Abstract are associated with emergence of resistance to many

Abstract

Objective: to investigate the genetic basis for quinolones and fluoroquinolones
resistance in E.coli isolated from
outpatients with urinary tract infections (UTIs) in Egypt.

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Methods: A total of 51 nalidixic acid resistant out of 155 E.coli was isolated from adult patients with UTIs from September
2014 to August 2015. Antimicrobial sensitivity testing was determined by disc
diffusion test, minimum inhibitory concentration (MIC) of nalidixic acid and
ciprofloxacin was determined by E. test strips. Mutations in the quinolone
resistance determining region (QRDR) of
gyrA and parC genes, and
screening for other plasmid mediated quinolone resistance (PMQR) determinants (qnr
, aac(6)-ib-cr, qepA and oqxAB) were done by PCR amplification and DNA
sequencing.

Results: Genetic analysis of the QRDR of
gyrA and parC genes revealed
presence of mutations in the codons 83 and 87 of the gyrA gene and at codons 80 and 84 of the parC gene. Mutations in parC were always found simultaneously
with mutations in gyrA gene specifically
in highly fluoroquinolones resistant isolates. Most of the isolates (94%) have
at least one of the PMQR genes. We found aac(6′)-Ib-cr variant in 88.2%
of the samples, qnrS in 23.5 % of the isolates, qepA in 47.1% of
the isolates and oqxAB was detected in 5.9% of the isolates.

Conclusion: QRDR mutations in chromosomal genes encoding gyrA and parC play an
essential role in quinolone and fluoroquinolone resistance in the clinical E.
coli strains studied. The accumulation of amino acid alterations in gyrA and the concurrent presence of the parC alterations play a principal role
in emerging high-level resistance to fluoroquinolones. The detected PMQR
determinants were aac(6′)-Ib-cr, qepA and qnrS and oqxAB
genes. These determinants have wide spread in the community and they are
associated with emergence of resistance to many other classes of antibiotics. To
the best of our knowledge, this is the first report to detect oqxAB in
Egypt.

Keywords: E.coli,
Resistance, Fluoroquinolones.

1.      Introduction

In 1885, Theodor Escherich isolated E.coli
from the feces of neonates and he described it as bacterium coli commune. It
was later named Escherichia coli (Todar, 2006). Although that there are some
strains of E.coli that are major pathogens of human and animals, there are
other strains that are harmless commensals of the intestinal tract .The
pathogenic strains are separated into two groups: those causing diseases inside
the intestinal tract and others capable of infection at extra-intestinal sites
as the uropathogenic E.coli which is the most common organism implicated in
UTIs (80-85%) (Kaper et al, 2004, Nicolle, 2008).

Quinolones are synthetic antibacterial
agents with excellent action against enterobacteriaceae. (Soto et al, 2003). In
1962 the first quinolone, Nalidixic acid was discovered, this discovery led to
the development of a variety of quinolones. Structural modification by the
addition of fluorine atom at C-6 position led to the innovation of
fluoroquinolones which are more systemically active. Quinolones act by
inhibiting the action of topoisomerase ? (DNA gyrAse) and topoisomerase 1V,
both of them are involved in DNA replication  
(Andriole, 2005). The extensive usage of quinolones was responsible for
spreading quinolone resistance in different microorganisms (Robicsek et al,
2006).

Quinolone resistance is caused mainly as a
result of mutations in the quinolone resistance-determining regions (QRDRs) of
DNA gyrase and topoisomerase IV, decreased membrane permeability and
overexpression of efflux pumps. Recently plasmid-mediated quinolone resistance
(PMQR) genes were reported (Cao et al, 2011).

 

The first PMQR mechanism the qnr gene
(currently qnrA1), was detected in 1998.Qnr determinants are supposed to bind
to and protect DNAgyrAse and/or topoisomerase IV from fluoroquinolone action.
Five families of this gene have been described (qnrA, qnrB, qnrS, qnrC and D)
(Martinez-Martinez et al, 1998, Baudry-Simner et al., 2012).

The second PMQR mechanism is the
aac(6′)-Ib-cr which is a variant from the AAC(6?)-Ib enzyme. This enzyme act by
N-acetylation of the amino nitrogen on the piperazinyl substitute of the
fluoroquinolones with an unsubstituted piperazinyl group, ciprofloxacin and
norfloxacin (Robiscek et al, 2006).

The third PMQR mechanism includes active
efflux pumps: the OqxAB multidrug efflux pump and the QepA efflux pump, which
causes decreased sensitivity to hydrophilic fluoroquinolones such as
ciprofloxacin (Kim et al 2009, Yamane et al, 2008).This study aimed to detect
the quinolones resistance mechanisms among E.coli isolates from outpatients
with UTIs in Egypt.

2.      Materials and methods

2.1.   
Clinical isolates

Total of 280 urine
samples from patients suspected with with UTIs from outpatient sections at
clinical microbiological laboratory in Fayoum university hospital and other
private laboratories of medical analysis at Fayoum governorate, Egypt, were
screened forE.coli by conventional
bacterial identification tests and confirmed by microbact identification test, (oxoid,
UK).  The patient’s data were
recorded (the gender, age, recurrences of UTI, previous treatment with
antibiotics and the presence of chronic diseases.

 

2.2.    Antibiotic sensitivity
testing

The output isolates
were subjected to antimicrobial sensitivity tests according to routine
laboratory protocols of clinical and laboratory standards institute (CLSI,
2013).Susceptibility testing to Nalidix acid, Ciprofloxacin, Ampicillin,
Amoxicillin/Clavulanate, Ceftazidime, Cefoxitin, Cefotaxime, Ceftriaxone,
Gentamicin, Amikacin, and Imipenem (oxoid, UK) according to the CLSI
guidelines. The MIC of nalidixic acid and ciprofloxacin was determined
by E.test (Liofilchem, Italy). it has been suggested that  MIC of nalidixic acid could be used as a
genetic marker of resistance for quinolones in Gram-negative bacteria (Ruiz,
2003) so the recovering  of the
isolates was according to it? s nalidixic acid resistance (MIC). E.coli
ATCC 25922 was used as a control strain.

 

2.3.    Phenotypic detection of
extended spectrum beta-lactamases (ESBLs)

ESBLs
were detected phenotypically by the double-disc potentiation test (Jarlier et al., 1988).

 

2.4.    Genetic analysis of
resistance mechanisms

2.4.1. DNA extraction

Extraction
of DNA was performed using genomic purelink extraction kits according to
the manufacture? s extraction guidelines (Invitrogen, USA).

 

2.4.2.
PCR of the quinolone resistance-determining region (QRDR) of the gyrA and parC genes

QRDR
of the gyrA and parC genes of selected isolates with different
ciprofloxacin MICs were amplified by pcr using the primers showed in table (1)
to give a 620-bp p and 559-bp products respectively.5ul DNA was
added to 20 ul reaction mixture 
containing  12.5 ul PCR reaction
mix (sigma, USA), 1ul of each primer and 5.5 ul dH2O.PCR conditions for both
genes were as follows:  initial
denaturation step of 5 min at 94 ?C, 1 min at 94 ?C, 1min at the annealing
temperature 55 ?C and 1min at 72 ?C for 30 cycles, final extension step was 10
min at 72 ?C. reaction mixes without DNA template served as negative controls.
(Weigel et al., 1998, Qiang et al., 2002) reaction mixes
without a DNA template served as negative controls.

 

2.4.3.
PCR and for detection of PMQR genes
(using primers in (table 1)):

For
all PMQR genes the following reaction mixture was used: 1ul template DNA was
added to 24 ul PCR msatermix containing 12.5ul PCR reaction mix, 1ul of each
primer and completed to 25ul with dH2O.

 

2.4.3.1. Multiplex
pcr of qnrA, qnrB , and  qnrS
genes was performed to give 516-bp, 469-bp, and 417 bp products
respectively.The pcr conditions were 94 ?C for 45s, 53 ?C for 45s, and 72 ?C
for 60s for 30 cycles and a final extension at 72 ?C for 10 min (Robicsek et al, 2006).

