72 MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY Zdrav Vestn | January – February 2024 | Volume 93 | https://doi.org/10.6016/ZdravVestn.3448 Copyright (c) 2024 Slovenian Medical Journal. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Antimicrobial susceptibility of bacteria causing uncomplicated cystitis in Slovenia Občutljivost za antibiotike pri povzročiteljih nezapletenega cistitisa v Sloveniji Helena Ribič,1 Ingrid Berce,2 Tjaša Žohar Čretnik,3 Urška Dermota,4 Marica Lugovski,3 Tatjana Harlander5 Abstract Background: Acute uncomplicated cystitis in women is a common reason for antibiotic treatment in the outpatient clinic of family doctors. Microbiological diagnostics is not indicated, treatment is generally empirical and based on guidelines and recommendations. Therefore, we do not have representative data on the incidence and sensitivity of pathogens from regular laboratory work. Special targeted research is needed. Methods: We present the results of a prospective study conducted in four regional laboratories of the Centre for Medical Microbiology NLZOH between 15 September 2017 and 31 December 2019. We included 110 female patients with sus- pected acute uncomplicated cystitis who completed a questionnaire and submitted urine samples for microbiological examination. Results: The result of urine examination was positive in 79 patients (71.8%). Among all isolates, the most common bacte- rium was Escherichia coli (74%), followed by Staphylococcus saprophyticus (10%), and others. The susceptibility of E. coli to trimethoprim with sulfamethoxazole was 85.5%, to nitrofurantoin 98.4%, and to fosfomycin 100%. S. saprophyticus isolates were susceptible to the tested antibiotics except for fosfomycin, against which the bacterium is intrinsically re- sistant. The total susceptibility of all isolates was 85.8% - 88.2% for trimethoprim with sulfamethoxazole, 88.1% - 95.2% for nitrofurantoin, 73.8% - 80.8% for fosfomycin, 72.6% for ampicillin and amoxicillin, 97.7% for cefadroxil and 98.9% for amoxicillin with clavulanate acid. A comparison with the results of monitoring bacterial resistance as part of the regular work of microbiological laboratories shows significant differences for most of the tested antibiotics. Conclusion: The research results show a relatively good susceptibility of the causative agents of acute uncomplicated cystitis to oral antibiotics, the only exception being amoxicillin. 1 Oddelek za medicinsko mikrobiologijo Kranj, Nacionalni laboratorij za zdravje, okolje in hrano, Kranj, Slovenija 2 Oddelek za medicinsko mikrobiologijo Nova Gorica, Nacionalni laboratorij za zdravje, okolje in hrano, Nova Gorica, Slovenija 3 Oddelek za medicinsko mikrobiologijo Celje, Nacionalni laboratorij za zdravje, okolje in hrano, Celje, Slovenija 4 Inštitut za mikrobiologijo in imunologijo, Medicinska fakulteta, Univerza v Ljubljani, Ljubljana, Slovenija 5 Oddelek za medicinsko mikrobiologijo Novo mesto, Nacionalni laboratorij za zdravje, okolje in hrano, Novo mesto, Slovenija Correspondence / Korespondenca: Helena Ribič, e: helena.ribic@nlzoh.si Key words: Escherichia coli; Staphylococcus saprophyticus; trimethoprim sulfamethoxazole; nitrofurantoin; fosfomycin Ključne besede: Escherichia coli; Staphylococcus saprophyticus; trimetoprim sulfametoksazol; nitrofurantoin; fosfomicin Received / Prispelo: 5. 5. 2023 | Accepted / Sprejeto: 26. 9. 2023 Cite as / Citirajte kot: Ribič H, Berce I, Žohar Čretnik T, Dermota U, Lugovski M, Harlander T. Antimicrobial susceptibility of bacteria causing uncomplicated cystitis in Slovenia. Zdrav Vestn. 2023;92(1–2):72–80. DOI: https://doi.org/10.6016/ZdravVestn.3448 eng slo element en article-lang 10.6016/ZdravVestn.3448 doi 5.5.2023 date-received 26.9.2023 date-accepted Microbiology, bacteriology, virology, mycology Mikrobiologija, bakteriologija, virologija, mi-kologija discipline Professional article Strokovni članek article-type Antimicrobial susceptibility of bacteria caus- ing uncomplicated cystitis in Slovenia Občutljivost za antibiotike pri povzročiteljih ne- zapletenega cistitisa v Sloveniji article-title Antimicrobial susceptibility of bacteria caus- ing uncomplicated cystitis in Slovenia Občutljivost za antibiotike pri povzročiteljih ne- zapletenega cistitisa v Sloveniji alt-title Escherichia coli, Staphylococcus saprophyti- cus, trimethoprim sulfamethoxazole, nitrofu- rantoin, fosfomycin Escherichia coli, Staphylococcus saprophyticus, trimetoprim sulfametoksazol, nitrofurantoin, fosfomicin kwd-group The authors declare that there are no conflicts of interest present. Avtorji so izjavili, da ne obstajajo nobeni konkurenčni interesi. conflict year volume first month last month first page last page 2024 93 1 2 72 80 name surname aff email Helena Ribič 1 helena.ribic@nlzoh.si name surname aff Ingrid Berce 2 Tjaša Žohar Čretnik 3 Urška Dermota 4 Marica Lugovski 3 Tatjana Harlander 5 eng slo aff-id Department for medical microbiology Kranj, Nacionalni laboratorij za zdravje, okolje in hrano, Kranj, Slovenia Oddelek za medicinsko mikrobiologijo Kranj, Nacionalni laboratorij za zdravje, okolje in hrano, Kranj, Slovenija 1 Department for medical microbiology Nova Gorica, Nacionalni laboratorij za zdravje, okolje in hrano, Nova Gorica, Slovenia Oddelek za medicinsko mikrobiologijo Nova Gorica, Nacionalni laboratorij za zdravje, okolje in hrano, Nova Gorica, Slovenija 2 Department for medical microbiology Celje, Nacionalni laboratorij za zdravje, okolje in hrano, Celje, Slovenia Oddelek za medicinsko mikrobiologijo Celje, Nacionalni laboratorij za zdravje, okolje in hrano, Celje, Slovenija 3 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Inštitut za mikrobiologijo in imunologijo, Medicinska fakulteta, Univerza v Ljubljani, Ljubljana, Slovenija 4 Department for medical microbiology Novo mesto, Nacionalni laboratorij za zdravje, okolje in hrano, Novo mesto, Slovenia Oddelek za medicinsko mikrobiologijo Novo mesto, Nacionalni laboratorij za zdravje, okolje in hrano, Novo mesto, Slovenija 5 Slovenian Medical Journallovenian Medical Journal 73 PROFESSIONAL ARTICLE Antimicrobial susceptibility of bacteria causing uncomplicated cystitis in Slovenia 1 Introduction Acute uncomplicated cystitis (AUC) is an infection in women aged 18 to 65 who do not have risk factors for a more severe course or complicated infection, such as an- atomical or functional abnormalities of the urinary tract, pregnancy, chronic diseases (e.g. diabetes) or treatments that lower the body’s defences, previous urinary tract sur- gery, urinary catheterisation, etc. (1). It is one of the most common reasons for women to visit their family doctor (1,2). Treatment of AUC is usually empirical. In Slovenia, ni- trofurantoin and fosfomycin are the recommended drugs, the same as provided in European guidelines (3,4). If the proportion of resistant Escherichia coli isolates is less than 20%, trimethoprim/sulfamethoxazole (TMP/SMX) can be used; we can also use amoxicillin with clavulanic acid or cephalosporins, regardless of the proportion of resistant E. coli isolates (1,3-5). Amoxicillin without clavulanic acid is discouraged by European guidelines because of the high proportion of resistant E. coli, and the use of fluoroquino- lones is very limited because of the potential for serious side effects (3,6). Microbiological diagnosis is not indicated in AUC, and infection treatment is empirical and based on na- tional recommendations. Exceptions are patients with atypical symptoms or if the initial antibiotic was ineffec- tive (3). Therefore, the routine work of microbiology lab- oratories does not provide us with representative data on the pathogens, their antimicrobial susceptibility, and the Izvleček Izhodišče: Akutni nezapleteni cistitis pri ženskah je pogost razlog za zdravljenje z antibiotiki v ambulanti družinskih zdrav- nikov. Mikrobiološke preiskave niso indicirane, zdravljenje je praviloma izkustveno in temelji na smernicah in priporo- čilih. Zato reprezentativnih podatkov o pojavnosti in občutljivosti povzročiteljev iz rednega dela laboratorijev nimamo. Potrebne so posebne usmerjene raziskave. Metode: V prispevku obravnavamo rezultate prospektivne raziskave, ki smo jo v štirih regionalnih laboratorijih Centra za medicinsko mikrobiologijo NLZOH izvedli v času od 15. 9. 2017 do 31. 12. 2019. V raziskavo smo vključili 110 bolnic s su- mom na akutni nezapleteni cistitis, ki so izpolnile vprašalnik in oddale vzorec seča za urinokulturo. Rezultati: Rezultat preiskave seča je bil pozitiven pri 79 bolnicah (71,8 %). Med vsemi izolati je bila najpogostejša bakterija Escherichia coli (74 %), sledila je bakterija Staphylococcus saprophyticus (10 %) in druge. Občutljivost E. coli za trimetop- rim s sulfometoksazolom je bila 85,5 %, za nitrofurantoin 98,4 % in za fosfomicin 100 %. Izolati S. saprophyticus so bili dobro občutljivi za vse testirane antibiotike razen za fosfomicin, proti kateremu je bakterija naravno odporna. Skupna občutljivost vseh izolatov je bila za trimetoprim s sulfometoksazolom 85,8– 88,2 %, za nitrofurantoin 88,1–95,2 %, za fosfo- micin 73,8–80,8 %, za ampicilin in amoksicilin 72,6 %, za cefadroksil 97,7 % in za amoksicilin s klavulansko kislino 98,9 %. Primerjava z rezultati spremljanja odpornosti bakterij v okviru rednega dela mikrobioloških laboratorijev kaže velike razli- ke pri večini testiranih antibiotikov. Zaključek: Rezultati raziskave kažejo razmeroma dobro občutljivost povzročiteljev akutnega nezapletenega cistitisa za peroralne antibiotike, izjema je le amoksicilin. appropriateness of the recommendations for empirical treatment. For this reason, studies involving patients with AUC have been carried out in several countries (5,7-9). The results showed large differences between countries, es- pecially in the proportions of antibiotic-resistant isolates. Thus, an AUC survey is needed in every country. To our knowledge, one has not yet been carried out in Slovenia. The Slovenian recommendations for TMP/SMX are based on data from routine work of microbiology laboratories, which includes diagnostics of all clinical samples; the data is available on the website of the Slovenian National An- timicrobial Susceptibility Testing Committee (SKUOPZ) (10). In September 2017, the Centre for Medical Microbi- ology of the National Laboratory of Health, Environment and Food (NLZOH), with the participation of physicians from general and family outpatient clinics in the regions of Nova Gorica and Gorenjska, started a targeted prospective study involving patients with a clinical picture that met the definition of AUC. The design and implementation of the study followed the example of studies carried out in Euro- pean countries (7-9). In April 2018, the study was extend- ed to the region of Novo mesto, and two months later, to the region of Celje (11). This paper presents the results of the tests carried out until the end of the study, i.e., from 15 September 2017 to 31 December 2019. The study results were compared with the results of resistance monitoring in samples from routine work of microbiological laboratories. 74 MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY Zdrav Vestn | January – February 2024 | Volume 93 | https://doi.org/10.6016/ZdravVestn.3448 Bacteria number prevalence Escherichia coli 62 0.74 Staphylococcus saprophyticus 8 0.10 Proteus mirabilis 4 0.05 Streptococcus agalactiae 3 0.04 Klebsiella spp. 3 0.04 Citrobacter koseri 2 0.02 Enterococcus faecalis 2 0.02 Total 84 Table 1: Number and prevalence of isolates from urine samples in women with acute uncomplicated cystitis. 