Multilocus sequence analysis, biofilm production, antibiotic susceptibility and synergy tests of burkholderia species in patients with and without cystic fibrosis

Burkholderia spp. emerged as important pathogens in the airways of immunocompromised humans, especially those with cystic fibrosis (CF). Failure of identification with conventional techniques, high intrinsic resistance to most antibiotics and biofilm formation can cause treatment of these infections very difficult. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with with routine microbiological methods and multilocus sequence analysis (MLSA), to determine of the antibiotic resistance and synergies, and to evaluate biofilm formation of these isolates. A total of 38 Burkholderia spp. (25 CF, 13 non-CF) isolates identified and sequence types revealed by multilocus sequence analysis (MLSA). Sequence types of isolates were identified using the PubMLST database. Characteristics of biofilm formation of clinical isolates were evaluated by microplate method. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim-sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI (2017) guidelines. Synergy tests are performed by checkerboard method. Clinical isolates identified as B. cenocepacia (n=16), B. contaminans (n=11), B. gladioli (n=4), B. dolosa (n=4), B. multivorans (n=2) and B. seminalis (n=1). Sequence types of these isolates were determined as ST-19, ST-72, ST-102, ST-180, ST-270, ST-482, ST-602, ST-629, ST-740, ST-839 and ST-1392. Correct species-level identification of MALDI-TOF MS was %94-100 for all isolates when B. contaminans (%0) was excluded. Biofilm formation in these species was determined as 53% (n=20). There was no statistical difference in biofilm production among Burkholderia species and biofilm production rates between CF (56%, 14/25) and non-CF (46%, 6/13) Burkholderia isolates when examined separately. (Chi-square test, p > 0.05). Overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin of the isolates was 35%, 66%, 50% and 40%, respectively. In antibiotic resistance against Burkholderia spp., isolates obtained from CF patients were more susceptible to ceftazidime, but no significant difference was found for other antibiotics. Synergy has shown between meropenem and TMP-SXT at two isolates. Antagonism has detected at 15 isolates, 12 of them were between meropenem and ceftazidime, three of them were between ceftazidime and TMP-SXT. A variety of molecular approaches have been developed for the identification of closely related species and MLSA was found to be the most reliable method for Burkholderia isolates. Numerous resistance mechanisms may lead to higher resistance in this bacteria, whereas the antagonism between meropenem and ceftazidime in this study might be attributed to the expression of beta-lactamases. This study identifies the distinctness of sequence types between Burkholderia spp. isolated from CF and non-CF patient, provides a better understanding about the importance of biofilm formation for the infections with these bacteria and emphasizes the management of therapy should be driven by the antibiotic test results.