Frequency distribution of genes encoding aminoglycoside modifying enzymes in uropathogenic E. coli isolated from Iranian hospital
BMC Research Notes
Frequency distribution of genes encoding aminoglycoside modifying enzymes in uropathogenic E. coli isolated from Iranian hospital
Neda Soleimani 1 3
Mahdi Aganj 2
Liaqat Ali 0 5
Leili Shokoohizadeh 4
Türkân Sakinc 0
0 Division of Infectious Diseases, Department of Internal Medicine II, University Hospital Freiburg , Freiburg 79106 , Germany
1 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University , Po Box:14115-158, Tehran , Iran
2 Department of Microbiology, Mashhad University of medical sciences, Faculty of Medical Sciences, student research committee , Mashhad , Iran
3 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University , Po Box:14115-158, Tehran , Iran
4 Department of laboratory medical Sciences, Faculty of Para Medical Sciences , Ahvaz Jondishapour , University of medical sciences , Ahvaz , Iran
5 Faculty of Biology, Albert Ludwigs University of Freiburg , Schänzlestraße 1, Freiburg 79104 , Germany
Background: Escherichia coli is considered as the most common cause of urinary tract infection (UTI) and acquired multiple resistances to a wide range of antibiotics such as aminoglycosides. Enzymatic alteration of aminoglycosides (AMEs) by aminoglycoside- modifying enzymes is the main mechanism of resistance to these antibiotics in E. coli. The aim of this study was detection and investigation of frequency of genes encoding aminoglycoside modifying enzymes (aac(3)-IIa and ant(2′′)-Ia) in UPEC isolated from hospitalized patients in teaching hospital of Tehran, Iran. Findings: A total of 276 UPEC were obtained from Urine samples in a hospital from Tehran. Antibiotic susceptibility to aminoglycosides was determined by disk diffusion method according CLSI guidelines in UPEC isolates. MICs of target antibiotics were determined by agar dilution method. All isolates were screened for the presence of the AMEs genes using the PCR. The results of disk diffusion showed 21%, 24.6%, 23.18%, 3.62% and 6.15% of isolates were resistant to Gentamicin, Tobramycin, Kanamicin, Amikacin and Netilmicin respectively. The agar dilution's results (MICs) were high, 66.19% for Gentamicin. The aac (3)-IIa and ant(2″)-Ia genes were detected in (78.87%) and 47.88% of isolates respectively. Conclusions: This study shows the high frequency of genes encoding (AMEs) aac(3)-IIa and ant(2”)-Ia genes and their relationship between different aminoglycoside resistance phenotypes.
UPEC; Aminoglycoside modifying enzymes and UTI
The aminoglycosides are potent bactericidal agents that
inhibit bacterial protein synthesis by binding to the 30S
ribosomal subunit. They are often used in combination
with either a b-lactam or a glycopeptide, especially in
the treatment of Escherichia coli UTI, as these drugs act
synergically [1,2]. The application may be limited by the
appearance of resistant strains in treatment. Various
mechanisms are playing a role in the development of
aminoglycoside resistance but the presence of
aminoglycoside modifying enzymes is the most clinical and
epidemiological importance [3,4]. These enzymes are
divided into three classes: aminoglycoside acetyltrans- ferases
(AACs), aminoglycoside phosphotransferases (APHs) and
aminoglycoside nucleotidyltransferases (ANTs) .
Urinary tract infection is one of the most common
human infections, especially in young women and frequently
influenced by sex and age and 20-30% of young women
experienced this infection [6,7]. Due to the importance of
the resistance to aminoglycosides and the role of ant(
)II-a genes in mechanism, the main purpose of this
study is the detection of resistance genes ant(2”)-Ia, aac(
II-a in clinical isolates of aminoglycoside resistant E. coli
isolated from urine of hospitalized patients in teaching
hospital of Tehran, Iran, to know the prevalence and
frequency of distribution of genes encoding
aminoglycoside in Iranian population.
Materials and methods
A total of 276 clinical isolates of E. coli from urine
specimens were randomly collected from Tehran Heart
center. All isolates were then identified as E.coli using
conventional microbiological tests. Informed written
consent was obtained from the patients and the study
was approved by the institutional ethics committee of
Department of Bacteriology, Faculty of Medical Sciences,
Tarbiat Modares University, Tehran. Pure stock cultures
of all isolates were stored frozen at −80°C in tryptic soy
broth, containing 15% glycerol.
