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27 (); 56-62
doi:
10.1016/j.jor.2021.08.012

Bacterial DNA screening to characterize surgical site infection risk in orthopaedic patients

, , , , , ,
a University of Missouri, Thompson Laboratory for Regenerative Orthopaedics, Missouri Orthopaedic Institute, 1100 Virginia Ave., Columbia, MO, 65212, USA
b University of Missouri, Department of Orthopaedic Surgery, Missouri Orthopaedic Institute, 1100 Virginia Ave., Columbia, MO, 65212, USA
c Geisinger Commonwealth School of Medicine, Geisinger Musculoskeletal Institute, Danville, PA, USA

∗Corresponding author: Aaron M. Stoker. stokera@health.missouri.edu

Received: , Accepted: ,
Professor P K Surendran Memorial Education Foundation
Disclaimer:
This article was originally published by Reed Elsevier India Pvt. Ltd. and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Abstract

To provide an initial characterization of relevant bacterial DNA profiles for patients undergoing closed-fracture fixation or total joint arthroplasties.

Swabs were collected and analyzed using Polymerase Chain Reaction from adult patients undergoing closed-fracture fixation or total shoulder, knee, or hip arthroplasties.

Bacterial DNA profiles varied across the different orthopaedic patient populations, and produced uncharacteristic profile shifts with direct relevance to each clinical infection.

Findings provide a foundational dataset regarding bacterial colonization of relevant anatomic sites that can act as sources of surgical site infections for patients.

Keywords

Fractures
Arthroplasty
Bacteria
Surgical site infection
DNA
Polymerase chain reaction
1

1 Introduction

Surgical site infection (SSI) is widely recognized as a risk factor for complications, morbidity, and poor outcomes after orthopaedic surgery.1–4 Bacterial infections occur with consistent frequency in association with fracture repair and arthroplasty. While incidences for SSIs associated with closed fracture repair and elective arthroplasty are relatively low, the rare infections that do occur can culminate in devastating complications including nonunion, implant failure, need for revision surgery, or even amputation.5–10 Currently, health care costs related to these complications exceed $1.6 billion in the United States alone.11

Orthopaedic SSIs can result from a variety of bacterial sources that may be present from the pre-operative period through recovery.12 Unfortunately, routine patient screening procedures and health optimization protocols, such as surgical skin preparation, timely prophylactic antibiotic administration, intra-operative lavage, meticulous skin closure, and surgical wound containment, do not comprehensively address potential risks or sources for infections. In addition, early identification of orthopaedic infections can be difficult, as physical examination, diagnostic imaging, and routine laboratory testing data are often insensitive or non-specific.1,13–15

While clinical diagnosis of an orthopaedic SSI may occur from a synthesis of patient history, examination findings, and elevated systemic inflammatory markers, the current gold standard for diagnosis of orthopaedic infections is microbial culture of samples obtained from swabs, deep aspirates, biopsies, or surgery.16,17 However, this method is invasive, time-consuming, and may still have poor sensitivity due to contamination, sampling error, the presence of biofilms, low virulence of some organisms, inadequate operative tissue sampling, and/or concurrent use of antibiotics.12,16,18 As such, there is a false negative rate of 10–30% for orthopaedic-related infections, which can delay treatment, provoke use of empirical treatment, lead to improper treatment, and detrimentally affect outcomes.19

Polymerase chain reaction (PCR) for detection of bacterial DNA may provide an earlier and more sensitive and specific method for determining risk and identifying source and type of bacteria involved in orthopaedic infections. Recently, PCR technology has been increasingly employed in orthopaedics to assess for indolent infections.16,20,21 While this method is highly sensitive for detecting the presence of bacteria, correlations for bacterial DNA with risk for, or diagnosis of, clinical infections associated with many of the most common orthopaedic surgeries have not been determined to the authors’ knowledge.16,22–24 These correlations are critical to a better understanding of pre-operative patient screening and preparation, prophylactic antibiotic use, and diagnosis and management of orthopaedic SSIs. Therefore, this study sought to provide an initial characterization of bacterial DNA profiles for patients undergoing open reduction with internal fixation (ORIF) for closed radius or ankle fractures, or undergoing total shoulder arthroplasty (TSA), total hip arthroplasty (THA), or total knee arthroplasty (TKA).

