Abstract
Background: The genus Enterococcus contains gram-positive cocci. Before the widespread use of antibiotics, enterococci were known causes of endocarditis, urinary tract infections (UTIs), and intra-abdominal infections. The three most important enterococcal virulence agents discovered are cytolysin (cylA), enterococcal surface protein (esp), and gelatinase (gelE). Serious concerns about infectious illnesses are raised by the increasing number of genes for antibiotic resistance.
Materials and methods: The study was granted permission by the Institutional Ethical Committee (MU/Research/EC/Ph.D./2021/58), and every single sample was taken from patients who were hospitalized at the Index Medical College Hospital and Research Center in Indore, India. One hundred and six enterococcal isolates were collected between December 2021 and August 2024, and their species distribution was examined. Standard molecular techniques were used to identify virulence genes, such as cytolysin (cylA), enterococcal surface protein (esp), and gelatinase (gelE).
Results: Among 106 Enterococcus isolates analyzed by reverse transcription polymerase chain reaction (RT-PCR), the esp gene was the most prevalent (74.53%), followed by cylA (55.66%) and gelE (45.28%). Enterococcus faecalis showed higher frequencies of virulence genes compared to Enterococcus avium. High resistance was observed to erythromycin, penicillin, chloramphenicol, and tetracycline, whereas most isolates remained sensitive to linezolid and vancomycin. These findings indicate the presence of virulence-associated genes and variable antimicrobial resistance patterns among the Enterococcus isolates.
Conclusion: The study reveals a significant pathogenic potential of Enterococcus strains, evidenced by the high prevalence of major virulence genes (esp, cylA, and gelE).
Keywords: cytolysin (cyla), enterococcal infection, enterococcal surface protein (esp), enterococcus species, gram-positive cocci
Introduction
Enterococcus was primarily reported in the year 1899 as a new Streptococcus of gut organism. The same cause of identical gram-positive cocci was found in a patient with endocarditis in 1906 [1]. The genus Enterococcus contains gram-positive cocci that can be found individually, in pairs, or in short chains. When Gram stains are made from solid media growth, cells might become coccobacillus. When preparing Gram stains from thioglycolate broth, cells tend to be ovoid and in chains [2,3]. Prior to the widespread use of antibiotics, enterococci were known causes of endocarditis, urinary tract infections (UTIs), and intra-abdominal infections. The widespread use of antimicrobial drugs was associated with increased acknowledgment of the problem of enterococcal infection and an increase in the prevalence of enterococci in hospital-acquired illnesses [4,5]. Furthermore, the expansion of genes for antibiotic resistance raises serious concerns regarding infectious diseases. Cytolysin (cylA), enterococcal surface protein (esp), and gelatinase (gelE) are the most significant enterococcal infectious agents that have been found [6]. Certain virulence factors, including the genes for agg, esp, and cylA, have been discovered to be contained on a 153-kb pathogenicity island [7]. The virulence factors of enterococci may be linked to their persistence in a hospital environment [8]. Determining whether the resistance in the genetic elements is effectively transferred or if the isolates' resistance increased in a clonal is crucial. The potential of controlling a microbe that can both effectively disperse and effectively transfer resistance represents the worst-case scenario. Enterococcus is the best illustration of the latter phenomenon, which has become a major nosocomial infection within the past ten years [9-11].
Materials and methods
The study was granted permission by the Institutional Ethical Committee (MU/Research/EC/Ph.D./2021/58), and all samples were collected from patients hospitalized at the Index Medical College Hospital and Research Center, Indore, India. A total of 106 Enterococcal isolates were collected between December 2021 and August 2024, and their species distribution was analyzed.
Inclusion criteria
Clinical isolates of Enterococcus species obtained during the study period were included. These isolates were recovered from various clinical specimens, such as blood, urine, pus, aspirated fluids, cerebrospinal fluid (CSF), and other body fluids collected from admitted patients. Only those patients who provided written informed consent for participation in the study were included. Relevant clinical and demographic details were recorded using a standardized patient proforma.
Exclusion criteria
Isolates of Enterococcus species obtained from patients who did not provide consent to participate in the study were excluded. Additionally, duplicate isolates from the same patient and sputum and stool samples, and incomplete clinical information or inadequate quality for analysis were excluded.
Enterococci strains were obtained from a variety of clinical specimens submitted to the microbiology laboratory, including blood, urine, exudates (surgical and non-surgical wound swabs and pus), catheters, ascitic fluid, synovial fluid, pleural fluid, and other body fluids. Depending on the specimen type, samples were cultured on appropriate media, including Trypticase Soy Agar with 5% sheep blood, MacConkey agar, and Bile Esculin Azide agar (HiMedia Laboratories Pvt. Ltd., Mumbai, India), followed by incubation at 37°C overnight [12]. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar and Mueller-Hinton agar supplemented with 5% sheep or human blood (HiMedia Laboratories Pvt. Ltd., Mumbai, India). The antibiotics tested included penicillin (10 units), chloramphenicol (30 µg), erythromycin (15 µg), tetracycline (30 µg), doxycycline (30 µg), ciprofloxacin (5 µg), high-level gentamicin (120 µg), vancomycin (30 µg), linezolid (15 µg), nitrofurantoin (300 µg), and norfloxacin (for urinary isolates only) (HiMedia Laboratories Pvt. Ltd., Mumbai, India). Interpretation of results was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines (2022-2023) [12].
