Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota

Sewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales. The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyrA (codons S83L and D87N) and parC (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment

The significance of peroxisomes in secondary metabolite biosynthesis in filamentous fungi

Peroxisomes are ubiquitous organelles characterized by a protein-rich matrix surrounded by a single membrane. In filamentous fungi, peroxisomes are crucial for the primary metabolism of several unusual carbon sources used for growth (e.g. fatty acids), but increasing evidence is presented that emphasize the crucial role of these organelles in the formation of a variety of secondary metabolites. In filamentous fungi, peroxisomes also play a role in development and differentiation whereas specialized peroxisomes, the Woronin bodies, play a structural role in plugging septal pores. The biogenesis of peroxisomes in filamentous fungi involves the function of conserved PEX genes, as well as genes that are unique for these organisms. Peroxisomes are also subject to autophagic degradation, a process that involves ATG genes. The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells

The Nuclear Protein Sge1 of Fusarium oxysporum Is Required for Parasitic Growth

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth

Impacts of chemicals and microbiota from a hospital on the emergence of resistance in sewer systems

International audienceHospital wastewaters are composed of antibiotic resistant bacteria (ARB) and a cocktail of chemical agents, including antibiotics (ATB) exerting selective pressures. The aim of this study was to clarify the role of ARB and chemicals originating from hospitals in the emergence / dissemination of antimicrobial resistance in mixed municipal wastewater. As part of a field approach, domestic wastewater, hospital wastewater and mixed wastewater (domestic + hospital) were sampled and compared in terms of ATB concentrations and antibiotic resistance. Controlled microcosms were also conducted to assess the contribution of the microbiological versus chemical component of the hospital wastewater exposome. They consisted in mixing domestic wastewaters with either full hospital effluent, its chemical constituents, or its microbiota. They were incubated for 96 hours at 21°C and 100 rpm. Antibiotic resistance was monitored by: (i) assessment of percentage of ARB to ciprofloxacin and cefotaxime, (ii) quantification of antibiotic resistance genes (ARGs) by qPCR, (iii) determination of the sequence variants of ARGs from various antibiotic classes (fluoroquinolones, beta-lactamases, polymyxins, and others) by multiplex amplicon sequencing. The proportions of ARB to ciprofloxacin were similar in microcosms that received full hospital effluent or only its chemical component, suggesting that selective pressure rather than community coalescence/immigration was the main factor giving rise to ARB in mixed municipal wastewaters. Some ARG variants were unique to each wastewater type, whilst others were shared. Their profiles in the microcosms suggested that hospital-derived variants could persist and coexist along domestic wastewater variants in some instances (e.g., blaMIR, mdtg). In other cases, hospital variants were not maintained in the final microcosm communities (e.g., blaFOX). This research sheds light on the complex interplay between ARGs and chemical agents form hospitals in municipal wastewater, providing insights into the potential dissemination routes and persistence patterns

Impacts of chemicals and microbiota from hospitals on the emergence of resistance in sewer systems

International audienceIn the fight against the growing threat of antibiotic resistance, hospital wastewater is considered a hotspot, as it harbours a complex mixture of antibiotic-resistant bacteria, pathogenic bacteria and a cocktail of chemicals. The aim of this study was to clarify the respective roles of microbiota and chemicals from hospital wastewater in the emergence and dissemination of antibiotic resistance in mixed wastewater (hospital + domestic). As part of a field approach, domestic wastewater (DWW), hospital wastewater (HWW) and mixed wastewater were sampled and compared in terms of antibiotic concentrations, antibiotic resistance and taxonomic composition. In controlled microcosms, DWW was mixed with either full HWW, its chemical components alone, or its microbiota, and monitored for 96 hours. Antibiotic resistance was assessed by the percentage of bacteria resistant to ciprofloxacin and cefotaxime and the quantification of antibiotic resistance genes (ARGs). Multiplex sequencing of amplicons of ARG sequence variants from different classes and characterization of taxonomic composition were also used to study microbiota immigration. Finally, the SELECT method [1] was adapted in order to determine the lowest HWW proportion that significantly reduced the net growth of DWW microbiota. The percentage of bacteria resistant to ciprofloxacin was similar in the microcosms that received full HWW or only its chemical component, suggesting that selective pressure rather than community immigration was the main factor increasing antibiotic resistant bacteria. Indeed, the SELECT method showed a delay in the growth of DWW microbiota exposed to 20-fold diluted HWW chemicals, confirming their impact, even at really low concentrations. Moreover, the relative abundance of various genes was higher in microcosms exposed to HWW than the ones with DWW only. Certain ARG variants were specific to each type of wastewater. Their profiles in the microcosms suggest that hospital-derived variants can persist and coexist with domestic wastewater variants in some cases (blaMIR, mdtg...), while others were not maintained in the final microcosm communities (blaFOX, ermB). This research sheds light on the complex interplay between ARGs and chemical agents from hospitals in municipal wastewater, providing insights into the potential dissemination routes and persistence patterns

