Journal of Radiation Research and Applied Sciences

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Prevalence and sequence of aminoglycosides

modifying enzymes genes among E.coli and
Klebsiella species isolated from Egyptian hospitals

Mervat Aly Mohamed Abo-State, Youssry El-Sayed Saleh & Hazem

Mahmmoud Ghareeb

To cite this article: Mervat Aly Mohamed Abo-State, Youssry El-Sayed Saleh & Hazem
Mahmmoud Ghareeb (2018) Prevalence and sequence of aminoglycosides modifying enzymes
genes among E.coli and Klebsiella species isolated from Egyptian hospitals, Journal of Radiation
Research and Applied Sciences, 11:4, 408-415, DOI: 10.1016/j.jrras.2018.08.005

To link to this article: https://doi.org/10.1016/j.jrras.2018.08.005

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 Journal of Radiation Research and Applied Sciences 11 (2018) 408–415

HOSTED BY Contents lists available at ScienceDirect

Journal of Radiation Research and Applied Sciences

journal homepage: www.elsevier.com/locate/jrras

Prevalence and sequence of aminoglycosides modifying enzymes genes T

among E.coli and Klebsiella species isolated from Egyptian hospitals
Mervat Aly Mohamed Abo-Statea,∗, Youssry El-Sayed Salehb, Hazem Mahmmoud Ghareebc
a
Department of Radiation Microbiology, National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority(EAEA), Cairo, Egypt
b
Department of Plant and Microbiology, Faculty of Science, CairoUniversity, Egypt
c
National Center for Nuclear Safety and Radiation, Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: The WHO and CDC have expressed serious concern regarding the continued increase in the development of
Gram negative bacilli multidrug resistance among bacteria. Associated with the rise in antibiotic resistance is the lack of new anti-
GNB microbials. Bacteria have developed many ways by which they become resistant to antimicrobials among those
Aminoglycoside are enzymes. Aminoglycosides play an important role in treatment of serious infections that threat human life
PCR
caused by Gram-negative bacteria (GNB). In our study, clinical bacterial isolates(210) were collected from
Aminoglycoside modifying enzyme genes
AME-Genes
different Egyptian hospitals. Eight aminoglycoside antibiotics were used in the present study. The most prevalent
Sequencing pathogen were E. coli (36.66%),E.coli-ESBL(5.23%),Klebsiella spp. (25.33%), Klebsiella spp.–ESBL
(1.90%),Pseudomonas spp.(17.61%), Acinetobacter spp. (8.09%),Proteus spp. (2.85%),Enterobacter spp.(1.42%)
and (0.47%) for both of Citrobacter sp. and Morganella sp. The most efficiency aminoglycoside antibiotic was
Amikin(AK) and the resistance pattern increased over the last years in Egypt. The results of AME-genes indicated
that non of aac(3′)-Ia and Rmt(55) genes were detected in any of the thirty Klebsiella spp. and E. coli of Egyptian
isolates. The most prevalent AME-genes were aac(3′)-IIa(40%),aac(6′)-Ib (30%) followed by aph(3′)-Ia
(23.3%),ant(2″)-Ia(20.0%),aph(3′)-VI(13.3%),and aac(3′)-Ih(6.6%). Also, the results revealed that isolate
Klebsiella sp. MAM-16 acquired five AME genes aac(3′)-IIa (accession no. MF495896); acc(3′)-Ih (accession no.
MF 495898); aph(3′)-VI(accession no. MF495899); ant(2″)-Ia (accession no. MF495901) and aph(3′)-Ia (acces-
sion no. MF495903). E. coli MAM-24 also acquired five AME-genes aac(3′)-IIa (accession no. MF495897); aph
(3′)-VI(accession no. MF495900); ant(2″)-Ia (accession no. MF495902); aph(3′)-Ia(accession no. MF495904) and
acc(6′)-Ib (accession no. MF495905).

1. Introduction Resistance to aminoglycosides in GNB occurs by three different me-

Infectious diseases remain on the leading causes of morbidity and
mortality worldwide. The antibiotic resistance crisis is one of the most
pressing issue in global public health as a result of increasing in the
development of multidrug resistance (Baptista et al., 2018). The
emergence of antibiotics resistance bacteria has threaten our health
worldwide. This has triggered initiatives worldwide to develop novel
and more effective antimicrobial compounds and develop novel de-
livery and targeting strategies(Baptista et al., 2018; Chen, Su, Kuo,
Lauderdale, & Shih, 2018). Antimicrobial resistance has become a ser-
ious global health concern causing complication in treatment strategies,
consequently increasing the cost of health care (Mohammed, Gadzama,
Zailani, & Aboderin, 2016). The prevalence of extended spectrum Beta-
lactamase (ESBL is a worldwide problem (Alyamani et al., 2017).
chanisms: (a)production of enzyme that modifies aminoglycosides,(b)
impaired entry of aminoglycoside into cell by altering outer membrane
permeability (OMP),(c) alteration of receptor protein on the 30S ribo-
somal subunit via mutation (Park, 2009).
Aminoglycosides modifying enzyme AME-genes that encode the
aminoglycosides acetyl-transferase (AAC), aminoglycoside nucleoti-
deyl-transferase (ANT) and adenyl-transferase (AAD)enzymes. AAC and
ANT cause resistance to aminoglycoside antibiotic through modifica-
tion of the drugs. Expression of the AAC and ANT resistant genes is
regulated by aminoglycoside binding to 5′ leader RNA of aac/aad genes
(Chen & Munchie, 2014) and also phosphotransferase(phosphorelation
of a hydroxyl group-APH) (Park, 2009). The genes encoding ami-
noglycoside-modifyingenzymes (AMEs)were determined in isolates of
Acinetobacter baumannii and Pseudomonas aeruginosa. Nine AMEs genes

Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
∗
Corresponding author. NCRRT, 3Ahmed EL-Zomour St., Nasr City, Cairo, Egypt.
E-mail addresses: abostatem@yahoo.com (M.A.M. Abo-State), Salehyoussry@yahoo.com (Y.E.-S. Saleh), Hazem_ghareeb@yahoo.com (H.M. Ghareeb).

