Improved Detection of Biofilm-formative Bacteria by Vortexing and Sonication: A Pilot Study

Abstract

Bacteria such as staphylococci commonly encountered in orthopaedic infections form biofilms and adhere to bone implants and cements. Various methods to disrupt the biofilm and enhance bacterial detection have been reported. We will describe the effectiveness of vortexing and sonication to improve the detection of biofilmformative bacteria from polymethylmethacrylate by conventional quantitative bacterial culture and real-time quantitative PCR. We used a single biofilm-formative Staphylococcus aureus strain and 20 polymethylmethacrylate coupons as an in vitro biofilm model; four coupons were used for each of two control groups or three experimental sonication times (1, 5, and 30 minutes). Vortexing the cement without sonication increased the yield of adherent bacteria to a considerable extent. The combination of vortexing and sonication further enhanced the yield regardless of the duration of sonication. Quantitative conventional cultures correlated with quantitative PCR assay. The combination of vortexing and sonication to disrupt the bacterial biofilm followed by quantitative PCR and/or culture seems to be a sensitive method for detecting bacteria adherent to bone cement.

Does Dithiothreitol Improve Bacterial Detection from Infected Prostheses? A Pilot Study

“…We found limited data regarding reproducibility of methods used to dislodge bacteria from prosthetic implants [4,13]. Our data suggest that sonication and DTT treatment seem to provide reproducible bacterial counts, whereas high variability was observed for NAC and scraping.…”

“…Another problem the microbiologist faces is that the presence of bacteria in the biofilms makes them difficult to remove with traditional sampling techniques and subsequently to cultivate and identify. Sonication of explanted implants has notably improved sensitivity of microbiologic diagnosis of PJIs by dislodging bacteria from biofilm adhered to prosthetic surfaces, as documented in numerous studies [1,4,12,13,23,[28][29][30]. Since NAC exerts activity against bacterial biofilms [2,10,16,26,33], we presumed NAC and DTT could be used to remove bacteria from a prosthetic biofilm.…”

“…First, we evaluated four species, S aureus, P aeruginosa, S epidermidis, and E coli, and we did not include fastidious bacteria, such as the Haemophilus species, which has been reported as a frequent cause of PJIs [5], or anaerobic bacteria. S aureus and S epidermidis were chosen because they had been used in similar studies [4,13], being the most frequent pathogens involved in PJIs and known biofilm producers, whereas P aeruginosa and E coli were chosen as representative of gram-negative bacteria able to produce biofilm. Second, we used an in vitro model of bacterial biofilm, which could not fully reflect conditions occurring on clinical samples.…”

“…Bjerkan et al reported only range of bacterial counts, not means and SDs, therefore it is not possible to compare their data with ours. Kobayashi et al [13] reported means and SDs of bacterial counts after sonication and vortexing from which we extrapolated a CV% of approximately 2%. This value is less than the interassay variability we observed for S aureus, ranging from 2.8% to 3.5%, whereas it is greater than our intraassay CV% of 0.5% to 0.8%.…”

“…Given a frequency of false negatives of 7% to 20% and sensitivity of approximately 40% to 70% associated with tissue cultures [3,13,30], methods have been developed to dislodge the pathogens causing the infection from the biofilm [1,3,12,13,23,30]. Sonication reportedly yields rates of successful bacterial recovery of 70% to 100% compared with 10% to 100% when scraping the prosthetic surface [3] and sensitivity of approximately 65% to 80% depending on prior antibiotic therapy [13,30]. Combining vortexing and sonication has further improved an increase in sensitivity greater than 10% with respect to sonication only (78.5% versus 60.8%) [28,29].…”