Antimicrobial resistance patterns and virulence factors of enterococci isolates in hospitalized burn patients
Shokoohizadeh et al. BMC Res Notes
Antimicrobial resistance patterns and virulence factors of enterococci isolates in hospitalized burn patients
Leili Shokoohizadeh 0 2
Alireza Ekrami 0 2
Maryam Labibzadeh 1
Liaqat Ali 3 4
Seyed Mohammad Alavi 0
0 Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences , P.O. Box: 61357-15794, Ahvaz , Iran
1 Infertility Research and Treatment Center of Jahad Daneshgahi , Khuzestan, Ahvaz , Iran
2 Department of Medical Laboratory Sciences, Faculty of Para Medicine, Ahvaz Jundishapur University of Medical Sciences , P.O. Box: 61357-15794, Ahvaz , Iran
3 Department of Molecular Medicine, National University of Medical Sciences , Rawalpindi/Islamabad , Pakistan
4 Department of Internal Medicine II, University Hospital Freiburg , Freiburg , Germany
Objective: The objective of this study was to determine the frequency of the antimicrobial resistance and genes encoding virulence factors of enterococci isolated in hospitalized burn patients in a major burn center in Ahvaz, southwest of Iran. A total of 340 bacterial isolates were collected from the burn center from February 2014 to February 2015. The antimicrobial susceptibility and MIC of vancomycin were determined using the disk diffusion and microagar dilution techniques. The genus and species-specific genes, potential virulence genes, and vanA and vanB genes were detected by polymerase chain reaction. Results: According to our results, out of the 340 bacterial isolates, 16.4% (n = 56) were identified as enterococci. Out of the 56 enterococcal isolates, 35 (62.5%) were Enterococcus faecalis and 21 (37.5%) were Enterococcus faecium. More than 20% (n = 5) of E. faecium demonstrated resistance to vancomycin. The gelE and asa genes were the most prevalent virulence genes in E. faecalis (48.5%) and E. faecium (43%) isolates. The emergence of vancomycin resistant E. faecium strains which have several virulence factors should be considered as a major cause of concern for burn centers. Control and management of infections induced by enterococci should be regarded as highly important in burn patients.
Enterococci; Vancomycin resistant; Virulence genes; Burn
Nosocomial infections are the most common factors in
increasing morbidity, mortality, long-term treatment,
and cost in hospitalized patients [
]. Burn patients are
more at the risk of complications of nosocomial
infections due to their weakened immune system . In many
countries, particularly developing countries including
Iran, burn infections are one of the most serious issues in
the field of health [
]. Organisms associated with
nosocomial infections in burn patients include the patient’s own
normal flora organisms and the hospital environment or
the staff. Bacterial pathogens are the most common cause
of burn infections such as wounds in burn patients. In
addition to wound infections, bacterial pneumonia and
bloodstream infections are also common causes of death
in burn patients [
Among Gram-positive and Gram-negative bacteria,
Staphylococcus aureus and Pseudomonas aeruginosa are
considered as the most important factors causing burn
wound infections [
]. According to reports published in
recent years, enterococci have become important factors
in acquiring nosocomial infections [
Grampositive bacteria are part of the normal flora of the human
digestive system and were considered to be commensal
bacteria in the past [
]. Since enterococci have the ability
to cause infection in wounds, including burn wounds, and
are intrinsically resistant to many antibiotics due to their
ability to acquire antibiotic resistance genes, they are
considered as one of the most important causes of
nosocomial infections. Enterococci are now regarded as the third
most common cause of hospital infections. Enterococci
can cause urinary tract, intra-abdominal, pelvic, wound,
and super infections (including those caused by burns)
in patients [
]. The emergence of multidrug-resistant
enterococci strains (resistant to two or more classes of
antibiotics) or MDR has caused numerous problems for
patients in hospitals all over the world . In
Enterococcus species, E. faecium and E. faecalis are regarded as
important nosocomial bacterial pathogens. The published
reports of some countries indicated the prevalence of
burn wound infections caused by VRE strains in hospitals,
particularly in the intensive care unit (ICU) [
on bacteria and burn infections have been conducted in
Taleghani Hospital of Ahvaz as the main burn center in
Khuzestan Province, southwest of Iran [
]. Only a
few studies have been published on the role of enterococci
in burn patients in Iran, and given the increasing
importance of VRE-related infections, the aim of the present
study was to evaluate the frequency of enterococcal
infections, virulence factors, and antibiotic resistance patterns
in enterococci strains isolated from clinical samples of
burn patients hospitalized in one of the largest burn
centers in the southwest of Iran.
