Introduction

Enterococci are Gram-positive bacteria naturally inhabited in the GIT of most terrestrial mammals, including humans. As a group, they are responsible for many infections, including difficult-to-treat infections in people whose immune systems are compromised. Two enterococcal species, E. faecium and E. faecalis, account for at least 75 per cent of all enterococcal infections (1). E. faecalis is a common cause of UTIs as well as deeper abscesses. Resistant strains of E. faecalis are the main culprits behind infections that people acquire while receiving care in hospitals, including bloodstream infections, infections of the heart valves, UTIs, infections within the abdomen, surgical site infections, and infections from medical devices that are implanted in the body, such as catheters and intraocular lenses (2). What brought enterococci to the modern hospital? On one hand, they’re naturally resistant to many antibiotics that doctors use. Secondly, they’re tough to survive in extreme places. Thirdly, many can acquire mobile genetic elements that bring along drug resistance and other waste genes that help them cause disease. Finally, they biofilm on medical devices implanted in the body, such as catheters and intraocular lenses (3-6). Bacterial toxins are virulence determinants involved in pathogenesis – a host-pathogen interaction where toxins play a major role in transforming commensals into pathogens (i.e., the infection process). More specifically, these factors have unique functions to inhibit protein synthesis, assist the pathogen with evading host immune responses, and disrupt the membrane integrity of the target cell (7-10). Bacterial toxins can be grouped into numerous functional classes based on their biophysical properties, structures, and known modes of action. The most diverse protein toxins class is pore-forming toxins (PFTs). PFTs are proxies of the process known as pore-forming as they assemble on the membrane of a selected cell and create pores within it, thus increasing anion or cation permeability of the target membrane. In so doing, PFTs induce catastrophic alterations inside the cell, leading ultimately to cell lysis and cell death (11-14). Based on the secondary structures of the domains that construct the transmembrane pore, PFTs have been classified into two structural families: alpha-PFTs and beta-PFTs which, respectively, form helical and sheet-like pores. Well-studied examples of alpha-PFTs are the colicins produced by Escherichia coli, whereas Staphylococcus aureus produces a beta-PFT known as alpha-hemolysin (15-17). Cytolysin A (ClyA) (also called HlyE or SheA) is a 34-kilodalton α-PFT produced by some members of the Enterobacteriaceae family such as E. coli and Salmonella enterica. This toxin has been exhaustively studied in terms of its structure, its activity, and its importance as a virulence factor (18).

Food safety is an important issue that concerns scientific and commercial communities worldwide for its direct impact on people’s health. The current study used conventional methods to isolate Enterococcus faecalis from dairy products. The study also evaluated its pathogenicity (cytolysin) and detected the genes responsible for the production of cytolysin (cylM, cylB, asa, cylA, and cylL_s) using PCR.

Materials and methods

Ethical approve

The current study was approved by the Ethical Committee at the College of Veterinary Medicine, University of Al-Qadisiyah, Al-Diwaniyah City, Iraq, under the No.4414 in 23/11/2023

Samples

The study first started with collecting of 72 dairy product samples (21 cheese, 31 yogurts, and 20 curd samples). These samples were purchased from local stores in Al-Diwaniyah City, Iraq.

Bacterial cultivation and identification

Conventional methods were used to cultivate and identify bacterial species (9). The identification process included using selective media, such as Bile Esculin Agar, Enterococcsel Agar, and Slanetz and Bartley medium.

Hemolysis activity

The isolated bacteria of E. faecalis were grown on three different types of blood agar, horse blood agar (HnBA), HsBA and SBA. The HnBA is provided by the Health Clinic Centre situated in UKM, Bangi while HsBA and SBA are provided by Era Bumi Sains Sdn Bhd, Malaysia. After the overnight incubation (at 37ºC), the data were collected regarding the examination of hemolysis zone surrounding the colonies (19).

