Maria de Fátima Silva Lopes

Virulence factors in food, clinical and reference enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannons index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.

Virulence of Enterococcus faecalis dairy strains in an insect model: the role of fsrB and gelE

Despite the existence of various virulence factors in the Enterococcus genus, enterococcal virulence is still a debated issue. A main consideration is the detection of the same virulence genes in strains isolated from nosocomial or community-acquired infections, and from food products. The goal of this study was to evaluate the roles of two well-characterized enterococcal virulence factors, Fsr and gelatinase, in the potential virulence of Enterococcus faecalis food strains. Virulence of unrelated Enterococcus isolates, including dairy strains carrying fsr and gelE operons, was compared in the Galleria mellonella insect model. E. faecalis dairy strains were able to kill larvae and were as virulent as strain OG1RF, one of the most widely used for virulence studies. In contrast, Enterococcus durans and Enterococcus faecium strains were avirulent or poorly virulent for G. mellonella. To evaluate the role of fsrB and gelE in virulence of E. faecalis dairy strains, both genes were deleted independently in two strains. The Delta fsrB and Delta gelE deletion mutants both produced a gelatinase-negative phenotype. Although both mutations significantly attenuated virulence in G. mellonella, the Delta fsrB strains were more strongly attenuated. These results agree with previous findings suggesting the involvement of fsrB in the control of other cell functions relevant to virulence. Our work demonstrates that the presence of functional fsrB, and to a lesser extent gelE, in dairy enterococci should be considered with caution.

Chloramphenicol·cyclodextrin inclusion compounds: co-dissolution and mechanochemical preparations and antibacterial action

β-Cyclodextrin (βCD) inclusion compounds of chloramphenicol in the solid-state were prepared via two different procedures, co-crystallisation from aqueous solution and the solvent-free method of co-grinding. Chloramphenicol inclusion into permethylated βCD (TRIMEB) was also performed by co-dissolution in ethanol. The co-crystallisation procedure was the best for preparing chloramphenicol·βCD in 1:1 stoichiometry in high purity, while with the co-grinding treatment and the same starting proportion, a complete inclusion was not achieved. Microcrystals of chloramphenicol·βCD inclusion compound presented polymorphism, crystallising simultaneously in the triclinic P1 or monoclinic C2 space groups. Only crystals of the latter were suitable for single-crystal diffraction and data for the guest atoms was comprised of a smeared-out electron cloud, so theoretical calculations were used to propose their plausible geometry and location inside the host molecules. The co-grinding procedure curbed polymorphism and allowed the preparation of chloramphenicol·βCD in the amorphous state; chloramphenicol·TRIMEB prepared by co-dissolution of the components in a 1:1 proportion was also an amorphous material. The influence of inclusion with βCD and TRIMEB on the antimicrobial performance of chloramphenicol was evaluated, and both inclusion compounds demonstrated selective action against Enterococcus faecalis strains ATCC 29212 and A33562 and Listeria monocytogenes ATCC 7644. Chloramphenicol·TRIMEB also had higher activity against the E. faecalis strains A35906, 9308 and E4856 and against Listeria inocua.

Study on the dissemination of the bcrABDR cluster in Enterococcus spp. reveals that the BcrAB transporter is sufficient to confer high-level bacitracin resistance

Bacitracin is used both in clinical practice and as an animal growth promoter in some countries. Recent reports revealed the dissemination of high-level bacitracin resistance (HLBR) in the genus Enterococcus. The bcrABD operon confers HLBR in an Enterococcus faecalis strain, and the presence of the regulator BcrR was stated as essential for this phenotype. Our objectives were to investigate the spread of the bcrABDR genes in the genus Enterococcus and to evaluate the role of this genotype in the HLBR phenotype. Fifty-eight enterococcal isolates from different environments and species were screened for the presence of the bcrABDR genes, and minimum inhibitory concentrations for bacitracin were determined. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to study the expression of bacitracin resistance genes in strains with HLBR following growth in the presence and absence of the antibiotic. The bcrABDR genotype was found associated with E. faecalis strains from clinical settings bearing a HLBR phenotype. The most disseminated mechanism of bacitracin resistance was the BcrAB ABC transporter, encoded by the bcrAB genes. This transporter, which is constitutively expressed in the absence of bcrR, was sufficient to confer HLBR. Our findings stress the need for further evaluation of bacitracin resistance in this genus.

Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors

Enterococcus faecalis V583 is a vancomycin-resistant clinical isolate which belongs to the hospital-adapted clade, CC2. This strain harbours several factors that have been associated with virulence, including the fsr quorum-sensing regulatory system that is known to control the expression of GelE and SprE proteases. To discriminate between genes directly regulated by Fsr, and those indirectly regulated as the result of protease expression or activity, we compared gene expression in isogenic mutants of V583 variously defective in either Fsr quorum sensing or protease expression. Quorum sensing was artificially induced by addition of the quorum signal, GBAP, exogenously in a controlled manner. The Fsr regulon was found to be restricted to five genes, gelE, sprE, ef1097, ef1351 and ef1352. Twelve additional genes were found to be dependent on the presence of GBAP-induced proteases. Induction of GelE and SprE by GBAP via Fsr resulted in accumulation of mRNA encoding lrgAB, and this induction was found to be lytRS dependent. Drosophila infection was used to discern varying levels of toxicity stemming from mutations in the fsr quorum regulatory system and the genes that it regulates, highlighting the contribution of LrgAB and bacteriocin EF1097 to infection toxicity. A contribution of SprE to infection toxicity was also detected. This work brought to light new players in E. faecalis success as a pathogen and paves the way for future studies on host tolerance mechanisms to infections caused by this important nosocomial pathogen.

Evaluation of antimicrobial resistance and virulence of enterococci from equipment surfaces, raw materials, and traditional cheeses.

Forty enterococci isolated along the production chains of three traditional cheeses (PDO Pecorino Siciliano, PDO Vastedda della Valle del Belìce, and Caciocavallo Palermitano) made in Sicily (southern Italy) were studied for the assessment of their antibiotic resistance and virulence by a combined phenotypic/genotypic approach. A total of 31 Enterococcus displayed resistance to at least one or more of the antimicrobials tested. The strains exhibited high percentages of resistance to erythromycin (52.5%), ciprofloxacin (35.0%), quinupristin-dalfopristin (20.0%), tetracycline (17.5%), and high-level streptomycin (5.0%). The presence of tet(M), cat(pC221), and aadE genes for resistance to tetracycline, chloramphenicol, and streptomycin, respectively, was registered in all strains with resistance phenotype. The erm(B) gene was not detected in any erythromycin-resistant strain. The Enterococcus strains were further tested by PCR for the presence of virulence genes, namely, gelE, asa1, efaA, ace, and esp. Twenty strains were positive for all virulence genes tested. Among the enterococci isolated from final cheeses, three strains (representing 33.3% of total cheese strains) were sensible to all antimicrobials tested and did not carry any virulence factor. Although this study confirmed that the majority of dairy enterococci are vectors for the dissemination of antimicrobial resistance and virulence genes, only two strains showed a high resistance to aminoglycosides, commonly administered to combat enterococci responsible for human infections. Furthermore, the presence of the strains E. casseliflavus FMAC163, E. durans FMAC134B, and E. faecium PON94 without risk determinants, found at dominating levels over the Enterococcus populations in the processed products, stimulates further investigations for their future applications in cheese making. All strains devoid of the undesired traits were isolated from stretched cheeses. Thus, this cheese typology represents an interesting environment to deepen the studies on the risk/benefit role of enterococci in fermented foods for their qualified presumption of safety (QPS) assessment.

The incongruent gelatinase genotype and phenotype in Enterococcus faecalis are due to shutting off the ability to respond to the gelatinase biosynthesis-activating pheromone (GBAP) quorum-sensing signal.

The concomitant presence of a complete fsr quorum-sensing system and gelE-sprE operons in Enterococcus faecalis is known to be essential for the detection of gelatinase activity. However, there are reports of the absence of gelatinase activity despite the presence of complete fsr and gelE loci. In order to understand this incongruence between genotype and phenotype we sequenced fsr and gelE loci of the E. faecalis LN68 strain, which was previously found to carry both operons but to lack gelatinase activity. Of the 59 nucleotide differences detected compared with the gelatinase-positive V583 strain, we found a nonsense mutation (a premature STOP codon) predicted to truncate the ATPase sensor domain of the FsrC protein, responsible for sensing and transducing the signal from the quorum-sensing molecule. Strain LN68 was highly affected in the expression of the gelE and sprE genes, further supporting the lack of Fsr-dependent gelE induction. When we constructed a V583 mutant with the same premature stop mutation in the fsrC gene the resulting strain was no longer able to degrade gelatin. We conclude that the reduced ability to transduce the quorum-sensing signal of the prematurely truncated FsrC protein is sufficient to explain the negative gelatinase phenotype. As the incongruent genotype and phenotype is detected in natural isolates, we believe that the silencing of the quorum-sensing system Fsr may be beneficial for some E. faecalis strains.

