| Abstract|| |
Context: Multidrug resistant (MDR) Enterococcus faecium is a nosocomial pathogen and clonal complex 17 (CC17) is the main genetic subpopulation of E. faecium in hospitals worldwide. Aims: There has thus far been no report of major E. faecium clones in Iranian hospitals. Subjects and Methods: The present study analyzed strains of MDR E. faecium obtained from patients and the Intensive Care Unit environments using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to determine the antibiotic resistance patterns and genetic features of the dominant. Results: clones of E. faecium. PFGE and MLST analysis revealed the presence of 17and 15 different subtypes, respectively. Of these, 18 (86%) isolates belonged toCC17. Most strains in this clonal complex harbored the esp gene and exhibited resistance to vancomycin, teicoplanin, ampicillin, ciprofloxacin, gentamicin, and erythromycin. The MLST results revealed 12 new sequence types (ST) for the first time. Approximately 50% of the STs were associated with ST203. Conclusion: Detection of E. faecium strains belonging to CC17 on medical equipment and in clinical specimens verified the circulation of high-risk MDR clones among the patients and in hospital environments in Iran.
Keywords: Enterococcus faecium, multidrug resistant, multilocus sequence typing, pulsed field gel electrophoresis
|How to cite this article:|
Shokoohizadeh L, Mobarez AM, Alebouyeh M, Zali MR, Ranjbar R. Genotyping of clinical and environmental multidrug resistant Enterococcus faecium strains. Indian J Pathol Microbiol 2017;60:74-8
|How to cite this URL:|
Shokoohizadeh L, Mobarez AM, Alebouyeh M, Zali MR, Ranjbar R. Genotyping of clinical and environmental multidrug resistant Enterococcus faecium strains. Indian J Pathol Microbiol [serial online] 2017 [cited 2017 Mar 28];60:74-8. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/74/200048
| Introduction|| |
Enterococci are harmless microbiota residing in the human gastrointestinal tract. These commensal organisms can become major nosocomial pathogens with the acquisition of genetic markers presenting virulence properties or multidrug resistance (MDR). This can pose serious concerns, especially when they colonize on indwelling medical devices.,Enterococcus faecium is the dominant species of enterococci that can cause nosocomial infections. The emergence of pathogenic strains of E. faecium with resistance to vancomycin, ampicillin, and gentamicin has created serious challenges to hospitals.,
Epidemiological studies have revealed the key role of health-care workers, medical equipment, and the environment surrounding the patient in the spread of E. faecium., This has been confirmed by molecular epidemiological approaches such as multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE).E. faecium clonal complex-17 (CC17) has been designated as a distinct high-risk enterococcal clonal complex and is associated with the majority of hospital outbreaks and clinical infections worldwide., This clonal complex is a genetic line of E. faecium that has adapted to prolonged colonization in hospital settings. It is associated with ampicillin and quinolone resistance and contains a putative pathogenicity island.,,,
Several recent epidemiological studies have suggested the spread of high-risk E. faecium strains in Middle Eastern hospitals.,, Studies in Iran have shown the distribution of MDR E. faecium strains in different hospitals, but there is no information on the genetic entity of the main E. faecium clones or their primary hospital reservoirs.,, The present study characterized the dominant sequence types (STs) of MDR E. faecium strains in a number of hospitals in Iran. The investigation of clonal relatedness of the isolates from both clinical and environmental samples was also determined using PFGE and MLST.
| Materials and Methods|| |
Enterococcus faecium isolates
The samples were collected from September 2013 to June 2014. The clinical specimens were collected from urine, wounds, blood, body fluids, sputum, and endotracheal tube aspiration. The environmental samples were obtained from the Intensive Care Unit (ICU) environment surfaces including beds, patient bed sheets, ventilators, intravenous catheters, floors, patient charts, syringe pumps, soap dispensers, monitors, stations, and trolleys by rubbing with cotton wool swabs that had been moistened in phosphate-buffered saline. The cotton swabs were then inoculated in Enterococcus enrichment broth (EEB, BBL, USA) and incubated overnight at 37°C for 48 h. Both clinical and environmental samples were cultured on Enterococcus enrichment agar at 37°C for 24–48 h.
