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Virulence factors, antimicrobial resistance pattern and molecular analysis of Enterococcal strains isolated from burn patients
Microbial Pathogenesis 90 (2016) 93e97
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Virulence factors, antimicrobial resistance pattern and molecular analysis of Enterococcal strains isolated from burn patients Hamid Heidari a, Mohammad Emaneini a, Hossein Dabiri b, Fereshteh Jabalameli a, * a b
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Tehran, Iran Department of Medical Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2015 Received in revised form 10 November 2015 Accepted 19 November 2015 Available online 24 November 2015
The enterococci are emerging as a significant cause of hospital acquired infections. The pathogenesis of enterococci is attributed to the production of virulence factors and resistance to antibiotics. The purpose of the study was to assess the prevalence of genes encoding virulence factor, antimicrobial resistance determinant and molecular characteristic of enterococci isolated from burn patients. A total of 57 enterococci isolated from wound specimens of patients with burn injury were characterized by phenotypic and genotypic methods. The efaA was the most frequently detected gene (100%), followed by ace (89.1%), asa1 (54.3%), gelE (50%), cylA (30.4%), esp (23.9%) and hyl (8.7%) among Enterococcus faecalis isolates. The Enterococcus faecium strains carried asa1 and ace genes. All isolates were susceptible to tigecycline and vancomycin. Inducible resistance to clindamycin was not observed and 64% of isolates had resistance to erythromycin. High-level gentamicin resistance (HLGR) was seen in 65.2% of E. faecalis strains. The aac(60 )-Ie-aph(200 )-Ia gene was found in 47.8% of E. faecalis isolates. Our data indicated that the efaA, ace and asa1 were most frequent genes encoding virulence factors among Enterococci isolated from burn wound infection and the incidence of virulence factor genes was higher in E. faecalis rather than other isolates. The molecular analysis demonstrated high genetic diversity among Enterococcus populations from burn patients. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Enterococci Virulence factors Burn
1. Introduction Enterococci are Gram-positive cocci and common inhabitants of the human intestine and genitourinary tract [1,2], However they are cause of nosocomial infections, particularly bacteremia, sepsis, endocarditis, urinary tract infection (UTI) and wound infections [3,4]. Skin damage, immunodeficiency situation and hospitalization makes burns patients at a high risk for acquiring nosocomial infections such as Enterococci infections [5]. A collection of different factors including resistance to antibiotics and virulence determinants are involved in the success of enterococci in the hospital setting [6]. Presence of various virulence determinants such as, gelatinase (GelE), aggregation substance (AS) proteins (Asa1), enterococcal surface protein (Esp), collagen adhesine (Ace), cytolysin (CylA) and hyaluronidase (Hyl) can enhance bacterial colonization on hospitalized patients [1,6].
* Corresponding author. Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, 100 Poursina St., Keshavarz Blvd., Tehran, Iran. E-mail address: [email protected] (F. Jabalameli). http://dx.doi.org/10.1016/j.micpath.2015.11.017 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
Treatment of enterococcal infections could be difficult because they can survive in exposure to antimicrobial agents noticeably, they have intrinsically resistant to several antimicrobial agents including; b-Lactams, fluoroquinolones and trimethoprimsulfamethoxazole and moreover they can acquire resistance to antibiotics such as, aminoglycosides, macrolides and glycopeptides [7e9]. Resistance to vancomycin is encoded by the Van gene clusters which are carried on transposon [10]. Resistance to high concentrations of aminoglycoside antibiotics is usually due to aminoglycoside-modifying enzymes (AMEs) is encoded within mobile genetic elements [11]. Enterococcal isolates has emerged as important pathogen in Iran as in other countries, which presents serious challenges for hospital infection control practitioners and clinicians treating infected patients. There are several reports on the endemicity of vancomycin resistant enterococci (VRE) in Iran [12,13], but there is limited amount of information regarding the virulence determinants in hospitalized burn patients. Therefore, the aim of the current study was to evaluate the prevalence of genes encoding virulence factors and antimicrobial resistance and molecular
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characterization of Enterococcal strains isolated from burn patients.
2. Materials and methods 2.1. Bacterial isolates During June 2013 to June 2014, 57 Enterococcal isolates were collected from wound specimens of patients with burn injury from a burn hospital in Tehran. Only one isolate per patient was included. Identification of enterococci was performed based on a series of conventional microbiological tests [13]. The ddlE gene was targeted by Polymerase chain reaction (PCR) using specific primers (ddlE. faecalis F- 50 - ATCAAGTACAGTTAGTCT-30 and R-50 - ACGATTCAAAGCTAACTG-30 and ddlE. faecium F-50 - TAGAGACATTGAATATGCC30 and R-50 -TCGAATGTGCTACAATC-30 ) in order to confirm the identity of isolate as Enterococcus faecalis or Enterococcus faecium [14]. The PCR conditions consisted of a pre-denaturation step at 95 C for 5 min, followed by 30 cycles of 60 s at 95 C, 45 s at 45 C for ddlE. faecalis and 47 C for ddlE. faecium and 45 s at 72 C. A final extension step was performed at 72 C for 5 min.
