bioRxiv Science⌕ Search

Biology subjects

Calderon-Franco, D.

Publications and source records attributed to Calderon-Franco, D..

9 recordsLinked to original sources

Predicted meta-omics: a potential solution to multi-omics data scarcity in microbiome studies

Imbalances in the gut microbiome have been linked to conditions such as inflammatory bowel disease, diabetes, and cancer. While metagenomics and amplicon sequencing are commonly used to study the microbiome, they do not capture all layers of microbial functions. Other meta-omics data can provide more insights, but these are more costly and laborious to procure. The growing availability of paired meta-omics data offers an opportunity to develop machine learning models that can infer connections between metagenomics data and other forms of meta-omics data, enabling the prediction of these other forms of meta-omics data from metagenomics. We evaluated several machine learning models for predicting meta-omics features from various meta-omics inputs. Simpler architectures such as elastic net regression and random forests generated reliable predictions of transcript and metabolite abundances, with correlations of up to 0.77 and 0.74, respectively, but predicting protein profiles was more challenging. We also identified a core set of well-predicted features for each meta-omics output type, and showed that multi-output regression neural networks performed similarly when trained using fewer output features. Lastly, our experiments demonstrated that predicted features can be used for the downstream task of inflammatory bowel disease classification, with performance comparable to that of experimental data.

microbiology↗

The fate of sulfamethoxazole and trimethoprim in a micro-aerated anaerobic membrane bioreactor: implications for antibiotic resistance spreading

Interest in reusing treated wastewater drives efforts to eliminate antibiotics from water sources to prevent antibiotic resistance. Micro-aerated anaerobic membrane bioreactors (MA-AnMBR) promote wastewater reuse with high organic matter conversion to biogas, under a small footprint. However, the fates of antibiotics, antibiotic-resistant bacteria (ARB), and their antibiotic-resistance genes (ARGs) are not known in these systems. We studied the effects, conversions, and resistance induction, following the addition of 150 g{middle dot}L-1 of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), in a laboratory-scale MA-AnMBR. TMP and SMX were removed at 97 and 86%, indicating that micro-aeration did not hamper the removal of the antibiotics. These antibiotics only affected the pH and biogas composition of the process, with a significant change in pH from 7.8 to 7.5, and a decrease in biogas CH4 content from 84 to 78%. TMP was rapidly adsorbed onto the sludge and subsequently degraded during the long retention of the solids of 27 days. SMX adsorption was minimal, but the applied hydraulic retention time of 2.6 days was sufficiently long to biodegrade SMX. The levels of three ARGs (sul1 and sul2 for SMX, dfrA1) and one mobile genetic element biomarker (intI1) were analysed by qPCR, in combination with ARB tracked by plating. Additions of the antibiotics increased the relative abundances of all ARGs and intI1 in the MA-AnMBR sludge, with the sul2 gene folding 15 times after 310 days of operation. The MA-AnMBR was able to reduce the concentration of ARB in the permeate by 3 log. HighlightsO_LIAdditions of SMX and TMP had a negligible effect on the MA-AnMBR performance. C_LIO_LIThe laboratory-scale MA-AnMBR removed 86% of SMX and 97% of TMP. C_LIO_LIA 3 log removal of ARB was achieved between sludge and UF permeate. C_LIO_LIRelative abundances of ARGs were similar in sludge and permeate. C_LIO_LITMP and SMX resistance is better assessed by the heterotrophic plate count of ARB. C_LI

bioengineering↗

Influence of DNA extraction methods on microbiome and resistome analysis in activated sludge

Amplicon sequencing, metagenomics, and quantitative polymerase chain reaction (qPCR) are commonly used techniques to analyse microorganisms and antibiotic resistance genes (ARGs) in activated sludge from wastewater treatment plants (WWTPs). However, the lack of workflow harmonisation poses challenges in comparing measurements across studies and research groups. To address this issue, we examined the impact of DNA extraction procedures on 16S rRNA gene amplicon sequencing, shotgun metagenomics, and qPCR analyses of activated sludge by combining two widely used DNA extraction kits (PowerSoil and FastDNA) and two commonly employed disruption instruments (bead-beater and vortex) through a 2x2 factorial experimental design involving four groups of three analysts performing DNA extractions in triplicates. Our findings revealed significant differences in DNA yield, purity, and reproducibility of amplicon sequencing profiles among the extraction kits. Operator variability also influenced the results. We compared microbiome profiles obtained by amplicon sequencing and metagenomics and observed that bead-beating introduced more variability among triplicates compared to vortexing. The combinations of extraction kits and disruption instruments impacted the relative abundances of specific phyla such as Actinobacteriota, Bacteroidota, and Nitrospirota. For resistome analysis, we employed metagenomics for high-resolution profiling and qPCR for high-sensitivity detection of ARGs. The compositions and diversities of resistome datasets were not significantly affected by the choice of extraction kits and disruption instruments. Although using the same method is ideal for accurate comparisons, our results suggest that acceptable reproducibility can still be achieved when using different methods. This finding encourages the implementation of ARG monitoring in wastewater treatment processes. However, it is important to consider biases introduced by DNA extraction workflows when designing analytical studies, interpreting their results, and comparing their findings. Striving for more harmonised molecular workflows is crucial in the field of wastewater microbiology and engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/546617v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@caa36eorg.highwire.dtl.DTLVardef@1afabfdorg.highwire.dtl.DTLVardef@448b9aorg.highwire.dtl.DTLVardef@296404_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