All enterococci isolates were identified according to Collin and Facklam guidelines. PCR was conducted using ddl gene specific primers for all E. faecium and E. faecalis strains. The virulence gene (esp) and more prevalent vancomycin-resistant enterococci (VRE) genes (van A and van B) were also confirmed by PCR.,E. faecium ATCC 51559 and E. faecalis ATCC 51229 were used as the standard strains encoding van A and van B.
Antibiotic susceptibility tests
Antimicrobial susceptibility to vancomycin (30 µg), teicoplanin (30 µg), gentamicin (120 µg), ampicillin (10 µg), erythromycin (15 µg), ciprofloxacin (5 µg), tetracycline (30 µg), chloramphenicol (30 µg), nitroforantoin (300 µg), quinupristin-dalfopristin (synercid) (15 µg), and linezolide (30 µg) (Mast; UK) were determined using disk diffusion. The minimum inhibitory concentration (MIC) of vancomycin, ampicillin, and gentamicin was determined using agar dilution methods. Both tests were conducted according to Clinical and Laboratory Standards Institute criteria.
Pulsed-field gel electrophoresis
PFGE was used to analyze the clonal relatedness of MDR E. faecium strains in the clinical and environmental isolates. The genomic DNAs of the samples were digested with Sma I. PFGE was performed using a CHEF-DRIII (Bio-Rad, USA) apparatus as described in previous articles.
Multilocus sequence typing analysis
For the characterization and detection of common colonies of MDR E. faecium isolates, 21 isolates were selected for analysis by MLST based on PFGE pattern differences, antibiotic resistance profiles, location of isolation, and type of sample. MLST for E. faecium was performed using the scheme suggested by Homan et al. Internal fragments of seven housekeeping genes (atpA, ddl, gdh, purK, gyd, pstS, and adk) were amplified using specific primers according to conditions described in the online MLST database (http://efaecium.mlst.net/) and were then directly sequenced. Allele numbers and STs were assigned based on instructions in the online E. faecium database and eBURST (http://eburst.mlst.net/v3/instructions/) was used for phylogenetic analysis.
| Results|| |
A total of 162 enterococci strains were isolated from clinical and environmental specimens in four university hospitals in Tehran. Of these isolates, 84 (51.8%) were identified as E. faecium. A total of 72 (85.7%) E. faecium isolates were taken from clinical specimens and 12 (14.2%) from an ICU environment. The MDR phenotype was found in 69 (95%) clinical and 6 (50%) environmental isolates. All VRE isolates harbored the van A gene and showed simultaneous resistance to teicoplanin, ampicillin, gentamicin, ciprofloxacin, and tetracycline. MIC values for vancomycin, ampicillin, and gentamicin were 64–1024 µg/ml, 64–128 µg/µl, and 1024 µg/ml, respectively. A total of 26 (81%) E. faecium isolates showed amplified fragments of esp gene. This gene was only identified in VRE isolates.
Analysis of 45 MDR E. faecium strains by PFGE showed different patterns [Figure 1] among 17 pulsotypes, 3 pulsotypes were shared by clinical and environmental isolates. Pulsotype C was the most common and included 7 isolates from clinical and ICU environments. As for the PFGE results, diversity was also detected among STs. The MLST results showed that 15 different STs were identified [Figure 1]. The majority of isolates (80%, n = 17) belonged to CC17. ST739, ST419, ST742, and ST747 were singletons. A total of 12 STs (739, 740, 741, 742, 743, 744, 745, 746, 747, 748, and 762) were new and were being reported herein for the first time.
|Figure 1: Detection of diversity among multiresistant Enterococcus faecium by pulsed field gel electrophoresis. Gel image after pulsed field gel electrophoresis well 1 is Salmonella More Details cholerasuis serotype Braenderup H9812 as the molecular size marker, well 2 is from kidney transplant ward of Mofid Hospital, wells 2–7 are from gastroenterology ward in Taleghani Hospital, well 8 is from kidney transplant ward of Mofid Hospital, wells 9–12 are from kidney transplant ward of Labbafinejad Hospital, wells 13 and 14 are from the Intensive Care Units of Taleghani and Loghman Hospitals, respectively|
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The three largest ST groups were ST203 (19%), ST 740 (14.2%), and ST17 (14.2%) [Table 1] and [Figure 1]. According to eBURST analysis, more than 50% of STs (STs 740, 741, 743, 745 and 762) were single locus variants of ST203 [Figure 2]. ST203 and ST740 were identified in both clinical and environmental samples. ST 203 was detected in patient bed sheets and urine samples by different PFGE patterns (e.g. pulsotypes C and F). ST740 was detected in both clinical (blood and urine) and environmental samples (catheter tip) collected from the same ICU. It should be noted that the catheter and urine samples showed the same PFGE pattern [Table 1].