The present study was ethically approved by Ahvaz
Jundishapur University of Medical Sciences, Institutional
Review Board (Code No. 93146).
A total of 340 bacterial strains were isolated from
clinical samples of hospitalized burn patients (wound
biopsies, blood, and urine) in a burn center in Ahvaz from
February 2014 to February 2015. All the enterococci
isolates were identified in the genus and species levels by
Gram staining, catalase reaction, growth in 6.5% NaCl
solution, motility assessment, use of arabinose, bile, and
esculin hydrolysis tests [
]. Furthermore, a PCR-based
study was conducted using specific primers (ddlE. faecium
and ddlE. faecalis) for all E. faecium and E. faecalis isolates
]. Antimicrobial susceptibility test for enterococci
isolates was performed against vancomycin (30 μg),
teicoplanin (30 μg), gentamicin (120 μg), ampicillin (10 μg),
ciprofloxacin (5 μg), tetracycline (30 μg),
chloramphenicol (30 μg), and linezolid (30 μg) (Mast, UK) by the disk
diffusion technique. Vancomycin minimum inhibitory
concentration (MICs) was detected by micro-agar
dilution method based on the CLSI guidelines [
from different enterococcal isolates were extracted using
an appropriate DNA Extraction Kit (Cinagene, Iran).
Specific primers of ddlE. faecium, ddlE. faecalis vancomycin
resistant genes (vanA and vanB), and virulence genes
including hyaluronidase (hyl), enterococci surface
protein (esp), haemolysin activator (cyl), gelatinase (gelE),
and aggregation substance asaI (Metaboin, Germany)
were used for PCR amplification as described previously
14, 16, 18
Results and discussion
Prevalence of enterococci isolates
Enterococci were isolated from 16.4% (n = 56) of clinical
specimens of burn hospitalized patients. The frequency
of enterococci in wound, blood, and urine specimens
were 37.5% (n = 21), 30.3% (n = 17), and 35% (n = 18),
respectively. Among the 56 enterococci strains, 62.5%
(n = 35) were identified as E. faecalis and 37.5% (n = 21)
were identified as E. faecium. Table 1 shows the
comparison of the frequency distribution of E. faecium and
E. faecalis in different clinical specimens. Among
Enterococcus species, E. faecalis was identified as the major
cause of enterococcal infections. E. faecalis caused
enterococcal infections approximately ten times more than
other Enterococcus species [
]. E. faecalis has more
virulence factors than E. faecium; consequently, it has
higher pathogenicity [
]. Nevertheless, in recent years
a microbial shift from E. faecium to E. faecalis has been
observed which could be due to the emergence of MDR
strains resistant to vancomycin (VRE) species in
]. One of the influential factors in increasing
nosocomial infections resulted from enterococci is the
emergence of VREs in hospitals. According to the
studies conducted in Iran, nosocomial infections caused by
enterococci, particularly the resistant strains are highly
]. In 2001, the National Nosocomial
Infection Surveillance System (NNISS) reported that
the frequency of burn wound infections caused by
Enterococcus was 11% [
] which is close to the results of the
current study (16%). There have been few studies
investigating the existence of infection caused by enterococci in
burn patients. Our results showed an increase in the
contribution of E. faecium to infections which is consistent
with our results on the reported frequency ratio of E.
faecium to E. faecalis in Tehran’s hospitals in 2014. Although
these results are different from other results reported in
Iran and other countries, more than 80% of burn wound
infections caused by Enterococcus was due to E. faecalis
Antimicrobial resistance patterns
Figure 1 presents the comparison of the results of
antimicrobial susceptibility analysis of E. faecium and E.