2.2. Antimicrobial susceptibility testing Antimicrobial sensitivity tests were done by disc diffusion method on the Mueller- Hinton Agar (Merck Co., Germany) based on Clinical and Laboratory Standards Institute (CLSI) guideline [15]. The tested antibiotics (Mast Group Ltd., Merseyside, UK.) were ciprofloxacin (5 mg), erythromycin (15 mg), clindamycin (2 mg), tigecycline (15 mg), vancomycin (30 mg) and gentamicin (120 mg). E. faecalis ATCC 29212 was used as reference strain for antibiotic susceptibility testing. All of isolates were evaluated for inducible clindamycin resistance by double disc synergy test (D-test) accordance with CLSI guideline [15]. High-level gentamicin resistance (HLGR) was determined by the broth microdilution method using Brain Heart Infusion broth (BHI) (Conda S.A., Madrid, Spain) and measurement of minimal inhibitory concentration (MICs) of vancomycin was performed by the standard agar dilution on the BHI agar [15].
2.3. DNA extraction and gene detection Genomic DNA was extracted from overnight grown colonies as described previously [16]. PCR assay was performed for detection of the genes encoding virulence factor (gelE, asa1, cylA, hyl, esp, efa, ace) and the genes encoding to resistance vancomycin (vanA, vanB), aminoglycoside (aac(60 )-Ie aph(200 )Ia) and macrolide (ermA, ermB, msrA, mefA) [13,16e19].
2.4. Enterobacterial repetitive intergenic consensus (ERIC)-PCR For molecular analysis of isolates, ERIC-PCR was done as described previously [21]. Briefly, the PCR protocol consisted of a predenaturation step at 95 C for 5 min, followed by 30 cycles of 60 s at 95 C, 60 s at 36 C, and 60 s at 72 C. A final extension step was performed at 72 C for 10 min. PCR products were separated by electrophoresis in 1% agarose gels with 0.5X TBE (Tris/Borate/EDTA) buffer. DNA bands were observed by staining with KBC power load dye (Kawsar Biotech Co. Iran) and visualized under UV (ultraviolet) illumination. ERIC patterns were analyzed using GelCompar II. Isolates with a similarity coefficient equal or above 80% were considered as the same genotype. Only the dominant species (E. faecalis) were included in the analysis.
2.5. Statistical analysis Differences in the incidence of virulence genes among HLGR and non HLGR E. faecalis isolates were calculated by Fisher's test for each gene. A p-value of 0.05 was considered as statistically significant. 3. Results 3.1. Antimicrobial resistance pattern During one year study, a total of 57 Enterococcus strains, including E. faecalis 46 (80.7%), E. faecium 2 (3.5%) and other species 9 (15.8%) were isolated from burn wound. No isolates were found resistant to tigecycline and vancomycin (Table 1). Resistance to ciprofloxacin and clindamycin were more than 50% and 90% respectively. Inducible resistance to clindamycin was not observed and 64% of isolates were resistant to erythromycin. HLGR was seen in 65.2% of E. faecalis strains. The aac(60 )-Ie-aph(200 )-Ia gene was found in 47.8% of E. faecalis isolates. HLGR was found in one of the E. faecium strains which was negative for the aac(60 )-Ie-aph(200 )-Ia gene amplification. The erm(A), mef(A) and msr(A) genes were not detected in any of the isolates, and 25 (54.3%) of E. faecalis strains carried erm(B) gene. The vanA, and vanB genes were not seen in any of the enterococcal isolates. 3.2. Distribution of virulence genes The efaA was the most frequently detected gene (100%) among E. faecalis isolates, followed by ace (89.1%), asa1 (54.3%), gelE (50%), cylA (30.4%), esp (23.9%) and hyl (8.7%). The E. faecium strains carried asa1 and ace genes and one of them had gelE gene. The Prevalence of ace and efaA genes among other isolates were 22%, each and cylA was found in 11% of them. The hyl gene was significantly higher in HLGR isolates compared to non HLGR isolates (P 0.03). 