[Show abstract][Hide abstract] ABSTRACT: In nature, biofilms are the most common form of bacterial growth. In biofilms, bacteria display coordinated behaviour to perform specific functions. Here, we investigated electrical signalling as a possible driver in biofilm sociobiology. Using a multi-electrode array system that enables high spatio-temporal resolution, we studied the electrical activity in two biofilm-forming strains and one non-biofilm-forming strain. The action potential rates monitored during biofilm-forming bacterial growth exhibited a one-peak maximum with a long tail, corresponding to the highest biofilm development. This peak was not observed for the non-biofilm-forming strain, demonstrating that the intensity of the electrical activity was not linearly related to the bacterial density, but was instead correlated with biofilm formation. Results obtained indicate that the analysis of the spatio-temporal electrical activity of bacteria during biofilm formation can open a new frontier in the study of the emergence of collective microbial behaviour.
Journal of the Royal Society, Interface / the Royal Society. 01/2015; 12(102).
[Show abstract][Hide abstract] ABSTRACT: Standard protocols are available in order to apply Phenotype MicroArray (PM) technology to characterize different groups of microorganisms. Nevertheless, there is the need to pay attention to several crucial steps in order to obtain high-quality and reproducible data from PM, such as the choice of the Dye mix, the type and concentration of the carbon source in metabolic experiments, the use of a buffered medium. A systematic research of auxotrophies in strains to be tested should be carefully evaluated before starting with PM experiments. Detailed protocols to obtain defined and reproducible phenotypic profiles for bacteria and yeasts are shown. Moreover, the innovative software opm R packages and DuctApe suite for the analysis of kinetic data produced by PM and panphenome description are reported.
[Show abstract][Hide abstract] ABSTRACT: Isolates of Pseudomonas aeruginosa from an outbreak of ovine mastitis were analysed.•Two clonal isolates representing distinct colony morphology types were compared.•Variations in key growth and virulence-related characteristics were found.•Both minor (SNP) and major (large deletion) variations in the genomes were identified.•Our analyses suggest that the bacterial population diverged during the outbreak.
[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: Flor yeasts of Saccharomyces cerevisiae have an innate diversity of FLO11 which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling FLO11 alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to FLO11 expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air-liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the FLO11 gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts.
PLoS ONE 01/2014; 9(11):e112141. · 3.53 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.