https://doi.org/10.1016/j.jrras.2018.08.005
Received 4 June 2018; Received in revised form 14 August 2018; Accepted 17 August 2018
Available online 04 September 2018
1687-8507/ © 2018 The Egyptian Society of Radiation Sciences and Applications. Published by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M.A.M. Abo-State et al.
and three RNA methylases were investigated in all isolates, aph(3′)-Via
(90.6%) and aph(3′)IIb(61.8%) were the most prevalent AME genes in
A. baumannii and P. aeruginosa respectively (Aghazadeh, Rezaee,
Nahaei, & Mahdian, 2013).
Many of AME genes are wide spread in Pseudomonas aeruginosa and
A. baumanni isolates (Hujer, Hujer, Hulten, Bajaksouzian, & Adams,
2006; Doi & Arakowa, 2007; Dubois, Arpin, Dupart, Scavelli, &
Coulange, 2008; Moniri, Farahani, Shajar, NazemShirazi, & Ghasemi,
2010; Aghazadeh et al., 2013) in Escherichia coli and Klebsiella spp.
(Haldorsen, Simonsen, & Sundsfiord, 2014) and in Acinetobater
spp.,E.coli, Klebsiella spp. and Pseudomonas spp.(Chaudhary & Payasi,
2014). Methylation of 16 S rRNA is an important mechanism of ami-
noglycoside resistance among GNB. In Changchun area of China the
16 S rRNA methylases (arm A,rmt B,rmt A, rmt C and rmt D and npm A)
were identified by direct sequencing. Fifty of the isolates harboring
(43.1%) 16 S rRNAmethylase genes (Romanowska, McCammon, &
Trylska, 2011; Zhao, Shi, Jinghua, Zhou, & Sun, 2013). E. coli has been
reported to show a steadily increasing gentamycin resistance during
2004–2007 in Norway. The gene aac(6′)-Ib-cr in Norwegian clinical
isolates is reported to be the most prevalent of all aminoglycoside
acetyltransferase (AACs) and AAC(3′)-IIc as the second largest group of
aminoglycoside acetyltransferase confirming resistance to the ami-
noglycosides (Risberg, 2010).
The prevalence of AME-genes among ESBL-positive Proteus mirabilis
clinical strains indicated that nine strains showed the presence of more
than one AME-gene. The most frequent combination was ant(2″)-Ia
with aph(3″)-Ib(5strains) followed by aac(6″)-Ib with aph(3″)-Ib
(2strain) (Michalska, Sacha, Kaczynska, & Tryniszewska, 2014).
The present study emerged as ''there is a great need for research
related to aminoglycoside resistance in Egypt to understand about the
mechanisms on a molecular level which cause the increasing resistance
among Egyptian clinical GNB isolates, especially there is no studies in
that area of research''.
2. Materials and methods
2.1. Study area and collection time
A total of 210 clinical isolates were collected from Cleopatra
Hospital (Heliopolis), WadiEL-Nile Hospital(Hadayk El-Kobba), Abd-
Elkader Fahmy Hospital(Nasr city),Central health laboratories (EL-
Monera),between May 2012 to March 2014. The samples collection
followed the instructions and recommendations of Research Ethics
Committee (REC-NCRRT 2H/18).
2.2. Samples collection and culturing
The isolates were obtained from various clinical specimens (urine,
wound, sputum,ETT, pus and blood). The samples were inoculated and
incubated on the surface of blood agar Cystine-Lactose electrolyte de-
ficient (CLED) and MacConky agar plates using standard calibrated loop
(Cheesbrough, 2006). Single isolate was selected from each sample.
2.3. Identification of isolates
Isolates inoculated on MacConkey agar plates were incubated at
44.5 °C for 24–48 h, while isolates inoculated on blood agar and CLED
agar plates were incubated at 37 °C for 24–48 h. Isolates with colonial
appearance of Escherichia coli and Klebsiella spp. Were subjected to
Gram staining and rapid tests(catalase, oxidase, coagulase) and bio-
chemical tests(indol, citrate, triple sugar iron, urease, oxidation/fer-
mentation and hemolysin production) according to Cheesbrough
(2006).
409
Journal of Radiation Research and Applied Sciences 11 (2018) 408–415
2.4. Antibiotics discs
The antibiotic discs used in the present study were obtained from
Bioanalyse LTD, Tibbi, Malzemeler,San.VeTicLTD, Ankara,Turkey.The
antibiotic discs of family aminoglycosides were Amikin (AK,30 μg);
Tobramycin(TOB,10 μg); Kanamycin (K,30 μg); Gentamycin
(CN,10 μg); Neomycin(N,30 μg) and Netilimicin (NET,30 μg).
2.5. Antibiotic susceptibility test
Antibiotic susceptibility pattern for each E. coli and Klebsiella spp.
was determined according to the Clinical and Laboratory Standard
Institute (CLSI, 2012) recommended modified Kirby-Bauer disc diffu-
sion method on Mueller Hinton agar plates (Oxoid manual, 2006). A
suspension of each isolate was transferred using standard loop to 3 ml
sterile Mueller Hinton broth (Oxoid manual, 2006) and adjusted the
count to 1 × 103 CFU/ml using McFarland Standards (CLSI, 2012).
Mueller Hinton agar plates were inoculated uniformly on the surface by
100 μl of isolate suspension. The antibiotic discs were placed gently on
the surface of each inoculated plate using sterile forceps. The plates
were incubated for 24 h at 37 °C. The inhibition zone diameter was
determined for each antibiotic against each isolated strain.
2.6. Phenotypic identification of ESBL producing isolates
Phenotypic identification of ESBL producing isolates have been
carried out according to double disk synergy test(DDST) screening
method (CLSI, 2012). Antibiogram disks containing Ceftazidime
(CAZ,30 μg); Ceftazidime(CAZ,30 μg)+ Clavulanic acid (CA,10 μg);
Cefotaxime (CEF,30 μg) and Cefotaxime(CEF,30 μg)+ Clavulanic acid
(CA,10 μg) were used. Pairs of disks(CAZ with CAZ/CA) and (CEF with
CEF/CA)were placed on the surface of Muller-Hinton agar plates with
20 mm space between them. According to the CLSI and manufacturer
instruction, the ≥ 5 mm inhibition zone of growth in case of CAZ/CA
and CEF/CA than CAZ and CEF was regarded as isolate producing
ESBLs.
2.7. Extraction of bacterial DNA
DNA was extracted from bacterial colonies using (GeneJET™
Genomic DNA purification kit, Fermentas, Bur-lington, ON, Canada) as
described in the kit guidelines. Briefly, 2 × 109 bacterial cells was
harvested in 180 μL of Digestion Solution with addition of 20 μL of
proteinase K Solution and mixed thoroughly by vortex to obtain a
uniform suspension, then the sample incubated in shaking water bath at
56 °C until cells are completely lyses. After that 20 μL of RNase A so-
lution was added and mixed by vortex then incubated for 10 min at
room temperature and followed with addition of 200 μL of lyses solu-
tion and mixed thoroughly by vortex until a homogeneous mixture is
obtained. Before transferring, the prepared lysate to the purification
column 400 μL of 50% ethanol was added and mixed by pipeting. Then,
the column was centrifuged for 1 min at 6000 × g and washed twice by
adding 500 μL of wash buffer I and centrifuged for 1 min at 8000 × g,
and 500 μL of wash buffer II and centrifuged for 3 min at maximum
speed (≥12,000 × g). Finally, we added 200 μL of elution buffer to the
center of the purification column membrane to elute genomic DNA and
centrifuged for 1 min at 8000 × g. The DNA extracts was stored in
−20 °C.
2.8. PCR and gel electrophoresis
The oligonucleotide primers used for PCR amplification were com-
mercially synthesized (Invitrogen, Carlsbad, CA, USA) and are listed in
Table 1. No positive controls were used in all PCR reactions. For de-
tection of Aminoglycosides resistance genes standard PCR was per-
formed via using eight specific sets of primers to aac(3′)-Ia, aac(3′)-IIa,
M.A.M. Abo-State et al. Journal of Radiation Research and Applied Sciences 11 (2018) 408–415

