Analyzing pattern of microorganisms isolated from synovial fluid in patients with chronic orthopedic infection of large joints of extremities

Danir D. Ismatullin; Исматуллин Данир Дамирович; Andrei V. Kozlov; Козлов Андрей Владимирович; Artem V. Lyamin; Лямин Артем Викторович; Dmitriy S. Kudashev; Кудашев Дмитрий Сергеевич; Maria Yu. Sefedinova; Сефединова Мария Юрьевна; Andrey A. Knyazev; Князев Андрей Андреевич; Varvara V. Marchenko; Марченко Варвара Вячеславовна; Viktoria A. Ulivanova; Уливанова Виктория Александровна

doi:10.15789/2220-7619-APO-18016

Analyzing pattern of microorganisms isolated from synovial fluid in patients with chronic orthopedic infection of large joints of extremities

Authors: Ismatullin D.D.¹, Kozlov A.V.¹, Lyamin A.V.¹, Kudashev D.S.¹, Sefedinova M.Y.¹, Knyazev A.A.¹, Marchenko V.V.¹, Ulivanova V.A.¹
Affiliations:
1. Samara State Medical University of the Ministry of Health of the Russian Federation
Issue: Vol 16, No 2 (2026)
Pages: 319-325
Section: ORIGINAL ARTICLES
Submitted: 19.09.2025
Accepted: 05.01.2026
Published: 01.06.2026
URL: https://iimmun.ru/iimm/article/view/18016
DOI: https://doi.org/10.15789/2220-7619-APO-18016
ID: 18016

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Abstract

Total arthroplasty is a key method for treating degenerative and dystrophic diseases of large joints. The most serious complication related to endoprosthesis is periprosthetic infection, the treatment of which involves significant economic costs and a high risk of recurrence. Therefore, during endoprosthetics, it is necessary to conduct a preliminary microbiological examination, which allows to choose the most effective etiotropic therapy especially important in case of antibiotic-resistant microorganisms, fungi and mixed infections. The aim of the study was to analyze the pattern of microorganisms isolated from synovial fluid in patients with chronic orthopedic infection of large joints of the extremities. The study included 79 patients with suspected periprosthetic infection and 15 patients with chronic arthritis combined with osteonecrosis. Synovial fluid samples were delivered to the microbiological laboratory of SamSMU Clinics, where positive samples were cultured and microorganisms were identified using MALDI-ToF mass spectrometry (Bruker, Germany). The study found that among Gram-positive pathogens, S. aureus was the most common — 45.9% (56 strains), S. epidermidis — 29.5% (36 strains) and C. striatum — 13.1% (16 strains) of cases. Among S. aureus, 17.9% were methicillin-resistant. K. pneumoniae was the dominant Gram-negative species (50%), E. coli was the second most common, with 33.2%. 9 strains of K. pneumoniae produced beta-lactamase, and carbapenemases were detected in 4 cases. All E. coli strains were producers of extended-spectrum beta-lactamases. In patients with chronic arthritis and osteonecrosis, Gram-positive pathogens were mainly isolated (73.3%), including methicillin-resistant S. aureus (25%). Gram-negative pathogens accounted for 26.7%, and extended-spectrum beta-lactamase producers prevailed. Hence, Gram-positive bacteria predominate in the pattern of pathogens, especially S. aureus and coagulase-negative Staphylococcus spp. Gram-negative microorganisms act as an etiological agent much less frequently, however, the frequency of prevalence for strains with multiple antibiotic resistance is increasing among them. Thus, conducting a preliminary microbiological examination to determine antibiotic sensitivity becomes a necessary step during endoprosthetics.

Keywords

synovial fluid, periprosthetic infection, bacterial arthritis, joints, endoprosthetics, antibiotic resistance, microbiome

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Introduction

An increase in the life expectancy of the population, in the prevalence of obesity, diabetes mellitus, as well as in the long-term consequences of infection caused by the SARS-CoV-2 virus (including post-COVID-19 syndrome) are significant factors contributing to an increase in the incidence of destructive dystrophic diseases of the musculoskeletal system [1, 2, 19]. Nowadays, the most effective and clinically successful surgical method for the treatment of degenerative-dystrophic diseases of large joints of the extremities in terminal stages, in case of ineffectiveness of conservative therapy, is total arthroplasty. Currently, in Russia and worldwide, the number of primary endoprosthetic surgeries of large joints of the extremities continues to increase along with associated number of complications. The most severe one of them in terms of prevalence, characterized by prolonged and expensive treatment, accompanied by unforeseen consequences and high risks of recurrence, is periprosthetic joint infection (PJI) [9, 14]

In some cases, with timely diagnosis of acute PJI, an artificial joint can be preserved by performing a one-step revision of the endoprosthesis — DAIR procedure (debridment, antibiotics and implant retention), which is based on thorough surgical sanitation with tissue irrigation and replacement of modular and polyethylene components of the endoprosthesis [15].