 

2.4.3.2. Screening
for the aac(6′)-Ib-by pcr to give a 482bp product. pcr conditions were
94 ?C for 45s, 55 ?C for 45s, and 72 ?C for 45s for 30 cycles and a final
extension at 72 ?C for 10 min (Park et
al, 2006).

 

2.4.3.3. Screening
for the qepA gene by pcr to give 199 bp products. The pcr conditions
were denaturation at 96 ?C for 1 min, annealing at 60 ?C for 1 min, and
extension at 72 ?C for 1 min for 30 cycles and final extension step was at 72
?C for 5 min (Yamane et al.,
2008).

 

2.4.3.4. Screening
for OqxAB efflux pump by firstly amplification of oqxA gene of
membrane fusion protein with conditions: 94 ?C for 45s, 57 ?C for 45s, and 68
?C for 45s for 34 cycles to obtain 392bp products. To amplify oqxB gene
of inner-membrane pump, PCR conditions were: 94 ?C for 45s, 64 ?C for 45s and
72 ?C for 60s for 32 cycles to obtain 512bp products (Kim et al, 2009).

All
pcr products were electrophoresed in 1% agarose gel containing ethidium bromide
0.5 mg/L. Amplified fragments were purified and 
Nucleotide sequences were determined on both strands of PCR
amplification products at the Macrogen sequencing facility (Macrogen Inc.,
Seoul, Korea) Analysis and comparisons of nucleotide sequence were carried out
using programs available at the NCBI web interface (http://www.ncbi.nlm.nih.gov).

 

3.      Results

A total of 280 urine samples from UTI patients
were screened for E.coli. Out of which 155 were positive for it, of which 51
were nalidixic acid resistant isolates were recovered.

 

3.        
Results

3.1.        
Antibiotic sensitivity test results

Fifty one of all of the UPEC was nalidixic acid resistant.
Sensitivity pattern of these isolates is shown in table (2) and distribution of
MIC values of ciprofloxacin is shown in table (3).

 

3.2.        
Phenotypic detection of ESBLs

The disk diffusion test showed synergy between
Amoxicillin/Clavulanic and at least one of the following antibiotics:
Cefotaxime, Ceftazidime, Aztreonam and ceftriaxone in all isolates suggesting
the production of ESBLs.

 

3.3.        
PCR and sequencing of the quinolone resistance-determining region
(QRDR) of thegyrA andparC genes

Sequencing
of QRDR of the gyrA and parC genes revealed mutations at the
codon 83 (ser?Leu) and 87 (Asp?Asn) in gyrA
gene and at the codon 80 (Ser?Ile) and codon  
84 (Glu?Val) in parC gene. At
least one amino acid substitution in the
gyrA at the codon 83 (Ser83Leu) was detected in E. coli isolates (table 4).

 

3.4.        
PCR and sequencing for detection of PMQR genes

Among
the 51-quinolone resistant isolates, 48(94%) were positive for at least one
PMQR gene. QnrS gene was present in 12 (23.5 %) isolate. The sequencing of the
qnrS gene was identical to that of the qnrS.the qnrA or qnrB genes were not
detected in any of the isolates. Forty-five of the 51 isolates (88.2%) were
positive to aac (6)-Ib. the sequencing of the aac (6)-Ib was identical to the
–cr variant. qepA gene was detected in twenty-four of the 51 isolates (47.1%).
The oqxAB gene was detected in three isolates of  the 51 isolates (5.9 %).This is the first
report for detecting oqxAB gene in quinolone resistant urinary isolates in
EGYPT. the sequencing of the oqxAB gene was identical to that of the oqxAB1
(figure 1).

 

4.     
Discussion

Resistance
in E. coli to one of the most widely used medicines for the treatment of
urinary tract infections (fluoroquinolone antibiotics) is very widespread.
There are countries in many parts of the world where this treatment is now
ineffective in more than half of patients (WHO, 2017).

