Introduction Clostridioides difficile is a pathogen toxin producer, responsible for opportunistic infections in patients treated with broad-spectrum antibiotics. The annual incidence of C. difficile associated diarrhea (CDAD), affecting hospitalized and immunocompromised patients, has showed a worldwide significant increase in recent decades. Since pathogens’ antibiotic resistance is rapidly changing under the therapies’ selective pressure, we assessed whether the antibiotic susceptibility and toxin profile of C. difficile strains circulating at Padua University Hospital has modified over a 14-years period. Materials and methods C. difficile strains were isolated from fresh stool samples of patients with CDAD between 2010 and 2018 (before fidaxomicin introduction in Padua Hospital) and between January 2023 and March 2024 at the Microbiology Unit of Padua University Hospital. Isolates were identified by MALDI-TOF analysis (99.9% confidence). Susceptibility to vancomycin, clindamycin, moxifloxacin, metronidazole, ertapenem, imipenem, meropenem, piperacillin/tazobactam, amoxicillin/clavulanic acid, ampicillin and penicillin was determined by broth microdilution method with a commercial kit (MICRONAUTS-S Anaerobes MIC, Bruker). Resistances were confirmed with the E-test method. Moreover, fidaxomicin sensitivity was tested by agar diffusion method (20 micrograms/disk). CLSI and EUCAST Breakpoint were used to define susceptibility profile. Furthermore, multiplex PCR and related electrophoretic runs on agarose gel were set up to define the toxigenic profile of the 67 strains. Results Thirty C. difficile strains from 2010-2018 and 37 from 2023-2024 were analyzed. All isolates resulted susceptible to amoxicillin/clavulanic acid, ertapenem, meropenem, piperacillin/tazobactam, metronidazole and vancomycin. While the resistance rate to moxifloxacin and clindamycin significantly decreased (p<0.01), the incidence of resistance to ampicillin significantly increased in strains isolated in 2023-2024 compared to 2010-2018 strains (p<0.05). Contrary to, incidence of resistance to imipenem and penicillin was unaffected. The inhibition zones of fidaxomicin ranged between 15 mm and 30 mm in the 2010-2018 strains, whereas in the 2023-2024 group the inhibition zones varied between 15 and 35 mm (20.48±0.5 versus 20.60±0.5, respectively, p= n.s.). Discussion and Conclusions This study shows a modification in the sensitivity/resistance profile of C. difficile clinical isolates at Padua University Hospital. Although all strains retain sensitivity to metronidazole and vancomycin, a significant increase of ampicillin resistance was revealed. Although a wide range of isolates’ sensitivity to Fidaxomicin was revealed, no changes in overall sensitivity to fidaxomicin was detected following introduction of this antibiotic in clinical practice. These data support the need of continuous antibiotic susceptibility monitoring for this nosocomial-acquired pathogen.
Introduzione Clostridioides difficile è un patogeno anaerobio produttore di tossine, responsabile di infezioni opportunistiche in pazienti trattati con antibiotici ad ampio spettro. L’incidenza annuale della diarrea associata a C. difficile (CDAD), che colpisce pazienti ospedalizzati e immunocompromessi, ha mostrato un aumento significativo a livello mondiale negli ultimi decenni. Poiché la resistenza agli antibiotici dei patogeni sta rapidamente cambiando sotto la pressione selettiva delle terapie, abbiamo valutato se la sensibilità agli antibiotici e il profilo delle tossine dei ceppi di C. difficile circolanti presso l'Ospedale Universitario di Padova si sono modificati in un periodo di 14 anni. Materiali e metodi Ceppi di C. difficile sono stati isolati da campioni di feci fresche di pazienti con CDAD tra il 2010 e il 2018 (prima dell'introduzione della fidaxomicina nell'Ospedale di Padova) e tra gennaio 2023 e marzo 2024 presso l'Unità di Microbiologia dell'Ospedale Universitario di Padova. Gli isolati sono stati identificati mediante analisi MALDI-TOF (confidenza al 99,9%). La sensibilità a vancomicina, clindamicina, moxifloxacina, metronidazolo, ertapenem, imipenem, meropenem, piperacillina/tazobactam, amoxicillina/acido clavulanico, ampicillina e penicillina è stata determinata mediante il metodo della microdiluizione in brodo con un kit commerciale (MICRONAUTS-S Anaerobes MIC, Bruker). Le resistenze sono state confermate con il metodo E-test. Inoltre, la sensibilità alla fidaxomicina è stata testata mediante il metodo di diffusione in agar (20 microgrammi/disco). CLSI ed EUCAST Breakpoint sono stati utilizzati per definire il profilo di suscettibilità. Inoltre, sono state effettuate PCR multiplex e relative corse elettroforetiche su gel di agarosio per definire il profilo tossigenico dei 67 ceppi. Risultati Sono stati