3.3. Molecular genotyping ERIC-PCR of genomic DNA from E. faecalis strains amplified 4 to 10 bands with molecular weight ranging from 100 bp to 1.5 kb. Among all strains, six isolates did not show any product in reactions and thereby were non typable. Thirty four ERIC types obtained from 40 isolates. Of the 34 ERIC types, there were 6 types each containing 2 isolates separately while, 28 isolates had unique banding pattern and were classified as a distinct genotypes (Fig. 1). 4. Discussion Since the 1990s, Enterococci have been emerged as a significant cause of nosocomial infections [22]. Considering clinically important species of Enterococci, E. faecalis is the most common species cause of Enterococcal infections [1,23]. In our study, the most prevalent species was E. faecalis (80.7%), which is similar to result of other study in which the distribution of Enterococcal species was evaluated [13,16,24]. In the current study, the 65.2% of Enterococcus strains were HLGR and most of them (57.5%) carried the aac(60 )-Ie-aph(200 )-Ia gene. This finding is in accordance with previous studies in which have been shown that the aac(60 )-Ie-aph(200 )-Ia, aph(30 ), ant(40 ) and ant(6) genes encode aminoglycoside-modifying enzymes leading to high level resistance to gentamicin [16,25]. In the present study, there was no vancomycin resistant isolate. This result was different with previous studies in which the VRE strains have been isolated from burn infections [22,26]. Although our finding supports the vancomycin as an efficient choice against
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Table 1 The distribution of antimicrobial resistance genes and virulence genes among Enterococcus faecalis isolates. Isolate
Date of isolation
Resistance pattern
Resistance genes
Virulence genes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
4/6/2013 4/6/2013 11/6/2013 20/6/2013 27/6/2013 27/6/2013 29/6/2013 11/7/2013 18/7/2013 22/7/2013 27/7/2013 1/8/2013 9/8/2013 15/8/2013 23/8/2013 25/8/2013 2/9/2013 9/9/2013 14/9/2013 21/9/2013 23/9/2013 5/10/2013 16/10/2013 20/10/2013 10/11/2013 11/11/2013 11/11/2013 23/11/2013 23/11/2013 23/11/2013 26/11/2013 9/12/2013 11/12/2013 14/12/2013 24/12/2013 24/12/2013 13/1/2014 13/1/2014 25/1/2014 25/1/2014 22/2/2014 25/2/2014 3/3/2014 11/3/2014 19/5/2014 1/6/2014
GMa,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CD,E GM,CIP,CD,E GM,CIP,CD,E CD,E GM,CD,E GM,CIP,CD,E GM,CIP,CD GM,CIP,CD GM,CIP,CD GM,CIP,CD,E GM,CD GM,CIP,CD,E GM,CD GM,CIP,CD,E GM,CIP,CD,E GM,CD GM,CD,E GM,CIP,CD,E GM,CD,E GM,CD GM,CIP,CD GM,CIP,CD,E GM,CD GM,CIP,CD,E GM,CIP,CD,E GM,CD,E GM,CIP,CD,E GM,CIP,CD GM,CIP,CD GM,CIP,CD,E GM,CD,E GM,CD GM,CD,E GM,CIP,CD,E GM,CD GM,CD,E GM GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E
ermB, aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia e ermB,aac(60 )-Ie-aph(200 )-Ia ermB ermB ermB ermB e e aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia e aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia ermB aac(60 )-Ie-aph(200 )-Ia e e e ermB e ermB, aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia ermB ermB, aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia e ermB ermB e ermB ermB, aac(60 )-Ie-aph(200 )-Ia ermB ermB ermB, aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia ermB ermB aac(60 )-Ie-aph(200 )-Ia
cylA,gelE,ace,efa cylA,gelE,efa cylA,gelE,ace,efa esp,efa efa cylA,ace,efa cylA,ace,efa asa1,cylA,gelE,ace,efa asa1,cylA,gelE,esp,ace,efa cylA,ace,efa asa1,cylA,gelE,efa gelE,esp,ace,efa asa1,gelE,ace,efa asa1,gelE,ace,efa gelE,ace,efa hyl,gelE,ace,efa asa1,gelE,ace,efa gelE,ace,efa asa1,gelE,ace,efa asa1,gelE,ace,efa hyl,cylA,gelE,ace,efa asa1,hyl,gelE,esp,ace,efa ace,efa hyl,gelE,esp,ace,efa asa1, ace,efa asa1,cylA,gelE,ace,efa gelE,ace,efa asa1, ace,efa ace,efa asa1, ace,efa ace,efa asa1,gelE,ace,efa asa1, ace,efa asa1, ace,efa asa1,cylA,esp,ace,efa asa1, ace,efa ace,efa asa1, ace,efa asa1, ace,efa gelE,esp,ace,efa asa1,cylA,esp,ace,efa asa1,gelE,ace,efa asa1,gelE,ace,efa asa1,esp,ace,efa cylA,esp,ace,efa esp,efa
a
GM, Gentamicin; CIP, Ciprofloxacin; CD, Clindamycin; E, Erythromycin.
