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V prvem delu magistrskega naloge smo zbrali in analizirali podatke o odpornosti klinično pomembnih anaerobnih bakterij izoliranih v obdobju 2018-2020 v Sloveniji. Za anaerobne izolate, ki so bili pridobljeni iz vseh področnih oddelkov Centra za medicinsko mikrobiologijo Nacionalnega laboratorija za zdravje, okolje in hrano, smo preverili odpornost proti penicilinu, klindamicinu, metronidazolu, imipenemu in kombinaciji amoksicilin-klavulanska kislina. Podatke o minimalnih inhibitornih koncentracijah, interpretiranih v skladu z najnovejšim standardom EUCAST, smo zbrali v tabeli v programu Excel in s pomočjo standarda CLSI M39 pripravili kumulativni antibiogram anaerobnih bakterij. Največji delež bakterij je bil odporen proti penicilinu (40 %) in klindamicinu (24 %). Najbolj odporni bakterijski sevi spadajo v rodove Bacteroides, Prevotella in Parabacteroides. Ugotovili smo, da ostaja delež odpornih, klinično pomembnih anaerobnih bakterij v zadnjih letih v Sloveniji nespremenjen, in da je primerljiv z deležem po svetu. V drugem delu naloge smo validirali mikrodilucijsko metodo za ugotavljanje protimikrobne odpornosti, MICRONAUT-S Anaerobes (Merlin Diagnostika, Berlin, Nemčija) z izbranim naborom izolatov anaerobnih bakterij iz posameznih najpogostejših klinično pomembnih skupin. Naš namen je bil ugotoviti, ali je omenjena metoda primerna za rutinsko diagnostiko v kliničnih mikrobioloških laboratorijih. Ugotovili smo, da je metoda MICRONAUT-S primerljiva s standardno gradient-difuzijsko metodo z uporabo Etestov in zato primerna za uporabo v rutinski diagnostiki. In the first part of the study we collected and analysed antimicrobial resistance data of clinically important anaerobic bacteria in Slovenia isolated in the years 2018, 2019, and 2020. The anaerobic izolates were acquired from the laboratories of the National Laboratory of Health, Environment and Food. We checked the resistance to penicillin, clindamycin, metronidazole, imipenem, and amoxicillin-clavulanic acid. We then collected minimal inhibitory concentration values interpreted according to the latest EUCAST standard edition. We analysed and presented cumulative antimicrobial susceptibility test data in Excel according to the latest CLSI M39 standard edition. The highest resistance rates were for penicillin and clindamycin (40 % and 20% total resistance of tested isolates, respectively). The most resistant isolates were Bacteroides, Prevotella, and Parabacteroides. We found that resistance rates in anaerobes in Slovenia have remained stable in recent years and are comparable to global resistance rates. In the second part of the study, we validated the MICRONAUT-S Anaerobes microdilution method (Merlin Diagnostics, Berlin, Germany) for antimicrobial resistance testing. We tested some of the most clinically important anaerobic bacterial isolates. The aim of this study was to determine whether the MICRONAUT-S Anaerobes microdilution method is suitable for rutine diagnostics in clinical microbiology laboratories. We found that the MICRONAUT-S Anaerobes microdilution method is comparable to the standard gradient-diffusion Etest method and suitable for use in rutine diagnostics.

Odpornost proti antibiotikom med anaerobnimi bakterijami je močno narasla v zadnjih tridesetih letih. Najobsežnejša slovenska raziskava o protimikrobni odpornosti anaerobnih bakterij iz leta 2015 je pokazala, da je odpornost proti amoksicilinu s klavulansko kislino pri najpogosteje izolirani skupini B. fragilis znašala 8 %, odpornost proti imipenemu pa 1 %. Podatkov o občutljivosti za ostale betalaktamske antibiotike v kombinaciji z zaviralci betalaktamaz ni, saj se v rutinski diagnostiki pri nas testiranje zanje ne izvaja. Zato smo se odločili, da v našem magistrskem delu z gradient difuzijskim testom preverimo občutljivost izbranih anaerobnih bakterij, iz rodu Bacteroides in Parabacteroides, za amoksicilin s klavulansko kislino, piperacilin s tazobaktamom in ceftolozan s tazobaktamom. Skupno smo testirali 120 prospektivno zbranih izolatov (104 izolati rodu Bacteroides, 16 izolatov rodu Parabacteroides), ki smo jih izolirali iz kužnin in 87 izbranih izolatov iz zbirke IMI z znano odpornostjo proti amoksicilinu s klavulansko kislino (77 izolatov rodu Bacteroides, 10 izolatov rodu Parabacteroides). Odpornost prospektivno zbranih izolatov rodu Bacteroides za amoksicilin s klavulansko kislino je bila 17,3 %, med izolati rodu Parabacteroides je bilo odpornih 31,3 % testiranih izolatov. Odpornost izolatov rodu Bacteroides proti piperacilinu s tazobaktamom je znašala 11,5 %, za izolate rodu Parabacteroides pa kar 43,8 %. Odstotek odpornosti pri izbranih izolatih z znano odpornostjo proti amoksicilinu s klavulansko kislino je bil še višji in je za izolate rodu Bacteroides znašal 58,4 %, za izolate rodu Parabacteroides pa 50 %. Ker o ceftolozanu s tazobaktamom še ni smernic za interpretacijo mejnih vrednosti, smo določili distribucijo vrednosti MIK. Za prospektivno zbrane izolate je bila vrednost MIK50 16 μg/mL, MIK90 pa 256 μg/mL. Vsi izbrani izolati rodu Parabacteroides z znano odpornostjo proti amoksicilinu s klavulansko kislino so imeli vrednost MIK 256 μg/mL, medtem ko je bil razpon vrednosti za