Microbiological Profile and Resistance Patterns in Periprosthetic Joint Infections: A Regional Multicenter Study in Spain

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study analyzed the epidemiological and microbiological characteristics of 156 periprosthetic joint infections (PJI) in Navarra, Spain (2019–2023). The results revealed that 23% of cases were culture-negative, with 56% of these patients receiving antibiotics prior to sampling, significantly reducing pathogen detection rates. The predominant pathogens were methicillin-resistant Staphylococcus epidermidis (MRSE) and Cutibacterium acnes. Among gram-positive cocci (GPC), 40% were multidrug-resistant organisms (MDRO), while Staphylococcus aureus prevalence was low. Rising resistance trends were observed for quinolones (46%) and rifampicin (9%) in staphylococci. The study highlights regional resistance patterns (e.g., 40% third-generation cephalosporin resistance in Enterobacteriaceae), challenges of culture-negative results, and proposes optimizing diagnostics via molecular techniques (e.g., metagenomic sequencing), adjusting empirical antibiotics, and exploring non-antibiotic therapies. Limitations include insufficient sample size, multicenter data heterogeneity, and lack of molecular diagnostics. Future work should expand sample size, validate novel techniques prospectively, and strengthen methodological rigor. While providing valuable regional PJI data, the study requires supplemental methodological details and extended analyses to enhance clinical applicability.

Specific Recommendations:

1.Inadequate sample size and data scale.
The inclusion of only 156 PJI cases over 5 years may limit the detection of significant trends in resistance or microbial distribution. Extending the study duration or expanding geographic coverage (e.g., multi-regional collaboration in Spain) is recommended to improve generalizability, particularly given potential resistance variations across Spanish regions.

2.Optimization of sonication fluid culture protocols.
While sonication is mentioned (lines 89–92), recent advancements (e.g., enzymatic treatment or novel preprocessing) are not thoroughly discussed. Cite recent studies (e.g., Mei J et al., J Clin Med. 2023; Cai Y et al., J Clin Med. 2023) to contextualize current limitations and propose optimized workflows.

3.Lack of molecular biological techniques.
With a 23% culture-negative rate, molecular methods (e.g., PCR or metagenomic next-generation sequencing [mNGS]) are absent. This omits detection of uncultivable/dormant pathogens (e.g., small-colony variant Staphylococcus). Discuss the value of these techniques and cite relevant studies (e.g., Hu H et al., Bone Joint Res. 2024).

4.Insufficient statistical analysis.
Resistance trends were analyzed solely via χ² tests, neglecting confounding factors (e.g., antibiotic pretreatment vs. hospital tier). Apply multivariate logistic regression to identify independent risk factors (e.g., preoperative antibiotics and culture-negative rates).

5.Incomplete discussion of resistance mechanisms.
While rpoB mutations causing rifampicin resistance are noted (lines 157–160), genomic insights (e.g., virulence/biofilm-related genes) are missing. Expand whole-genome sequencing (WGS) data analysis and cite resistance evolution studies.

6.Inadequate control of multicenter heterogeneity.
The study involves four hospitals but lacks standardization details (e.g., variations in culture/susceptibility protocols). Discuss methodological harmonization or its impact on results.

7.Unsupported clinical recommendations.
The proposal to replace ceftazidime with carbapenems (lines 255–257) lacks evidence from susceptibility simulations or efficacy comparisons. Strengthen recommendations with data-driven references.

8.Underdeveloped discussion of limitations.
The absence of molecular diagnostics (e.g., 16S rRNA sequencing) likely underestimates pathogen diversity. Expand the "Limitations" section and cite comparative studies to address this gap.

Author Response

We sincerely thank the reviewer for the detailed and insightful comments, which have helped us improve the quality and scientific rigor of our manuscript. Below we provide a point-by-point response, outlining the changes made and clarifications added.

This study analyzed the epidemiological and microbiological characteristics of 156 periprosthetic joint infections (PJI) in Navarra, Spain (2019–2023). The results revealed that 23% of cases were culture-negative, with 56% of these patients receiving antibiotics prior to sampling, significantly reducing pathogen detection rates. The predominant pathogens were methicillin-resistant Staphylococcus epidermidis (MRSE) and Cutibacterium acnes. Among gram-positive cocci (GPC), 40% were multidrug-resistant organisms (MDRO), while Staphylococcus aureus prevalence was low. Rising resistance trends were observed for quinolones (46%) and rifampicin (9%) in staphylococci. The study highlights regional resistance patterns (e.g., 40% third-generation cephalosporin resistance in Enterobacteriaceae), challenges of culture-negative results, and proposes optimizing diagnostics via molecular techniques (e.g., metagenomic sequencing), adjusting empirical antibiotics, and exploring non-antibiotic therapies. Limitations include insufficient sample size, multicenter data heterogeneity, and lack of molecular diagnostics. Future work should expand sample size, validate novel techniques prospectively, and strengthen methodological rigor. While providing valuable regional PJI data, the study requires supplemental methodological details and extended analyses to enhance clinical applicability.

