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

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.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Suspended Materials in River Waters Differentially Enrich Class 1 Integron- and IncP-1 Plasmid-Carrying Bacteria in Sediments.

Aquatic ecosystems are frequently considered as the final receiving environments of anthropogenic pollutants such as pharmaceutical residues or antibiotic resistant bacteria, and as a consequence tend to form reservoirs of antibiotic resistance genes. Considering the global threat posed by the antibiotic resistance, the mechanisms involved in both the formation of such reservoirs and their remobilization are a concern of prime importance. Antibiotic resistance genes are strongly associated with mobile genetic elements that are directly involved in their dissemination. Most mobile genetic element-mediated gene transfers involve replicative mechanisms and, as such, localized gene transfers should participate in the local increase in resistance gene abundance. Additionally, the carriage of conjugative mobile elements encoding cell appendages acting as adhesins has already been demonstrated to increase biofilm-forming capability of bacteria and, therefore, should also contribute to their selective enrichment on surfaces. In the present study, we investigated the occurrence of two families of mobile genetic elements, IncP-1 plasmids and class 1 integrons, in the water column and bank sediments of the Orne River, in France. We show that these mobile elements, especially IncP-1 plasmids, are enriched in the bacteria attached on the suspended matters in the river waters, and that a similar abundance is found in freshly deposited sediments. Using the IncP-1 plasmid pB10 as a model, in vitro experiments demonstrated that local enrichment of plasmid-bearing bacteria on artificial surfaces mainly resulted from an increase in bacterial adhesion properties conferred by the plasmid rather than an improved dissemination frequency of the plasmid between surface-attached bacteria. We propose plasmid-mediated adhesion to particles to be one of the main contributors in the formation of mobile genetic element-reservoirs in sediments, with adhesion to suspended matter working as a selective enrichment process of antibiotic resistant genes and bacteria.

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

Background: Some antibiotics induce the dissemination of their own resistance genes by interfering with the regulation of specific mobile genetic elements. In Tn916, subinhibitory concentrations of tetracycline activate the transfer of the element through an anti-attenuation mechanism that relies on the Tet(M) resistance protein, itself encoded by the element. Objectives: This work explores the effects of a broad range of antibiotics on the transfer of Tn916 and for which the element does not provide any selective advantage. Methods: A sensitive promoter-reporter fusion approach was developed to test the effects of full antibiotic concentration gradients on gene promoter expression. Sixty molecules, covering most classes of antibiotics, were screened for their ability to modulate the activity of promoter Porf12 controlling the transfer of Tn916. Induction of Tn916 transfer was further demonstrated in mating assays with Enterococcus faecalis donors pre-exposed to subinhibitory concentrations of modulating antibiotics. Results: Several antibiotics, other than tetracyclines, were identified as interfering with Tn916 regulation. Macrolides, lincosamides and streptogramins appeared to activate the transfer of Tn916 at unprecedented levels, in a Tet(M)-independent way that implies a yet undescribed regulatory mechanism for controlling the mobility of the element. Conclusions: These results demonstrate that some ribosome-targeting antibiotics can induce the transfer of a given mobile genetic element, here Tn916, although it does not provide any resistance determinant for most of the triggering drugs. This implies that specific antibiotic therapies can have dramatic impacts on the dissemination of unexpected and unlinked resistance genes, with the clear risk of reducing our therapeutic potential for later treatments.

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

Salmonellosis 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.

Natural microbial communities supporting the transfer of the IncP-1ß plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group.

Antibiotic resistance gene transfer mediated by plasmids is a matter of concern for public health, but permissive environments supporting plasmid dissemination are still quite difficult to identify. Lately, we have reported a molecular approach based on quantitative PCR (qPCR) to monitor the fate of the IncP-1ß plasmid pB10 in natural microbial communities maintained in microcosms. Such plasmid transfer experiments were carried out with 13 different environmental matrices, and demonstrated that the transfer of the conjugative-proficient plasmid pB10 in complex environments is relatively rare and is strongly matrix dependent. An attempt to link the microbial community structure and the matrix permissiveness showed that TTGE analysis is not resolutive enough to point out common features among comparable communities supporting pB10 transfer. However, an estimation of the IncP-1α/IncP-1ß plasmids abundance by qPCR demonstrated that pB10 transfer tends to be supported by environmental matrices exhibiting a higher content of IncP-1 plasmids. We suggest that the relative abundance of IncP-1 plasmids in a given microbial community reflects its permissiveness to the transfer of plasmids belonging to the same incompatibility group, which prevails over transfer limitation due to a phenomenon known as superinfection immunity.

