Vancomycin-resistant enterococci among clinical isolates from north-west Iran: identification of therapeutic surrogates.
ABSTRACT Global emergence and dissemination of vancomycin resistance among enterococci is a serious concern especially in developing countries, requiring progressive research efforts. Present investigation was carried out on clinical isolates of enterococci obtained from three tertiary hospitals located in northwest of Iran. Multiplex PCR was performed on 220 enterococcal isolates for the presence of vanA, vanB, genus - species specific targets. Subsequently, alternative therapeutic options were evaluated for vancomycin resistant enterococci (VRE) strains. From isolated enterococci, 152 (69.1%) and 68 (30.9%) were E. faecalis and E. faecium, respectively. Of 48 VRE strains detected genotypically, vanA genotype was the predominant and three strains were found to possess vanB genes. One hundred and thirty three isolates (60.45%) revealed high level resistance to gentamicin. Amongst alternative agents, resistance towards quinipristin/dalfopristin was distinctly revealed by VRE isolates while, all were found sensitive to linezolid and except one strain to daptomycin, rendering latter antibiotics better therapeutic options. Clinicians and microbiologists should thus, be aware of the increasing prevalence of VRE and alternative agents should be evaluated against them.
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ABSTRACT: 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. 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). Overall, total of 92 (41.4%) isolates were identified as E. faecium, 45 (49%) were resistant to vancomycin with an MIC50 of [greater than or equal to] 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. 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.Diagnostic Pathology 10/2013; 8(1):163. · 1.85 Impact Factor
Vancomycin-resistant enterococci among clinical isolates from
north-west Iran: identification of therapeutic surrogates
Enterococci rank among the three major
pathogens isolated from the bloodstream,
surgical sites and urinary tract infections.
Studies from many countries, including
Iran (Cetinkaya et al., 2000; Feizabadi
et al., 2004), have shown that
vancomycin-resistant enterococci (VRE)
are formidable pathogens and a serious
concern for both physicians and patients.
Therefore, it is essential to explore
alternative therapeutic options for treating
patients with VRE infections. New
antibiotics were recently licensed in the
UK, Europe and the USA, including
linezolid, daptomycin and quinupristin/
dalfopristin (Lentino et al., 2008).
However, the paucity of literature available
on strains isolated from high-risk
hospitalized patients in North and West
Azarbaijan, Iran, prompted us to analyse
vancomycin resistance in Enterococcus
faecalis and Enterococcus faecium using
phenotypic and molecular methods. To
explore alternative treatments, the
efficacies of the aforementioned antibiotics
were evaluated prior to use.
This laboratory-based study was
conducted using sequential isolates of
enterococci obtained from different
clinical specimens at university teaching
hospitals in Tabriz (Imam Reza and Sina
Hospitals) and Orumieh (Imam Khomeini
Hospital), Iran, from April 2008 to June
2010. Multiplex PCR was performed on all
isolates for the simultaneous detection of
vanA, vanB, and genes encoding D-alanine-
D-alanine ligases specific to E. faecalis
(ddlE. faecalis) and E. faecium (ddlE. faecium),
as described previously (Kariyama et al.,
2000). Each PCR assay included vanA- and
vanB-positive control strains (kindly
provided by Dr M. Emaneini, Department
of Microbiology, Tehran University of
Medical Sciences) and a negative control,
which contained all the reagents but not
the template DNA.
The antimicrobial susceptibility of the
strains was determined using the disc
diffusion method, according to the Clinical
and Laboratory Standards Institute
guidelines (CLSI, 2006).
The MICs of vancomycin and gentamicin
were determined using the agar dilution
method and Etest (bioMe ´rieux) based on
the CLSI (2006) guidelines and the
manufacturer’s instructions, respectively.
Susceptibility of the VRE strains to
linezolid, daptomycin and quinupristin/
dalfopristin was determined using the
Etest. E. faecalis ATCC 29212 was used
as the quality-control strain.