With the chronization of an acute infectious process or the onset of PJI months or years after total joint replacement, surgical treatment is always aimed at removing all components of the endoprosthesis, which may be performed during one or two stages [17]. In the first case, surgical intervention includes removal of the endoprosthetic components, radical surgical treatment, sanitation, implantation of new endoprosthetic components and subsequent long-term course of parenteral and oral antibacterial therapy. For performing revision of endoprosthesis in one stage, preliminary microbiological examination is important to select the most effective antibacterial drug for therapy, especially in cases of mixed infections or isolation of infectious agents, which are difficult to treat (DTT) [9].

Another difficult group in terms of verifying the diagnosis and determining treatment tactics is patients with a combined course of osteonecrosis and chronic septic (bacterial) arthritis. Septic arthritis is an urgent clinical situation characterized by invasion of microorganisms (MO), mostly bacteria, into the synovial membrane of the joint. In this case, infection with the subsequent inflammatory process in the joint always leads to the destruction of articular surfaces [10]. Timely surgical treatment of purulent arthritis is aimed at urgent arthrotomy, exudate evacuation, drainage of the joint cavity, subsequent antibacterial therapy and extremity immobilization. This tactic is fundamental in urgent surgery, but the subsequent destruction of articular cartilage with changes in articular surfaces leads to the progression of osteonecrosis, the emergence of persistent contractures, limited joint movement, impaired extremity support and a sharp deterioration in the quality of patients’ life. The only possible way to restore the anatomical and physiological characteristics of the joint is total arthroplasty [13].

Gram-positive MO, mainly Staphylococcus aureus, is most often isolated in septic arthritis. Individual studies have shown that phagocytes have the ability to internalize S. aureus and provide its long-term intracellular survival [11, 14]. Over time, an increase in intracellular proliferation of the pathogen is observed against the background of a decrease in the inflammatory response in the host cell. Therefore, even in case of clinical regression of septic inflammation symptoms, intracellular microbial persistence remains (in osteocytes, particularly). This key pathogenetic mechanism indicates a high probability of infectious complications during total arthroplasty.

As a result, this category of patients has a clinical situation with mutually exclusive positions: on the one hand, the need for joint replacement; on the other hand, the impossibility of performing this operation due to the obvious and extremely high risk of septic complications in the postoperative period.

Despite the significant advantages, the microbiological diagnosis procedure faces a number of limitations, including the risk of samples contamination, the lack of the necessary amount of biological material for the study (the dry tap), difficulties in detecting bacteria forming biofilms and decreased diagnostic sensitivity after previous antibiotic therapy [19].

Consequently, such patients have a pronounced need for timely diagnosis and verification of the causative infectious agent in order to make a reasonable decision on the scope and terms of surgical intervention. Microbiological diagnosis with the microbial identification and the antimicrobial susceptibility tests is the fundamental step in determining treatment tactics. The data obtained makes it possible not only to adequately select etiological (local and systemic) therapy, but also to objectively assess the possible risks of surgical treatment [4, 7].

Materials and methods

The study was conducted in the Center for Revision Arthroplasty of the Joints of the Extremities and in the Microbiological Department of the Clinical Diagnostic Laboratory of the Clinics of the Samara State Medical University of the Ministry of Health of the Russian Federation. All patients attended a consultative and diagnostic appointment at the Center for Revision Arthroplasty with complaints of pain and/or the presence of a fistula in the area of the artificial joint. Based on the criteria of the International Consensus Meeting, 2018 (ICM, 2018), the presence of a fistula that reliably communicates with the joint cavity and/or visualization of the components of the endoprosthesis indicate the presence of PJI.

In other cases, a double microbiological examination of the synovial fluid was performed to confirm the diagnosis or refute it. All patients without fistula but with elevated laboratory markers of systemic inflammatory response (leukocyte count > 12 × 10⁹/L, ESR > 30 mm/hour, CRP > 10 mg/L) in combination with an X-ray picture of instability of the endoprosthesis components underwent a double outpatient puncture. The study also included patients with osteonecrosis of the knee and hip joints in combination with unverified synovitis and chronic septic arthritis, who attended consultative appointment.

The punctate was taken in the aseptic conditions of a surgical bandage room. The obtained material was inoculated into a vial for microbial cultivation Yunona^®, containing a nutrient medium and antibiotic neutralizers. The vial was designed to detect bacteria from normally sterile biological fluids. The material was taken in accordance with methodological guidelines 4.2.2039-05 “Techniques for collecting and transporting biomaterials to microbiological laboratories”. After that, the vial was delivered to a microbiological laboratory and installed in a Yunona^®LABSTAR 100 blood culture instrument (SCENKER, China).

