Low metabolic activity of biofilm formed by Enterococcus faecalis isolated from healthy humans and wild mallards (Anas platyrhynchos)

It is widely known that Enterococcus faecalis virulence is related to its biofilm formation. Although Enterococci are common commensal organisms of the gastrointestinal tract, the difference between commensal and pathogen strains remain unclear. In this study, we compare the biochemical profile of the biofilms formed by two groups of medical and two groups of commensal strains. The medical strains were isolated as pathogens from infections of urinary tract and other infections (wounds, pus and bedsores), and the commensal strains were taken from faeces of healthy volunteers and faeces of wild mallards (Anas platyrhynchos) living in an urban environment. The properties of biofilms formed by medical and commensal strains differed significantly. Commensal strains showed lower metabolic activity and glucose uptake and higher biofilm biomass than the medical ones. Consistent with glucose uptake experiments, we found that the glucose dehydrogenase gene was more expressed in medical strains. These results indicate that higher metabolic activity and lower protein concentration of E. faecalis cells within biofilms are formed during infections.This work was supported by the Medical University of Gdansk research grant (GUMed W-65) and was financed partly by University of Gdansk research grant (BW 1440-5-0099-7). We are grateful to Katarzyna Zolkos for her help in catching mallards and Magdalena Remisiewicz for correcting the English. Catarina Seabra helped in preparing assays

Comparative structural bioinformatics analysis of Bacillus amyloliquefaciens chemotaxis proteins within Bacillus subtilis group

Chemotaxis is a process in which bacteria sense their chemical environment and move towards more favorable conditions. Since plant colonization by bacteria is a multifaceted process which requires a response to the complex chemical environment, a finely tuned and sensitive chemotaxis system is needed. Members of the Bacillus subtilis group including Bacillus amyloliquefaciens are industrially important, for example, as bio-pesticides. The group exhibits plant growth-promoting characteristics, with different specificity towards certain host plants. Therefore, we hypothesize that while the principal molecular mechanisms of bacterial chemotaxis may be conserved, the bacterial chemotaxis system may need an evolutionary tweaking to adapt it to specific requirements, particularly in the process of evolution of free-living soil organisms, towards plant colonization behaviour. To date, almost nothing is known about what parts of the chemotaxis proteins are subjected to positive amino acid substitutions, involved in adjusting the chemotaxis system of bacteria during speciation. In this novel study, positively selected and purified sites of chemotaxis proteins were calculated, and these residues were mapped onto homology models that were built for the chemotaxis proteins, in an attempt to understand the spatial evolution of the chemotaxis proteins. Various positively selected amino acids were identified in semi-conserved regions of the proteins away from the known active sites

Genes Important for Catalase Activity in Enterococcus faecalis

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly

In Vitro Evaluation of Enterococcus faecalis Adhesion on Various Endodontic Medicaments

E. faecalis in endodontic infection represents a biofilm type of disease, which explains the bacteria’s resistance to various antimicrobial compounds and the subsequent failure after endodontic treatment. The purpose of this study was to compare antimicrobial activities and bacteria kinetic adhesion in vitro for three endodontic medicaments with a clinical isolate of E. faecalis. We devised a shake culture which contained the following intracanalar preparations: CPD, Endoidrox (EIX), PulpCanalSealer (PCS); these were immersed in a liquid culture medium inoculated with the microorganism. The shake system velocity was able to prevent non-specific bacteria adhesion and simulated the salivary flow. Specimens were collected daily (from both the medium and medicaments) for 10 days; the viable cells were counted by plate count, while the adhesion index AI° [E. faecalis fg DNA] /mm2 was evaluated in the pastes after DNA extraction, by quantitative real time PCR for the 16S rRNA gene. A partial growth inhibition, during the first 24 hours, was observed in the liquid medium and on the medicaments for EIX and subsequently for CPD (six logs). EIX showed the lowest adhesion coefficient (5*102 [fg DNA]/mm2) for nine days and was similar to the control. PCS showed no antimicrobial/antibiofilm properties. This showed that “calcium oxide” base compounds could be active against biofilm progression and at least in the short term (2-4 days) on E. faecalis cells growing in planktonic cultures

Bistability versus Bimodal Distributions in Gene Regulatory Processes from Population Balance

In recent times, stochastic treatments of gene regulatory processes have appeared in the literature in which a cell exposed to a signaling molecule in its environment triggers the synthesis of a specific protein through a network of intracellular reactions. The stochastic nature of this process leads to a distribution of protein levels in a population of cells as determined by a Fokker-Planck equation. Often instability occurs as a consequence of two (stable) steady state protein levels, one at the low end representing the “off” state, and the other at the high end representing the “on” state for a given concentration of the signaling molecule within a suitable range. A consequence of such bistability has been the appearance of bimodal distributions indicating two different populations, one in the “off” state and the other in the “on” state. The bimodal distribution can come about from stochastic analysis of a single cell. However, the concerted action of the population altering the extracellular concentration in the environment of individual cells and hence their behavior can only be accomplished by an appropriate population balance model which accounts for the reciprocal effects of interaction between the population and its environment. In this study, we show how to formulate a population balance model in which stochastic gene expression in individual cells is incorporated. Interestingly, the simulation of the model shows that bistability is neither sufficient nor necessary for bimodal distributions in a population. The original notion of linking bistability with bimodal distribution from single cell stochastic model is therefore only a special consequence of a population balance model

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments

Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism

Intra- and Interspecies Genomic Transfer of the Enterococcus faecalis Pathogenicity Island

Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transfered. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens