|Year : 2012 | Volume
| Issue : 2 | Page : 175-181
Molecular screening of virulence genes in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens in Northwest Iran
A Hasani1, Y Sharifi1, R Ghotaslou2, B Naghili3, A Hasani4, M Aghazadeh2, M Milani5, A Bazmani6
1 Research Center of Infectious Diseases and Tropical Medicine; Faculty of Medicine Department of Clinical Microbiology, Tabriz University of Medical Sciences, Tabriz, Iran
2 Faculty of Medicine Department of Clinical Microbiology, Tabriz University of Medical Sciences, Tabriz, Iran
3 Research Center of Infectious Diseases and Tropical Medicine, Department of Infectious Diseases, Tabriz University of Medical Sciences, Tabriz, Iran
4 Research Center of Infectious Diseases and Tropical Medicine; Department of Clinical Biochemistry and Biotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
5 Faculty of Medicine Department of Clinical Microbiology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
6 Research Center of Infectious Diseases and Tropical Medicine, Tabriz University of Medical Sciences, Tabriz, Tabriz, Iran
|Date of Submission||29-Nov-2011|
|Date of Acceptance||06-Mar-2012|
|Date of Web Publication||28-May-2012|
Research Center of Infectious Diseases and Tropical Medicine; Faculty of Medicine Department of Clinical Microbiology, Tabriz University of Medical Sciences, Tabriz
Purpose: The present study screened clinical isolates of Enterococcus faecalis and Enterococcus faecium to determine the prevalence of high-level gentamicin-resistant enterococci and the potential virulence genes among them. Materials and Methods: Clinical enterococcal isolates were obtained from three university teaching hospitals in Northwest Iran. Isolated enterococci were identified phenotypically followed by antibiotic susceptibility testing. Multiplex PCR was performed for the detection of genus, species-specific targets, gentamicin resistance, and potential virulence genes. Results: Of 220 enterococcal isolates, 133 (60.45%) isolates were identified as high-level gentamicin-resistant. Of these isolates, 79 (59.4%) and 54 (40.6%) were E. faecalis and E. faecium, respectively. All high-level gentamicin-resistant strains carried aac(6′)Ie-aph(2″)Ia. Of 220 isolates, 65.9% were positive for gelE, and 55%, 53.6%, 51.8%, and 49.5% of isolates were positive for cpd, asa1, ace, and esp, respectively. Phenotypically detected β-haemolytic strains (19.54%) were found to possess cylL ls MAB. Conclusion: The study revealed that high-level gentamicin-resistance was related to the presence of aac(6′)Ie-aph(2″)Ia. Isolated enterococci harboured potential virulence determinants, which were more common among E. faecalis than among E. faecium strains.
Keywords: Enterococci, gentamicin resistance, Multiplex PCR, virulence genes
|How to cite this article:|
Hasani A, Sharifi Y, Ghotaslou R, Naghili B, Hasani A, Aghazadeh M, Milani M, Bazmani A. Molecular screening of virulence genes in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens in Northwest Iran. Indian J Med Microbiol 2012;30:175-81
|How to cite this URL:|
Hasani A, Sharifi Y, Ghotaslou R, Naghili B, Hasani A, Aghazadeh M, Milani M, Bazmani A. Molecular screening of virulence genes in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens in Northwest Iran. Indian J Med Microbiol [serial online] 2012 [cited 2013 May 23];30:175-81. Available from: http://www.ijmm.org/text.asp?2012/30/2/175/96687
| ~ Introduction|| |
The past three decades have witnessed a gradual increase in the emergence of enterococcal nosocomial infections from diverse geographical regions, including many developing countries. , The organism causes serious infections, including bacteremia and endocarditis. The treatment of choice for such infections is usually the synergistic combination of penicillin or a glycopeptide with an aminoglycoside, primarily gentamicin. The efficacy of such combination therapy has declined due to the emergence and spread of multidrug-resistant enterococci.  The emergence of high-level gentamicin-resistant (HLGR) enterococci is related to the acquisition of genes, including aac(6′)Ie-aph(2″)Ia, that mediate the production of aminoglycoside-modifying enzymes. These genes are located either on a plasmid or in the chromosomal DNA and encode the bifunctional enzyme that confers resistance to all clinically useful aminoglycosides (gentamicin, tobramycin, amikacin, and netilmicin) excluding streptomycin and eliminates synergism between aminoglycosides and cell wall-active agents. Other additional genes that confer gentamicin resistance include aph(2′)-Ib, aph(2′)-Ic, and aph(2′)-Id. 
