High frequency distribution of heterogeneous vancomycin resistant Enterococcous faecium (VREfm) in Iranian hospitals
High frequency distribution of heterogeneous vancomycin resistant Enterococcous faecium (VREfm) in Iranian hospitals
Leili Shokoohizadeh 0
Ashraf Mohabati Mobarez 0
Mohammad Reza Zali
0 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University , P. O. Box: 14115-111, Tehran , Iran
Background: Enterococcus faecium is a multi-resistant nosocomial pathogen causing infection in debilitated patients. Vancomycin-resistant enterococcus faecium (VREfm) are a major concern and increased dramatically worldwide especially in hospitals environment. The current study focused on determining the high prevalence and distribution patterns of antibiotic resistance and also its genetic linkages among various VREfm strains isolated from indoor hospitalized patients in four major Iranian teaching hospitals of Tehran. Methods: The clinical samples were obtained from hospitalized patients during September 2010 to June 2011 from different teaching hospitals of Tehran. Antibiotics Resistance patterns, minimum inhibition concentration (MIC) value for vancomycin, ampicillin, gentamicin and presence of genetic linkage among the isolates were determined by pulsed-field gel electrophoresis (PFGE). Results: Overall, total of 92 (41.4%) isolates were identified as E. faecium, 45 (49%) were resistant to vancomycin with an MIC50 of 128 mg/L. The results showed that simultaneous resistance to teicoplanin, ampicillin, gentamicin, ciprofloxacine, tetracycline and erythromycin were observed the most frequent pattern. All the vancomycin resistant E. faecium isolates carried the vanA gene. intensive care units (ICUs) and Kidney transplantation, are most probably the wards with highest risk of infection by VRE. 17 pulsotypes were also detected by PFGE, most of the related pulsotypes belongs to the same hospitals. Conclusions: This study shows the high alarming prevalence of Enterococcus faecium infection and similar clones of VREfm strains in Iranian hospitals with threatening resistance phenotypes. Virtual slides: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/ 1270863903102282
E; faecium; Vancomycin resistant; Hospital and PFGE
Enterococci have been regarded as low grade pathogens
in the past, but in recent years it has rehabilitated into
major pathogen in nosocomial infections . Urinary
tract and surgical site infections, bacteremia and
endocarditis in immunocomprised patients are frequently caused
by these bacteria in hospital . The best therapy for
enterococcal infection is the combination of aminoglycoside
antibiotic (e.g. gentamicin) with a -lactam antibiotic
(e.g. ampicillin) or glycopeptides (e.g. vancomycin).
However, vancomycin resistant enterococci (VRE) do not
respond to these medications and thus there is limited
option of treatment . Although there is no report yet
available of vancomycin resistant E. faecium (VREfm)
outbreak in Iran but there is ample evidence showing that the
frequency of these strains are increasing in hospitals and
therefore caused a major therapeutic concern in recent
Characterization of VREfm strains is crucial for the
effective management of infection caused by this organism
. This characterization could be achieved with simple
assays such as screening for resistance against antibiotics
or more complex methods like Pulsed-field gel
electrophoresis (PFGE) which is a powerful genotyping method
used to study enterococcal distribution . Routes of
transmission and genetic linkage of these hospital
isolates were examined in many epidemiological and
molecular studies. Clonal spread of VREfm strains has been
associated with disease outbreak in hospitals .
However, different studies in Iran suggest the heterogeneity
and polyclonal distribution of nosocomial infection in
hospital environments [8-10].
Detection of antibiotic resistance patterns of VRE
isolates against a vast spectrum of antibiotics and
understanding how these strains transmitted based on their
genetic features could be useful in eliminating the
infection in hospital. Therefore, the main objective of this study
to investigating high frequency prevalence and antibiotic
resistance patterns of VREfm isolates. We also analyzed
the clonality and the genetic linkage between the strains
by comparing their resistance patterns and pulsotypes in
four major teaching hospitals in capital city Tehran, Iran.
Materials and methods
Identification of bacterial strains
The sampling was carried out in four major teaching
hospitals in Tehran from September 2010 to June 2011.
The isolates were obtained from clinical samples of
indoor hospitalized patients (urine, wound, blood, abscess,
sputum, bile, body fluid, intravenous catheter and trachea).
