Title: Antibacterial resistances in uncomplicated urinary tract infections in women: ECO * SENS II data from primary health care in Austria

Author's response to reviews Title: Antibacterial resistances in uncomplicated urinary tract infections in women: ECO*SENS II data from primary health care in Austria Authors: Gustav Kamenski (kamenski@aon.at) Gernot Wagner (gernot.wagner@meduniwien.ac.at) Sonja Zehetmayer (sonja.zehetmayer@meduniwien.ac.at) Waltraud Fink (waltraud.fink@gmx.at) Wolfgang Spiegel (wolfgang.spiegel@meduniwien.ac.at) Kathryn Hoffmann (kathryn.hoffmann@meduniwien.ac.at) Author's response to reviews: Dear Sir or Madam, Here are the details of the changes of our manuscript (MS: 9505271406706141) in response to the valuable comments of the two reviewers: First reviewer: MILAN CIZMAN: Version: 2 Date: 1 August 2012 1. Comment: Resistance of E. coli to nitrofurantoin and fosfomycin which are drugs of choice for empiric therapy of lower UTI were in both less than 1%. Indeed, the resistance of E. coli to nitrofurantoin and fosfomycin were both less than 1% and these two antibiotics are recommended in the new S3 guideline (Wagenlehner et al, Dtsch Arztebl Int 2011; 108 (24):415-23) for the treatment of uncomplicated UTI as first-line drugs but do GPs really prescribe these drugs for UTIs? For instance the Austrian ABSGROUP (Arzt&Praxis, Juni 2012, 986: Unkomplzierte Harnwegsinfekte aktuelles Therapiewissen im Überblick ) recommend pivmecillinam, amoxicillin/clavulanic acid and sultamicillin as first-line drugs. For amoxicillin/clavulanic acid the resistance rate gained by the ECO SENSE II study for Austria was 8.9% which is not yet alarming but a negative trend. We guess that in other countries other recommendations like the Austrian one exist and that, therefore, our study can promote further research for antibiotic resistances in the primary health care sector not only to check the currentness of guidelines but also to make the GPs think about their own prescribing behaviour. We added the following part into the Discussion section, second break, page 12: Fortunately, the resistance of E. coli to nitrofurantoin and fosfomycin were both less than 1% since these two antibiotics are recommended in the S3-guideline for the treatment of uncomplicated UTI as first-line drugs [22] but how is the prescribing behaviour of GPs and what do local guidelines recommend? For instance the Austrian ABSGROUP recommend pivmecillinam, amoxicillin/clavulanic acid and sultamicillin as first-line drugs [23]. For amoxicillin/clavulanic acid the resistance rate in the ECO SENSE II study for Austria was 8.9% which is not yet alarming but a negative trend. Therefore, 1

our study can promote further surveillance of antibiotic resistance, especially, in the primary health care sector not only to check the currentness of guidelines but also the make the GPs think about their own prescribing behaviour, not only in Austria. 2. Comment: The study is of national interest only. As already stated in our answer to comment 1, national resistance data can - together with the knowledge about current guidelines - be of international interest. For the GPs these data are important to reassess their prescribing behaviour. In addition, our data could motivate physicians from other countries to request resistance data from the ambulatory sector to be collected and implemented in their own national guidelines too. Moreover, antibiotic resistances are not limited to the borders of one country. An alarming situation in Austria could be an indicator for problematic situations in the surrounding countries as well. Especially, the comparison of the resistance rates of the ECO*SENS study - which was performed exclusively in the primary care sector for uncomplicated UTIs - with the resistance rates of the Austrian resistance Report 2008 (AURES) demonstrated that it is not possible to obtain comprehensive resistance data for special diseases with samples sent to the laboratories from the ambulatory sector by chance like it is conducted for the resistance report. To obtain comprehensive resistance data for uncomplicated UTIs that have to find their way into the guidelines, clearly structured studies have to be performed on a regular base. We think that to realise this fact is of main importance for other countries as well. To highlight this important finding - that was discussed by now only in the Discussion section - we made some adaptions in the text. Background, last sentence: In addition, these results were contrasted with the resistance data for E. coli in the Austrian resistance report 2008 (AURES) for the ambulatory sector. Methods, Data analysis, last sentence: Moreover, these results were contrasted with the resistance data for E. coli in the Austrian resistance report 2008 (AURES) for the ambulatory sector to show possible differences. Results, third subheading: Comparison of the results from ECO SENS, ECO SENS II and the results from the Austrian resistance report 2008 for E. coli for the ambulatory sector Table 3: We adapted the table by including the resistance data for E. coli in the Austrian resistance report 2008 (AURES) for the ambulatory sector into the table. Results, Comparison, last break, page 11: By contrasting the results of the ECO SENS II study with the results from the Austrian resistance report 2008 for the ambulatory sector, the resistance results in the Austrian resistance report 2008 were much higher. Ampicillin (39.8%), trimethoprim/sulphamethoxazole (24.6%) and ciprofloxacin (15.7%) were the ABs with the highest 2

resistance rates for E. coli but also mecillinan (12.2%), cefadroxil (8.5%) and nitrofurantoin (2.2%) showed much higher resistance rates compared to the ECO SENS II study. Discussion, page 13, last beak: In a routine primary health care setting, samples from the primary care sector are sent predominantly to a laboratory if a complicated UTI occurred. Therefore, these data do not fit as reference data for local or national guidelines. However, in a lot of countries data collected like these are the only available which should be questioned. To obtain comprehensive resistance data for uncomplicated UTIs that have to find their way into the guidelines, clearly structured studies have to be performed on a regular base. Conclusion, page 14, last break: It is not enough to sample and test all specimens coming to a laboratory from the ambulatory sector by chance to obtain comprehensive resistance data for the primary health care sector for special diseases. Structured studies have to be performed on a regular base. 3. Comment: Needs some English language corrections before being published. The revised manuscript was checked for English language again by the authors and, in addition, by another fluent English speaking person, Martina Gröblacher. She was added in the acknowledgement section. 1. Abstract, Background, page 2, second sentence: but these data are scarce for the Austrian primary health care sector. 2. Abstract, Conclusions, page 3, first sentence: The resistance data for E.coli in uncomplicated UTIs in women gained by this study are the most recent data for this disease in Austria at the moment. 3. Background, page 4, first beak: Approximately 80% of the cases with a significant positive urine culture for pathogens an infection with Escherichia coli (E.coli) can be expected; 4. Background, page 4, second break: Since UTIs are a frequent reason for prescribing antibiotics (AB) the decision which AB should be prescribed has to be considered prudently. 5. Background, page 4, second break: Therefore, it is necessary to gain susceptibility data 6. Background, page 4, third break: Some studies on this topic have been performed. One has been the ECO SENS project. 7. Background, page 5, third break: A total of 3,278 women (69.2%) had culture proven lower UTIs with E. coli responsible for 77% of all culture proven pathogens. 8. Methods, page 5, Patients and Investigational Centres, third sentence: The investigational centres that had to subscribe an informed consent form too, were located in three geographical regions in Austria each separated from the other by at least 150 km. 9. Methods, page 6, Inclusion and exclusion criteria, fourth sentence: Only patients with a symptom score of 2 were included in the study. 10. Discussion, page 11, first break, first sentence: The resistance data for E. coli in uncomplicated UTIs in 18-65 year old women gained by this study are the most recent data for this disease in Austria at this moment. 3

11. Discussion, page 11, first break, fourth sentence: The chance to conduct such a comparison was due to the number given by the centres... 12. Discussion, page 11, first break last sentence: Nevertheless, it has to be considered that this comparison is between two cross-sectional studies and, therefore, it has limitations which were discussed below. 13. Discussion, page 11, third break, third and fourth sentence: Resistance rates of mecillinam (0%), nitrofurantoin (0.7%), fosfomycin (0.7%), gentamycin (1.4%), cefotaxime (2.7%) and ceftazidine (2.7%) remained very low. Based on the literature we could confirm the persisting very low resistance rate for mecillinam. Second reviewer: TERESITA MAZZEI: Version: 2 Date: 13 July 2012 1. Comment: We would like to clarify our numbers: 1. All in all, a total of 313 patients were included in the study and they delivered 313 urine samples because they had a symptom score of 2 or more. 2. 200 (=64%) of the total of 313 urine samples showed a significant bacterial growth. 3. 50 (=16%) of the total of 313 urine samples showed a significant bacterial growth of other pathogens than E. coli and Klebsiella pneumoniae and were not further tested (e.g. Entobacteriacea and Staphylococcus saprophyticus). 4. 3 (=1%) of the total of 313 urine sample showed a significant growth of Klebsiella pneumoniae. 5. 147 (=47%) of the total of 313 urine sample showed a significant growth of E. coli. 6. The antimicrobial resistance data of one E. coli culture was missing; therefore, the resistance calculations took place with 146 E.coli specimens. This calculation is important to show the numbers and percentages of the E.coli specimens that occur in all samples of women with symptoms of an uncomplicated UTI and not only in samples with a significant culture growth. Also in other international publications like the ECO*SENSE I publication by Kahlmeter in the Journal of Antimicrobial Chemotherapy (2003);51,69-76, table 2 and result section, the percentages of the E.coli found were in relation to all samples (53.3%). Nevertheless, the percentages related to the positive cultures only are of importance and interest for the reader too, therefore, we added these results into table 1. Of course, the calculation of the resistance rates of E.coli took place in samples with a significant E.coli growth only (n=146) as stated in table 2. 1. We adapted figure 1 by describing the process in the flow-chart boxes more in detail: Box 1: 327 patients signed the informed consent form 4

Box3: 313 dip slides from primary care centers were sent to the central laboratory Box 4: 113 negative and mixed cultures were excluded from further analysis Box 5: 200 positive cultures were further analyzed Box 6: 50 cultures showed other pathogens than E. coli or K. pneumonia and were excluded from antimicrobial resistance testing Box 7: 147 E. coli and 3 K. pneumoniae cultures were tested for antimicrobial resistances Box 8: The antimicrobial resistance data of one E. coli culture was missing and excluded from analysis Box 9: 146 E. coli and 3 K. pneumoniae strains underwent complete antimicrobial susceptibility and MIC testing 2. We added in the Method section under Data Analysis, first break, the sentences: The prevalence of the bacteria were described first in relation to all included urine samples and then in relation to all significant positive cultures. Statistically significant differences in the frequency of E.coli in the different age subgroups were performed by using the Pearson s Chi- Square test (alpha 5%; CI 95%). The resistance rates for E.coli and K.pneumoniae were described for each of the 14 antibiotics by conduction descriptive statistical methods. 3. We adapted the text in the Result section under Patients characteristics pages 8 and 9 into: Figure 1 shows the flow chart for the sampling and analysing process for patients, urine samples and bacterial specimens for Austria. In Austria 23 primary care investigational centres recruited 327 patients of which 14 had to be refused due to the exclusion criteria. The 313 remaining patients had a mean age of 40.2 (SD 14.8) years and a mean total symptom score of 5.5 (SD 2.7). All 313 urine samples of these patients were included in the further analyses. Overall, there has been a significant bacterial growth in 64% (n=200) of the 313 urine samples. Table 1 shows the distribution of all culture test results, first, in relation to all 313 samples and, in addition, in relation to the 200 samples with bacterial growth. Concerning the two age cluster no statistically significant difference in the prevalence of E. coli in relation to all 313 samples (48.8% in age group 18-50 years vs. 44.0% in age group 51-65 years; p>0.05) or in relation to the 200 samples with a positive culture growth (75.2% vs. 71.3%; p>0.05) could be found. 4. Table 1: We adapted table 1 to describe the results more in detail: First, for the percentages of the bacteria related to all included samples and then for the percentages of the bacteria related to all positive cultures. In addition, we took the age group calculations out of table 1 and described them in the text (page 9, Result section, Patients characteristics, last break): Concerning the two age cluster no statistically significant difference in the prevalence of E. coli in relation to all 313 samples (48.8% in age group 18-50 years vs. 44.0% in age group 51-65 years; p>0.05) or in relation to the 200 samples with a positive culture growth (75.2% vs. 71.3%; p>0.05) could be found. 2. Comment: Table 2: This table shows samples with positive growth of E. coli (n=146) only because the resistance rates related to the 14 antibiotics can be calculated only with these data. 5

To clarify our calculations, a column with the absolute numbers in addition to the percentages was inserted. Since there are resistances against more than one antibiotic, the sum of the absolute numbers exceeds 146 which is explained in the legend now. 3. Comment: Figure 1: We adapted the text in the flow chart (see answer to comment 1, change 1) and adapted the heading with more detailed description: Flow chart for the recruiting, sampling and testing process for patients, urine samples and bacterial specimens Figure 2: We adapted the heading with more detailed description: Distribution of the 55 single and multi-resistant E. coli isolates. The bars represent the absolute numbers of isolates in which resistance against 1 to 10 different antibiotics were observed at the same time. The percentages indicate the distribution within this sample. In addition we revised the text in the Result section under Resistance Data, second break (page 9/10): In total, 91 (62.3%) of the 146 isolates were susceptible to all tested antibiotics, 16 (10.9%) isolates were resistant to one antibiotic, 12 (8.2%) were resistant to two, 8 (5.5%) isolates to three and 19 (13.1%) isolates to four and more antibiotics. Figure 2 describes the distribution of the 55 (37.7%) single and multi-resistant E. coli isolates. 4. Comment: The study is of limited interest. See answer to the second comment of reviewer 1. 5. Comment: Needs English language corrections before being published. See answer to the third comment of reviewer 1. We hope that these revisions meet your expectations and that the manuscript is now acceptable for publication. Sincerely yours, Kathryn Hoffmann, Gustav Kamenski and Gernot Wagner 6