The possession of diverse virulence factors have been important benefit to enterococci, since owing any of them may change the severity of infections caused by these bacteria. On the other hand, it is believed that nosocomial enterococci might have virulence elements that increase their ability to colonize hospitalized patients.  This study was designed to identify the genes that contribute to high-level gentamicin resistance in Enterococcus faecalis nterococcus faecium, the species most frequently isolated from hospital infections, and to perform genotypic characterization of virulence genes (VGs) in these isolates, including aggregation substances (asa1), enterococcal surface protein (esp), gelatinase (gelE), collagen adhesine (ace), sex pheromones (cpd), and cyolysin (cyl), using multiplex PCR. To our knowledge, this is the first report on the virulence of enterococci isolated from high-risk patients in Northwest Iran.
| ~ Materials and Methods|| |
The study utilized 220 isolates of enterococci obtained from different clinical specimens in three university teaching hospitals located in Northwest Iran, from April 2008 to June 2010. Isolates were identified to the species level according to standard biochemical tests, and their identities were later confirmed by PCR. Only one isolate was analysed from each patient. Isolates other than E. faecalis and E. faecium were excluded from this study.
Production of haemolysin
To determine haemolysin production, isolates were inoculated on human blood agar plates and incubated at 37°C for 24 h. A clear zone of haemolysis around the colonies was considered a positive reaction.
Antibiotic susceptibility testing
The susceptibility pattern of isolates was determined using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI)  guidelines. Initially, HLGR strains were identified using high-content gentamicin (120 μg) disks (Mast Diagnostics, Merseyside-UK). For all isolates, the minimum inhibitory concentration (MIC) of gentamicin was determined using the agar dilution method according to the CLSI  guidelines, and HLGR isolates were confirmed by the E-test (BioMerieux, SA). E. faecalis ATCC 29212 was used as a quality control strain for performing antimicrobial tests.
DNA extraction and molecular approach
The DNA of clinical isolates was extracted using a commercial kit (DNG TM -Plus; CinnaGen, Tehran-Iran). Multiplex PCRs were performed on enterococcal isolates for simultaneous detection of: (i) genes encoding d-alanine-d-alanine ligases specific for E. faecalis and E. faecium,  (ii) genes related to gentamicin resistance,  and (iii) potential VGs, as described previously  but with slight modifications in the primer combinations and concentrations.
Multiplex PCR for VGs was performed in four groups as follows: Group I: esp, gelE, and asa1; group II: cpd and ace; group III: cylA, cylM, and cylL ls ; and group IV: cylB. Briefly, for group I, the 25 μL PCR mixture contained 2.5 μL of bacterial DNA, 15 pM of each primer for asa1 and gelE and 30 pM of each primer for esp, 1× PCR buffer, 2.0 mM MgCl 2 , 0.2 mM of each dNTP, and 2.5 U of Taq DNA polymerase (CinnaGen). The second mixture contained 10 pM of each primer for cpd and 4 pM of each primer for ace, 1.5 mM MgCl 2 , and the reagents mentioned for the first mixture. The primer concentrations for group III were 10 pM for cylA and cylL ls and 11 pM for cylM, and for group IV were 15 pM for cylB. The primers (Eurofins MWG Operon, Ebersberg-Germany) used in this study are shown in [Table 1].
Reactions were performed in a thermal cycler (ASTEC, Nagano-Japan) with an initial denaturation step of 10 min at 95°C; 30 cycles of 60 s at 94°C, 60 s at 56°C, and 60 s at 72°C; and a final extension step of 10 min at 72°C.
PCR products were analysed using gel electrophoresis on a 1.5% agarose gel with 0.5× Tris-borate-EDTA buffer. The gels stained with ethidium bromide were photographed under UV illumination (Uvitec, Cambridge-UK). Each PCR assay was performed with a negative control containing all of the reagents without template DNA. E. faecium SF11770 (Ib + ), E. faecalis SF350 (Ia + ), and E. faecium 899 (Ia + and Id + ), kindly provided by Dr. Jensen L. B., and Dr. Zarrilli R., were used as positive controls.
The prevalence of the species, gentamicin resistance, and VGs were compared using Fisher's test. Statistical analysis was performed with the SPSS (version 18) statistical program. P<0.05 was regarded as statistically significant.
| ~ Results|| |
Bacterial isolates and susceptibility testing
In total, 152 (69.1%) E. faecalis and 68 (30.9%) E. faecium were collected from different clinical specimens; urine was the most common source (85.5%) followed by blood (7.7%), body fluids (4.1%), wounds (1.8%), and intravenous catheters (0.9%). In total, 43 (19.54%) strains exhibited β-haemolytic activity in human blood agar.