2

2 Patients and Methods

2.1

2.1 Patient population

With IRB approval (IRB #2012861, 2007772) and informed patient consent, samples were collected from adult patients (n = 76) with distal radius fractures (n = 10) or peri-articular ankle fractures (n = 10), or undergoing primary or revision THA (n = 24), TKA (n = 24), or TSA (n = 8). Exclusion criteria included open fractures, fractures undergoing planned staged procedures (temporary external fixation with delayed ORIF), compartment syndrome, talus fractures, percutaneous treatment, arthroplasty for fracture, conversion of previous surgery to arthroplasty, inflammatory arthropathies, those with a history of infection involving the affected joint, pregnant or lactating females, incarcerated patients, or incompetent patients.

2.2

2.2 Operative procedures

For fracture patients, preoperative antibiotic treatment using weight-based intravenous cefazolin, or vancomycin in cases of known allergies to cefazolin, were administered within 30 min prior to incision. The skin on the affected extremity was prepared for aseptic surgery using sequential treatments of 70% isopropyl alcohol (IPA), chlorhexadine digluconate scrub, and chlorhexidine gluconate-IPA solution.

TJA patients underwent standard preoperative protocols to minimize risk of infection including a skin decolonization protocol with chlorhexidine showers (3–5 days), nasal mupirocin, preoperative chlorhexidine wipes, and intraoperative alcohol/chlorhexidine skin prep. Perioperative antibiotic prophylaxis consisted of intravenous cefazolin administered in the operating room between 10 and 20 min before incision and intravenous vancomycin initiated in the preoperative holding area and administered over 60–90 min with an intent for at least 80% infusion before surgical incision for TKA and THA patients, and intravenous or ceftriaxone administered in the operating room between 10 and 20 min before incision for TSA patients.

At the time of operation, all standard surgical protocols were followed. Standard-of-care protocols for surgical site preparation and draping, aseptic technique, intra-operative lavage, meticulous skin closure, and postoperative wound management were followed. Surgical approach was dictated by the patient's indication and the treating surgeon's discretion.

2.3

2.3 Sampling procedures

Swabs were placed in the operating rooms used for these procedures for a representative proportion of cases. Swabs were left exposed during the surgery to determine the potential ambient operating room bacterial load profile.

For fracture patients, two intraoperative swabs (Remel™ BactiSwab™ Amies Charcoal and Amies Clear, ThermoFischer Scientific) were obtained for PCR analysis. One swab was obtained as soon as the fracture was exposed. The operation continued until ORIF was achieved and the wound was ready to be closed. Prior to irrigation and closure, another swab of the fracture site with fixation implants in place was obtained.

For TJA patients, swabs were used to collect a sample from the patient's nasal cavity and oral cavity prior to induction of anesthesia; the skin surrounding the surgical site after intraoperative preparation; and the joint immediately after arthrotomy.

After sampling, all PCR swabs were immediately transported to an on-site laboratory and stored at −80 °C until DNA extraction and purification were performed.

For all patients, incision closure, wound management, postoperative care, and follow-up procedures were dictated by standard protocols under the discretion of the attending surgeon.

2.4

2.4 PCR analysis

Bacterial DNA was extracted from each swab using a PureLink Microbiome DNA Purification Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacture's protocol. The extracted DNA was stored at −80 °C until used for analysis of bacterial genome. The purified DNA was sent to the University of Missouri DNA Core Facility where Sanger sequencing services were performed to create the 16S ribosomal RNA library for each sample. A reverse primer with a unique 12-base index was used in each PCR reaction with a single primer. Extracted genomic DNA was quantitated. PCR reactions contained 100 ng of genomic DNA, forward and reverse primers, deoxynucleotide triphosphates, and Phusion High-Fidelity DNA Polymerase. PCR amplification was performed for 35 cycles. Amplified product from each PCR reaction was combined to make a single pool and were purified by beads to an equal volume of 50 μl of amplicons. The plate was incubated at room temperature for 15 min. Products were placed on a magnetic stand while supernatant was removed and discarded. Each well was washed by 80% ethyl alcohol, incubated, and the supernatant was removed. Wash steps were repeated, and the plate could dry on a magnetic stand. The dried pellet was resuspended, incubated, and then placed on the magnetic stand. Supernatant was transferred and stored in micro centrifuge tubes. The final pool was evaluated, quantified, and diluted for sequencing.

