Effects of different pretreatment methods on microbial recovery of infected tissues


 This study aimed to evaluate the effects of different pretreatment methods on the degree of microbial recovery in infected tissues. Standard 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), ultrasonic lysis (UL), dithiothreitol (DTT), and direct culture (DC) were used to pretreat infection tissues, the ability of the different pretreatment methods to achieve pathogen recovery in the different bacterial infection models was compared. At the same time, periprosthetic tissues collected from periprosthetic joint infection (PJI) were pretreated with the same methods. We showed that regardless of whether the single-surface or full-surface infection model was used in SA, EC, and CA infection, the microbial acquisition in the MH group was significantly higher than that in the MM (P <0.01) and UL groups (P <0.01). In the internal infection model, the microbial acquisition of the MH group was significantly higher than that of the MM (P <0.01), UL (P <0.01), DTT (P <0.01), and DC groups (P <0.01). In the PJI cases, the number of bacterial colonies obtained by MH was significantly higher than that obtained by other pretreatment methods (P = 0.004). The effects of MH and DTT in microbial recovery were significantly better than that of DC, UL and MM, and these methods can be used to process multiple tissue samples at the same time, which can further improve the efficiency of clinical microbial diagnosis.


Effects of different pretreatment methods on microbial recovery of 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. 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, UL, 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 full-surface infection models by various methods
In respectively. The amount of microorganisms obtained in the MH group was signi cantly higher than that in the MM (P <0.01), UL (P <0.01) and DC groups (P <0.01), but there was no difference compared with that of the DTT group (P = 0.924). (Fig 2B).

Culture results incontrol 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 UL 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

Discussion
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 UL. 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, and UL. 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: 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.

Conclusion
Mechanical homogenization and dithiothreitol released the bacteria in tissues signi cantly better than ultrasonic lysis, 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. Tables Table 1 Detection