Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
Survey of Antibiotic susceptibility of Escherichia
coli Isolated from Patient with Significant
Bacteriurea
Jenan Mohammed Hussein
Ali Almohana
Department of Biology
Department of Microbiology
College of Education for Girls
College of Medicin
Eman Jar-Allah
Kufa University, Department of biology
sciences college / Babylon University
Abstract
Multiple resistances to antimicrobial drugs arising in Escherichia coli isolates may complicate therapeutic
management of urinary tract infection (UTI) by this organism. In order to assess the multidrug resistance
(MDR) among urinary E. coli isolates, we have tested 27 antimicrobial drugs against 192 isolates from
patients with significant bacteriurea from 3 hospitals in Najaf during june to Augest 2011. The number of
patients with urinary tracts infection was higher in females, 136 (70.9%) compared to males, 56 (29.1%).
The antibiotic susceptibility patterns of isolates were determined by disk diffusion method. Among the
isolates from the hospital patients, 95.3%, 94.3%, 93,8%, 92.2%, 91.7%, and 90.1% were resistant to
carpencillin, ticarcillin, ampicillin, cefepim, cefamandol, and pipracillin, respectively; Resistant profile for
cefoxitin, and amoxicillin was 85.4% and 80.2%, respectively. Uoropathogenic E. coli was resistant to
cefotaxime, cefetriaxon, and Aztreonam (71.4%), and resistance to trimetheoprim, doxicycllin, ceftazidim,
amoxiclave, and nalidixic acid were 69.8%, 68.2%, 68.8%, 67.2%, and 61%, respectively. susceptibility
prfile to aminoglycosid antibiotics (tobramycin, gentamycin, and amikacin) were 56.8%, 53.1%, and
10.4%, respectively. And for fluoroquinolons (ciprofloxacin, norfloxacin, ofloxacin, and levofloxacin) were
49%, but for gatifloxacin, resistant was 40.6%. E. coli was resistance to chloramphenicol and nitroforantoin
17.7% and 12%, respectively.The high prevalence of drug resistance among UTI patients calls for
continuous surveillance to assure effective control of this infection.
1. Introduction
Urinary tract infections (UTIs) having as etiologic agent Escherichia coli are
common infections with an estimated annual global incidence of at least 250 million
cases, being costly to both patients and health care funding system (Ronald et al., 2001).
Non-complicated infections, particularly in women, account for the highest number of
UTIs. Women diagnosed for acute uncomplicated cystitis are usually treated as
outpatients: the microbiologic characteristics of this infection are highly predictable even
in otherwise healthy subjects. Physicians have therefore been advised that empirical
antimicrobial treatment not requiring culture is appropriate in such cases (Hooton et al.,
1997) Therefore, the empirical therapy has been so widely used that a few UTIs were
routinely cultured. However studies clearly demonstrated an increasing antibiotic
resistance in E. coli causing both community- and
nosocomially acquired UTIs (Gupta et al.,1999 ; Gales et al., 2000).
Updated knowledge of causal bacteria and their susceptibility patterns are
important for proper selection and use of antibiotics as well as for an appropriate
prescribing policy. The aim of the present study was to define current occurrence and
phenotypes of multidrug-resistant (MDR) E. coli among UTI isolates from Najaf
hospitals.
267
2 Material and Methods
2.1 Bacterial Isolates
Escherichia coli isolates were identified depending on the traditional
morphological and biochemical tests according to the methods of Cowan (1985), Holt et
al. (1994), and MacFaddin (2000). (Table 2-1), and Media used in this study listed in
Table (2-2) were prepared in accordance with the manufacturer's instructions fixed on
their containers. All the media were sterilized in the autoclave at 121°C for 15 min
Table (2-1): Phenotypic characterization of 117 E. coli isolated from urine samples
of patients with significant bacteriuria
% of positive
results
100%
100%
0
0
100%
100%
0
0
3%
95%
100%
98%
36%
99%
100%
Test
Gram-negative bacilli
Catalase
Oxidase
H2S
Indole
Methyl red
Vogas-Proskaur
Citrate utilization
Urease
Motility
Acid from Glucose
D-mannose
Sucrose
D-xylose
Lactose
Triple sugar iron agar
acid/acid
100%
Table (2-2): Culture Media
Medium
Manufacturer (Origin)
Brain heart infusion broth and agar
CHROMagar orintation
Mast Diagnostic (UK)
ChroMagar (France)
Eosin methylen blue agar
Himedia
MacConkey agar
Biolife (Italy)
Muller Hinton agar
Oxoid (UK)
MR-VP broth
Oxoid
Peptone water
Biolife
Simmons citrate agar
Mast Diagnostic
Triple sugar iron agar
Biolife
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Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
Table (2-3): Antibiotic disk
Antibiotic
name
symbol
Content
Origin
Ampicillin
Am
10 µg
Himedia
(India)
Aminopenicillin
Amoxicillin
Ax
25 µg
Himedia
Carboxypenicillin
Carbenicillin
PY
100 µg
Himedia
Piperacillin
PRL
100 µg
Bioanalyse
(Turkey)
Ticarcillin
TIC
75 µg
Bioanalyse
Amoxi-clavc
Ac
Cefotaxime
CTX
30µg
Bioanalyse
Ceftazidime
CAZ
30µg
Bioanalyse
Ceftriaxone
CRO
30 µg
Bioanalyse
Cephalosporin IV
Cefepime
FEP
30µg
Bioanalyse
Cephamycin
Cefoxitin
FOX
30µg
Bioanalyse
Cephalosporin I
Cefamandole
MA
30µg
Bioanalyse
Aztreonam
AZT
30µg
Bioanalyse
Imipenem
IPM
10µg
Bioanalyse
Amikacin
AK
30µg
Himedia
Tobramycin
TOB
10µg
Himedia
Gentamicin
CN
10µg
Bioanalyse
Nalidixic acid
NA
30µg
Himedia
Ciprofloxacin
CIP
5µg
Himedia
Norfloxacin
NX
10µg
Himedia
Ofloxacin
OFX
5µg
Himedia
Levofloxacin
Lev
5µg
Himedia
Gatifloxacin
GT
5µg
Himedia
Folate pathway
inhibitors
Trimethoprim
TMP
5µg
Bioanalyse
Tetracyclines
Doxycyclin
DO
30µg
Bioanalyse
Nitrofurans
Nitrofurantoin
F
300µg
Bioanalyse
Phenicols
Chloramphenicol
C
30µg
Bioanalyse
Antibiotic class
Penicillin
Antibiotic subclass
Ureidopencillin
-lactame/ß-lactamaseß
inhibitor combinations
Cephalosporin III
Cephems (parenteral)
Cephems (parenteral)
Monobactams
Penems
Carbapeneme
Aminoglycosides
Quinolone
30µg
Himedia
Fluoroquinolone
269
2. 2 Susceptibility Testing
Antimicrobial susceptibility was determined by the Kirby-Bauer disk diffusion
method following the definition of the National Committee of Clinical Laboratory
Standards (NCCLS) for agar diffusion tests (CLS, 2010) using antibiotic containing
disks. Antibiotic disks listed in Table(2-3). Quality control was performed using E. coli
ATCC 25922. An isolate was considered multidrugresistant (MDR) if it was resistant to
at least three of the antimicrobial agents tested.
3 Results and Discusions
3.1 Survey and Etiological Agent
According to demographic data, it was observed that the number of patients with
urinary tracts infection was higher in females, 136 (70.9%) compared to males, 56
(29.1%) and statistical analysis showed high significance at (P ≤ 0.01) in female patients
(Figure 3-1). The finding that females had higher prevalence of urinary tracts infection
than males agrees with earlier studies (Anochie et al., 2001; Hadi, 2008; Mordi et al.,
2010). The reason may be due to the higher number of the females than the males in the
present study populations, or may be because those males are less potent UTI, possibly
their longer urethra and the presence of antimicrobial substances in prostatic fluid
(Orhiosefe et al., 2009). The high frequency of UTI in females, which might be due to
diversity of factors related with females, such as the close proximity of the female
urethral meatus to the anus and they lack the bacteriostatic properties of prostatic
secretions (Lipsky, 1989; Akinloye et al., 2006). Alterations in vaginal microflora that
play a critical role in encouraging vaginal colonization with coliforms that may lead to
UTI (Hooton et al., 1997; Aiyegoro et al., 2007). In Nigeria, UTI is prevalent among men
and women, but is more common among women, especially during pregnancy (Johnson
et al., 1995). In agreement with present study, Todar (2002) reported that the UTIs are
fourteen times more common in females than males by virtue of the abbreviated urethra.