Assembly, binning, and annotation of DNA sequences was performed at the University of Missouri Informatics Research Core Facility. Briefly, contiguous DNA sequences were assembled using FLASH software, and culled if found to be short after trimming for a base quality less than 31. Qiime v1.841 software was used to perform de novo and reference-based chimera detection and removal, and remaining contiguous sequences were assigned to operational taxonomic units (OTUs) via de novo OTU clustering and a criterion of 97% nucleotide identity. Taxonomy was assigned to selected OTUs using BLAST against the SILVA database of 16S rRNA sequences and taxonomy.

Samples with >1000 RNA sequences after PCR from each swab were identified and further analyzed. Mean #RNA sequences per collection site were calculated for each sample, the top three bacteria identified at each site, and the mean percent of total bacterial load was determined. In addition, the percent of samples that contained the respective bacteria from samples >1000 RNA sequences of the top three bacteria from each location was reported. Further, a One-Way ANOVA followed by a Tukey post-hoc analysis was performed in R version 4.1.0 (R Core Team, 2021) to determine significant differences among sites and time of collection for number of DNA sequences for each bacterium identified. Figures were produced using the package ggplot2 (Wickham, 2009)

2.5

2.5 Clinical outcomes

Standard postoperative follow-up was performed. Enrolled patients were followed to determine and record outcomes, including infection-related complications. For the stated purpose of the present study, the end point was defined a priori as 1-year postoperatively. Clinical and radiographic healing and all complications were documented. Any patient requiring operative debridement had additional samples collected at the time of surgery for quantitative microbial cultures. Swabs for microbial culture were used to inoculate chocolate agar, blood agar, phenylethyl alcohol agar, MacConkey agar, anaerobic blood agar, and laked kanamycin-vancomycin agar plates as well as in thioglycolate broth. The plates and bottles were incubated at 35 °C in a 5% CO2 incubator. The incubated cultures and bottles were examined at 24 h, 48 h, and 96 h post-incubation. If no growth was seen after 96 h, “no growth day 4” was reported. If growth was detected, the isolate was identified, when possible.

3

3 Results

Operating room samples had the lowest number of rRNA sequences identified per swab. The top 3 bacteria identified from operating room samples were Brevibacillus, Cutibacterium, and Bacillus in descending % load (2514, 1884, and 476 #RNA sequences, respectively). Bacterial DNA from all three of these species were recovered from all samples from operating rooms used for this study. Bacterial DNA profiles for the patient cohorts are presented in Table 1.

