Effects of different pretreatment methods on microbial yield from infected tissues


 Background: This study aimed to evaluate the effects of different pretreatment methods on the microbial yield in infected tissues. Methods: Strains of Staphylococcus aureus (SA), Escherichia coli (EC) and Candida albicans (CA) were used to construct single-surface, full-surface, and internal infection models in sterile pork tissue. Manual milling (MM), mechanical homogenization (MH), sonificated (SF), dithiothreitol (DTT), and direct culture (DC) were used to pretreat tissues, the microbial yield from different pretreatment methods were compared. At the same time, periprosthetic tissues collected intraoperatively from periprosthetic joint infection (PJI) patients were pretreated with the same methods. Results: The study showed that the microbial yield from MH pretreatment was significantly higher than that of MM (P <0.01) and SF pretreatment method (P <0.01). Furthermore, in the internal infection model, the microbial yield from MH group was also significantly higher than that of SF (P <0.01), DTT (P <0.01), and DC group (P <0.01). Moreover, the number of bacterial colonies obtained from periprosthetic tissues pretreated by MH was significantly higher than pretreated by other pretreatment methods (P = 0.004). Conclusions: The effects of MH and DTT in microbial yield were significantly higher than that of DC, SF and MM, and these methods can be used to process multiple tissue samples at the same time, which might further improve the sensitivity of infectious disease.


Effects of different pretreatment methods on microbial yield from infected tissues
Background Periprosthetic joint infection (PJI) is a serious complication after joint arthroplasty, which brings a heavy economic burden to patients and society 1 . Microbial culture is essential for the diagnosis and treatment of PJI 2,3 . At present, the traditional laboratory diagnostic methods include synovial uid, sonication uid and periprosthetic tissues, but in many cases, synovial/sonication are insu cient, resulting in poor sensitivity of microbial culture. But it is relatively easy to obtain tissue samples intraoperatively, but the positive rate is not high if tissues are not pretreated properly 4 . Due to different various levels toughness of tissues and different distribution of bacteria on the tissue surface, if proper pretreatment is not performed, it will affect the release and recovery of pathogenic bacteria. Therefore, optimizing the pretreatment method of tissue specimens to make full use of PJI tissue samples is of great signi cance for improving the sensitivity of microbial culture as a diagnostic tool. China) for 15 min at room temperature, specimens were transferred to a 2 mL EP tube and 1 mL of LB liquid medium was added to seal the tubes. SF group: tissues were transferred to 2 mL EP tubes with 1 mL LB culture medium, vortexed and shaken for 30 s and placed in an ultrasonic cleaner (Wuxi Woxin Instrument Co., Ltd., Jiangsu, China), sonicated at 40 Hz for 5 min, vortexed for 30 s, centrifuged at 4 000 r/min for 15 min, the supernatant was discarded, and the pellet was resuspended in sterile saline. DC group: tissues were transferred to 2 mL EP tubes with 1 mL LB culture medium and vortexed for 15 min.
After pretreatment, the inoculated specimens were centrifuged at 5500 rpm/min after the above four tissue processing methods for 20 s to spin down impurities. After centrifugation, 100 μL of each solution was inoculated onto Columbia blood agar plates (Thermo Fisher Scienti c, USA) and cultured overnight at 37 °C in a biochemical incubator (Shanghai Qixin Scienti c Instrument Co., Ltd, China). And CFUs were obtained and calculated.

Various tissues pretreatment methods performed on periprosthetic tissues from PJI
A total of 30 original specimens were collected from 8 PJI patients according to the American Society for Musculoskeletal Infection (MSIS) criteria 10 . Each original specimen was divided into 5 equal portions (each = 50 mg) and treated with the above methods (MH, MM, DTT, SF, DC) to obtain inoculated specimens.
One hundred microliters of each of the inoculated samples was inoculated onto Columbia blood agar plates (Qingdao Haibo Biotechnology Co., Ltd., HBPM0153) and CDC anaerobic blood agar plates (Qingdao Haibo Biotechnology Co., Ltd., HBPM16) under aerobic and anaerobic conditions, respectively.
Samples were incubated in a biochemical incubator (Shanghai Qixin Scienti c Instrument Co., Ltd., China) at37 ℃ for 14 days, and colony growth was observed 2, 4, 7, and 14 days after incubation. If there was colony growth, the CFU per ml were calculated.
This study was approved by institutional review board, and all patients signed informed consent forms.

Statistical analysis
The continuity variables were expressed as mean±standard deviation. One-way ANOVA was used to compared the differences between groups. All statistical analysis was performed on GraphPad Prism 8 .0. P <0.05 was considered statistically signi cant.

Microbial biomass recovered from single-surface infection models by various methods
In  Fig 2B).

Culture results in control samples
Negative (sterile) control samples consisted of 30 cubes of pork tissue (10 for each single-surface/fullsurface/internal model). In the MM group, growth of Streptococcus viridans and Staphylococcus epidermidis was found in the negative control of the single-surface and full-surface models, respectively. In the negative control of the SF group, a pork cube was found to have growth of the bacterial contaminant Corynebacterium (Table 1).

