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Cai et al. BMC Musculoskeletal Disorders (2021) 22:209 https://doi.org/10.1186/s12891-021-04071-5 RESEARCH ARTICLE Open Access Microbial yield from infectious tissues pretreated by various methods: an invitro study Yuanqing Cai1†, Xinyu Fang1†, Lvheng Zhang1†, Xurong Yang1, Lixiong Nie1, Zida Huang1, Wenbo Li1, Chaofan Zhang1, Bin Yang2, Zhenpeng Guan3* and Wenming Zhang1* Abstract Background: This study aimed to evaluate the effects of different pretreatment methods on the microbial yield from infectious 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 these tissues, the microbial yield from different pretreatment methods were recorded and compared. Moreover, periprosthetic tissues collected intraoperatively from periprosthetic joint infection (PJI) patients were used as a verification. 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 diagnostic sensitivity of infectious disease. Keywords: Pretreatment methods, Tissues, Microbial recovery, Periprosthetic joint infection, Periprosthetic tissues Background [2, 3]. At present, the laboratory diagnostic methods Periprosthetic joint infection (PJI) is a serious complica- mainly based on synovial fluid, sonication fluid and peri- tion after joint arthroplasty, which brings a heavy eco- prosthetic tissues culture, but in many cases, synovial/ nomic burden to patients and society [1]. Microbial sonication fluid are insufficient. However, It is feasible to culture is essential for the diagnosis and treatment of PJI obtain sufficient tissue samples intraoperatively, but the positive microbial culture rates are frustrating if tissues are not properly pretreated [4]. Therefore, optimizing * Correspondence: guanzhenpeng@qq.com; zhangwm0591@fjmu.edu.cn † Yuanqing Cai, Xinyu Fa

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positive microbial culture rates are frustrating if tissues are not properly pretreated [4]. Therefore, optimizing * Correspondence: guanzhenpeng@qq.com; zhangwm0591@fjmu.edu.cn † Yuanqing Cai, Xinyu Fang and Lvheng Zhang contributed equally to this the pretreatment methods of tissue specimens to make work. 3 full use of PJI tissue samples is of great significance for Department of Orthopedic Surgery, Peking University Shougang Hospital, improving the positive culture rate. Beijing, China 1 Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian The current mainstream tissue pretreatment methods Medical University, Fuzhou, China reported in clinical microbiology laboratories are direct Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Cai et al. BMC Musculoskeletal Disorders (2021) 22:209 tissue culture (DC) and manual milling (MM) [5]. How- ever, MM is a tedious process in which contamination can easily be introduced. Studies have shown that mech- anical homogenization (MH), dithiothreitol (DTT), and sonificated (SF) can increase the release of bacteria from tissue [6–8]. However, wether these methods are clinical available are still in the air, because currently there are no reports that directly compared these different speci- men pretreatment methods in vitro. In this study, the abovementioned pretreatment methods were used to pretreat infectious tissues models constructed in vitro and tissue specimens collected from PJIs. The recovery colony-forming units (CFU) were re- corded and compared to evaluate the efficiency of differ- ent pretreatment methods. Methods This study was approved by institutional review board. For PJI cases, the demographic data, medical boratory tests and microbial culture results were pro- spective collected from January 2018 to December 2019 in our center, and all patients signed informed consent forms. PJI was diagnosed based on the American Society for Musculoskeletal Infection (MSIS) criteria for PJI. Establishment of infection tissues model Fresh pork was frozen and cut into cubes at a size of 0.5 × 0.5 × 0.5 cm3, each weighing approximately 40 mg. After Page 2 of 7 infection model in vitro. In order to simulate PJI infec- tion as much as possible, in which pathogen may be dis- tributed on a single surface or multiple surfaces even inside tissues [6], therefore, three tissues infection models were established (Fig. 1): ① Single surface infec- tion: pipette aspirate 10 μL (200 CFU) bacterial dilution (S. aureus /E. coli/Candida albicans) to colonized a sin- gle surface of the pork cube. ② Full surface infection model: Microorganisms were colonized on the entire surface of pork cubes, with 10 μL (200 CFU) for each surface. ③ Internal infection model: Ten microliters (200 CFU) of bacterial dilution was injected into the cen- ter of the pork cube with a syringe for colonization. And the control group which have an incision on the surfaces of the pork was injected with the same amount of saline, to simulated a surgical wound was also established. Fifty pieces of each pathogen model were made, and the pork samples were inoculated for an hour. history, la- Various pretreatment methods on infectious tissues model in vitro Fifty samples of each infection model were randomly assigned to MH group, MM group, DTT group, SF group, and DC group. For pretreatment: ① MH group: tissues were transferred to 2 mL Eppendorf (EP) tubes with 1 mL LB culture medium (Haibo Biotechnology Co., Ltd., Qingdao, Chian), vortexed for 15 min, and put into a fully automatic rapid grinder (Jingxin Ind