Table 1 Bacterial DNA profiles for respective site samples among fracture and total joint arthroplasty Patients.
Orthopaedic Cases (n = 76) Top 3 #RNA Sequences %Bacterial Sample %Samples
Radial Fracture (n = 10) Pre 1. Staphylococcus 154,341 71.0 100
2. Corynebacterium 11,782 5.4 100
3. Propionibacterium acnes 8,615 4.0 100
Post 1. Staphylococcus 155,380 64.6 100
2. Corynebacterium 14,291 5.9 100
3. Propionibacterium acnes 13,532 5.6 100
Ankle Fracture (n = 10) Pre 1. Corynebacterium 8,712 42.3 100
2. Staphylococcus 2,183 7.7 100
3. Rothia mucilaginosa 1,100 4.6 100
Post 1. Corynebacterium 23,261 30.1 100
2. Staphylococcus 10,750 13.2 100
3. Anaerococcus 251 5.5 100
TSA (n = 8) Skin 1. Brevibacillus 1,384 32.7 100
2. Cutibacterium 1,302 21.4 100
3. Streptococcus 367 12.8 100
Joint 1. Ochrobactrum 771 21.6 50
2. Cutibacterium 774 19.1 100
3. Corynebacterium 710 16.7 100
Oral 1. Streptococcus 346,621 57.2 100
2. Haemophilus 54,371 8.8 100
3. Rothia 35,826 6.8 100
Nasal 1. Corynebacterium 107,250 36.6 100
2. Staphylococcus 60,107 32.0 100
3. Cutibacterium 14,452 9.4 100
TKA (n = 24) Skin 1. Corynebacterium 2,853 10.5 100
2. Bacillus 1,511 10.3 92.9
3. Staphylococcus 1,590 5.8 100
Joint 1. Acinetobacter 1,358 23.4 100
2. Bacillus 70 7.7 100
3. Acidithiobacillus 910 6.5 100
Oral 1. Streptococcus 146,342 73.3 100
2. Actinomyces 9,468 6.444 94.7
3. Haemophilus 5,409 2.4 94.7
Nasal 1. Corynebacterium 50,296 35.6 100
2. Staphylococcus 14,188 19.2 100
3. Streptococcus 16,037 16.6 100
THA (n = 24) Skin 1. Pseudomonas 7,265 15.7 100
2. Acidithibacillus thiooxidans 3,669 8.7 100
3. Ralstonia 3,543 6.4 16.7
Joint 1. Actinetobacter 6,015 24.9 100
2. Diaphorobacter 6,249 15.7 100
3. Streptococcus 1,494 7.0 100
Oral 1. Streptococcus 122,621 66.6 100
2. Gemella 8,630 4.6 100
3. Rothia 5,011 3.3 100
Nasal 1. Corynebacterium 43,396 33.3 100
2. Staphylococcus 52,324 32.3 100
3. Dolosigranulum 22,000 12.1 100
3.1

3.1 Fracture patients

Study participants included 8 males and 12 females with an average age of 41.9 ± 16.7 years and an average body mass index (BMI) of 29.6 ± 9.3 kg/m2. Quantifiable amounts of bacterial DNA of numerous species were identified in all intraoperative samples. Staphylococcus, Proprionibacteriacea, Streptococcus, and Corynebacterium were the most common species identified in both fracture type populations. There were no significant differences in #RNA sequences per swab between pre- and post-treatment collection times for overall bacterial load.

One fracture patient (5%) experienced wound dehiscence prior to ankle fracture healing. Cultures obtained during debridement demonstrated growth of Enterobacter cloacae. Interestingly, percent of bacterial DNA from the Enterobacteriaceae family increased and was the second most common bacteria present in this sample. The pre-treatment (Fig. 1a) swab contained 10.9% of its bacterial DNA from Enterobacteriaceae for which the cohort average was 0.13%, and the post-treatment swab (Fig. 1b) contained 18.6% of its bacterial DNA from the Enterobacteriaceae for which the cohort average was 0.05%. No other fracture in this cohort was documented to have infection-related complications during the study period.

a) Pre-treatment and b) post-treatment surgical site bacterial DNA profiles for the ankle fracture patient (right) with a documented Enterobacter cloacae SSI compared to the surgical site average bacterial DNA profiles for the ankle fracture cohort (left).
Fig. 1 a) Pre-treatment and b) post-treatment surgical site bacterial DNA profiles for the ankle fracture patient (right) with a documented Enterobacter cloacae SSI compared to the surgical site average bacterial DNA profiles for the ankle fracture cohort (left).
3.2

3.2 Total shoulder arthroplasty

Study participants included 7 males and 1 female with an average age of 67.5 ± 6.5 years and an average BMI of 29.1 ± 3.6 kg/m2. The oropharynx was found to have a significantly higher (p < 0.05) bacterial DNA load compared to all other sites tested. The joint site had the lowest number of samples, 50%, with >1000 rRNA sequences identified. Ochrobactrum was the most abundant bacterial DNA for the joint site but was only found in 50% of the samples containing >1000 DNA sequences. One joint sample had a bacterial DNA load of ~99% Ochrobactrum, which may have skewed these data. No patient (0%) in this cohort was documented to have SSI-related complications during the study period.

