Department of Pathology and Microbiology, Faculty of Medicine,
University of Jordan, Amman, Jordan.
مقاومة مضادات المكروبات ومُرْتَسَمات البلاسميدات
الإشريكيات القولونية من المرضى الأردنيِّين
عاصم الشهابي، عزمي محافظة، خالد الخليلي
قام الدارسون باستقصاء أنماط المقاومة لمضادات المكروبات
ومُرْتَسَمات البلاسميدات في مستفردات الإشريكيات القولونية من
المرضى الأردنيِّين الداخليِّين والخارجيِّين فـي المستشفـى
الجامعـي الأردنـي عامَـيْ 2000 و2001. وقد كانت الإشريكية
القولونية تمثِّل 32.4% إلى 37.4% من المستفردات جميعها. وقد كان
أقل التأثـُّر بالأمبيسلين (11%)، ثم بالكوتريموكسازول (23%) ثم
بالتتراسيكليـن (26%) ثم بالأوغمنتين (33%). وقد وجد أن معدل
الوقوعات النسبية للمستفردات من الإشريكيات القولونية المقاوِمة
للأوغمنتين وحمض الناليديكسيك والجنتاميسين والنورفلوكساسين
والسيفوروكسيم والنيترفورانين، أكثر بكثير لدى المرضى الداخليِّين
منه لدى المرضى الخارجيِّين (مَنْسَب الدقَّة 0.05 >
وقد لوحظ وجود بلاسميد المقاومة الكبير المؤلَّف من 26 كيلو قاعدة
في معظم (67% من) مستفردات الإشريكيات القولونية، المقاوِِمة
للأمبيسيلين والكوتريموكسازول والتتراسيكلين على الأقل. ويدلُّ ذلك
على أن مستودع بلاسميد المقاومة هذا قد يساهم في انتشار المقاومة
المتعدِّدة للمضادات الحيوية في إقليمنا.
Abstract: We investigated antimicrobial resistance
patterns and plasmid profiles of uropathogenic Escherichia coli
isolates from inpatients and outpatients at Jordan University
Hospital in 2000 and 2001. E. coli accounted for 32.4% and 37.4%
of all isolates respectively. The lowest susceptibility was for
ampicillin (11%), cotrimoxazole (23%) and tetracycline (26%).
The relative incidence of resistant isolates of E. coli to
nalidixic acid, gentamicin, norfloxacin, cefuroxime and
nitrofurantoin was significantly greater for inpatients than for
outpatients (P < 0.05). A large, transferable R-plasmid of 28 kb
was found in most E. coli isolates (67%) that were resistant to
at least ampicillin, cotrimoxazole and tetracycline. This
R-plasmid reservoir may contribute to the spread of multiple
antibiotic resistance in our Region.
Résistance aux antimicrobiens et profils plasmidiques des
isolats urinaires d’Escherichia coli de patients jordaniens
RESUME Nous avons étudié la résistance aux antimicrobiens et les
profils plasmidiques des isolats d’Escherichia coli
uropathogénique chez des malades hospitalisés et des malades
externes à l’Hôpital universitaire jordanien en 2000 et 2001. E.
coli représentait 32,4 % et 37,4 % de tous les isolats. La
sensibilité la plus faible concernait l’ampicilline (11 %), le
cotrimoxazole (23 %) et la tétracycline (26 %). L’incidence
relative des isolats d’E. coli résistants à l’acide nalidixique,
à la gentamicine, à la norfloxacine, à la céfuroxime et à la
nitrofurantoïne était significativement plus élevée chez les
malades hospitalisés que chez les malades externes (p < 0,05).
On a trouvé un grand plasmide R, de 28 kb, dans la plupart des
isolats d’E. coli (67 %) qui étaient résistants à l’ampicilline,
au cotrimoxazole et à la tétracycline tout au moins. Ce
réservoir de plasmide R peut contribuer à la propagation de
l’antibiorésistance multiple dans notre Région.
Urinary tract infections occur in all age groups and are caused
by a wide variety of pathogens with antimicrobial susceptibility
patterns that have changed over time [1–6]. Increased age of
patients and presence of underlying disease or immunosuppressed
conditions render hospitalized patients susceptible to urinary
tract infections with multiresistant bacterial strains [7–9].