Molecular detection of virulence genes by real-time polymerase chain reaction (PCR)
Genomic DNA was extracted using the HiPurA® Bacterial Genomic DNA Purification Kit (MB505) (HiMedia Laboratories Pvt. Ltd., Mumbai, India) according to the manufacturer’s protocol. The extracted DNA was used for the detection of target genes (esp, cylA, gelE) using real-time PCR. The HiPurA® kit provides a rapid and efficient method for isolating high-quality genomic DNA suitable for molecular applications. It employs a silica-based HiElute Miniprep Spin Column system for DNA purification. Cell lysis is achieved using lysozyme and Proteinase K, followed by purification through centrifugation. DNA quality and quantity were assessed using agarose gel electrophoresis and spectrophotometric analysis.
Binding step
Two hundred microliters of ethanol (95-100%) was added to the lysate, mixed, and transferred to the HiElute Miniprep Spin Column (HiMedia Laboratories Pvt. Ltd., Mumbai, India) [13].
Prewash step
Five hundred microliters of Prewash Solution and Wash Solution (HiMedia Laboratories Pvt. Ltd., Mumbai, India) were added sequentially, and the mixture was centrifuged as per protocol [13].
Elution step
DNA was eluted using 200 µl Elution Buffer (HiMedia Laboratories Pvt. Ltd., Mumbai, India) [13].
Real-time polymerase chain reaction (PCR) conditions
Amplification was carried out using the Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). The cycling conditions included initial denaturation at 95°C for 15 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 1 minute, and final extension at 72°C for 10 minutes [13].
Statistical analysis
Data were compiled and analyzed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and Minitab 17 (Minitab LLC, State College, PA, USA). Descriptive statistics and Chi-square test were applied, with significance set at p < 0.05.
Results
The distribution of virulence genes among Enterococcus isolates is depicted. A total of 106 isolates were analyzed for the presence of virulence genes using real-time PCR. Out of 106 isolates, 79 (74.53%) were positive for the esp gene, while 27 (25.47%) were negative. For the cylA gene, 59 (55.66%) isolates were positive, and 47 (44.34%) were negative. Similarly, 48 (45.28%) isolates were positive for the gelE gene, whereas 58 (54.72%) were negative. Overall, the esp gene was the most prevalent virulence factor, followed by cylA and gelE genes among the studied Enterococcus isolates (Table 1).
Table 1. Association of virulence genes with gender among Enterococcus isolates.
Data are presented as frequency (percentage). The association between gender and virulence genes (esp, cylA, and gelE) was analyzed using the chi-square test. Pairwise chi-square analysis demonstrated that esp (χ² = 23.68, p < 0.001) and gelE (χ² = 9.93, p = 0.002) genes showed strong significant associations, while the cylA gene showed a modest but statistically significant association (χ² = 4.31, p = 0.038).
| Gene | Group | Overall n (%) | Chi-square | p-value |
| esp gene | Positive | 79 (74.53%) | 23.68 | 0.0001 |
| Negative | 27 (25.47%) | |||
| cylA gene | Positive | 59 (55.66%) | 4.31 | 0.038 |
| Negative | 47 (44.34%) | |||
| gelE gene | Positive | 50 (47.17%) | 9.93 | 0.002 |
| Negative | 56 (52.83%) |
The distribution of virulence genes among Enterococcus species across various clinical samples is depicted. All 106 Enterococcus isolates, the distribution of virulence genes among Enterococcus isolates was analyzed according to gender. A total of 106 isolates were included, comprising 50 males (47.16%) and 56 females (52.83%). The presence of esp, cylA, and gelE genes was determined using RT-PCR. Among male patients (n = 50), 39 (78%) isolates were positive for the esp gene, 24 (48%) for the cylA gene, and 25 (50%) for the gelE gene. The corresponding negative isolates were 11 (22%), 26 (52%), and 25 (50%), respectively. Among female patients (n = 56), 40 (71.43%) isolates were positive for the esp gene, 35 (62.50%) for the cylA gene, and 25 (44.65%) for the gelE gene. The negative isolates were 16 (28.57%), 21 (37.50%), and 31 (55.35%), respectively (Table 2).
Table 2. Table shows the total percentage of virulence genes between gender wise in the Enterococcus species.
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. Statistical analysis using the chi-square test showed no significant association between gender and the presence of virulence genes (esp: χ² = 0.60, p = 0.44; cylA: χ² = 2.25, p = 0.13; gelE: χ² = 0.85, p = 0.36), indicating that the distribution of these genes was not significantly different between male and female patients (p > 0.05).
| Genes | Groups | Gender | Chi-square | p-value | |
| Male 50 (47.16%) | Female 56 (52.83%) | ||||
| esp gene | Positive | 39 (78%) | 40 (71.43%) | 0.60 | 0.44 |
| Negative | 11 (22%) | 16 (28.57%) | |||
| cylA gene | Positive | 24 (48%) | 35 (62.50%) | 2.25 | 0.13 |
| Negative | 26 (52%) | 21 (37.50%) | |||
| gelE gene | Positive | 25 (50%) | 25 (44.65%) | 0.85 | 0.36 |
| Negative | 25 (50%) | 31 (55.35%) | |||
The association between the esp gene and different clinical samples among Enterococcus infection isolates was analyzed using a total of 106 samples. The distribution of samples included urine (n = 77), blood (n = 12), pus (n = 11), body fluids (n = 4), and swab samples (n = 2). Out of the total isolates, 79 (74.53%) were positive for the esp gene, while 27 (25.47%) were negative. Among urine samples, 60 (75.94%) were esp gene positive and 17 (62.96%) were negative. In blood samples, 8 (10.13%) isolates were positive and 4 (14.81%) were negative. Similarly, pus samples showed 8 (10.13%) positive and 3 (11.11%) negative isolates. Body fluid samples demonstrated 2 (2.53%) positive and 2 (7.41%) negative isolates, whereas swab samples showed 1 (1.26%) positive and 1 (3.70%) negative isolate (Table 3).
