Идентификација, антимикробна осетливост и молекуларна типизација на Clostridioides difficile

Abstract

Introduction Clostridioides difficile is one of the most important intra-hospital pathogens. This sporogenic anaerobic bacteria has commonly been isolated from feces, mostly from older hospitalized patients on antibiotics and has been associated with few clinical manifestations from diarrhea to pseudomembranous colitis. Aims Aims of this study were to investigate the presence of Clostridioides difficile in samples from suspected patients, to determine the toxicity of the strains directly from the samples and also from the cultures, to determine the genotype of the isolates, to determine their antibiotic susceptibility and eventual associations of such genotypic and phenotypic characteristics between the isolates. Material and methods All fecal samples accepted from 1.2015-1.2019 at the Institute of microbiology and parasitology, Medical faculty in order to diagnose Clostridioides difficile infection (CDI), were subject to immunochromatographic detection of glutamate dehydrogenase (GDH) antigen and toxins A and B of Clostridioides difficile. In order to cultivate them, same samples were planted on two plates: directly on Cycloserine-Cefoxitin-Fructose agar (CCFA) and on Columbia blood agar after doing the alcohol shock test. Such planted plates were incubated anaerobically for 48 hours on 370C in order to isolate Clostridioides difficile. Grown colonies were identified by characteristic macroscopic appearance and also microscopically by Gram staining. Definitive identification was done by using the automated system VITEK 2. Grown cultures were also used to determine the presence of toxins A and B with the same immunochromatographic kits. Eighty isolates of Clostridioides difficile from as many patients were collected from the cultures and were later typed using the PCR ribotyping method as the most used one in Europe for typing of this bacterium. The antimicrobic susceptibility towards the eight antibiotics: vancomycin, metronidazole (according to EUCAST breakpoints) and tetracycline, clindamycin, erythromycin, imipenem, ciprofloxacin and moxifloxacin (according to CLSI break points) by using the E test on all eighty isolates was also determined. Results Analisis of the 1380 fecal samples received from suspected patients for CDI at the Institute of microbiology and parasitology in the period of january 2015- january 2019, showed presence of the bacterium in 13% of the samples, while laboratory diagnosis of CDI (confirmed toxin in the feces) was confirmed in 12,1% of the samples. Number of fecal samples sent from different clinics requesting laboratory diagnosis of CDI wasn’t proportional with the number of positive diagnosis. The biggest such discrepancy was observed within samples from surgery clinics. Percentual difference in terms of isolating C. difficile between genders in the research sample (80 isolates) showed no statistical significance. Average age of the patients which the 80 isolates were taken from was 54. 56,25% from the patients were above 60 years old. None of the isolates were taken from infants younger than 1 year. Percentage of toxigenic strains among the isolates was 92,5%. In 93% of the toxigenic isolates both toxins (A and B) were detected and in 7% only toxin B was detected. Presence of only toxin A was not detected in any of the isolates. Ribotyping of the 80 isolates of C.difficile showed that they belong to 20 different ribotypes. The most common one was 001/072, with 40 % (32) of the isolates. Most of the isolates from this ribotype were from patients hospitalized at the surgery clinics and none of them was from patients outside of the clinical centar “Mother Theresa”. Ten isolates belonged to the ribotype 014/020(12,5%). Five isolates belonged to each of the ribotypes 002, 017 and 027. Ribotypes 005, 255/258, SLO 046 and SLO 047 were represented by 3 isolates each and ribotypes 003, 012, 015, 023, 046, 070, SLO 069, SLO 110, SLO 120, SLO 160, SLO 187 were represented by 1 isolate each. Isolates of all ribotypes produced bout of the toxins (A and B), except all isolates from ribotype 017 which produced only toxin B and all isolates from ribotypes SLO 046 and SLO 047 which were non toxigenic. All 80 of the C.difficile isolates in this study showed good susceptibility towards vancomycin and metronidazole. Resistance percentages towards tetracycline, clindamycin, erythromycin, imipenem, ciprofloxacin and moxifloxacin were 1,25%, 49%, 55%, 57%, 100% and 45% respectively. Highest antimicrobial resistance percentages were detected in isolates taken from patients of surgery clinics. Highest antimicrobial resistance percentages were detected in isolates belonging to the dominant ribotype 001/072 and hypervirulent ribotypes 017 and 027. Discussion and conclusions There is a selectivity on some of the clinics in the “Mother Teresa” complex in terms of sending specimens for laboratory diagnosis of CDI, resulting in underdiagnosis of this infection. Laboratory diagnosis of CDI should be based on the two-step algorithm, including detecting of GDH and toxins A and B of C.difficile , directly from the fecal samples. Additional searching of toxins from the culture of C. difficile, lowers the number (%) of false negative results nonsignificantly. Vancomycin and metronidazole should remain first option therapy for CDI. Therapy with clindamycin, erythromycin, imipenem, ciprofloxacin and moxifloxacin could be a risk factor for acquiring CDI. Patients on ciprofloxacin are at especially high risk. The most dominant ribotype in our hospitals is 001/072. Results mentioned above are indicating connection between ribotypes and antimicrobial resistance in C. Difficile. Acquiring antimicrobial resistance is one of the main contributors of distribution and mobility of ribotypes, especially in the hospital environment and also a big factor in emerging of new types. Tracking of these genotypic and phenotypic characteristics of the isolates can be of great epidemical value.