1 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Pozuelo a Majadahonda, 28220 Majadahonda, Madrid; 2 Servicio de Microbiología, H. Ramón y Cajal, Madrid, Spain
Received 22 July 2002; returned 10 September 2002; revised 19 September 2002; accepted 20 September 2002
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Abstract |
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Keywords: antimicrobial resistance, invasive infections, EARSS, Escherichia coli
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Introduction |
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Escherichia coli is the most common cause of Gram-negative bacillus infections4 and the first or second most frequently isolated microorganism from blood.58 E. coli has a relatively large potential for developing resistance. Indeed, antimicrobial resistance to ß-lactams and other antibiotics has been reported from many countries.4,6,9 Resistance to fluoroquinolones has been emerging in recent years,10 even in countries where antimicrobial resistance rates are low,11 and multidrug resistance has been reported.9 Careful monitoring of the emerging antimicrobial resistance among E. coli strains is needed to highlight potential and future problems and may help to formulate intervention strategies.
Spain has one of the highest rates of antibiotic consumption in Europe.12,13 Antibiotic resistance is a major public health problem in this country.14 The goal of this study is to describe and analyse E. coli antibiotic resistance data collected by the Spanish hospitals participating in the EARSS in 2001.
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Materials and methods |
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To fulfil the goal of obtaining representative data, participating hospitals were chosen in order to meet the following criteria: (i) combined coverage of at least 20% of the Spanish population; (ii) different areas of the country covered; and (iii) different kinds of hospital (size and category) represented.
Strains studied
All clinical isolates of E. coli obtained from blood and cerebrospinal fluid (CSF) in Spanish laboratories participating in the EARSS in 2001 were included. Only the first invasive isolate per patient was reported.
Data collection
A questionnaire concerning some hospital characteristics (coverage, number of beds, number of patients admitted per year and hospital departments) and methods of antimicrobial susceptibility study and interpretation criteria was submitted to each participating centre. One isolate record form per patient was filled in. This form included patients personal data (code, age and sex), hospital and departmental data and antibiotic susceptibility data.
Hospitals sent prospectively standardized results to the Centro Nacional de Microbiología of the Instituto de Salud Carlos III, where results were analysed and validated using the laboratory-based Whonet.5 program. All records were carefully analysed by a clinical microbiologist.
Duplicated isolates, more than one isolate per patient, were identified and deleted. Discrepancies and atypical results were resolved by telephone and the corresponding database records updated if necessary. At the end of 2001, an annual report with all the data stored in the central database was sent to each participating laboratory to avoid potential disagreement.
Strain identification and susceptibility studies
Each laboratory identified the strains and tested their susceptibility using its own routine methods and breakpoints.
The protocol for E. coli susceptibility testing included the following antibiotics: ampicillin or amoxicillin, aminoglycoside (gentamicin and/or tobramycin), fluoroquinolone (ciprofloxacin and/or ofloxacin) and third-generation cephalosporins (cefotaxime or ceftriaxone and/or ceftazidime). In addition, data on antimicrobial susceptibility to other antibiotics were also considered, in case a participating laboratory routinely tested them. The number of strains studied, by antibiotic and method, was not always equal to the total number of strains.
Extended-spectrum ß-lactamase (ESBL) production was suspected when the MIC of ceftazidime and/or cefotaxime was >1 mg/L. This was considered to be confirmed when the activity of cefotaxime and/or ceftazidime tested with clavulanic acid was greater (defined as a >two-fold decrease in MIC or a >4 mm increase in zone diameter) than when tested alone.15
Multidrug resistance was defined as resistance to three or more of the antimicrobials tested.
Quality control
To assess the comparability of susceptibility test results, a quality assurance exercise was performed in September 2001 among the 27 laboratories participating in the Spanish EARSS. This exercise was designed by the UK National External Quality Assurance Scheme (NEQAS) and included three E. coli strains with different resistance phenotypes. The first control was susceptible to all antibiotics; the second was an ESBL producer with an MIC of cefotaxime higher than that to ceftazidime and was gentamicin resistant; the third was also an ESBL producer but with an MIC of ceftazidime higher than that of cefotaxime. Data on susceptibility to ampicillin, gentamicin, ciprofloxacin, cefotaxime and ceftazidime were required.