Worldwide, Escherichia coli is still the most common causative
microorganism of urinary tract infection, accounting for 30%–70%
of all uropathogens [1–9]. Many recent studies from Europe and
North America and a few from Arab countries have documented the
development of resistance to fluoroquinolones among
uropathogenic E. coli isolates [1–14].
The aim of our study was to investigate prospectively
antimicrobial resistance patterns and plasmid profiles of
uropathogenic E. coli isolates simultaneously from community and
hospitalized patients at a major teaching hospital in Jordan.
In the first phase of our study, we collected the culture
results of all midstream urine specimens submitted to the
clinical bacteriology laboratory at the Jordan University
Hospital in Amman for culture and susceptibility testing from
January 2000 to December 2001. Specimens were cultured on
MacConkey and blood agar (Oxoid, Basingstoke, UK) plates and
incubated for 24 hours at 37 °C. Bacterial species were
identified using standard biochemical tests . Urine
cultures, mostly midstream, were recorded as positive if their
bacterial growth showed the presence of > 105 colony forming
units/mL unless clinical data indicated urinary tract infection,
in which case urine cultures with lower counts (102–104 colony
forming units/mL) were also included. Positive cultures were
submitted for antibiotic susceptibility using the disk diffusion
method and results were expressed as susceptible or resistant
according to the National Committee for Clinical Standards
guidelines of 1997 .
The second phase of our study included 53 of 160 representative
multidrug- resistant E. coli single isolates that were randomly
obtained from positive urine cultures of inpatients (23
isolates) and outpatients (22 isolates) of all age groups.
Isolates were multidrug resistant if they displayed resistance
to 3 or more different antibiotic classes. Eight to 10 colonies
of each pure culture of E. coli were taken and inoculated in 1
mL of brain-heart infusion broth (Oxoid, Basingstoke, UK)
containing 40% glycerol and then stored at –20 °C for further
We included 53 multidrug-resistant E. coli isolates that were
susceptible to nalidixic acid in conjugation experiments using 2
methods [17,18]; the recipient E. coli strain (K-12) was
resistant to nalidixic acid only. All isolates and control
strains were tested for antibiotic susceptibility prior to use.
Minimum inhibitory concentrations (MICs) were determined by the
agar dilution method . Serial 2-fold dilutions of
antibiotics were made in Mueller–Hinton agar (Difco, USA). E.
coli ATCC 25922 (Difco, USA) was the standard control strain
throughout the study.
We used 2 methods for the extraction of plasmid DNA: the
alkaline lysis method of Birnboim and Doly  and the DNA
purification system, A7500 (Promega, Madison, Wisconsin, USA).
Plasmid DNA and lambda DNA marker were restricted by Hind III
restriction enzyme. Gel electrophoresis using 0.7% agarose was
run for 2 hours at 95 volts in horizontal electrophoresis
All data were entered in a computer database and analysed with
SPSS software to compare variables, using 2-tailed paired
samples and Student t-test with a significant cut-off value of
Table 1 shows the distribution of E. coli isolates from urine
cultures examined in the clinical bacteriology laboratory of
Jordan University Hospital. A total of 3583 of 12 021 (29.8%)
(2000) and 3140 of 11 328 (27.7%) (2001) urine cultures were
positive for significant bacterial growth. E. coli isolates were
32.4% (2000) and 37.4% (2001) of the total microorganisms
isolated. The ratio of E. coli isolates for outpatients
(community-acquired) to inpatients was approximately 3:1 in both
years (Table 1). The ratio of male patients to female patients
Table 2 shows the antimicrobial susceptibility patterns of 2335
E. coli isolates from inpatients and outpatients as well as the
overall susceptibility rates. The rates of resistance to
amoxicillin/clavulanate nalidixic acid, gentamicin, norfloxacin,
cefuroxime and nitrofurantoin in isolates from inpatients were
significantly greater (P < 0.05) than for isolates from
Table 3 shows the 12 resistance phenotypes of 53 randomly
selected multiresistant E. coli isolates. Resistance phenotype
of 4-drugs (AGmTTs) accounted for almost half these isolates
(26/53, 49%). Other resistance phenotypes were less common and
ranged between 2% and 9%. The MIC90 of 20 multiresistant E. coli
isolates (resistant to 4 drugs or more) were: ampicillin > 128
mg/L, cotrimoxazole > 128 mg/L, tetracycline > 32 mg/L,
gentamicin > 16 mg/L and nalidixic acid > 8 mg/L (Table 2).