Table 1
Oligonucleotides used as primers for PCR amplification of aminoglycoside resistance genes.
Target gene Direction Primer Sequence No. of bp Ref.

aac(3′)-Ia F 5-GACATAAGCCTGTTCGGTT-3` 372bp Akers et al. (2010)

R 5-TCCGAACTCACGACCGA -3`
aac(3′)-IIa F 5-ATGCATACGCGGAAGGC-3` 822bp Akers et al. (2010)
R 5-TGCTGGCACGATCGGAG-3`
aac(3′)-Ih F 5-TGCCGATATCTGAATC-3` 407bp Akers et al. (2010)
R 5-ACACCACACGTTCAG-3`
aph(3′)-VI F 5-CGGAAACAGCGTTTTAGA-3` 717bp Akers et al. (2010)
R 5-TTCCTTTTGTCAGGTC-3`
ant(2″)-Ia F 5-ATCTGCCGCTCTGGAT-3` 404bp Akers et al. (2010)
R 5-CGAGCCTGTAGGACT-3`
Rmt55 F 5-CCCTAGCGTCCATCCTTTCC-3` 701bp Akers et al. (2010)
R 5-ATGTACACAAGCTCTATTCC-3`
aph(3′)-Ia F 5-CGAGCATCAAATGAAACTGC-3` 623bp Akers et al. (2010)
R 5-GCGTTGCCAATGATGTTACAG-3`
aac(6′)-Ib F 5-TATGAGTGGCTAAATCGAT-3` 395bp Akers et al. (2010)
R 5-CCCGCTTTCTCGTAGCA-3`

aac(3′)-Ih, aph(3′)-VI, ant(2″)-Ia, Rmt55, aph(3′)-Ia and aac(6′)-Ib. Re-
action was done in a total volume of 25 μl using 12.5 μl of Emerald
Amp® MAX PCR master mix (Takara, Dalian, China), 5 μl DNA template,
1 μl of each upstream and downstream primers (10 pmol\ml) and vo-
lume was completed with 5.5 μl free RNA water. The reaction thermal
profile was initial denaturation at 95 °C for 5 min; followed by 40 cycles
of denaturation at 95 °C for 30 s,annealing at 50 °C for 30 s to aac(3′)-Ih
and aph(3′)-VI, 53 °C for 30 s to ant(2″)-Ia and aac(6′)-Ib and 55 °C for
30 s to aac(3′)-Ia, aac(3′)-IIa, Rmt55 and aph(3′)-Ia and extension 72 °C
for 40 s,followed by terminal extension at 72 °C for 10 min. Amplified
DNA products were visualized on 2% agarose gels (Bio Basic INC, Ca-
nada) stained with ethidium bromide, and photographed by the GBOX-
F3 gel documentation system Syngene (Syngene, MD, USA). All PCR
amplifications were conducted using sterilized tubes and tips in a bio-
safety hood that was not previously exposed to any bacterial work.
2.9. DNA purification
PCR product was purified using Gene Jet PCR purification kit
(Thermo K0701,Germany) by Sigma Scientific services company as the
following,45 μl of binding buffer was added to complete PCR mixture
and mixed thoroughly. The mixture was transferred to the Gene JET
purification column and centrifuged for 30–60 s at more than
12000 × g.The flow through was discarded.
Wash buffer(100 μl) was added to the Gene Jet purification column
and centrifuged for 30–60s.The follow-through was discarded and the
purification column was placed back into the collection tube. The
empty Gene Jet purification column was centrifuged for additional
1min.to completely remove any residual wash buffer. Gene Jet pur-
ification column was transferred to a clean 1.5 ml micro centrifuge tube
and 25 μl of elution buffer was added to the center of Gene Jet pur-
ification column membrane and centrifuged for 1min. The Gene Jet
purification column was discarded and the purified DNA was stored at
−20 °C.
2.10. DNA sequencing
Sequence of DNA was conducted by using purified PCR product in
GATC company(ABI 3730xI DNA sequencer, Germany) by using for-
warded and reverse primers. Only by combining the traditional Sanger
technology with the new 454 technology, genomes now can be se-
quenced and analyzed in half the usual project time, with a consider-
able reduction in number of coatings and gaps.
2.11. Deposition of the DNA sequences in GenBank
The nucleotide sequences have been submitted to GenBank/EMBL/
DDBJ database. All the DNA sequences were queried against the
National Center for Biotechnology Information (NCBI) database using
BLAST algorithm search analysis to determine the similarity percentage
with other sequences in GenBank. The sequences of the present study
was accessed in GenBank to determine the accession No.
3. Results
3.1. The most prevalent pathogen and most efficient antibiotic against
clinical bacterial isolates
A total of clinical isolates (210) were isolated from four different
hospitals and laboratories during period from May 2012 to March 2014
in Cairo, Egypt. Out of 210 isolates,88 isolates were identified as E. coli
(41.89%) and 57 isolates were Klebsiella spp.(27.13%) as indicated in
Fig. 1. The results of ESBL screening revealed that 11 isolates of E. coli
(5.23%) were positive ESBL, while four isolates out of 57 Klebsiella spp.
(1.90%) were positive ESBL as indicated in Table 2. The resistance
percentage of clinical bacterial isolates to each aminoglycoside anti-
biotic were indicated in Fig. 2.
The results revealed that the most efficient aminoglycoside anti-
biotic against the Egyptian clinical isolates was amikin, where 56.17%
of the clinical isolates were resistant to amikin,12.35% were inter-
mediate while 31.4% were sensitive. However, the lowest antibiotic
efficiency was kanamycin, about 74% of the clinical isolates were re-
sistant,11.41% were intermediates and only 14.28% were sensitive.
3.2. The most prevalent AME-genes among clinical bacterial isolates
The results of PCR as summarized in Table 3 revealed that, thirty
isolates were used in the present study. The first four isolates were E.
coli sensitive(MAM-1,2,3,4)and three Klebsiella isolates were also sen-
sitive (MAM-6,8,9), i. e no PCR product have been detected (negative).
The results of AME-genes indicated that two out of the eight AME-
genes investigated aac(3′)-Ia and Rmt(55) have not been detected in
any of the thirty isolates. The most prevalent AME-gene resistance was
aac(3′)-IIa found in 12 out of 30 isolates(40%),followed by aac(6′)-Ib
(30%),followed by aph(3′)-Ia(23.3%),ant(2″)-Ia(20.0%),aph(3′)-VI
(13.3%)and aac(3′)-Ih (6.6%). The results also revealed that 8 out of 15
Klebsiella spp. acquired aac(3′)-IIa(53.3%)while E. coli isolates recorded
4 out of 15 E. coli(26.6%) acquired the same gene aac(3′)-IIa. In case of
aac(3′)-Ih gene, only two Klebsiella spp. acquired this gene but none of
E. coli acquired aac(3′)-Ih gene. While aac(6′)-Ib gene was detected in 6
out of 15 Klebsiella spp.(40%) and 3 out of 15 E. coli isolates(20.0%).
The results of aph(3′)-VI genes recorded that 3 out of 15 Klebsiella
spp. (20.0%) was positive while one out of 15 E coi isolates(6.6%) was
positive too. Also the results of PCR for aph(3′)-Ia revealed that three