In addition, each laboratory filled in a questionnaire concerning the methods used for determining susceptibility and the interpretation criteria applied.
Statistical analyses
Differences in the prevalence of antibiotic resistance between different groups were assessed by the 2 test, with the Yates correction when necessary. Association was determined by calculation of odds ratios (OR) with 95% confidence intervals (CI). The null hypothesis was rejected for values of P < 0.05. Statistical analyses were performed using EPI-Info version 6.04.
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Results |
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During 2001, 27 laboratories reported data on invasive E. coli isolates. The estimated average coverage of the Spanish population was 21%, corresponding to 7 500 000 persons. The number of hospital beds and patients admitted were
13 000 and
495 000, respectively. Three hospitals out of 27 (11.11%) had >1000 beds; five (18.52%) had between 500 and 1000; 14 (51.85%) had between 250 and 500; and five (18.52%) had <250.
Identification methods and antimicrobial susceptibility testing
Identification and antimicrobial susceptibility tests were performed with commercial microdilution systems in 25 laboratories: 12 MicroScan (Dade-Behring, Deerfield, IL, USA), six Wider (Fco. Soria Melguizo S.A., Madrid, Spain), five Vitek (bioMérieux, Marcy lÉtoile, France) and two Sensititre (Radiometer/Copenhagen Company, Copenhagen, Denmark); in two laboratories, no commercial systems were used.
Results were scored as susceptible, intermediate or resistant, according to the NCCLS15 criteria in 26 laboratories and according to the Neosensitab system (Rosco Diagnostics, Taastrup, Denmark; users guide available at http://www.rosco.dk/uk/userguides/neo-sensitabs_o.pdf) in one. No significant differences in susceptibility results between the laboratory using Neosensitab system criteria and the laboratories using the NCCLS criteria were found; also, quality control results of this laboratory agreed with those obtained by reference laboratories.
Quality control results
Among participating laboratories, the overall concordance of susceptibility to ampicillin, gentamicin and ciprofloxacin in the three different E. coli control strains was 100%, 89100% and 100%, respectively. The ESBL production in quality control strain number 2 (MIC of cefotaxime higher than that of ceftazidime) and in quality control strain number 3 (MIC of ceftazidime higher than that of cefotaxime) was detected by 85.2% and 92.6% of the laboratories, respectively.
Antimicrobial susceptibility
During 2001, the 27 hospitals reported data on 1962 invasive isolates of E. coli, 1959 from blood and three from CSF, corresponding to the same number of patients. Of these, 996 strains (50.76%) were isolated from women and 955 (48.67%) from men. One hundred and eight (5.50%) of the isolates were from children (14 years). Hospital-acquired infections accounted for 1081 (55.09%) cases and community-acquired infections for 864 (44.03%).
The antibiotic susceptibility of the 1962 E. coli isolates is provided in Table 1. Resistance to ampicillin, ciprofloxacin and co-trimoxazole was detected in 58.46%, 17.19% and 32.91% of the strains, respectively.
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Aminoglycosides tested were gentamicin (1958 isolates), tobramycin (1625) and amikacin (874), with resistance rates of 6.39%, 4.93% and 0%, respectively.
Isolates from men were more resistant to ciprofloxacin than those from women (Table 2), but there were no sex differences for ampicillin, gentamicin, co-trimoxazole and third-generation cephalosporins (Table 2).
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Comparisons of antibiotic susceptibility in strains isolated in intensive care units (ICUs) and other medical departments revealed no statistical differences for any antibiotic.
As shown in Table 4, resistance to co-trimoxazole, ciprofloxacin and gentamicin was more prevalent in ampicillin-resistant strains than in ampicillin-susceptible strains. Besides, ESBL-producing strains (n = 30) were more resistant than non-producing strains to other non-ß-lactam antibiotics, such as ciprofloxacin, co-trimoxazole and gentamicin (Table 4).
