1 Department of Clinical Microbiology, Central Hospital, SE-351 85 Växjö, Sweden; 2 Microbiology and Tumor Biology Center, Karolinska Institute, SE-171 77 Stockholm, Sweden
Received 11 December 2004; returned 18 January 2005; revised 15 March 2005; accepted 15 March 2005
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Abstract |
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Methods: The 2481 E. coli, typed with the PhenePlateTM (PhP) System utilizing the dynamics and end result of 11 biochemical reactions in a microplate system, were clustered and the Simpson's index of diversity calculated.
Results: Seventy-four Common PhP Types (CT) comprising 2067 isolates and 414 Single Types (Si) were identified. Of these, 916 isolates (37%) belonged to one of the four most frequent CT (arbitrarily numbered CT48, 10, 26 and 20). CT48 with 400 isolates and 11 different susceptibility patterns, was widely disseminated across Europe and Canada and was the most frequent type in 13 countries and the second most frequent in the remaining four countries. Sixty-four per cent of the E. coli were susceptible to all eight investigated antimicrobials (CT48: 73%, CT10: 77%, CT26: 62% and CT20: 37%). Forty-six different susceptibility patterns were seen, the three most common being isolated resistance to ampicillin, resistance to ampicillin and trimethoprim, and isolated resistance to trimethoprim. Multiresistance, here defined as resistance to four or more of the investigated antibiotics, was distributed among E. coli belonging to several PhP types.
Conclusions: There was no obvious correlation between the phenotypes identified with the PhP System and the susceptibility pattern. The data did not indicate clonal dissemination within or between countries as a major reason for differences in antimicrobial resistance rates.
Keywords: E. coli , clonality , typing , PhP , UTI
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Introduction |
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In a previous study (the ECO·SENS project), the prevalence and antimicrobial resistance of pathogens causing community-acquired acute uncomplicated urinary tract infections in women was investigated. Resistance rates in E. coli from UTIs in women in 16 European countries and Canada were determined.7,8 Cross-resistance and associated resistance9 and the relationships between resistance rates and the use of antimicrobials in the European countries10 were described. Using the E. coli isolates from the ECO·SENS survey, we have determined the clonal diversity with a commercial typing system, the PhenePlateTM (PhP) system. It utilizes not only the biochemical reaction as such but also the rate at which it occurs and performs a computerized numerical analysis of the kinetics of a set of biochemical tests in wells in microplates.1113 The system was first developed for typing of E. coli, but has since been developed for many other species and groups of bacteria. For different bacteria, different sets of reagents and culture methods are used to give a high level of discrimination.11,12,14
The aim of this study was to determine whether a clonal dissemination of E. coli within and/or between countries could help explain the varied and sometimes high rates of antimicrobial resistance in the 16 European countries and Canada that were part of the ECO·SENS antimicrobial resistance project.
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Materials and methods |
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E. coli strains (n=2481) from the ECO·SENS project, a survey of pathogens causing community-acquired lower urinary tract infections in otherwise healthy non-pregnant women in 16 European countries and Canada during 1999 and 2000, were typed. Information about the isolates has been published previously,7,8 and further information is given in Table 1.
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Antimicrobial susceptibility testing
From the 12 antibiotics used in the ECO·SENS study, we chose eight that were deemed representative of their respective class (ampicillin, cefadroxil, nalidixic acid, ciprofloxacin, trimethoprim, gentamicin, nitrofurantoin, fosfomycin). The antimicrobial susceptibility testing followed the recommendation of the Swedish Reference Group for Antibiotics15 and has been described previously.7
PhP typing
The PhenePlateTM System (PhPlate Microplate Techniques AB, Stockholm, Sweden; http://www.phplate.se) consists of microplates containing dehydrated reagents. In the PhP-RE system, as used in this study, the microplates contain 96 wells with 11 dehydrated reagents (cellobiose, lactose, rhamnose, deoxyribose, sucrose, sorbose, tagatose, D-arabitol, melbionate, gal-lacton and ornithine) repeated in eight rows. The technique for typing of the E. coli isolates with the PhP system has been previously described.11,12,16,17
Test results were read after 8, 24 and 48 h incubation with a flatbed scanner (HP Scanjet 7400c scanner). The images were converted to numerical absorbance data by the PhPWIN4 software (PhPlate AB, Stockholm, Sweden). The value for each well was set by the software to vary between 0 (yellow, acid reaction) and 26 (blue, alkaline reaction). After the last reading, the mean values from the readings of each well were calculated to give the biochemical fingerprint of each tested isolate. The biochemical fingerprints of all isolates were then compared to each other and the similarity between each pair was calculated. The PhPWIN4 software was used to analyse the data, calculate similarities and create dendrograms.
The reproducibility of the assay was used to determine the identity (ID) level. The ID level was defined as the mean correlation coefficient between multiple assays of the same isolates minus two standard deviations (SD), corresponding to a 95% confidence level. Isolates with a correlation coefficient higher than the ID level were regarded as identical and were assigned to the same PhP type. PhP types with two or more isolates exhibiting the same biochemical fingerprint were designated Common Types (CT), whereas Single PhP types (Si) contained one isolate only.12,14 Experiences from earlier studies11,14,18,19 have shown that an ID level of 0.975 is appropriate for comparisons within a population of E. coli where all assays were performed simultaneously, whereas an ID level of 0.965 is appropriate when comparing assays performed consecutively.
The diversity was calculated using Simpson's index of diversity, which measures the probability for two randomly selected isolates to be assigned to different phenotypes. It gives a measure of the distribution of isolates among the phenotypes.18 A value close to 1 indicates a high degree of diversity whereas a value close to 0 means that few types are dominant in the population.12
Definition of common types and single types
To determine the number of biochemical groups of isolates in the material, the PhP results were clustered in two steps. The software used clusters of 1000 isolates at a time. The analysis was performed in the following way:
The isolates were clustered country by country. When the software could match two or more isolates within a country, a tentative Common Type (CT) was automatically defined. The software also automatically selects the best representative from each CT.
In a second step, the tentative CT representatives from all countries were clustered and 74 PhP profiles (CT01-CT74) with one or several matches within one (n=6) or more (n=68) countries were found.
In a third step, all 2481 isolates were compared with the 74 Common Type profiles. Each E. coli isolate was classified as belonging to a Common Type or, if no match was found, to a Single Type.
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Results |
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Two E. coli strains and one K. pneumoniae and one A. hydrophila were included in every analysis (n=21). Dendrogram analysis showed a complete separation to the species level and between the two E. coli strains. A clinical isolate of E. coli was tested on 14 occasions and on each occasion the system assigned it to the same PhP type.
PhP types
The 2481 E. coli strains from the ECO·SENS project could be subdivided into 488 different types. Of these, 414 types were Single Types (Si). The remaining 2067 isolates belonged to 74 Common Types (CTs), i.e. types that were found in several isolates per country and most often (68 of the 74 types) in more than one country. PhP typing yielded a diversity of 0.94 among all isolates. Four of the CTs contained 100 or more isolates each. CT48 contained 400 (16%) of the 2481 E. coli. Nine hundred and sixteen (37%) belonged to one of the four most frequent CTs (CT48, 10, 26 and 20) and 1287 (52%) belonged to one of the 10 most frequent CTs (Tables 2 and 3).
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Diversity index and similarity
The PhP type diversity index varied between countries (Table 1). The lowest (index 0.908), corresponding to a low degree of variation, was recorded in France (where 337 patients attended 41 primary care centres), and the highest in Germany (index 0.967) (where 337 patients attended 11 centres). In France, 63% of the E. coli and in Germany 43% were found among the 10 most frequent CTs in the respective countries (Table 3).
Antimicrobial susceptibility patterns
Sixty-four per cent (n=1585) of the E. coli did not exhibit resistance to any of the drugs investigated. For the most frequent CTs, i.e. CT48, 10, 26 and 20, the corresponding percentages were 73, 77, 62 and 37. For the 414 single types, the figure was 60%. CT20 had the highest rates of both ampicillin (56%) and trimethoprim (42%) resistance.
None of the 74 Common Types exhibited a homogeneous susceptibility pattern. Forty-six different susceptibility patterns were identified, yielding a diversity of 0.55 among all isolates. Resistance to ampicillin alone (n=408 E. coli distributed among 55 CTs) and to ampicillin in combination with trimethoprim (n=214 belonging to 44 CTs) were the most common susceptibility patterns followed by single resistance to trimethoprim (n=70 in 29 CTs) and single resistance to nalidixic acid (n=29 in 15 CTs) (Table 2).
In almost all countries, the most common susceptibility pattern was isolated resistance to ampicillin followed by resistance to both ampicillin and trimethoprim and isolated resistance to trimethoprim or nalidixic acid (Table 4).
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There were 51 E. coli resistant to four or more of the eight investigated antibiotics. Sixteen CTs and 11 Single Types were represented. Fifteen different susceptibility patterns were recorded, seven of which occurred in two or more isolates (Tables 2 and 5).
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Discussion |
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The PhP System was first developed in 1985 for E. coli11 and since then has been used in many investigations. It has been used to determine whether recurrent infections after acute pyelonephritis were relapses or reinfections and whether E. coli strains causing reinfections in one patient could be found in other patients.19 Almost half of the E. coli strains causing initial acute pyelonephritis in that study were found to belong to a single PhP cluster, thus probably representing a common, widespread and potentially virulent clone. The stability of the biochemical properties/markers of E. coli strains used in the PhP System have been found to be good also after storage and repeated subculturing.20 In comparison with other numerical typing methods for E. coli, the reproducibility of the PhP system has been shown to be as good and the discriminatory ability better than with other biochemical typing systems.18
Comparisons between PhP and PFGE have shown that PFGE is more discriminative than the PhP system. In enterococci,21 the diversity was 0.92 for PhP and 0.94 for PFGE (DNA typing discriminated 25% more types than PhP typing), while for K. pneumoniae and K. oxytoca,22 the diversity was 0.94 for PhP and 0.96 for PFGE. PhP is especially useful for screening large numbers of isolates. PFGE can be used to verify the results and/or increase the discrimination of the system.21,22
We found the PhP system suitable for handling the large number of E. coli in the ECO·SENS study. The reagents are inexpensive and the analysis not overly labour- or time consuming. The fact that the reactions are read several times and that the speed of the reaction is part of the numerical value means that the system is more sensitive than a traditional qualitative biochemical typing system. With other typing methods, the results are read just once and the dynamics of the reaction is lost to the analysis.
The PhP typing divided the 2481 E. coli isolates into 488 types, of which 414 contained only one isolate each (Single Types), probably representing rare clonal groups, and 74 contained more than one isolate (Common Types). One PhP type (CT48, n=400), belonging to serotype O6:H+, was frequent in all 17 countries and this type comprised 16% of all isolates. An interesting finding was that these isolates were closely similar to those of a uropathogenic clone previously named DS-17, which was first isolated from an outbreak among infants in Swedish neonatal wards in 198123
and which since then has been the subject of several investigations.24,25
This specific PhP pattern was only rarely found among the PhP types of 12 000 isolates from other sources (human and animal faeces, water and sewage) stored in our database. Thus, this PhP cluster probably represents a uropathogenic clonal group of long standing.
Clonal dissemination of community-acquired Staphylococcus aureus and E. coli as the cause of high rates of antimicrobial resistance has previously been demonstrated. Österlund et al.5 showed that an epidemic impetigo S. aureus strain harbouring fusidic acid resistance rapidly increased fusidic acid resistance from 5% to more than 50% in S. aureus isolated from children 012 years of age. Manges et al.6 showed that 11% of the urinary tract infections in a cohort in California were caused by a clonal group of E. coli resistant to trimethoprimsulfamethoxazole. Most of the isolates belonged to serotypes O11:H(nt) or O77:H(nt), neither of which occurred among our 10 most common types. Trimethoprim resistance in our study varied from 42% in CT20 (n=119 isolates; serotype O17:H+), 28% in CT49 (n=25 isolates) and 22% in CT34 (n=63 isolates; serotype O172:H+) to 0% in CT42 which also exhibited the lowest ampicillin resistance (7%). The Manges study was performed in a few selected cohorts where the individuals had a common background. In this study, the strains were collected from patients admitted to 252 different centres in 17 countries and very few of the patients had a common background except their nationality.
Our study could detect neither a national nor an international dissemination of a resistant E. coli clone or clones as responsible for antimicrobial resistance. However, several PhP types exhibited characteristic, but as yet unexplained, findings in their antimicrobial susceptibility. One of our arbitrary types (CT34) exhibited the highest rate of isolated trimethoprim resistance and at the same time the lowest rate of isolated ampicillin resistance. It was present in 15 of the 17 countries and was among the most common types in Canada and Spain. Thirty-six per cent of the 2481 E. coli exhibited resistance to one or more of the eight antimicrobials. The corresponding figures for the most frequent PhP types were 27% (CT48), 23% (CT10), 38% (CT26) and 63% (CT20) and for Single Types 40%. Thus, antimicrobial resistance was decidedly more common in CT20 than in the others. This was evident when resistance to ampicillin and trimethoprim was compared. Ampicillin resistance was 56% in CT20 compared to 25% in CT48, 19% in CT10 and 30% in CT26. Trimethoprim resistance in CT20 was 42% compared with 5% in the frequently isolated CT48, 4% in CT10 and 10% in CT26. CT20 appeared in all countries, but was more common in some (Denmark 9.6%) than in others (Austria 1.6%). Thus, although there was no straightforward match between antimicrobial resistance and certain E. coli types, there was evidence that antimicrobial resistance was related to certain types and that some types were more common in some countries than others.
Austria, Sweden and Finland exhibited the lowest diversity in susceptibility patterns. Spain followed by Portugal exhibited the highest diversity in susceptibility patterns. These findings correlate well to the resistance rates as reported in the ECO·SENS study8 where the highest resistance rates were recorded in Spain and Portugal and the lowest rates in Austria and the Nordic countries. The diversity index measures how isolates are distributed into different types. The low diversities obtained from susceptibility testing are due to the fact that in most countries, a majority of the isolates were susceptible to all antibiotics tested and thus were assigned to one main type. A low diversity index based on PhP typing in a certain population means that the population contains many identical isolates and is thus an indication that certain clonal groups dominate in the population. The isolates from France and Sweden show the lowest diversity with PhP typing. In both these countries, CT48 was the dominating PhP type (Tables 1, and 4).
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Conclusions |
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Some PhP types were more frequently involved in urinary tract infections than others. All types exhibited diverse antimicrobial susceptibility patterns. This together with the dominating role of resistance to the older drugs ampicillin and trimethoprim suggests that these represent clonal groups that have been around for a long time. Our data did not indicate that antimicrobial resistance in E. coli causing urinary tract infections in Europe was primarily caused by the dissemination of specific clones carrying specific antimicrobial resistance, neither within nor between countries. However, there was good evidence that some types were prone to be decidedly more resistant than others and that some types were more common in some countries than others.
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Acknowledgements |
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