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with 1 mL LB culture medium (Haibo Biotechnology Fresh pork was frozen and cut into cubes at a size of 0.5 × Co., Ltd., Qingdao, Chian), vortexed for 15 min, and put 0.5 × 0.5 cm3, each weighing approximately 40 mg. After into a fully automatic rapid grinder (Jingxin Industrial immersion in diluted penicillin G (500 U/ml), strepto- Development Co., Ltd., Shanghai, China) at 40 Hz for mycin (600 μg/ml) and amphotericin B (2.5 μg/ml) for 1 h, 60–90 s until the tissue specimen was homogenized. ② the samples were washed three times with PBS. MM group: tissues were transferred to 2 mL EP tubes The strain of Staphylococcus aureus (S. aureus) ATCC with 1 mL LB culture medium and manually ground 25923 (American Type Culture Collection, USA) the with a disposable sterile grinding rod until a homogenate strain of Escherichia coli (E. coli) ATCC 25922 (Ameri- appeared. ③ DTT group: the shock time and DTT con- can Type Culture Collection, USA), and the strain of centration established in previous in vitro studies was Candida albicans (C. albicans)ATCC 90029 (American used [9]. After soaking in 1 g/L DTT (Haibo Biotechnol- Type Culture Collection, USA) were used to establish ogy Co., Ltd., Qingdao, China) for 15 min at room Fig. 1 Establishment of infection tissues in vitro

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Cai et al. BMC Musculoskeletal Disorders (2021) 22:209 Page 3 of 7 temperature, specimens were transferred to a 2 mL EP CFU/ml (95% CI: 840–1035), and 533 ± 108 CFU/ml tube and 1 mL of LB liquid medium was added to seal (95% CI: 455–611), respectively. The CFUs obtained in the tubes. ④ SF group: tissues were transferred to 2 mL MH group was significantly higher than that in the MM EP tubes with 1 mL LB culture medium, vortexed and (P < 0.01), SC (P < 0.01), and SF groups (P < 0.01), but shaken for 30 s and placed in an ultrasonic cleaner there was no significant difference with that of the DTT (Wuxi Woxin Instrument Co., Ltd., Jiangsu, China), son- group (P = 0.26) (Supplementary file; Fig. 2a). icated at 40 Hz for 5 min. ⑤ DC group: tissues were In the EC infection groups, the average CFU of the transferred to 2 mL EP tubes with 1 mL LB culture MM, MH, SF, DTT, and DC groups were 451 ± 192 medium and vortexed for 15 min. CFU/ml (95% CI: 313–588), 1373 ± 132 CFU/ml (95% After pretreatment, the inoculated specimens were CI: 1278–1468), 480 ± 135 CFU/ml (95% CI: 383–576), centrifuged at 5500 rpm/min after the above four tissue 1347 ± 162 CFU/ml (95% CI: 1231–1463), and 393 ± 178 pretreatment methods for 20 s to spin down impurities. CFU/ml (95% CI: 265–520), respectively. The CFUs ob- After centrifugation, 100 μL of each solution was inocu- tained in the MH group was significantly higher than lated on Columbia blood agar plates (Thermo Fisher Sci- that in the MM (P < 0.01), SF (P < 0.01), and DC groups entific, USA) and CDC anaerobic blood agar plates (P < 0.01), but there was no significant difference from (Qingdao Haibo Biotechnology Co., Ltd., HBPM16) that of the DTT group (P = 0.71). (Supplementary file; under aerobic and anaerobic conditions, respectively, Fig. 2a). overnight at 37 °C in a biochemical incubator (Shanghai In the CA infection groups, the average CFU of the Qixin Scientific Instrument Co., Ltd., China). Finally, MM, MH, SF, DTT, and DC groups were 440 ± 70 CFU/ CFUs were obtained and calculated. ml (95% CI: 391–488), 818 ± 122 CFU/ml (95% CI: 730– 905), 435 ± 68 CFU/ml (95% CI: 386–484), 771 ± 108 Various tissues pretreatment methods on periprosthetic CFU/ml (95% CI: 694–849), and 360 ± 85 CFU/ml (95% tissues collected from PJIs CI: 298–421), respectively. The CFUs obtained in the A total of 30 tissue specimens were collected from 8 PJI MH group was significantly higher than that in the MM patients who were diagnosed according to MSIS criteria (P < 0.01), SF (P < 0.01) and DC groups (P < 0.01), but for PJI [10]. Each tissue specimen was divided into 5 there was no significant difference with that of the DTT equal portions (each = 50 mg) and treated with the above group (P = 0.945). (Supplementary file; Fig. 2a). methods (MH, MM, DTT, SF, DC). One hundred microliters of each of the inoculated Microbial yield recovered from full-surface infection samples was inoculated onto Columbia blood agar plates