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M.A.M. Abo-State et al. Journal of Radiation Research and Applied Sciences 11 (2018) 408–415

Fig. 1. Clinical bacterial isolates collected during four periods.

Table 2 out of 15 Klebsiella spp.(20.0%)were positive, while four out of 15 E.

Prevalence of E.coli and Klebsiella spp. among clinical isolates. coli(26.6%)were positive. AME genes ant(2″)-Ia have been detected in 4
Organism No. of isolates
out of 15 Klebsiella spp.(26.6%) and two out of 15 E. coli isolates
(13.3%) as indicated in Fig. 3. The results revealed that Klebsiella sp.
20/5/ 1/3/2013 5/9/2013 1/1/2014 Total % MAM-16 acquired five AME genes aac(3′)-IIa,aac(3′)-Ih,aph(3′)-VI,ant
2012 to to 1/5/ to 5/11/ to 1/3/ (2″)-Ia and aph(3′)-Ia. Mainwhile, E. coli MAM-24 was the only E. coli
20/7/ 2013 2013 2014
isolate that acquired five AME-genes aac(3′)-IIa,aph(3′)-VI,ant(2″)-
2012
Ia,aph(3′)-Ia and aac(6′)-Ib as indicated in Figs. 4,5.
E.coli 21 19 18 19 77 36.66 Consequently, the two strains Klebsiella sp. MAM-16 and E. coli
E.coli(ESBL) 5 - - 6 11 5.23 MAM-24 were chosen to be sequenced as a product of PCR and these
Total E.coli 26 19 18 25 88 41.89
sequences(10 sequences for 10 genes)were deposited in GenBank and
Klebsiella spp. 8 11 18 16 53 25.23
Klebsiella - 1 - 3 4 1.90 accessed.
(ESBL)
Total Klebsiella 8 12 18 19 57 27.13 3.3. Similarity of DNA sequences of AME-genes among E.coli and Klebsiella
spp.
spp

Sequence of each resistance gene of the two clinical pathogenic

isolates (Klebsiella sp. MAM-16 and E. coli MAM-24) have been de-
posited in GenBank to determine the similarity between the sequences

Fig. 2. Pattern of Aminoglycosides resistance among clinical bacterial isolates.

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M.A.M. Abo-State et al. Journal of Radiation Research and Applied Sciences 11 (2018) 408–415

Table 3
Prevalence of aminoglycoside modifying enzyme(AMEs) genes among E.coli and Klebsiella spp. I = E.coli (negative) III&V= Klebsiella(positive) II=Klebsiella(nega-
tive) IV = E.coli (positive).
Sample code aac(3′)-Ia aac(3′)-IIa aac(3′)-Ih Aph(3′)-VI ant(2″)-Ia Rmt (55) aph(3′)-Ia aac(6′)-Ib

I 1 Negative Negative Negative Negative Negative Negative Negative Negative

2 Negative Negative Negative Negative Negative Negative Negative Negative
3 Negative Negative Negative Negative Negative Negative Negative Negative
4 Negative Negative Negative Negative Negative Negative Negative Negative
II 6 Negative Negative Negative Negative Negative Negative Negative Negative
8 Negative Negative Negative Negative Negative Negative Negative Negative
9 Negative Negative Negative Negative Negative Negative Negative Negative
III 10 Negative Positive Negative Negative Negative Negative Negative Positive
11 Negative Positive Negative Negative Negative Negative Negative Negative
12 Negative Negative Negative Negative Negative Negative Negative Positive
13 Negative Positive Negative Positive Positive Negative Positive Positive
14 Negative Negative Positive Negative Negative Negative Negative Negative
15 Negative Negative Negative Negative Negative Negative Negative Positive
16 Negative Positive Positive Positive Positive Negative Positive Negative
17 Negative Negative Negative Negative Positive Negative Negative Positive
18 Negative Positive Negative Negative Negative Negative Negative Negative
19 Negative Positive Negative Negative Negative Negative Negative Negative
20 Negative Positive Negative Positive Positive Negative Positive Positive
IV 21 Negative Negative Negative Negative Negative Negative Negative Negative
22 Negative Negative Negative Negative Negative Negative Positive Positive
23 Negative Positive Negative Negative Negative Negative Negative Negative
24 Negative Positive Negative Positive Positive Negative Positive Positive
25 Negative Negative Negative Negative Negative Negative Negative Negative
26 Negative Negative Negative Negative Negative Negative Negative Negative
27 Negative Positive Negative Negative Positive Negative Positive Negative
28 Negative Positive Negative Negative Negative Negative Positive Positive
29 Negative Negative Negative Negative Negative Negative Negative Negative
30 Negative Negative Negative Negative Negative Negative Negative Negative
31 Negative Negative Negative Negative Negative Negative Negative Negative
V 32 Negative Positive Negative Negative Negative Negative Negative Negative