3.3

3.3 Total knee arthroplasty

Study participants included 9 males and 15 females with an average age of 62.4 ± 12.1 years and the average BMI was 33 ± 8.2 kg/m2. In the nasopharynx, Corynebacterium was found to be most abundant followed by Staphylococcus. Overall, the samples from the skin were found to be diverse in bacterial DNA species identified compared to the other locations tested. The joint location was found to have the lowest bacterial DNA load. The most common bacterial DNA in joint swabs were Acinetobacter and Bacillus. The nasal and oral swabs were found to contain significantly more total bacterial DNA compared to the joint (p < 0.001) and the skin (p < 0.001), with the oral swabs containing significantly more bacterial DNA than the nasal swabs (p = 0.049) as well.

One patient (4%) had superficial wound dehiscence with unidentifiable rare gram-positive cocci growth from a wound swab. Interestingly, the nasal swab (Fig. 2) from this patient produced high numbers of Streptococcus bacterial RNA which represented 95.3% of the overall bacteria present while the average for this nasal site among patients in this cohort was 16.6%.

Pre-operative nasal swab bacterial DNA profile for the TKA patient (right) with a documented gram-positive cocci SSI compared to the surgical site average bacterial DNA profiles for the TKA cohort (left).
Fig. 2 Pre-operative nasal swab bacterial DNA profile for the TKA patient (right) with a documented gram-positive cocci SSI compared to the surgical site average bacterial DNA profiles for the TKA cohort (left).
3.4

3.4 Total hip arthroplasty

Study participants included 8 males and 16 females with an average age of 59.6 ± 11.7 years and the average BMI was 32 ± 6.9 kg/m2. THA patients had very similar nasopharynx and oropharynx bacterial DNA profiles in comparison with TKA patients. The average bacterial DNA loads were comparable to TKA patients at all sites tested as well, with nasopharynx and oropharynx being most abundant (p < 0.001) compared to the joint and skin sites. Also similar to the TKA cohort, THA joints were found to have the lowest bacterial DNA loads among patient sites tested. The bacterial DNA profile from skin swabs for the THA population differed from the TKA cohort in terms of including low loads of bacterial DNA commonly associated with SSI.

Of the patients undergoing THA, one (4%) had symptoms of surgical site infection. For this patient, a wound swab produced growth of a rare Staphylococcus sp. Interestingly, this patient's nasal swab (Fig. 3a) contained a relatively high percentage (98%) of Staphylococcus bacterial DNA compared to the average for this site among patients in this cohort (32.3%). Further, the bacterial DNA profile for this patient at the joint site (Fig. 3b) contained a high percentage of Staphylococcus bacterial DNA, 33.6% of the bacteria content identified, compared to the average for this site among patients in this cohort, which was 4.5%.

a) Pre-operative nasal swab and b) joint swab bacterial DNA profiles for the THA patient (right) with a documented Staphylococcus sp SSI compared to the surgical site average bacterial DNA profiles for the THA cohort (left).
Fig. 3 a) Pre-operative nasal swab and b) joint swab bacterial DNA profiles for the THA patient (right) with a documented Staphylococcus sp SSI compared to the surgical site average bacterial DNA profiles for the THA cohort (left).

Surgical site infection data for the patients included in the present study are summarized in Table 2.

Table 2 Incidence and Type of Surgical Site Infections among Fracture and Total Joint Arthroplasty Cohorts with Possible Sources of Infection based on Corresponding Bacterial DNA Isolates.
Cohort SSI Incidence SSI Type Possible Source
Fractures 5% Enterobacter cloacae Enterobacter sp at surgical site (ankle)
TSA 0% na na
TKA 4% Gram-positive cocci Streptococcus in nasopharynx
THA 4% Staphylococcus sp. Staphylococcus in nasopharynx and/or surgical site
4

