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.

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.

Vancomycin-susceptible dairy and clinical enterococcal isolates carry vanA and vanB genes.

A total of 109 enterococcal isolates from dairy food products and from human and dog infections, isolated in Portugal, and 26 type and reference strains of the genus Enterococcus were screened for vancomycin resistance. MIC values, both for vancomycin and teicoplanin, were determined. The genetic relatedness of isolates carrying either vanA and/or vanB was determined using Pulsed Field Gel Electrophoresis. For vanA carrying isolates, transposon Tn1546 was partially mapped using PCR. None of the 59 dairy isolates was resistant to vancomycin. Among the 50 clinical isolates, only one, carrying vanB, behaved as resistant, with a MIC value of 256 microg/mL. The type and reference strains used were susceptible both to vancomycin and teicoplanin. vanA was found in 37% of the dairy isolates and 40% of the clinical isolates. vanB was only detected in 18% of the clinical, both human and dog, isolates. PCR partial mapping of Tn1546 revealed 23 different patterns among 42 isolates. Some patterns were shared between dairy and clinical isolates. Using Pulsed Field Gel Electrophoresis six groups of isolates were found to be genetically undistinguishable and grouping was found to be geographically and location specific/related. No genetic relatedness was found between isolates from dairy, human and veterinary sources. These results show that an incomplete and/or unfunctional Tn1546 element may explain the absence of resistant behaviour in the studied isolates, even when vanA gene is present. Moreover, the work reported shows that both clinical (human and animal) and dairy isolates have been in contact with VanA genotype of resistance and suggest that dissemination of vanA gene has been through transposable elements, like Tn1546, and not by clonal dissemination of a resistant strain. Therefore, a national strategy should be implemented to survey both vancomycin resistance and its genetic dissemination.

Activity and expression of a virulence factor, gelatinase, in dairy enterococci.

In order to understand the virulence potential of dairy enterococci, 35 isolates from raw ewe's milk and traditionally fermented cheeses, identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus dispar and Enterococcus hirae, were screened for their capacity to produce gelatinase and for the presence of the genes gelE, sprE, fsrA, fsrB and fsrC. Studies correlating gelatinase production with maintenance and subculture of the isolates in the Laboratory environment, and growth in different media were performed. These studies were conducted with two dairy isolates identified as E. faecalis and E. durans, and one clinical isolate, E. faecalis OG1-10. RT-PCR was used for detection of transcripts of the above mentioned genes. Results demonstrated that the virulence factor gelatinase is disseminated among the genus Enterococcus and that dairy isolates are capable of producing gelatinase at comparable levels with clinical isolates, although this capacity is easily lost during conservation by freezing in the laboratory. Therefore, gelatinase production potential of dairy enterococci may be underestimated. The gene gelE was present in all studied isolates. The same was observed for the fsr operon, either complete or incomplete, revealing that the gelatinase genetic determinants, so far only described in E. faecalis, are a common trait in the genus. This work describes for the first time the detection of the complete Fsr-GelE operon in other species than E. faecalis, namely E. faecium and E. durans. The loss of expression of this virulence factor under laboratory culture conditions correlated with the loss of one or more genes of the regulatory fsr operon, although the gene gelE was maintained, demonstrating that a complete fsr operon is required for a positive GelE phenotype. Independent of the detection of any gelatinase activity, if both gelE and the complete fsr operon are present in the cell, all genes are transcribed, as revealed by RT-PCR, suggesting that regulation of gelatinase activity can also be post-transcriptional. The silent behavior of gelE was only observed in E. faecalis, but not in E. durans, suggesting different modulation mechanisms of gelatinase activity in these two species. Overall, these findings reopen the issue of food safety of enterococci and reinforce the need to further study the mechanisms responsible for triggering the virulence factor gelatinase in non-pathogenic enterococcal environmental isolates.

Gentamicin resistance in dairy and clinical enterococcal isolates and in reference strains.

Enterococci isolated from Portuguese dairy products (milk and cheese) and clinical settings (hospitals and veterinary clinics), together with reference strains from the genus Enterococcus, were screened for low- and high-level gentamicin resistance using the standard disc diffusion method (10 and 120 microg gentamicin discs). MICs were also determined using both the macrodilution method and the Etest. Four genes [aac(6')-Ie-aph(2")-Ia, aph(2")-Ib, aph(2")-Ic and aph(2")-Id] responsible for high- and mid-level gentamicin resistance were sought using PCR. Although enterococci generally are regarded as being intrinsically resistant to low levels of gentamicin, results revealed that many dairy enterococci (around 30% of the isolates used) are not intrinsically resistant to gentamicin, showing MICs of < or = 4 mg/l. High-level gentamicin resistance was not detected in any of the dairy isolates studied, except for aph(2")-Ib, which was found in one. Therefore, gentamicin resistance should be monitored in dairy enterococci, although it does not seem to be a problem at present. In contrast, all clinical isolates studied were, as expected, intrinsically resistant to low levels of gentamicin, presenting MICs > 8 mg/l. Fifteen percent of these clinical isolates showed high-level gentamicin resistance (MICs > 512 mg/l), with the bifunctional gene aac(6')-aph(2") being detected in four of them. However, discs with gentamicin 120 microg failed to detect some isolates with high-level gentamicin resistance.

Characterization of a highly thermostable extracellular lipase from Lactobacillus plantarum.

After screening for the presence of lipase activity in lactobacilli isolated from "chouriço", a traditional Portuguese dry fermented sausage, a strain of Lactobacillus plantarum (DSMZ 12028) was chosen for extracellular lipase characterisation and purification. Proteinase K did not significantly affect lipolytic activity, as opposed to trypsin, which completely eliminated this activity. Among NaCl, Ca2+, EDTA, BSA, glycerol, Mn2+ and Mg2+, only Mn2+ and Mg2+ stimulated the lipase. Purification by gel filtration chromatography and gel electrophoresis revealed four bands, between 98 and 45 kDa, all with lipolytic activity against olive oil.