Fig. 3. Prevalence of Aminoglycoside modifying enzymes(AMEs) resistance genes among E. coli and Klebsiella spp.

of the present study and that previously deposited in GenBank by other
investigators for the same genes.
Sequence of acc(3′)-IIa gene of Klebsiella sp. MAM-16 was similar by
99% to gentamicin(3′)N-acetyl transferase of Acinetobacter pittii 42F
with accession no. CD12826.1. However,the sequence of the same gene
aac(3′)-IIa of E. coli MAM-24 was similar by 100% with aminoglycoside
N-acetyl transferase III of mixed culture bacterium GE-Gf1DD01-08
with accession no. ACT97805.1.
Sequence of aph(3′)-VI gene of Klebsiella sp. MAM-16 indicated that
this gene sequence was similar by 100% with aminoglycoside phos-
photransferase APH(3′) of Acinetobacter calcoaceticus/baumannii com-
plex with accession no. WP000422638.1. Sequence of aph(3′)-VI gene
of E. coli MAM-24 of the present study was similar by 100% with multi
species aminoglycoside phosphotransferase APH(3′) of Acinetobacter
calcoaceticus/baumannii complex with accession no. WP000422638.1.
From the previous studies, it was clear that sequences of AME-gene
(aph(3′)-VI) of both Egyptian bacterial isolates (Klebsiella sp. MAM-16
and E. coli MAM-24)were identical (the same sequence) and similar by
100% of the sequence of aph(3′)-VI of aminoglycoside phospho-
transferase APH(3′) of Acinetobacter calcoaceticus/baumannii with ac-
cession no. WP000422638.1. Sequence of ant(2″)-Ia gene of Klebsiella
sp. MAM-16 showed that more than 50 aminoglycoside(2″) adenyl-
transferase of different bacterial species was similar by 100%. Among
these genes of aminoglycoside (2″) adenyltransferase of Pasteurella

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M.A.M. Abo-State et al. Journal of Radiation Research and Applied Sciences 11 (2018) 408–415

Table 4
Determination of accession numbers by GenBank.
Sequence_ID Identifier Organism GenBank accession number

Seq-1 aac(3′)-IIa Klebsiella MF495896

Seq-2 aac(3′)-IIa Escherichia coli MF495897
Seq-3 aac(3′)-Ih Klebsiella MF495898
Seq-4 aph(3′)-VI Klebsiella MF495899
Seq-5 aph(3′)-VI Escherichia coli MF495900
Seq-6 ant(2″)-Ia Klebsiella MF495901
Seq-7 ant(2″)-Ia Escherichia coli MF495902
Seq-8 aph(3′)-Ia Klebsiella MF495903
Seq-9 aph(3′)-Ia Escherichia coli MF495904
Seq-10 aac(6′)-Ib Escherichia coli MF495905

Fig. 4. Agarose gel electrophoresis showing PCR products. M, DNA Ladder; c-
negative control; Lane2, aac(3′)-IIa; Lane3, aac(3′)-Ih; Lane4, aph(3′)-VI; Lane5,
ant(2″) –Ia; Lane7, aph (3′)-Ia of Klebsiella spp.(Sample16).
WP036934288.1.
Sequence of ant(2″)-Ia gene of E. coli MAM-24 is similar by 99% of
2-deoxyribose-5-phosphate aldolase Proteus mirabilis with accession no.
WP017827555.1 and similar by 97% of deoxyribose-phosphate aldolase
Proteus vulgaris with accession no. WP072064360.1.

3.4. Determination of AME-genes accession no

The results of deposition of DNA sequences in GenBank and de-

termination of the similarity percentage between the sequences of the
present study and the sequences of another investigators and comparing
the data by GenBank led to determination of the accession no. for each
one of the 10 genes of AME-genes in the present study as indicated in
Table 4.

