[Show abstract][Hide abstract] ABSTRACT: Efflux pumps are membrane proteins involved in the active extrusion of a wide range of structurally dissimilar substrates from cells. A multidrug efflux pump named TetA belonging to the major facilitator superfamily (MFS) of transporters was identified in the Streptococcus thermophilus DSM 20617T genome. The tetA-like gene was found in the genomes of a number of S. thermophilus strains sequenced to date and in S. macedonicus ACA-DC 198, suggesting a possible horizontal gene transfer event between these two Streptococcus species, which are both adapted to the milk environment. Flow cytometry (single-cell) analysis revealed bistable TetA activity in the S. thermophilus population, and tetA-like gene over-expression resulted in a reduced susceptibility to ethidium bromide, tetracycline, and other toxic compounds even when the efflux pump was over-expressed in a strain naturally lacking tetA-like gene.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Staphylococcus aureus strains harboring QacA, QacB, QacC, QacG transporters and norA promoter up-regulating mutations were characterized by Phenotype Microarray (PM), standard methods for susceptibility testing, and ethidium bromide efflux assays, in order to increase knowledge on phenotypes associated to efflux pumps and their substrates. PM data and standard susceptibility testing lead to the identification of new potential efflux targets, such as guanidine hydrochloride or 8-hydroxyquinoline for QacA and QacC pumps, respectively. The identification of compounds to which the presence of efflux pumps induced increased susceptibility opens new perspectives for potential adjunct anti-resistance treatment (i.e. strains bearing QacB transporters showed increased susceptibility to thioridazine, amitriptyline and orphenadrine). Although the tested isolates were characterized by high degree of heterogeneity, a hallmark of clinical isolates, direct ethidium bromide efflux assays were effective in highlighting differences in efflux efficiency among strains. These data add to characterization of substrate specificity in the different classes of staphylococcal multi drug efflux systems conferring specific substrate profiles and efflux features to each of them.
Microbiological Research 01/2014; · 1.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hexavalent chromium [Cr(VI)] contamination is one of the main problems of environmental protection because the Cr(VI) is hazardous to human health. Cr(VI) form is highly toxic, mutagenic and carcinogenic, and it spreads widely beyond the site of initial contamination because of its mobility. Cr(VI), crossing the cellular membrane via the sulfate uptake pathway, generates active intermediates Cr(V) and/or Cr(IV), free radicals, and Cr(III) as the final product. Cr(III) affects DNA replication, causes mutagenesis, and alters the structure and activity of enzymes, reacting with their carboxyl and thiol groups. To persist in Cr(VI)-contaminated environments, microorganisms must have efficient systems to neutralize the negative effect of this form of chromium. The systems involve detoxification or repair strategies such as Cr(VI) efflux pumps, Cr(VI) reduction to Cr(III) and activation of enzymes involved in the ROS detoxifying processes, repair of DNA lesions, sulfur metabolism, and iron homeostasis. This review provides an overview of the processes involved in bacterial and fungal Cr(VI) resistance that have been identified through "omics" studies. A comparative analysis of the described molecular mechanisms is offered and compared to the cellular evidence obtained with classical microbiological approaches. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.
Antimicrobial Agents and Chemotherapy 08/2013; 57:3488-97. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: The acquisition of resistance may produce fitness costs (FC) impacting the possibility of spread of resistant microorganisms. Thus, measuring these fitness costs is relevant for predicting the emergence of resistance.
Objectives: To determine FC of Escherichia coli (Ec), Klebsiella pneumoniae (Kpn) and Salmonella enterica (Sal) mutants selected with biocides (triclosan, TRI; benzalkonium chloride, BZC; chlorhexidine, CHX) or antibiotics (ampicilin, Amp; ciprofloxacin, Cip)
Methods: 21 mutants exhibiting different phenotypes of biocides or antibiotics resistance were studied. FC was determined by calculating their relative growth rates in comparison with wild-types stains. Expression of gapA, acrB, acrF, acrD, marA, ramA, and soxS was measured by quantitative RT-PCR. Phenotype microarrays of carbon sources were performed for Ec.
Results and conclusions: Two of the 5 Ec mutants, presenting changes in TRI and ceftazidime susceptibility, showed high FC (26 and 75%). Six out of 9 Kpn mutants with decreased susceptibility to TRI and BKC had relevant FC (4-66%). The Kpn mutant with the highest cost exhibited reduced susceptibility to several antibiotics. Four of the 7 Sa mutants presented relevant FC. Mutants with the highest FC showed increased expression of marA (Ec) or ramA transcriptional regulators (Kpn and Sal). Ec mutants with the highest MIC to TRI and high FC metabolize carboxylic acids, aminoacids, and carbohydrates better than wild-type. FC of biocide resistance depends on the type of mutation involved. The finding that mutants with high FC can metabolize better some carbon sources indicate that FC may depend on the environment and the available nutrients.