izolate rodu Bacteroides od 1,5 do 256 μg/mL. Antibiotic resistance among anaerobic bacteria has risen sharply in the last thirty years. The most extensive Slovenian research about antimicrobial resistance of anaerobic bacteria from 2015 showed that resistance to amoxicillin/clavulanic acid of the most frequently isolated B. fragilis group was 8%, and the resistance to imipenem was 1%. There is no data about susceptibility for the other beta-lactam antibiotics in combination with beta-lactamase inhibitors as testing for them is not performed in our routine diagnostics. For this reason, we decided to test the susceptibility of selected anaerobic bacteria from the genus Bacteroides and Parabacteroides to amoxicillin/clavulanic acid, piperacilin/tazobactam, and ceftolozan/tazobactam with a gradient diffusion method. We tested a total of 120 prospectively collected clinically significant anaerobic bacteria (104 isolates of the genus Bacteroides, 16 isolates of the genus Parabacteroides) and 87 selected isolates from the IMI collection with known resistance to amoxicillin/clavulanic acid (77 isolates of the genus Bacteroides, 10 isolates of the genus Parabacteroides). The resistance of prospectively collected Bacteroides isolates to amoxicillin/clavulanic acid was 17.3%, and 31.3% for the Parabacteroides isolates. The resistance of isolates of the genus Bacteroides to piperacillin/tazobactam was 11.5%, and among isolates of the genus Parabacteroides resistance was as high as 43.8%. The percentage of resistance of the selected isolates with known resistance to amoxicillin with clavulanic acid was even higher: 58.4% for Bacteroides isolates and 50% for isolates of the genus Parabacteroides. As there are no guidelines for interpretive breakpoints for ceftolozane/ tazobactam, we only determined MIC distribution. For prospectively collected isolates, the MIC50 value was 16 μg/mL, and MIC90 was 256 μg/mL. All selected isolates of the genus Parabacteroides with known resistance to amoxicillin/clavulanic acid had a MIC value of 256 μg/mL, while the value range for isolates of the genus Bacteroides ranged from 1.5 to 256 μg/mL.

Svrha: Svrha studije bila je utvrditi ima li razlike u prisutnosti potencijalno patogenih anaerobnih mikroorganizama oko implantata i homolognog zuba kod pacijenata koji su nakon postavljanja dentalnih implantata bili upućeni u individualni pristup u održavanju oralne higijene. Materijal i postupci: U istraživanju je sudjelovalo 30 ispitanika (10 muškaraca i 20 žena) prosječne dobi 49,6 godina (22 – 78 godina). Implantati su bili protetički opskrbljeni metalkeramičkim krunicama prosječne starosti 5,26 godina (2 – 14 godina). Na kontrolnom pregledu parodontnom sondom zabilježeni su sljedeći indeksi i mjere: aproksimalni indeks plaka (API), indeks krvareće papile (PBI), dubina sondiranja parodontnih džepova (PD) i recesija gingive. Vestibularno se uzorkovala tekućina oko implantata i gingivalna sulkusna tekućina oko homolognog zuba na kontralateralnoj strani. Rezultati: Rezultati naše studije pokazali su pozitivan API na 30 % implantata, a na 70 % bio je negativan. Vrijednosti PBI-ja bile su identične vrijednostima API-ja. Izmjerena je prosječna retrakcija mukoze oko implantata od 0,15 mm i prosječna vrijednost dubine sondiranja oko implantata od 2,25 mm. Na homolognim zubima API je bio pozitivan na 78,3 % zuba, kao i PBI. Izmjerena je prosječna retrakcija gingive od 1,06 mm i prosječna vrijednost dubine sondiranja od 1,85 mm. U skupini od 30 ispitanika, anaerobne bakterije pronađene su kod njih 12 (40 %). Kod sedam ispitanika anaerobne bakterije izolirane su samo na implantatu, kod tri samo na homolognom zubu, a kod dva i na implantatu i na homolognom zubu. Zaključak: Zapaženo je više anaerobnih bakterija na implantatu u odnosu prema homolognom zubu., Objective: The objective of the study was to establish whether there is a difference in the presence of potentially pathogenic anaerobic microorganisms around the implant and the homologous tooth in implant-prosthetic patients who received individual information about maintaining their oral hygiene. Material and methods: The study included 30 subjects with dental implants and metal-ceramic crowns. A periodontal probe was used to record the approximal plaque index (API), the papilla bleeding index (PBI), the periodontal pocket probing depth (PD) and the gingival recession. The fluid around the implant and the gingival sulcus fluid around the homologous tooth on the opposite lateral side were sampled. Results: The results have shown a positive API and PBI on 30% of the implants and a negative one on 70% of the implants. The average mucosal retraction measured around the implants was 0.15 mm, and the average probing depth was 2.25 mm. The API and PBI were positive on 78.3% of the homologous teeth. The average gingival retraction measured was 1.06 mm, and the average probing depth was 1.85 mm. Anaerobic bacteria were found in 12 out of 30 subjects (40%). Anaerobic bacteria were isolated only on the implant in 7 subjects, only on the homologous tooth in 3 subjects and both on the implant and the homologous tooth in 2 subjects. Conclusions: Anaerobic bacteria were more abundantly present on implants than on homologous teeth.