The disk diffusion test indicated that 128 (58.2%) isolates were resistant to gentamicin (120 μg). HLGR isolates were resistant to penicillin (117 [88%]), ampicillin (57 [42.9%]), ciprofloxacin (127 [95.5%]), and streptomycin (87 [67.96%]).
The MICs of gentamicin ranged from 8 to 2048 μg/mL using the agar dilution method. In total, 133 (60.45%) isolates were HLGR strains with MICs≥512 μg/mL. Of non-HLGR (87 [39.5%]) isolates; Three isolates presented MICs of 16 μg/mL, one presented an MIC of 8 μg/mL, and the remaining isolates (83 [37.73%]) had MICs<8 μg/mL. The MIC results obtained using the E-test was compatible with those obtained using the agar dilution method.
Forty-three (32.33%) HLGR isolates exhibited resistance to vancomycin (MIC≥256 μg/mL excluding 1 isolate with MIC=8 μg/mL). Among these isolates, 33 (76.74%) strains were E. faecium, whereas 10 (23.26%) were E. faecalis.
PCR analysis of gentamicin resistance genes and virulence genes
A 348-bp fragment of aac(6′)Ie-aph(2″)Ia was identified in all E. faecalis (79 [59.4%]) and E. faecium (54 [40.6%]) isolates with phenotypic resistance to gentamicin [Figure 1]; however, aph(2′)-Ib, aph(2′)-Ic,or aph(2′)-Id were not detected in any isolate.
|Figure 1: Agarose gel electrophoresis of amplifi ed gentamicin resistance genes. Lane 1: 1-kb DNA ladder; Lanes 2-5: Isolates positive for the aac(6′)Ie-aph(2′)Ia gene; Lane 6: Negative control (without DNA)|
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We tested all isolates for the presence of 5 VGs and the cyl operon (cylL ls MAB). The numbers of VGs in isolates were as follows: 1 VG (45 [20.45%]); 2 VGs (17 [7.72%]); 3 VGs (46 [20.90%]); 4 VGs (28 [12.72%]); 5 VGs (14 [6.36%]); 6 VGs (7 [3.18%]); 7 VGs (7 [3.18%]); 8 VGs (17 [7.72%]); and 9 VGs (20 [9.09%]). Nineteen (8.63%) E. faecium strains did not harbour any of the tested genes.
Of 220 isolates, 145 (65.9%) were positive for gelE, and 55%, 53.6%, 51.8%, and 49.5% of isolates were positive for cpd, asa1, ace, and esp, respectively [Figure 2]. The distribution of VGs among HLGR, non-HLGR isolates, and entirely was shown in [Table 2].
|Figure 2: Agarose gel electrophoresis of amplifi ed asa1, esp, gelE, cpd and ace by multiplex PCR. Lanes 1 and 12: 1-kb DNA ladder; Lanes 2, 3, 7 and 10: Isolates positive for asa1 and gelE; Lanes 4 and 6: Isolates|
positive for asa1, esp and gelE; Lanes 5, 8 and 9: Isolates positive for esp; Lanes 13, 14 and 18: Isolates positive for cpd; Lanes 15, 17 and 21: Isolates positive for cpd and ace; Lanes 16, 19 and 20: Isolates negative for cpd and ace; Lanes 11 and 22: Negative control (without DNA)
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|Table 2: Distribution of virulence genes related to high-level gentamicin-resistant (HLGR) and non-HLGR E. faecalis and E. faecium Isolates|
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Among 43 (32.33%) concomitant HLGR and vancomycin-resistant enterococcus isolates, the most prevalent virulence determinant was esp (75.8%). Moreover, esp-positive E. faecium strains exhibited high resistance to ampicillin and penicillin (97.5%), ciprofloxacin (100%), and vancomycin (67.5%), and 92.5% of these strains were HLGR. We detected this gene in 52.1% of isolates from urinary tract infection samples, 29.4% of blood, 25% of wound, and 33.3% of body fluid specimens.
All β-haemolytic strains were E. faecalis strains exhibiting the cylL ls MAB genotype. Interestingly, we found various combinations of cyl genes in 16 (7.27%) strains that did not exhibit β-haemolytic activity in blood agar [Table 3]. Among the detected cytolysin genes, cylL ls [59 (26.8%)] was the most common.
| ~ Discussion|| |
Since the turn of the century, enterococci have become important hospital-acquired pathogens, and the most isolation of E. faecalis and E. faecium ranked them the third most prevalent nosocomial bacteria worldwide. Acquired resistance, most obviously to penicillin, ampicillin, glycopeptides, and aminoglycosides, has been reported in an increasing number of isolates, limiting the therapeutic spectrum in these cases.  The emergence and spread of high-level resistance to aminoglycosides, in particular to gentamicin, has been reported worldwide. ,
In the present study, the results obtained for HLGR strains using the disk diffusion and agar dilution methods were compatible; however, a minor discrepancy was observed between these two methods that indicated the possibility of false susceptibility being detected by the disk diffusion test. The disk diffusion test is easy to perform; however, the gentamicin results should be replaced with an MIC determination, which has been demonstrated to be more reliable. 