Table 3. Distribution of the esp gene among different clinical sample types.
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. Overall, although higher esp gene positivity was observed in pus, body fluids, and swab samples, the association between the esp gene and different clinical samples was not statistically significant (χ² = 2.78, p = 0.597; p > 0.05).
| Sample | esp Gene positive n=79 (74.53%) | esp Gene negative n=27 (25.47%) | Chi-square | P-value |
| Urine (77) | 60 (75.94%) | 17 (62.96%) | 2.78 | 0.597 |
| Blood (12) | 8 (10.13%) | 4 (14.81%) | ||
| Pus (11) | 8 (10.13%) | 3 (11.11%) | ||
| Body fluids (04) | 2 (2.53%) | 2 (7.41%) | ||
| Swab (02) | 1 (1.26%) | 1 (3.70%) |
The association between the cylA gene and different clinical sample types among Enterococcus isolates was analyzed using a total of 106 isolates. The samples included urine (n = 77), blood (n = 12), pus (n = 11), body fluids (n = 4), and swab samples (n = 2). Among these, urine samples showed 41 isolates (69.49%) positive for the cylA gene, while 36 (76.60%) were negative. In blood samples, 5 isolates (8.47%) were positive, whereas 7 (14.89%) were negative. Pus samples demonstrated 7 isolates (11.86%) positive and 4 (8.51%) negative. All isolates from body fluids showed 4 (6.78%) positive and none (0%) negative. Similarly, swab samples showed 2 isolates (3.39%) positive, with no negative isolates (0%) observed. Overall, 59 (55.66%) isolates were positive for the cylA gene, while 47 (44.34%) were negative (Table 4).
Table 4. Distribution of the cylA gene among different clinical sample types.
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. Overall, although higher cylA gene positivity was observed in pus, body fluids, and swab samples, the association between esp gene presence and type of clinical sample was statistically not significant (chi-square = 6.20, p = 0.185; p > 0.05).
| Sample | cylA gene positive n=59 (55.66%) | cylA gene negative n=47 (44.34%) | Chi-square | P-value |
| Urine (77) | 41 (69.49%) | 36 (76.60%) | 6.20 | 0.185 |
| Blood (12) | 5 (8.47%) | 7 (14.89%) | ||
| Pus (11) | 7 (11.86%) | 4 (8.51%) | ||
| Body fluids (04) | 4 (6.78%) | 0 (0%) | ||
| Swab (02) | 2 (3.39%) | 0 (0%) |
The association between the studied parameter and different clinical samples among Enterococcus isolates was analyzed using a total of 106 isolates. The samples included urine (n = 77), blood (n = 12), pus (n = 11), body fluids (n = 4), and swab samples (n = 2). Among these, urine samples showed 37 isolates (77.08%) positive, while 40 (68.97%) were negative. In blood samples, 5 isolates (10.42%) were positive, whereas 7 (12.07%) were negative. Pus samples demonstrated 4 isolates (8.33%) positive and 7 (12.07%) negative. Body fluid samples showed 1 isolate (2.08%) positive and 3 (5.17%) negative. Similarly, swab samples showed 1 isolate (2.08%) positive and 1 (1.72%) negative. Overall, 48 (45.28%) isolates were positive, while 58 (54.72%) were negative (Table 5).
Table 5. Distribution of gelE gene among different clinical sample types.
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. The association between gelE gene presence and type of clinical sample was analyzed using the chi-square test and was found to be statistically non-significant (χ² = 1.34, p = 0.855; p > 0.05).
| Sample | gelE gene positive 48 (45.28%) | gelE gene negative 58(54.72%) | Chi-square | P-value |
| Urine (77) | 37 (77.08%) | 40 (68.97%) | 1.34 | 0.855 |
| Blood (12) | 5 (10.42%) | 7 (12.07%) | ||
| Pus (11) | 4 (8.33%) | 7 (12.07%) | ||
| Body fluids (04) | 1 (2.08%) | 3 (5.17%) | ||
| Swab (02) | 1 (2.08%) | 1 (1.72%) |
The distribution of the esp gene among different Enterococcus species was analyzed based on a total of 79 positive and 27 negative isolates (n = 106). Among the esp gene-positive isolates, E. faecalis and E. faecium each accounted for 30 cases (37.97%), followed by E. durans with 16 cases (20.25%) and E. avium with 3 cases (3.80%). In contrast, among the esp gene-negative isolates, E. faecalis represented the highest proportion with 13 cases (48.15%), followed by E. faecium with 10 cases (37.04%), E. durans with 3 cases (11.11%), and E. avium with 1 case (3.70%). Overall, when considering both positive and negative isolates together, E. faecalis was the most prevalent species, followed by E. faecium, while E. durans and E. avium contributed relatively fewer isolates (Table 6).