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tions (each = 50 mg) and treated with the above group (P = 0.945). (Supplementary file; Fig. 2a). methods (MH, MM, DTT, SF, DC). One hundred microliters of each of the inoculated Microbial yield recovered from full-surface infection samples was inoculated onto Columbia blood agar plates models pretreated by various methods (Thermo Fisher Scientific, USA) and CDC anaerobic In the SA infection groups, the average CFU of the MM, blood agar plates (Qingdao Haibo Biotechnology Co., MH, SF, DTT, and DC groups were 4014 ± 888 CFU/ml Ltd., HBPM16) under aerobic and anaerobic conditions, (95% CI: 3378–4650), 6268 ± 1019 CFU/ml (95% CI: respectively. Samples were incubated in a biochemical 5539–6997), 3960 ± 875 (95% CI: 3334–4568) CFU/ml, incubator (Shanghai Qixin Scientific Instrument Co., 6227 ± 1000 CFU/ml (95% CI: 5512–6942), and 3255 ± Ltd., China) at37 °C for 14 days, and colony growth was 1048 CFU/ml (95% CI: 2505–4005), respectively. The observed 2, 4, 7, and 14 days after incubation. If there CFUs obtained in the MH group was significantly higher was colony growth, the CFU per ml were calculated. than that in the MM (P < 0.01), SF (P < 0.01) and DC groups (P < 0.01), but there was no significant difference Statistical analysis compared with that of the DTT group (P = 0.924). (Sup- The continuous variables were expressed as mean ± plementary file; Fig. 2b). standard deviation. One- way ANOVA and post-test In the EC infection groups, the average CFU of the were used to compared the differences between groups. MM, MH, SF, DTT, and DC groups were 3079 ± 934 All statistical analysis was performed on GraphPad CFU/ml (95% CI: 2410–3747), 7058 ± 920 CFU/ml (95% Prism 8 .0. P < 0.05 was regarded as statistically CI: 6400–7717), 3060 ± 902 CFU/ml (95% CI: 2415– significance. 3706), 6853 ± 858 CFU/ml (95% CI: 6239–7466), and 2533 ± 637 CFU/ml (95% CI: 2077–2989), respectively. Results The CFUs obtained in the MH group was significantly Microbial yield recovered from single-surface infection higher than that in the MM (P < 0.01), the SF (P < 0.01), models pretreated by various methods and DC groups (P < 0.01), but there was no significant In the SA infection groups, the average CFU of the MM, difference compared with that of the DTT group (P = MH, SF, DTT, and DC groups were 611 ± 101 CFU/ml 0.964). (Supplementary file; Fig. 2b). (95% CI: 538–683), 999 ± 141 CFU/ml (95% CI: 898– In the CA infection groups, the average CFU of the 1100), 609 ± 96 CFU/ml (95% CI: 540–679), 938 ± 136 MM, MH, SF, DTT, and DC groups were 3080 ± 842