In this study, HLGR isolates exhibited multidrug resistance against the tested antibiotics. These findings were more obvious in E. faecium strains than in E. faecalis strains. HLGR E. faecium and E. faecalis strains displayed resistance to ampicillin (96.3% vs. 6.3%) and vancomycin (61.1% vs. 12.7%), and this difference was statistically significant (P<0.001). Resistance to ciprofloxacin (96.3% vs. 94.9%) and penicillin (98.1% vs. 81%) was also high among E. faecium strains; however, these differences were not significant. By contrast, when resistance to streptomycin was compared between HLGR isolates, E. faecalis strains (86.1%) were more resistant than E. faecium strains (40.7%) (P<0.05). Although the clinical use of streptomycin has long been limited, findings of fairly resistant (45.9%) strains bring the vulnerability of this drug into question.
The presence of high-level gentamicin resistance and concurrent resistance to penicillin or ampicillin and vancomycin were reported in some studies. , A study from the USA demonstrated that the gentamicin-resistance plasmid might co-transfer vancomycin-resistance plasmids.  Considering such a finding, the detection of HLGR strains collectively with vancomycin-resistant enterococci (32.33%) in this research represents an alarming situation in our region.
Our study revealed that all HLGR strains carried aac(6′)-Ie-aph(2″)-Ia. This is consistent with other studies that indicated that aac(6′)-Ie-aph(2″)-Ia is the most prevalent gene among the gentamicin-resistant enterococci. , Although aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id are also known to mediate resistance to gentamicin in enterococci, none of the isolates carried these genes.
The presence of diverse virulence factors has been commonly of the most distresses related to enterococci, as any of them may change the severity of enterococcal infections. In this study, E. faecalis and E. faecium exhibited significant differences in their harboured virulence factors. All E. faecalis strains carried two or more virulence determinants, whereas 72.1% of E. faecium isolates were positive for one of the tested virulence factors, most frequently esp (P<0.001). Other studies also reported that more virulence determinants were detected in E. faecalis isolates. ,
The pheromone-inducible surface protein aggregation substance, encoded by asa1, has been the subject of many studies that have found additional roles of this protein in enterococcal virulence. In our study, asa1 was detected in 92.4% of E. faecalis strains and 7.6% of E. faecium strains. A high incidence of this gene in E. faecalis was reported in previous studies.  One study reported that 13% of clinical E. faecium isolates carried asa1.  By contrast, in some studies, this gene was not found in E. faecium isolates. , In this study, the distribution of asa1 among HLGR E. faecalis isolates (84.8%) was significantly higher than that among E. faecium isolates (9.3%) (P<0.001). This gene was also found at a higher frequency among HLGR isolates (61%) than among non-HLGR strains (39%). From these results, a relationship between asa1 and the presence of gentamicin-resistance genes can be suggested. This gene was found in 11 of 17 isolates (64.7%) recovered from blood. It appears that these isolates are more likely to carry asa1 than isolates of other origins (urine [53.3%], body fluids [44.4%]).
A previous study reported that asa1 was always associated with the presence of pheromone determinants and detected this determinant in all E. faecalis strains.  In our study, 91 (75.2%) of 121 cpd-positive strains were concomitantly asa1-positive. Of these, all except two strains were E. faecalis (97.8%). The results from the present study indicate a very high incidence of cpd in HLGR E. faecalis, whereas, it was detected in three HLGR E. faecium isolates. The isolates possessing sex-pheromone genes have the potential to acquire the relevant sex-pheromone plasmids (the plasmids carrying the HLGR determinants) and therefore the associated virulence and resistance determinants. 
