Virulence Factor Genes Incidence among Enterococci from Sewage Sludge in Eastern Slovakia following Safety Aspect

The sewage sludges represent a potential health hazard because of the quantity of different microbiota detected in sewages. Among microbiota detected in sewages, also belong representatives of the phylum Firmicutes. In the past, environmental enterococci in addition to coliforms were widely used as indicators of faecal contamination. Regarding the enterococcal strains as potential pathogenic bacteria, their pathogenicity is mainly caused by production of virulence factors. Therefore, the aim of the study was to analyse incidence of virulence factors in enterococci from cows' dung water. Species identification of 24 enterococci using MALDI-TOF MS system allotted 23 strains to the species Enterococcus faecium with highly probable species identification and E. faecalis EEV20 with a score value meaning secure genus identification/probable species identification. Enterococci were absent of cytolysin A gene, hyaluronidase gene, and element IS gene. It can be concluded that they are not invasive which is very important from safety aspect. The most frequently detected gene was adhesin E. faecium (efaAfm, in 22 E. faecium strains and in one E. faecalis). Adhesin efaAfs gene was detected in E. faecalis EEV20 and in two E. faecium. GelE gene was present in three strains. E. faecium EF/EC31 was absent of virulence factor genes.


Introduction
Stabilized animal sewage sludge, including cow's dung water is frequently used for agricultural purposes to farmland application due to its high organic matter content serving, e.g., as a source of nutrients for plants [1]. However, it has brought the hygienic and/or safety aspect into focus. at is, the sewage sludges represent a potential health hazard. e hazards are mainly associated with the amount of different microbiota detected in sewages, especially pathogenic species [2]. Stiborová et al. [1] assessed sewage sludges in Czech Republic through both taxonomic and phylogenetic approaches. ere, the bacterial community dominated was affiliated with Proteobacteria including the phyla Deinococcus-ermus and ermotogae. e most frequently detected genera in the sludge in Czech Republic were Mycobacterium and Streptomyces [1]. However, they also detected the phylum Firmicutes. In the framework of the phylum Firmicutes, Lauková et al. [3] reported representatives of the genus Enterococcus. Previously, environmental enterococci in addition to coliforms were widely used as indicators of faecal contamination [4]. Regarding the enterococcal strains as potential pathogenic bacteria, their pathogenicity is mainly caused by production of virulence factors and/or resistance to antibiotics [5]. erefore, the aim of this study was to analyse incidence of virulence factors in enterococci isolated from cow's dung water (in Eastern Slovakia), which is useful to know from at least two aspects-characteristic and properties of enterococci from different niches in the framework of the basic research and from safety aspect. at is, in sewage sludge could perform possible conjugative transfer or another way of virulence factor gene transfer among strains which can threaten human population.

Sampling, Strain Management, and Identification.
Bacterial strains tested (24) were isolated from cow's dung water (sewage sludge) as previously described by Lauková et al. [3]. ey were collected from the basins of 25 cattle farms in 15 North-Eastern Slovakian districts. Forty-five samples were transported in bottles and analysed. ey were treated according to ISO (International Organization for Standardization), diluted in Ringers solution (Oxoid), and appropriate dilutions were spread plated onto M-Enterococcus agar (Becton and Dickinson, Cockeysville, USA). Incubation was performed in a CO 2 atmosphere at 37ºC for 24-48 h. Isolated strains were phenotyped with the API 20 Strep system (API, Biomerieux, L'Etoile, France). en, they were stored using a freeze dryer (MicroModulyo, ermo corp., Asheville, Nebraska, USA). However, before testing of virulence factor genes, strains were re-covered in MRS broth (De Man-Rogosa-Sharpe, Merck, Germany) by cultivating at 37ºC for 24 h following their plating on Brain heart agar enriched with sheep's blood (5%), and then plated on M-Enterococcus agar (Difco, Detroit, Michigan, USA). Pure strains were stored for next analyses with the Microbank system (Pro-Lab Diagnostic, Richmond, Canada).
Besides phenotypization, species identification was performed using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, Billerica, Maryland, USA); [6], meaning Biotyper TM identification system (Bruker Daltonics, USA [7]). is system is used especially for research microbiology. e method is based on analysis of bacterial proteins (fingerprints) using a Microflex MALDI-TOF mass spectrometer. Briefly, a single colony from M-Enterococcus agar was mixed with the matrix (α-cyano-4-hydroxycinnamic acid and trifluoroacetic acid) and the suspension was spotted onto a MALDI plate and ionized with a nitrogen laser (wave-length 337 nm, frequency 20 Hz). Lysates of bacterial cells were prepared according to the producer's instructions (Bruker Daltonics). e results were evaluated using the MALDI Biotyper 3.0 (Bruker Daltonics) identification database. Taxonomic allocation was evaluated on the basis of highly probable species identification-score 2.300-3.000, then secure genus identification/probable species identification (value score 2.000-2.299) and probable genus identification associated with score value of 1.700-1.999. Positive controls were Enterococcus faecium CCM4231 [8] and Enterococcus faecalis DSM 20478 (Bruker Daltonics database 2008).