analizzati trenta ceppi di C. difficile del periodo 2010-2018 e 37 del periodo 2023-2024. Tutti gli isolati sono risultati sensibili ad amoxicillina/acido clavulanico, ertapenem, meropenem, piperacillina/tazobactam, metronidazolo e vancomicina. Mentre il tasso di resistenza alla moxifloxacina e alla clindamicina è diminuito significativamente (p<0,01), l’incidenza della resistenza all’ampicillina è aumentata significativamente nei ceppi isolati nel 2023-2024 rispetto ai ceppi 2010-2018 (p<0,05). Al contrario, l’incidenza della resistenza all’imipenem e alla penicillina non è stata influenzata. Le zone di inibizione della fidaxomicina variavano tra 15 mm e 30 mm nei ceppi 2010-2018, mentre nel gruppo 2023-2024 le zone di inibizione variavano tra 15 e 35 mm (20,48±0,5 contro 20,60±0,5, rispettivamente, p= n.s.) . Discussione e conclusioni Questo studio mostra una modifica nel profilo di sensibilità/resistenza degli isolati clinici di C. difficile presso l'Ospedale Universitario di Padova. Sebbene tutti i ceppi mantengano la sensibilità al metronidazolo e alla vancomicina, è stato rivelato un aumento significativo della resistenza all'ampicillina. Sebbene sia stata rilevata un’ampia gamma di sensibilità degli isolati alla fidaxomicina, non è stato rilevato alcun cambiamento nella sensibilità complessiva alla fidaxomicina dopo l’introduzione di questo antibiotico nella pratica clinica. Questi dati supportano la necessità di un monitoraggio continuo della sensibilità agli antibiotici per questo patogeno acquisito in ambito nosocomiale.
Analisi del profilo tossigenico e di farmaco resistenza di ceppi di Clostridioides difficile isolati presso la UOC di Microbiologia dell'Azienda Ospedale Università di Padova. 2010 - 2018 versus 2022 - 2024
ASA'AD, SHIRIN
2022/2023
Abstract
Introduction Clostridioides difficile is a pathogen toxin producer, responsible for opportunistic infections in patients treated with broad-spectrum antibiotics. The annual incidence of C. difficile associated diarrhea (CDAD), affecting hospitalized and immunocompromised patients, has showed a worldwide significant increase in recent decades. Since pathogens’ antibiotic resistance is rapidly changing under the therapies’ selective pressure, we assessed whether the antibiotic susceptibility and toxin profile of C. difficile strains circulating at Padua University Hospital has modified over a 14-years period. Materials and methods C. difficile strains were isolated from fresh stool samples of patients with CDAD between 2010 and 2018 (before fidaxomicin introduction in Padua Hospital) and between January 2023 and March 2024 at the Microbiology Unit of Padua University Hospital. Isolates were identified by MALDI-TOF analysis (99.9% confidence). Susceptibility to vancomycin, clindamycin, moxifloxacin, metronidazole, ertapenem, imipenem, meropenem, piperacillin/tazobactam, amoxicillin/clavulanic acid, ampicillin and penicillin was determined by broth microdilution method with a commercial kit (MICRONAUTS-S Anaerobes MIC, Bruker). Resistances were confirmed with the E-test method. Moreover, fidaxomicin sensitivity was tested by agar diffusion method (20 micrograms/disk). CLSI and EUCAST Breakpoint were used to define susceptibility profile. Furthermore, multiplex PCR and related electrophoretic runs on agarose gel were set up to define the toxigenic profile of the 67 strains. Results Thirty C. difficile strains from 2010-2018 and 37 from 2023-2024 were analyzed. All isolates resulted susceptible to amoxicillin/clavulanic acid, ertapenem, meropenem, piperacillin/tazobactam, metronidazole and vancomycin. While the resistance rate to moxifloxacin and clindamycin significantly decreased (p<0.01), the incidence of resistance to ampicillin significantly increased in strains isolated in 2023-2024 compared to 2010-2018 strains (p<0.05). Contrary to, incidence of resistance to imipenem and penicillin was unaffected. The inhibition zones of fidaxomicin ranged between 15 mm and 30 mm in the 2010-2018 strains, whereas in the 2023-2024 group the inhibition zones varied between 15 and 35 mm (20.48±0.5 versus 20.60±0.5, respectively, p= n.s.). Discussion and Conclusions This study shows a modification in the sensitivity/resistance profile of C. difficile clinical isolates at Padua University Hospital. Although all strains retain sensitivity to metronidazole and vancomycin, a significant increase of ampicillin resistance was revealed. Although a wide range of isolates’ sensitivity to Fidaxomicin was revealed, no changes in overall sensitivity to fidaxomicin was detected following introduction of this antibiotic in clinical practice. These data support the need of continuous antibiotic susceptibility monitoring for this nosocomial-acquired pathogen.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/81470