Table 6. Distribution of virulence gene esp among Enterococcus species.
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. For the esp gene (chi-square = 1.45), a p-value of 0.69 was obtained, indicating no statistically significant difference in esp gene distribution across species (p > 0.05).
| Species | esp gene positive (79) | esp gene negative (27) | Chi-square | P-value |
| Enterococcus faecalis | 30 (37.97%) | 13 (48.15%) | 1.45 | 0.69 |
| Enterococcus faecium | 30 (37.97%) | 10 (37.04%) | ||
| Enterococcus durans | 16 (20.25%) | 3 (11.11%) | ||
| Enterococcus avium | 3 (03.80%) | 1 (3.70%) |
The distribution of the cylA gene among different Enterococcus species was analyzed based on a total of 59 positive and 47 negative isolates (n = 106). Among the cylA gene-positive isolates, E. faecium accounted for the highest number of cases with 23 (38.98%), followed closely by E. faecalis with 22 cases (37.28%), E. durans with 11 cases (18.64%), and E. avium with 3 cases (5.08%). In contrast, among the cylA gene-negative isolates, E. faecalis represented the highest proportion with 21 cases (44.68%), followed by E. faecium with 17 cases (36.17%), E. durans with 8 cases (17.02%), and E. avium with 1 case (2.12%). Overall, when combining both positive and negative isolates, E. faecalis was the most prevalent species, followed by E. faecium, while E. durans and E. avium contributed comparatively fewer isolates (Table 7).
Table 7. Distribution of virulence gene cylA among Enterococcus species.
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. For the cylA gene (chi-square = 1.05), a p-value of 0.79 was obtained, indicating no statistically significant difference in cylA gene distribution across species (p > 0.05).
| Species | cylA gene positive (59) | cylA gene negative (47) | Chi-square | P-value |
| Enterococcus faecalis | 22 (37.28%) | 21 (44.68%) | 1.05 | 0.79 |
| Enterococcus faecium | 23 (38.98%) | 17 (36.17%) | ||
| Enterococcus durans | 11 (18.64%) | 8 (17.02%) | ||
| Enterococcus avium | 3 (5.08%) | 1 (2.12%) |
The distribution of the gelE gene among different Enterococcus species was analyzed based on a total of 48 positive and 58 negative isolates (n = 106). Among the gelE gene-positive isolates, E. faecalis accounted for the highest number of cases with 19 (39.58%), followed by E. faecium with 17 cases (35.41%), E. durans with 11 cases (22.91%), and E. avium with 1 case (2.08%). In contrast, among the gelE gene-negative isolates, E. faecalis again represented the highest proportion with 24 cases (41.37%), followed by E. faecium with 23 cases (39.65%), E. durans with 8 cases (13.79%), and E. avium with 3 cases (5.17%). Overall, when combining both positive and negative isolates, E. faecalis was the most prevalent species, followed by E. faecium, while E. durans and E. avium contributed comparatively fewer isolates (Table 8).
Table 8. Distribution of virulence gene gelE among Enterococcus species.
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. For the gelE gene (chi-square = 2.03), a p-value of 0.57 was obtained, indicating no statistically significant difference in gelE gene distribution across species (p > 0.05).
| Species | gelE gene positive (48) | gelE gene negative (58) | Chi-square | P-value |
| Enterococcus faecalis | 19 (39.58%) | 24 (41.37%) | 2.03 | 0.57 |
| Enterococcus faecium | 17 (35.41%) | 23 (39.65%) | ||
| Enterococcus durans | 11 (22.91%) | 8 (13.79%) | ||
| Enterococcus avium | 1 (2.08%) | 3 (5.17%) |
The association between virulence genes and vancomycin resistance among Enterococcus isolates was analyzed using a total of 106 isolates, categorized into vancomycin-sensitive (VSE) and vancomycin-resistant (VRE) groups. Among the esp gene-positive isolates (n = 79), 51 (64.55%) were vancomycin-sensitive, while 28 (35.44%) were vancomycin-resistant. In comparison, among the esp gene-negative isolates (n = 27), 18 (66.66%) were sensitive and 9 (33.33%) were resistant. Similarly, for the cylA gene, among positive isolates (n = 59), 38 (64.40%) were sensitive and 21 (35.59%) were resistant, whereas among negative isolates (n = 47), 31 (65.95%) were sensitive and 16 (34.04%) were resistant. In contrast, for the gelE gene, among positive isolates (n = 48), 24 (50%) were sensitive and 24 (50%) were resistant, while among negative isolates (n = 58), 45 (77.58%) were sensitive and 13 (22.41%) were resistant. Overall, these findings indicate that while esp and cylA genes do not show any significant relationship with vancomycin resistance, the gelE gene is significantly associated with resistance patterns among Enterococcus isolates (Table 9).
Table 9. Distribution of virulence genes (esp, cylA, and gelE) in relation to VRE antibiotic susceptibility (sensitive vs. resistant).