faecalis. More than 20% (n = 5) of E. faecium exhibited
Table 1 Frequency distribution of E. faecalis and E. faecium
isolates in clinical specimens of burn patients
resistance to vancomycin and teicoplanin. This can be the
reason for the increased presence of E. faecium which is
resistant to vancomycin and can survive in the hospital
environment. E. faecium strains exhibited higher
levels of resistance to antibiotics. Higher levels of
resistance to tetracycline and ampicillin were observed in 71%
(n = 15) and 66% (n = 14) of E. faecium isolates,
respectively. According to the findings, this can be an alarm for
burn patients owing to the fact that more than 50% of E.
faecium strains and 100% of VRE strains were resistant
to ampicillin and gentamicin. No resistance to linezolid
and vancomycin was observed in E. faecalis isolates. The
most frequent resistance pattern was simultaneous
resistance to ampicillin/tetracycline/gentamicin (AMP/TET/
GEM) in 43% of E. faecium and tetracylin/gentamicin
(TET/GEM) in 46% of E. faecalis isolates. All vancomycin
resistant E. faecium (VREfm) possessed vanA gene, while
no vanB gene was observed in VREfm strains. On the
other hand, all VRE strains which possessed vanA gene
showed resistance both to vancomycin and teicoplanin.
MIC values for vancomycin ranged from 32 to 1024 µg/
ml. In addition, there was no correlation between the
presence of vancomycin resistance, virulence factors, and
the clinical samples. So far, no documented results for the
presence of VRE in burn patients have been published in
Iran which may be attributed to the lack of expensive and
purposive studies on enterococci in the burn units.
However, some countries have reported VREs in burn units
]. According to the results of this study and similar
studies, linezolid was identified as the most effective
antibiotic against enterococcal infections [
10, 22, 23
Distribution of virulence genes
Table 2 presents the frequency and distribution of
virulence genes among clinical specimens. The gelE and asa
genes were the most frequent virulence genes in E.
faecalis (48.5%) and E. faecium (43%) isolates, respectively.
This is consistent with the results reporting gelE as the
most frequent virulence factor in E. faecalis strains [
], while some reports have indicated the absence or
low rate of gelE gene in enterococcal isolates [
25, 30, 31
Some reports have indicated the absence or low rate of
asa gene in E. faecium strains [
]. The cyl gene was
not detected in any E. faecium isolates. The hyl gene was
found in 1.7% (n = 1) of E. faecium isolates. A total of
68.5% (n = 24) of E. faecalis isolates and 42.8% (n = 9) of
E. faecium isolates were positive for more than one
virulence gene. There was no relationship between virulence
genes and clinical specimens.
In conclusion, based on the results of the current study,
it was demonstrated that the detection of enterococci
in clinical samples of burn patients is highly important.
In addition, the presence of E. faecium strains resistant
to vancomycin with several virulence factors can be a
source of alarm for burn centers. Considering the
possible transmission of antibiotic resistance genes among
enterococci, and staphylococci, the control and
management of infections caused by enterococci as well as the
appropriate administration of antibiotics in burn patients
can be highly effective in treating nosocomial infections
in burn centers.
In this study, no significant differences exist between the
presence of virulence genes and resistance to
vancomycin. One of the reasons for this result may be the low
number of vancomycin resistant strains. Therefore, more
samples must be collected from hospitals in general and
from burn centers in particular.
MIC: minimum inhibition concentration; E. faecium: Enterococcus faecium; E.
faecalis: Enterococcus faecalis; MDR: multi-drug resistant; VRE: vancomycin
resistant enterococci; PCR: polymerase chain reaction; hyl: hyaluronidase; esp:
enterococci surface protein; cyl: haemolysin activator; gleE: gelatinase; capD:
capsular polysaccharides biosynthesis protein; asaI: aggregation substance;
VAN: vancomycin; TEI: teicoplanin; AMP: ampicillin; GEM: gentamicin; CHL:
chloramphenicol; LZD: linezolid; IMP: imipenem; CIP: ciprofloxacin; TET:
LS and AE conceived the study. LS and ML conducted the experiments and
analyzed the results. LA and LS drafted the manuscript and made substantial
contributions to the design of the study. LA, AE, and SMA critically reviewed
the manuscript. LS, SMA and ML, participated in data analysis. All authors read
and approved the final manuscript.
