[Show abstract][Hide abstract] ABSTRACT: In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.
[Show abstract][Hide abstract] ABSTRACT: Background and aims
The establishment of a successful symbiosis between the nitrogen-fixing bacterium Ensifer meliloti and compatible host legumes (Medicago spp.) depends on a complex molecular signal exchange. The early stage of signaling involves the release from plant roots of the flavonoid luteolin, which in turn induces the expression of rhizobia nodulation (nod) genes required for root infection and nodule development. To date, the bacterial response to the luteolin perception has been characterized in detail as far as gene expression is concerned. Nevertheless, despite this molecular information, a global view on E. meliloti phenotypes affected by the plant signal luteolin is still lacking. Therefore, an extensive phenotypic investigation of luteolin effect on the nitrogen-fixing E. meliloti 3001 has been performed.
A thousand different growth conditions, including sensitivity to osmolites, pH stresses, antibiotics and toxic compounds, were tested by the application of the high-throughput Phenotype MicroArray (PM) technology, as well as by several dedicated assays to evaluate growth stimulation, motility, biofilm formation, N-acyl homoserine lactones and Indole-3- acetic acid (IAA) production.
Results revealed that the plant signal luteolin affected a wide spectrum of E. meliloti 3001 phenotypes. E. meliloti 3001 displayed an enhanced resistance phenotype in the presence of luteolin toward a broad set of chemicals including several antibiotics, toxic ions, respiration inhibitors, membrane damagers, DNA intercalants and other potential antimicrobial agents. Moreover, the presence of luteolin significantly reduced overall AHLs production, as well as the lag phase in relation to the starting cellular density, the motility and biofilm formation under nutrient-limited growth conditions. An effect on E. meliloti indole-3-acetic acid (IAA) production was also detected in vitro as a response to luteolin.
Overall, these findings suggest that the plant signal luteolin triggers a broad response in E. meliloti 3001, which was shown to be dependent on nutritional conditions sensed by the bacterium, pointing out a wide role in modifying rhizobial phenotypes, possibly in relation to plant root association and then symbiotic interaction.
[Show abstract][Hide abstract] ABSTRACT: Biocides are used without restriction for several purposes. As a consequence, large amounts of biocides are released without any control in the environment, a situation that can challenge the microbial population dynamics, including selection of antibiotic resistant bacteria. Previous work has shown that triclosan selects Stenotrophomonas maltophilia antibiotic resistant mutants overexpressing the efflux pump SmeDEF and induces expression of this pump triggering transient low-level resistance. In the present work we analyze if two other common biocides, benzalkonium chloride and hexachlorophene, trigger antibiotic resistance in S. maltophilia. Bioinformatic and biochemical methods showed that benzalkonium chloride and hexachlorophene bind the repressor of smeDEF, SmeT. Only benzalkonium chloride triggers expression of smeD and its effect in transient antibiotic resistance is minor. None of the hexachlorophene-selected mutants was antibiotic resistant. Two benzalkonium chloride resistant mutants presented reduced susceptibility to antibiotics and were impaired in growth. Metabolic profiling showed they were more proficient than their parental strain in the use of some dipeptides. We can then conclude that although bioinformatic predictions and biochemical studies suggest that both hexachlorophene and benzalkonium chloride should induce smeDEF expression leading to transient S. maltophilia resistance to antibiotics, phenotypic assays showed this not to be true. The facts that hexachlorophene resistant mutants are not antibiotic resistant and that the benzalkonium chloride resistant mutants presenting altered susceptibility to antibiotics were impaired in growth suggests that the risk for the selection (and fixation) of S. maltophilia antibiotic resistant mutants by these biocides is likely low, at least in the absence of constant selection pressure.
[Show abstract][Hide abstract] ABSTRACT: There is a growing concern by regulatory authorities for the selection of antibiotic resistance caused by the use of biocidal products. We aimed to complete the detailed information on large surveys by investigating the relationship between biocide and antibiotic susceptibility profiles of a large number of Staphylococcus aureus isolates using four biocides and antibiotics commonly used in clinical practice. The minimal inhibitory concentration (MIC) for most clinically-relevant antibiotics was determined according to the standardized methodology for over 1600 clinical S. aureus isolates and compared to susceptibility profiles of benzalkonium chloride, chlorhexidine, triclosan, and sodium hypochlorite. The relationship between antibiotic and biocide susceptibility profiles was evaluated using non-linear correlations.