Analysis of the microcrystalline inclusion compounds of triclosan with β-cyclodextrin and its tris-O-methylated derivative.

Solid 1:1 inclusion compounds of triclosan with native and permethylated β-cyclodextrin (β-CD and TRIMEB) were prepared by co-crystallisation and co-evaporation, respectively, and studied by FT-IR and (13)C{(1)H} CP/MAS NMR spectroscopies, thermogravimetric analysis, X-ray diffraction and theoretical calculations. Results showed that triclosan inclusion into TRIMEB afforded an amorphous solid, whilst β-CD·triclosan is composed of microcrystals belonging to two different phases. In the phase featuring larger crystals, X-ray diffraction was carried out and the β-CD host units, packing head-to-head in infinite channels, were refined; the geometry for the included but highly disordered triclosan molecules was assessed by theoretical calculations. The bacterial growth inhibitory action of the inclusion compounds was studied in comparison to that of pure triclosan on Gram-negative (Salmonella, Escherichia) and Gram-positive strains (Bacillus, Listeria, Enterococcus and Staphylococcus) typically associated with human pathologies, and also on environmental bacteria isolated from different soil and water sources. The antimicrobial activities obtained in the present work showed that, of the two CD hosts, TRIMEB brings the most favourable carrier effect: it reduced the toxicity of triclosan against some of the environmental strains and afforded slightly higher action against virulent strains.

Incongruence between the cps type 2 genotype and host-related phenotypes of an Enterococcus faecalis food isolate

Enterococcus faecalis is a nosocomial opportunistic pathogen, but is also found in fermented food products where it plays a fundamental role in the fermentation process. Previously, we have described the non-starter E. faecalis cheese isolate QA29b as harboring virulence genes and proven to be virulent in Galleria mellonella virulence model. In this study, we further characterized this food strain concerning traits relevant for the host-pathogen relationship. QA29b was found to belong to sequence type (ST) 72, a common ST among food isolates, and thus we consider it as a good representative of food E. faecalis strains. It demonstrated high ability to form biofilms, to adhere to epithelial cells and was readily eliminated by J774.A1 macrophage cells. Despite carrying the cps locus associated with the capsular polysaccharide CPS 2 type, cps genes were not expressed, likely due to an IS6770 inserted in the cpsC-cpsK promoter region. This work constitutes the first study of traits important for interaction, colonization and infection in the host performed on a good representative of E. faecalis food isolates. Reported results stress the need for a reliable serotyping assay of E. faecalis, as cps genotyping may not be reliable. Overall, QA29b characterization shows that despite its virulence potential in an insect model, this food strain is readily eliminated by mammalian macrophages. Thus, fine tuned approaches combining cellular and mammalian models are needed to address and elucidate the multifactorial aspect of virulence potential associated with food isolates.

Identification of a new gene, vanV, in vanB operons of Enterococcus faecalis

Subinhibitory concentrations of vancomycin are known to induce a cell-wall stimulon in some Gram-positive pathogens, but this has never been studied in the genus Enterococcus. In this study, Enterococcus faecalis V583 strain was submitted to a subinhibitory concentration of vancomycin. DNA microarray technology was used to analyse the transcriptomic changes induced by this antibiotic. EF2292, annotated as a hypothetical protein in the E. faecalis V583 genome, was highly induced in response to vancomycin exposure, to similar levels as the vanB operon genes. We investigated further and provide evidence for co-transcription of ef2292 with vanY(B)WH(B)BX(B) genes. It was also demonstrated that expression of ef2292 is under the control of vanR(B)S(B) and it is proposed to name it vanV. This gene was found not to be required for vancomycin resistance under the conditions tested, thus coding for another accessory protein in the vanB operon. vanV was detected in some, but not all, E. faecalis carrying the vanB operon, suggesting that this operon can have different composition amongst E. faecalis isolates.