The data were analysed using the
chi-squared test by the SPSS
statistical software (version 18.0). A
P-value ,0.05 was considered statistically
A total of 220 isolates were obtained from
different wards. Most were isolated from
intensive care units (n543; 19.5 %),
followed by nephrology (n538; 17.3%)
and internal wards (n527; 12.3%). Urine
was the most common source (85.5%),
followed by blood (7.7%), body fluids
(4.1%), wounds (1.8%) and intravenous
Of the isolated enterococci, 152 (69.1%)
were E. faecalis and 68 (30.9%) were
Antibiotic susceptibility testing using disc
diffusion revealed high resistance to fusidic
acid (89.5%), followed by rifampicin
(85%) and erythromycin (75%),
irrespective of species. Nearly 70% of the
isolates were also resistant to ciprofloxacin
and penicillin. Furthermore, the isolates
were resistant to teicoplanin (21.8%),
imipenem (31.4%) and high-content
(300 mg) streptomycin (45.9%).
Vancomycin resistance was observed in
20.5% of the isolates. In vancomycin disc
diffusion tests, intermediate resistance was
observed in 63.8% of E. faecalis and 7.4%
of E. faecium isolates, with 15 mm
(20.6%) or 16 mm (79.4%) zone
The agar dilution method indicated that
45 (20.45%) strains were vancomycin
resistant. Among these, the MIC was
256 mg ml21for 31 isolates and
¢512 mg ml21for 13 isolates; a MIC of
8 mg ml21was seen for only one isolate.
In addition, 133 (60.45%) strains showed
high-level gentamicin resistance (MIC
¢512 mg ml21). The results of the agar
dilution were confirmed by Etests.
VRE strains were tested for alternative
therapeutic options using the Etest. These
isolates were found to be susceptible to
linezolid (MIC ¡0.05–2 mg ml21). VRE
isolates also were sensitive to daptomycin,
with the exception of an E. faecium strain
(MIC ¢6 mg ml21).
The MIC of quinupristin/dalfopristin
ranged from 0.75 to 32 mg ml21. Among
the ten vancomycin-resistant E. faecalis
strains, the MIC was ¡1.5 mg ml21for
one strain, whereas the remaining nine
strains showed high resistance (MIC
¢16 mg ml21). Three vancomycin-
resistant E. faecium strains also showed
resistance to this agent (MICs ¢4, 8 and
32 mg ml21), while six strains showed
Among the 45 VRE strains with
phenotypic resistance, 43 (8 E. faecalis and
35 E. faecium) carried vanA, whereas vanA
and vanB were not detected in two E.
faecalis strains. None of the vancomycin-
susceptible isolates carried vanA.
Surprisingly, vanB was detected in three
vancomycin-sensitive isolates that
produced 18–19 mm inhibition zones in
the disc diffusion test and for which the
MIC was ¡4 mg ml21in the agar dilution
test. These isolates were E. faecium from
Tabriz (Fig. 1). Based on the phenotypic
and genotypic analyses, we identified
48 VRE isolates.
The prevalence of VRE is obvious in
developed countries, and the burden has
been partially lifted; however, periodic
evaluations of antibiotic susceptibility and
G2012 SGMPrinted in Great Britain
the early detection of VRE are a
prerequisite in developing countries. In
this study, the analysis of distribution of
VRE species in the two cities revealed a
higher frequency of VRE isolates from
Tabriz (28.2%) than Orumieh (15.5%),
although this difference was not
statistically significant. The majority of
enterococcal isolates were identified as
E. faecalis; however, most of the VRE were
E. faecium (77.8%; P,0.001).
A decline in the prevalence of E. faecalis
(69.1% versus 90.5%) and an almost
fivefold increase in the prevalence of
E. faecium (30.1% versus 5.84%) and VRE
isolates (21.8% versus 4.38%) in the
present investigation were remarkable
compared with those in a previous report
from Tabriz, Iran (Akhi et al., 2009). This
is an alarming situation because the
prevalence of VRE has increased almost
fivefold over an 8 year time period.
In this study, antibiotic susceptibility
testing by disc diffusion revealed 46.4%
strains with intermediate vancomycin
resistance; however, the vancomycin MICs
were ¡2 mg ml21for these strains using
the agar dilution and Etest methods,
indicating that they were susceptible to this
antibiotic. This implies that vancomycin
susceptibility testing should be replaced by
MIC determination, which has been shown
to be more reliable (CLSI, 2006).