The main outcome evidenced was an absence of any strong or moderate statistically significant correlation when susceptibilities of either triclosan or sodium hypochlorite were compared for any of the tested antibiotics. On the other hand, correlation coefficients for MICs of benzalkonium chloride and chlorhexidine were calculated above 0.4 for susceptibility to quinolones, beta-lactams, and also macrolides.
Our data do not support any selective pressure for association between biocides and antibiotics resistance and furthermore do not allow for a defined risk evaluation for some of the compounds. Importantly, our data clearly indicate that there does not involve any risk of selection for antibiotic resistance for the compounds triclosan and sodium hypochlorite. These data hence infer that biocide selection for antibiotic resistance has had so far a less significant impact than feared.
Full-text · Article · Mar 2015 · Current pharmaceutical design
[Show abstract][Hide abstract] ABSTRACT: We performed a longitudinal study (repeated observations of the same sample over time) to investigate both the composition and structure of temporal changes of bacterial community composition in soil mesocosms, subjected to three different treatments (water and 5 or 25 mg kg(-1) of dried soil Cd(2+)). By analogy with the pan genome concept, we identified a core bacteriome and an accessory bacteriome. Resident taxa were assigned to the core bacteriome, while occasional taxa were assigned to the accessory bacteriome. Core and accessory bacteriome represented roughly 35 and 50 % of the taxa detected, respectively, and were characterized by different taxonomic signatures from phylum to genus level while 15 % of the taxa were found to be unique to a particular sample. In particular, the core bacteriome was characterized by higher abundance of members of Planctomycetes, Actinobacteria, Verrucomicrobia and Acidobacteria, while the accessory bacteriome included more members of Firmicutes, Clamydiae and Proteobacteria, suggesting potentially different responses to environmental changes of members from these phyla. We conclude that the pan-bacteriome model may be a useful approach to gain insight for modeling bacterial community structure and inferring different abilities of bacteria taxa.
Full-text · Article · Jan 2015 · Antonie van Leeuwenhoek
[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.
Full-text · Article · Jan 2015 · Journal of The Royal Society Interface
[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.
No preview · Article · Jan 2015 · Methods in Molecular Biology
[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.
Full-text · Article · Dec 2014 · Microbiological Research
[Show abstract][Hide abstract] ABSTRACT: Background
The stoned olive pomace (SOP), which represents approximately 50% of the conversion process of olive in oil, is largely not-utilized and creates costs for its disposal and problems related to environmental impact. In-vitro trial experiments were employed to study the effect of feeds integrated with this bio-waste rich in polyphenols on rumen biohydrogenation, using sheep rumen liquor as inoculum.ResultsFatty acid (FA) analysis and polymerase chain reaction denaturing gradient gel electrophoresis (DGGE) approach aimed to microbial community characterization showed that including SOP in feeds at the level of 50 g/kg and 90 g/kg induced changes in FA profile and in microbial populations. The contemporary decrease of Butyrivibrio proteoclasticus and the accumulation of vaccenic acid was observed. A depression of Neisseria weaveri, Ruminobacter amylophilus and other unclassified bacteria related to members of the Lachnospiraceae and Pasteurellaceae families was detected, suggesting that these microbial groups may be involved in rumen biohydrogenation.Conclusions
Supplementation of feeds with SOP changes rumen bacterial community, including bacteria responsible for vaccenic acid hydrogenation to stearic acid, thus modifying FA profile of rumen liquor. Hence, an use of SOP aimed to produce meat or dairy products enriched in functional lipids could be hypothesized.
Full-text · Article · Nov 2014 · BMC Veterinary Research
[Show abstract][Hide abstract] ABSTRACT: Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections.
Full-text · Article · Nov 2014 · Veterinary Microbiology
[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.
[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: 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.
Preview · Article · Nov 2013 · FEMS microbiology reviews
[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.
Full-text · Article · Aug 2013 · Antimicrobial Agents and Chemotherapy
[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.
[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.
Full-text · Article · Jan 2013 · Genome Announcements