Cytotoxic activity

The isolated bacteria were grown in 5 mL of brain heart infusion (BHI) medium with 1% glucose and 0.03% L-arginine at a temperature of 37ºC for 18 hrs, as previously described by Booth et al. (20). The colonies were centrifuged in 6000 xg for 15 mins at 4ºC. The liquid above the sediment was taken and filtered through the 0.22 μm Millipore membrane, then later used to test Vero cells, originating from the African green monkey kidney, for identifying the ability of strains of E. faecalis to produce cytolysin. The survivability of the Vero cell to detects the toxicity caused by the strains was done in an MTT assay. Method for MTT assay as described by Raheel et al. (21). Vero cell lines gained from a stock of Virology Laboratory Faculty of Science and Technology UKM. Negative control uses sterilized broth without culture. The samples DMEM was taken, and 20 µL per well of the MTT was added (5 mg/ml in PBS) then the plates were incubated for another 4 hrs. The OD was measured in an ELISA reader (BioRad, Chapan) at 540 nm. Percentages of cell death were calculated using the formula as follows:

Cells Die % = [Mean OD control / Mean OD treated cell] × 100

Extraction of DNA

Ten bacterial contents of genomic DNA were extracted using the Wizard® Genomic DNA purification kit (Promega, USA). According to the manufacturer's protocol, the bacteria grown overnight in BHI broth was harvested by centrifuging the solution at 13,000 RPM for 1 minute. Then the pellet formed was suspended in 480 μL of 50 mM EDTA solution. The solution was then centrifuged at 13,000 rpm for 2 minutes, and the liquid portion above the sediment was separated and eluded.

PCR

The 50 μL of PCR reaction mixture contained 10 μL of 5X Go Taq Flexi buffer, 1 μL of 0.2 mM dNTPs, 8 μL of 4 mM MgCl2, 1 μL of 1 μM of each primer, 1 μL of 0.5 μg DNA, 0.25 μL of 1.25 U DNA polymerase, and 27.75 μL water. The PCR was performed in a Bio-Rad MyCycler thermal cycler (Bio-Rad, USA). The program was set for 2 minutes at 95ºC for an initial denaturation, followed by 35 cycles of 95ºC for 1 minute (denaturation), annealing at 40ºC to 60ºC for 1 minute and extension at 72ºC for 1 minute, and a final extension product of 5 minutes at 72ºC. The amplified DNA product was separated on 1.5% agarose gel with an applied voltage of 80 V for 45 minutes. Before loading the gel, a DNA stain (FloroSafe stain) was added. Finally, all gels were observed and recorded using a UV trans-illuminator (syngene, UK).

Results

The results of the cultivation revealed the presence of the bacterium E. faecalis in 43/72 (59.7%) samples, which is distributed over 20/21 (95.2%) cheese, 14/31 (45.2%) yogurt, and 9/20 (45%) curd samples. The findings of the hemolysis analysis demonstrated that the hemolytic capability of the isolated bacteria of types (γ, α, and β) differed, depending on the type of blood agar employed HnBA, SBA, and HsBA. On HBA plates, 17 isolates (39.5%) exhibited γ hemolytic action; however, 21 isolates (48.8%) exhibited β hemolytic activity. The results revealed that only 5 isolates showed α blood hemolysis. Overall, 39.5% of the isolates (n=17) showed γ counts, 11.6% (n=5) were α and 48.8% (n=21) were β hemolysis colonies on human blood agar. For SBA, the report revealed differences in some of the ratios. Notably, none showed β in SBA. γ blood hemolysis was recorded in 39.5% (n=17) of the isolated pluses. While α blood, in this case, was 60.5% (n=26). Interestingly, some isolates were unable to show β blood hemolysis on SBA even though they had already demonstrated hemolysis on HnB and HsB agars. The second was the delay in the process, as some of the isolates were able to show it after 72 hours (Table 1 and Figure 1). The cytotoxic activity on Vero cells showed various rates of dead cells and ranged from 0.2% (for the control) to 50.1% for certain isolates (Figure 2). The result of the PCR revealed the amplification of the examined genes in the bacterial isolates of dairy products (Figure 3).