Results and Discussion
Species identification of 24 enterococcal strains, done using MALDI-TOF MS system alloted, 23 strains to the species E. faecium and one strain to the species E. faecalis. irteen strains of 23 E. faecium (56.5%) were identified with a score value ranging from 2.300 to 3.000, meaning highly probable species identification. Ten strains (43.5%) were alloted to the species E. faecium with a score value ranging from 2.000 to 2.299, indicating secure genus identification/probable species  identification. e strain E. faecalis EEV20 was alloted taxonomically with a score value ranging from 2.000 to 2.299 ( Table 2).
All tested enterococci from sewage sludge were absent of cytolysin A-cylA gene, hyaluronidase-hylEfm gene and element IS (IS16) gene. e most frequently detected virulence factor gene was adhesin E. faecium (efaAfm). is efaAfm adhesin gene was present in 22 E. faecium strains and even in one E. faecalis strain (EEV20, Table 2); altogether in 95.8% strains out of all tested. Only E. faecium EF/EC31 strain was absent of efaAfm gene. On the other hand, adhesin efaAfs gene was detected not only in E. faecalis strain EEV20 but also in two E. faecium strains EF20 and EF/ED21. GelE gene was present in three strains, two E. faecium (EFV10 and EF20) as well as in the strain E. faecalis EEV20 (Table 2). Regarding the strains, only one strain-E. faecium EF/EC31 was absent of virulence factor genes. E. faecium EFV10 had gelE gene and efaAfm gene; E. faecium EF/ED21 had also two virulence factor genes: efaAfm and efaAfs. ree genes (gelE, efaAfm, efaAfs) were detected in the strains E. faecium EF20 and E. faecalis EEV20.
Mass spectrometry is an analytical technique in which chemical compounds are ionized into charged molecules and the ratio of their mass to charge (m/z) is measured. e development of electron spray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) in 1980s increased the applicability of MS to large biological molecules like proteins. In both spectrometries, peptides are converted into ions by either addition or loss of one or more than one proton [10]. MALDI-TOF is now considered to be a real alternative for bacterial identification due to the provision of rapid and specific determination analogous to molecular sequencing techniques, with benefit of significant time and cost savings. Comparing previously identified species using phenotypization, E. faecium was predominant species [3] as was also confirmed using MALDI-TOF system here. In addition, the species E. faecalis was also identified among those strains previously. However, phenotypization also indicated the representatives of the other species such as E. casseliflavus and E. durans among those strains; this was not confirmed using MALDI-TOF MS but a higher percentage of strains identified belonged to the species E. faecium. Our experience confirmed a high identification score using MALDI-TOF MS technology associated with phenotypization not only in species E. faecium [11] but also E. faecalis [12]. High quality MALDI-TOF mass spectra were also obtained in identification of environmental bacteria [13]. E.g., Cherkaoui et al. [14] compared MALDI-TOF technology with conventional phenotypic identification of clinical bacterial strains and they found high confidence identifications for 680 isolates, of which 674 (99.1%) were correct with phenotypization. So, implementation of MS as an identification strategy would improve its efficacy in further analyses to study additional properties of strains e.g., in the framework of basic research. Only one limitation of MALDI-TOF MS technology is that the spectral database containing peptide mass fingerprints of the type strains has to be upgraded to involve new species [10,13]. As formerly indicated, potential pathogenicity of enterococci is associated with the presence of virulence factor determinants/genes or virulence factor production. Gene IS16 is a marker specific for clinical E. faecium/E. faecalis strains associated with nosocomial infection. None of our environmental enterococci contained IS16 gene. Similarly, enterococci tested were absent of hyl (hyaluronidase) gene. Hyaluronidase acts on hyaluronic acid and increases bacterial invasion [15]; it can play a role in different inflammations in host organism, e.g., ear inflammation. It was described to be a part of a genomic island located on a plasmid and this was shown to be enriched in hospital-associated, polyclonal subpopulation of E. faecium strains [16]. Enterococcal surface protein (esp) supports adhesion of bacteria; enterococci tested were also absent of esp gene. Similarly, Kubašová et al. [9] described no occurrence or only rare occurrence of those genes in faecal canine enterococci. In addition, faecal rabbits' enterococci from Pannon White breed of rabbits were free of hylefm and IS16 genes [12]. On the other hand, the most frequently detected determinants were those encoding adhesin efaAfm (95.8%) which is again similar as reported in canine enterococci by Kubašová et al. [9]. is is also in association with the most frequently detected species E. faecium in our study because adhesin efaAfm is typical for E. faecium species [17]. Cyl A, the cytolysin activator (bacterial toxin with hemolytic activity against eukaryotic cells encoded by cylA gene [18]; it can even induce tissue damage. Also this cylA gene was not present in tested enterococci. Although our strains were not tested to form a biofilm, we would be interested in further parameters because these factors not only participate in invasion and colonization of host but may also contribute to biofilm formation [19]. Gelatinase, extracellular metalloprotease is able to hydrolyze gelatin, collagen and hemoglobin, which has contributed to bacterial adherence and biofilm formation [20]. Occurrence of virulence factor determinants can be influenced with a source of tested strain or also with a species; that is, Eaton and Gasson [17] reported that E. faecium strains and E. faecalis showed significantly different patterns in the incidence of virulence determinants. Abouelnaga et al. [21] found three strains out of 88 from fermented food free from virulence determinants, and 16% strains from unfermented food were free of virulence factor determinants. Because enterococci tested were mostly absent of virulence factor determinants such as cylA gene, IS16 element, hylefm gene esp gene and rare in efaAfs, gelE gene detection; it can be concluded that they are not invasive which is very important from safety aspect. It can be supposed that their occurrence in environment did not represent health risk. However, here no antibiotic profile was shown; but resulting from previous studies, they were mostly susceptible to antimicrobials (bacteriocins) [22].

Conclusion
To conclude from our results, prevalence of the species E. faecium was detected in cow's dung water (sewage sludge from samples in Eastern Slovakia) with few species of E. faecalis. Detected strains were, however, mostly absent virulence factor determinants. is indicates that they did not present invasive character and pathogenicity in environment and host regarding safety aspect. Of course, other studies are underway. Moreover, this study is also a contribution to the basic knowledge regarding the environmental enterococci.
Data Availability e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.