Enterococcal infections with no resistance against, however the vancomycin prescription should be under control for prevention of VRE strain emerges. Resistance to erythromycin and clindamycin were more than 60% and 90% respectively. Decreased susceptibility to erythromycin is likely associated to the wide use of these classes of antibiotics [23]. Similar to various reports, the ermB was present in most erythromycin resistant enterococci in this study [16,27,28]. The ermB gene in Enterococcus spp has a main role in getting resistance against macrolides, lincosamides, and streptogramin B (MLSB phenotype) [23]. Similar to previous studies, we found out that E. faecalis had more virulence genes in compare to other isolates [20,23]. Our findings indicated that the efaA and ace genes were most prevalent virulence genes in Enterococcal strains. The efaA gene was found in all of studied E. faecalis isolates and similar frequencies of the efaA have been reported in various studies [19,29,30]. It seems that, the efaA gene is always present in E. faecalis strains. The frequency of gelE, cylA and esp genes were 42%, 26% and 19% respectively. These results are in contrast to other studies in which
have reported that gelE, cylA and esp are most frequent Enterococcal virulence determinants in non-invasive infections of enterococci such as mucosal and urinary tract infections [29,30]. The presence of the hyl gene in HLGR E. faecalis isolates was significant and this may be due to co-presence of the hyl and aac(60 )-Ie-aph(200 )-Ia genes in a common transmissible plasmid. The asa1, ace and gelE genes were found in our E. faecium strains. Nevertheless, previous study has reported that E. faecium strains are generally clear of virulence determinants [30]. Enterococci possess efficient mechanisms to gene transfer such as conjugation and conjugative transposition [30,31]. Thus, this strain could acquire virulence genes by conjugation. Analysis of banding profiles of ERIC-PCR result showed high degree of genetic heterogeneity. The strains that classified in the same ERIC types, presented similar virulence genes or drug resistance pattern. Isolates no. 20 and 21 with similar ERIC profile were isolated from the patients within a close period of time. These data may confirm the genotypic correlations. High genetic diversity among isolates may contribute to the survival of various enterococci strains in hospital. Hospital procedures or equipment might
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Conflicts of interest statement All contributing authors declare no conflicts of interest. Acknowledgment This research has been supported by Tehran University of Medical Sciences & health Services grant 93-03-30-25139. References
Fig. 1. Dendrogram showing relatedness between ERIC-PCR patterns of Enterococcus faecalis strains isolated from burn wound infection. M, size marker (100 bp).
have led to spread of these strains among admitted patients. In conclusion, our study indicated that the efaA, ace and asa1 were most frequent genes encoding virulence factors among Enterococci isolated from burn wound infection and the prevalence of virulence factor genes was higher in E. faecalis rather than other isolates. The ERIC-PCR analysis demonstrated high genetic diversity among Enterococcus populations from burn patients.
[1] K. Fisher, C. Phillips, The ecology, epidemiology and virulence of Enterococcus, Microbiology 155 (2009) 1749e1757. [2] Y. Cetinkaya, P. Faik, C.G. Mayhall, vancomycin-resistant Enterococci, Clin. Microbiol. Rev. 13 (2000) 680e707. [3] A. Ronald, The etiology of urinary tract infection: traditional and emerging pathogens, Dis. Mon. 49 (2003) 71e82. [4] L. Baldassarri, R. Creti, L. Montanaro, et al., Pathogenesis of implant infections by enterococci, Int. J. Artif. Organs 28 (2005) 1101e1109. [5] L.K. Branski, A. Al-Mousawi, H. Rivero, et al., Emerging infections in burns, Surg. Infect. (Larchmt) 10 (2009) 389e397. [6] C.A. Arias, B.E. Murray, The rise of the Enterococcus: beyond vancomycin resistance, Nat. Rev. Microbiol. 10 (2012) 266e278. [7] M.M. Huycke, D.F. Sahm, M.S. Gilmore, Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future, Emerg. Infect. Dis. 4 (1998) 239e249. [8] K.S. Kaye, D. Kaye, Multidrug-resistant pathogens: mechanisms of resistance and epidemiology, Curr. Infect. Dis. Rep. 2 (2000) 391e398. [9] T. O'Driscoll, C.W. Crank, Vancomycin-resistant enterococcal infections: epidemiology, clinical manifestations, and optimal management, Infect. Drug Resist. 8 (2015) 217e230. [10] M. Emaneini, F.B. Hashemi, M. Aligholi, et al., Detection of vanB genotype enterococci in Iran, Int. J. Antimicrob. Agents 26 (2005) 98e99. [11] K.J. Shaw, P.N. Rather, R.S. Hare, et al., Molecular genetics of aminoglycosid resistance genes and familial relationships of aminoglycoside-modifying enzymes, Microbiol. Rev. 57 (1993) 138e163. [12] M.M. Feizabadi, S. Asadi, A. Aliahmadi, et al., Drug resistant patterns of enterococci recovered from patients in Tehran during 2000-2003, Int. J. Antimicrob. Agents 24 (2004) 521e522. [13] B. Fatholahzadeh, F.B. Hashemi, M. Emaneini, et al., Detection of vancomycin resistant nterococci (VRE) isolated from urinary tract infections (UTI) in Tehran, Iran, Daru 14 (2006) 141e145. [14] S. Dutka-Malen, S. Evers, P. Courvalin, Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR, J. Clin. Microbiol. 