The Esp protein encoded by esp is thought to promote primary surface attachment, contributing to colonization and persistence in the urinary tract and biofilm formation.  This gene was first isolated from E. faecalis but not from any E. faecium isolate.  Thereafter, other studies revealed that esp is more prevalent in E. faecium isolates. , In this study, esp was the only gene with a higher incidence in E. faecium isolates than in E. faecalis (58.8% vs. 45.4%). Moreover, esp-positive E. faecium strains exhibited multidrug resistance against the tested antibiotics. This gene was also significantly more common (P<0.005) among E. faecium strains with coresistance to vancomycin and gentamicin (75.8%), whereas 10 vancomycin-resistant HLGR E. faecalis strains did not carry esp. Considering these results, it appears that the presence of esp may make it easier for E. faecium isolates to acquire additional antibiotic-resistance genes, and there is an association between esp and gentamicin-resistance genes (P<0.05).The high rate of this gene in urine isolates in comparison to other specimens indicated that the presence of esp is an advantage for the establishment of urinary tract infections, as has been suggested by others. 
Gelatinase is an extracellular zinc metalloendopeptidase encoded by gelE, and it was also demonstrated to be enriched in clinical E. faecalis isolates. , In this study, the most prevalent VG was gelE (65.9%) and we found it in higher frequency among clinical E. faecalis isolates. This gene was also commonly detected among HLGR (96.2%) and vancomycin-resistant HLGR (100%) E. faecalis strains (P<0.05). According to these results, there is an association between a high level of resistance to gentamicin and the presence of gelE among E. faecalis strains. In agreement with our findings, a lower frequency of gelE-positive E. faecium isolates was reported in clinical isolates.  Meanwhile, 128 (68%) isolates recovered from urinary tract infection samples carried gelE, and this association was statistically significant (P<0.05).
Ace, encoded by ace, mediates binding to certain collagens and may play a role in the pathogenesis of enterococci. A diverse incidence of ace has been reported in clinical isolates, irrespective of species. , In the present study, ace was more commonly detected in E. faecalis than in E. faecium (98.2% vs. 1.8%). It is interesting to note that ace had a higher incidence among non-HLGR than HLGR E. faecalis strains (91.8% vs. 57%). This is in contrast to another study that found that all HLGR E. faecalis strains were positive for ace.  Although this gene was most commonly detected in urine isolates (56.4%), no important differences regarding other origins were observed.
Haemolysin production has been detected on the basis of lysed human erythrocytes. All phenotypic β-haemolytic (19.54%) strains were E. faecalis and carried entire cyl genes. This is in agreement with previous studies on E. faecalis in which 11-60% of clinical isolates produced haemolysin. ,, The observation of β-haemolysis and detection of cyl genes in this study revealed a significant association between the phenotypic occurrence of β-haemolysis and the presence of cylL ls MAB genotypes. Additionally, we identified 16 strains with different combinations of cyl genes without β-haemolytic activity, suggesting the presence of silent genes and/or necessities for the presence of the complete cyl genes for β-haemolytic activity. Previously, a study explained the absence of β-haemolytic activity in the presence of all or some cyl genes by the low levels or downregulation of gene expression or by inactive gene products, and these genotypes may be affected by environmental factors.  In some studies, haemolysin production was observed in E. faecium isolates, , but we did not find such activity in any E. faecium strains. We did not find a significant difference between HLGR [26/133 (19.5%)] and non-HLGR [17/87 (19.5%)] strains regarding β-haemolytic activity, and no association was observed between origin and the presence of cyl genes. The lower frequency of haemolysin production renders the role of this factor as the virulence determinant of these species unlikely.
| ~ Conclusion|| |
Among the gentamicin-resistance genes investigated, aac(6′)Ie-aph(2″)Ia was responsible for the emergence of HLGR strains in our region. Our study demonstrated that clinical enterococcal isolates harbour potential virulence determinants, and these determinants are more common in E. faecalis than in E. faecium which may explain the higher frequency of E. faecalis among clinical isolates. According to these results, there is an association between a high level of resistance to gentamicin and the presence of gelE and asa1 among E. faecalis strains. The predominant virulence determinant in E. faecium was esp, and a significant correlation was found between the presence of esp-positive strains and resistance to the tested antibiotics; nevertheless, an increasing tendency of acquiring other virulence factors was also observed among E. faecium strains.
Different cyl genotypes and their silent forms revealed by our study indicate that the phenotypic assay should be used for the assessment of haemolytic activity in enterococci along with the molecular assay for cyl genes in nonhaemolytic strains to evaluate their virulence potential.
| ~ Acknowledgment|| |
This work was supported fully by Research Center of Infectious Diseases and Tropical Medicine (grant No.89/5), Tabriz University of Medical Sciences, Tabriz, Iran. We wish to thank Dr. Jensen LB and his colleagues from DK and Dr. Zarrilli R. from Italy for providing positive controls strains. In addition, we are grateful to Ms. Mitra Nojavan and Ms. Leila Deighani for their assistance in specimen collection. This work has been done as part of Ph.D. thesis (No. 88/4-4/5) of corresponding author.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]