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Cai et al. BMC Musculoskeletal Disorders (2021) 22:209 Page 4 of 7 Fig. 2 Quantitative recovery of microorganisms from inoculated tissues. Tissue cubes were separately inoculated on 1 surface (a), on six surfaces (b), or on the inside of the samples (c) with 2 × 102 CFU of S. aureus, E. coli, or C. albicans, respectively. Number of colony forming units (CFU) obtained from PJI patient specimens by various pretreatment methods (d).CFU = colony forming units; *P < 0.01 compared to the DTT group, #P < 0.01 compared to the MM group, & P < 0.01 compared to the SF group, ^P < 0.01 compared to the DC group. α P < 0.05 compared to the DTT group, β P < 0.05 compared to the MM group, γ P < 0.05 compared to the SF group, ε P < 0.05 compared to the DC group CFU/ml (95% CI: 2477–3682), 5274 ± 966 CFU/ml (95% was significantly higher than that in the MM group (P < CI: 4583–5966), 2971 ± 890 CFU/ml (95% CI: 2334– 0.01), SF group (P < 0.01), DTT group (P < 0.01), and DC 3608), 5269 ± 853 CFU/ml (95% CI: 4658–5879), and group (P < 0.01). (Supplementary file; Fig. 2c). 2287 ± 851 CFU/ml (95% CI: 1678–2896), respectively. In the CA infection groups, the average CFU of the The CFUs obtained in the MH group was significantly MM, MH, SF, DTT, and DC groups were 169 ± 60 CFU/ higher than that in the MM (P < 0.01), SF (P < 0.01), and ml (95% CI: 125–212), 359 ± 55 CFU/ml (95% CI: 319– DC groups (P < 0.01), but there was no significant differ- 399), 164 ± 58 CFU/ml (95% CI: 122–206), 172 ± 58 CFU/ ence compared with that of the DTT group (P = 0.989). ml (95% CI: 130–213), and 119 ± 47 CFU/ml (95% CI: 85– (Supplementary file; Fig. 2b). 154), respectively. The CFUs obtained in the MH group was significantly higher than that in the MM group (P < Microbial yield recovered from internal infection models 0.01), SF group (P < 0.01), DTT group (P < 0.01), and DC pretreated by various methods group (P < 0.01). (Supplementary file; Fig. 2c). In the SA infection groups, the average CFU of the MM, MH, SF, DTT, and DC groups were 267 ± 68 CFU/ml Microbial culture results of control samples (95% CI: 218–316), 636 ± 90 CFU/ml (95% CI: 571–701), Negative (sterile) control samples consisted of 30 cubes 300 ± 73 CFU/ml (95% CI: 247–353), 270 ± 63 CFU/ml of pork tissue (10 for each single-surface/full-surface/in- (95% CI: 224–315), and 208 ± 95 CFU/ml (95% CI: 140– ternal model). In the MM group, Streptococcus viridans 277), respectively. The CFUs obtained in the MH group and Staphylococcus epidermidis were isolated from nega- was significantly higher than that in the MM (P < 0.01), tive control of single-surface and full-surface models, re- SC (P < 0.01), DTT (P < 0.01), an

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), respectively. The CFUs obtained in the MH group and Staphylococcus epidermidis were isolated from nega- was significantly higher than that in the MM (P < 0.01), tive control of single-surface and full-surface models, re- SC (P < 0.01), DTT (P < 0.01), and SF groups (P < 0.01). spectively. And Corynebacterium, which was considered (Supplementary file; Fig. 2c). as a contaminant, was isolated from negative control In the EC infection groups, the average CFU of the MM, pretreated by SF (Table 1). MH, SF, DTT, and DC groups were 313 ± 78 CFU/ml (95% CI: 257–369), 698 ± 134 CFU/ml (95% CI: 601–794), Microbial yield of tissue samples collected from PJIs 298 ± 72 CFU/ml (95% CI: 246–351), 306 ± 93 CFU/ml intraoperatively pretreated by various methods (95% CI: 239–373), and 234 ± 82 CFU/ml (95% CI: 175– The numbers of CFUs obtained from tissues collected 293), respectively. The CFUs obtained in the MH group from PJIs subjected to various pretreatment methods