molecular biology↗

Microbiome, resistome and mobilome of chlorine-free drinking water treatment systems

Drinking water treatment plants (DWTPs) are designed to remove physical, chemical, and biological contaminants. However, until recently, the role of DWTPs in minimizing the cycling of antibiotic resistance determinants has got limited attention. In particular, the risk of selecting antibiotic-resistant bacteria (ARB) is largely overlooked in chlorine-free DWTPs where biological processes are applied. Here, we combined high-throughput quantitative PCR and metagenomics to analyze the abundance and dynamics of microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) across the treatment trains of two chlorine-free DWTPs involving dune-based and reservoir-based systems. The microbial diversity of the water being treated increased after all biological unit operations, namely rapid and slow sand filtration (SSF), and granular activated carbon filtration. Both DWTPs reduced the concentration of ARGs and MGEs in the water by about 2.5 log gene copies mL-1, despite their relative increase in the disinfection sub-units (SSF in dune-based and UV treatment in reservoir-based DWTPs). The total microbial concentration was also reduced (2.5 log units), and none of the DWTPs were enriched for antibiotic resistant bacteria. Our findings highlight the effectiveness of chlorine-free DWTPs in supplying safe drinking water while reducing the concentration of antibiotic resistance determinants. To the best of our knowledge, this is the first study that monitors the presence and dynamics of antibiotic resistance determinants in chlorine-free DWTPs.

microbiology↗

Antibiotic resistance response to sulfamethoxazole from the intracellular and extracellular DNA fractions of activated sludge

In activated sludge, the antibiotic resistance genes (ARGs) can be present either in the intracellular (iDNA) or extracellular DNA fraction (exDNA). Recent advances in the exDNA extraction methodology allow a better profiling of the pool of ARGs. However, little is known about how stress conditions modify the distribution of ARGs between both DNA fractions. Here, we performed two batch tests for analyzing the effects of two different stress conditions, namely nutrient starvation and high concentrations of sulfamethoxazole (1, 10 and 150 mg L-1) in activated sludge. We tracked by qPCR the resulting relative abundances of four target genes, namely the universal 16S rRNA gene, the class 1 integron-integrase gene intI1, and the sulfonamide resistance genes sul1 and sul2 in both the iDNA and exDNA fractions. In the exDNA pool, unlike starvation, which provoked a decrease of 1-2 log10 [copies] ng DNA-1 in the concentration of sul1 and intI1, the presence of sulfamethoxazole did not influence the abundances of sul1 and sul2. However, high concentrations of sulfamethoxazole (150 mg L- 1) selected for microorganisms harboring sul1 and, more remarkably, sul2 genes in their iDNA during their exponential growth phase. The abundances of intI1 and sul1 were positively correlated in the exDNA fraction (r>0.7), whereas no significant correlation (p<0.05) between the abundance of these two genes was found in the iDNA fraction of the sludge. High SMX concentrations influenced the abundance of ARGs in the iDNA; their abundance in the exDNA was influenced by nutrient limitations. Further studies should consider the profiling of exDNA fractions because of the relationship between ARGs and mobile genetic elements. Besides, the surveillance of antimicrobial resistance is encouraged in wastewater treatment plants facing high antibiotic concentrations. HighlightsO_LIStarvation caused a decrease in the exDNA concentration of the activated sludge. C_LIO_LIA positive correlation in the abundances of intI1 and sul1 was found in the exDNA fraction. C_LIO_LIHigh concentrations of SMX selected for antibiotic resistant microorganisms. C_LI

bioengineering↗

Catch me if you can: Capturing extracellular DNA transformation in mixed cultures via Hi-C sequencing