Antibiotic susceptibility testing
Antimicrobial susceptibility test for different E.coli isolates
was performed against Gentamicin (10 μg), Tobramycin
(10 μg), Kanamycin (30 μg), Amikacin (30 μg) and
Nethelmicin 30 μg, (Mast, UK) by disc diffusion method. Sizes
were interpreted using standard recommendations of
CLSI . As the Gentamicin is the most applicable
antibiotics to treat the infections due to gram positive and
gram negative bacteria in Iranian patients, thus,
Gentamicin MIC values were detected and the results were
interpreted according to the CLSI guidelines.
DNA extraction and Polymerase Chain Reaction
Total DNAs were extracted from bacteria isolates using
the extraction kit (Bioneer, korea). The DNA was then
extracted following the manufacturer’s instructions and
electrophoresed on 0.8% agarose gel stained with
ethidium bromide and visualized by UV-transillumination
and gel documentation (Biometra Germani). Two sets of
specific oligonucleotide primers for (aac(
)-IIa and ant
(2′′)-Ia) genes were used as listed in Table 1 (designated
by primer 3 software). The PCR mixture was prepared
in a final volume of 25 μl. The amplification mixture
consisted of template DNA (2 μl), 0.1 μM of the
respective primers, 2.5 μl of a 10-fold concentrate PCR
buffer, 200 μM of deoxynucleotide triphosphates, 2.5 μM
MgCl2, and 1.5 U of Taq DNA polymerase (Cinna Gene).
A thermocycler (Mastercycler gradient; Eppendorf,
Hamburg, Germany) was programmed with the following
parameters: after an initial denaturation for 5 min at 95°C,
30 cycles of amplification were performed with
denaturation at 95.8°C for 1 min, annealing at 62°C for 1 min, and
DNA extension at 72°C for 1 min, followed by a final
extension at 72°C for 10 min. Then, 5 to 10 μl of the PCR
products was analyzed by electrophoresis on o.8-1% (w/v)
TAE agarose gel (Fermentas UAB, Vilnius, Lithuania)
containing 0.5 μl/μl of ethidium bromide. Stained amplicons
were then viewed on a UV transilluminator at 260 nm,
(BioDoc- Analyse; Biometra, Goettingen, Germany).
klebsiella pneumoniae 23823 [possessing (aac(
E. coli 85085 [possessing (ant(2″)-Ia+)] by Statens Serum
Institute of Denmark served as positive controls and
E. coli ATCC 25922 as negative control (Table 1).
Antibiotic resistance analysis showed among 276 E.coli
isolated from clinical specimens, simultaneous resistance
to gentamicin, nethelmicin and kanamicin in 39% of
isolates was the most common antibiotic resistance pattern
among isolates under study (Table 2). The range of MIC
for 5% Gentamicin was from 1 μg/ml to 512 μg/ml.
MIC ≥ 64 were detected in more than 35% of isolates.
Some isolates showed MIC of >512ug/ml. All antibiotic
sensitivity/resistance of E. coli strains isolated from the
urine clinical specimens are shown in Figure 1.
Amplification and screening of genes encoding AME by
All isolates were screened for the presence of genes
encoding the two AMEs, enzymes (AAC(
ANT(2′′)-Ia). Amplified DNA fragments of two
different sizes (700 and 740 bp) were subjected to agarose gel
electrophoresis and snapped by gel picture (Figures 2
GM, gentamicin; AK, amikacin; N, netilmicin; TN, tobramycin; K, kanamycin.
and 3). Based on PCR results, The prevalence of
)IIa gene and ant(2”)-Ia gene were 47.88% and 78.87%
respectively 32.39% of isolates only harbored the aac(
and 7.04% ant(2”)-Ia. In addition our results,
demonstrated the relationship between AME genes and different
aminoglycoside resistance phenotypes (Table 3).