Specific Recommendations:

1. Inadequate sample size and data scale.
   The inclusion of only 156 PJI cases over 5 years may limit the detection of significant trends in resistance or microbial distribution. Extending the study duration or expanding geographic coverage (e.g., multi-regional collaboration in Spain) is recommended to improve generalizability, particularly given potential resistance variations across Spanish regions.

We acknowledge the limited sample size and geographic scope as potential limitations. This study was designed as a regional analysis to provide a preliminary characterization of PJI in Navarre. We have now included this limitation more explicitly in the “Limitations” section and discussed the importance of future multi-regional or national studies to enhance generalizability and detect broader resistance patterns (lines 311-313).

Thank you very much.

2. Optimization of sonication fluid culture protocols.
   While sonication is mentioned (lines 89–92), recent advancements (e.g., enzymatic treatment or novel preprocessing) are not thoroughly discussed. Cite recent studies (e.g., Mei J et al., J Clin Med. 2023; Cai Y et al., J Clin Med. 2023) to contextualize current limitations and propose optimized workflows.

Thank you for this valuable suggestion. We have expanded the Discussion section to further elaborate on recent developments in sonication protocol optimization, including enzymatic treatment and preprocessing enhancements. We have incorporated relevant recent literature, including Mei J et al., J Clin Med 2023, and Cai Y et al., J Clin Med 2023, to contextualize current practices and opportunities for improvement (lines 201-203).

 

3. Lack of molecular biological techniques.
   With a 23% culture-negative rate, molecular methods (e.g., PCR or metagenomic next-generation sequencing [mNGS]) are absent. This omits detection of uncultivable/dormant pathogens (e.g., small-colony variant Staphylococcus). Discuss the value of these techniques and cite relevant studies (e.g., Hu H et al., Bone Joint Res. 2024).

We agree that the use of molecular diagnostics is crucial, particularly in the context of a high culture-negative rate. We have added a paragraph in the Discussion highlighting the potential benefits of PCR-based methods and metagenomic next-generation sequencing (mNGS), especially for detecting fastidious or dormant pathogens like small-colony variants. We also referenced relevant studies such as Hu H et al., Bone Joint Res 2024 (lines 221-225).

4. Insufficient statistical analysis.
   Resistance trends were analyzed solely via χ² tests, neglecting confounding factors (e.g., antibiotic pretreatment vs. hospital tier). Apply multivariate logistic regression to identify independent risk factors (e.g., preoperative antibiotics and culture-negative rates).

This is an important observation. We have re-analyzed the data using multivariate logistic regression to evaluate independent associations between potential risk factors (prior antibiotic use) and culture-negative results. These results are now included in the Results section and further discussed in the revised manuscript (Results, lines 141-145; Discussion, lines 191-196).

5. Incomplete discussion of resistance mechanisms.
   While rpoB mutations causing rifampicin resistance are noted (lines 157–160), genomic insights (e.g., virulence/biofilm-related genes) are missing. Expand whole-genome sequencing (WGS) data analysis and cite resistance evolution studies.

We appreciate this suggestion. Although genomic data were limited in this study, we have now expanded the discussion on resistance mechanisms, including a broader overview of genes associated with biofilm formation, methicillin resistance, and rifampicin resistance in the Results section (lines 162-170). We have also discussed how these factors impact the management of PJI in the Discussion section, including supporting references from the literature (lines 255-268)

6. Inadequate control of multicenter heterogeneity.
   The study involves four hospitals but lacks standardization details (e.g., variations in culture/susceptibility protocols). Discuss methodological harmonization or its impact on results.

Microbiological processing of samples was performed centrally at the Hospital Universitario de Navarra, the reference center for the region of Navarre. This ensured consistent methodology across all samples from the three participating hospitals, thereby minimizing potential biases related to microbiological processing. This clarification has also been included in the Materials and Methods section (lines 85-88) to prevent any potential misinterpretation

 

7. Unsupported clinical recommendations.
   The proposal to replace ceftazidime with carbapenems (lines 255–257) lacks evidence from susceptibility simulations or efficacy comparisons. Strengthen recommendations with data-driven references.

