HME, NFE, and HAE-1 efflux pumps in Gram-negative bacteria: a comprehensive phylogenetic and ecological approach.

The three primary resistance-nodulation-cell division (RND) efflux pump families (heavy metal efflux [HME], nodulation factor exporter [NFE], and hydrophobe/amphiphile efflux-1 [HAE-1]) are almost exclusively found in Gram-negative bacteria and play a major role in resistance against metals and bacterial biocides, including antibiotics. Despite their significant societal interest, their evolutionary history and environmental functions are poorly understood. Here, we conducted a comprehensive phylogenetic and ecological study of the RND permease, the subunit responsible for the substrate specificity of these efflux pumps. From 920 representative genomes of Gram-negative bacteria, we identified 6205 genes encoding RND permeases with an average of 6.7 genes per genome. The HME family, which is involved in metal resistance, corresponds to a single clade (21.8% of all RND pumps), but the HAE-1 and NFE families had overlapping distributions among clades. We propose to restrict the HAE-1 family to two phylogenetic sister clades, representing 41.8% of all RND pumps and grouping most of the RND pumps involved in multidrug resistance. Metadata associated with genomes, analyses of previously published metagenomes, and quantitative Polymerase Chain Reaction (qPCR) analyses confirmed a significant increase in genes encoding HME permeases in metal-contaminated environments. Interestingly, and possibly related to their role in root colonization, genes encoding HAE-1 permeases were particularly abundant in the rhizosphere. In addition, we found that the genes encoding these HAE-1 permeases are significantly less abundant in marine environments, whereas permeases of a new proposed HAE-4 family are predominant in the genomes of marine strains. These findings emphasize the critical role of the RND pumps in bacterial resistance and adaptation to diverse ecological niches.

Versatility of Stenotrophomonas maltophilia: Ecological roles of RND efflux pumps.

S. maltophilia is a widely distributed bacterium found in natural, anthropized and clinical environments. The genome of this opportunistic pathogen of environmental origin includes a large number of genes encoding RND efflux pumps independently of the clinical or environmental origin of the strains. These pumps have been historically associated with the uptake of antibiotics and clinically relevant molecules because they confer resistance to many antibiotics. However, considering the environmental origin of S. maltophilia, the ecological role of these pumps needs to be clarified. RND efflux systems are highly conserved within bacteria and encountered both in pathogenic and non-pathogenic species. Moreover, their evolutionary origin, conservation and multiple copies in bacterial genomes suggest a primordial role in cellular functions and environmental adaptation. This review is aimed at elucidating the ecological role of S. maltophilia RND efflux pumps in the environmental context and providing an exhaustive description of the environmental niches of S. maltophilia. By looking at the substrates and functions of the pumps, we propose different involvements and roles according to the adaptation of the bacterium to various niches. We highlight that i°) regulatory mechanisms and inducer molecules help to understand the conditions leading to their expression, and ii°) association and functional redundancy of RND pumps and other efflux systems demonstrate their complex role within S. maltophilia cells. These observations emphasize that RND efflux pumps play a role in the versatility of S. maltophilia.

Growth of Stenotrophomonas maltophilia and expression of Sme efflux pumps encoding genes in the presence of supernatants from amoebal and bacterial co-cultures: towards the role of amoebal secondary metabolites.

Resistance-Nodulation-Division (RND) efflux pumps are relevant determinants of Stenotrophomonas maltophilia multidrug resistance as they can extrude a broad range of antibiotics and compounds involved in virulence and physiological functions. S. maltophilia, an environmental bacterium, was shown to be associated with amoebae and able to multiply inside them. To explore whether S. maltophilia RND efflux pumps play a role when interacting with amoebae, we evaluated the effect of amoebal culture and co-culture supernatants on the growth of S. maltophilia and the expression of sme efflux pump genes. Acanthamoeba castellanii and Willaertia magna were used as amoebal models and strain S. maltophilia BurE1 as bacterial one. Our data showed that both bacterial growth and sme gene expression were not modified by amoebal culture supernatants. On the contrary, co-culture supernatants negatively impacted the growth of BurE1 and induced the expression of three out of eight efflux pump genes, i.e. smeE, smeN and smeZ. Finally, we evidenced the production of A. castellanii secondary metabolites, putatively belonging to the diterpene family, in the amoebal supernatant and in the co-culture supernatant of A. castellanii and BurE1. Whether these compounds act directly as substrates of the efflux pumps and/or inducers of the sme genes need further investigations.

Comparative Genomics of Environmental and Clinical Burkholderia cenocepacia Strains Closely Related to the Highly Transmissible Epidemic ET12 Lineage.

The Burkholderia cenocepacia epidemic ET12 lineage belongs to the genomovar IIIA including the reference strain J2315, a highly transmissible epidemic B. cenocepacia lineage. Members of this lineage are able to cause lung infections in immunocompromised and cystic fibrosis patients. In this study, we describe the genome of F01, an environmental B. cenocepacia strain isolated from soil in Burkina Faso that is, to our knowledge, the most closely related strain to this epidemic lineage. A comparative genomic analysis was performed on this new isolate, in association with five clinical and one environmental B. cenocepacia strains whose genomes were previously sequenced. Antibiotic resistances, virulence phenotype, and genomic contents were compared and discussed with an emphasis on virulent and antibiotic determinants. Surprisingly, no significant differences in antibiotic resistance and virulence were found between clinical and environmental strains, while the most important genomic differences were related to the number of prophages identified in their genomes. The ET12 lineage strains showed a noticeable greater number of prophages (partial or full-length), especially compared to the phylogenetically related environmental F01 strain (i.e., 5-6 and 3 prophages, respectively). Data obtained suggest possible involvements of prophages in the clinical success of opportunistic pathogens.

Survival and growth of Stenotrophomonas maltophilia in free-living amoebae (FLA) and bacterial virulence properties.

Stenotrophomonas maltophilia is found ubiquitously in the environment and is an important emerging nosocomial pathogen. S. maltophilia has been recently described as an Amoebae-Resistant Bacteria (ARB) that exists as part of the microbiome of various free-living amoebae (FLA) from waters. Co-culture approaches with Vermamoeba vermiformis demonstrated the ability of this bacterium to resist amoebal digestion. In the present study, we assessed the survival and growth of six environmental and one clinical S. maltophilia strains within two amoebal species: Acanthamoeba castellanii and Willaertia magna. We also evaluated bacterial virulence properties using the social amoeba Dictyostelium discoideum. A co-culture approach was carried out over 96 hours and the abundance of S. maltophilia cells was measured using quantitative PCR and culture approach. The presence of bacteria inside the amoeba was confirmed using confocal microscopy. Our results showed that some S. maltophilia strains were able to multiply within both amoebae and exhibited multiplication rates up to 17.5 and 1166 for A. castellanii and W. magna, respectively. In contrast, some strains were unable to multiply in either amoeba. Out of the six environmental S. maltophilia strains tested, one was found to be virulent. Surprisingly, this strain previously isolated from a soil amoeba, Micriamoeba, was unable to infect both amoebal species tested. We further performed an assay with a mutant strain of S. maltophilia BurA1 lacking the efflux pump ebyCAB gene and found the mutant to be more virulent and more efficient for intra-amoebal multiplication. Overall, the results obtained strongly indicated that free-living amoebae could be an important ecological niche for S. maltophilia.

Diversity of free-living amoebae in soils and their associated human opportunistic bacteria.

