Evaluation of antibiotic susceptibility of haemophilus influenzae isolates

Objective: In this study, antibiotic susceptibility of 84 H.influenzae isolates grown in the cultures of lower respiratory tract samples sent to Ankara University School of Medicine İbn-i Sina Training and Research Hospital Central Microbiology Laboratory between 01.01.2020-01.06.2021 was investigated by disc diffusion method. It was aimed to evaluate the susceptibility results of imipenem and meropenem with disc diffusion and gradient strip test methods and to examine their compatibility with the reference method, the broth microdilution method. Materials and Methods: Lower respiratory tract samples were evaluated microscopically. Microscopically suitable samples were cultured on 5% sheep blood agar (SBA), MacConkey agar, and chocolate agar media. SBA and MacConkey agar plates were incubated at 37 oC in ambient air, chocolate agar plates were incubated in CO2 for 24-48 hours. Bacteria were identified by Gram stain and MALDI-TOF MS. Disk diffusion method was used to determine the antibiotic susceptibility of the isolates. Beta-lactamase enzyme production of the isolates was carried out using a chromogenic nitrocefin disc. The presence of the resistance mechanism of isolates against beta-lactam drugs was screened with a penicillin disc containing 1 unit of benzylpenicillin. Gradient strip test and broth microdilution test methods were used to determine the minimum inhibitory concentrations (MIC) of carbapenem group antibiotics. Results: In our study, more than half of the isolates were isolated from patients aged 50 years and older. Male patients in the study comprised 70.23% of all patients. Beta-lactamase negative ampicillin-susceptible (BLNAS) isolates were the most abundant phenotype with 45.23%. This was followed by beta-lactamase negative ampicillin-resistant (BLNAR) with 39.28%, beta-lactamase positive ampicillin-resistant (BLPAR) with 10.71%, and beta-lactamase positive amoxicillin-clavulanic acid resistant (BLPACR) isolates with 4.76%. In our study, resistance to imipenem, meropenem, and cefotaxime was not detected by the disc diffusion method. The highest resistance was seen in ampicillin with 54.76%. The lowest resistance was seen in quinolone group antibiotics with 13.09%. Resistance rates; cefuroxime 39.28%, amoxicillin/clavulanic acid 28.57%, ciprofloxacin, levofloxacin, and moxifloxacin 13.09%, tetracycline 16.66%, trimethoprim/sulfamethoxazole 29.76%. Disc diffusion and broth microdilution susceptibility results were found to be compatible with each other for imipenem and meropenem. The categorical agreement of the gradient strip test method and the broth microdilution method for meropenem was 100% (n=84), the essential agreement was 70.23% (n=59), while the categorical agreement for imipenem was 70.23% (n=59), the essential agreement was found to be 41.66% (n=35). The major error rate for imipenem was found to be 29.76%(n=25) In the study, in-zone growths were observed in 17 (20.23%) of the isolates tested for imipenem in the disk diffusion test. In the gradient strip test, a double zone with heteroresistance was observed in 12 of these isolates (70.58%). When the phenotype distributions of 17 isolates were examined, thirteen BLNAR, two BLPAR, one BLPACR, and one BLNAS isolate were found. The MIC values in BLNAR isolates >32 µg/ml in six isolates, 12 µg/ml in one isolate, 8 µg/ml in one isolate, 2 µg/ml in two isolates, 0.5 µg/ml in one isolate, 0.75 µg/ml in one isolate, and 0.094 µg/ml in one isolate. The MIC value was found to be >32 µg/ml in two BLPAR isolates, the MIC value was >32 µg/ml in one BLPACR isolate and 4 µg/ml in one BLNAS isolate. Conclusion: In our study, the highest antimicrobial resistance was determined as ampicillin and certain rates of resistance were detected to the routinely tested antibiotics. Resistance to imipenem, meropenem, and cefotaxime was not detected by the disc diffusion test. BLNAR isolates have started spreading all over the world, raising concerns about the development of resistance to beta-lactam group antibiotics. In our study, although no resistance was detected in either carbapenem with the broth microdilution method, 25 isolates were found resistant to imipenem by the gradient strip test. Consistent with previous studies, in our study, it was determined that the broth microdilution method is important in determining the carbapenem susceptibility of H.influenzae isolates, especially in terms of imipenem susceptibility. Considering the compatibility of the disc diffusion method with the broth microdilution method for imipenem and meropenem, the disc diffusion method maintains its importance in terms of routine laboratory applications. The appearance of heteroresistant isolates may be a warning for the emergence of resistant isolates. It is important to conduct surveillance studies in order to reveal resistance rates and mechanisms in H.influenzae isolates. Keywords: H. influenzae, imipenem, meropenem, broth microdilution, gradient strip test