4 Discussion

The results of this study suggest that PCR can detect a wide spectrum of bacterial DNA species from nasopharynx, oropharynx, skin, and surgical site swabs that may aid in assessing orthopaedic surgery patients for relative risk and may correspond to some types of subsequent surgical site infections. These data provide an initial characterization of bacterial DNA profiles for patients undergoing open reduction with internal fixation (ORIF) for closed distal radius or ankle fractures, total shoulder arthroplasty (TSA), total hip arthroplasty (THA), or total knee arthroplasty (TKA), which have important implications for pre-operative patient screening and preparation, prophylactic antibiotic use, and diagnosis and management of orthopaedic infections. For each of the surgical site infections documented in the present study, the PCR testing for bacterial DNA identified an uncharacteristic bacterial DNA profile that had direct relevance to the clinical infection. As such, bacterial DNA screening may be a consideration in high-risk settings, aberrant infection rates, and/or unusual SSI culprits.

Previous studies have supported the application of PCR-based methods for clinical use in orthopaedic surgery, primarily in prosthetic joint infections.22,25,26 These previous studies have reported that microbial complexity is often underestimated, especially in biofilms, which may explain the common difficulties for completely resolving SSIs in orthopaedic patients. Additionally, Clarke et al.27 performed a study in 40 THA patients to identify the effects that the type of operating room (standard vs. ultra-clean) had on wound contamination during the procedure. These authors reported that ultra-clean operating rooms had a significantly lower rate of positive cultures and bacterial DNA load when compared to standard operating rooms. However, they did not correlate contamination with clinical outcomes. Palmer et al.16 evaluated 34 fracture nonunions comparing intraoperative culture and bacterial DNA analyses. Cultures were positive in eight nonunions (23%), while PCR identified bacteria in 26 out of 30 nonunions (88%). Importantly, that study also implemented fluorescence in situ hybridization (FISH) to verify the specific bacterium identified by PCR, allowing the authors to conclude that molecular analysis was more sensitive and specific for examining potential infection-related causes for nonunion. These studies demonstrate the potential for bacterial DNA analyses for clinically relevant preventative and diagnostic applications in orthopaedic surgery. However, while PCR techniques for bacterial analysis can be effective, there are still barriers and limitations to their routine use in a clinical setting. Some of these barriers include access to the necessary resources, expertise, and time required to perform and report these analyses. As such, labor-intensity and cost-effectiveness must be considered when determining clinical applicability for each indication and institution. Furthermore, a limitation of PCR techniques for bacterial analysis is that these techniques only indicate the presence of the bacterial DNA, and not the presence of viable and virulent bacteria. As also supported by the results of the present study, bacterial DNA profiles may vary widely across patient populations based on a number of factors such that screening may need to be location-, indication-, or even patient-specific as part of a precision medicine-based approach.

For the fracture cohort in the present study, the number and types of bacteria present based on PCR were similar between pre- and post-treatment surgical wound swabs, suggesting that significant surgical contamination did not occur. Even still, the data suggest that “clean” fracture sites contain types and amounts of bacterial DNA that could contribute to clinical infections and related nonunions. Further, changes in percent of bacterial DNA load detectable at the time of surgery may be predictive of later SSIs such that routine bacterial DNA profiling could be justified in higher-risk cases or when aberrant infection rates or types are noted.

For the TSA cohort in the present study, there were no complications involving bacterial infections. However, relatively high loads of Cutibacterium DNA were detected in skin and joint swabs. As this organism is well documented to be a common source for SSIs associated with TSA,28,29 the bacterial DNA profiles noted in this cohort are likely representative of this patient population such that further characterization and tracking of SSIs are warranted.