4. Discussion

Antibiotic resistance is a problem of deep scientific concern both in

Fig. 5. Agarose gel electrophoresis showing PCR products. M, DNA Ladder; c-
negative control; Lane 2,aac(3′)-IIa; Lane4,aph(3′)-VI; Lane5, ant(2″) –Ia;
Lane7,aph (3′)-Ia; Lane8, aac (6′)-Ib of E. coli (Sample24).
multocida with accession no. AEG 42370.1 and Salmonella enterica
subsp. enterica serover typhimurium with accession no. CAG34229.1.
The previous comparison between AME-gene ant(2″)-Ia of Klebsiella
sp. MAM-16 with other aminoglycoside(2″) adenyltransferase genes
revealed that more than 50 different bacterial species were similar by
100%. These data proved that a horizontal transfer of that gene be-
tween different bacterial species have been occurred in nature.
Sequence of aph(3′)-Ia gene of Klebsiella sp. MAM-16 revealed that
this gene was similar by 99% with 17 bacterial strains having aph(3′)-Ia
genes and more than 65 bacterial strains recorded 98% similarity with
aph(3′)-Ia gene of Klebsiella sp. MAM-16. Among the similar gene(99%),
aminoglycoside (3′) phosphotransferase of Acinetobacter baumannii with
accession no. WP052776689.1. and Klebsiella pneumonia with accession
no. WP002003952.1. Meanwhile, sequence of aph(3′)-Ia gene of E. coli
MAM-24 of the present study indicated that this gene was similar by
100% with APH(3′)-Ia family aminoglycoside O- phosphotransferase of
Enterobacteries homaechei with accession no. WP072034762.1.
However,in case of sequence of acc(6′)-Ib gene of E. coli MAM-24,it
was found that 46 bacterial sequences of aminoglycoside(6′)N-acetyl-
transferase of different bacterial species were similar by 100% with aac
(6′)-Ib gene of E. coli MAM-24. Among these genes, aminoglycoside N
(6′) acetyltransferase of Klebsiella pneumonia with accession no.
QJJ88941.1. and E. coli with accession no. QJL44526.1.
Sequence of aac(3′)-Ih of Klebsiella MAM-16 is similar by 99% of the
sequence2-deoxyribose-5-phosphate aldolase Proteus mirabilis with ac-
cession no. WP017827555.1 and similar by 96% of 2-deoxyribose-5-
phosphate aldolase Proteus vulgaris. with accession no.
hospital and community setting. The most prevalent Egyptian clinical
bacterial isolates were E. coli (n = 88/210,41.89%)followed by
Klebsiella spp.(n = 57/210,27.13%). The most efficient aminoglycoside
antibiotic was Amikin(AK) against GNB (Saleh, Abo-State, Helal, &
Ghareeb, 2017).
The previous results was in agreement with the results of other in-
vestigators. Abo-State, Khatab, and Ghareeb (2010) reported that E. coli
represented 26.66% of clinical isolates (210 isolates,139 were Gram
negative bacilli,70 were Gram positive cocci and one was Candida) and
Klebsiella spp. (20.47%). The most efficient antibiotic was imipenem
(IMP).
Meanwhile, Gram negative bacilli(GNB) were144 isolates(72%) of
the clinical isolates collected from EL-Gharbia Governorate. The most
frequent pathogen were Klebsiella spp.(42%) followed by E. coli(15.5%).
The most efficient antibiotics were aminoglycosides against Egyptian
clinical isolates. The most efficient aminoglycoside antibiotic was
amikacin(AK). The result of MIC50 revealed that AK had the lowest
MIC50 against MDR-isolates (Abo-State, Abd EL-Salam, & Assi, 2017).
The combination of (AK/IMP/FEP/TIG) was the only combination
which achieved > 90% killing against all the MDR-isolates isolated
from urinary tract infection (UTI) and hemodialysis patients (Abo-State,
Saleh, & Fathy, 2016).
The most prevalent AME-genes among the Egyptian GNB (E.coli and
Klebsiella spp.,30 isolates) were aac(3′)-IIa(40%),followed by aac(6′)-Ib
(30%). One isolate (Klebsiella sp. MAM-16) and one isolate E. coli MAM-
24,each one of them acquired five AME-genes. The previous results of
the present study could be compared with the results of other in-
vestigators in different countries. Akers, Chaney, Barsoumian, Beskius,
and Zera (2010) reported that all seven AME genes included in multi-
plex PCR were present singly or in combination. Fourty two isolates
(39.3%) had one AME gene,60 isolates(56.1%) had two AME genes,4
isolates(3.7%) had three AME genes. The ant(2″)-Ia gene was statisti-
cally significantly associated with aminoglycoside resistance for