Thank you for this important point. The recommendation regarding ceftazidime and carbapenem use has been revised. We now clarify that this is a theoretical consideration based on the high resistance rates observed and stress the need for local antimicrobial susceptibility data and prospective clinical validation. We have removed overly prescriptive language and added references to support our suggestion (Lines 282-285).

• Antony S, Khan I, Chowdhury O, Heydemann J, Antony N, Heydemann J, et al. Proposed Guidelines for the Management of ESBL in Prosthetic Joint Infections. Infect Disord Drug Targets [Internet]. 2020 Jun 16 [cited 2025 May 26];20(5):563–9. Available from: https://pubmed.ncbi.nlm.nih.gov/31203809/
• Triffault-Fillit C, Mabrut E, Corbin K, Braun E, Becker A, Goutelle S, et al. Tolerance and microbiological efficacy of cefepime or piperacillin/tazobactam in combination with vancomycin as empirical antimicrobial therapy of prosthetic joint infection: a propensity-matched cohort study. J Antimicrob Chemother [Internet]. 2020 Aug 1 [cited 2025 May 26];75(8):2299–306. Available from: https://pubmed.ncbi.nlm.nih.gov/32407512/

8. Underdeveloped discussion of limitations.
   The absence of molecular diagnostics (e.g., 16S rRNA sequencing) likely underestimates pathogen diversity. Expand the "Limitations" section and cite comparative studies to address this gap.

We have expanded the “Limitations” section to address the absence of molecular diagnostics and its potential impact on pathogen detection and resistance analysis. We also discuss the need for advanced molecular techniques, such as 16S rRNA sequencing or mNGS, to improve microbiological yield. A recent meta-analysis has been cited to support this point, concluding that NGS has demonstrated higher sensitivity and diagnostic accuracy compared to conventional culture methods, although with a slightly lower specificity.  (lines 313-322).

• Su S, Wang R, Zhou R, Bai J, Chen Z, Zhou F. Higher diagnostic value of next-generation sequencing versus culture in periprosthetic joint infection: A systematic review and meta-analysis. Knee Surg Sports Traumatol Arthrosc [Internet]. 2024 Sep 1 [cited 2025 May 26];32(9):2277–89. Available from: https://pubmed.ncbi.nlm.nih.gov/38713871/

Reviewer 2 Report

Comments and Suggestions for Authors

The work “Incidence and Resistance Patterns in Periprosthetic Joint Infections: A Regional Multicenter Study in Spain” is devoted to the study of the causes of periprosthetic joint infections in Spain.

The high number of microbiologically negative periprosthetic joint infections (PJI) detected each year highlights the need to use resistance models to properly manage the infection. In Spain, there is a paucity of published data on this subject. This study analyzed data from patients diagnosed with PJI over the past 5 years in Navarre in 2019-2023. It was found that methicillin-resistant Staphylococcus epidermidis was the predominant etiologic agent, followed by Staphylococcus aureus and Cutibacterium acnes, which in itself indicates a strong difference from other nearby European countries. The high percentage of resistance in microorganisms causing PJI confirms the limitations of empirical treatment in cases of suspected PJI. It is worth paying attention to the choice of therapeutic approaches during infection after surgery, as well as to actively pursue the development of non-antibiotic treatments.

There are some points in the work that require corrections:

1. The paper states that the high percentage of resistance in the region under consideration to the main antibiofilm agents raises the question of the future benefit of new non-antibiotic treatment strategies that may prove promising in the treatment of PJI. However, the text of the paper does not indicate which alternative treatment methods can be considered. For example, such possible treatment methods include stem cell-AMPs, CRISPR-Cas, probiotics, nanobiotics, and a number of others. What methods, in the authors' opinion, should be considered in the near or long term?
2. The excessive use of antibiotics as growth promoters in some food animals is a major factor in the spread of antibiotic resistance genes. Could this be a factor in the difference from other nearby European countries where the main pathogen of PJI is Aureus?
3. The choice of round brackets for formatting references to literature is unclear. Square brackets are usually used for this purpose.
4. What impact do drug-resistant forms of etiologic agents have on the results of the work? For example, drug-resistant staphylococci may in some cases be the main pathogens that increase morbidity, mortality and health care costs.
5. In my opinion, a separate section “Conclusions” should be added to the work to make the work more accessible to readers.

Author Response

We thank the reviewer for their thoughtful and constructive feedback, which has greatly contributed to improving the quality and clarity of our manuscript. Below, we provide our detailed responses to each of the points raised:

The work “Incidence and Resistance Patterns in Periprosthetic Joint Infections: A Regional Multicenter Study in Spain” is devoted to the study of the causes of periprosthetic joint infections in Spain.