Besides the side effects and increasing resistance,
aminoglycosides play an important role in curing bacterial
infections. Modification of aminoglycosides by
aminoglycosides modifying enzymes is the common resistance
mechanism against aminiglycosides in E.coli as these
enzymes are not capable of binding to ribosomes of the
cell [9,10]. Resistance against Gentamicin, Kanamycin,
Cizomycin and Tobramycin is mediated by ANT(2”)-Ia
enzyme which is coded by ant(2”)-Ia gene in E.coli and
also simultaneous resistance to Gentamycin and
Tobramycin, mediated by AAC(
)-IIa enzyme which is coded
)-IIa gene .
In this study the prevalence of ant(2”)-Ia,aac(
resistance genes in 71 aminoglycosides resistant E.coli
isolates among 276 UTI isolates was determined by
PCR. It is implied that 24.63% of isolates were resistant
to tobramycin and the resistance rate against other 130
antibiotics were as following; Kanamycin 23.18%,
Gentamicin 21.01%, Netilmicin 6.15% and Amikasin 3.62%. In
1999 Van hoof R and his colleagues reported that among
897 blood 132 isolates of Entrobacteriacea, 5.9% of isolates
were resistance against Gentamycin, whereas 7.7% of
isolates were resistant against Tobramycin,7.5% against
netilmicin and 8.2% against Amikacin [11,12]. In 2006 Kong
and 2010 Lang Hoo and colleagues reported: 44
clinicalisolates of E.coli, the resistance rate against aminiglycosides
were: Amikasin 18.18%, Gentamicin 56.82% and
Tobramycin 63.36% and among 249 clinical isolates of E.coli
83.83% were resistant to Gentamicin respectively [13,14].
The respective studies suggested an increasing
resistance against aminoglycosides but the contradiction in
results is due to different geographical areas and various
numbers of different isolates. PCR results showed that
78.87% of isolates contained aac(
)-IIa resistance gene.
In 2004, Minard showed that 17% of animal and 33% of
human isolates contained the aac(
)-IIa resistance gene,
)-Ia was detected in 6.97% and 4% of human
isolates of Kanamycin resistant. Also, E.coli in 8% of animal
isolates and 7.04% of human isolates of neomycin
resistant while ant(2”)-Ia gene was not detected in this study
. Jaconson et al.  studied 120 isolates of E. coli
for occurrence of amino glycoside modifying enzymes
namely ant(2”)-Ia, aac(
)-IIa and aac(
)-IV and also
subjected the isolates to MIC for Gentamicin by dilution
GM, N, TN, K
GM, N, TN, K
GM, TN, K
method. The E.coli isolates having aac(
)-IIa gene had
high MIC’s, 32–512 mgs/ltr suggesting that, there is a
correlation between MIC and specific ame production,
but still not cleared . Jacobson et al. also studied 76
isolates of Gentamicin resistant E. coli which are 63.15%
and contained aac(
)-IIa gene, although, ant(2”)-Ia gene
was not screened in this study . In an
epidemiological study in 2010 it was concluded that aac(
(aaC2) gene was present in 84.1% of human isolates and
75.5% of animal isolates,while it was the common gene
among the studied isolates .
Therefore, our results shows high frequency
prevalence of aac(
)-IIa and ant(2”)-Ia genes, which were
47.88% and 78.87% respectively and also, demonstrated
the relationship between AME genes and different
aminoglycoside resistance phenotypes. According to
the reviewed studies the prevalence of the respective
genes has been increasing over time in various
geographical patterns, which needs regular attention and
In conclusion, our data show high frequency distribution
)-IIa and ant(2”)-Ia genes and their relationship
between AME genes and different aminoglycoside
resistance phenotypes. Further experiments will be needed to
clarify the exact mechanisms and functions of these
genes to controlled high prevalence of urinary tract
infections caused by EPEC strains, increasing resistance
against antibiotics in order to select the best medicine to
avoid this confrontation.
The authors declare that they have no competing interests.
NS and LA conceived the study. NS conducted the experiments and
analyzed the results. LA drafted the manuscript and made substantial
contributions to the design of the study. TS, MA and LS critically reviewed
the manuscript and participated in data analysis. All the authors studied and
approved the final manuscript.
This study was generously funded by tarbiat Modares University. The author
LA sincerely thanks to DAAD for awarding a PhD Fellowship.
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