Environmental microorganisms evolve constantly under various stressors using different adaptive mechanisms, including horizontal gene transfer. Microorganisms benefit from transferring genetic information that code for antibiotic resistance via mobile genetic elements (plasmids). Due to the complexity of natural microbial ecosystems, quantitative data on the transfer of genetic information in microbial communities remain unclear. Two 1-L chemostats (one control and one test) were inoculated with activated sludge, fed with synthetic wastewater, and operated for 45 days at a hydraulic retention time of 1 day to study the transformation capacity of a rolling-circle plasmid encoding GFP and kanamycin resistance genes, at increasing concentrations of kanamycin (0.01-2.5-50-100 mg L-1) representing environmental, wastewater, lab-selection, and gut or untreated pharmaceutical wastewater discharge environments. The plasmid DNA was spiked daily at 5 {micro}g L-1 in the test chemostat. The evolution of the microbial community composition was analyzed by 16S rRNA gene amplicon sequencing and metagenomics, and the presence of the plasmid by quantitative PCR. We used Hi-C sequencing to identify natural transformant microorganisms under steady-state conditions with low (2.5 mg L-1) and high (50 mg L-1) concentrations of kanamycin. Both chemostats selected for the same 6 predominant families of Spirosomaceae, Comamonadaceae, Rhodocyclaceae, Rhizobiaceae, Microbacteriaceae, and Chitinophagaceae, while biomass formation in the presence of kanamycin was higher with the plasmid. Hence, the antibiotic exerted the main pressure on microbial selection, while the plasmid helped these populations better resist the antibiotic treatment and grow. The kanamycin resistance gene increased in both reactors (log 7 gene copies g VSS-1). When higher antibiotic concentrations were applied, the GFP/16S ratio was increased, highlighting plasmids accumulation in the test reactor over time. The plasmid transformed mainly inside populations of Bosea sp., Runella spp., and Microbacterium sp.. This study made one significant step forward by demonstrating that microorganisms in enrichments from activated sludge biomasses can acquire exogenous synthetic plasmids by transformation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/508255v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1002657org.highwire.dtl.DTLVardef@d535f6org.highwire.dtl.DTLVardef@1c9e045org.highwire.dtl.DTLVardef@a2fcfc_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

Metagenomic profiling and transfer dynamics of antibiotic resistance determinants in a full-scale granular sludge wastewater treatment plant

In the One Health context, wastewater treatment plants (WWTPs) are central to safeguard water resources. Nonetheless, many questions remain about their effectiveness to prevent the dissemination of antimicrobial resistance (AMR). Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. In this study, we analyzed the transfer of ARGs and MGEs in a full-scale Nereda(R) reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%), tetC (1.6%), sul1 (1.5%), oqxB (1.2%), and aph(3")-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas, Acinetobacter, Aeromonas, Acidovorax, Rhodoferax, and Streptomyces populations were the main hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. These can pose a risk for AMR dissemination by transformation into microorganisms of receiving water bodies. Total intracellular ARGs decreased 3-42% as a result of wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL-1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/482492v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1a5eedcorg.highwire.dtl.DTLVardef@e4da78org.highwire.dtl.DTLVardef@c324bforg.highwire.dtl.DTLVardef@1a18138_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA DNA database from an AGS reactor was constructed to study the system resistome, mobilome, and microbiome. C_LIO_LIThe genera Pseudomonas and Rhodoferax were the predominant ARG carriers in the system. C_LIO_LIMGEs and ARGs often co-localize on contigs recovered from the exDNA of the effluent. C_LIO_LIAGS plants are efficient at reducing ARB. C_LIO_LIThe exDNA is an underestimated DNA fraction containing ARGs in the effluent. C_LI

bioengineering↗

Antibiotic resistance genes and mobile genetic elements removal from treated wastewater by sewage-sludge biochar and iron-oxide coated sand

