Chemotherapy Research and PracticeVolume 2011 (2011), Article ID 218431, 6 pagesdoi:10.1155/2011/218431

Research Article

Escherichia coli Isolated from Urinary Tract Infections of Lebanese Patients between 2000 and 2009: Epidemiology and Profiles of Resistance

Ziad Daoud¹ and Claude Afif²

¹Faculty of Medicine and Medical Sciences, University of Balamand, P.O. Box 100, Tripoli, Lebanon²Faculty of Medicine and Medical Sciences, University of Balamand, P.O. Box 166378, Beirut, Lebanon

Received 1 July 2011; Revised 10 August 2011; Accepted 13 August 2011

Academic Editor: Spyros Pournaras

Copyright © 2011 Ziad Daoud and Claude Afif. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The purpose of this study was to investigate the bacterial etiology of urinary tract infections in one of the busiest hospitals of Lebanon and to examine the epidemiologic and microbiologic properties of Escherichia coli isolated from urinary tract infections of Lebanese patients over a 10-year period. Methods. This retrospective study analyzed the data generated between 2000 and 2009 (10,013 Gram-positive and Gram-negative bacteria). Bacterial identification was based on standard culture and biochemical characteristics of isolates. Antimicrobial susceptibility was tested by the disk diffusion method, and ESBL production was detected by synergy with third-generation cephalosporins and amoxiclav. Results. E. coli was the most frequent isolate throughout the ten years (60.64% of the total isolates). It was followed by Klebsiella pneumoniae and Proteus sp., Pseudomonas aeruginosa, Enterococcus sp., and Streptococcus agalactiae. E. coli occurred more frequently in women (69.8%) than in men (61.4%). The lowest percentage of susceptibility of E. coli was manifested against piperacillin and ampicillin. An increase in the production of ESBL was observed (2.3% in 2000 to 16.8% in 2009). Conclusions. The etiology of urinary tract infections and their susceptibility profiles are important to be evaluated in countries like Lebanon where a severe misuse of antibiotics at all levels is observed.

1. Introduction

Urinary tract infections (UTIs) are one of the most common infectious diseases [1–3]. They may be symptomatic or asymptomatic, and either type of infection can result in serious sequelae if not appropriately treated [4]. Although different causative agents can be responsible for UTIs, bacteria are the major cause being responsible for more than 95% of UTI cases [5]. In this context, E. coli is the most prevalent organism and is solely responsible for the majority of these infections. An accurate and prompt diagnosis is important in shortening the disease course and for preventing the ascent of the infection to the upper urinary tract [6]. Treatment of UTI is often started empirically. UTIs are often treated with different broad-spectrum antibiotics when one with a narrow spectrum of activity may be appropriate because of concerns about infection with resistant organisms, and antimicrobial susceptibility testing of the urinary pathogens constitutes the basis for antibiotic therapy. However, in view of the increasing bacterial resistance, regular monitoring of resistance patterns is necessary to improve guidelines for empirical antibiotic therapy [6–8]. The present study aims at analyzing the infectious epidemiology of UTIs in a general university hospital located in Beirut over a period of ten years. In addition, it examines the susceptibility profiles of E. coli between 2000 and 2009.

2. Material and Methods

2.1. Study Design

This is a retrospective study conducted at the Microbiology section of the Medical Laboratories of the Saint George Hospital-University Medical Center in Beirut and covering ten years (2000 to 2009). The population included all in- and outpatients with documented UTI. This included 10,013 different Gram-positive and Gram-negative bacteria in addition to Candida albicans and Candida sp. There were 6,708 (66.99%) samples from female patients and 3,305 (33.01%) from male patients. Adult patients were sampled by clean catch midstream urine, and children aged less than 3 years were sampled using sterile urine bags. Only a single positive culture per patient was included in the analysis within the period of three months.

2.2. Isolation and Identification of Organisms

Samples for urine culture were tested within half an hour of sampling. All samples were inoculated on blood agar as well as Mac Conckey agar and incubated at 37°C for 24 hours, and for 48 hours in negative cases. A specimen was considered positive for UTI in the light of the number of yielded colonies (≥10⁵ cfu/mL) and the cytology of the urine through microscopic detection of bacteriuria and PMNs (≥8 leukocytes/mm³). However, lower colony counts associated with significant pyuria or low PMN count associated with significant colony counts was considered and analyzed in the light of the clinical picture and the patient’s immunological status. Bacterial identification was based on standard culture and biochemical characteristics of isolates.

Gram-negative bacteria were identified by standard biochemical tests [5, 6]. Gram-positive microorganisms were identified with the corresponding recommended laboratory tests.

2.3. Susceptibility Testing

Antimicrobial susceptibility of E. coli was tested by the disk diffusion method according to the CLSI recommendations, using the Mueller-Hinton agar [6]. Antimicrobial agents tested were ampicillin, amoxicillin-clavulanic acid, aztreonam, cephalothin, cefoxitin, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefipime, piperacillin, piperacillin-tazobactam, imipenem, gentamycin, tobramycin, norfloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, and tetracycline. The CLSI-ESBL phenotypic confirmatory test with ceftazidime, cefotaxime, ceftriaxone, and cefixime was performed for all the isolates by disk diffusion method on the Mueller-Hinton agar plates with and without 10 μg of amoxiclav. Susceptibility test results were interpreted according to the criteria established by the Clinical & Laboratory Standard Institute (CLSI). A minimum of 5 mm increase in the zone of diameter of third-generation cephalosporins, tested in combination with amoxiclav versus its zone when tested alone, was considered indicative of ESBL production. E. coli ATCC 25922 was used as ESBL-negative and K. pneumoniae 700603 was used as ESBL-positive reference strain. Statistical analysis: variables were expressed as percentages.

3. Results

Over a 10-year period, a total of 10,013 positive urine isolates including 6,071 E. coli were analyzed.

Tables 1 and 2 show the microorganisms most frequently isolated from these positive urine cultures. As expected, E. coli was the most frequent isolate throughout the ten years (average of 60.64% of the total isolates). It was followed by Klebsiella pneumoniae where rate of isolation ranged between 6.1 and 9.9%. The next most frequently isolated bacteria were Proteus sp., Pseudomonas aeruginosa, Enterococcus sp., and Streptococcus agalactiae. Candida albicans and Candida sp. were commonly isolated; however, their clinical significance was not always evident. If Candida and minor bacterial isolates are not included, Gram-negative bacteria accounted for 92% of the UTI, while Gram-positive infections were responsible only for 8%.

Table 1: The organisms most commonly isolated from Lebanese patients with UTI between 2000 and 2004 at Saint George Hospital University Medical Center, Beirut.

The microorganisms most commonly isolated from Lebanese patients with UTI between 2000 and 2004

2000
2001
2002
2003
2004

Nb
%
Nb
%
Nb
%
Nb
%
Nb
%

Acinetobacter sp.
3
0.5
4
0.5
2
0.2
0
0.0
14
1.3

Candida albicans
15
2.4
12
1.6
23
2.4
26
2.4
31
2.9

Candida sp.
16
2.5
22
2.9
28
2.9
47
4.3
34
3.1

Citrobacter sp.
6
0.9
11
1.5
19
2.0
17
1.5
15
1.4

Enterobacter sp.
4
0.6
12
1.6
14
1.5
6
0.5
16
1.5

Enterococcus faecalis
17
2.7
18
2.4
28
2.9
48
4.4
20
1.9

Enterococcus faecium
6
0.9
18
2.4
20
2.1
30
2.7
35
3.2

Escherichia coli
395
62.5
440
58.3
575
59.8
637
57.9
661
61.1

Klebsiella sp.
48
7.6
75
9.9
59
6.1
85
7.7
79
7.3

Morganella morganii
5
0.8
4
0.5
11
1.1
14
1.3
14
1.3

Proteus sp.
45
7.1
50
6.6
72
7.5
74
6.7
65
6.0

Pseudomonas aeruginosa
30
4.7
38
5.0
45
4.7
35
3.2
51
4.7

Pseudomonas sp.
0
0.0
2
0.3
1
0.1
1
0.1
0
0.0

Staphylococcus aureus
3
0.5
6
0.8
3
0.3
7
0.6
9
0.8

Staphylococcus saprophyticus
6
0.9
7
0.9
5
0.5
2
0.2
3
0.3

Streptococcus agalactiae
20
3.2
15
2.0
14
1.5
25
2.3
18
1.7

Streptococcus, Group D
1
0.2
2
0.3
4
0.4
1
0.1
6
0.6

Other minor organisms
12
1.9
19
2.5
38
4.0
45
4.1
10
0.9

Total isolates
632
755
961
1100
1081

Table 2: The organisms most commonly isolated from Lebanese patients with UTI between 2000 and 2004 at Saint George Hospital University Medical Center, Beirut.