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Cai et al. BMC Musculoskeletal Disorders (2021) 22:209 Page 5 of 7 Table 1 Microbial culture results of control samples MH MM DTT SF DC Single surface infection model 0/10 1/10 0/10 0/10 0/10 Streptococcus viridans (10 CFU) Full surface infection model 0/10 1/10 0/10 0/10 0/10 Staphylococcus epidermidis (20 CFU) Internal infection model 0/10 0/10 0/10 1/10 0/10 Corynebacterium (20 CFU) were listed in Table 2. Among 30 tissue samples, from 8 of contamination, the pork cubes were immersed in PBS PJI patients, 10 tissue samples showed at least one posi- containing antibodies (penicillin, streptomycin and tive microbial culture result, which collected from 7 amphotericin B) for 1 h before further experiment. cases. Only one patient’s microbial culture results were Thereafter, tissue samples collected from PJI cases were all negative from tissues samples pretreated by various pretreated by different methods used in pork tissues to methods. The total number of CFUs obtained by MH further verify the effects of different methods on micro- was significantly higher than that obtained by the other bial yield. pretreatment methods (Fig. 2d, P = 0.004). To study the effects of different pretreatment methods on different microbial species, S. aureus, E. coli, and C. Discussion albicans were chosen as representatives of gram- In the present work, various infection tissues models positive, gram-negative bacteria, and fungi to construct were established in vitro, together with tissues collected infectious tissues models in vitro. The concentration from PJI patients, were pretreated by different methods, used in single-surface infection was based on previous finally the microbial yield were recorded and compared. reports to avoided massive bacterial growth and while This study showed that the effects of MH and DTT in convenient for CFUs counting [12]. microbial yield from tissues were significantly higher There are still some issues about optimal pretreatment than that of DC, SF and MM, and these methods can be methods on t

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T in convenient for CFUs counting [12]. microbial yield from tissues were significantly higher There are still some issues about optimal pretreatment than that of DC, SF and MM, and these methods can be methods on tissues, because insufficient pretreatment used to process multiple tissue samples at the same might result in low bacteria yield released from tissues, time, which can further improve the diagnostic efficiency while excessive pretreatment might reduce bacterial via- of clinical infectious diseases. bility. Mohamed Askar reported that the use of mecha- This study differs from other studies in that it provides nized steel ball grinding and homogenization might a more comprehensive assessment of different currently reduce the recovery of bacteria from tissue samples [6]. reported pretreatment methods. In this study, it was im- However, this study showed there were no significant ef- possible to obtain sufficient tissues from a single patients fects on the viability of bacteria pretreated by various to established infectious model in vitro [11], thus, fresh methods. This might be attributed to the usage of pre- pork was used to established different infectious tissues cooled working solution for various pretreatment, which models, which simulated tissues samples collected from could reduce heat generation during processing and PJI patients to a large extent. To reduce the possibility thereby avoid reduce of bacterial vitality. Table 2 Microbial yield of tissue samples collected from PJIs intraoperatively pretreated by various methods Original specimen Pathogenic bacteria MH MM DTT SF DC 1 (case 1) Staphylococcus epidermidis 10 0 0 1 0 2 (case 2) Staphylococcus aureus 360 50 179 60 34 3 (case 3) E.coli 187 23 45 8 10 4 (case 3) E.coli 68 40 20 23 30 5 (case 5) Staphylococcus epidermidis 96 22 36 28 16 6 (case 6) Enterococcus faecalis 46 0 33 0 0 7 (case 6) Enterococcus faecalis 89 17 46 34 0 8 (case 7) Staphylococcus epidermidis 147 0 36 28 0 9 (case 7) Staphylococcus epidermidis

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89 17 46 34 0 8 (case 7) Staphylococcus epidermidis 147 0 36 28 0 9 (case 7) Staphylococcus epidermidis 15 0 0 0 0 10 (case 8) Staphylococcus aureus 283 12 16 26 10 Total 1041 164 329 208 100