For the TKA and THA cohorts, the joint site contained the lowest levels of bacterial DNA of the sites studied, and bacterial species commonly associated with SSI were not consistently identified on swabs from individuals without complications. Interestingly, in each of these cohorts, the SSI was associated with changes in the bacterial DNA profiles in joint and/or nasopharynx at the time of surgery that corresponded to subsequent infection-associated bacteria. These data suggest that relative bacterial DNA load in the nasal cavity and/or surgical-site skin may be indicative for risk of subsequent arthroplasty-related SSI. In contrast, many of the most abundant bacteria identified by PCR analyses of swabs from these patients were species noted to have positive commensal competition effects in reducing risk for SSI, including Corynebacterium, Dolosigranulum, Streptococcus, and Gemella species.30–32,32,33,33

It is important to note that the utility of molecular testing for SSI prevention and diagnosis is still in question based on relative expense and labor-intensity16 in conjunction with the clinical relevance of bacterial DNA with respect to microbial viability or virulence.22 However, these modalities have had clinical impact in improving screening, diagnosis, and/or treatment for chronic otitis media,34 adenoiditis,35 surgical site infections,36 infected arthroplasties,37 and chronic venous insufficiency wounds.38 In addition, the data from the present study suggest that bacterial DNA profiles may be useful in identifying potential risk for at least some surgical site infections associated with closed fractures and total joint arthroplasties. As such, further research in this area should be pursued to determine the effectiveness of these screening protocols to predict patient risk for SSI with the aim of optimizing patient screening, diagnostic, and preparation protocols that improve outcomes for these common orthopaedic procedures.

With this in mind, there are several limitations that must be considered when interpreting the data from this study including the relatively small number of patients in each cohort and the single method for assessing bacterial DNA. Patient numbers were limited based on the design of the study as an initial exploration into this emerging area of orthopaedic healthcare. In addition, bacterial DNA were only assessed via PCR from swabs, which provide quantitative data for relative amounts (#rRNA sequences per swab) for each species identified from which percent bacterial load for each bacterium was calculated. However, presence of viable bacteria at each tested site was not confirmed through standard culture methodologies, and other methods for verifying the presence of bacteria, such as in situ hybridization (FISH) or confocal microscopy assessments were not included. Importantly, Tuttle et al.36 demonstrated that while the potential bias for detection of nonviable bacteria with molecular techniques exists, the underestimation of bacterial presence via standard cultures is more likely. Therefore, future validation studies in this important area of clinical research should include swabs from a larger population of patients as well as tissue samples for PCR, in situ hybridization (FISH), or confocal microscopy assessments, and quantitative microbial cultures with correlation to longer term outcomes.

5

5 Conclusion

This study was designed to characterize bacterial DNA profiles from the nasopharynx, oropharynx, skin, surgical site, and operating room for patients undergoing a spectrum of common orthopaedic surgical procedures. The findings from this study provide a foundational dataset regarding bacterial colonization of relevant anatomic sites that can act as sources of surgical site infections in the large and growing population of patients undergoing orthopaedic surgeries. It is now possible to expand this database to more fully characterize bacterial DNA profiles, assess the influences of demographic factors and co-morbidities, and carefully track long-term outcomes to determine the clinical significance of the presence of bacterial DNA at each site. Pursuit of this research path has the potential to accurately determine risk for surgical site infection that can be used to optimize prevention and treatment strategies.

Conflict of interest disclosure

No external funding was used in the execution of this study. Portions of these data were presented at the Austin, TX, the Orthopaedic Research Society Annual Meeting, February 8–11, 2020, Phoenix, AZ, the American Association of Orthopaedic Surgeons, March 12–13, 2019, Las Vegas, NV, annual conference of the Orthopaedic Research Society Annual Meeting, February 2–5, 2019, and the Orthopaedic Trauma Association's Annual Meeting, Oct. 17–20, 2018, Kissimmee, FL.

Author contributions statement

All authors were instrumental in study design, acquisition and analysis of study data, manuscript drafting and final approval of all manuscript-related items.

CRediT authorship contribution statement

Preston N. Wolfe: Formal analysis, Writing. Brian D. Campfield: Conceptualization, Data collection, Writing, Writing – review & editing. Brett D. Crist: Conceptualization, Data collection, Writing – review & editing. James A. Keeney: Data collection, Writing – review & editing. Matthew J. Smith: Data Collection, Writing – review & editing. James L. Cook: Experimental Design, Writing, Writing – review & editing. Aaron M. Stoker: Experimental design, Formal analysis, Supervision, Writing, Writing – review & editing.

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