|Table 1: Genotypic characteristics of multidrug resistant Enterococcus faecium isolates from four Iranian hospitals based on pulsed field gel electrophoresis and multilocus sequence typing|
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|Figure 2: eBurst algorithm of multilocus sequence typing data, ring shapes showed the sequence types founded in Iranian hospitals. Sequence types 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 762 are new sequence types of E. faecium. The full algorithm is available at http://efaecium.mlst.net|
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All strains except the isolate with ST742 showed the allele of the type 1 purK gene which has been repeatedly reported to be related to epidemic VRE strains., Results obtained by MLST showed a clonal relationship among VRE E. faecium strains isolated from the four hospitals in Tehran and these same STs (17, 203 and 740) [Table 1]. Similar STs showed different antibiotic resistance levels and MIC values.
| Discussion|| |
E. faecium is well-known as a pathogen infecting hospitalized patients and is frequently linked to resistance against multiple antibiotics. Acquisition of antibiotic resistance and virulence genes play key roles in the emergence of new and high-risk clones of E. faecium in hospitals. The incidence of E. faecium in nosocomial infections has increased in Iran and many other countries in recent years, largely because of the increase in resistance to vancomycin among E. faecium strains.,,, Recent reports suggest a polyclonal endemicity distribution of E. faecium in Iranian hospitals.,,,, In the present study, diversity among E. faecium strains was confirmed by both MLST and PFGE (17 pulsotypes and 15 STs).
CC17 is a major clone in hospitals throughout the world and has been recognized as a successful hospital-associated E. faecium clonal complex exhibiting high levels of resistance to vancomycin, ampicillin, and quinolone although it has remained primarily vancomycin-susceptible in most European countries.,, CC17 E. faecium strains isolated in Asia and the Middle East are resistant to vancomycin.,,,,, The findings of the present study showed that CC17 is not limited to just VRE isolates, one VSE strain and 81% (n = 17) of VRE strains belonged to CC17 [Figure 1] and [Table 1].
In the current study, most strains were derivatives of ST203 and ST17. Consistent with our findings, recent studies from Australia and Malaysia have reported ST203 as the major E. faecium ST.,,, Lam et al. reported significant differences in the genome between strains ST17 and ST203. The ST203 strains grow at a significantly faster rate than ST17 strains and possess additional plasmids and genes that encode for resistance to other antimicrobials and metabolic pathways.
Investigations have confirmed the importance of the inanimate environment as a reservoir in the spread of E. faecium. The contamination of the hospital environment and the ability of enterococci to survive outside the human body for prolonged periods as factors for the occurrence of cross-contamination either through the patient or hospital environment., In the present study, STs 203 and 740 were found in environmental and clinical VRE isolates [Table 1]. The isolates with the same ST and similar resistance phenotypes could be considered as related clones with close genetic relationships that show possible transmission and evolution from single clones over the years.
| Conclusion|| |
Our results indicate that we are faced with threatening multiresistant E. faecium strains such as ST203 which belongs to CC17 in the clinical and environmental sections of Iranian hospitals. Genetic relatedness of clinical and hospital environmental samples shows that more attention should be directed toward controlling the environmental sources of infection in hospital settings. Our findings can be the basis of administrative decision-making in hospitals and as national and international roadmaps for controlling nosocomial infections and antimicrobial resistance.
This work was funded in part by a grant from Tarbiat Modares University and Research Center of Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Financial support and sponsorship
There is no financial and sponsorship.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Willems RJ, Top J, van Schaik W, Leavis H, Bonten M, Sirén J, et al.
Restricted gene flow among hospital subpopulations of Enterococcus faecium
. MBio 2012; 3:e00151-12.
Edwards DD. Enterococci attract attention of concerned microbiologists. ASM News 2000; 66:540-5.
Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, et al.
NHSN annual update: Antimicrobial-resistant pathogens associated with healthcare-associated infections: Annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect Control Hosp Epidemiol 2008; 29:996-1011.
Sood S, Malhotra M, Das BK, Kapil A. Enterococcal infections & antimicrobial resistance. Indian J Med Res 2008; 128:111-21.
Drees M, Snydman DR, Schmid CH, Barefoot L, Hansjosten K, Vue PM, et al.
Prior environmental contamination increases the risk of acquisition of vancomycin-resistant enterococci. Clin Infect Dis 2008;46:678-85.
Willems RJ, van Schaik W. Transition of Enterococcus faecium
from commensal organism to nosocomial pathogen. Future Microbiol 2009; 4:1125-35.
Willems RJ, Top J, van Santen M, Robinson DA, Coque TM, Baquero F, et al.
Global spread of vancomycin-resistant Enterococcus faecium
from distinct nosocomial genetic complex. Emerg Infect Dis 2005;11:821-8.
Leavis HL, Willems RJ, van Wamel WJ, Schuren FH, Caspers MP, Bonten MJ. Insertion sequence-driven diversification creates a globally dispersed emerging multiresistant subspecies of E. faecium
. PLoS Pathog 2007; 3:e7.
Werner G, Fleige C, Ewert B, Laverde-Gomez JA, Klare I, Witte W. High-level ciprofloxacin resistance among hospital-adapted Enterococcus faecium
(CC17). Int J of Antimicrob Agents 2010; 28; (2):119-25.
Leavis HL, Willems RJ, Top J, Bonten MJ. High-level ciprofloxacin resistance from point mutations in gyrA and parC confined to global hospital-adapted clonal lineage CC17 of Enterococcus faecium
. J Clin Microbiol 2006; 44:1059-64.
Leavis H, Top J, Shankar N, Borgen K, Bonten M, van Embden J, Willems RJ. A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium
and associated with epidemicity. J Bacteriol2004; 186:672-82.
Kirdar S, Sener AG, Arslan U, Yurtsever SG. Molecular epidemiology of vancomycin-resistant Enterococcus faecium
strains isolated from haematological malignancy patients in a research hospital in Turkey. J Med Microbiol 2010; 59(6):660-4.
Khan MA, van der Wal M, Farrell DJ, Cossins L, van Belkum A, Alaidan A, et al.
Analysis of VanA vancomycin-resistant Enterococcus faecium
isolates from Saudi Arabian hospitals reveals the presence of clonal cluster 17 and two new Tn1546 lineage types. J Antimicrob Chemother 2008; 62:279-83.
Saifi M, Pourshafie MR, Dallal MM, Katouli M. Clonal groups of high-level gentamicin-resistant Enterococcus faecium
isolated from municipal wastewater and clinical samples in Tehran, Iran. Lett Appl Microbiol 2009; 49:160-5.
Pourshafie MR, Talebi M, Saifi M, Katouli M, Eshraghi S, Kühn I, et al.
Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium
with unique vanS. Trop Med Int Health 2008; 13:722-7.
Shokoohizadeh L, Mobarez AM, Zali MR, Ranjbar R, Alebouyeh M, Sakinc T, et al.
High frequency distribution of heterogeneous vancomycin resistant Enterococcous faecium
(VREfm) in Iranian hospitals. Diagn Pathol 2013; 8:163.
Facklam RR, Collins MD. Identification of Enterococcus
species isolated from human infections by a conventional test scheme. J Clin Microbiol 1989; 27:731-4.
Kariyama R, Mitsuhata R, Chow JW, Clewell DB, Kumon H. Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. J Clin Microbiol 2000; 38:3092-5.
Shepard BD, Gilmore MS. Differential expression of virulence-related genes in Enterococcus faecalis
in response to biological cues in serum and urine. Infect Immun 2002; 70:4344-52.
CLSI – Clinical Laboratory Standards Institute. Performance Standard for Antimicrobial Susceptibility Testing. Document M100-S21. Wayne: Clinical Laboratory Standards Institute; 2011. p. 172.
Homan WL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, et al.