413
M.A.M. Abo-State et al.
amikacin and tobramycin. Antibiotic reistance rates in Pseudomonas
aeruginosa in France revealed that of the 10 genes encoding ami-
noglycoside resistance mechanisms, only 4 were detected. The aac(6′)-
Ib gene was the most frequently encountered in 19 strains(36.5%) of
resistant strains. The presence of this gene was associated with three
different phenotypes: GEN-TOB-NET for 1 isolate, GEN-TOB-NET-AMK
for 12 and GEN-TOB-NET-AMK for 6 isolates (Dubois et al., 2008).
In Poland PCR assays revealed the presence of aac(6′)-Ib among 26
(59.2%) strains, aph(3′)-Ib among 16 (36.2%), aac(3′)-Ia among 7
(15.9%), and ant(2″)-Ia among 2 (4.6%) strains, aac(6′)-Ib and aph(3′)-
Ib genes were common among ESBL non-producers, and were detected
among 4 (26,7%) and 3 (20%) strains, respectively; whereas, among
ESBL producers, the most frequently detected genes encoding AMEs
were aac(6′)-Ib and aph(3′)-Ib, observed in 22 (75.9%) and 13 (44.8%)
of isolates, respectively. In addition, few ESBL-producing strains pre-
sented aac(3′)-Ia and ant(2″)-Ia genes. Additionally, they noticed that
one isolates harbored three genes encoding AMEs: aph(3′)-Ib, aac(3′)-Ia,
aac(6′)-Ib (Ojdana et al., 2018).
Genes sequencing showed the presence of a Leucine at position 119
for 12 strains exhibiting the GEN-TOB-NET-AMK phenotype (aac(6′)-Ib)
and of serine at the same position for strain of phenotypeGEN-TOB-NET
(aac(6′)-Ib) for six remaining strains with GENTOB-NET-AMK pheno-
type (Dubois et al., 2008). The most prevalent gene encoding AME
among ESBL-positive P. mirabilis was ant(2″)-Ia, present in(80.3%) of
AME-positive isolates from Poland (Michalska et al., 2014). This is a
very interesting results, because the most common variant of AME
among Gram-negative and Gram-positive was aac(6′)-Ib (Almaghrabi,
Clancy, & Doi, 2014; Ramirez & Tolmasky; 2010; Wen, Zhon, Yang, &
Xu, 2014).
In another study on P. mirabilis and P. aeruginosa isolates from
Poland,the aac(6′)- Ib gene was detected in 71.43% and 58.3% of strain,
respectively (Sacha et al., 2012; Wleczorek, Sacha, & Hauschild, 2008).
However,in Iran high rate(71.0%) of AME among CTX-M-15 producing
K. pnumonia (Peerayeh, Rostoni, Siadat, & Derakhshan, 2014). With
increasing prevalence of ESBL-positive strains resistant to Beta-lacta-
mase,the rate of isolate resistant to aminoglycosides increasing also
(Michalska et al., 2014). More than 60% of Acinetobacter sp. isolates in
Iran contained phosphotransferase aphA6 and acetyltransferase genes
aacC1,but adenyltransferase genes aadA(41.7%) and aadB(3.3%) were
less prominent 21.7% of the strains contain three aminoglycoside re-
sistance genes(aphA6,aacC1 and aadA1) (Moniri et al., 2010).
Military medical facilities treating patient injured in Iraq and
Afghanistan having a multidrug resistant A. baumanmii isolates. Genes
encoding AMEs represented 97% of the isolates. These isolates were
Amikacin resistant harboring phosphotransferase aphA6,while other
genes encoding AMEs involved adenyltransferase genes aad A1(39%)
and aad B(48%) and acetyltransferase genes aacC1(56%) and aac C2
(5%). Seventy-eight percent (40/51) of Tobramycin-resistant isolates
contained either the aacC2 or aad B gene (Hujer et al., 2006).
But in Norway,the prevalence of aminoglycoside resistance was
increased among clinical E. coli and Klebsiella spp. isolates.This is
mainly due to the presence of AME AAC(3′)-II and AAC(6′)-Ib
(Haldorsen et al., 2014). AMEs genes aph(3′)-VI a (90.6%) and aph(3″)-
IIb(61.8%) were the most prevalent in A. baumanmii and P. aeruginosa
respectively. Eight(26%) amikacin highly resistant A. baumanmii iso-
lates were positive for armA methylase in Iran (Aghazadeh et al., 2013).
In Egypt susceptibility testing revealed that carbapenems and tige-
cycline were the most effective agents. Investigation of genes encoding
AMEs revealed that acc(6′) -Ib was the most prevalent, followed by aac
(3′)-IIa, aph(3′)-IV, and ant(3″)-I (El-Badawy, Tawakol, El-Far,
Maghrabi, & Al-Ghamdi, 2017).
In China molecular identification of the 162 isolates obtained from
the hospital showed that the positive rates of AMEs genes, such as aac
(3′)-II, aac (6′)-Ib, ant (3″)-I and ant (2″)-I, were 30.2%, 19.8%, 13.6%
and 4.3%, respectively. Also 16S rRNA methylase genes were also
identified with positive rates of arm A and rmt B of 11.1% and 6.2%,
414
Journal of Radiation Research and Applied Sciences 11 (2018) 408–415
respectively. All sequences of the detected amplicons were aligned and
it was shown that there was over 99% identity with the reported target
genes accessed from NCBI. The distribution of AMEs and 16S rRNA
methylase gene were in K. pneumoniae. Among them, 28 strains carried
both AMEs and 16S rRNA methylase genes. A total of 62 strains car-
rying resistance genes included 16 strains with 1 genotype, 26 strains
with 2 genotypes, 12 strains with 3 genotypes, 6 strains with 4 geno-
types and 2 strains with 5 genotypes. The most common genotype was
aac (3′)-II + aac (6′)-Ib, and the positive rate was 12.9% (8/62); this
was followed by aac (3′)-II with the a positive rate of 11.3% (7/62)
(Liang et al., 2015).
5. Conclusion
A total of 210 clinical isolates have been isolated from Egyptian
hospitals and laboratories (May 2012 to March, 2014). Out of 210
bacterial isolates,88 isolates were E. coli(41.89%) and 57 isolates were
Klebsiella spp.(27.13%). The most efficient aminoglycoside antibiotic
among Egyptian clinical bacterial isolates was Amikin. PCR results re-
vealed that two out of eight AME-genes investigated at the present
study have not been detected aac (3′)-Ia and Rmt55 among E. coli and
Klebsiella spp.
Each strain of E. coli MAM-24 and Klebsiella sp. MAM-16 acquired
five resistance genes. The most prevalent AME-gene resistance was aac
(3′)-IIa(40%)followed by aac(6′)-Ib (30%) among E. coli and Klebsiella
spp. The results proved that the same gene of resistance was transferred
(Horizontal transfer) between different bacterial species as determined
by comparing the sequences with that of GenBank.
Acknowledgment
We acknowledge the members of Egyptian atomic Authority for
support this research.
References
Abo-State, M. A. M., Abd EL-Salam, S. S., & Assi, R. (2017). Prevalence of multidrug
resistant (MDR) among clinical bacterial isolates in EL Gharbia Governorate and ef-
ficiency of antibiotic combinations. World Journal of Pharmacy and Pharmaceutical
Sciences, 6, 116–133.
Abo-State, M. A. M., Khatab, O., & Ghareeb, H. M. (2010). Trends in antimicrobial sus-
ceptibility of pathogenic strains isolated from different hospitals in Egypt. Egypt.
Journal of Biotechnology, 36 98-11.
Abo-State, M. A. M., Saleh, Y. E., & Fathy, S. (2016). Efficiency of antibiotic combinations
on multidrug resistant bacterial strains isolated from urinary tract infection and he-
modialysis patients. Journal of Ecology Health Environment, 4, 61–65.
Aghazadeh, M., Rezaee, M. A., Nahaei, M. R., & Mahdian, R. (2013). Dissemination of
