Author: Yau, Chong Yee Miranda
Title: New strategies for culture recovery, identification, and susceptibility testing of bacteroides fragilis group organisms
Advisors: Leung, Hang Mei Polly (HTI)
Degree: DHSc
Year: 2018
Subject: Hong Kong Polytechnic University -- Dissertations
Department: Faculty of Health and Social Sciences
Pages: 174 pages : color illustrations
Language: English
Abstract: The Bacteroides fragilis group (BFG) includes the most frequently isolated anaerobic bacteria. Routine culture plates are usually used for anaerobic culture investigation. The treatment for infection by anaerobes is mainly empirical, and routine antimicrobial susceptibility testing is not conducted except in the case of a serious or life-threatening infection. In recent years, the increasing resistance of these bacteria to several antibiotics has been reported, and this resistance has shown great variation in different geographic areas and at various institutions. This bacterial resistance may lead to treatment failure and adverse clinical outcomes. Aims: The aims of this study are to initiate a new strategy for culture recovery, identification, and susceptibility testing of B. fragilis group organisms to tackle the afore-mentioned drawbacks of existing practice. First, a novel B. fragilis selective (BFS) medium that can improve the sensitivity of recovery of B. fragilis from clinical samples is validated. Using a combination of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and CarbaNP test, the insertion sequence, (IS) element-activated cfiA gene in B. fragilis can be detected within several hours and can then guide therapy for a B. fragilis infection more rapidly. Finally, by adopting the tentative European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion method, the antimicrobial susceptibility pattern of B. fragilis group organisms in Hong Kong can be studied.
Methods: First, validation of a novel BFS medium was performed by prospective comparison with a routinely used medium (neomycin blood agar) on 1209 clinical specimens. Second, we used the matrix-assisted laser desorption ionization Biotyper system and ClinProTools software to generate support vector machine models that were then used to differentiate cfiA-positive (division II) and cfiA-negative B. fragilis (division I). The cfiA-positive B. fragilis were then tested by CarbaNP assay. The performance parameters of these tests were assessed by genotypic analysis as reference methods. Finally, a recently developed EUCAST disk diffusion method was used to measure the susceptibility of the 741 B. fragilis group and Parabacteroides species to six antibiotics. Results: Sixty B. fragilis isolates were detected on BFS agar, and 46 were detected on neomycin blood agar (McNemar's test, p = 0.008). The sensitivities of routine and BFS plates were 70.8% (46 of 65) and 92.3% (60 of 65), respectively. In this study, the CarbaNP test for detection of an IS element in the cfiA upstream region demonstrated 100% sensitivity, 80.4% specificity, a 75.0% positive predictive value, and a 100% negative predictive value. In our B. fragilis group collection, more than 90% of isolates were susceptible to metronidazole followed by imipenem and amoxicillin-clavulanate using the tentative disk breakpoints. The E-test confirmed that 8.2% (61 of 741) and 1.6% (12 of 741) of the isolates were not susceptible to imipenem and metronidazole, respectively. Significance: In conclusion, this novel BFS medium can support the growth of B. fragilis and facilitate its differential identification in clinical specimens especially in a mixed culture. This study reveals a newly developed strategy for rapid (within 3 hours) detection of B. fragilis with the IS element-activated cfiA gene using a combination of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and the CarbaNP test in a clinical laboratory. This study provides a pattern of antimicrobial susceptibility of the clinical isolates of B. fragilis and Parabacteroides species in Hong Kong and demonstrates the usefulness of the recently developed tentative EUCAST disk diffusion method for testing of resistance to imipenem, metronidazole, clindamycin, and moxifloxacin.
Rights: All rights reserved
Access: restricted access

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