All Enterococci isolates were identified according to their
genus and species levels by gram staining, catalase
reaction, growth in 6.5% NaCl, motility assessment, use of
arabinose, bile and esculin hydrolysis and also pigment
production after their growth on enterococcus selective
agar (BBL, USA) all based on Falkman and Collins criteria
. PCR based study was conducted by using specific
primer (ddl E.faecium) for each E.faecium species strains .
Protocols conformed to the ethical guidelines of the 1975
Declaration of Helsinki and were approved by Research
Ethics Committee of the Tarbiat Modares University.
DNA extraction and polymerase chain reaction
DNAs from different bacterial isolates were extracted by
using appropriate DNA extraction Kit (Sinagene, Iran).
For PCR amplification assay, specific primers of ddlE.faecium
and vancomycin resistance genes (vanA and vanB) were
used as describe previously . E. faecium ATCC 51559
and E. faecalis ATCC 51229 as vanA and vanB standard
encoding strains were used.
Antimicrobial susceptibility test
Antimicrobial susceptibility test for isolates of E. faecium
was performed against vancomycin (30 g), teicoplanin
(30 g), gentamicin (120 g), ampicillin (10 g), erythromycin
(15 g), ciprofloxacin (5 g), tetracycline (30 g),
chloramphenicol (30 g), nitroforantoin (300 g),
quinopristindalfopristin (synercid) (15 g) and linezolide (30 g)
(Mast, UK), by the disc diffusion method. Vancomycin,
ampicillin and gentamicin MICs (Minimum inhibitory
concentration) were determined by the agar dilution
method. The results were interpreted according to the
Clinical and Laboratory Standards Institute guidelines
Pulsed-field gel electrophoresis
Genomic typing of isolates was performed by PFGE.
Genomic DNA was prepared in low melting agarose
plugs as described by Saifi et al. . Restriction enzyme
SmaI (Roche, Manheim, Germany) was used to digest
the DNAs in small slices of the agarose plugs. The plugs
were placed in 1% agarose (Invitrogen, USA) that was in
0.5% TBE and were electrophoresed with switch times
ramped from 5 s to 35 s at 6 V with a run time of 23 hours
at 14C and an angel 120 in the Bio-Rad CHEF-DRIII
system. Salmonella cholerasuis serotype Branderup
H9812 was used as the molecular size marker. The gels
were stained with ethidium bromide and photographed
under ultraviolet light. The banding patterns were
clustered by unweighted paired group (UPGMA) method by
Gelcompar II software version 4.0. Interpretation was
done by using the guidelines set out previously .
Prevalence of E. faecium species
A total of 222 enterococcal isolates samples were
collected from clinical hospitalized patients. Overall, 41.4%
(n = 92), 51.3% (n = 114) and 7.2% (n = 16) of the isolates
were confirmed as E. faecium, E. faecalis and other
species of enterococci respectively. Results of PCR for the
ddl gene confirmed the biochemically identification in
E. faecium isolates. Most of the clinical isolates belonged
to urine 70.6% (n = 65) followed by wound samples 9.7%
(n = 9) (Figure 1).
Antimicrobial susceptibility testing
Antibiotic resistance analysis showed a high rate of
vancomycin resistance 48.9% (n = 45) in the isolates under
study. More (35.5%) VREfm were isolated from hospitals
by local code number 1. ICUs (n = 17, 38%) and kidney
transplant wards (n = 9, 20%) have the larger number of
these strains. All (100%) VRE isolates were also
resistant to ampicillin, gentamicin, ciprofloxacin erythromycin
and teicoplanin, this was followed by tetracycline (n = 36,
80%), nitroforantoin (n = 32, 71%), choleramphenicol (n =
8, 18%), quinopristin-dalfopristin (synercid) (n = 6, 13.3%)
and linezolide (n = 1, 2%). MIC values for vancomycin,
ampicillin and gentamicin were from 64 to 1024, 32 to
Figure 1 The rate (%) of Enterococcus faecium strains isolated from clinical samples and (%) of VRE.
256 and 512 to 1024 g/mL respectively. MIC50 for
vancomycin and ampicillin was 128 mg/L and for gentamicin
was 1024 mg/L. All VRE isolates harbored the vanA
gene. Resistance to vancomycin, ampicillin, gentamicin,
ciprofloxacin, tetracycline, erythromycin and teicoplanin
was the dominant antibiotic resistance phenotype (77.7%)
(n = 35). All VRE isolates were selected for genotyping by
PFGE (Figure 2).