In
the present study 55.4% of all uropathogens was E.coli .this result agrees with
that reported in previous studies (Niranjan and Malini, 2014, Daoud and Afif,
2011 Nicolle, 2008).The sensitivity pattern of the E. coli isolates in our
study was similar to previous studies (El-Sokkary and Abdelmegeed, 2015, Dash
et al, 2012) All the isolates were sensitive to 
imipenem  , this result is
consistent with that reported by (Mendonça et al, 2007).imipenem  appear to have wider range of activity than
other tested antimicrobial agents. The explanation for that is probably the
fact that these are very powerful drugs used only in hospital settings and not
as first-line so they are of limited use.

Ciprofloxacin
resistance observed in this study was higher than that reported in previous
studies from Egypt (El-Kholy et al, 2003). This increase in Ciprofloxacin
resistance rate may be due to the prolonged use of low dose of the more potent
fluoroquinolones such as ciprofloxacin. Also the inappropriate use of
fluoroquinolones   may lead to the
acquisition of resistance. In our country Wide-spectrum antibiotics and
quinolones are often prescribed in primary health care and there is an affinity
for using antibiotics for most symptoms, also antibiotics could be easily
obtained from pharmacies easily without the approval of a physician.

In
this study All quinolone resistant 
isolates  collected were ESBL
producers, this finding  is consistent
with that reported by (Kim et al, 2008) and also a study conducted in Europe
reported quinolone resistance in 92% of ESBL-producing isolates(Kilinc et al,
2015). The high prevalence of uropathogenic quinolone-resistant ESBL-producing
E. coli strains has become a major health problem, making treatment of
bacterial infections  more challenging
and resulting in higher morbidity and mortality rates .

 

In
this study all isolates collected were multi drug resistant (MDR). (MDR)
bacterial infections are now becoming quite common, not only in hospital
settings but also in the community (Levy et al, 2004).due to the limits on
selection of the isolates to be nalidixic acid resistant, this may suggest
previous treatment of the patient by antibiotics and also may suggest the
recurrence of UTIs that leaded to the evolution of this high percentage of MDR
isolates. This High prevalence of MDR isolates of E. coli has been observed in
many developing countries (Ibrahim et al, 2012, Malhotra et al, 2016).This high
prevalence of MDR and ESBL production among these uropathogenic E.coli isolates
may be attributed to many reasons. The most abundant reason is plasmids which
carry the genes encoding ESBL, and also carry the genes encoding multi
resistance to several antibiotics, including quinolones, further contributing
to the spreading of resistance (Kilinc et al, 2015).

 

Resistance
to fluoroquinolones occurs mainly as a result of mutations in bacterial gyrA
and parC genes that code for DNA gyrase and topoisomerase IV, respectively (Ito
et al, 2008)

 

Sequencing
of QRDR of the gyrA and parC genes revealed mutations at the codon 83 (ser?Leu)
and 87 (Asp?Asn) in gyrA gene and at the codon 80 (Ser?Ile) and 84 (Glu?Val) in
parC gene. at least one amino acid substitution in the gyrA at the codon 83
(Ser83Leu was detected in  E. coli
isolates. A single amino acid substitution in the gyrA protein at the codon 83
(Ser83Leu) leads to high levels of resistance to nalidixic acid (MIC ranging
from 32mg/Lto256mg/L) and a reduced susceptibility to fluoroquinolones (MIC of
ciprofloxacin ?4mg/L).An additional mutation in theAsp87codon of gyrA is
associated with high increase in fluoroquinolone resistance (MIC of
ciprofloxacin ?32mg/L).

parC
gene is the secondary target for quinolone resistance, Point mutations at
codon80 in parC QRDRs (Ser80 Ile) and at the position 84 (Glu84 Val) were found
. Mutations in parC were always found together with mutations in gyrA. This is
due to topoisomerase IV being a secondary target for quinolones in E. coli
However mutations in parC play an important role in the formation of highly
resistant strains (Hopkins et al, 2005),

Differences
in MICs values for fluoroquinolones were observed (MICs of ciprofloxacin
ranging from 4mg/L to 32mg/L), Among E. coli isolates with the same types of
mutation (Ser83Leu/Asp87Asn in gyrA and Ser80Ile/Glu84Val in parC). This may be
attributed to other mechanisms involved in fluoroquinolone resistance such as
decreased membrane permeability and over expression of efflux pumps (Kim et al,
2012).