33 (1995) 1434. [15] Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing; Twenty-third Informational Supplement, 2013. M100eS23. [16] M. Emaneini, M. Aligholi, M. Aminshahi, Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran, Pol. J. Microbiol. 57 (2008) 173e178. [17] M. Emaneini, R. Bigverdi, D. Kalantar, et al., Distribution of genes encoding tetracycline resistance and aminoglycoside modifying enzymes in Staphylococcus aureus strains isolated from a burn center, Ann. Burns Fire Disasters 30 (2013) 76e80. [18] J.M. Choi, G.J. Woo, Molecular characterization of high-level gentamicinresistant Enterococcus faecalis from chicken meat in Korea, Int. J. Food Microbiol. 165 (2013) 1e6. €m, B. Lund, A. Sullivan, et al., Virulence and antimicrobial resistance [19] H. Billstro in clinical Enterococcus faecium, Int. J. Antimicrob. Agents 32 (2008) 374e377. n, S. Blomqvist, A. Almståhl, et al., Virulence factors and antibiotic [20] G. Dahle susceptibility in enterococci isolated from oral mucosal and deep infections, J. Oral. Microbiol. 4 (2012) 1e7. [21] A.M. Martín-Platero, E. Valdivia, M. Maqueda, et al., Characterization and safety evaluation of enterococci isolated from Spanish goats' milk cheeses, Int. J. Food Microbiol. 132 (2009) 24e32. [22] U. Altoparlak, O. Koca, Z. Ozkurt, et al., Incidence and risk factors of vancomycin-resistant enterococcus colonization in burn unit patients, Burns 37 (2011) 49e53. [23] L.K. Zou, H.N. Wang, B. Zeng, et al., Erythromycin resistance and virulence genes in Enterococcus faecalis from swine in China, New Microbiol. 34 (2011) 73e80. [24] E.E. Udo, N. Al-Sweih, O.A. Phillips, et al., Species prevalence and antibacterial resistance of enterococci isolated in Kuwait hospitals, J. Med. Microbiol. 52 (2003) 163e168. kova, H. Drahovska , et al., Occurrence of aminoglycoside[25] M. Filipov a, H. Bujda modifying-enzyme genes aac(60 )-aph(2”), aph(30 ), ant(4') and ant(6) in clinical isolates of Enterococcus faecalis resistant to high-level of gentamicin and amikacin, Folia Microbiol. (Praha) 51 (2006) 57e61.
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Virulence factors, antimicrobial resistance pattern and molecular analysis of Enterococcal strains isolated from burn patients Hamid Heidari a, Mohammad Emaneini a, Hossein Dabiri b, Fereshteh Jabalameli a, * a b
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Tehran, Iran Department of Medical Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2015 Received in revised form 10 November 2015 Accepted 19 November 2015 Available online 24 November 2015
The enterococci are emerging as a significant cause of hospital acquired infections. The pathogenesis of enterococci is attributed to the production of virulence factors and resistance to antibiotics. The purpose of the study was to assess the prevalence of genes encoding virulence factor, antimicrobial resistance determinant and molecular characteristic of enterococci isolated from burn patients. A total of 57 enterococci isolated from wound specimens of patients with burn injury were characterized by phenotypic and genotypic methods. The efaA was the most frequently detected gene (100%), followed by ace (89.1%), asa1 (54.3%), gelE (50%), cylA (30.4%), esp (23.9%) and hyl (8.7%) among Enterococcus faecalis isolates. The Enterococcus faecium strains carried asa1 and ace genes. All isolates were susceptible to tigecycline and vancomycin. Inducible resistance to clindamycin was not observed and 64% of isolates had resistance to erythromycin. High-level gentamicin resistance (HLGR) was seen in 65.2% of E. faecalis strains. The aac(60 )-Ie-aph(200 )-Ia gene was found in 47.8% of E. faecalis isolates. Our data indicated that the efaA, ace and asa1 were most frequent genes encoding virulence factors among Enterococci isolated from burn wound infection and the incidence of virulence factor genes was higher in E. faecalis rather than other isolates. The molecular analysis demonstrated high genetic diversity among Enterococcus populations from burn patients. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Enterococci Virulence factors Burn
1. Introduction Enterococci are Gram-positive cocci and common inhabitants of the human intestine and genitourinary tract [1,2], However they are cause of nosocomial infections, particularly bacteremia, sepsis, endocarditis, urinary tract infection (UTI) and wound infections [3,4]. Skin damage, immunodeficiency situation and hospitalization makes burns patients at a high risk for acquiring nosocomial infections such as Enterococci infections [5]. A collection of different factors including resistance to antibiotics and virulence determinants are involved in the success of enterococci in the hospital setting [6]. Presence of various virulence determinants such as, gelatinase (GelE), aggregation substance (AS) proteins (Asa1), enterococcal surface protein (Esp), collagen adhesine (Ace), cytolysin (CylA) and hyaluronidase (Hyl) can enhance bacterial colonization on hospitalized patients [1,6].