PFGE was performed in order to study the genetic linkage
analysis among the clinical isolates. These isolates
were divided into 17 pulsotype groups (A-Q) according
to a similarity cutoff of 95%. The predominant
pulsotype (C) comprised 7 isolates (15%). Six isolates (35.2%)
showed unique PFGE pattern as shown in Figure 2.
Generally, E. faecalis has been known as the major cause
of enterococcal infections, 10 times more prevalent than
other enterococcus species. However, in recent years a
remarkable change has seen from E. faecalis to E. faecium
probably because of the emergence of VRE strains among
members of this species in hospital environments .
The prevalence of E. faecium has been increased in
Iranian hospitals during the last few years. In the present
study, the ratio of infections due to E. faecalis to those
caused by E. faecium was 1.2: 1 (51.3% versus 41.4%)
which is still higher than some previous reports published
from Iran and some other countries [4,15,16]. The
increased ratio was supported by enhancement of VREfm
strains. The emergence of E. faecium isolates with a high
level of resistance to three main classes of antibiotics
(i.e. glycopeptides, -lactams and amino glycosides)
against enterococci spp is a major concern in hospitals.
Congruent with the results from the USA hospitals, all the
studied VRE isolates in Iran are resistant to ampicillin,
whereas European hospital-derived clones are reported to
be vancomycin susceptible but resistant to ampicillin and
Our current study clearly shows that in Iran the
frequency distribution of VRE is high compared to rest of
the countries of the world . Also, all of VRE
isolates showed resistance to more than 6 antibiotics, 60%
of VRE isolates showed threatening resistance phenotype
to vancomycin, teicoplanin, ampicillin, gentamicin,
ciprofloxacin, tetracycline, erythromycin, nitrofourantoin
(Va/Tei/Am/Gm/Cip/Te/E//Ni). Moreover high MIC
values (MIC50 128) were also found for vancomycin
and ampicillin as well as gentamicin MIC50 1024.
It is also vibrant from the present study that all VRE
isolates harbored vanA gene as describes previously. The
wards related to kidney transplantation and nephrology,
and ICU was estimated to be the ones with the highest
risk of infection by VRE (Figure 2). The present study
also reveals that linezolid and quinopristin- dalfopristin
(synercid) were the most effective agents against the
E. faecium isolates.
The dissemination of VREfm, studies conducted in
Iranian hospitals have found the dominancy in
polyclonal among clinical isolates instead of clonal spreading
as reported in USA and Europe but has consistency with
Saudi Arabian hospitals [7,18-20].
The relative congruence of antibiotic resistance patterns
and the specific PFGE patterns among the studied isolates
demonstrates the presence of E. faecium strains with
similar clone types in each of the hospitals e.g. pulsotypes C,
H and L in hospital 1, pulsotype A in hospital 2, pulsotype
I in hospital 3 and J in hospital 4 were identified.
The pulsotype patterns and resistance profiles suggest
that there was an inter-hospital dissemination of pulsotype
F and D (isolates were obtained from different sources
Figure 2 Dendrogram cluster analysis of PFGE data for 45 VRE isolates with Tei/amp/Gm/Cip/E resistance phenotypes, based on
hospitals, wards, source of infection, resistance to other antibiotics and pulsotypes. a; Gastroenterology, b; Kidney Transplant, c; Nephrology,
d; Onchology e; Abscess, h; catheter, g; Cardiology, f; Surgery.
and wards). Most of the other pulsotypes (e.g. Pulsotypes
A, C and I) were related to the same hospitals. The best
example for intra hospital dissemination was pulsotype C,
because all of these strains were isolated from urine
samples and from the same ward of a hospital.
study was most probably due to the selective antibiotic
pressures. The highlighted results need a strong
attention for surveillance programs to continuously monitor
the nosocomial changes in bacterial resistance and the
exchange of information about pathogens.
In conclusion, the increasing of polyclonal VREfm with
threatening resistance phenotypes is a serious concern
in Iranian hospitals. High resistance to antibiotics in our
LS and AMM conceived the study. LS conducted the experiments and
analyzed the results. LA and LS drafted the manuscript and made substantial
contributions to the design of the study. LA, AMM, and TS critically reviewed
the manuscript. MRZ, RR, TS and MA participated in data analysis. All the
authors studied and approved the final manuscript.
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. We would like to thank all the
staff at Food Born Disease Department of Taleghani hospital, ICUs in studied
hospitals (Taleghani, Labbafinejad, Mofid and Loghman), and Molecular
Biology Research Center, Baqiyatallah University of Medical Sciences of
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