Magali Casellas

Integron involvement in environmental spread of antibiotic resistance.

The spread of antibiotic-resistant bacteria is a growing problem and a public health issue. In recent decades, various genetic mechanisms involved in the spread of resistance genes among bacteria have been identified. Integrons – genetic elements that acquire, exchange, and express genes embedded within gene cassettes (GC) – are one of these mechanisms. Integrons are widely distributed, especially in Gram-negative bacteria; they are carried by mobile genetic elements, plasmids, and transposons, which promote their spread within bacterial communities. Initially studied mainly in the clinical setting for their involvement in antibiotic resistance, their role in the environment is now an increasing focus of attention. The aim of this review is to provide an in-depth analysis of recent studies of antibiotic-resistance integrons in the environment, highlighting their potential involvement in antibiotic-resistance outside the clinical context. We will focus particularly on the impact of human activities (agriculture, industries, wastewater treatment, etc.).

Techno-economic evaluation of thermal treatment, ozonation and sonication for the reduction of wastewater biomass volume before aerobic or anaerobic digestion.

Aerobic and anaerobic digestions were compared with different sludge reduction processes such as ultrasonic, ozone, and thermal treatments. Each treatment was tested under the following conditions to improve batch aerobic or anaerobic digestion: ultrasound (200,000kJkgTS(0)(-1)), thermal (40 degrees C, 60 degrees C, 90 degrees C for 90 min, 120 degrees C 15 min, 1 bar), and ozonation (0.1gO(3)gTS(0)(-1)). The different pretreatments induced organic matter solubilisation and intrinsic sludge reduction (total suspended solids): ultrasound (47%), thermal 90 degrees C (16%), ozone (15%), thermal 60 degrees C (9%), thermal 40 degrees C (5%), autoclave (120 degrees C) (4.2%). TSS (and also VSS) solubilisation were found to be highly correlated to the pretreatment ability to break the flocs rather than to specific energy input. The total values of TSS reduction ranged from 57% to 71% under aerobic conditions and from 66% to 86% under anaerobic conditions. TSS solubilisation after pretreatment can be considered as a predictive parameter of sludge volume reduction enhancement after aerobic or anaerobic digestion while specific energy input did not show anything or negligible impact. In our experimental conditions, ultrasound and ozone led to the best TSS removal improvement after both aerobic (30% and 20%) and anaerobic digestion (20%). Ultrasonic and ozone pretreatments prior to aerobic or anaerobic digestion led to the best reduction of the specific energy required for removing 1 kg of TSS compared to the control. Anaerobic digestion was globally more effective (compare to aerobic digestion) in enhancing sludge production reduction.

Quantitative and qualitative impact of hospital effluent on dissemination of the integron pool

There is increasing evidence that human activity, and especially the resulting effluent, has a major role in the dissemination of bacterial antibiotic-resistance determinants in the environment. Hospitals are the major antibiotic consumers and thus facilitate the spread of antibiotic resistance. Questions are increasingly being raised about the management of hospital effluents, but their involvement in antibiotic-resistance dissemination has never been assessed. Integrons are a paradigm of genetic transfer between the environmental resistome and both commensal and pathogenic bacteria. In order to assess the impact of hospital activities on antibiotic-resistance dissemination in the environment, we monitored integrons and their gene cassettes in hospital effluents, and their release in the environment. We found that bacterial communities present in a hospital effluent contained a high proportion of integrons. In terms of both their gene cassette diversity and gene cassette arrays, the urban effluent and municipal wastewater treatment plant (WWTP) influent were most similar, whereas the hospital effluent and recirculation sludge exhibited very specific patterns. We found that anthropogenic activities led to the release of abundant integrons and antibiotic-resistance gene cassettes, but we observed no specific impact of hospital activities on the receiving environment. Furthermore, although the WWTP did not reduce the normalized integron copy number, it reduced the diversity of gene cassette arrays contained in the raw wastewater, underlining the effect of the biological treatment on the anthropogenic integron pool arriving at the WWTP.

Flocs surface functionality assessment of sonicated activated sludge in relation with physico-chemical properties

Flocs surface functional groups evolutions due to an ultrasonic treatment were investigated in respect with the mechanisms involved during sonication. Activated sludge surface functional groups changes were studied after treatment of a sample at different ultrasonic specific energies. Sludge functionality was qualitatively assessed by recording the infrared (FT-IR) spectra of centrifugation pellets. Potentiometric titration coupled with proton surface complexation modeling was used to assess the nature and quantity of ionizable functional groups present at the floc surface and in the aqueous phase. These evolutions were linked to changes of both mixed liquor biochemical composition (TSS, VSS, COD, proteins, humic like substances, polysaccharides) and physical properties (floc size and settleability). Observations carried out showed that activated sludge flocs were essentially mechanically disintegrated by ultrasonic waves: the nature of chemical bonds observed by FT-IR did not shift after ultrasonic treatment. Moreover, the total number of ionizable functional groups measured by potentiometric titrations remained constant during sonication. However, due to the solubilization of organic components induced by cavitation process, the corresponding ionizable functional groups (carboxyl, hydroxyl, amine) were transferred from particulate to soluble fraction. Moreover, due to the variable amount of proteins, humic like substances and polysaccharides solubilised, the relative contributions of carboxyl, hydroxyl and amine groups varied at floc surface.

Heavy metals uptake by sonicated activated sludge: Relation with floc surface properties

The effects of sonication of activated sludge on heavy metal uptake were in a first time investigated in respect with potential modifications of floc surface properties. The treatment led to the simultaneous increase of specific surface area and of the availability of negative and/or hydrophilic sites. In parallel, organic matter was released in the soluble fraction. Sorption isotherms of cadmium and copper showed that uptake characteristics and mechanisms were highly dependent on both heavy metal species and specific energy supplied. The increase of both specific surface area and fixation sites availability led to the increase of Cd(II) uptake. For Cu(II), organic matter released in soluble phase during the treatment seemed to act as a ligand and to limit adsorption on flocs surface. Three different heavy metals uptake mechanisms have been identified: proton exchange, ion exchange and (co)precipitation.

Fate of cadmium in activated sludge after changing its physico-chemical properties by thermal treatment

The effect of thermal treatment of activated sludge on cadmium uptake was investigated in respect with potential modifications of floc surface properties. Soluble fraction biochemical composition and floc size measurements evidenced floc destructuration and the release of (in)organic ligands in solution. Characterization of sludge samples by potentiometric titrations and IR spectra showed the transfer of functional groups from particulate to soluble fraction as well as the higher availability of phosphate groups originating from cell membrane phospholipids after thermal treatment. Batch biosorption tests demonstrated that cadmium uptake was highly affected by sludge modifications due to thermal treatment. For temperatures below 95 degrees C, floc size decrease allowed a better availability of binding sites, resulting in a higher sorption capacity. At temperatures above 95 degrees C, the effect of released soluble ligands and of the lower total number of surface functional groups limited cadmium uptake. Uptake mechanisms were also affected by sludge thermal treatment as surface complexation involving ion exchange tends to become predominant over precipitation.

Dynamic Assessment of the Floc Morphology, Bacterial Diversity, and Integron Content of an Activated Sludge Reactor Processing Hospital Effluent

The treatment of hospital effluents (HE) is a major concern, as they are suspected of disseminating drugs and antibiotic resistance determinants in the environment. In order to assess HE influence on wastewater treatment plant biomass, lab-scale conventional activated sludge systems (CAS) were continuously fed with real HE or urban effluent as a control. To gain insights into the main hurdles linked to HE treatment, we conducted a multiparameter study using classical physicochemical characterization, phase contrast and confocal laser scaning microscopy, and molecular biology (i.e., pyrosequencing) tools. HE caused erosion of floc structure and the production of extracellular polymeric substances attributed to the development of floc-forming bacteria. Adaptation of the sludge bacterial community to the HE characteristics, thus maintaining the purification performance of the biomass, was observed. Finally, the comparative metagenomic analysis of the CAS showed that HE treatment resulted in an increase of class 1 resistance integrons (RIs) and the introduction of Pseudomonas spp. into the bacterial community. HE treatment did not reduce the CAS process performance; nevertheless it increases the risk of dissemination into the environment of bacterial species and genetic determinants (RIs) involved in antibiotic resistance acquisition.

Monitoring of slaughterhouse wastewater biodegradation in a SBR using fluorescence and UV-Visible absorbance.

The aim of this study was to demonstrate that the effectiveness of slaughterhouse wastewater treatment by activated sludge could be enhanced through the use of optical techniques, such as UV-Visible absorbance and fluorescence spectroscopy, to estimate the hydraulic retention time necessary to remove the biodegradable chemical oxygen demand (COD). Two experiments were conducted. First, a batch aerobic degradation was performed on four wastewater samples collected from four different cattle processing sites in order to study the changes in the spectroscopic properties of wastewater during biodegradation. Second, a sequencing batch reactor was used in order to confirm that the wastewater fluorescence could be successfully used to monitor wastewater biodegradation in a pilot-scale experiment. Residual blood was the main source of organic matter in the wastewater samples. The absorbance at 416 nm, related to porphyrins, was correlated to the COD during wastewater biodegradation. The tryptophan-like/fulvic-like fluorescence intensity ratio was related to the extent of biodegradation. The COD removal efficiency ranged from 74% to 94% with an hydraulic retention time (HRT) of 23 h. A ratio of tryptophan-like/fulvic-like fluorescence intensities higher than 1.2 indicated incomplete biodegradation of the wastewater and the need to increase the HRT.

Comparing the effects of three pre-treatment disintegration techniques on aerobic sludge digestion: biodegradability enhancement and microbial community monitoring by PCR–DGGE

The objectives of this work were to compare and investigate the effect of three activated sludge disintegration processes before aerobic sludge digestion on 1) aerobic biodegradability enhancement and 2) microbial community evolution using the polymerase chain reaction–denaturant gel gradient electrophoresis (PCR–DGGE) technique. The comparison of three disintegration processes: thermal treatment (95 °C, 2 h), sonication (100,000 kJ/kg TS) and ozonation (0.108 g O3/g TS) showed that the disintegration processes acted differently according to the composition of the soluble phase and to the DNA damage. Thermal treatment led to significant protein solubilization and to DNA modification. Sonication and ozonation resulted in similar soluble phase compositions and did not lead to any DNA modifications. During activated sludge aerobic digestion, intrinsic biodegradability enhancement was observed for thermal and ozone activated sludge pre-treatments. The analysis of the DGGE patterns at the end of aerobic digestion showed that population diversity was affected by both the aerobic digestion and the pre-treatment. The dissimilarity percentages measured at the end of aerobic digestion in the control sample and in the treated sludge were equal to 22, 25 and 20% for thermal treatment, sonication and ozonation respectively. This study indicated that PCR–DGGE could be a useful tool for the comparison of disintegration processes before and after aerobic digestion.

Cadmium biosorption by ozonized activated sludge: the role of bacterial flocs surface properties and mixed liquor composition.

Cadmium uptake by activated sludge was studied following modifications of sludge composition and surface properties induced by ozone treatment. Ozone leads to the solubilization of sludge compounds as well as their mineralization, especially humic like substances. Small particles were formed following floc disintegration, leading to a decrease of average floc size. The study of surface properties underlined the mineralization as the number of surface binding sites decreased with the increase of ozone dose. Depending on ozone dose, cadmium uptake by activated sludge flocs was either increased or decreased. Different mechanisms were involved: below 10 mg O(3)/g TS, the increase of floc specific surface area following floc size decrease as well as the release of phosphate ions yielded an increase by 75% of cadmium uptake, due to the better availability of biosorption sites and the increase of precipitation. Inversely, at higher ozone doses, the number of biosorption sites decreased due to oxidation by ozone. Moreover, dissolved organic matter concentration increased and provided ligands for metal complexation. Cadmium uptake was therefore limited for ozone doses ranging from 10 to 16.8 mg O(3)/g TS.