Table 3 also shows transferable resistance and plasmid profiles
of 16 multiresistant E. coli strains, and 15 E. coli
transconjugants strains. The results showed 13 different plasmid
profiles associated with 12 resistance phenotypes and plasmid
sizes ranged from 2.0 kb (kilobases) to 59.4 kb. A common large
plasmid (28.0 kb) with similar digest patterns to Hind III
restriction enzyme was in 10 of 15 E. coli donor strains (67%)
with mostly similar antimicrobial resistance patterns. Also, 11
of 15 E. coli strains (73%) contained more than 1 plasmid, but
no isolate contained more than 6 plasmids. R- plasmids were
detected in E. coli isolates from both hospitalized patients (7)
and non-hospitalized patients (6) with nearly similar rates (47%
and 53% respectively).
The prevalence of uropathogens and their susceptibility to
certain antimicrobial drugs has been reported in a few studies
in Jordan over the last 2 decades [20–24]. These studies, which
covered major regions of the country over a period of 21 years
(1978–1999), showed that 42%–82% of all positive urine cultures
were E. coli and that community-acquired urinary tract
infections were mostly caused by E. coli (75%–82%). These
studies along with our 2-year study (2000–2001) demonstrated
that uropathogenic E. coli isolates were highly resistant to
ampicillin (82%–95%), tetracycline (83%–86%) and cotrimoxazole
(48%–77%). In addition, moderate to high rates of resistance to
other commonly prescribed antibiotics in our country have
recently developed. Examples include cefuroxime and norfloxacin
in uropathogenic E. coli isolates, as demonstrated in our study.
A comparison of antibiotic-resistance patterns of uropathogenic
E. coli isolates in 1978 and 2000–2001 in the same hospital
(Jordan University Hospital, Amman) revealed that gentamicin
resistance increased significantly (P < 0.05), from 13% to 48%,
nalidixic acid resistance increased from 16% to 47%, and
resistance rates to cotrimoxazole, ampicillin and tetracycline
were very high (48%–83%) in 1978 and remained high throughout
the 23-year period. Nitrofurantoin resistance increased
slightly, from 14% to 24% . In our study, however, a
significantly higher rate of resistance to amoxicillin/clavulanate,
nalidixic acid, gentamicin, norfloxacin, cefuroxime and
nitrofurantoin was detected in urinary E. coli strains from
hospitalized patients than strains from outpatients (P < 0.05).
In a 1997 study at our hospital, there was a slight increase in
the incidence of E. coli resistance to extended-spectrum b-lactam
drugs over a period of 3 years, but there was a significant
increase in the resistance of Klebsiella pneumoniae to
ceftazidime and aztreonam among patients in intensive care units
Recent studies published in Europe and North America during the
past 3 years have demonstrated an increasing antibiotic
resistance among uropathogenic E. coli isolated from either
community or hospitalized patients. In particular, increased
resistance up to 37% to various penicillins, cephalosporins and
cotrimoxazole have been reported in uropathogenic E. coli in
certain countries [2,4–6,12,14]. Although most of these studies
have observed low rates of fluoroquinolone resistance in
uropathogenic E. coli (2%–12%), 2 studies from Spain have
recently reported that 22%–27% of uropathogenic E. coli strains
were resistant to ciprofloxacin [4,11]. In our study plasmid
analysis of representative E. coli isolates showed the presence
of a wide range of plasmid sizes, and there were no consistent
relationships between plasmid profiles and resistance
phenotypes. Partial resistance was mostly transferred in
association with a common large plasmid of molecular size 28 kb.
The frequency of conjugative R-plasmids was high in association
with resistance to 3–7 drugs in E. coli strains. These
R-plasmids were found in E. coli isolates from both hospitalized
patients and non-hospitalized patients at similar rates (47% and
In conclusion, the results of our study indicate a high-level of
antimicrobial resistance to drugs commonly prescribed for
urinary tract infections (including norfloxacin) in
uropathogenic E. coli isolates in Jordan, particularly in
hospitalized patients. Additionally, the common presence of
transferable R-plasmids in these E. coli isolates is a potential
reservoir for the spread of multiple antibiotic resistance in
We wish to thank our colleagues at the bacteriology laboratory,
Jordan University Hospital in Amman for their support in
collecting the urine culture results and the E. coli isolates.
This work was supported by a grant from the Deanship of
Postgraduate Studies, University of Jordan.
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