Table 1: Pattern of hemolysis of E. Faecalis from cheese, yogurt, and curd

Figure 1: Hemolysis activity on different blood agars of Enterococcus faecalis bacteria isolated from cheese, yogurt, and curd samples. (a) Gamma hemolysis on sheep blood agar (E. faecalis S21), (b) alpha hemolysis on sheep blood agar (E. faecalis H57), (c) beta hemolysis on human blood agar (E. faecalis S2), (d) beta hemolysis on horse blood agar (E. faecalis S133, H85, H87 and M1), (e) alpha hemolysis on horse blood agar (E. faecalis S29)

Figure 2: Cytotoxicity activity of E. faecalis isolated from cheese, yogurt, and curd tested on Vero cell line. A. Optical density. B. Dead cell percentage.

Figure 3: Agarose gel electrophoresis of A. cylM, cylB, asa, B. cylA, C. and cylL_s genes responsible for cytolysin coding in E. faecalis isolated from cheese, yogurt, and curd.

Discussion

Blood agar is used to identify and test the antibiotic susceptibility of many different bacterial pathogens. The high cost and harsh atmosphere required to raise wool sheep or horses to collect the blood lead microbiologists in developing countries to go for the cheaper and more suitable human blood agar. Several pathogens do not grow or exhibit morphologies and hemolytic profiles so it is difficult to differentiate their colonies (22). Hence, this study intended to use different types of erythrocytes other than those obtained from humans such as horse blood and sheep blood. This study used three different types (human, sheep and horse) of blood agar to evaluate E. faecalis isolates (n=43) for the activity of hemolysis (22). As shown in Figure 1, the results from the hemolysis test done on the erythrocytes showed that the three isolates (γ, α and β) have varied degrees of hemolysis depending on the type of blood agar used among the isolates sampled, whether human, sheep, or horses. The overall, a total of 17 isolates (39.5%) showed γ hemolysis on horse blood agar while a total of 21 isolates (48.8%) were β hemolysis. Only 5 isolates showed α blood hemolysis (22).

Cell proliferation (cell growth is an augmentation in cell viability. Cell death (cell loss) is deterioration in cell viability as characterized by cell death caused by the toxic effects of the test material (23). The predominance of E. faecalis in infections can be partially attributed to its ability to inhibit the growth of other bacteria in the same environment. This inhibition is likely due to the Enterococcal cytolysin (encoding genes of E. faecalis, as they all act against both Gram-positive and Gram- negative species (24). Furthermore, the presence of cytolysin also increases the likelihood of E. faecalis dominating a mixed infection and providing an ecological advantage that potentially can lead to diseases in humans and animals.

No cytolysin genes were found in the phenotypic non-γ hemolysin isolates. Moreover, the data show differences in toxicity between the bacterial isolates, with the cells infected with E. faecalis mediated γ hemolysin and lacking cytolysin genes showing a decreased toxicity level compared with the other isolates. The isolates exhibiting γ hemolysin capability and lacking cytolysin genes such as S21, S37, S46, S52, S88, S98, S131, H79 and M10 had, respectively, dead cell percentages of 2.2, 0.7, 0.8, 5.3, 2.2, 1.3, 1.6, 4.4, and 4.4%. Since cytolysin may cause hemolysis and cytotoxicity, the absence of the cytolysin genes could also account for these results (25-34).

Enterococci are Gram-positive bacteria naturally inhabited in the GIT of most terrestrial mammals, including humans. Food safety is an important issue that concerns scientific and commercial communities worldwide for its direct impact on people’s health (35-48).

Conclusion

The current data demonstrate the presence of Enterococcus faecalis in dairy products with the indications of its pathogenicity due to the presence of cytolysin-coding genes and the cytotoxic activity of this protein on Vero cells.

Acknowledgement

Not applicable

Conflict of interest

The study has no conflict of interest as it is declared by the authors.