The microorganisms most commonly isolated from Lebanese patients with UTI between 2005 and 2009

2005
2006
2007
2008
2009

Nb
%
Nb
%
Nb
%
Nb
%
Nb
%

Acinetobacter sp.
19
1.9
14
1.2
18
1.6
16
1.3
5
0.5

Candida albicans
14
1.4
37
3.3
27
2.4
29
2.4
25
2.5

Candida sp.
29
2.9
57
5.1
34
3.0
31
2.6
35
3.5

Citrobacter sp.
15
1.5
18
1.6
19
1.7
16
1.3
9
0.9

Enterobacter sp.
16
1.6
22
2.0
14
1.2
19
1.6
6
0.6

Enterococcus faecalis
14
1.4
15
1.3
20
1.8
14
1.2
15
1.5

Enterococcus faecium
32
3.2
25
2.2
34
3.0
34
2.8
6
0.6

Escherichia coli
609
60.2
711
63.4
688
60.7
727
60.0
628
62.5

Klebsiella sp.
92
9.1
84
7.5
100
8.8
118
9.7
90
9.0

Morganella morganii
13
1.3
6
0.5
10
0.9
24
2.0
7
0.7

Proteus sp.
57
5.6
53
4.7
70
6.2
73
6.0
63
6.3

Pseudomonas aeruginosa
39
3.9
27
2.4
40
3.5
44
3.6
41
4.1

Pseudomonas sp.
0
0.0
6
0.5
0
0.0
6
0.5
3
0.3

Staphylococcus aureus
7
0.7
4
0.4
6
0.5
4
0.3
10
1.0

Staphylococcus saprophyticus
0
0.0
5
0.4
1
0.1
2
0.2
3
0.3

Streptococcus agalactiae
27
2.7
14
1.2
16
1.4
16
1.3
8
0.8

Streptococcus, Group D
1
0.1
1
0.1
1
0.1
0
0.0
1
0.1

Other minor organisms
28
2.8
23
2.0
36
3.2
38
3.1
50
5.0

Total isolates
1012
1122
1134
1211
1005

Analysis of the results according to patient gender (not shown) indicated that although E. coli is the predominant isolated pathogen from both sexes, it occurred more frequently in women (69.8% in women compared to 61.4% in men.) The trend of infectious etiology in the population did not really differ over the 10-year period.

The percentages of susceptibility (and subsequently of nonsusceptibility including both resistant and intermediately resistant strains) of E. coli isolates to the panel of antibiotics which are commonly used to treat Escherichia infections are shown in Table 3. The lowest percentage of susceptibility was manifested against piperacillin (between 9 and 24%) followed by ampicillin (between 26 and 38%), whereas an absolute susceptibility was observed with imipenem (100%). In general, the urinary isolates showed a slightly better susceptibility profile in comparison to all the hospital isolates of E. coli. Over the successive years, the susceptibility to cephalosporins, including generations 3 and 4, tends to decrease; this is coupled by an increase in the production of ESBL as shown in Table 4 where ESBL production goes from 2.3% in 2000 to 16.8% in 2009 for the urinary isolates.

Table 3: Susceptibility profiles of urinary and nonurinary isolates of E. coli collected from Lebanese patients between 2000 and 2009 at Saint George Hospital University Medical Center, Beirut.

Percentages of susceptibility of E. coli isolated from Lebanese patients

Year
Site of isolation
Number
Antibacterial Agent

Ampicillin
Amox-Clav
Aztreonam
Cephalothin
Cefoxitin
Cefuroxime
Cefotaxim
Ceftriaxone
Ceftazidime
Cefepime
Piperacillin
Pipera-Tazo
Imipenem
Gentamycin
Tobramycin
Norfloxacin
Ciprofloxacin
Trimeth-Sulfa