However, Ryan and Ray (2004) statement that UTI is more widespread in females, 40.0%
of women have an episode in their lifetime. Sexually active females are also more
predisposed to UTI than their male counterparts (Reid et al., 1987; Younis et al., 2009).
In young sexually active women, sex is the cause of 75-90% of bladder infections, with
the risk of infection related to the frequency of sex (Nicolle, 2008). The term
"honeymoon cystitis" has been applied to this phenomenon of frequent UTI during early
marriage.
Male
Female
70.9
29.1
Figure (3-1): The percentage incidence rate of urinary tract infection according to
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Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
sex.
Nevertheless, in male UTI most commonly occur in older men with prostatic
disease, outlet obstruction, or urinary tract instrumentation. In later life, UTI is more
common among men until the age of prostatic hypertrophy (above 40 years of age)
(Schroeder et al., 1990). An enlarged prostate means the gland has grown bigger. As the
gland grows, it can press on the urethra causing obstructs the urethra, leading to increased
difficulty in micturition. Because there is less urin flushing the urethra, there is a higher
incidence of E. coli colonization (Nicolle, 2008). However, the infection occurs in all
persons regardless of sex or age, with particular impact on the young and the very elderly
(Rubin et al., 1986).
3.2 Survey of Susceptibility to Antibiotics
Problems associated with the development and spread of antibiotic resistance in
clinical practice have been increasing since the early 1960s and are currently viewed as a
major threat to the public health on a global level (Ashraf et al., 2007; Levy et al.,
2004). The prevalence of resistance among pathogenic bacteria also varies widely, among
bacterial species and geographical regions, but it is raising enough to be considered a
health threat (Amábile et al., 2009).
In this investigation, all the 192 isolates of E. coli were tested for their antibiotic
susceptibility to 27 antibiotics using Kirby-Bauer disk diffusion method on MullerHinton agar (Bauer et al, 1966), and were interpreted according to Clinical and
Laboratory Standard Institute guidelines (CLSI, 2010). The antimicrobial potency of
selected antibiotics against the E. coli is summarized in Figure (3-2).
E. coli isolates from urine samples were frequently resistant to aminopenicillins
including drugs commonly prescribed in the outpatients setting, including ampicillin
(93.8%) and amoxicillin (80.2%) Several reports have indicated that these drugs were
also less effective against E. coli and other bacterial agents isolated in previous studies in
Najaf (Almohana, 2004; Hadi, 2008; Al-Hilali, 2010), largely because they are
inexpensive and can be obtained easily without a medical prescription. Resistance is
probably due to indiscriminate antibiotics usage (drug abuse) which could result in
plasmid-mediated antibiotic resistance found to be common in E. coli (Taneja et al.,
2008). In this investigation, the resistant effect of isolates to carboxypenicillins including
carbenicillin and ticarcillin are comparable, the rates of resistance were 95.3% and
94.3%, respectively.
The present results revealed high rate of resistant in E. coli isolates to
carboxypenicillins, this rate is consistent with those reported by Al-Hili (2010) who
found that 95% of E. coli isolates obtained from Merjan teaching hospital in Hilla to be
resistant to carbenicillin. In recent study in Najaf, 90.9% and 86.4% of enteropathogenic
E. coli isolates were found resistant to carbenicillin and ticarcillin, respectively (AlHilali, 2010). This high rate of resistant is probably due to the extensive use of β-lactam
antibiotics in Najaf hospitals and the current empirical antibiotic therapy.
Present study observed a high rate of E. coli isolates resistant to piperacillin
(90.1%) E. coli is typically resistant to extended-spectrum penicillins, such as ampicillin
and piperacillin, due to production of β-lactamase(s). The first plasmid-mediated βlactamase isolated from Gram-negative bacteria was TEM-1, described in E. coli, and
mediates resistance to ampicillin, piperacillin, ticarcillin and first generation
cephalosporins.
271
Unfortunately, resistance to amoxycillin clavulanic acid appeared first in E. coli isolates,
then in other species of enterobacteria (Speldooren et al., 1998). This study permitted an
evaluation of the frequencies of amoxyclave resistance in urinary isolates of E. coli in
Najaf hospitals. The present result found that the majority of isolates (67.2%) were
resistant to amoxyclave. This is often due to the production of excessive amounts of
TEM-1 β-lactamase that swamp the activity of clavulanic acid (Bradford, 2001).