Data are presented as counts (percentages). S: sensitive; R: resistant. Statistical analysis was performed using the Pearson chi-square test to evaluate the association between virulence gene positivity and vancomycin resistance (VRE 37). For the esp gene (chi-square = 0.04), a p-value of 0.84 was obtained, indicating no statistically significant association between the esp gene and antibiotic resistance pattern (p > 0.05). For the cylA gene (chi-square = 0.03), a p-value of 0.87 was obtained, indicating no statistically significant association between the cylA gene and antibiotic resistance pattern (p > 0.05). For the gelE gene (chi-square = 8.79), a p-value of 0.001 was obtained, indicating a statistically significant association between the gelE gene and antibiotic resistance pattern (p < 0.05).
| Genes | Groups | VRE 37 | Chi-square | p-value | |
| S | R | ||||
| esp gene | Positive (79) | 51 (64.55%) | 28 (35.44%) | 0.04 | 0.84 |
| Negative (27) | 18 (66.66%) | 9 (33.33%) | |||
| cylA gene | Positive (59) | 38 (64.40%) | 21 (35.59%) | 0.03 | 0.87 |
| Negative (47) | 31 (65.95%) | 16 (34.04%) | |||
| gelE gene | Positive (48) | 24 (50%) | 24 (50%) | 8.79 | 0.001 |
| Negative (58) | 45 (77.58%) | 13 (22.41%) | |||
The relationship between antimicrobial susceptibility patterns and virulence genes (esp, cylA, and gelE) was analyzed among 106 Enterococcus isolates. For the esp gene (n = 106; positive = 79, negative = 27), the highest sensitivity was observed for linezolid (71/79 (89.8%)) and vancomycin (51/79 (64.5%)), whereas the highest resistance was noted for erythromycin (75/79 (94.9%)). Statistically significant associations were observed between the esp gene and penicillin (χ² = 6.87, p = 0.009), chloramphenicol (χ² = 5.25, p = 0.022), erythromycin (χ² = 4.69, p = 0.030), and nitrofurantoin (χ² = 7.21, p = 0.007). For the cylA gene (positive = 59, negative = 47), isolates showed high sensitivity to linezolid (52/59 (88.1%)) and vancomycin (38/59 (64.4%)). High resistance was observed for erythromycin (53/59 (89.8%)), penicillin (49/59 (83.0%)), and tetracycline (48/59 (81.3%)). However, no statistically significant association was found between the cylA gene and antimicrobial susceptibility (p > 0.05). For the gelE gene (positive = 48, negative = 58), the highest sensitivity was observed for linezolid (43/48 (89.6%)) and moderate sensitivity for vancomycin (24/48 (50.0%)). High resistance was observed for erythromycin (45/48 (93.7%)) and tetracycline (41/48 (85.4%)). A statistically significant association was observed for high-level gentamicin (χ² = 4.49, p = 0.034) and vancomycin (χ² = 8.80, p = 0.003). Overall, linezolid was the most effective antibiotic across all virulence gene groups, while erythromycin exhibited the highest resistance. Statistical significance was considered at p < 0.05 (Table 10).
Table 10. Association of virulence genes (esp, cylA, and gelE) with antimicrobial susceptibility patterns among Enterococcus isolates (n=106).
Values are expressed as n (%). Percentages are column-wise. P-values were calculated using the chi-square test where appropriate. A p-value <0.05 was considered significant. S: sensitive; R: resistant. Data are presented as a count (percentage). χ²: chi-square value; p: p-value.
*n=77 for norfloxacin and nitrofurantoin. Statistical analysis was performed using the Pearson chi-square test to evaluate the association between virulence gene positivity and antimicrobial susceptibility. Highlighted p-values indicate statistically significant associations (p < 0.05).
For the esp gene: significant associations were observed with penicillin (p=0.009), chloramphenicol (p=0.022), erythromycin (p=0.030), nitrofurantoin (p=0.007), high-level gentamicin (p=0.034), and vancomycin (p=0.003); all other antimicrobials showed no significant association (p > 0.05).
For the cylA gene: no statistically significant associations were observed with any of the tested antimicrobial agents (all p > 0.05).
For the gelE gene: significant associations were observed with high-level gentamicin (p=0.034) and vancomycin (p=0.003); all other antimicrobials showed no significant association (p > 0.05).