2000
General
620
37
58
95
54
92
89
96
96
95
97
20
93
100
89
88
75
78
51

Urinary
395
38
59
98
57
97
95
96
96
97
99
24
94
100
92
89
88
83
54

Nonurinary
225
35
56
91
52
81
82
95
95
94
96
17
93
100
85
88
59
70
52

2001
General
697
28
56
92
49
91
82
93
93
92
95
18
93
100
89
88
75
78
48

Urinary
440
32
63
94
51
92
88
96
96
97
97
22
94
100
92
89
88
83
51

Nonurinary
257
22
49
91
47
89
74
91
89
87
92
13
91
100
87
86
61
73
45

2002
General
901
30
62
92
60
96
84
94
94
92
96
12
96
100
83
79
64
76
50

Urinary
699
34
69
94
62
97
90
96
97
97
97
16
97
100
86
80
77
81
53

Nonurinary
202
26
54
89
56
93
77
90
91
87
94
7
93
100
81
77
50
73
45

2003
General
1221
28
58
81
54
96
83
83
83
82
83
13
95
100
78
74
60
77
48

Urinary
637
32
65
83
56
97
89
89
90
89
90
17
96
100
81
75
73
82
51

Nonurinary
516
24
50
78
51
94
77
76
80
74
77
10
94
100
75
74
48
72
44

2004
General
1094
28
65
52
59
95
77
82
82
82
82
9
99
100
78
81
62
65
64

Urinary
661
32
72
54
61
96
83
88
89
89
89
13
100
100
81
82
75
70
67

Nonurinary
433
25
59
50
56
93
71
75
75
75
74
7
97
100
78
79
52
63
60

2005
General
998
26
61
53
49
95
76
80
80
80
81
10
98
100
76
99
58
62
51

Urinary
609
30
68
55
51
96
82
86
87
87
88
14
99
100
79
100
71
67
54

Nonurinary
389
24
55
51
48
93
72
75
73
72
74
8
97
100
74
99
51
60
49

2006
General
1072
29
64
79
54
95
80
83
83
83
83
10
99
100
79
99
43
71
49

Urinary
711
33
71
81
56
96
86
89
90
90
90
14
100
100
82
100
56
76
52

Nonurinary
361
25
60
79
55
94
75
79
78
78
78
8
99
100
76
99
39
74
48

2007
General
1049
29
65
81
59
95
78
81
81
81
81
10
99
100
80
99
44
48
47

Urinary
688
34
72
83
61
96
84
87
88
88
88
14
100
100
83
100
57
53
50

Nonurinary
361
23
60
80
58
93
73
78
77
77
77
7
99
100
78
99
36
45
44

2008
General
1098
28
66
77
55
89
73
78
80
80
80
9
99
100
75
93
42
47
45

Urinary
727
30
67
80
55
96
78
82
88
88
89
15
99
100
82
98
62
52
51

Nonurinary
371
25
65
76
54
84
69
75
73
73
73
5
98
100
70
89
39
45
40

2009
General
1011
26
60
79
46
96
76
79
79
79
79
13
98
100
79
99
44
46
47

Urinary
628
31
69
81
47
96
84
86
86
87
53
18
99
100
83
99
63
52
51

Nonurinary
383
23
53
79
45
96
69
74
74
75
80
11
99
100
76
99
35
41
44

Table 4: ESBL production in E. coli collected from Lebanese patients between 2000 and 2009 at the Saint George Hospital University Medical Center, Beirut. U: urinary isolates, NU: nonurinary isolates.

ESBL production in E. coli isolated between 2000 and 2009

2000
2001
2002
2003
2004

Isolates
All
U
NU
All
U
NU
All
U
NU
All
U
NU
All
U
NU

Number of isolates
620
395
225
697
440
257
901
699
202
1221
637
584
1094
661
433

% of ESBL
2.3
2.1
2.3
4
3.9
4.3
9.8
9.9
9.5
13.6
14.5
12.1
12.9
11.0
14.1

2005
2006
2007
2008
2009

Isolates
All
U
NU
All
U
NU
All
U
NU
All
U
NU
All
U
NU

Number of isolates
998
609
389
1072
711
361
1049
688
361
1098
727
371
1011
628
383

% of ESBL
20.3
15.7
26.1
17.4
15.6
19.1
19.45
19.2
19.8
19.4
17.1
22.0
18.6
16.8
21.8

Table 5 shows the susceptibility profiles of ESBL producing E. coli to families of antibiotics other than beta-lactams. Tigecycline, amikacin, and piperacillin-tazobactam seem to have the highest antibacterial activity on these organisms.

Table 5: Susceptibility profiles of ESBL producing E. coli collected from Lebanese patients between 2005 and 2009 at the Saint George Hospital-University Medical Center, Beirut.

