluting the bacterium in activated sludge. The protein patterns were compared and Sphingomonas proteins were discriminated against the ones from sludge itself on 2D-gels. The detection limit of the applied protocol was defined as 10(-3) g TTNP3 g(-1) total suspended solids (TSSs). The results proved that proteomics can be a promising methodology to assess the presence of specific enzymes in activated sludge samples, however improvements of its sensitivity are still needed.

Crystallization and preliminary X-ray crystallographic analysis of hydroquinone dioxygenase from Sphingomonas sp. TTNP3.

Hydroquinone dioxygenase (HQDO), a novel Fe(II) ring-fission dioxygenase from Sphingomonas sp. strain TTNP3 which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been crystallized. The enzyme is an α(2)ß(2) heterotetramer constituted of two subunits of 19 and 38 kDa. Diffraction-quality crystals of HQDO were obtained using the sitting-drop vapour-diffusion method at 277 K from a solution consisting of 16% PEG 4000, 0.3 M MgCl(2), 0.1 M Tris pH 8.5. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 88.4, b = 125.4, c = 90.8 Å, ß = 105.3°. The asymmetric unit contained two heterotetramers, i.e. four copies of each of the two different subunits related by noncrystallographic 222 symmetry. A complete data set extending to a maximum resolution of 2.5 Šwas collected at 100 K using a wavelength of 0.980 Å.

Formation of toxic 2-nonyl-p-benzoquinones from α-tertiary 4-nonylphenol isomers during microbial metabolism of technical nonylphenol.

In many environmental compartments, microbial degradation of α-quaternary nonylphenols proceeds along an ipso-substitution pathway. It has been reported that technical nonylphenol contains, besides α-quaternary nonylphenols, minor amounts of various α-H, α-methyl substituted tertiary isomers. Here, we show that potentially toxic metabolites of such minor components are formed during ipso-degradation of technical nonylphenol by Sphingobium xenophagum Bayram, a strain isolated from activated sewage sludge. Small but significant amounts of nonylphenols were converted to the corresponding nonylhydroquinones, which in the presence of air oxygen oxidized to the corresponding nonyl-p-benzoquinones-yielding a complex mixture of potentially toxic metabolites. Through reduction with ascorbic acid and subsequent analysis by gas chromatography-mass spectrometry, we were able to characterize this unique metabolic fingerprint and to show that its components originated for the most part from α-tertiary nonylphenol isomers. Furthermore, our results indicate that the metabolites mixture also contained several α, ß-dehydrogenated derivatives of nonyl-p-benzoquinones that originated by hydroxylation induced rearrangement, and subsequent ring and side chain oxidation from α-tertiary nonylphenol isomers. We predict that in nonylphenol polluted natural systems, in which microbial ipso-degradation is prominent, 2-alkylquinone metabolites will be produced and will contribute to the overall toxicity of the remaining material.

An unexpected gene cluster for downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3.

In silico analysis of nucleotide sequences flanking the recently found hydroquinone dioxygenase in Sphingomonas sp. strain TTNP3 revealed a gene cluster that encodes a hydroquinone catabolic pathway. In addition to the two open-reading frames encoding the recently characterized hydroquinone dioxygenase, the cluster consisted of six open-reading frames. We were able to express the three open-reading frames, hqdC, hqdD, and hqdE, and demonstrated that the three gene products, HqdC, HqdD, and HqdE had 4-hydroxymuconic semialdehyde dehydrogenase, maleylacetate reductase, and intradiol dioxygenase activity, respectively. Surprisingly, the gene cluster showed similarities to functionally related clusters found in members of the ß- and γ-proteobacteria rather than to those found in other members of the genus Sphingomonas sensu latu.