Impacts of chemicals and microbiota from a hospital on the emergence of resistance in sewer systems

International audienceHospital wastewaters are composed of antibiotic resistant bacteria (ARB) and a cocktail of chemical agents, including antibiotics (ATB) exerting selective pressures. The aim of this study was to clarify the role of ARB and chemicals originating from hospitals in the emergence / dissemination of antimicrobial resistance in mixed municipal wastewater. As part of a field approach, domestic wastewater, hospital wastewater and mixed wastewater (domestic + hospital) were sampled and compared in terms of ATB concentrations and antibiotic resistance. Controlled microcosms were also conducted to assess the contribution of the microbiological versus chemical component of the hospital wastewater exposome. They consisted in mixing domestic wastewaters with either full hospital effluent, its chemical constituents, or its microbiota. They were incubated for 96 hours at 21°C and 100 rpm. Antibiotic resistance was monitored by: (i) assessment of percentage of ARB to ciprofloxacin and cefotaxime, (ii) quantification of antibiotic resistance genes (ARGs) by qPCR, (iii) determination of the sequence variants of ARGs from various antibiotic classes (fluoroquinolones, beta-lactamases, polymyxins, and others) by multiplex amplicon sequencing. The proportions of ARB to ciprofloxacin were similar in microcosms that received full hospital effluent or only its chemical component, suggesting that selective pressure rather than community coalescence/immigration was the main factor giving rise to ARB in mixed municipal wastewaters. Some ARG variants were unique to each wastewater type, whilst others were shared. Their profiles in the microcosms suggested that hospital-derived variants could persist and coexist along domestic wastewater variants in some instances (e.g., blaMIR, mdtg). In other cases, hospital variants were not maintained in the final microcosm communities (e.g., blaFOX). This research sheds light on the complex interplay between ARGs and chemical agents form hospitals in municipal wastewater, providing insights into the potential dissemination routes and persistence patterns

Impacts of chemicals and microbiota from hospitals on the emergence of resistance in sewer systems

International audienceIn the fight against the growing threat of antibiotic resistance, hospital wastewater is considered a hotspot, as it harbours a complex mixture of antibiotic-resistant bacteria, pathogenic bacteria and a cocktail of chemicals. The aim of this study was to clarify the respective roles of microbiota and chemicals from hospital wastewater in the emergence and dissemination of antibiotic resistance in mixed wastewater (hospital + domestic). As part of a field approach, domestic wastewater (DWW), hospital wastewater (HWW) and mixed wastewater were sampled and compared in terms of antibiotic concentrations, antibiotic resistance and taxonomic composition. In controlled microcosms, DWW was mixed with either full HWW, its chemical components alone, or its microbiota, and monitored for 96 hours. Antibiotic resistance was assessed by the percentage of bacteria resistant to ciprofloxacin and cefotaxime and the quantification of antibiotic resistance genes (ARGs). Multiplex sequencing of amplicons of ARG sequence variants from different classes and characterization of taxonomic composition were also used to study microbiota immigration. Finally, the SELECT method [1] was adapted in order to determine the lowest HWW proportion that significantly reduced the net growth of DWW microbiota. The percentage of bacteria resistant to ciprofloxacin was similar in the microcosms that received full HWW or only its chemical component, suggesting that selective pressure rather than community immigration was the main factor increasing antibiotic resistant bacteria. Indeed, the SELECT method showed a delay in the growth of DWW microbiota exposed to 20-fold diluted HWW chemicals, confirming their impact, even at really low concentrations. Moreover, the relative abundance of various genes was higher in microcosms exposed to HWW than the ones with DWW only. Certain ARG variants were specific to each type of wastewater. Their profiles in the microcosms suggest that hospital-derived variants can persist and coexist with domestic wastewater variants in some cases (blaMIR, mdtg...), while others were not maintained in the final microcosm communities (blaFOX, ermB). This research sheds light on the complex interplay between ARGs and chemical agents from hospitals in municipal wastewater, providing insights into the potential dissemination routes and persistence patterns

Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota

International audienceSewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales . The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyr A (codons S83L and D87N) and par C (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment