Demonstrating plasmid-based horizontal gene transfer in complex environmental matrices: A practical approach for a critical review

International audiencePlasmid-based dissemination of antibiotic resistance genes in environmental microbial communities is a matter of concern for public health, but it remains difficult to study for methodological reasons. In this study, we used the broad host range plasmid pB10 to compare and to point out the main drawbacks of the three different approaches currently used to evaluate plasmid transfer in natural communities. Culture-based selection of transconjugants appeared to be compromised by high prevalence of antibiotic resistances among natural communities, unless high loads of initial pB10-donor inocula were used. Fluorescence-based detection of transconjugants reached a dead-end consequently to the narrow host range of bacteria expressing fluorescent proteins from a genetically modified pB10 plasmid, in addition to the relatively high background level of fluorescence exhibited by some environmental matrices. The molecular-based approach was the only one to provide a mean to detect rare plasmid transfer events following a low but realistic initial pB10-donor inoculation. Whatever the method, culture-based or molecular-based, the detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events

Comparing TiO2 photocatalysis and UV-C radiation for inactivation and mutant formation of Salmonella typhimurium TA102

International audienceSalmonellosis is one of the most common causes of foodborne bacterial human disease worldwide, and the emergence of multidrug-resistant (MDR) strains of Salmonella enterica serovar Typhimurium (S. typhimurium) was associated to the incidence of invasive salmonellosis. The objective of the present work was to investigate the effects of the TiO2 photocatalysis process in terms of both bacteria inactivation and the emergence of mutants, on S. typhimurium TA102 water suspensions. The TiO2 photocatalysis was compared with a conventional disinfection process such as UV-C radiation. In spite of the faster bacterial inactivation obtained in UV-C disinfection experiments (45, 15, and 10 min for total inactivation for initial cell density 109, 108, and 107 CFU mL-1, respectively), photocatalytic disinfection (60, 30, and 15 min) was more energy efficient because of a lower energy requirement (2-20 mWs cm-2) compared to the UV-C disinfection process (5-30 mWs cm-2). During the photocatalytic experiments, the mutation frequency increased up to 1648-fold compared to background level for a 108 CFU mL-1 initial bacterial density, and mutants were inactivated after 1-10-min treatment, depending on initial bacterial cell density. In UV-C disinfection experiments, the mutation frequency increased up to 2181-fold for a 108 CFU mL-1 initial bacterial cell density, and UV-C doses in the range of 0.5-4.8 mWs cm-2 were necessary to decrease mutation frequency. In conclusion, both disinfection processes were effective in the inactivation of S. typhimurium cells, and mutants released into the environment can be avoided if cells are effectively inactivated

Inducibility of Tn916 conjugative transfer in Enterococcus faecalis by subinhibitory concentrations of ribosome-targeting antibiotics

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Atypical stress response to temperature and NaOCl exposure leading to septation defect during cell division in Cupriavidus metallidurans CH34

International audienceCupriavidus metallidurans CH34 has long been known for its temperature-induced mutagenesis and mortality phenotype (TIMM), for which a genetic origin has been suggested repeatedly. In this report, we present microscopic-based evidences that the TIMM process actually starts with a septation defect, leading to aberrant cell morphologies. Moreover, the septation defect of CH34 could be induced by NaOCl, thus showing that the TIMM phenotype may be part of a more general stress response. Sequence analysis of a TIMM survivor exhibiting a recurrent recognizable lysA mutation ruled out the possibility of a genetic ground linking TIMM survival and peptidoglycan synthesis

Exposure of bacteria to CdTe quantum dots : the importance of surface chemistry on cytotoxicity

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Identification of antibiotics triggering the dissemination of antibiotic resistance genes by SXT/R391 elements using a dedicated high-throughput whole-cell biosensor assay

International audienceAbstract Background Mobile genetic elements (MGEs) are widely involved in the dissemination of antibiotic resistance genes and some of them, such as the integrative and conjugative element SXT, are even induced by specific antibiotics at sub-lethal concentrations. Objectives This work explores collateral effects of a broad range of antibiotics on the mobility of the SXTMO10 element using a specifically designed high-throughput screening test. Methods Twenty-five promoters involved in the mobility of SXT and six artificial constitutive promoters were transcriptionally fused to luxCDABE bioluminescent genes and introduced into Escherichia coli strains with or without SXT to build whole-cell biosensors for a large-scale screening involving 48 antibiotics. A bioluminescent assay implementing a classical agar diffusion approach was coupled to an automated data processing pipeline developed to extract and analyse luminescence data from over 2000 antibiotic/biosensor combination profiles. Results In addition to quinolones previously reported as inducing the expression of SXT mobility genes, we found that specific antibiotics belonging to other classes, such as imipenem and azithromycin, also behave as inducers. The use of a control set of constitutive biosensors also revealed an unexpected intricate relationship between cell respiration and light production that allowed the identification of antibiotics interfering with the respiration process. Conclusions The effect of antibiotics goes beyond the interaction with their primary cell targets and may lead to adverse effects such as triggering the dissemination of resistance by MGEs, sometimes in unpredictable ways. Identifying such MGE-triggering antibiotics is of prime importance for better controlling collateral effects during therapy