aminoglycoside modifying enzymes and 16srRNA methylases among Acinetobacter
baumannii and Pseudomonas aeruginosa isolates. Microbial Drug Resistance, 19,
282–288.
Akers, K. S., Chaney, C., Barsoumian, A., Beskius, M., & Zera, W. (2010). Aminoglycoside
resistance and susceptibility testing errors in Acinetobacter baumannii- calcoaceticus
complex. Clinical Microbiology Journal, 48, 1132–1138.
Almaghrabi, R., Clancy, C. J., & Doi, Y. (2014). Carbapenem-resistantKlebsiella pneumonia
strains exhibit diversity in aminoglycoside modifying enzymes,which exert differing
effects on plazomicin and other agents. Antimicrobial Agents and Chemotherapy, 58(8),
4443–4451.
Alyamani, E. J., Anamil, M. K., Rayan, Y. B., Majed, A. M., Fayez, S. B., & Elena, R.
(2017). The occurrence of ESBL-producing Escherichia coli carrying aminoglycoside
resistance genes in urinary tract infections in Saudi Arabia. Annals of Clinical
Microbiology and Antimicrobials, 16, 1–13.
Baptista, P., McCusker, M., Carvalho, A., Ferreira, D., Mohan, N., Martins, M., et al.
(2018). Nano-strategies to fight multidrug resistant bacteria "A Battle of the Titans".
Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.01441.
Chaudhary, M., & Payasi, A. (2014). Resistance patterns and prevalence of the ami-
noglycoside modifying enzymes in clinical isolates of Gram negative pathogens.
Global Pharmacology Journal, 8, 73–79.
Cheesbrough, M. (2006). District laboratory practice in tropical counteries part (2)Egyptian
edition. UK: Cambridge University Press.
Chen, D., & Munchie, A. I. H. (2014). An aminoglycoside sensing ribswitch controls the
expression of aminoglycoside resistance acetyltransferase and adenyltransferases.
Biochimica et Biophysica Acta, 1839, 951–958.
Chen, H., Su, P., Kuo, S., Lauderdale, T., & Shih, C. (2018). Adding a c-terminal cysteine
(CTC)can enhance the bactericidal activity of three different antimicrobial peptides.
Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.01440.
M.A.M. Abo-State et al.
Clinical Laboratories Standard Institute(CLSI) (2012). Performance standard for anti-
microbial disc susceptibility tests: Approved Standard.11 thed. document Mo2-A11.
Doi, Y., & Arakowa, Y. (2007). 16 S ribosomal RNA methylation: Emerging resistance
mechanism against aminoglycoside. Antimicrobial Resistance, 45, 88–94.
Dubois, V., Arpin, C., Dupart, V., Scavelli, A., & Coulange, L. (2008). Beta lactam and
aminoglycoside resistance rate and mechanisms among Pseudomonas aeruginosa in
French general practice (community and private healthcare centers). Antimicrobial
Chemotherapy, 62, 316–323.
El-Badawy, M. F., Tawakol, W. M., El-Far, S. W., Maghrabi, I. A., & Al-Ghamdi, S. A.
(2017). Molecular identification of aminoglycoside-modifying enzymes and plasmid-
mediated quinolone resistance genes among Klebsiella pneumoniae clinical isolates
recovered from Egyptian patients. International Journal of Microbiology, 2017, 1–12.
Haldorsen, B., Simonsen, G. S., & Sundsfiord, A. (2014). Increased prevalence of ami-
noglycoside resistance in clinical isolates of Escherichia coli and Klebsiella spp. in
Norway is associated with the acquisition of AAC(3')-II and AAC(6')-Ib. Diagnostic
Microbiology and Infectious Disease, 78, 66–69.
Hujer, K. M., Hujer, A. M., Hulten, E. A., Bajaksouzian, S., & Adams, J. M. (2006).
Analysis of antibiotic resistance genes in multidrug resistant Acinetobacter sp. isolates
from military and civilian patients treated at the Walter Reed Army Medical Center.
Antimicrobial Agents and Chemotherapy, 50, 4114–4123.
Liang, C., Xing, B., Yang, X., Fu, Y., Feng, Y., & Zhang, Y. (2015). Molecular epidemiology
of aminoglycosides resistance on Klebsiella pneumonia in a hospital in China.
International Journal of Clinical and Experimental Medicine, 8(1), 1381–1385.
Michalska, A. D., Sacha, P. T., Kaczynska, K., & Tryniszewska, E. A. (2014). The diversity
of aminoglycoside modifying enzymes among ESBL positive Proteus mirabilis clinical
strains. MEDtube Science, 4, 16–20.
Mohammed, Y., Gadzama, G. B., Zailani, S. B., & Aboderin, A. O. (2016). Characterization
of extended spectrum beta-lactamase from Escherichia coli and Klebsiella species from
north eastern Nigeria. Clinical Diagnostic Research Journal, 10(2), 7–10.
Moniri, R., Farahani, R. K., Shajar, G.h, NazemShirazi, M. H., & Ghasemi, A. (2010).
Molecular epidemiology of aminoglycosides resistance in Acinetobacter spp.with
emergence of multidrug resistant strains. Iranian Journal of Public Health, 39(2),
63–68.
Ojdana, D., Sienko, A., Sacha, P., Majewski, P., Wieczorek, P., Wieczorek, A., et al.
415
Journal of Radiation Research and Applied Sciences 11 (2018) 408–415
(2018). Genetic basis of enzymatic resistance of E.coli to aminoglycosides. Advances in
Medical Sciences, 63, 9–13.
Oxoid manual (2006). Manual of culture media, ingredients and other laboratory services (4th
ed.). (reprint).Compiled by E. Y. Bridson 9th Edition Published by OXOID Limited,
Wade Road, Basingstoke, Hampshire RG24 8PW, UK.
Park, Y. (2009). Aminoglycoside resistance in Gram-negative bacilli. Korean Journal
Clinical Microbiology, 12, 57–61.
Peerayeh, S. N., Rostoni, E., Siadat, S. D., & Derakhshan, S. (2014). High rate of ami-
noglycoside resistance in CTX-M-15 producing Klebsiella pneumonia isolates in
Tehran. Iran Laboratory Medicine, 45(3), 231–237.
Ramirez, M. S., & Tolmasky, M. E. (2010). Aminoglycoside modifying enzymes. Drug
Resistance Updates, 13(6), 151–171.
Risberg, K. (2010). Aminoglycoside resistance mechanisms in EnterobacteriaceaeMSc.Thesis.
University of Tromso, Faculty of Medicine. Department of Pharmacy.
Romanowska, J., McCammon, J. A., & Trylska, J. (2011). Understanding the origins of
bacterial resistance to aminoglycosides through molecular dynamics mutational
study of the ribosomal A-Site. PLoS Computational Biology, 7, 1–18.
Sacha, P., Jaworowska, J., Ojdana, D., Wieczorek, P., Czaban, S., & Tryniszewska, E.
(2012). Occurrence of the aacA4 gene among multidrug resistant strains of
Pseudomonas aeruginosa isolated from bronchial secretions obtained from the
Intensive Therapy Unit at University Hospital in Bialystok, Poland. Folia Histochemica
et Cytobiologica, 50, 322–324.
Saleh, Y. E., Abo-State, M. A. M., Helal, N. L., & Ghareeb, H. M. (2017). Aminoglycoside
and Beta-Lactam resistance pattern among Gram negative bacilli (GNB) isolated from
Egyptian hospitals. Asian Journal Microbiology Biotechnology, 2, 20–29.
Wen, J. T., Zhon, Y., Yang, L., & Xu, Y. (2014). Multidrug resistant genes of aminogly-
coside modifying enzymes and 16S rRNAmethylases in Acinetobacter baumannii-
strains. Genetics and Molecular Research, 13(2), 3842–3849.
Wleczorek, P., Sacha, P., & Hauschild, T. (2008). The acc(6')-Ib gene in Proteus mirabilis
strains resistant to aminoglcosides. Folia Histochemica et Cytobiologica, 46(4),
531–533.
Zhao, F., Shi, H., Jinghua, L., Zhou, J., & Sun, Y. (2013). Detection of 16S rRNA me-
thylases genes in Gram negative bacilli isolated from hospitals in Changchun, China.
Advances in Infectious Diseases, 3, 290–294.