The high number of microbiologically negative periprosthetic joint infections (PJI) detected each year highlights the need to use resistance models to properly manage the infection. In Spain, there is a paucity of published data on this subject. This study analyzed data from patients diagnosed with PJI over the past 5 years in Navarre in 2019-2023. It was found that methicillin-resistant Staphylococcus epidermidis was the predominant etiologic agent, followed by Staphylococcus aureus and Cutibacterium acnes, which in itself indicates a strong difference from other nearby European countries. The high percentage of resistance in microorganisms causing PJI confirms the limitations of empirical treatment in cases of suspected PJI. It is worth paying attention to the choice of therapeutic approaches during infection after surgery, as well as to actively pursue the development of non-antibiotic treatments.

There are some points in the work that require corrections:

1. The paper states that the high percentage of resistance in the region under consideration to the main antibiofilm agents raises the question of the future benefit of new non-antibiotic treatment strategies that may prove promising in the treatment of PJI. However, the text of the paper does not indicate which alternative treatment methods can be considered. For example, such possible treatment methods include stem cell-AMPs, CRISPR-Cas, probiotics, nanobiotics, and a number of others. What methods, in the authors' opinion, should be considered in the near or long term?

 

We appreciate this insightful suggestion. In response, we have expanded the discussion to include a brief overview of emerging non-antibiotic strategies that could be promising for the treatment of PJI. Specifically, we now mention approaches such as antimicrobial peptides (AMPs), stem cell-derived AMPs, CRISPR-Cas-based therapies, probiotics, phage therapy, and nanobiotics and references addressing these new therapies. Highlighting that phage therapy is currently in the most promising stages of study and therapeutic application. The updated text can be found in the Discussion section (lines 286-295).

 

• Semeshchenko, P. A. Slullitel, A. Farinati, A. F. Albani-Forneris, N. S. Piuzzi, and M. A. Buttaro, “Unconventional Therapies in Periprosthetic Joint Infections: Prevention and Treatment: A Narrative Review,” J Clin Med, vol. 14, no. 8, Apr. 2025, doi: 10.3390/JCM14082610.
• Ponce Benavente et al., “Targeted enhancement of bacteriophage activity against antibiotic-resistant Staphylococcus aureus biofilms through an evolutionary assay,” Front Microbiol, vol. 15, p. 1372325, Jul. 2024, doi: 10.3389/FMICB.2024.1372325/BIBTEX.
• Wang et al., “Phage-liposome nanoconjugates for orthopedic biofilm eradication,” Journal of Controlled Release, vol. 376, pp. 949–960, Dec. 2024, doi: 10.1016/J.JCONREL.2024.09.049.

2. The excessive use of antibiotics as growth promoters in some food animals is a major factor in the spread of antibiotic resistance genes. Could this be a factor in the difference from other nearby European countries where the main pathogen of PJI is Aureus?

This is a valuable observation. While our study was not designed to assess the origin of resistance gene dissemination, we agree that the use of antibiotics in livestock could potentially contribute to regional variations in resistance patterns. We appreciate the reviewer’s thoughtful observation. While the use of antibiotics in food animals is indeed a well-known factor in the spread of antibiotic resistance genes, it is not specific to prosthetic joint infections (PJIs) and has been extensively discussed in broader contexts. As our study was focused on the clinical and microbiological aspects of PJIs within a specific healthcare setting, we did not explore ecological or agricultural contributors to resistance patterns. For this reason, we did not include a discussion of this factor in the manuscript.

3. The choice of round brackets for formatting references to literature is unclear. Square brackets are usually used for this purpose.

Thank you for pointing this out. We have revised the manuscript to use square brackets for all in-text citations, in accordance with the journal's formatting guidelines.

 4. What impact do drug-resistant forms of etiologic agents have on the results of the work? For example, drug-resistant staphylococci may in some cases be the main pathogens that increase morbidity, mortality and health care costs.

We fully agree with this important point. We have expanded the Discussion to emphasize the clinical implications of drug-resistant pathogens, particularly methicillin-resistant coagulase-negative staphylococci (MRCoNS) and methicillin-resistant Staphylococcus aureus (MRSA). These organisms are associated with increased treatment failure, longer hospital stays, and higher healthcare costs. We believe this addition strengthens the practical relevance of our findings. The new text appears on lines 255-264.

 5. In my opinion, a separate section “Conclusions” should be added to the work to make the work more accessible to readers.

Thank you for this helpful recommendation. We have added a dedicated “Conclusions” section at the end of the manuscript to clearly summarize the main findings and their clinical implications. We believe this enhances the readability and overall structure of the paper