However, carbapenemase and AmpC β-lactamases producing E. coli are not inhibited by
the presence of clavulanic acid (Mirela and Ariana, 2011; Peter et al., 2011).
Antibiotic susceptibility testing of isolates revealed high rate of resistance
(91.7%) to second-generation cephalosporin including cefamandole. In the mid 1970s,
the cefamandole (β-lactamase-stable cephalosporin compound) was marketed
(Moellering et al., 1978). This antibiotic is a potent inducer, when E. coli isolates are
exposed to two β-lactam antibiotics when of which is cefamandole, antagonism between
the two antibiotics may result (Rao, 2006).
In regards to third generation cephalosporins, results from Figure (3-2) revealed that
E. coli isolates were highly resistant to cefotaxime (71.4%), ceftriaxone (71.4%), and
ceftazidime (68.8%). The resistance to third generation cephalosporins was caused
mainly by mutations in the common group of class A β-lactamases, which consisting of
TEM, SHV and CTX-M β-lactamases that has extended hydrolytic spectrum activity on
cephalosprins, The excessive use of expanded-spectrum cephalosporins in clinical
practice is the main factor responsible for the appearance of ESBLs in the enteric bacteria
and several studies have found a relationship between third generation cephalosporin use
and acquisition of ESBL producing strains (Livermore et al., 1996; Kjerulf et al., 2008).
Therefore, clinicians should be familiar with the clinical importance of these enzymes
and potential strategies for dealing with them.
In this study, the vast majority of E. coli isolates recovered from patients with
significant bacteriuria was resistant to cefepime; the resistance rate was 92.2%. The
present result was much higher than result recently conducted by Al-Muhannak (2010)
who revealed that 63.5% of E. coli clinical isolates were resistant to cefepime. In Turkey,
Kiremitci et al. (2009) found that high resistance rates for both E. coli and K. pneumoniae
against cefepime (81.7%). However, an Australian study confirmed that more than 40%
of Enterobacter spp. isolates from humans were resistant to cefepime. Therefore, present
study concluded that treatment using fourth generation cephalosporins is also not
recommended before the susceptibility testing is known.
Aztreonam is used primarily as an alternative to aminoglycosides and for the
treatment of aerobic Gram-negative infections. Approved indications for its use include
infections of the urinary tract or lower respiratory tract, intra-abdominal and gynecologic
infections, septicemia, and cutaneous infections caused by susceptible organisms (Scully
and Neu, 1985). Beside, the present study revealed that 137 (71.4%) of 192
uropathogenic E. coli isolates recovered from patients with significant bacteriuria were
resistant to aztreonam. The high rates of resistance might be as marker for the production
of ESBLs by these isolates. Generally, an isolate is suspected to be an ESBL producer,
when it shows in vitro resistance to the third-generation cephalosporins and to aztreonam
(Samaha-Kfoury and Araj, 2003). However, clinical failure of cephalosporin and
aztreonam therapy due to ESBLs is a growing problem in Najaf hospitals (Hadi, 2008;
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Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
Al-Muhannak, 2010; AL-Sehlawi, 2012) as they go undetected by current isolation and
susceptibility tests.
The major causes of cefoxitin resistant in E. coli are encoding of plasmid
mediated AmpC β-lactamase or other factors might cause the reduce susceptibility of E.
coli to cefoxitin (Tan et al., 2009). Present results revealed, the cefoxitin resistant E. coli
isolates are disseminated in Najaf hospitals. This may due to the presence of contributed
factors like; over expression of the chromosomeal ampC genes, acquisition of plasmidic
ampC genes, porin or permeability mutations, or a combination of these factors (Mulvey
et al., 2005)
Carbapenems are considered the most potent agents for treating multi-drug
resistant (MDR) Gram-negative bacteria the present study revealed that the most effective
antibiotic in vitro among the uropathogenic E. coli isolates was imipenem. None of the
isolates demonstrated resistance to this antibiotic (100% susceptible). The high efficiency
of this antibiotic may be due to rarely usage in Najaf hospitals.