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Discussion |
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EARSS methodology has previously been evaluated as a research project and is currently considered the official European network of national surveillance systems. It aims to collect comparable and reliable antimicrobial resistance data (EARSS website published by RIVM at http://www.earss.rivm.nl).
The main source of EARSS information is the primary laboratory, which receives the clinical specimens and performs routine antimicrobial susceptibility testing. Major disadvantages of the use of routine results might be that antimicrobial susceptibility testing could be performed by different methods in each laboratory, although, in our experience, the vast majority used NCCLS-recommended methodologies. To resolve this matter, external quality control evaluation is needed. Nevertheless, exploitation of routine susceptibility data may be an important source of information for targeting and evaluating interventions in order to reduce the problem of antimicrobial resistance. Cross-validation of routine data-gathering and centralized surveys has been performed.16 For example, in invasive E. coli, the same resistance trends to ampicillin, ciprofloxacin, gentamicin and trimethoprim were found in routine results reported and for a centrally tested sample.16
Despite the clinical relevance of E. coli isolated from blood and CSF, there are few published data concerning the antibiotic susceptibility of this pathogen. Ampicillin resistance was very prevalent (58.44%) in invasive isolates in this study; this is a higher value than the 46.7% reported in a recent study with European clinical blood isolates,6 but very similar to the 62% non-susceptibility described among 472 bacteraemic strains in one Spanish hospital more than 10 years ago.7 Of the countries participating in the EARSS in 2001, only Israel had a higher percentage of resistance to ampicillin than that obtained in Spain. Resistance to ampicillin was 54% in Portugal, 48% in Belgium, 46% in Germany, 46% in Greece and 39% in the Netherlands (EARSS website). Overall results of antibiotic susceptibility obtained in our study were similar to those of urinary tract infection (UTI) isolates described recently in Spain,1719 in spite of the belief that more-resistant strains are less virulent19 but in agreement with a recent article20 that described a high frequency of ampicillin resistance in more virulent E. coli.
Resistance (>8 mg/L) to third-generation cephalosporins was infrequent (<2%), of a similar prevalence to that reported by Fluit et al.6 in European blood isolates. However, nearly 5% showed an increased MIC of cefotaxime (MIC > 1 mg/L), although one-third of them were ESBL producers. Although it was not studied, the rest were probably overproducing an AmpC ß-lactamase. Among ESBL producers, cefotaxime-non-susceptible strains were more prevalent than ceftazidime-non-susceptible strains (20 versus 11). In recent years, CTX-M ß-lactamases (ceftazidime-susceptible and cefotaxime-non-susceptible) have been described in Spain and other countries worldwide.2124 CTX-M-producing E. coli is spreading and replacing other ESBL producers derived from TEM and SHV ß-lactamase types (ceftazidime non-susceptible).23,24 Our results are consistent with the replacement described previously in some hospitals in Spain (authors experience, unpublished data). This has an important implication for the detection of E. coli ESBL producers in clinical microbiology laboratories. Susceptibility to ceftazidime or cefpodoxime (principally ceftazidime in Spain) has been considered the most sensitive routine test for detecting ESBL production because they are the best third-generation cephalosporin substrates for most TEM- and SHV-derived ESBLs.25 Testing ceftazidime alone as a representative of third-generation cephalosporins could be considered a correct and effective routine strategy when TEM- and SHV-derived ESBLs were the most frequent. However, with CTX-M ß-lactamase spreading, presently it would be necessary to test both cefotaxime and ceftazidime to detect all types of ESBL.
The association of ampicillin resistance and decreased susceptibility to third-generation cephalosporins with resistance to other non-ß-lactam antibiotics (co-trimoxazole, ciprofloxacin and gentamicin) was the cause of multidrug resistance in nearly 14% of strains. This multidrug resistance has important implications for the empirical therapy of infections caused by E. coli.