* Corresponding author. Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, 100 Poursina St., Keshavarz Blvd., Tehran, Iran. E-mail address: [email protected] (F. Jabalameli). http://dx.doi.org/10.1016/j.micpath.2015.11.017 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
Treatment of enterococcal infections could be difficult because they can survive in exposure to antimicrobial agents noticeably, they have intrinsically resistant to several antimicrobial agents including; b-Lactams, fluoroquinolones and trimethoprimsulfamethoxazole and moreover they can acquire resistance to antibiotics such as, aminoglycosides, macrolides and glycopeptides [7e9]. Resistance to vancomycin is encoded by the Van gene clusters which are carried on transposon [10]. Resistance to high concentrations of aminoglycoside antibiotics is usually due to aminoglycoside-modifying enzymes (AMEs) is encoded within mobile genetic elements [11]. Enterococcal isolates has emerged as important pathogen in Iran as in other countries, which presents serious challenges for hospital infection control practitioners and clinicians treating infected patients. There are several reports on the endemicity of vancomycin resistant enterococci (VRE) in Iran [12,13], but there is limited amount of information regarding the virulence determinants in hospitalized burn patients. Therefore, the aim of the current study was to evaluate the prevalence of genes encoding virulence factors and antimicrobial resistance and molecular
94
H. Heidari et al. / Microbial Pathogenesis 90 (2016) 93e97
characterization of Enterococcal strains isolated from burn patients.
2. Materials and methods 2.1. Bacterial isolates During June 2013 to June 2014, 57 Enterococcal isolates were collected from wound specimens of patients with burn injury from a burn hospital in Tehran. Only one isolate per patient was included. Identification of enterococci was performed based on a series of conventional microbiological tests [13]. The ddlE gene was targeted by Polymerase chain reaction (PCR) using specific primers (ddlE. faecalis F- 50 - ATCAAGTACAGTTAGTCT-30 and R-50 - ACGATTCAAAGCTAACTG-30 and ddlE. faecium F-50 - TAGAGACATTGAATATGCC30 and R-50 -TCGAATGTGCTACAATC-30 ) in order to confirm the identity of isolate as Enterococcus faecalis or Enterococcus faecium [14]. The PCR conditions consisted of a pre-denaturation step at 95 C for 5 min, followed by 30 cycles of 60 s at 95 C, 45 s at 45 C for ddlE. faecalis and 47 C for ddlE. faecium and 45 s at 72 C. A final extension step was performed at 72 C for 5 min.
2.2. Antimicrobial susceptibility testing Antimicrobial sensitivity tests were done by disc diffusion method on the Mueller- Hinton Agar (Merck Co., Germany) based on Clinical and Laboratory Standards Institute (CLSI) guideline [15]. The tested antibiotics (Mast Group Ltd., Merseyside, UK.) were ciprofloxacin (5 mg), erythromycin (15 mg), clindamycin (2 mg), tigecycline (15 mg), vancomycin (30 mg) and gentamicin (120 mg). E. faecalis ATCC 29212 was used as reference strain for antibiotic susceptibility testing. All of isolates were evaluated for inducible clindamycin resistance by double disc synergy test (D-test) accordance with CLSI guideline [15]. High-level gentamicin resistance (HLGR) was determined by the broth microdilution method using Brain Heart Infusion broth (BHI) (Conda S.A., Madrid, Spain) and measurement of minimal inhibitory concentration (MICs) of vancomycin was performed by the standard agar dilution on the BHI agar [15].
2.3. DNA extraction and gene detection Genomic DNA was extracted from overnight grown colonies as described previously [16]. PCR assay was performed for detection of the genes encoding virulence factor (gelE, asa1, cylA, hyl, esp, efa, ace) and the genes encoding to resistance vancomycin (vanA, vanB), aminoglycoside (aac(60 )-Ie aph(200 )Ia) and macrolide (ermA, ermB, msrA, mefA) [13,16e19].
2.4. Enterobacterial repetitive intergenic consensus (ERIC)-PCR For molecular analysis of isolates, ERIC-PCR was done as described previously [21]. Briefly, the PCR protocol consisted of a predenaturation step at 95 C for 5 min, followed by 30 cycles of 60 s at 95 C, 60 s at 36 C, and 60 s at 72 C. A final extension step was performed at 72 C for 10 min. PCR products were separated by electrophoresis in 1% agarose gels with 0.5X TBE (Tris/Borate/EDTA) buffer. DNA bands were observed by staining with KBC power load dye (Kawsar Biotech Co. Iran) and visualized under UV (ultraviolet) illumination. ERIC patterns were analyzed using GelCompar II. Isolates with a similarity coefficient equal or above 80% were considered as the same genotype. Only the dominant species (E. faecalis) were included in the analysis.