Free-living amoebae (FLA) are ubiquitous protozoa found worldwide in the environment. They feed by phagocytosis on various microorganisms. However, some bacteria, i.e., amoebae-resistant bacteria (ARB) or bacterial endocytobionts, can resist phagocytosis and even multiply inside FLA. This study investigated the diversity of culturable FLA in various soils from agricultural and mining sites and their bacterial endocytobionts. FLA were cultured on non-nutrient agar with alive Escherichia coli and identified by PCR and sequencing. Amoebae were lysed and bacterial endocytobionts were cultured on TSA 1/10 and Drigalski medium. Bacterial isolates were identified by PCR and 16S rDNA sequencing and characterized for their antibiotic resistance properties. To measure bacterial virulence, the amoebal model Dictyostelium discoideum was used. The analysis of FLA diversity showed that Tetramitus was the most prevalent genus in agricultural soil from Burkina Faso (73%) and garden soil from Vietnam (42%) while Naegleria and Acanthamoeba were dominant genera in mining soil from Vietnam (55%) and French alpine soil (77%). Some genera were only present in one out of the four soils analyzed. The bacterial endocytobiont included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Human opportunistic pathogens identified as Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Burkholderia cepacia were found associated with amoebae including Micriamoeba, Tetramitus, Willaertia, or Acanthamoeba. Some of these bacteria showed various antibiotic resistance phenotypes and were virulent. Our study confirms that the occurrence of these opportunistic bacteria with FLA in soils may be important for the survival, multiplication, and spread of pathogens in the environment.

Impact of untreated urban waste on the prevalence and antibiotic resistance profiles of human opportunistic pathogens in agricultural soils from Burkina Faso.

This study examined the long-term effects of the landfill disposal of untreated urban waste for soil fertilization on the prevalence and antibiotic resistance profiles of various human opportunistic pathogens in soils from Burkina Faso. Samples were collected at three sites in the periphery of Ouagadougou during two campaigns in 2008 and 2011. At each site, amendment led to changes in physico-chemical characteristics as shown by the increase in pH, CEC, total C, total N, and metal contents. Similarly, the numbers of total heterotrophic bacteria were higher in the amended fields than in the control ones. No sanitation indicators, i.e., coliforms, Staphylococci, and Enterococci, were detected. Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) were detected at a low level in one amended field. Stenotrophomonas maltophilia was detected from both campaigns at the three sites in the amended fields and only once in an unamended field. Diversity analysis showed some opportunistic pathogen isolates to be closely related to reference clinical strains responsible for nosocomial- or community-acquired infections in Northern countries. Antibiotic resistance tests showed that P. aeruginosa and Bcc isolates had a wild-type phenotype and that most S. maltophilia isolates had a multi-drug resistance profile with resistance to 7 to 15 antibiotics. Then we were able to show that amendment led to an increase of some human opportunistic pathogens including multi-drug resistant isolates. Although the application of untreated urban waste increases both soil organic matter content and therefore soil fertility, the consequences of this practice on human health should be considered.

Draft Genome Sequences of Stenotrophomonas maltophilia Strains Sm32COP, Sm41DVV, Sm46PAILV, SmF3, SmF22, SmSOFb1, and SmCVFa1, Isolated from Different Manures in France.

Stenotrophomonas maltophilia is a major opportunistic human pathogen responsible for nosocomial infections. Here, we report the draft genome sequences of Sm32COP, Sm41DVV, Sm46PAILV, SmF3, SmF22, SmSOFb1, and SmCVFa1, isolated from different manures in France, which provide insights into the genetic determinism of intrinsic or acquired antibiotic resistance in this species.

Occurrence of Stenotrophomonas maltophilia in agricultural soils and antibiotic resistance properties.