High antibiotic consumption selects for resistance in microorganisms of commensal flora.26 Most E. coli bacteraemias involve organisms originating from the patients own gut flora,27 and the resistance of these is very likely to reflect previous patterns of prescribing in a community. This could be the cause of more prevalent resistance to ampicillin and co-trimoxazole in children, who probably consume more of these classes of antibiotics than adults. Similar results have been described recently in the USA.9
Resistance of E. coli to quinolones has remained rare until recently, until their use increased. Fluoroquinolone consumption in Spain has increased from 1.26 defined daily doses (DDD)/1000 population/day in 1987 to 2.4 DDD/1000 population/day in 2000.13,28 Currently, ciprofloxacin resistance in UTI E. coli isolates in Spain exceeds 1520%10,17,18 (similar to the level in invasive isolates in our experience) and is increasing in other European countries.11 Ciprofloxacin resistance was present in 8.1% of 1918 invasive E. coli strains isolated in 19971998 in Europe.6 Among the EARSS countries participating in 2001, only Israel (21%) and Portugal (18%) had higher rates of ciprofloxacin resistance than Spain (EARSS website); the majority of European countries had levels <10% (EARSS website).
Resistance to ciprofloxacin varied between the sexes, whereby isolates from males were more resistant than those from women. Similar trends have been described recently in the USA9 and the Netherlands.11 These data probably reflect the tendency for men to present more often with complicated UTIs, the principal source of invasive E. coli, which may be associated with more antibiotic treatments, many of which involve fluoroquinolones. Nosocomial isolates were more resistant than community ones, as described in a recent study in South Korea.29 Admitted patients are probably more likely to have chronic pathologies that make more antibiotic treatments necessary. Lastly, the most important and determining factor in ciprofloxacin resistance is the consumption of this group of antibiotics, as described previously.30,31
It is important to emphasize the high prevalence of ciprofloxacin resistance in isolates from children, in which we found no significant differences from isolates from adults, despite ciprofloxacin not being consumed by children. This resistance could be due to environmental conditions, such as transmission of resistant isolates between adults and children in families or in day-care and school settings10 or to the prophylactic use of fluoroquinolones (enrofloxacin) in populations of chicken and poultry.32
In a comparative European study in 1997, antibiotic consumption in Spain was only surpassed by that of France.12 Spain was the country with the second-highest consumption of broad-spectrum penicillins (18.01 DDD/1000 inhabitants/day) and quinolones (2.48 DDD/1000 inhabitants/day), behind France and Portugal, respectively.12
Extended-spectrum penicillin and co-trimoxazole consumption have decreased in recent years in Spain, from 10.3 DDD/1000 inhabitants/day in 1985 to 5.2 DDD/1000 inhabitants/day in 2000 (extended-spectrum penicillins) and from 3.3 DDD/1000 inhabitants/day in 1985 to 0.4 DDD/1000 inhabitants/day in 2000 (co-trimoxazole).28 However, resistance rates to these antibiotics in E. coli have remained constant and at the top of the league table of antibiotic resistance in Europe.6,7,18 Resistance prevalence can increase very rapidly in a population; however, the rate of decline in this prevalence is much slower when antibiotic consumption is reduced.33 Furthermore, in areas with very high resistance rates, reduction in antibiotic pressure may have an even slower effect, especially in the presence of multidrug resistance.34,35 There are various explanations for this phenomenon: the possibility of resistance to different classes of antibiotics and co-selection with the usage of only one of them;33,36 the reservoir of molecular mechanisms in species of the commensal flora and its exchange between these and pathogenic species.37,38
Antimicrobial patterns are continually evolving, and multidrug resistance among some of the most important human pathogens is increasing. Therefore, properly designed and conducted surveillance systems will continue to be essential to ensure the provision of safe and effective empirical therapies. Moreover, results obtained from these surveillance systems must be used to implement prevention programmes and policy decisions to prevent the emergence and spread of antimicrobial resistance.
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Acknowledgements |
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Footnotes |
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Members of the European Antimicrobial Resistance Surveillance System are listed in the Acknowledgements.
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References |
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