Tissue culture results of periprosthetic joint infection specimens
The numbers of colonies obtained from PJI patient specimens subjected to various pretreatment methods are listed in Table 2. Out of a total of 30 original samples from 8 patients, 10 original samples received at least one positive result, distributed in 7 cases. Only one patient's 4 original specimens were negative with the various pretreatment methods. The total number of pathogenic bacterial colonies obtained by MH was signi cantly higher than that obtained by the other pretreatment methods (Fig 2D, P  = 0.004).

Discussion
In the present work, various infection models were established with pork samples, then this samples and tissue samples collect from PJI patients were both treated by different methods, nally the microbial yield were compared. This study showed that the effects of MH and DTT in microbial recovery were signi cantly better than that of DC, SF and MM, and these methods can be used to process multiple tissue samples at the same time, which can further improve the e ciency of clinical microbial diagnosis.
This study differs from other studies in that it provides a more comprehensive assessment of all currently reported pretreatment methods, with recovery capabilities for different representative pathogens and different distribution models. This study used fresh pork as a specimen for in vitro experiments since multiple infection methods were analyzed, and a large amount of sample was required. It was not possible to obtain enough soft tissue from a single human case for the experiment to ensure sample uniformity 11 . To reduce the possibility of contamination, the pork cubes were immersed in PBS containing double antibodies (mixture of penicillin and streptomycin) for 1 hour and then washed for later use. Thereafter, we used tissue samples from clinically con rmed infection cases to further verify the ability of the above methods to recover bacteria.
To study the effects of different pretreatment methods on different microbial species, we selected S. aureus, E. coli, and C. albicans as representatives of gram-positive bacteria, gram-negative bacteria, and fungi to construct infection models. The single-surface inoculation of 2 × 10 2 bacteria avoided massive bacterial growth and facilitated counting 12 . The inoculation volume of 10 μL was based on our experimental experience. This amount of inoculum was stable enough for colonization but the amount of uid was not excessive enough for it to slip off the surface of the tissue specimen.
A contradiction exists in using pretreatment methods, because insu cient pretreatment can result in not enough bacteria being released, and excessive pretreatment may reduce bacterial viability. Mohamed Askar reported that the use of mechanized steel ball grinding and homogenization may reduce the recovery of bacteria from tissue samples 6 . The results of this study showed that the various pretreatment methods had no signi cant effect on the viability of various bacteria. This may have been related to our use of precooled working uid for various pretreatment operations to reduce heat generation from processing and thereby avoid loss of bacterial vitality.
This study found that the average CFU acquired with MH in every model was more than that with DC and MM, which is basically consistent with the results of Sylvio Redanz et al. 11 . The pretreatment methods of DTT and SF were added in this study because the literature reports that the mucolytic agent DTT can homogenize tissues and release the microorganisms in tissue samples 7 . The application of SF to break the bio lm on the surface of a prosthesis and to thus release bacteria has been recognized by many researchers [13][14][15][16] . The results of this study show that DTT's ability to separate bacteria on the surface is similar to that of MH, while SF's ability to elute bacteria from tissue surfaces is not as good as that of MH and DTT. In the internal infection model, only MH facilitated high levels of bacterial isolation, and DTT's bacterial recovery ability dropped to the same level as that of MM and SF. The above results con rm that MH has a good ability to recover pathogens under various complex conditions.
Finally, we collected tissue specimens from PJI patients diagnosed according to MSIS standards and processed the specimens with MH, MM, DTT, SF and DC. The results were consistent with the animal experiments. The number of colonies obtained by MH was signi cantly greater than that obtained by other treatment methods, which further con rms the superiority of MH compared with other methods under clinical conditions. This is also consistent with the reports of Mohamed Askar, Sylvio Redanz and others 611 .
Because tissue sample processing takes longer than synovial uid processing, false positives caused by contamination during processing are also a concern. This study found that the MH and DTT methods were not associated with false positives in the negative control samples. Two samples were contaminated with the MM method, and one sample was contaminated with the SF method. The reason may be that, with MH and DTT, the samples are basically sealed during processing, and MM exposes the samples to air during grinding, while the use of a water bath in the SF method increases the chance of contamination.
This study has the following limitations: 1) This study only selected common pathogens, such as S. aureus, E. coli, and C. albicans, and did not study rare pathogens associated with PJI, such as Mycobacterium tuberculosis and non-tuberculous mycobacteria, mycoplasma, etc. ; 2).The purpose of this study was to evaluate the recovery rate of bacteria by different tissue treatment methods, and it was not possible to clearly determine their actual diagnostic e ciency in clinical applications; 3) The study design was transversal, so it was insu cient to measure the effect. Further longitudinal study comparing the pre and post application steps of the proposed methods should be performed.

Conclusion
Mechanical homogenization and dithiothreitol released the bacteria in tissues signi cantly better than soni cated, manual milling, and direct culture methods. In internal infection models, the effect of mechanical homogenization was better than that of dithiothreitol. The mechanical homogenization and dithiothreitol methods were not conducive to contamination, and multiple tissue samples could be processed at the same time with these methods, which may improve the e ciency of clinical microbial diagnosis.

Declarations
Ethics approval and consent to participate: This study was approved by the Ethics Committee and Institutional Review Board of our institution. An informed consent was signed by each patient before the data was collected.
Consent for publication: Participants gave informed consent for publication.
Availability of data and materials: The datasets of the present work were available in supplementary les.