2.5. Statistical analysis Differences in the incidence of virulence genes among HLGR and non HLGR E. faecalis isolates were calculated by Fisher's test for each gene. A p-value of 0.05 was considered as statistically significant. 3. Results 3.1. Antimicrobial resistance pattern During one year study, a total of 57 Enterococcus strains, including E. faecalis 46 (80.7%), E. faecium 2 (3.5%) and other species 9 (15.8%) were isolated from burn wound. No isolates were found resistant to tigecycline and vancomycin (Table 1). Resistance to ciprofloxacin and clindamycin were more than 50% and 90% respectively. Inducible resistance to clindamycin was not observed and 64% of isolates were resistant to erythromycin. HLGR was seen in 65.2% of E. faecalis strains. The aac(60 )-Ie-aph(200 )-Ia gene was found in 47.8% of E. faecalis isolates. HLGR was found in one of the E. faecium strains which was negative for the aac(60 )-Ie-aph(200 )-Ia gene amplification. The erm(A), mef(A) and msr(A) genes were not detected in any of the isolates, and 25 (54.3%) of E. faecalis strains carried erm(B) gene. The vanA, and vanB genes were not seen in any of the enterococcal isolates. 3.2. Distribution of virulence genes The efaA was the most frequently detected gene (100%) among E. faecalis isolates, followed by ace (89.1%), asa1 (54.3%), gelE (50%), cylA (30.4%), esp (23.9%) and hyl (8.7%). The E. faecium strains carried asa1 and ace genes and one of them had gelE gene. The Prevalence of ace and efaA genes among other isolates were 22%, each and cylA was found in 11% of them. The hyl gene was significantly higher in HLGR isolates compared to non HLGR isolates (P 0.03). 3.3. Molecular genotyping ERIC-PCR of genomic DNA from E. faecalis strains amplified 4 to 10 bands with molecular weight ranging from 100 bp to 1.5 kb. Among all strains, six isolates did not show any product in reactions and thereby were non typable. Thirty four ERIC types obtained from 40 isolates. Of the 34 ERIC types, there were 6 types each containing 2 isolates separately while, 28 isolates had unique banding pattern and were classified as a distinct genotypes (Fig. 1). 4. Discussion Since the 1990s, Enterococci have been emerged as a significant cause of nosocomial infections [22]. Considering clinically important species of Enterococci, E. faecalis is the most common species cause of Enterococcal infections [1,23]. In our study, the most prevalent species was E. faecalis (80.7%), which is similar to result of other study in which the distribution of Enterococcal species was evaluated [13,16,24]. In the current study, the 65.2% of Enterococcus strains were HLGR and most of them (57.5%) carried the aac(60 )-Ie-aph(200 )-Ia gene. This finding is in accordance with previous studies in which have been shown that the aac(60 )-Ie-aph(200 )-Ia, aph(30 ), ant(40 ) and ant(6) genes encode aminoglycoside-modifying enzymes leading to high level resistance to gentamicin [16,25]. In the present study, there was no vancomycin resistant isolate. This result was different with previous studies in which the VRE strains have been isolated from burn infections [22,26]. Although our finding supports the vancomycin as an efficient choice against
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Table 1 The distribution of antimicrobial resistance genes and virulence genes among Enterococcus faecalis isolates. Isolate
Date of isolation
Resistance pattern
Resistance genes
Virulence genes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
4/6/2013 4/6/2013 11/6/2013 20/6/2013 27/6/2013 27/6/2013 29/6/2013 11/7/2013 18/7/2013 22/7/2013 27/7/2013 1/8/2013 9/8/2013 15/8/2013 23/8/2013 25/8/2013 2/9/2013 9/9/2013 14/9/2013 21/9/2013 23/9/2013 5/10/2013 16/10/2013 20/10/2013 10/11/2013 11/11/2013 11/11/2013 23/11/2013 23/11/2013 23/11/2013 26/11/2013 9/12/2013 11/12/2013 14/12/2013 24/12/2013 24/12/2013 13/1/2014 13/1/2014 25/1/2014 25/1/2014 22/2/2014 25/2/2014 3/3/2014 11/3/2014 19/5/2014 1/6/2014
GMa,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CD,E GM,CIP,CD,E GM,CIP,CD,E CD,E GM,CD,E GM,CIP,CD,E GM,CIP,CD GM,CIP,CD GM,CIP,CD GM,CIP,CD,E GM,CD GM,CIP,CD,E GM,CD GM,CIP,CD,E GM,CIP,CD,E GM,CD GM,CD,E GM,CIP,CD,E GM,CD,E GM,CD GM,CIP,CD GM,CIP,CD,E GM,CD GM,CIP,CD,E GM,CIP,CD,E GM,CD,E GM,CIP,CD,E GM,CIP,CD GM,CIP,CD GM,CIP,CD,E GM,CD,E GM,CD GM,CD,E GM,CIP,CD,E GM,CD GM,CD,E GM GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E GM,CIP,CD,E