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

Plasmid-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.

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

Cupriavidus 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.

Physicochemical properties and cellular toxicity of (poly)aminoalkoxysilanes-functionalized ZnO quantum dots.

Luminescent ZnO nanocrystals were synthesized by basic hydrolysis of Zn(OAc)(2) in the presence of oleic acid and then functionalized with (poly)aminotrimethoxysilanes in the presence of tetramethylammonium hydroxide to render the QDs water-dispersible. The highest photoluminescence quantum yield (17%) was achieved using N(1)-(2-aminoethyl)-N(2)-[3-(trimethoxysilyl)propyl]-1,2-ethanediamine as surface ligand. Transmission electron microscopy and powder x-ray diffraction showed highly crystalline materials with a ZnO nanoparticle diameter of about 4 nm. The cytotoxicity of the different siloxane-capped ZnO QDs towards growing Escherichia coli bacterial cells was evaluated in MOPS-minimal medium. Although concentrations of 5 mM in QDs caused a complete growth arrest in E. coli, siloxane-capped ZnO QDs appeared weakly toxic at lower doses (0.5 or 1 mM). The concentration of bioavailable Zn (2+) ions leaked from ZnO QDs was evaluated using the biosensor bacteria Cupriavidus metallidurans AE1433. The results obtained clearly demonstrate that concentrations of bioavailable Zn(2+) are too low to explain the inhibitory effects of the ZnO QDs against bacteria cells at 1 mM and that the siloxane shell prevents ZnO QDs from dissolution contrary to uncapped ZnO nanoparticles. Because of their low cytotoxicity, good biocompatibility, low cost and large number of functional amine end groups, which makes them easy to tailor for end-user purposes, siloxane-capped ZnO QDs offer a high potential as fluorescent probes and as biosensors.

The exposure of bacteria to CdTe-core quantum dots: the importance of surface chemistry on cytotoxicity.

A series of water-soluble CdTe-core quantum dots (QDs) with diameters below 5.0 nm and functionalized at their surface with polar ligands such as thioglycolic acid (TGA) or the tripeptide glutathione (GSH) were synthesized and characterized by UV-vis absorption spectroscopy, their photoluminescence measurements, atomic force microscopy (AFM) and transmission electron microscopy (TEM). Because cell elongations and growth inhibitions were observed during labeling experiments, the cytotoxicity of CdTe-core QDs was investigated. Using growth inhibition tests combining different bacterial strains with different CdTe-core QDs, it was possible to demonstrate that the cytotoxicity of QDs towards bacteria depends on exposure concentrations, surface chemistry and coating, and that it varied with the strain considered. Growth inhibition tests carried out with heavy-metal-resistant bacteria, as well as ICP-AES analyses of cadmium species released by CdTe@TGA QDs, demonstrated that the leakage of Cd2+ is not the main source of QD toxicity. Our study suggests that QD cytotoxicity is rather due to the formation of TeO2 and probably the existence of CdO formed by surface oxidation. In this respect, QDs possessing a CdO shell appeared very toxic.

Nucleic acid fluorochromes and flow cytometry prove useful in assessing the effect of chlorination on drinking water bacteria.

Flow cytometry (FCM), combined with staining using two fluorochromes (propidium iodide, PI, or SYBR Green II RNA gel stain, SYBR-II), was used to assess nucleic acid injuries to chlorinated drinking water bacteria. Highly fluorescent SYBR-II-stained bacteria were converted to bacteria with low fluorescence after chlorination. PI staining of bacteria exposed to different doses of chlorine showed membrane permeabilisation ([Cl2] < 0.2 mg L(-1)) and nucleic acid damage at higher doses ([Cl2] > 0.3 mg L(-1)). Above a threshold dose (between 1.5 and 3 mg Cl2 L(-1)), nucleic acids appeared severely damaged and incapable of being stained by PI or SYBR-II. These results constitute evidence that FCM is a promising tool for assessing drinking water bacteria injuries and for controlling chlorine disinfection efficiency much more rapidly than the standard sensitive but time-consuming heterotrophic plate count method.