Present results revealed that amikacin was the most prescribed empirical
antibiotic followed by gentamicin and tobramycin. The results of this study also support
the recommendation of the aminoglycoside, amikacin as suitable antibiotic for treating
uropathogenic E. coli infections. In this study, the better activity of amikacin may be due
to its less vulnerability to bacterial enzymes than other aminoglycosides. However,
aminoglycosides like gentamicin and amikacin, which are known to have good coverage
against P. aeruginosa, and other bacteria that resistant to other antibiotics are usually
reserved for serious urinary tracts infection and used in combination with other
antibiotics (Farrell et al., 2003).
Nitrofurantoin is considered one of the oldest urinary anti-infective drugs in use,
acts by inhibiting several protein synthesis-and-assembly steps at the ribosomal level.
This drug remained the most active agent and as it can be administered orally and is
highly concentrated in urine, it may therefore be the most appropriate agent for empirical
use in uncomplicated UTI (Younis et al., 2009). Surprisingly, resistance to this drug
remains minimal as seen in Figure (3-2), it seems that nitrofurantoin, because of the
lower resistance in E. coli isolates as compared with other antibiotics,
273
Percentage
93.8
Am
80.2
AX
95.3
PY
94.3
TIC
90.1
PRL
67.2
AC
91.7
MA
71.4
CTX
71.4
CRO
68.8
CAZ
92.2
Fep
85.4
FOX
71.4
AZT
0
IPM
10.4
AK
56.8
TOB
53.1
CN
12
F
69.8
TMP
17.7
C
68.2
DO
61
NA
49
CIP
49
NX
40.6
GT
49
OFX
49
Levo
0
20
40
60
80
100
Figure (3-2): Susceptibility profil of E. coli for different antibiotics.
Am: Ampicillin; Ax: Amoxicillin; PY: Carpencillin; TIC: Ticarcillin; PRL: Pipracillin; Ac: Amoxicillin
MA: Cefamandol; CTX: Cefotaxime; CRO: Cefetriaxon; CAZ: Ceftazidim; Fep: Cefepim; FOX: Cefoxitin
AZT: Aztreonam; IPM: Imipenim; AK: Amikacin; TOB: Tobramycin; CN: Gentamycin; F: Nitrforantoin
TMP: Trimetheoprim; C: Chloramphenicol; DO: Doxicycllin; NA: Nalidixic acid; CIP: Ciprofloxacin;
NX: Norfloxacin; GT: Gatifloxacin; OFX: Ofloxacin; Levo: Levofloxacin.
can be considered as a good alternative therapy in E. coli urinary tracts infection in Najaf.
This investigation also confirmed high resistant rate (69.8%) against
trimethoprim. In agreement with present study, Hadi (2008) found that 60.5% of E. coli
isolates recovered from patients with significant bacteriuria in Najaf were resistant to
trimethoprim. In the UK, trimethoprim is usually recommended for empirical treatment
of episodes of uncomplicated cystitis in the community (Baerheim et al., 2001).
However, the major cause of trimethoprim resistance in Gram-negative bacteria is
plasmid-borne dihydrofolate reductase (dfr) genes, which are commonly found as gene
cassettes in class 1 integrons (Ashraf et al., 2007).
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Present investigation showed that the other active antibiotic to treat infection
caused by uropathogenic E. coli was chloramphenicol, with a resistance percentage of
17.7%. The low rate of resistance against chloramphenicol probably due to rarely use of
this antibiotic to treatment of urinary tracts infection and other infections caused by E.
coli.
In this study, a relatively high occurrence of resistance in E. coli isolates was
observed for nalidixic acid (61%). The resistant effect of isolates to fluoroquinolones
including; ciprofloxacin, norfloxacin, ofloxacin, levofloxacin, and gatifloxacin are
comparable, the rates of resistance were 49 % for ciprofloxacin, norfloxacin, ofloxacin,
levofloxacin, whereas 40.6% of the isolates were resistant to gatifloxacin (Figure 3-2).
Many factors may have contributed to such high rates of nalidixic acid resistance. These
including misuse of antibiotic by health care professionals or non-skilled practitioners,
misuse of antibiotic by the general public (antibiotic can be purchased in Najaf without a
prescription), and may be inadequate surveillance due to lack of information arising from
routine antimicrobial susceptibility testing, like reports from other developing countries
(Oðuz and Bünyamin, 2004; Pittet et al., 2008).
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