The occurrence of Stenotrophomonas maltophilia was monitored in organic amendments and agricultural soils from various sites in France and Tunisia. S. maltophilia was detected in horse and bovine manures, and its abundance ranged from 0.294 (±0.509) × 10(3) to 880 (±33.4) × 10(3) CFU (g drywt)(-1) of sample. S. maltophilia was recovered from most tested soil samples (104/124). Its abundance varied from 0.33 (±0.52) to 414 (±50) × 10(3) CFU (g drywt)(-1) of soil and was not related to soil characteristics. Antibiotic resistance properties of a set of environmental strains were compared to a clinical set, and revealed a high diversity of antibiotic resistance profiles, given both the numbers of resistance and the phenotypes. Manure strains showed resistance phenotypes, with most of the strains resisting between 7 and 9 antibiotics. While French soil strains were sensitive to most antibiotics tested, some Tunisian strains displayed resistance phenotypes close to those of clinical French strains. Screening for metal resistance among 66 soil strains showed a positive relationship between antibiotic and metal resistance. However, the prevalence of antibiotic resistance phenotypes in the studied sites was not related to the metal content in soil samples.

Comparative Genomics of Environmental and Clinical Stenotrophomonas maltophilia Strains with Different Antibiotic Resistance Profiles.

Stenotrophomonas maltophilia, a ubiquitous Gram-negative γ-proteobacterium, has emerged as an important opportunistic pathogen responsible for nosocomial infections. A major characteristic of clinical isolates is their high intrinsic or acquired antibiotic resistance level. The aim of this study was to decipher the genetic determinism of antibiotic resistance among strains from different origins (i.e., natural environment and clinical origin) showing various antibiotic resistance profiles. To this purpose, we selected three strains isolated from soil collected in France or Burkina Faso that showed contrasting antibiotic resistance profiles. After whole-genome sequencing, the phylogenetic relationships of these 3 strains and 11 strains with available genome sequences were determined. Results showed that a strain's phylogeny did not match their origin or antibiotic resistance profiles. Numerous antibiotic resistance coding genes and efflux pump operons were revealed by the genome analysis, with 57% of the identified genes not previously described. No major variation in the antibiotic resistance gene content was observed between strains irrespective of their origin and antibiotic resistance profiles. Although environmental strains generally carry as many multidrug resistant (MDR) efflux pumps as clinical strains, the absence of resistance-nodulation-division (RND) pumps (i.e., SmeABC) previously described to be specific to S. maltophilia was revealed in two environmental strains (BurA1 and PierC1). Furthermore the genome analysis of the environmental MDR strain BurA1 showed the absence of SmeABC but the presence of another putative MDR RND efflux pump, named EbyCAB on a genomic island probably acquired through horizontal gene transfer.

Brilliant glyconanocapsules for trapping of bacteria.

Nanoprecipitation of miglyol into droplets surrounded by a functional glycopolymer generates nanocapsules of biointerest. Fluorophores are trapped in situ or post-grafted onto the crosslinked polymer shell for efficient imaging. The resulting colloids induce aggregation of bacteria through strong specific interactions and promote their facile removal.

Antibiotic and metal resistance among hospital and outdoor strains of Pseudomonas aeruginosa.

Phenotypic analyses of antibiotic and metal resistance of a collection of 130 strains of Pseudomonas aeruginosa from various outdoor (i.e. soil, water, animals) and hospital (environment, patients, individuals with cystic fibrosis) settings were performed. Resistance was scored according to the origin of the strains and their likely exposure to antibiotics and chemicals. Most of the 76 outdoor strains showed a wild-type antibiotic resistance phenotype, i.e. resistance to minocycline and trimethoprim-sulfamethoxazole. Sixty percent of hospital strains showed a multiresistance phenotype (from 3 to 16 antibiotics) and confirmed that frequent exposure to antibiotics favored selection and maintenance of antibiotic resistance in P. aeruginosa. Twelve percent of outdoor strains naturally exposed to antiseptics and hydrocarbons showed significant resistance profiles, suggesting that chemical contaminants could contribute to selection of antibiotic resistance. For metal resistance, outdoor strains were more frequently resistant to zinc and cadmium, whereas hospital strains were more frequently resistant to mercury and copper. Differences in metal resistance between the 130 strains investigated were not related to previously characterized processes such as those implicating czcA, involved in cadmium, zinc, and cobalt resistance, or copA and copB, involved in copper resistance. Regulatory or new processes were likely to have contributed to the observed variations. Strains showing strong resistance to antibiotics were the least resistant to metals, and inversely. The lack of significant correlations between antibiotic and metal resistance suggests involvement of distinct processes that are rarely co-selected. The effects of the P. aeruginosa collection size and multi-factorial selective pressure on data sets are discussed.