We would like to thank all members of microbiology laboratory of Taleghani
Hospital in Ahvaz.
The authors declare that they have no competing interests.
Availability of data and materials
All the information supporting our conclusions and appropriate references are
included in the manuscript.
Consent for publication
Ethics approval and consent to participate
The present study was ethically approved by the Ahvaz Jundishapur University
of Medical Sciences, Institutional Review Board (Code No. 93146). The Samples
were accessed retrospectively. Permission was granted from the hospital and
Ahvaz Jundishapur University of Medical Sciences to access the samples.
This research was funded by a grant from infection and tropical disease
research centers, Jundishapur University of Medical Sciences, Ahvaz.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Haas JP . Measurement of infection control department performance: state of the science . Am J Infect Control . 2006 ; 34 ( 9 ): 543 - 9 .
2. Mayhall GC . The epidemiology of burn wound infections: then and now . Clin Infect Dis . 2003 ; 37 : 543 - 50 .
3. Azimi L , Motevallian A , Ebrahimzadeh Namvar A , Asghari B , Lari AR . Nosocomial infections in burned patients in Motahari Hospital , Tehran, Iran. Dermatol Res Pract . 2011 .
4. D 'Avignon LC , Hogan BK , Murray CK , Loo FL , Hospenthal DR , Cancio LC , et al. Contribution of bacterial and viral infections to attributable mortality in patients with severe burns: an autopsy series . Burns . 2010 ; 36 : 73 .
5. Lawrence J . Burn bacteriology during the last 50 years . Burns. 1994 ; 18 : 23 - 9 .
6. Sood S , Malhotra M , Das BK , Kapi A . Enterococcal infections & antimicrobial resistance . Indian J Med Res . 2008 ; 128 : 111 - 21 .
7. Lindenstrau AG , Pavlovic M , Bringmann A , Behr J , Ehrmann MA , Vogel RF . Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium . Syst Appl Microbiol . 2011 ; 34 : 553 - 60 .
8. Arshadi M , Douraghi M , Shokoohizadeh L , Moosavian SM , Pourmand MR . High prevalence of diverse vancomycin resistance Enterococcus faecium isolates in clinical and environmental sources in ICU wards in southwest of Iran . Microb Pathog . 2017 ; 111 : 212 - 7 .
9. Willems RJ , Hanage WP , Bessen DE , Feil EJ . Population biology of Grampositive pathogens: high-risk clones for dissemination of antibiotic resistance . FEMS Microbiol Rev . 2011 ; 35 ( 8 ): 72 - 900 .
10. Shokoohizadeh L , Mobarez AM , Zali MR , Ranjbar R , Alebouyeh M , Sakinc T , et al. High frequency distribution of heterogeneous vancomycin resistant Enterococcus faecium (VREfm) in Iranian hospitals . Diagn pathol . 2013 ; 8 : 163 .
11. May AK , Melton SM , McGwin G , Cross JM , Moser SA , Rue LW . Reduction of vancomycin-resistant enterococcal infections by limitation of broadspectrum cephalosporin use in a trauma and burn intensive care unit . Shock . 2000 ; 14 : 259 - 64 .
12. Ekrami A , Hemadi A , Kalantar E , Latifi M , Kayedani A . Epidemiology of hospitalized burn patients during 5 years in Khuzestan province , Iran. Iran J Clin Infect Dis . 2010 ; 5 ( 1 ): 40 - 4 .
13. Ekrami A , Montazeri EA , Kaydani GA , Shokoohizadeh L . Methicillin resistant staphylococci: prevalence and susceptibility patterns in a burn center in Ahvaz from 2013-2014 . Iran J Microbiol. 2015 ; 7 ( 4 ): 208 .
14. Falkman RR , Collin MD . Identification of Enterococcus species isolated from human infections by a conventional test scheme . J Clin Microbiol . 1989 ; 27 : 731 - 4 .
15. 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 .
16. Performance standards for antimicrobial susceptibility testing , vol. 31 . 21th ed. Wayne: Clinical and Laboratory Standards Institute; 2011 . p. 84 - 7 .