Most (90.4%) of our clinical isolates were
resistant to at least three of the antibiotics
tested, and antibiotic resistance was more
common among E. faecium isolates than
E. faecalis, which is in agreement with
previous studies (Simonsen et al., 2003;
Top et al., 2007). For example, ampicillin
resistance was detected in 31.4% of the
isolated enterococci, but nearly 93% of
these were E. faecium (P,0.001).
In this study, vanA was the predominant
vancomycin-resistance type, which is
consistent with studies from Iran
(Feizabadi et al., 2004; Emaneini et al.,
2008) and other countries (Abele-Horn
et al., 2006).
We detected one vanA-positive strain
(OEfm-71; isolated from a urine specimen)
for which the vancomycin MIC was 8 mg
ml21that was susceptible to teicoplanin
(MIC 1.5 mg ml21) and thus showed the
VanB+phenotype but vanA genotype. To
the best of our knowledge, this is the first
report of this type of clinical enterococcal
isolate from north-west Iran. VanB+-vanA
enterococci were previously reported from
south-east Asia (Eom et al., 2004).
All the VRE were found to be susceptible
to linezolid and daptomycin, except one
E. faecium isolate that was resistant to
daptomycin. These data are in agreement
with previous reports (Hsueh et al., 2005).
An efflux pump that confers resistance to
dalfopristin appears to be intrinsic in E.
faecalis (Eliopoulos, 2003). Accordingly,
nine vancomycin-resistant E. faecalis
isolates showed resistance to quinupristin/
dalfopristin. Unexpectedly, nine
vancomycin-resistant E. faecium strains
also were non-susceptible to this agent.
Quinupristin/dalfopristin is not yet
used in our hospital settings because
resistance prior to exposure remains a
possibility that requires further
investigation of the resistance mechanism.
A likely explanation is the application of
antibiotics (for example, virginiamycin)
as growth promoters in animal feed and
the emergence of cross-resistance to
quinupristin/dalfopristin. A study from
Iran showed that more than 50%
of meat samples contained detectable
antibiotic residues (Tajick & Shohreh,
In conclusion, our findings highlight the
incremental emergence of VREs in our
clinical setting. This necessitates
mandatory testing of isolates for
vancomycin resistance, by the phenotypic
and genotypic methods alongside each
other because some VRE strains (two
vanA- and vanB-negative strains; three
vanB-positive strains) would have been
Among the new therapeutic regimes,
linezolid and daptomycin exhibited better
in vitro activity than quinupristin/
dalfopristin against the VRE isolates.
Nonetheless, careful and continuous
monitoring of the effectiveness of these
antibiotics will be required to detect any
changes in their current status or
occurrence of resistance to new agents.
This work was financially supported by the
Research Center of Infectious Diseases and
Tropical Medicine, Tabriz University of
Medical Sciences, Tabriz, Iran (grant no.
89/3). The authors wish to thank Dr M.
Emaneini for providing the vanA- and vanB-
positive isolates. They are also grateful to Ms
Mitra Nojavan and Ms Leila Deighani for
their assistance in specimen collection. This
work was done as part of the PhD thesis (no.
88/4-4/5) of the first author.
Yaeghob Sharifi,1,2Alka Hasani,1,2
Mojtaba Varshochi,1,3Akbar Hasani,1,4
Mohammad Hossein Soroush,2
and Morteza Milani2,5
Fig. 1. Agarose gel electrophoresis of amplified vanA, vanB, and E. faecalis- and E.
faecium-specific genes by multiplex PCR. Lanes: 1, 1 kb DNA ladder; 2 and 4, E. faecalis
positive for vanA; 3 and 5, E. faecium positive for vanA; 6, E. faecium positive for vanB; 7,
negative control (without DNA); 8, 100 bp DNA ladder.
1Research Center of Infectious Diseases
and Tropical Medicine, Faculty of
Medicine, Tabriz University of Medical
Science, Tabriz, Iran
2Department of Clinical Microbiology,
Faculty of Medicine, Tabriz University of
Medical Science, Tabriz, Iran
3Department of Infectious Diseases,
Faculty of Medicine, Tabriz University of
Medical Science, Tabriz, Iran
4Department of Clinical Biochemistry and
Biotechnology, Tabriz University of
Medical Science, Tabriz, Iran
5Faculty of Pharmacy, Tabriz University of
Medical Science, Tabriz, Iran
Correspondence: Alka Hasani
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