Autoinducer production and quorum-sensing dependent phenotypes of Pseudomonas aeruginosa vary according to isolation site during colonization of intubated patients.

BACKGROUND: Pseudomonas aeruginosa frequently colonizes and is responsible for severe ventilator-associated pneumonia in intubated patients. A quorum-sensing (QS) circuit, depending on the production of the two QS-signaling molecules (autoinducers, AIs) 3-oxo-C12-HSL and C4-HSL, regulates the production by P. aeruginosa of several virulence factors and is required for biofilm formation. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies. However the precise role of QS during colonization and subsequent infections of intubated patients remains unclear. RESULTS: We wondered whether QS is active during colonization of intubated patients, and whether P. aeruginosa isolates growing inside the biofilm covering the intubation devices and those resident in the lungs of colonized patients differ in their QS-dependent phenotypes. We collected the intubation devices of eight patients colonized by P. aeruginosa. We detected 3-oxo-C12-HSL on eight, and C4-HSL on six of these devices. In three of these patients we also obtained P. aeruginosa isolates from tracheal aspirates at the time of extubation (n = 18), as well as isolates from the intubation devices (n = 25). We genotyped these isolates, quantified their AIs production, and determined three QS-dependent phenotypes (adherence capacity, biofilm and elastase production). The production of 3-oxo-C12-HSL was consistently increased for isolates from the intubation devices, whereas the production of C4-HSL was significantly higher for isolates from tracheal aspirates. Isolates from tracheal aspirates produced significantly higher amounts of elastase but less biofilm, and had a marginally reduced adhesion capacity than isolates from the intubation devices. Levels of 3-oxo-C12-HSL and elastase production correlated statistically for tracheal intubation isolates, whereas levels of 3-oxo-C12-HSL production and adhesion ability, as well as biofilm production, correlated weakly amongst intubation device isolates. CONCLUSION: Our findings demonstrate that autoinducers are produced during the colonization of intubated patients by P. aeruginosa. The microenvironment, in which P. aeruginosa grows, may select for bacteria with different capacities to produce autoinducers and certain QS-dependent phenotypes. QS-inhibition might therefore affect differently isolates growing inside the biofilm covering intubation devices and those resident in the lungs.

Characterization of a novel selenium methyltransferase from freshwater bacteria showing strong similarities with the calicheamicin methyltransferase.

A novel group of Se-methyltransferases is presented. The genetic determinant, named mmtA, which revealed this group was isolated from selenite and selenate-resistant freshwater bacteria. E. coli expressing mmtA and grown with a Se supplement emitted dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe). Phylogenetic analysis divided MmtA-like bacterial sequences into two clusters, one grouping MmtA with S- and O-methyltransferases, and one grouping UbiE C-methyltransferases. Se methylation by some of these MmtA phyletic neighbours was investigated.

Characterization of cell-to-cell signaling-deficient Pseudomonas aeruginosa strains colonizing intubated patients.