| Antimicrobial agent | S/R | esp gene (n=106) | cylA gene (n=106) | gelE gene (n=106) | |||||||||
| Positive 79 (%) | Negative 27 (%) | esp: χ² | esp: p | Positive 59 (%) | Negative 47 (%) | cylA: χ² | cylA: p | Positive 48 (%) | Negative 58 (%) | gelE: χ² | gelE: p | ||
| High-level gentamicin (120 µg) | S | 28 (35.4) | 17 (40.7) | 0.02 | 0.88 | 18 (30.5) | 20 (42.5) | 1.65 | 0.20 | 12 (25.0) | 26 (44.8) | 4.49 | 0.034 |
| R | 51 (64.5) | 17 (62.9) | 41 (69.4) | 27 (57.4) | 36 (75.0) | 32 (55.1) | |||||||
| Vancomycin (30 µg) | S | 51 (64.5) | 18 (66.6) | 0.04 | 0.84 | 38 (64.4) | 31 (65.9) | 0.03 | 0.87 | 24 (50.0) | 45 (77.5) | 8.80 | 0.003 |
| R | 28 (35.4) | 9 (33.3) | 21 (35.5) | 16 (34.0) | 24 (50.0) | 13 (22.4) | |||||||
| Linezolid (15 µg) | S | 71 (89.8) | 21 (77.7) | 2.57 | 0.11 | 52 (88.1) | 40 (85.1) | 0.21 | 0.65 | 43 (89.5) | 49 (84.4) | 0.60 | 0.44 |
| R | 8 (10.1) | 6 (22.2) | 7 (11.8) | 7 (14.8) | 5 (10.4) | 9 (15.5) | |||||||
| Doxycycline (30 µg) | S | 20 (25.3) | 9 (33.3) | 0.65 | 0.42 | 13 (22.0) | 16 (34.0) | 1.89 | 0.17 | 11 (22.9) | 18 (31.0) | 0.87 | 0.35 |
| R | 59 (74.6) | 18 (66.6) | 46 (77.9) | 31 (65.9) | 37 (77.0) | 40 (68.9) | |||||||
| Penicillin (10 units) | S | 9 (11.3) | 9 (33.3) | 6.87 | 0.009 | 10 (16.9) | 8 (17.0) | 0.00 | 1.00 | 7 (14.5) | 11 (18.9) | 0.36 | 0.55 |
| R | 70 (88.6) | 18 (66.6) | 49 (83.0) | 39 (82.9) | 41 (85.4) | 47 (81.0) | |||||||
| Ciprofloxacin (5 µg) | S | 22 (27.8) | 12 (44.4) | 2.54 | 0.11 | 21 (35.5) | 13 (27.6) | 0.76 | 0.38 | 17 (35.4) | 17 (29.3) | 0.45 | 0.50 |
| R | 57 (72.1) | 15 (55.5) | 38 (64.4) | 34 (72.3) | 31 (64.5) | 43 (74.1) | |||||||
| Chloramphenicol (30 µg) | S | 27 (34.1) | 16 (59.2) | 5.25 | 0.022 | 25 (42.3) | 18 (38.2) | 0.18 | 0.67 | 20 (41.6) | 23 (39.6) | 0.04 | 0.83 |
| R | 52 (65.8) | 11 (40.7) | 34 (57.6) | 29 (61.7) | 28 (58.3) | 35 (60.3) | |||||||
| Erythromycin (15 µg) | S | 4 (5.0) | 5 (18.5) | 4.69 | 0.030 | 6 (10.1) | 3 (6.3) | 0.48 | 0.49 | 3 (6.2) | 6 (10.3) | 0.57 | 0.45 |
| R | 75 (94.5) | 22 (81.4) | 53 (89.8) | 44 (93.6) | 45 (93.7) | 52 (89.6) | |||||||
| Tetracycline (30 µg) | S | 11 (13.9) | 7 (25.9) | 2.61 | 0.11 | 10 (16.9) | 8 (17.0) | 0.03 | 0.87 | 6 (12.5) | 12 (20.6) | 1.25 | 0.26 |
| R | 66 (83.5) | 20 (74.0) | 48 (81.3) | 38 (80.8) | 41 (85.4) | 45 (77.5) | |||||||
| Norfloxacin* (10 µg) | S | 2 (3.3) | 1 (3.7) | 1.91 | 0.17 | 2 (4.8) | 1 (2.7) | 0.89 | 0.34 | 1 (2.7) | 2 (5.0) | 1.14 | 0.29 |
| R | 58 (96.6) | 16 (94.1) | 39 (95.1) | 36 (97.2) | 36 (97.2) | 38 (95.0) | |||||||
| Nitrofurantoin* (300 µg) | S | 19 (31.6) | 11 (64.7) | 7.21 | 0.007 | 16 (39.0) | 14 (38.8) | 0.67 | 0.41 | 14 (50.0) | 15 (37.5) | 0.95 | 0.33 |
| R | 41 (68.3) | 6 (35.2) | 25 (60.9) | 22 (61.1) | 22 (59.4) | 25 (62.5) | |||||||
Discussion
In our study, Enterococcus strains were tested for virulence genes by the reverse transcription polymerase chain reaction (RT-PCR) method for molecular detection, such as the esp gene, cylA gene, and gelE gene. Out of 106 enterococcus isolates, 74.53% show positive esp gene, followed by 55.66% showing positive cylA gene, and 45.28% show gelE gene, respectively. According to similar findings by Weng et al. [14], human infection severity is significantly influenced by cytolysin. In another finding, phenotypic hemolytic activity was found in 30.8% of E. faecalis, whereas 43.5% had cylA. In support of the results of this investigation. In another study by Jahansepas et al. [15] demonstrated that 41% of Enterococci have the cylA gene, and hemolytic activity was found in 38% of cylA-positive isolates. The study's multiplex PCR procedure, which simultaneously detected five distinct virulence genes, turned out to be a dependable and quick substitute for uniplex PCR and phenotypic testing [16].
According to this, the main virulence factor is gelatinase. Virulence factor from catheter-induced infection, blood, and wounds. The presence of quiet genes may be the reason why more strains tested positive for the gelE gene by PCR than strains that tested positive in phenotypic assays.
For a more comprehensive characterization of the strains, both phenotypic and genotypic analyses are essential. No single gene was found to be significantly associated with biofilm formation, suggesting that this process is multifactorial and depends on the interaction of multiple genes along with various environmental factors [8].