5th Federation of European Microbiological Societies, Leipzig; 07/2013
[Show abstract][Hide abstract] ABSTRACT: We report the draft genome sequence of 34, a Cr(VI)-hyperresistant and biofilm-producing bacterium that might be used for the bioremediation of chromate-polluted soils. The genome sequence might be helpful in exploring the mechanisms involved in chromium resistance and biofilm formation.
[Show abstract][Hide abstract] ABSTRACT: The pneumococcal chromosome encodes about 140 transporters, many of which predicted to be involved in efflux. In order to critically evaluate pneumococcal efflux, a series of transporter mutants were constructed, and their phenotypes were assayed by disk diffusion, microdilution drug susceptibility testing (MIC), growth cultures at subMIC concentrations and Phenotype Microarray analysis. Mutants for seven ABC transporters, three MATE efflux pumps and one MFS transporter were obtained in S. pneumoniae strain DP1004. The susceptibility of these eleven mutants to over 250 different substances was compared to that of the parent strain. Out of the tested transporters, only the ABC transporter PatAB (SP2073-5) presented a clear MDR profile as the mutant showed significantly increased susceptibility to ethidium bromide, acriflavin and berberine. Among the other transporters analysed, the mutants devoid of the MATE efflux pump SP2065 exhibited reduced susceptibility to novobiocin and the DinF MATE family transport system (SP1939) exhibited increased susceptibility to moxifloxacin, ciprofloxacin and levofloxacin. This change in quinolone MIC was found to be independent form the competence mediated effect of quinolones on the cinA-recA-dinF operon. Furthermore the dinF mutant allowed, in contrast to the parental strain, to select for quinolone resistant mutants when exposed to moxifloxacin. These data confirm the clear MDR profile of the PatAB ABC transporter and propose for the MATE DinF a phenotype associated to quinolone susceptibility, in particular for moxifloxacin.
Antimicrobial Agents and Chemotherapy 10/2012; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The widely used biocide triclosan selectively targets FabI, the NADH-dependent trans-2-enoyl-acyl carrier protein reductase, which is an important target for narrow-spectrum antimicrobial drug development. In relation to the growing concern about biocide resistance, we compared in vitro mutants and clinical isolates of Staphylococcus aureus with reduced triclosan susceptibility. Clinical isolates of S. aureus as well as laboratory-generated mutants were assayed for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) phenotypes and genotypes related to reduced triclosan susceptibility. A potential epidemiological cut-off (ECOFF) MBC of >4 mg/L was observed for triclosan in clinical isolates of S. aureus. These showed significantly lower MICs and higher MBCs than laboratory mutants. These groups of strains also had few similarities in the triclosan resistance mechanism. Molecular analysis identified novel resistance mechanisms linked to the presence of an additional sh-fabI allele derived from Staphylococcus haemolyticus. The lack of predictive value of in-vitro-selected mutations for clinical isolates indicates that laboratory tests in the present form appear to be of limited value. More importantly, detection of sh-fabI as a novel resistance mechanism with high potential for horizontal gene transfer demonstrates for the first time that a biocide could exert a selective pressure able to drive the spread of a resistance determinant in a human pathogen.
International journal of antimicrobial agents 07/2012; 40(3):210-20. · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ecological sociobiology is an emerging field that aims to frame social evolution in terms of ecological adaptation. Here we explore the ecological context for evolution of quorum sensing diversity in bacteria, where social communication is limited to members of the same quorum sensing type (pherotype). We sampled isolates of Bacillus subtilis from soil on a microgeographical scale and identified three ecologically distinct phylogenetic groups (ecotypes) and three pherotypes. Each pherotype was strongly associated with a different ecotype, suggesting that it is usually not adaptive for one ecotype to 'listen' to the signalling of another. Each ecotype, however, contained one or more minority pherotypes shared with the other B. subtilis ecotypes and with more distantly related species taxa. The pherotype diversity within ecotypes is consistent with two models: first, a pherotype cycling model, whereby minority pherotypes enter a population through horizontal genetic transfer and increase in frequency through cheating the social interaction; and second, an occasional advantage model, such that when two ecotypes are each below their quorum densities, they may benefit from listening to one another. This is the first survey of pherotype diversity in relation to ecotypes and it will be interesting to further test the hypotheses raised and supported here, and to explore other bacterial systems for the role of ecological divergence in fostering pherotype diversity.