Percentages of susceptibility of ESBL producing E. coli

Antibacterial agent

Year
Isolates
Amikacin
Cefoxitin
Gentamycin
Pip + Tazo
TSM
Tigecycline
Ciprofloxacin

2005
All
90.5
85.6
29.8
97.6
39.4
ND
21.1

Urinary
92.3
87.6
35.2
97.5
41.1
ND
16.4

Nonurinary
88.2
83.5
25.1
97.8
37.1
ND
17.9

2006
All
97.9
80.7
27.3
93.3
24.1
ND
17.6

Urinary
95.6
84.4
33.5
94.1
40.8
ND
18.8

Nonurinary
98.3
76.9
22.1
93.5
18.9
ND
16.5

2007
All
93.6
81.4
30.1
92.2
23.5
89.6
23.1

Urinary
91.2
87.5
22.4
89.9
36.8
91.2
25.7

Nonurinary
90.2
75.6
36.8
95.4
41.3
93.2
19.1

2008
All
94.2
80.6
27.8
94.1
25.6
87.4
18.8

Urinary
89.1
82.4
24.4
87.9
26.6
91.4
21.1

Nonurinary
97.3
77.1
32.2
98.8
23.9
82.1
15.5

2009
All
93.6
83.4
30.8
94.2
19.5
91.6
19.6

Urinary
94.6
84.5
36.1
96.6
25.4
94.4
19.5

Nonurinary
91.8
82.2
26.3
92.2
10.6
89.9
19.2

Table 6 shows the percentages of simultaneous resistance in the urinary isolates of E. coli to various families of antimicrobial agents.

Table 6: Multiple resistance of urinary isolates of E. coli.

Percentage of resistance profiles

Resistance profile of urinary E. coli
2005
2006
2007
2008
2009

Resistant to aminoglycosides and fluoroquinolones
3.2
2.6
3.1
3
3.7

Resistant to 3rd-Generation cephalosporins and fluoroquinolones
3.6
3
4.9
8.2
9.6

Resistant to 3rd-Generation cephalosporins and aminoglycosides
3.9
3.4
3.5
2.5
3.1

Resistant to 3rd-Generation cephalosporins, aminoglycosides, and fluoroquinolones
8.7
9
9.8
13.8
7.3

4. Discussion

This study shows the distribution of microbial species and antibiotic susceptibility patterns of E. coli isolated from Lebanese patients with UTIs. Saint George Hospital is a 300-bed hospital located in Beirut; however, it is one of the busiest hospitals in the country and receives patients from different areas of Lebanon. In this retrospective study, no clinical data are provided, and this constitutes by itself a limitation. Subsequently, important information related to symptomatic versus asymptomatic, complicated versus uncomplicated UTI, and health-care-associated versus catheter-related UTIs could not be addressed and discussed. The majority of pathogens were isolated from women (69.8%). It has been extensively reported that adult women have a higher prevalence of UTI than men, principally owing to anatomic and physical factors [9].

Antibiotic resistance is a major clinical problem in treating infections caused by these microorganisms. The resistance to the antimicrobials has increased over the years. Resistance rates vary from country to country [10]. In Lebanon, there is an evidence for increase in ESBL producing Enterobacteriaceae. This was previously reported in our hospital as well as in other institutions in the country [11–15]

In this study, E. coli accounted for approximately 61% of all clinically significant urinary isolates and 76.8% of all Enterobacteriaceae. This is consistent with the findings of previous studies in which E. coli was the predominant pathogen isolated from patients with UTIs [16, 17]. The rate of isolation of Klebsiella pneumoniae might be described as high when compared to other studies [16, 17]; however, this can be explained by the fact that our study includes both hospital and acquired UTI.

E. coli isolates from urinary infections show a similar pattern of susceptibility to those isolated from all body site infections although with a more enhanced susceptibility percentages. Aminopenicillins do not constitute a therapeutic option in this population; even the combination amoxicillin-clavulanic acid does not seem to offer important alternative for treatment. In view of the increasing ESBL production, all cephalosporins activities are affected and challenged by these inactivating enzymes. Imipenem remains the only antibiotic with 100% of susceptibility.

It is pretty alarming to note that between 7.3 and 13.8% of the urinary isolates of E. coli show simultaneous resistance to third-generation cephalosporins, aminoglycosides, and fluoroquinolones. A genetic investigation on these isolates should be performed in order to identify the mechanisms of resistance and discover whether they are correlated to each other or independently occurring.

Conflict of Interests

The authors declare that they have no conflict of interests.

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