According to Strateva et al. [17], the cytolysin structural gene was never absent from β-hemolytic E. faecalis, although it was absent from E. faecium. Therefore, another cytotoxic factor must be responsible for the hemolysis of E. fecium. Similar to the results of this investigation in E. faecium isolates, 75 isolates with hemolytic activity in another study tested negative for the cylA gene, indicating a potential involvement of additional genes in hemolytic activity. By encouraging cell-to-cell contact and the conjugal transfer of plasmids carrying antibiotic resistance and virulence, the fecal VRE's capacity to create slime is especially crucial for resistance acquisition genes. These strains may aid in the spread of these bacteria in hospital environments and seem to be the entrance point for novel resistance genes into enterococcal species in the gastrointestinal tract [17,18].
According to our results, most esp virulence gene-positive and negative strains show sensitivity and resistance to antibiotics. Highly sensitive to linezolid and vancomycin (89.8% and 64.5%) and highly resistance in erythromycin 94.4% shown in esp gene because other study by Armin et al. [19] the presence of esp was higher in clinical infections and isolates linked to epidemics than in surveillance isolates, research revealed that the variant esp gene in vancomycin-resistant E. faecium and vancomycin-sensitive E. faecium was strongly associated with a particular epidemiologic source [20].
The most resistant antibiotic was ciprofloxacin. Only 5.21% of cases had vancomycin-resistant enterococci (VRE), and the majority of these cases had Van A-type VRE detected phenotypically. By encouraging cell-to-cell contact and the conjugal transfer of plasmids carrying antibiotic resistance and virulence, the fecal VRE's capacity to create slime is especially crucial for resistance acquisition genes. These strains may aid in the spread of these bacteria in hospital environments and seem to be the entrance point for novel resistance genes into enterococcal species in the gastrointestinal tract [6,21].
In addition, the prevalence of multidrug-resistant (MDR) Enterococcus spp., including vancomycin-resistant enterococci (VRE), has been reported to be increasing in India, posing a significant clinical challenge. This rising resistance trend enhances the ability of enterococci to survive under antimicrobial pressure and increases the likelihood of interactions with other drug-resistant pathogens.
Limitations of the study
This study has certain limitations that should be considered while interpreting the results. The sample size was relatively small, and the study was conducted at a single center, which may limit the generalizability of the findings. Only selected virulence genes (cylA, gelE, and esp) were analyzed, and other potentially relevant genes were not included. Additionally, advanced molecular techniques and detailed genetic characterization were not performed. Furthermore, clinical outcomes and long-term follow-up data were not assessed. Future studies with larger sample sizes, multi-center involvement, and comprehensive molecular analysis are recommended.
Conclusions
This study evaluated the prevalence, antibiotic susceptibility, and clinical significance of enterococcal infections in a hospital setting. Enterococcus faecalis and Enterococcus faecium were the most common isolates, but the emergence of less common species highlights the need for accurate species-level identification and further research into regional prevalence. These findings emphasize the importance of cautious use of critical antibiotics, such as vancomycin and linezolid, along with strict infection control measures to prevent the spread of resistant strains like VRE. Continuous monitoring of antibiotic susceptibility patterns, regular screening, and minimum inhibitory concentration (MIC) testing are essential for guiding effective treatment strategies and developing regional antibiotic policies.
Disclosures
Human subjects: Informed consent for treatment and open access publication was obtained or waived by all participants in this study. Malwanchal University Ethics Committee issued approval MU/Reseal/EC/Ph.D./2021/58.
Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.
Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following:
Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.
Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.
Other relationships: Financial Relationships: The authors declare that they have received no financial support, funding, grants, honoraria, consulting fees, or any other form of financial benefit from any commercial entity, pharmaceutical company, or organization that could be perceived to have influenced the conduct or reporting of this research. Non-Financial Relationships: The authors declare no non-financial competing interests including personal relationships, academic rivalries, institutional affiliations, or ideological biases that could have influenced the design, conduct, analysis, or reporting of this study.
Author Contributions
Concept and design: Bhawani Shankar Verma, Prashanth K. Guddeti, Ramanath Karicheri, Md Abdullah, Hemant B. Patil
Acquisition, analysis, or interpretation of data: Bhawani Shankar Verma, Prashanth K. Guddeti, Ramanath Karicheri, Md Abdullah, Hemant B. Patil
Drafting of the manuscript: Bhawani Shankar Verma, Prashanth K. Guddeti, Ramanath Karicheri, Md Abdullah, Hemant B. Patil
Critical review of the manuscript for important intellectual content: Bhawani Shankar Verma, Prashanth K. Guddeti, Ramanath Karicheri, Md Abdullah, Hemant B. Patil
References