PMQR
genes such as the Qnr family genes (qnrA, qnrB, qnrC, qnrD, and qnrS), aac
(6?)-Ib-cr, qepA, and oqxAB, are widely disseminated among Enterobacteriaceae
(Cao et al, 2015)

 

Qnr
determinants are believed to bind to and protect DNA gyrase and/or
topoisomerase IV from fluoroquinolone inhibition (Martinez-Martinez et al,
1998)

qnr
gene was first detected in Egypt in 
Providencia spp (Wiegand et al, 2004). In the current study qnrs1 was
the only detected qnr gene, it was detected in 23.5% of the isolates.This
result agrees with previous studies from Egypt that described that qnrs as the
most prevalent qnr gene in Egypt (El-Mahdy, 2016, El-Essawy et al, 2015).

The
aac(6′)-Ib-cr gene which is a variant of the gene AAC(6′)-Ib, was first
described in 2006 .this  enzyme reduces
only ciprofloxacin and norfloxacin activity by acetylation . Quinolones without
piperazinyl nitrogen are not affected by aac(6′)-Ib-cr(Robicsek et al, 2006)
Screening for aac(6′)-Ib-cr shows high prevalence in E. coli, 88.2% of the
isolates were positive for it .This result agrees with that reported by Yang et
al.(77.9%) (Yang et al, 2014).

aac(6?)Ib-cr
was the most  prevalent PMQR determinant
found in these isolates. This result was expected because all of the isolates
were ESBL producers, and many studies have shown its predominance among other
populations of E coli, especially among ESBL producers (Baudry-Simmer et al,
2012).It was found that all qnr positive isolates were also aac(6′)-Ib-cr
positive. This could be explained by the co-existence of the qnr and
aac(6`)-Ib-cr genes on the same plasmid (Jiang et al. 2008)and may suggest a
genetic association of quinolone resistance with aminoglycoside resistance.

Recently
qepA and oqxAB are multidrug efflux pumps were discovered and reported as the
third PMQR mechanism involved in quinolone resistance (Kim et al, 2009 Yamane
et al, 2008)).

In
2007, qepA was first reported in clinical E coli isolates from Japan (Yamane et
al, 2008)). In the present study, the prevalence of qepA among the isolates
(47.1%) was higher than that in previous study from Egypt(Hassan et al,
2012),This high prevalence may be attributed to the composition of the E. coli
collection. most of the isolates were 
ciprofloxacin resistant with high MIC.

OqxAB
efflux pump is encoded by the oqxA and oqxB genes, which are located in the
same operon. Plasmid-mediated OqxAB was first detected in a human clinical
isolate of E coli from Korea (Kim et al, 2009)

In
this study, we screened for oqxAB in clinical E.coli isolates for the first
time in Egypt and it was detected in (5.9) % in E. coli. This result agrees
with that reported by (Yuan et al, 2012).

 

This
study suggested that the rate of fluoroquinolne resistance is increasing in
Egypt. This data supports our understanding of the molecular mechanisms of FQ
resistance due to the accumulation of mutations in the QRDR of gyrA and parC
genes in and PMQR determinants due to the inappropriate and increased use of
wide-spectrum antibiotics and quinolones, it is essential to restrict the using
of these antibiotics to be prescribed only when necessary and only when
approved by a physician.

People,
not only patients should be aware that antibiotics not for all symptoms and
should be used only under medical supervision.

 

The
potential spread of PMQR determinants in Egypt because of uncontrolled oral
quinolone usage is troublesome, Furthermore plasmids usually harbor other
resistance genes causing multiple resistance among isolates which can
complicate therapeutic management of infections and also can transfer to other
bacterial species.

This
is the first report of the detection of oqxAB inE.coli isolate in Egypt.

 

5.     
Conclusion