Multilocus sequence typing scheme for Enterococcus faecium
. J Clin Microbiol 2002; 40:1963-71.
Leavis HL, Willems RJ, Top J, Spalburg E, Mascini EM, Fluit AC, et al.
Epidemic and nonepidemic multidrug-resistant Enterococcus faecium
. Emerg Infect Dis 2003; 9:1108-15.
Caplin JL, Hanlon GW, Taylor HD. Presence of vancomycin and ampicillin-resistant Enterococcus faecium
of epidemic clonal complex-17 in wastewaters from the south coast of England. Environ Microbiol 2008; 10:885-92.
Leavis HL, Bonten MJ, Willems RJ. Identification of high-risk enterococcal clonal complexes: Global dispersion and antibiotic resistance. Curr Opin Microbiol 2006;9:454-60.
Sharifi Y, Hasani A, Ghotaslou R, Varshochi M, Hasani A, Soroush MH, et al.
Vancomycin-resistant enterococci among clinical isolates from north-west Iran: Identification of therapeutic surrogates. J Med Microbiol 2012;61(Pt 4):600-2.
Talebi M, Rahimi F, Katouli M, Möllby R, Pourshafie MR. Epidemiological link between wastewater and human vancomycin-resistant Enterococcus faecium
isolates. Curr Microbiol 2008;56:468-73.
Ko KS, Baek JY, Lee JY, Oh WS, Peck KR, Lee N, et al.
Molecular characterization of vancomycin-resistant Enterococcus faecium
isolates from Korea. J Clin Microbiol 2005;43:2303-6.
Willems RJ, Hanage WP, Bessen DE, Feil EJ. Population biology of Gram-positive pathogens: High-risk clones for dissemination of antibiotic resistance. FEMS Microbiol Rev 2011; 35:872-900.
Liu Y, Cao B, Gu L, Liu K, Feng Z. Successful control of vancomycin-resistant Enterococcus faecium
nosocomial outbreak in a teaching hospital in China. Am J Infect Control 2012; 40:568-71.
Cho HH, Song JY, Kwon KC, Lim JS, Koo SH. Antimicrobial resistance and multilocus sequence typing of vancomycin-resistant Enterococcus faecium
isolated from the Chungcheong area. Korean J Clin Microbiol 2011; 14:60-6.
Hsieh YC, Lee WS, Ou TY, Hsueh PR. Clonal spread of CC17 vancomycin-resistant Enterococcus faecium
with multilocus sequence type 78 (ST78) and a novel ST444 in Taiwan. Eur J Clin Microbiol Infect Dis 2010; 29:25-30.
Kamolvit W, Sidjabat HE, Nimmo GR, Anuj SN, Bergh H, Richardson LJ, et al.
Predominance of VREfm ST203 subgroup in Queensland. Pathology 2013; 45:99.
Johnson PD, Ballard SA, Grabsch EA, Stinear TP, Seemann T, Young HL, et al.
A sustained hospital outbreak of vancomycin-resistant Enterococcus faecium
bacteremia due to emergence of vanB E. faecium
sequence type 203. J Infect Dis 2010;202:1278-86.
Getachew Y, Hassan L, Zakaria Z, Abdul Aziz S. Genetic variability of vancomycin-resistant Enterococcus faecium
and Enterococcus faecalis
isolates from humans, chickens, and pigs in Malaysia. Appl Environ Microbiol 2013; 79:4528-33.
Lam MM, Seemann T, Tobias NJ, Chen H, Haring V, Moore RJ, et al.
Comparative analysis of the complete genome of an epidemic hospital sequence type 203 clone of vancomycin-resistant Enterococcus faecium
. BMC Genomics 2013; 14:595.
Hayden MK, Bonten MJ, Blom DW, Lyle EA, van de Vijver DA, Weinstein RA. Reduction in acquisition of vancomycin-resistant Enterococcus
after enforcement of routine environmental cleaning measures. Clin Infect Dis 2006; 42:1552-60.
Cheah AL, Spelman T, Liew D, Peel T, Howden BP, Spelman D, et al.
Enterococcal bacteraemia: Factors influencing mortality, length of stay and costs of hospitalization. Clin Microbiol Infect 2013; 19:E181-9.
Ashraf Mohabati Mobarez
Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]