2 Material and methods This paper presents the results of the microbiological examination of urine samples from patients in four de- partments of the Centre for Medical Microbiology of the NLZOH in Nova Gorica, Kranj, Novo mesto, and Celje in the period from 15 September 2017 to 31 December 2019. Patients aged 18 to 65 years with AUC symptoms with- out factors for complicated infection were invited to partici- pate in the study at the time of examination in an outpatient clinic of family doctors. A total of 8 healthcare institutions participated: 6 health centres (ZD Nova Gorica, ZD Radovl- jica, ZD Tržič, ZD Kranj, ZD Trebnje, ZD Celje), the Vitali- ja General Medicine Outpatient Clinic and the RR Private Health Centre. There were 115 patients willing to partici- pate. Five patients did not meet the eligibility criteria; thus, 110 were included in the study. At the time of examination, patients signed a consent form and completed a question- naire with demographic data, data on signs and symptoms of infection, and exclusion criteria for AUC. The physician excluded complicated infections and upper urinary tract infections and completed the questionnaire with the antibi- otic prescribed at the time of examination (12). Before the start of treatment, patients submitted a clean- catch midstream urine sample for testing. The samples were examined using the semi-quantitative urine culture meth- od with standard procedures, and the results were discussed according to the European guidelines (13,14). In the study, we treated 110 patients who met the eligibil- ity criteria for inclusion. The patients were aged 18–65 years, with a mean age of 42.6 years. We treated 76 patients (69.1%) in the laboratory in Kranj, 19 patients (17.3%) in Nova Gori- ca, 11 patients (10%) in Celje, and 4 patients (3.6%) in No- vo mesto. Only isolates from the 79 patients with positive urine culture results were included for further analysis of the pathogens and their antimicrobial susceptibility. Antimicrobial susceptibility was determined by the disk diffusion method, and the results were interpreted accord- ing to the European Committee on Antimicrobial Suscep- tibility Testing (EUCAST) guidelines (15-17). According to the EUCAST guidelines, the result for nitrofurantoin is only valid for uncomplicated urinary tract infections; for Entero- bacteriaceae, the interpretation is only valid for E. coli, for enterococci only for Enterococcus faecalis, for staphylococci only for Staphylococcus saprophyticus, and for beta-haemo- lytic streptococci only for Streptococcus agalactiae (15-17). Under the EUCAST guidelines, interpretation for oral fosfo- mycin (fosfomycin trometamol) is only valid for uncompli- cated urinary tract infections, and there is no interpretation for other infections. Up to and including 2020, the interpre- tation was given for all Enterobacteriaceae, and from 2021 onwards, due to stricter criteria only for the E. coli species (15-18). There is no interpretation for fosfomycin for other bacteria (E. faecalis, S. agalacatiae, etc.), and S. saprophyticus is intrinsically resistant to fosfomycin (15-19). In some cases, the clinical efficacy of the antibiotic is not sufficiently known, so interpretation of the result according to EUCAST guidelines is not possible; these combinations of bacteria/ antibiotic were not tested: for E. faecalis, the ef- ficacy of TMP/SMX is not known; for Klebsiella spp. and Citrobacter spp., there is insufficient data to support the effi- cacy of nitrofurantoin, for Enterobacteriaceae other than E. coli, there is insufficient data to support the efficacy of oral fosfomycin. In order to establish the efficiency of particular antibiot- ics (expressed as the ratio of the number of isolates suscep- tible to a particular antibiotic to the number of all isolates), we looked at the prevalence of each bacterial species and grouping among all isolates and the proportion of each spe- cies or grouping that is susceptible to a particular antibiotic. The contribution to the susceptibility of a bacterial species is the product of the prevalence and the proportion of anti- microbial susceptibility of isolates of that species. For exam- ple, the proportion of E. coli among all isolates in the study was 0.738; the susceptibility to nitrofurantoin was 100%, which means that E. coli contributes the product of 0.738 and 100%, i.e., 73.8%, to the susceptibility to nitrofurantoin. For bacteria/antibiotic combinations for which the clinical efficacy is not sufficiently known and for which there is no interpretation, but according to the EUCAST documents, the effect is possible, and the proportion of susceptibility is indicated in a range of susceptibility from 0% to 100% (20-22). A positive opinion for conducting the study was ob- tained from the National Medical Ethics Committee of the Republic of Slovenia (No 0120-455/2016-2, KME 40/09/16). 75 PROFESSIONAL ARTICLE Antimicrobial susceptibility of bacteria causing uncomplicated cystitis in Slovenia Legend: yrs – years. * The proportion of isolates relative to the number of patients with a positive urine culture in percentage. The number of isolates is small, so proportions should be treated with caution. Ag e gr ou p N o. o f pa tie nt s N o. a nd pr op or tio n (% ) o f pa tie nt s w ith p os iti ve ur in al ys is N o. o f is ol at es to ta l No. and proportion of isolates by species E. coli N (%)* S. saprophyticus N (%)* S. agalactiae N (%)* P. mirabilis N (%)* Others N (%)* 18-30 yrs 25 21(84.0) 22 12 (57.1) 6 (28.6) 0 2 (9.5) 2 (9.5) 31-40 yrs 27 19(70.4) 20 16 (84.2) 1 (5.3) 1 (5.3) 0 2 (10.5) 41-50 yrs 22 12(54.5) 13 12 (100.0) 0 0 1 (8.3) 0 51-65 yrs 36 27(75.0) 29 22 (81.5) 1 (3.7) 2 (7.4) 1 (3.7) 3 (11.1) Total 110 79(71.8) 84 62 (78.5) 8 (10.1) 3 (3.8) 4 (5.1) 7 (8.9) Table 2: Number and proportion of isolates from urine samples in women with acute uncomplicated cystitis according to age groups. Legend: * According to the EUCAST guidelines, the result for ampicillin is also valid for amoxicillin. Antibiotic No. of tested E. coli isolates Proportion of E. coli isolates susceptible to the antibiotic % Proportion of E. coli isolates resistant to the antibiotic trimethoprim/sulfamethoxazole 62 85.5 14.5 nitrofurantoin 62 98.4 1.6 fosfomycin trometamol 58 100 0 norfloxacin 53 98.1 1.9 ciprofloxacin 62 98.4 1.6 ampicillin (and amoxicillin)* 62 72.6 27.4 amoxicillin with clavulanic acid – interpretation for uncomplicated cystitis 62 98.4 1.6 amoxicillin with clavulanic acid – interpretation for systemic infections 62 95.2 4.8 cefadroxil 58 100 0 cefuroxime axetil 62 100 0 cefixime 58 100 0 Table 3: Antimicrobial susceptibility and resistance of E. coli isolates from urine samples in women with acute uncomplicated cystitis. 3 Results The result of microbiological urinalysis was positive in 79 patients (71.8%) and negative in 24 patients (21.8%); in 7 patients (6.4%) three or more different types of bacteria were isolated, which was defined as probable contamina- tion of the sample at the time of collection; the investigation was completed without an antibiogram. Among the positive samples, 74 cases (93.7%) had one species of bacteria, and five cases (6.3%) had two species each. 76 MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY Zdrav Vestn | January – February 2024 | Volume 93 | https://doi.org/10.6016/ZdravVestn.3448 Legend: (1) Interpretation for uncomplicated cystitis. (2) The isolate is intrinsically resistant to the antibiotic. (3) In the EUCAST guidelines, there are no criteria for the interpretation of the antibiogram result, the efficacy of the antibiotic for the bacterium is not (sufficiently) known, the outcome of the treatment cannot be predicted, but according to the EUCAST documents, the effect is possible, the sensitivity is indicated in the interval (20-22). (4) The results for ampicillin, amoxicillin and amoxicillin with clavulanic acid are inferred from the result for benzylpenicillin. (5) The result for ampicillin is also valid for amoxicillin with clavulanic acid. (6) In the EUCAST guidelines, there are no criteria for the interpretation of the antibiogram result; the efficacy of the antibiotic for the bacterium is not sufficiently known and is not recommended for treatment (22). Bacteria / antibiotic N o of is ol at es Pr ev al en ce o f t he ba ct er ia l s pe ci es o r gr ou p Proportion of susceptible strains among all isolates in percent ni tr of ur an to in tr im et ho pr im / su lfa m et ho xa zo le am pi ci lli n an d am ox ic ill in am ox ic ill in w ith cl av ul an ic a ci d fo sf om yc in tr om et am ol ce fa dr ox il ce fu ro xi m e ax et il ce fix im e ci pr of lo xa ci n Escherichia coli 62 0.738 72.6 63.1 53.6 72.6(1) 73.8 73.8 73.8 73.8 72.6 Staphylococcus saprophyticus 8 0.095 9.5 9.5 9.5 9.5 (5) 0(2) 9.5 9.5 9.5 9.5 Streptococcus agalactiae 3 0.036 3.6 3.6 3.6 (4) 3.6(4) 0(6) 3.6 3.6 3.6 0(6) Proteus mirabilis 4 0.048 0(2) 3.6 3.6 4.8 0–4.6(3) 4.8 4.8 4.8 2.4 Klebsiella spp. 3 0.036 0–3.6(3) 3.6 0 3.6 0(6) 3.6 3.6 3.6 3.6 Citrobacter koseri 2 0.024 0–2.4(3) 2.4 0 2.4 0 –2.4(3) 2.4 0(6) 2.4 2.4 Enterococcus faecalis 2 0.024 2.4 0–2.4(3) 2.4 2.4(5) 0(6) 0(6) 0(6) 0(6) 2.4 Total 84 1 88.1–95.2 85.8 – 88.2 72.6 98.9 73.8 – 80.8 97.7 95.3 97.7 92.9 Table 4: Antimicrobial susceptibility of isolates from urine samples in women with acute uncomplicated cystitis. The number and prevalence of identified AUC patho- gens are shown in Table 1, while the number and propor- tion of isolates by age group are presented in Table 2. E. coli was the prevalent bacterium among the isolates (62 isolates, 74%), followed by S. saprophyticus (8 isolates, 10%) and P. mirabilis (4 isolates, 5%), with the proportion of the remain- ing bacteria being less than 5%. In all age groups, E. coli was predominant. In the age group of 18 to 30 years, the propor- tion of E. coli was the smallest, 57.1%, and the proportion of S. saprohyticus was the largest, 28.6%. In the other age groups, the proportion of E. coli exceeded 80%, and the pro- portion of S. saprohyticus was 5% or less. The mean age of E. coli patients was 44 years, and of S. saprophyticus patients, 30 years. Antimicrobial susceptibility is shown in Tables 3 and 4. The susceptibility of E. coli strains to TMP/SMX was 85.5%, to nitrofurantoin 98.4%, and to fosfomycin trometamol (oral fosfomycin) 100%. E. coli strains and other Enterobac- teriaceae with extended-spectrum beta-lactamases (ESBLs) were not detected. For E. coli, 42 isolates (67.7%) were susceptible to all tested antibiotics. Resistance to one antibiotic was found in 12 isolates (19.4 %). Resistance to two or more antibiotics was found in 8 isolates (12.9%): 6 isolates (9.7%) were resis- tant to both ampicillin and TMP/SMX, one isolate (1.6%) was resistant to both ampicillin and amoxicillin with clavu- lanic acid (interpretation for uncomplicated cystitis), and one isolate (1.6%) was resistant to TMP/SMX, ciprofloxacin and norfloxacin. All S. saprophyticus isolates (8 isolates) were susceptible to all tested antibiotics: TMP/SMX, nitrofurantoin, cipro- floxacin, cefoxitin, ampicillin, and tetracycline. The result for ampicillin also holds for amoxicillin and amoxicillin with clavulanic acid (17). All three isolates of Streptococcus 77 PROFESSIONAL ARTICLE Antimicrobial susceptibility of bacteria causing uncomplicated cystitis in Slovenia Three studies: 1) susceptibility monitoring of isolates from all clinical samples in 2017 in the context of the SKUOPZ (10), 2) data from urine isolates of patients of both genders aged 15–65 from the routine work of two microbiology laboratories of the NLZOH in 2016 (23), and 3) the results of the study on uncomplicated cystitis (women aged 18–65; 15 September 2017 to 31 December 2019). Legend: NDA – no data available ESBL – extended-spectrum beta-lactamases. Study SKUOPZ 2017 urine samples from both genders (2016) acute uncomplicated cystitis (2017–2019) No. of E. coli isolates 17,990–24,272 565 62 antibiotic proportion of susceptible strains in percent nitrofurantoin 99 99.6 98.4 fosfomycin trometamol NDA NDA 100 trimethoprim/sulfamethoxazole 70.2 62.7 85.5 ciprofloxacin 78.1 86.2 98.4 amoxicillin with clavulanic acid – interpretation for uncomplicated cystitis 89.4 92.4 98.4 amoxicillin with clavulanic acid – interpretation for systemic infections 79.9 82.3 95.2 proportion of ESBL strains in per cent 8.3 3.5 0 Table 5: Comparison of the proportions of antibiotic-susceptible E. coli in three studies. agalactiae were susceptible to TMP/SMX, nitrofuranto- in, ampicillin, amoxicillin, tetracycline, and vancomycin. There is no interpretation for fosfomycin and ciprofloxacin in S. agalactiae; the antibiotics are ineffective in this species (15-17). E. faecalis isolates were susceptible to nitrofuranto- in, ciprofloxacin, ampicillin, amoxicillin, and vancomycin. Among the Klebsiella spp. isolates, Citrobacter koseri and P. mirabilis (9 isolates in total), the proportion of susceptible strains was 88.9% for TMP/SMX, 85.7% for ciprofloxacin, 77.8% for norfloxacin, and 33.3% for ampicillin and amox- icillin; all tested strains were susceptible to the other anti- biotics, i.e. amoxicillin with clavulanic acid and oral ceph- alosporins (cefadroxil, cefuroxime axetil, and cefixime). P. mirabilis species are intrinsically (naturally) resistant to nitrofurantoin, whereas there is no interpretation for Kleb- siella spp. and Citrobacter spp. (15-18). Table 4 shows the overall susceptibility of all isolates to a specific antibiotic, taking into account the prevalence of each bacterial species or grouping among all isolates and the proportion of susceptibility of each species or grouping to a specific antibiotic. For bacteria naturally (intrinsically) resistant to a particular antibiotic (P. mirabilis to nitrofuran- toin and S. saprophyticus to fosfomycin), zero percent are indicated for the proportion of susceptible strains. The overall susceptibility of all isolates was 85.8%– 88.2% for TMP/SMX, 88.1%–95.2% for nitrofurantoin, 73.8%–80.8% for fosfomycin, 72.6% for ampicillin and amoxicillin, 97.7% for cefadroxil, and 98.9% for amoxicillin with clavulanic acid. Table 5 compares the E. coli susceptibility results of the present study with the results of the 2017 SKUOPZ suscep- tibility monitoring and the results of the routine work of NLZOH in 2016 (urine testing of patients of both genders aged 15 to 65) (10,23). The SKUOPZ monitoring includes bacterial susceptibility results from the routine work of mi- crobiology laboratories. It includes all clinical samples, and it is impossible to separate results from individual samples (e.g., urine) (10). 4 Discussion An average of one in two women will experience AUC at least once in their lifetime, with infections often recur- ring (24-26). AUC is thus a common reason for antibiotic treatment in primary healthcare (26). The infection is usu- ally treated empirically with as narrow-spectrum antibiotic as possible, which acts on the most common pathogens of AUC. The drugs of choice are nitrofurantoin and fosfomy- cin trometamol. An alternative treatment is TMP/SMX, provided that the proportion of resistant E. coli strains is less than 20% (1,3-5). If the above antibiotics cannot be pre- scribed to the patient or are poorly tolerated by the patient, 78 MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY Zdrav Vestn | January – February 2024 | Volume 93 | https://doi.org/10.6016/ZdravVestn.3448 AUC can be treated with beta-lactam antibiotics: amox- icillin with clavulanic acid or cephalosporins. These are broad-spectrum antibiotics that have an adverse ecological impact (3). Amoxicillin without clavulanic acid is discour- aged by European guidelines due to the high proportion of resistant E. coli (3). The European Medicines Agency has se- verely restriced the use of fluoroquinolones in recent years due to the severe side effects observed, and they can only be used to treat AUC in exceptional cases where treatment with another effective antibiotic is not possible (6). As microbiological testing is not indicated in patients with AUC, with a few exceptions, data from routine work of microbiology laboratories is not helpful in planning em- pirical treatment and monitoring the appropriateness of the recommendations for the treatment of AUC. Periodic targeted examinations of AUC patients are needed. To our knowledge, the present study conducted at the NLZOH from 15 September 2017 to 31 December 2019 is the first AUC study in Slovenia. Previously, results of urine samples for the diagnosis of urinary tract infections were collected periodically from routine laboratory work. The results from routine work of laboratories mainly include samples from patients with complicated upper and lower urinary tract infection, from patients with recurrent infection or from patients in whom initial treatment of urinary tract infec- tion has not been effective (23,27-29). Urine samples from patients with AUC are very rare in the routine work of mi- crobiology laboratories and cannot be distinguished from other samples. In the study, we found 84 bacterial isolates in 79 (71.8%) patients with a positive urine culture. E. coli was the most frequently detected bacterium (62 isolates; prevalence among all isolates 0.74), followed by S. saprophyticus (8 iso- lates; prevalence 0.10). Other bacteria were found in smaller numbers. The results are similar to those of studies carried out in other countries (7-9). In the ARESC study, involving 9 European countries and Brazil, the E. coli prevalence in 2009 was 68.1% in Austria, 72.6% in Italy, 78.8% in Hunga- ry, and ranged from 72.6% to 83.8% in the other participat- ing countries; the mean range was 76.7%. The proportion of S. saprohyticus was 2.2% in Austria, 0% in Italy and Hunga- ry, and ranged from 0% to 5.6% in other countries, with an average of 3.6% (9). The differences between the countries are relatively large and are probably mainly due to the dif- ferences in the patients’ age. Microbial susceptibility was relatively good: for E. coli, it was 98.4% for nitrofurantoin, 85.5% for TMP/SMX, 100% for oral fosfomycin, and the tested oral cephalosporins (ce- fadroxil, cefixime and cefuroxime-acetyl), 98.4% for amox- icillin with clavulanic acid as interpreted for uncomplicated urinary tract infections and ciprofloxacin. According to the EUCAST guidelines, cefadroxil, cefixime and oral cefurox- ime are only suitable for treating uncomplicated urinary tract infections (15-18). The results of a study conducted in Austria in 2007 and 2008 were very similar: the resistance of E. coli strains to nitrofurantoin was 0.7%, to fosfomycin 0.7%, to TMP/SMX 14.4%, and to ciprofloxacin 4.1% (8). However, compared with the large international ARESC study, the proportion of susceptibility in Slovenia was high- er. The ARESC study included 2,315 E. coli strains, with a 70.5% susceptibility to TMP/SMX, 95.2% susceptibility to nitrofurantoin, 98.1% susceptibility to fosfomycin, 91.8% susceptibility to ciprofloxacin, 45.1% susceptibility to am- picillin, and 82.1% susceptibility to amoxicillin with clavu- lanic acid (9). AUC is an infection occurring in the home environ- ment in otherwise healthy women, and the susceptibili- ty of pathogens is expected to be higher than in patients seen in the routine work of microbiology laboratories. This was confirmed by comparing the E. coli susceptibility re- sults of the present study with the results of susceptibility monitoring of isolates from all clinical samples in 2017 in the context of the SKUOPZ and the results of the survey of patients of both genders aged 15–65 from the routine work of the NLZOH in 2016 (10,23). Relatively large differences in susceptibility were found for TMP/SMX, ciprofloxacin, amoxicillin with clavulanic acid, and the proportion of E. coli strains with ESBLs. There was no data from routine lab- oratory work for fosfomycin trometamol; the susceptibility of E. coli in the present AUC study was 100%. S. saprophyticus isolates were susceptible to all antibi- otics included in the study except fosfomycin trometamol, to which this bacterium is intrinsically resistant (18). The number of isolates was small (8), so we are cautious in our interpretation. As S. saprophyticus is mainly responsible for causing AUC and very rarely for other urinary tract infec- tions, results from the routine work of microbiology labo- ratories are also suitable for susceptibility testing. In a study in which 174 S. saprophyticus isolates were tested from uri- nary tract samples in three microbiology laboratories of the NLZOH from 1 January 2016 to 31 December 2018, suscep- tibility was found to be from 98% to 100% for TMP/SMX, from 96% to 100% for nitrofurantoin, and from 93% to 96% for ampicillin; the results for ampicillin are also valid for amoxicillin (29). In order to determine the suitability of each antibiotic for the empirical treatment of AUC, the overall suscepti- bility of all isolates to oral antibiotics was determined. The overall susceptibility of all isolates was 85.8%–88.2% for TMP/SMX, 88.1%–95.2% for nitrofurantoin, 73.8%–80.8% for fosfomycin trometamol, 72.6% for ampicillin and amox- icillin, 98.9% for amoxicillin with clavulanic acid, 97.7% for 79 PROFESSIONAL ARTICLE Antimicrobial susceptibility of bacteria causing uncomplicated cystitis in Slovenia cefadroxil and cefixime, 95.3% for cefuroxime axetil, and 92.9% for ciprofloxacin. Due to their narrow spectrum of action and lower impact on microbial ecology than other antibiotics, nitrofurantoin, fosfomycin trometamol, and TMP/SMX have a significant advantage over cefadroxil, amoxicillin with clavulanic acid, cefuroxime axetil, and cefixime. Fosfomycin is a broad-spectrum intravenous antibiotic that has historically been used in combination with other antibiotics for treating pneumonia, other lower respiratory tract infections, upper respiratory tract infections, osteo- myelitis, and numerous other infections. The fosfomycin derivative, fosfomycin trometamol, is suitable for the oral treatment of AUC caused by E. coli. The interpretation of results for fosfomycin trometamol was available in the EUCAST guidelines for all Enterobacteriaceae up to and including 2020. However, it was discontinued in 2021 due to stricter requirements for evidence of efficacy. Oral fos- fomycin appears to be insufficiently effective for other bac- teria, or there is insufficient evidence of clinical efficacy (22). Susceptibility to fosfomycin trometamol was relatively good in this study. The exception is the age group of 18 to 30 years, in which the proportion of S. saprophyticus was relatively high (28.6%). Therefore, fosfomycin trometamol is less suitable for treating ANC in this age group.Ampicil- lin and amoxicillin for the empirical treatment of urinary tract infections are discouraged by the EUCAST due to the high proportion of resistant E. coli. Fluoroquinolones (cip- rofloxacin and others) are highly effective, but the Europe- an Medicines Agency has severely restricted their use due to severe and potentially permanent side effects; in AUC, treatment with fluoroquinolones is limited to cases where no other effective antibiotic is available (6). Treatment with fluoroquinolones also significantly impacts the human mi- crobiota and leads to the selection of resistant bacteria. Sci- entists have shown that ciprofloxacin treatment reduces the absolute number of Enterobacteriaceae in the gut microbi- ota and increases the proportion of ciprofloxacin-resistant Enterobacteriaceae. Colonisation with ciprofloxacin-resis- tant Enterobacteriaceae was also found in people who lived in the same household as ciprofloxacin-treated patients. No increase in the frequency of ciprofloxacin-resistant or ni- trofurantoin-resistant strains was detected in nitrofuranto- in-treated patients (30). A limitation of the present study is the small number of patients treated and, consequently, the long duration of the research and the low number of isolates of each spe- cies, except E. coli isolates. Family doctors in Slovenia have a heavy workload and find it challenging to take on addition- al responsibilities, which was a key barrier to participation in the study. Another reason family doctors gave was that there were not as many AUC patients in outpatient clinics as expected. The study was conducted in four regions, with the high- est proportion of patients seen in the Gorenjska region (69.1%). Due to the small number of isolates and so as to monitor trends and establish potential differences between regions, it would be reasonable to repeat the study and in- clude more or maybe even all regions in the country. 5 Conclusion The results of our study have shown that E. coli was the most frequently detected bacterium in patients with acute uncomplicated cystitis (74%), followed by S. saprophyticus (10%), and the proportion of other bacteria was 5% or less. The susceptibility of E. coli to trimethoprim- sulfamethox- azole was 85.5%, to nitrofurantoin 98.4%, and to fosfomy- cin 100%. S. saprophyticus isolates were susceptible to all tested antibiotics except fosfomycin, to which the bacte- rium is naturally resistant. The overall susceptibility of all isolates was 85.8%–88.2% for trimethoprim- sulfamethox- azole, 88.1%–95.2% for nitrofurantoin, 73.8%–80.8% for oral fosfomycin, 72.6% for ampicillin and amoxicillin, and 98.9% for amoxicillin with clavulanic acid. We believe that the results provide a sound basis for revising the national recommendations for the treatment of acute uncomplicated cystitis and will make an important contribution to the ra- tional use of antibiotics in Slovenia. Conflict of interest None declared. Acknowledgements We want to thank all the participating family doctors from ZD Radovljica, ZD Kranj, ZD Tržič, ZD Nova Gori- ca, ZD Trebnje and ZD Celje, the Vitalija General Medi- cine Outpatient Clinic, and the RR Private Health Centre for their cooperation, efforts and support: Tanja Leskovar, Metka Čepar, Maja Petrovič Šteblaj, Polona Žuber, Tina Tomšič, Alice Kikel, Damijana Pogačnik Peternel, Romana Pintar, Špela Grilc, Ana Skok, Andreja Štular, Dragana Pa- jčin Sarjanović, Metka Munih, Mateja Fišer, prim. Tatjana Primožič, Andreja Golnar, Simona Grasselli, Špela Jenkole, Simona Kajba Veninšek, Marija Petek Šter, and Jana Zajc. We would also like to thank the other participants from the above healthcare institutions who contributed in any way to the implementation of the study and all the employees in the microbiology laboratories of the NLZOH in Kranj, Nova Gorica, Celje, and Novo mesto. 80 MICROBIOLOGY, BACTERIOLOGY, VIROLOGY, MYCOLOGY Zdrav Vestn | January – February 2024 | Volume 93 | https://doi.org/10.6016/ZdravVestn.3448 References 1. Gupta K, Hooton TM, Naber KG, Wullt B, Colgan R, Miller LG, et al. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: A 2010 update by the Infectious Diseases Society of America and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis. 2011;52:103-29. DOI: 10.1093/cid/ciq257 PMID: 21292654 2. Car J, Marinko T. Zdravljenje nezapletene okužbe sečnega mehurja pri ženskah v družinski medicini. Zdrav Vestn. 2003;72:79-83. 3. Logar M, Nadrah K, Lindič J, Taskovska M, Smrkolj T, Beović B. Antibiotično zdravljenje okužb sečil v Sloveniji. In: Beović B, Lejko Zupanc T, Tomažič J, et al. Stopenjska diagnostika in zdravljenje pogostih okužb. Infektološki simpozij 2017. Ljubljana. 29–31st October 2020; Ljubljana: Klinika za infeckcijske bolezni in vročinska stanja; 2017. 4. Čižman M, Beović B. Kako predpisujemo protimikrobna zdravila v bolnišnicah. Ljubljana: Sekcija za protimikrobno zdravljenje Slovenskega zdravniškega društva; 2013. 5. Kahlmether G. An international survey of the antimicrobial susceptibility of pathogens from uncomplicated urinary tract infections: the ECO·SENS Project. J Antimicrob Chemother. 2003;51(1):69-76. DOI: 10.1093/jac/ dkg028 PMID: 12493789 6. European Medicines Agency. Disabling and potentially permanent side effects lead to suspension or restrictions of quinolone and fluoroquinolone antibiotics. Amsterdam: EMA; 2022 [cited 30 May 2022]. Available from: https://www.ema.europa.eu/en/documents/press- release/disabling-potentially-permanent-side-effects-lead-suspension- restrictions-quinolone-fluoroquinolone_en.pdf. 7. Kahlmether G, Menday P. Cross-resistance and associated resistance in 2478 Escherichia coli isolates from the Pan-European ECO·SENS Project surveying the antimicrobial susceptibility of pathogens from uncomplicated urinary tract infections. J Antimicrob Chemother. 2003;52(1):128-31. DOI: 10.1093/jac/dkg280 PMID: 12805266 8. Kamenski G, Wagner G, Zehetmayer S, Fink W, Spiegel W, Hoffmann K. Antibacterial resistances in uncomplicated urinary tract infections in women: ECO·SENS II data from primary health care in Austria. BMJ Infect Dis. 2012;12:222. DOI: 10.1186/1471-2334-12-222 PMID: 22989349 9. Schito GC, Naber KG, Botto H, Palou J, Mazzei T, Gualco L, et al. The ARESC study: an international survey on the antimicrobial reistance of pathogens involved in uncomplicated urinary tract infections. Int J Antimicrob Agents. 2009;34:407-13. DOI: 10.1016/j.ijantimicag.2009.04.012 PMID: 19505803 10. Štrumbelj I, Pirš M, Berce I, Bombek Ihan M, Fišer J, Golle A, et al. Pregled občutljivosti bakterih za antibiotike - Slovenija 2017. Ljubljana: Inštitut za mikrobiologijo in imunologijo; 2022 [cited 2022 Jun 16]. Available from: https://imi.si/wp-content/uploads/2022/03/skoupz_porocilo_2017_ CIP.pdf. 11. Ribič H, Dermota U, Štrumbelj I, Grmek Košnik I, Sarjanović L, Berce I, et al. Preliminarni rezultati spremljanja občutljivosti za antibiotike pri akutnem nezapletenem cistitisu. Ljubljana: eNBOZ; 2022 [cited 2022 Jun 16]. Available from: https://www.nijz.si/sites/www.nijz.si/files/uploaded/ enboz_julij-avgust_0.pdf. 12. Ribič H, Dermota U, Štrumbelj I, Grmek Košnik I, Berce I, Harlander T. Nezapletene okužbe sečil v Sloveniji. In: Pirš M, Maver-Vodičar P, ur. 9. Likarjev simpozij: Okužbe sečil: [zbornik predavanj]. Ljubljana. Med Razgl. 2019;58:39-46. 13. European Confederation of Laboratory Medicine (ECLM)European Urinalysis Guidelines. Scan J clin Lab Invest Suppl. 2000;231:1-86. PMID: 12647764 14. Hooton TM, Roberts PL, Cox ME, Stapleton AE. Voided midstream urine culture and acute cystitis in premenopausal women. N Engl J Med. 2013;369(20):1883-91. DOI: 10.1056/NEJMoa1302186 PMID: 24224622 15. The European Committee on Antimicrobial Susceptibility Testing. The Breakpoint tables for interpretation of MICs and zone diameters Version 7.1. EUCAST. 2017. Växjö: EUCAST; 2022 [cited 2022 Jun 6]. Available from: http://www.eucast.org. 16. The European Committee on Antimicrobial Susceptibility Testing. The Breakpoint tables for interpretation of MICs and zone diameters Version 8.0. EUCAST. 2018. Växjö: EUCAST; 2022 [cited 2022 Jun 6]. Available from: http://www.eucast.org. 17. The European Committee on Antimicrobial Susceptibility Testing. The Breakpoint tables for interpretation of MICs and zone diameters Version 9.0. EUCAST. 2019. Växjö: EUCAST; 2022 [cited 2022 Jun 6]. Available from: http://www.eucast.org. 18. The European Committee on Antimicrobial Susceptibility Testing. The Breakpoint tables for interpretation of MICs and zone diameters Version 11.0. EUCAST. 2021. Växjö: EUCAST; 2022 [cited 2022 Jun 6]. Available from: http://www.eucast.org. 19. The European Committee on Antimicrobial Susceptibility Testing. The Intrinsic resistance and exceptional phenotypes, Expert Rules version 3.1. EUCAST. 2016. Växjö: EUCAST; 2022 [cited 2022 Jun 6]. Available from: http://www.eucast.org. 20. The European Committee on Antimicrobial Susceptibility Testing. Nitrofurantoin: Rationale for the clinical breakpoints, version 1.0, 2010. Växjö: EUCAST; 2023 [cited 2023 Mar 15]. Available from: http://www. eucast.org. 21. The European Committee on Antimicrobial Susceptibility Testing. Trimethoprim – sulpha metoxazole. Rationale for the clinical breakpoints, version 1.0, 2017. Växjö: EUCAST; 2023 [cited 2023 Mar 15]. Available from: http://www.eucast.org. 22. The European Committee on Antimicrobial Susceptibility Testing. Fosfomycin trometamol: Rationale for the clinical breakpoints, version 1.0, 2013. Växjö: EUCAST; 2023[ cited 2023 Mar 15]. Available from: http:// www.eucast.org. 23. Ribič H, Lugovski M, Dermota U, Kavka D. Novosti v mikrobiološki diagnostiki okužbe sečil. In: Govc Eržen J. Aktualno v družinski medicini: zbornik predavanj. 7. Majhnov dan. Celje. 16. 11. 2017; Ljubljana: Zavod za razvoj družinske medicine; 2017. 24. Donnenberg M. Uncomplicated cystitis--not so simple. N Engl J Med. 2013;369(20):1959-60. DOI: 10.1056/NEJMe1312412 PMID: 24224629 25. Foxman B, Barlow R, D’Arcy H, Gillespie B, Sobel J. Urinary tract infection: self-reported incidence and associated costs. Ann Epidemiol. 2000;10(8):109-15. DOI: 10.1016/s1047-2797(00)00072-7 PMID: 11118930 26. Foxman B. Recurring urinary tract infection: incidence and risk factors. Am J Publ Health. 1990;80(3):331-3. DOI: 10.2105/ajph.80.3.331 PMID: 2305919 27. Križan-Hergouth V, Logar M. Diagnostika in etiologija okužb sečil v Slovenij. In: Beovič B, Lejko-Zupanc T, Tomažič J, ur. Stopenjska diagnostika in zdravljenje pogostih okužb. Infektološki simpozij 2017. Ljubljana. oktober 2017; Ljubljana: Sekcija za protimikrobno zdravljenje SZD. 28. Ribič H, Dermota U, Novak D, Harlander T, Kavčič M, Sarjanović L, et al. Odpornost povzročiteljev okužb sečil v Sloveniji. In: Beovič B, Strle F, 1949-, Čižman M, ur. Infektološki simpozij 2006. Zbornik predavanj. Ljubljana. 2006; Ljubljana: Sekcija za kemoterapijo SZD; 2006. 29. Maršič K, Jurišević Dodič A, Ribič H, Kavka D, Rak M, Lugovski M. Pomen koagulazno negativnih stafilokokov pri okužbah sečil. In: Pirš M, Maver- Vodičar P, ur. 9. Likarjev simpozij: Okužbe sečil: [zbornik predavanj]. Ljubljana. Med Razgl. 2019;58:95-101. 30. Stewardson AJ, Vervoort N, Adriaenssens N, Coenen S, Godycki- Cwirko M, Kowalczyk A, et al. Effect of outpatient antibiotics for urinary tract infections on antimicrobial resistance among commensal Enterobacteriaceae: a multinational prospective cohort study. Clin Microbiol Infect. 2018;24(9):972-9.