ermB, aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia e ermB,aac(60 )-Ie-aph(200 )-Ia ermB ermB ermB ermB e e aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia e aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia ermB aac(60 )-Ie-aph(200 )-Ia e e e ermB e ermB, aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia ermB ermB, aac(60 )-Ie-aph(200 )-Ia aac(60 )-Ie-aph(200 )-Ia e ermB ermB e ermB ermB, aac(60 )-Ie-aph(200 )-Ia ermB ermB ermB, aac(60 )-Ie-aph(200 )-Ia ermB, aac(60 )-Ie-aph(200 )-Ia ermB ermB aac(60 )-Ie-aph(200 )-Ia
cylA,gelE,ace,efa cylA,gelE,efa cylA,gelE,ace,efa esp,efa efa cylA,ace,efa cylA,ace,efa asa1,cylA,gelE,ace,efa asa1,cylA,gelE,esp,ace,efa cylA,ace,efa asa1,cylA,gelE,efa gelE,esp,ace,efa asa1,gelE,ace,efa asa1,gelE,ace,efa gelE,ace,efa hyl,gelE,ace,efa asa1,gelE,ace,efa gelE,ace,efa asa1,gelE,ace,efa asa1,gelE,ace,efa hyl,cylA,gelE,ace,efa asa1,hyl,gelE,esp,ace,efa ace,efa hyl,gelE,esp,ace,efa asa1, ace,efa asa1,cylA,gelE,ace,efa gelE,ace,efa asa1, ace,efa ace,efa asa1, ace,efa ace,efa asa1,gelE,ace,efa asa1, ace,efa asa1, ace,efa asa1,cylA,esp,ace,efa asa1, ace,efa ace,efa asa1, ace,efa asa1, ace,efa gelE,esp,ace,efa asa1,cylA,esp,ace,efa asa1,gelE,ace,efa asa1,gelE,ace,efa asa1,esp,ace,efa cylA,esp,ace,efa esp,efa
a
GM, Gentamicin; CIP, Ciprofloxacin; CD, Clindamycin; E, Erythromycin.
Enterococcal infections with no resistance against, however the vancomycin prescription should be under control for prevention of VRE strain emerges. Resistance to erythromycin and clindamycin were more than 60% and 90% respectively. Decreased susceptibility to erythromycin is likely associated to the wide use of these classes of antibiotics [23]. Similar to various reports, the ermB was present in most erythromycin resistant enterococci in this study [16,27,28]. The ermB gene in Enterococcus spp has a main role in getting resistance against macrolides, lincosamides, and streptogramin B (MLSB phenotype) [23]. Similar to previous studies, we found out that E. faecalis had more virulence genes in compare to other isolates [20,23]. Our findings indicated that the efaA and ace genes were most prevalent virulence genes in Enterococcal strains. The efaA gene was found in all of studied E. faecalis isolates and similar frequencies of the efaA have been reported in various studies [19,29,30]. It seems that, the efaA gene is always present in E. faecalis strains. The frequency of gelE, cylA and esp genes were 42%, 26% and 19% respectively. These results are in contrast to other studies in which
have reported that gelE, cylA and esp are most frequent Enterococcal virulence determinants in non-invasive infections of enterococci such as mucosal and urinary tract infections [29,30]. The presence of the hyl gene in HLGR E. faecalis isolates was significant and this may be due to co-presence of the hyl and aac(60 )-Ie-aph(200 )-Ia genes in a common transmissible plasmid. The asa1, ace and gelE genes were found in our E. faecium strains. Nevertheless, previous study has reported that E. faecium strains are generally clear of virulence determinants [30]. Enterococci possess efficient mechanisms to gene transfer such as conjugation and conjugative transposition [30,31]. Thus, this strain could acquire virulence genes by conjugation. Analysis of banding profiles of ERIC-PCR result showed high degree of genetic heterogeneity. The strains that classified in the same ERIC types, presented similar virulence genes or drug resistance pattern. Isolates no. 20 and 21 with similar ERIC profile were isolated from the patients within a close period of time. These data may confirm the genotypic correlations. High genetic diversity among isolates may contribute to the survival of various enterococci strains in hospital. Hospital procedures or equipment might
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Conflicts of interest statement All contributing authors declare no conflicts of interest. Acknowledgment This research has been supported by Tehran University of Medical Sciences & health Services grant 93-03-30-25139. References
Fig. 1. Dendrogram showing relatedness between ERIC-PCR patterns of Enterococcus faecalis strains isolated from burn wound infection. M, size marker (100 bp).
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