17. Eaton TJ , Gasson MJ . Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates . Appl Environ Microbiol . 2001 ; 67 : 1628 - 35 .
18. Vankerckhoven V , Van Autgaerden T , Vael C , Lammens C , Chapelle S , Rossi R , et al. 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 . J Clin Microbiol . 2004 ; 42 : 4473 - 9 .
19. Mundy LM , Sahm DF , Gilmore M. Relationships between enterococcal virulence and antimicrobial resistance . Clin Microbiol Rev . 2000 ; 13 : 513 - 22 .
20. Sharifi Y , Hasani A , Ghotaslou R , Varshochi M , Hasani A , Aghazadeh M , Milani M. Survey of virulence determinants among vancomycin resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens of hospitalized patients of northwest of Iran . Open Microbiol J. 2012 ; 6 : 34 - 9 .
21. Lester CH , Sandvang D , Olsen SS , Schønheyder HC , Jarløv JO , Bangsborg J . Emergence of ampicillin-resistant Enterococcus faecium in Danish hospitals . J Antimicrob Chemother . 2008 ; 62 : 1203 - 6 .
22. Sharifi Y , Hasani A , Ghotaslou R , Varshochi M , Hasani A , Soroush MH , Aghazadeh M , Milani M . Vancomycin-resistant enterococci among clinical isolates from north-west Iran: identification of therapeutic surrogates . J Med Microbiol . 2012 ; 61 : 600 - 2 .
23. Heidari H , Emaneini M , Dabiri H , Jabalameli F . Virulence factors, antimicrobial resistance pattern and molecular analysis of Enterococcal strains isolated from burn patients . Microb Pathog . 2016 ; 90 : 93 - 7 .
24. Emaneini MO , Aligholi MA , Aminshahi MA . Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran . Pol J Microbiol. 2008 ; 57 : 173 - 8 .
25. Sharifi Y , Hasani A , Ghotaslou R , Naghili B , Aghazadeh M , Milani M , Bazmany A. Virulence and antimicrobial resistance in enterococci isolated from urinary tract infections . Adv Pharm Bull . 2013 ; 3 : 197 - 201 .
26. Altoparlak U , Koca O , Ozkurt Z , Akcay MN . Incidence and risk factors of vancomycin-resistant enterococcus colonization in burn unit patients . Burns . 2011 ; 37 : 149 - 53 .
27. Wibbenmeyer L , Williams I , Ward M , Xiao X , Light T , Latenser B , et al. Risk factors for acquiring vancomycin-resistant Enterococcus and methicillinresistant Staphylococcus aureus on a burn surgery step-down unit . J Burn Res . 2010 ; 31 : 269 - 79 .
28. Falk PS , Winnike J , Woodmansee C , Desai M , Mayhall CG . Outbreak of vancomycin-resistant enterococci in a burn unit . Infect Control Hosp Epidemiol . 2000 ; 21 : 575 - 82 .
29. Sabia C , De Niederhäusern S , Guerrieri E , Messi P , Anacarso I , Manicardi G , et al. Detection of bacteriocin production and virulence traits in vancomycin-resistant enterococci of different sources . J Appl Microbiol . 2008 ; 104 ( 4 ): 970 - 9 .
30. Waar K , Muscholl-Silberhorn AB , Willems RJ , Slooff MJ , Harmsen HJ , Degener JE . Genogrouping and incidence of virulence factors of Enterococcus faecalis in liver transplant patients differ from blood culture and fecal isolates . J Infect Dis . 2002 ; 185 ( 8 ): 1121 - 7 .
31. Hällgren A , Claesson C , Saeedi B , Monstein HJ , Hanberger H , Nilsson LE . Molecular detection of aggregation substance, enterococcal surface protein, and cytolysin genes and in vitro adhesion to urinary catheters of Enterococcus faecalis and E. faecium of clinical origin . Int J Med Microbiol . 2009 ; 299 ( 5 ): 323 - 32 .
32. Billström H , Sullivan Å , Lund B . Cross-transmission of clinical Enterococcus faecium in relation to esp and antibiotic resistance . J Appl Microbiol . 2008 ; 15 : 2115 - 22 .