Identifying the range of sub-inhibitory concentrations of antibiotics triggering bacterial functions of relevant interest

International audienceBeyond their selective role, some antibiotics continue to exert an effect below their minimum inhibitory concentration level, where they can act as signaling molecules to modulate various bacterial activities: biofilm formation, exopolymers production, virulence, quorum sensing, cell mobility, and even the dissemination of specific antibiotic resistance genes. This clearly asks the question of the possible effects antibiotics can have once diluted after the disposal of reclaimed wastewater into the aquatic environment or during its reuse. Lately we developed a biosensor approach to identify antibiotics interfering with gene regulation. This approach lies on the use of bacteria genetically modified to express a bioluminescence once the promoter of a relevant function is induced by a modulating antibiotic. Rather than testing arbitrary chosen concentrations, the biosensor is exposed to diffusion gradients of antibiotics in agar plate (as for antibiograms) while luminescence profiles are recorded by CCD camera. With such approach we could detect several antibiotics able to trigger the activation of promoters controlling the mobility functions of "integrative and conjugative elements" (ICEs) such as Tn916 and SXT. An automated image analysis algorithm combined with a modelling of the diffusion process in the agar has been developed to convert the luminescence profile into a concentration profile information, enabling to estimate the level of induction and the range of sub-MIC concentration at which an antibiotic remain effective. The biosensor strains are first spread onto the agar of a square Petri dish before receiving one to sixteen antibiotics loaded disks deposited in an equispaced manner. The agar plate is incubated at 30°C allowing the antibiotics to diffuse and luminescence and transmitted light images are regularly acquired over time. An automated image processing system has been developed to detect the induction and inhibition zones around each antibiotic loaded disk. For the present experiment two antibiotics were used: ciprofloxacin (CIP) (broad spectrum) and nalidixic acid (NAL) (limited spectrum). Both inhibit the DNA gyrase while CIP inhibits also topoisomerase IV. Commercially available discs (BioRad) were used (30 µg for NAL and 5 µg for CIP). Additional discs were prepared by loading cellulose discs with antibiotic solutions. The MIC values were determined in separate experiments in liquid phase for each antibiotic. The diffusion of antibiotics in agar is modelled through a finite element computational model based on Fick's second law of diffusion. The simulations are performed using a Computational Fluid Dynamics software (Fluent by ANSYS). The mesh contains ≈ 80,000 cells. It is assumed that the antibiotics deposited in the thin top agar layer exhibit higher diffusivities in this layer than in the bulk solid agar. Bulk agar Top agar The key parameters are the antibiotic diffusivity in the top agar and the diffusivity ratio between the top and the bulk agar. Here D top /D bulk = 1000. Effect of clone and concentration with NAL Effect of Petri dish with NAL and CIP (same clone) Effect of concentration on CIP (same clone) MIC (CIP) = 0.015 µg/mL = 4.5310-5 mole/m 3 MIC (NAL) = 3 µg/mL = 1.2910-2 mole/m 3 D = 1. 10-14 m 2 /s D = 1. 10-11 m 2 /s %of total time D = 1. 10-14 m 2 /s D = 1. 10-13 m 2 /s D = 1. 10-12 m 2 /s D = 1. 10-11 m 2 /s D = 1. 10-10 m 2 /s Range of D for CIP (5 µg) D = 1. 10-10 m 2 /s D = 1. 10-11 m 2 /s D = 1. 10-11 m 2 /s Order of magnitude of D for NAL (30 µg) Simulated MIC paths The effect of experimental parameters such as clone and antibiotic type and concentration has been tested. An improvement of the spatial resolution would be useful for antibiotics such as NAL with a high MIC. First simulation results show that it is possible to estimate the diffusivity of an antibiotic in gelose media. A refinement of the simulation mesh is however necessary, especially for high-MIC antibiotics, to estimate properly sub-inhibitory concentrations. Such refinement is currently tested