Inoculation was performed from each positive vial on commercial universal chromogenic nutrient media and blood agar. Cultivation was carried out at a temperature of 37°C in a Sanyo “MCO-18AC” CO₂ incubator (Panasonic, Japan) for 24–48 hours and under anaerobic conditions using the “Bactron 300-2” anaerobic chamber (Sheldon Manufacturing Inc., USA). Microbial identification was performed using MALDI-ToF mass spectrometry on a “Microflex LT” (Bruker, Germany). In the absence of MO growth, the contents of the vials were microscoped to exclude a possible false negative result.

The antimicrobial drug susceptibility was determined in accordance with the Russian Guidelines “Determination of the susceptibility of microorganisms to antimicrobial drugs” (2021).

In total, during the study period from August 2023 to December 2024, microbiological analysis of synovial fluid obtained from 145 patients was performed. These patients underwent a double joint punсture (a total number of samples is 290). Among 290 examinations, 102 cases (51 patients) showed a negative result — no microbial growth was detected. MO were detected in 188 studies (94 patients). These 94 patients were included in our study.

According to the main diagnosis, the patients were divided into 2 groups: 79 (84%) patients with suspected PJI after primary arthroplasty and 15 (16%) patients with chronic arthritis combined with osteonecrosis.

General characteristics of the observed patients: 59 (62.8%) women and 35 (37.2%) men. The average age of the patients was 67.9 years (from 45 to 81 years).

The distribution of lesion localization by joints showed following results: hip joint — 51 (54.2%) patients, knee joint — 37 (39.4%) patients, shoulder joint — 6 (6.4%) patients. The characteristics of the examined groups of patients with joint lesions are presented in Table.

Table. Characterization of the examined groups of patients with joint lesions

	PJI patients (n = 79) abs. (%)	Patients with chronic arthritis combined with osteonecrosis (n = 15) abs. (%)
Lesion localization
Hip joint	43 (54.4)	7 (46.7)
Knee joint	33 (41.8)	4 (26.7)
Shoulder joint	3 (3.8)	4 (26.7)
Presence of fistula
Fistulous form	54 (68.4)	8 (53.3)
Fistula is absent	25 (31.6)	7 (46.7)
Previous surgical interventions
Present	38 (48.1)	11 (73.3)
Absent	41 (51.9)	4 (26.7)

69 (73.4%) patients had concomitant diseases such as diabetes mellitus, systemic diseases, obesity, HIV-infection, hepatitis B and C, etc., which are risk factors for the emergence of implant-associated infection.

Results

In the group of patients with suspected PJI, 158 MO were isolated and identified, among which 122 (77.2%) were Gram-positive bacteria and 36 (22.8%) were Gram-negative bacteria. In all cases, MO were isolated in monoculture, yeast and mycelial fungi were not identified.

Among the most common Gram-positive pathogens there were Staphylococcus aureus — 45.9% of cases (56 strains), Staphylococcus epidermidis — 29.5% (36 strains) and Corynebacterium striatum — 13.1% (16 strains).

In individual cases, another 7 species were identified: Enterococcus faecalis, Staphylococcus haemolyticus, Staphylococcus pasteuri, Staphylococcus hominis, Streptococcus pneumoniae, Streptococcus constellatus, Streptococcus cannis — 2 strains of each species, which together accounted for 11.5% of all Gram-positive microorganisms during the study period.

Analyzing the structure of Gram-negative MO, a lower species diversity was revealed. Among 36 bacteria identified, Klebsiella pneumoniae was identified in half of the cases — 50% (18 strains). Escherichia coli was the second most common species — 33.2% of all cases (12 strains). In individual cases another species were identified: Pseudomonas aeruginosa — 5.6% (2 strains), Serratia marcescens — 5.6% (2 strains strains) and Acinetobacter baumannii — 5.6% (2 strains).

After performing an antibiotic susceptibility test, 34 strains (60.7%) susceptible to methicillin (MSSA) were identified among isolated strains of Staphylococcus aureus. Another 12 strains (21.4%) showed selective resistance to benzylpenicillin and macrolides. The remaining 10 strains (17.9%) were resistant to methicillin (MRSA).

Among coagulase-negative staphylococci, only 12 strains of Staphylococcus epidermidis showed phenotypic signs of methicillin resistance.

All Corynebacterium striatum strains (16 strains) isolated from clinical material were resistant to benzylpenicillin and ciprofloxacin, but remained susceptible to vancomycin, linezolid, tetracycline and rifampicin.

In half of the cases, all identified Klebsiella pneumoniae strains (9 strains) were producers of extended-spectrum beta-lactamases (BLRS). In 4 more cases, we encountered highly resistant carbapenemase-producing strains: antimicrobial effectiveness in these cases was revealed only with colistin.