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Cai et al. BMC Musculoskeletal Disorders (2021) 22:209 Page 6 of 7 This study found that the average CFUs obtained from Conclusion tissues pretreated by MH in every model were higher Mechanical homogenization and dithiothreitol could sig- than that pretreated by DC and MM, which is basically nificantly facilitate bacteria released from tissues than consistent with the results of Sylvio Redanz et al [11]. sonificated, manual milling, and direct culture methods, The DTT and SF were also performed in this study be- which were recommended for tissues sample pretreat- cause previous reported that the DTT can homogenize ment in clinical, and potential improve the diagnostic ef- tissues and facilitate microorganisms release from tissue ficiency of infectious disease. samples [7], and the application of SF was reported to break the biofilm on the surface of a prosthesis and thus promote bacteria release [13–16]. And we Supplementary Information The online version contains supplementary material available at https://doi. showed that DTT’s ability to facilitate bacteria release org/10.1186/s12891-021-04071-5. from infectious tissues was similar to that of MH, while SF’s ability to promote bacteria release from tis- Additional file 1: Table 1. Microbial yield recovered from three sues was not as good as that of MH and DTT. In the infection models by various pretreatment methods. internal infection model, only MH could facilitate bacteria release, and DTT did not show this role, Abbreviations which was similar to MM and SF. All above results SA: Staphylococcus aureus; EC: Escherichia coli; CA: Candida albicans; demonstrated that MH had the best effect on high MM: Manual milling; MH: Mechanical homogenization; SF: Sonificated; DTT: Dithiothreitol; DC: Direct culture; ATCC: American Type Culture microbial yield from tissues of various models. Collection; PJI: Periprosthetic joint infection; CFU: Colony-forming units; Finally, tissue specimens collected from PJI patients MSIS: The American Society for Musculoskeletal Infection who was diagnosed according to MSIS criteria were pre- treated by MH, MM, DTT, SF and DC. And the results Acknowledgements were consistent with the infectious pork tissues. The Not applicable. number of colonies obtained from tissues pretreated by MH was significantly higher than that pretreated by Authors’ contributions other methods, which further confirms the superiority of YQC, XYF, LHZ: performed this study, analyzed data, wrote the manuscript; X MH under clinical conditions. This was also consistent RY, LXN: literature review, collected data; WBL, BY: revised manuscript; ZDH, C FZ: English edited; WMZ, ZPG: designed the study. The author(s) read and with the reports of Mohamed Askar and Sylvio Redanz approved the final manuscript. [6, 11].

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review, collected data; WBL, BY: revised manuscript; ZDH, C FZ: English edited; WMZ, ZPG: designed the study. The author(s) read and with the reports of Mohamed Askar and Sylvio Redanz approved the final manuscript. [6, 11]. It is worth noting that tissue sample processing Funding takes longer time and more tedious than synovial This work was supported by the National Natural Science Foundation of fluid, false positive induced during this process were China (81772251, 82072458). Joint Funds for the innovation of science and Technology, Fujian province (2019Y9136). The Middle-aged Backbone Project also a concern. This study showed that there were no of the Fujian Provincial Health and Family Planning Commission (2019-ZQN- false positive culture results in the negative control 55). The funding body had no role in the design and conduct of the study; samples pretreat by MH and DTT. The reason might collection, management, analysis, and interpretation of the data. be that, when tissues were pretreated by MH and DTT, the samples were basically sealed during the Availability of data and materials whole process, while samples were exposed to the air The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. when pretreated by MM and thus contaminations might be induced. Ethics approval and consent to participate There were some limitations in this study: 1) This This study was approved by the Ethics Committee and Institutional Review study constructed infectious tissues model in vitro only Board of our institution. An informed consent was signed by each patient by S. aureus, E. coli, and C. albicans, while other patho- before the data was collected. gens would also include in PJI, such as Mycobacterium tuberculosis and non-tuberculous mycobacteria, myco- Consent for publication plasma, etc.; 2) The purpose of this study was to evaluate Informed consent for publication were obtained from participants in written. the effect of different pretreatment methods on micro- bial yield, but it was impossible to determine their actual Competing interests diagnostic efficiency in clinical practice, further investi- The authors declare that they have no conflicts of interest. gation should be performed; 3) The study design was Author details transversal, so it was insufficient to measure the effect. 1 Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Further longitudinal study comparing the pre and post Medical University, Fuzhou, China. 2Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China. application steps of the proposed m

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longitudinal study comparing the pre and post Medical University, Fuzhou, China. 2Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China. application steps of the proposed methods should be 3 Department of Orthopedic Surgery, Peking University Shougang Hospital, performed. Beijing, China.

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0):1453–7. 14. Bellova P, Knop-Hammad V, Königshausen M, Mempel E, Frieler S, Gessmann J, Schildhauer TA, Baecker H. Sonication of retrieved implants improves sensitivity in the diagnosis of periprosthetic joint infection. BMC Musculoskelet Disord. 2019;20(1):623. 15. Portillo ME, Salvadó M, Trampuz A, Plasencia V, Rodriguez-Villasante M, Sorli L, Puig L, Horcajada JP. Sonication versus vortexing of implants for diagnosis of prosthetic joint infection. J Clin Microbiol. 2013;51(2):591–4. 16. Trampuz A, Piper KE, Jacobson MJ, Hanssen AD, Unni KK, Osmon DR, Mandrekar JN, Cockerill FR, Steckelberg JM, Greenleaf JF, Patel R. Sonication of removed hip and knee prostheses for diagnosis of infection. N Engl J Med. 2007;357(7):654–63. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.