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas corrugata 28 represents a microorganism that can potentially be applied for in situ bioremediation of Cr(VI) contaminated sites. This strain combines a high resistance toward toxic Cr(VI) with the ability to reduce Cr(VI) to Cr(III) under oxic conditions. In this study, the aerobic reduction of Cr(VI) by Pseudomonas corrugata 28 was examined under different carbon and sulfur supply conditions to assess the influence of microbial carbon and sulfur metabolism on Cr(VI) reduction. The fate of reduced chromium was elucidated by investigating the speciation of chromium in solution as well as the interaction of chromium with bacterial surfaces. Reduction of Cr(VI) was found to be a metabolic process resulting mainly in the formation of dissolved organic Cr(III)-complexes. Small amounts of reduced chromium were weakly associated with bacterial surfaces. The formation of inorganic Cr(III)-precipitates was not indicated.
[Show abstract][Hide abstract] ABSTRACT: The effect of long-term (8 years) compost treatments (compost or compost plus mineral fertilizer) on genetic structure of bacterial and fungal populations in both bulk soil and rhizosphere of grapevine (Vitis vinifera) was analyzed in respect to a control constituted by the soil treated with mineral fertilization. Soils were sampled in early summer (July), mid-summer (August), and before harvest (October). Bacterial and fungal populations were characterized by genetic fingerprints generated by the application of 16S rDNA and ITS rDNA Multiplex Terminal Fragment Length Polymorphism (M-TRFLP) technique. Compost induced no significant differences at any time on microbial communities from bulk soil samples, whereas seasonal variations significantly affected both bacterial and fungal populations as indicated by the Multi Dimensional Scaling (MDS) ordination method of the M-TRFLPs results. MDS analysis of grapevine rhizosphere M-TRFLPs showed that temporal separation was significant for the bacterial population only. Results suggested that soil microbial populations in vineyard productive ecosystems may be sensitive to environmental changes induced by seasonal variations and show a certain degree of resilience to different agricultural practices.
Geomicrobiology Journal - GEOMICROBIOL J. 01/2012; 29(6):506-519.
[Show abstract][Hide abstract] ABSTRACT: The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium:solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection.
PLoS ONE 01/2012; 7(3):e33320. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Characterization of bacterial communities in oil-contaminated soils and evaluation of their degradation capacities may serve as a guide for improving remediation of such environments. Using physiological and molecular methods, the aim of this work was to characterize 17 Acinetobacter strains (13 species) able to use diesel fuel oil as sole carbon and energy source. The strains were first tested for their ability to grow on different alkanes on minimal medium containing high NaCl concentrations. The envelope hydrophobicity of each strain was assessed by microbial adhesion to the hydrocarbon test (MATH) when grown in LB medium or minimal medium containing succinate or diesel fuel. Most strains were hydrophobic both in LB and minimal medium, except for strain Acinetobacter venetianus VE-C3 that was hydrophobic only in minimal medium. Furthermore, two A. venetianus strains, RAG-1(T) and LUH 7437, and strain ATCC 17905 (genomic species 13BJ) displayed biosurfactant activity. The alkM gene encoding alkane hydroxylase was detected in the chromosome of the 15 strains by PCR amplification, sequencing and Southern blot analysis. Phenotype microarray analysis performed on the five A. venetianus strains revealed that they differentially used purines as N-source and confirmed that they are unable to use carbohydrates.
Research in Microbiology 12/2011; 163(3):161-72. · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bacteria are known to adopt complex metabolic strategies in an effort to counteract the impact of numerous toxic compounds. In this study, a Cr(VI)-sensitive mutant of the Cr(VI)-hyperresistant bacterium Pseudomonas corrugata 28, obtained by insertional mutagenesis using the EZ-Tn5™ <R6Kγori/KAN-2>Tnp, was employed to gain a greater understanding of Cr(VI) resistance in bacteria. The insertion of the transposon, which occurred 16 bp upstream from the start codon of an ORF encoding a soluble pyridine nucleotide transhydrogenase (STH), negatively affected expression of the sth gene. The compromised expression of the sth gene in the mutant had two main effects on the pyridine nucleotide pools: (i) a decrease in NADPH and NADH fractions with a consequent shift in the redox state toward oxidation; and (ii) a decrease in the total concentration of the pyridine nucleotides. In the absence of a suitable pool of NADPH, the mutant failed to sustain an effective defense against the oxidative stress induced by Cr(VI).
Research in Microbiology 07/2011; 162(8):747-55. · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phenotype MicroArray (PM) permits the characterisation of bacteria under nearly 2000 culture conditions. The PM standard procedure for the chemical sensitivity analysis of Gram-positive bacteria failed in the analysis of Streptococcus thermophilus. Therefore, we developed an efficient and reproducible protocol to obtain a chemically sensitive profile of S. thermophilus using PM.
Journal of microbiological methods 06/2011; 86(2):258-61. · 2.43 Impact Factor