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

Authors: Ziad Daoud; Claude Afif
Journal: Chemotherapy Research and Practice
 (2011)

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## 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 ofEscherichia 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.


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## Body

## 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 toCandida 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 ofE. 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. coliATCC 25922 was used as ESBL-negative and K. pneumoniae700603 was used as ESBL-positive reference strain. Statistical analysis: variables were expressed as percentages.


## 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 toCandida 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 ofE. 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. coliATCC 25922 was used as ESBL-negative and K. pneumoniae700603 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 6,071E. coli were analyzed.Tables1 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.,andStreptococcus 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 200420002001200220032004Nb%Nb%Nb%Nb%Nb%Acinetobacter sp.30.540.520.200.0141.3Candida albicans152.4121.6232.4262.4312.9Candida sp.162.5222.9282.9474.3343.1Citrobacter sp.60.9111.5192.0171.5151.4Enterobacter sp.40.6121.6141.560.5161.5Enterococcus faecalis172.7182.4282.9484.4201.9Enterococcus faecium60.9182.4202.1302.7353.2Escherichia coli39562.544058.357559.863757.966161.1Klebsiella sp.487.6759.9596.1857.7797.3Morganella morganii50.840.5111.1141.3141.3Proteus sp.457.1506.6727.5746.7656.0Pseudomonas aeruginosa304.7385.0454.7353.2514.7Pseudomonas sp.00.020.310.110.100.0Staphylococcus aureus30.560.830.370.690.8Staphylococcus saprophyticus60.970.950.520.230.3Streptococcus agalactiae203.2152.0141.5252.3181.7Streptococcus, Group D10.220.340.410.160.6Other minor organisms121.9192.5384.0454.1100.9Total isolates63275596111001081Table 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 200920052006200720082009Nb%Nb%Nb%Nb%Nb%Acinetobacter sp.191.9141.2181.6161.350.5Candida albicans141.4373.3272.4292.4252.5Candida sp.292.9575.1343.0312.6353.5Citrobacter sp.151.5181.6191.7161.390.9Enterobacter sp.161.6222.0141.2191.660.6Enterococcus faecalis141.4151.3201.8141.2151.5Enterococcus faecium323.2252.2343.0342.860.6Escherichia coli60960.271163.468860.772760.062862.5Klebsiella sp.929.1847.51008.81189.7909.0Morganella morganii131.360.5100.9242.070.7Proteus sp.575.6534.7706.2736.0636.3Pseudomonas aeruginosa393.9272.4403.5443.6414.1Pseudomonas sp.00.060.500.060.530.3Staphylococcus aureus70.740.460.540.3101.0Staphylococcus saprophyticus00.050.410.120.230.3Streptococcus agalactiae272.7141.2161.4161.380.8Streptococcus, Group D10.110.110.100.010.1Other minor organisms282.8232.0363.2383.1505.0Total isolates10121122113412111005Analysis of the results according to patient gender (not shown) indicated that althoughE. 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) ofE. coli isolates to the panel of antibiotics which are commonly used to treat Escherichiainfections 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 ofE. coli collected from Lebanese patients between 2000 and 2009 at Saint George Hospital University Medical Center, Beirut.
Percentages of susceptibility ofE. coli isolated from Lebanese patientsYearSite of isolationNumberAntibacterial AgentAmpicillinAmox-ClavAztreonamCephalothinCefoxitinCefuroximeCefotaximCeftriaxoneCeftazidimeCefepimePiperacillinPipera-TazoImipenemGentamycinTobramycinNorfloxacinCiprofloxacinTrimeth-Sulfa2000General6203758955492899696959720931008988757851Urinary3953859985797959696979924941009289888354Nonurinary22535569152818295959496179310085885970522001General6972856924991829393929518931008988757848Urinary4403263945192889696979722941009289888351Nonurinary25722499147897491898792139110087866173452002General9013062926096849494929612961008379647650Urinary6993469946297909697979716971008680778153Nonurinary2022654895693779091879479310081775073452003General12212858815496838383828313951007874607748Urinary6373265835697898990899017961008175738251Nonurinary51624507851947776807477109410075744872442004General1094286552599577828282829991007881626564Urinary66132725461968388898989131001008182757067Nonurinary4332559505693717575757479710078795263602005General9982661534995768080808110981007699586251Urinary60930685551968286878788149910079100716754Nonurinary3892455514893727573727489710074995160492006General10722964795495808383838310991007999437149Urinary711337181569686899090901410010082100567652Nonurinary3612560795594757978787889910076993974482007General10492965815995788181818110991008099444847Urinary688347283619684878888881410010083100575350Nonurinary3612360805893737877777779910078993645442008General1098286677558973788080809991007593424745Urinary7273067805596788288888915991008298625251Nonurinary3712565765484697573737359810070893945402009General10112660794696767979797913981007999444647Urinary6283169814796848686875318991008399635251Nonurinary3832353794596697474758011991007699354144Table 4
ESBL production inE. 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 inE. coli isolated between 2000 and 200920002001200220032004IsolatesAllUNUAllUNUAllUNUAllUNUAllUNUNumber of isolates62039522569744025790169920212216375841094661433% of ESBL2.32.12.343.94.39.89.99.513.614.512.112.911.014.120052006200720082009IsolatesAllUNUAllUNUAllUNUAllUNUAllUNUNumber of isolates9986093891072711361104968836110987273711011628383% of ESBL20.315.726.117.415.619.119.4519.219.819.417.122.018.616.821.8Table5 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 producingE. coli collected from Lebanese patients between 2005 and 2009 at the Saint George Hospital-University Medical Center, Beirut.
Percentages of susceptibility of ESBL producingE. coliAntibacterial agentYearIsolatesAmikacinCefoxitinGentamycinPip + TazoTSMTigecyclineCiprofloxacin2005All90.585.629.897.639.4ND21.1Urinary92.387.635.297.541.1ND16.4Nonurinary88.283.525.197.837.1ND17.92006All97.980.727.393.324.1ND17.6Urinary95.684.433.594.140.8ND18.8Nonurinary98.376.922.193.518.9ND16.52007All93.681.430.192.223.589.623.1Urinary91.287.522.489.936.891.225.7Nonurinary90.275.636.895.441.393.219.12008All94.280.627.894.125.687.418.8Urinary89.182.424.487.926.691.421.1Nonurinary97.377.132.298.823.982.115.52009All93.683.430.894.219.591.619.6Urinary94.684.536.196.625.494.419.5Nonurinary91.882.226.392.210.689.919.2Table6 shows the percentages of simultaneous resistance in the urinary isolates of E. colito various families of antimicrobial agents.Table 6
Multiple resistance of urinary isolates ofE. coli.
Percentage of resistance profilesResistance profile of urinaryE. coli20052006200720082009Resistant to aminoglycosides and fluoroquinolones3.22.63.133.7Resistant to 3rd-Generation cephalosporins and fluoroquinolones3.634.98.29.6Resistant to 3rd-Generation cephalosporins and aminoglycosides3.93.43.52.53.1Resistant to 3rd-Generation cephalosporins, aminoglycosides, and fluoroquinolones8.799.813.87.3


## 4. Discussion

This study shows the distribution of microbial species and antibiotic susceptibility patterns ofE. 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. coliwas the predominant pathogen isolated from patients with UTIs [16, 17]. The rate of isolation of Klebsiella pneumoniaemight 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. coliisolates 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 ofE. colishow 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.



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