All 12 Escherichia coli strains isolated in the study were producers of BLRS, but no carbapenemase activity was detected. Amoxicillin/clavulanate, ampicillin/sulbactam, amikacin, and tigecycline were effective against these strains.

Serratia marcescens strains were resistant to ampicillin, ampicillin/sulbactam, and amoxicillin/clavulanate due to their natural resistance to these drugs.

Strains of Pseudomonas aeruginosa and Acinetobacter baumannii found in our study showed resistance to piperacillin/tazobactam, ciprofloxacin, amikacin, and carbapenems. Colistin was the only effective antibiotic in these cases.

In patients with chronic arthritis combined with osteonecrosis, Gram-positive pathogens were mainly isolated — 22 strains (73.3% of the total number of MO). Among these strains identified there were strains of Staphylococcus aureus, including 2 strains of MRSA, 5 strains of Staphylococcus epidermidis (all susceptible to methicillin), 5 strains of Corynebacterium striatum and 4 strains of Enterococcus faecalis.

Among 8 isolates of Gram-negative bacteria (26.7% of the total number of MO), 4 strains of Klebsiella pneumoniae were isolated, 3 of which were BLRS producers. Two strains of Escherichia coli and two strains of Pseudomonas aeruginosa were also detected.

Discussion

According to data, obtained worldwide, knee and hip arthroplasty is one of the most common surgeries [2, 3]. The ankle, wrist, shoulder and elbow joints are relatively less frequently replaced. At the same time, patients are at constant risk of developing PJI throughout their lives.

In our work, we conducted a study of synovial fluid from patients with suspected PJI (n = 79) and patients with chronic arthritis combined with osteonecrosis (n = 15).

The main causative agents of PJIs are MO with the ability to rapidly form biofilms synthesized from the extracellular matrix on artificial surfaces of prostheses [5]. In most cases, the causative agents of joint infections are Gram-positive bacteria, especially S. aureus and coagulase-negative staphylococci, followed by Streptococcus spp., Enterococcus spp. and Corynebacterium spp. [19, 20]. It is interesting to note that the growth of some microorganisms (Staphylococcus spp., E. coli) was detected under anaerobic conditions in the absence of growth during subcultivation in an aerobic environment [18, 21]. The isolation frequency of gram-negative bacteria is lower, however, in the case of their isolation in practice, we encounter a large number of polyresistant bacteria. And our research is generally consistent with global trends.

В Gram-positive microorganisms prevailed in both groups — 77.2% and 26.7%, respectively. 10 strains of S. aureus and 12 strains of S. epidermidis with methicillin resistance were detected in patients with suspected PJI, which together account for 18% of the total number of Gram-positive bacteria. In patients with chronic arthritis combined with osteonecrosis, 2 strains of MRSA (9% of the total number of Gram-positive bacteria) were detected, all strains of S. epidermidis were susceptible to methicillin.

It is worth noting the high isolation frequency of C. striatum in both groups of patients: 16 strains (13.1% of the total number of Gram-positive bacteria) in patients with suspected PJI and 5 strains (22.7% of the total number of Gram-positive bacteria) in patients with chronic arthritis combined with osteonecrosis. This pathogen belongs to non-diphtheria corynebacteria and is a relatively new pathogen, the clinical significance of which is certainly growing with the identification of patients with various risk factors. Following resistance genes have been described in multidrug resistant C. striatum strains: erm(X), encoding resistance to erythromycin, clindamycin; tetA and tetB, encoding resistance to tetracycline [6, 12].

Gram-negative pathogens were significantly less common in our study than Gram-positive ones, but cases of antibiotic resistance were more common among them. Therefore, in synovial fluid samples from patients with suspected PJI, 13 K. pneumoniae strains showed signs of resistance: 9 strains produced BLRS, and 4 were carbapenemase producers. All E. coli strains were also producers of BLRS.

A serious situation from clinical and microbiological assessment point of view is the isolation of polyresistant strains of P. aeruginosa and A. baumannii from synovial fluid.

Due to the fact that one of the main stages of the PJIs treatment is the installation of a cement spacer with an antibiotic, the assessment of susceptibility to antimicrobial drugs using various methods of microbiological diagnosis allows to apply targeted antibacterial therapy, taking into account the various mechanisms of causative agent’s resistance. This is mainly relevant due to the recent increase in resistant strains, especially in a number of Enterobacterales, among which the most problematic species is K. pneumoniae.

The problem of PJIs occupies one of the key places among the endoprosthetic complications in traumatology. Currently, there is a noticeable increase in such operations, therefore, the rate of revision interventions is also increasing [8, 16]. Under the current conditions, there is also a significant increase in cases of PJIs, which indicates the high importance of research aimed at studying the control of infectious agents, as well as improving methods of diagnosis, treatment and prevention.