Disinfection of treated wastewater in wastewater treatment plants (WWTPs) is used to minimize emission of coliforms, pathogens, and antibiotic resistant bacteria (ARB) in the environment. However, the fate of free-floating extracellular DNA (eDNA) that do carry antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) is overlooked. Water technologies are central to urban and industrial ecology for sanitation and resource recovery. Biochar produced by pyrolysis of sewage sludge and iron-oxide-coated sands recovered as by-product of drinking water treatment were tested as adsorbents to remove ARGs and MGEs from WWTP effluent. DNA adsorption properties and materials applicability were studied in batch and up-flow column systems at bench scale. Breakthrough curves were measured with ultrapure water and treated wastewater at initial DNA concentrations of 0.1-0.5 mg mL-1 and flow rates of 0.1-0.5 mL min-1. Batch tests with treated wastewater indicated that the adsorption profiles of biochar and iron-oxide coated sand followed a Freundlich isotherm, suggesting a multilayer adsorption of nucleic acids. Sewage-sludge biochar exhibited higher DNA adsorption capacity (1 mg g-1) and longer saturation breakthrough times (4 to 10 times) than iron-oxide coated sand (0.2 mg g-1). The removal of a set of representative ARGs and MGEs was measured by qPCR comparing the inlet and outlet of the plug-flow column fed with treated wastewater. ARGs and MGEs present as free-floating eDNA were adsorbed by sewage-sludge biochar at 85% and iron-oxide coated sand at 54%. From the environmental DNA consisting of the free-floating extracellular DNA plus the intracellular DNA of the cells present in the effluent water, 97% (sewage-sludge biochar) and 66% (iron-oxide coated sand) of the tested genes present were removed. Sewage-sludge biochar displayed interesting properties to minimize the spread of antimicrobial resistances to the aquatic environment while strengthening the role of WWTPs as resource recovery factories. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/302018v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@bdc78borg.highwire.dtl.DTLVardef@177a537org.highwire.dtl.DTLVardef@1ffefdeorg.highwire.dtl.DTLVardef@1dd1457_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISewage-sludge biochar and iron oxide coated sands were tested to adsorb DNA and cells. C_LIO_LIBiochar removed 97% of genes tested from environmental DNA of unfiltered effluent. C_LIO_LI85% of ARGs and MGEs of free-floating extracellular DNA were retained by biochar. C_LIO_LIBiochar is a WWTP by-product that can be re-used for public health sanitation. C_LI

bioengineering↗

A novel method to isolate free-floating extracellular DNA from wastewater for quantitation and metagenomic profiling of mobile genetic elements and antibiotic resistance genes

Antibiotic resistant genes (ARGs) and mobile genetic elements (MGEs) can be found in the free-floating extracellular DNA (eDNA) fraction of microbial systems. These xenogenic components can generate bacterial cells resistant to one or more antibiotics by natural transformation. Because of low concentration in wastewater, the obtaining of a high quality and a high yield of eDNA extract is challenging. We developed a method using chromatography to isolate eDNA without causing cell lysis (often unchecked) from complex wastewater matrices. The chromatographic step involved a diethylaminoethyl-cellulose-monolithic column to capture the eDNA found in cell-free filtered wastewater samples (e.g. influent wastewater, activated sludge and treated effluent wastewaster). Free-floating eDNA yields from 1 L of influent, activated sludge and treated effluent water reached 12.5 {+/-} 1.9 g, 12.3 {+/-} 1 g and 5.6 {+/-} 2.9 g of raw eDNA and 9.0 {+/-} 0.7 g, 5.6 {+/-} 0.46 g and 2.6 {+/-} 1.3 g of purified eDNA, respectively. In order to check the suitability of free-floating eDNA extracts for molecular analysis, qPCR and metagenomics were performed. eDNA extracts from treated effluent water were analyzed by qPCR to quantify a selected panel of ARGs and MGEs. Microbiome, resistome, and mobilome profiles from activated sludge free-floating eDNA were measured by metagenomic sequencing. Between iDNA and eDNA fractions, qPCR showed differences of 0.94, 1.11, 1.92 and 1.32 log10 gene copies mL-1 for sulfonamides resistant genes (sul1 and sul2), {beta}-lactamase resistance gene blaCTXM, and the class 1 integron-integrase (intI1) MGE, respectively. These differences highlighted the crucial need for an isolation method to discern both iDNA and eDNA to understand ARGs persistence and quantity in complex cultures. The eDNA yields obtained from 1 L of activated sludge (3.6 g of total suspended solids L-1) samples were substantially higher than the amount of DNA template needed for high-throughput sequencing (>1 g) in service facilities. Subsystems classification showed that the eDNA metagenome was mainly composed by MGEs (65.1%). The 35.9% rest related to traditional functional genetic signatures. It was the first time the resistome from the eDNA fraction was analyzed showing lower number of primary aligned reads when compared to the iDNA and a predominance of aminoglycosides and {beta}-lactamams. Metagenome results showed that eDNA can not be discarded as a pool of ARGs and MGEs for horizontal gene transfer. This novel isolation method was powerful to elucidate the molecular compositions of free-floating eDNA fractions in complex environmental samples such as wastewater environments at different microbial densities. Data obtained using this extraction method will foster xenogenic and microbial risk assessments across urban and natural water systems. This will support water authorities in the delineation of measures to adopt at wastewater treatment plants to remove them and safeguard environmental and public health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/072397v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1905305org.highwire.dtl.DTLVardef@3a774borg.highwire.dtl.DTLVardef@6e1262org.highwire.dtl.DTLVardef@790c42_HPS_FORMAT_FIGEXP M_FIG Picture created with BioRender C_FIG

molecular biology↗