Cell-to-cell signaling involving N-acyl-homoserine lactone compounds termed autoinducers (AIs) is instrumental to virulence factor production and biofilm development by Pseudomonas aeruginosa. In order to determine the importance of cell-to-cell signaling during the colonization of mechanically ventilated patients, we collected 442 P. aeruginosa pulmonary isolates from 13 patients. Phenotypic characterization showed that 81% of these isolates produced the AI-dependent virulence factors elastase, protease, and rhamnolipids. We identified nine genotypically distinct P. aeruginosa strains. Six of these strains produced AIs [N-butanoyl-homoserine lactone or N-(3-oxo-dodecanoyl)-homoserine lactone] and extracellular virulence factors (elastase, total exoprotease, rhamnolipid, hydrogen cyanide, or pyocyanin) in vitro. Three of the nine strains were defective in the production of both AIs and extracellular virulence factors. Two of these strains had mutational defects in both the lasR and rhlR genes, which encode the N-acyl-homoserine lactone-dependent transcriptional regulators LasR and RhlR, respectively. The third of these AI-deficient strains was only mutated in the lasR gene. Our observations suggest that most, but not all, strains colonizing intubated patients are able to produce virulence factors and that mutations affecting the cell-to-cell signaling circuit are preferentially located in the transcriptional regulator genes.

Biofilm formation by Pseudomonas aeruginosa: role of the C4-HSL cell-to-cell signal and inhibition by azithromycin.

OBJECTIVES: In Pseudomonas aeruginosa, biofilm formation is controlled by a cell-to-cell signalling circuit relying on the secretion of 3-oxo-C12-HSL and C4-HSL. Previous studies suggested that C4-HSL plays no significant role in biofilm formation. However the wild-type PAO1 strain PAO-BI, used as a control in these studies is itself impaired in the production of C4-HSL. We wondered therefore whether the role of C4-HSL in biofilm formation might have been underestimated, and whether azithromycin inhibits biofilm formation by interfering with cell-to-cell signalling. METHODS: We used isogenic mutants of wild-type PAO1 strains PAO-BI and PT5 in a static biofilm model. Biofilm formation was quantified using Crystal Violet staining and exopolysaccharide measurements. RESULTS: Wild-type strain PAO-BI, as a result of its reduced C4-HSL secretion, produced 40% less biofilm compared with the wild-type PAO1 strain PT5. Using isogenic mutants of strain PT5 we have shown that whereas a lasI mutant (deficient in 3-oxo-C12-HSL) produced similar amounts of biofilm to the wild-type, a rhlI mutant (deficient in C4-HSL) produced 70% less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C4-HSL. Azithromycin, known to reduce the production of both 3-oxo-C12-HSL and C4-HSL, inhibited biofilm formation of wild-type PT5 by 45%. This inhibition could be reversed by the addition of both cell-to-cell signals. CONCLUSIONS: Our results indicate that C4-HSL also plays a significant role in biofilm formation. Furthermore, we demonstrate the potential of using cell-to-cell signalling blocking agents such as azithromycin to interfere with biofilm formation.

Detection of Pseudomonas aeruginosa cell-to-cell signals in lung tissue of cystic fibrosis patients.

Chronic Pseudomonas aeruginosa infections lead to progressive lung tissue destruction in cystic fibrosis (CF) patients. Two bacterial cell-to-cell signals, 3-oxo-C(12)-HSL and C(4)-HSL are required for the production of several extracellular virulence factors. 3-oxo-C(12)-HSL is also required for the development of a differentiated biofilm, induces IL-8 production by epithelial cells and possesses immunomodulatory activities. These two signalling molecules are therefore believed to play a role in the pathogenesis of P. aeruginosa infections, but have never been isolated from infected human tissues. We extracted and quantified the two P. aeruginosa cell-to-cell signals from lung tissues of two CF patients infected by P. aeruginosa. 3-oxo-C(12)-HSL and C(4)-HSL were detected in the lung tissues in fmol/gram, respectively pmol/gram concentrations; the ratio C(4)-HSL/3-oxo-C(12)-HSL exceeded 100 in all tissue samples. Random Amplified Polymorphism DNA genotyping revealed that one genotype was present per lung. In vitro the P. aeruginosa isolates from the two lungs produced 3-oxo-C(12)-HSL, whereas some isolates did not produce detectable C(4)-HSL. Our results suggest that both P. aeruginosa cell-to-cell signals were produced in the lung tissue of these two cystic fibrosis patients.