- 1.Enterococcus faecium: evolution, adaptation, pathogenesis and emerging therapeutics. Wei Y, Palacios Araya D, Palmer KL. https://pubmed.ncbi.nlm.nih.gov/38890478/ Nat Rev Microbiol. 2024;22:705–721. doi: 10.1038/s41579-024-01058-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Enterococcus spp. and Streptococcus spp. bloodstream infections: epidemiology and therapeutic approach. Blanes Hernández R, Blanes Juliá M. https://pubmed.ncbi.nlm.nih.gov/37997861/ Rev Esp Quimioter. 2023;36:2–4. doi: 10.37201/req/s01.01.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Prevalence of Enterococcus species in various clinical samples and their antimicrobial susceptibility pattern. Mahajan M, Shinde R, Karande GS, Patil S. Cureus. 2024;16:0. doi: 10.7759/cureus.72836. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.The prevalence and the characterization of the enterococcus species from various clinical samples in a tertiary care hospital. Sreeja S, Sreenivasa Babu PR, Prathab AG. J Clin Diagn Res. 2012;6:1486–1488. doi: 10.7860/JCDR/2012/4560.2539. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Antibiotic resistance pattern of Enterococci isolates from nosocomial infections in a tertiary care hospital in Eastern India. Chakraborty A, Pal NK, Sarkar S, Gupta MS. J Nat Sci Biol Med. 2015;6:394–397. doi: 10.4103/0976-9668.160018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Genetic diversity, antimicrobial resistance, and biofilm-associated virulence in clinical Enterococcus faecalis isolates from Hamedan, Iran. Mokari S, YousefiMashouf R, Karami P, Taheri M. https://pmc.ncbi.nlm.nih.gov/articles/PMC12983561/ BMC Infect Dis. 2026;26:544. doi: 10.1186/s12879-026-12768-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Detection of virulence factors and antimicrobial susceptibility among clinical strains of Enterococcus faecalis. Shakhatreh MA, Rabaiah HH, Khabour OF, Jacob JH, Alzoubi KH. Curr Pharm Biotechnol. 2025;26:41029013. doi: 10.2174/0113892010388498250818114856. [DOI] [PubMed] [Google Scholar]
- 8.Improved detection of esp, hyl, asa1, gelE, cylA virulence genes among clinical isolates of Enterococci. Kiruthiga A, Padmavathy K, Shabana P, Naveenkumar V, Gnanadesikan S, Malaiyan J. BMC Res Notes. 2020;13:170. doi: 10.1186/s13104-020-05018-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Investigation of antibiotic resistance and virulence factors of Enterococcus faecium and Enterococcus faecalis strains isolated from clinical samples. Gök ŞM, Türk Dağı H, Kara F, Arslan U, Fındık D. https://pubmed.ncbi.nlm.nih.gov/32050876/ Mikrobiyol Bul. 2020;54:26–39. doi: 10.5578/mb.68810. [DOI] [PubMed] [Google Scholar]
- 10.High-level gentamicin resistance mediated by Aac(6')-Ie-aph(2")-Ia gene in Enterococcus species isolated from clinical samples in Northern India. Das AK, Dudeja M, Kohli S, Ray P, Nandy S. Indian J Pharmacol. 2022;54:171–176. doi: 10.4103/ijp.IJP_41_20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Multidrug-resistant enterococcal infection in surgical patients, what surgeons need to know. Farsi S, Salama I, Escalante-Alderete E, Cervantes J. Microorganisms. 2023;11:238. doi: 10.3390/microorganisms11020238. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Enterococcal infections in a tertiary care hospital, North India. Yadav RK, Agarwal L. Ann Afr Med. 2022;21:193–197. doi: 10.4103/aam.aam_110_20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.HiMedia Laboratories Pvt Ltd. Thane, India: HiMedia Laboratories Pvt Ltd; 2026. HiPurA® Bacterial Genomic DNA Purification Kit (MB505): Instruction Manual (Internet) [Google Scholar]
- 14.Antibiotic susceptibility patterns, biofilm formation and esp gene among clinical enterococci: is there any association? Weng PL, Ramli R, Hamat RA. Int J Environ Res Public Health. 2019;16:3439. doi: 10.3390/ijerph16183439. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Occurrence of Enterococcus faecalis and Enterococcus faecium in various clinical infections: detection of their drug resistance and virulence determinants. Jahansepas A, Aghazadeh M, Rezaee MA, Hasani A, Sharifi Y, Aghazadeh T, Mardaneh J. Microb Drug Resist. 2018;24:76–82. doi: 10.1089/mdr.2017.0049. [DOI] [PubMed] [Google Scholar]
- 16.Characteristics of vancomycin-resistant Enterococcus strains in the West Balkans: a first report. Jakovac S, Bojić EF, Ibrišimović MA, Tutiš B, Ostojić M, Hukić M. Microb Drug Resist. 2017;23:122–126. doi: 10.1089/mdr.2015.0355. [DOI] [PubMed] [Google Scholar]
- 17.Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria. Strateva T, Atanasova D, Savov E, Petrova G, Mitov I. Braz J Infect Dis. 2016;20:127–133. doi: 10.1016/j.bjid.2015.11.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. Vankerckhoven V, Van Autgaerden T, Vael C, et al. J Clin Microbiol. 2004;42:4473–4479. doi: 10.1128/JCM.42.10.4473-4479.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Genotyping, antimicrobial resistance and virulence factor gene profiles of vancomycin resistance Enterococcus faecalis isolated from blood culture. Armin S, Fallah F, Karimi A, Rashidan M, Shirdust M, Azimi L. Microb Pathog. 2017;109:300–304. doi: 10.1016/j.micpath.2017.05.039. [DOI] [PubMed] [Google Scholar]
- 20.Linezolid and vancomycin resistant enterococci: a therapeutic problem. Yadav G, Thakuria B, Madan M, Agwan V, Pandey A. J Clin Diagn Res. 2017;11:0. doi: 10.7860/JCDR/2017/27260.10474. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Molecular characterization of vancomycin-resistant Enterococcus faecium strains isolated from carriage and clinical samples in a tertiary hospital, Turkey. Gozalan A, Coskun-Ari FF, Ozdem B, et al. J Med Microbiol. 2015;64:759–766. doi: 10.1099/jmm.0.000088. [DOI] [PubMed] [Google Scholar]