Klinik Örneklerden İzole Edilen Haemophilus influenzae Suşlarının İn Vitro Antibiyotik Duyarlılıklarının EUCAST ve CLSI Kriterleri Doğrultusunda Değerlendirilmesi ve Beta-Laktam Direncinin Moleküler Analizi
Özet
Antibiotic resistance of Haemophilus influenzae isolates, that cause invasive and non-invasive infections, is increasing all over the world, particularly in beta-lactam group antibiotics. This study aimed to analyse the in vitro antibiotic susceptibility profiles of H. influenzae isolates according to the EUCAST and CLSI criteria, to examine the molecular mechanisms underlying resistance development against beta-lactam group antibiotics and to evaluate the concordance of EUCAST and CLSI results with the genomic analysis. Between January 2018 and June 2022, a total of 150 H. influenzae isolates, obtained from blood and respiratory tract samples, were included in the study. Identification of the isolates was performed by MALDI-TOF MS. Antimicrobial susceptibilities of ampicillin, amoxicillin-clavulanic acid, meropenem, cefotaxime, cefuroxime, tetracycline and trimethoprim-sulfamethoxazole were determined by EUCAST disc diffusion method. MICs of ampicillin and cefotaxime were determined by EUCAST broth microdilution method. In accordance with the EUCAST recommendations, the nalidixic acid screening test for fluoroquinolones was performed and the benzylpenicillin G screening test was performed according to the recommended flowchart for the detection of beta-lactam resistance mechanisms. The results were interpreted according to the EUCAST criteria. In addition, antibiotic susceptibility results of the isolates obtained using the disc diffusion method according to the CLSI criteria were extracted from the Laboratory Information Management System data. Beta-lactamase production was determined by the chromogenic nitrocefin disc. The presence of the blaTEM-1 and the blaROB-1 genes in beta-lactamase positive isolates was investigated by conventional PCR method. Mutations causing beta-lactam resistance were detected by sequence analysis of PCR products of the ftsI gene in the isolates determined as beta-lactamase negative ampicillin resistant (BLNAR) and beta-lactamase positive amoxicillin clavulanic acid resistant (BLPACR). The distribution of the H. influenzae isolates included to the study were as follows: 102 from sputum, 24 from bronchoalveolar lavage, 13 from deep tracheal aspiration and 11 from blood samples. According to the EUCAST disk diffusion method, 58.7% of the isolates were resistant to benzylpenicillin G, 35.3% to ampicillin, 32.7% to amoxicillin-clavulanic acid, 38.9% to cefuroxime, 1.3% to cefotaxime, 1.3% to meropenem, 6.7% to nalidixic acid, 2% to tetracycline, 37.6% to trimethoprim-sulfamethoxazole. EUCAST broth microdilution method revealed that 24.3% of the isolates were resistant to ampicillin and 4.7% to cefotaxime. While the resistance rate for ampicillin and amoxicillin-clavulanic acid was found to be higher in the antibiotic susceptibility tests performed according to the EUCAST, the resistance rate for tetracycline and trimethoprim-sulfamethoxazole was found to be higher in the susceptibility tests performed according to the CLSI. While cefotaxime susceptibility results were similar for cephalosporins, the resistance rate for cefuroxime was found to be significantly higher in EUCAST disk diffusion method. Susceptibility results for meropenem between the two guidelines were similar. Eighteen isolates were detected as beta-lactamase positive by the nitrocefin disc and all of them were found to be positive for the blaTEM-1 gene. Thirty-five (23.3%) of the isolates were identified as BLNAR and 16 (10.7%) as BLPACR. As a result of the sequence analysis of the ftsI gene performed on 49 of these 51 isolates, no mutation was detected in four isolates which were not included in the genotypic classification. In the sequence analysis, a total of 19 amino acid substitutions were detected in 18 positions and the most common mutations were determined as D350N, M377I, G490E, A502V and N526K. According to the genotypic classifications based on ftsI mutations, there was one isolate in group I, nine isolates in group IIa, 25 isolates in group IIb, two isolates in group IIc, three isolates in group IId, four isolates in group III like and one isolate in group III+. Of the 34 BLNAR isolates with resistance mutations, 31 were found to be low-level resistant BLNAR (group I and II), while three were found to be high-level resistant (group III). While nine of the 11 BLPACR isolates with resistance mutation were found to be low-level resistant BLPACR, two were found to be high-level resistant BLPACR. Detection of ftsI mutations associated with high-level BLNAR and BLPACR resistance reveals the need to review the performance of the nitrocefin test and antibiotic susceptibility tests used in routine laboratories in H. influenzae isolates.
