The following methods and laboratory precautions were used at the FNRCF to confirm the diagnosis of tularemia. For F. tularensis culturing, samples and strains were housed in a Biosafety Level 3 laboratory. Samples were seeded on chocolate agar and incubated at 37°C in a CO₂-enriched atmosphere for 10 days. Methods for identifying F. tularensis subsp. holarctica varied among the 3 patient cases described in this article. Before 2018 (case 1), specific real-time insertion sequence F. tularensis (ISFtu2) PCR and sequencing of the 16S 23S intergenic region (16) were used to confirm identification at the subspecies level. Since 2018 (cases 2 and 3), F. tularensis subsp. holarctica identification has been confirmed using in-house ISFtu2 PCR and F. tularensis subsp. holarctica–specific PCR, according to previously published protocols (17,18). Before 2018 (case 1), serologic testing was performed using in-house microagglutination test (MAT) and in-house indirect immunofluorescence assay (IFA). Since 2018, ELISA (Serion Diagnostics) (case 2) and chemiluminescent immunoassay (Vircell) have replaced the MAT method.

For this study, we performed retrospective antibiotic susceptibility testing to determine the MIC of 7 antibiotics (gentamicin, ciprofloxacin, levofloxacin, rifampin, erythromycin, azithromycin, and doxycycline). We used an in-house broth microdilution method, as previously described (19).

For the retrospective case search, we performed a PubMed search for published cases of PJI caused by F. tularensis using the terms “Francisella AND joint AND infection” with no restriction of date or language. In total, 21 articles matched the searched criteria, but only 5 cases corresponded to PJI.

Prosthetic joint replacement is a common surgery increasingly used to improve quality of life (22,23). Several recommendations to limit infection at the surgical site exist (24–26), which are either preoperative (skin disinfection by shower with antiseptic or antimicrobial soap, nasal and cutaneous decolonization of S. aureus), perioperative (perioperative antiseptic use, intraoperative systemic antimicrobial prophylaxis, asepsis of the operative environment), or postoperative (postoperative antimicrobial prophylaxis). Despite those preventive measures, prosthetic joint infections occur in 1%–2% of patients (27–29). The most common pathogens described are staphylococci (S. aureus and coagulase-negative staphylococci), isolated in 50%–60% of the cases; streptococci; and enterococci (together accounting for 10%). Aerobic gram-negative rods and anaerobic bacteria are isolated from <10% of knee and hip prosthetic joint infections. However, 5%–34% of prosthetic infections remain culture-negative because of previous antimicrobial treatment or because fastidious or low-inoculum pathogens can remain undetectable by classic culture or PCR methods (27,28,30).

Prosthetic joint infection is an extremely rare form of tularemia; we found only 5 cases reported in the literature to date (Tables 1, 2) (10–13). Of the 8 cases of prosthetic infections summarized in this article, 7 (88%) occurred in men >49 years of age (range 49–84 years). The knee prosthesis was infected in 5 (62.5%) patients and hip prostheses in 3 (37.5%) patients. In all but 2 patients, clinical symptoms appeared >6 months after joint placement (7 days–19 years, median 4 years) and were unspecific to tularemia. Fever, joint pain, and joint exudate were often described, and inflammatory blood markers, where reported, usually increased (CRP range 16–100 mg/L). Inoculation of F. tularensis at the surface of the prosthesis seems to be possible in 3 different ways: through direct transfer from contaminated skin wounds or ulcers during surgery (case 1), after surgery through direct skin contamination close to the surgical site scar, or by hematologic spread of the bacteria from an initial infection site distant from the prosthesis (cases 2 and 3). In the reported BJI cases, the initial contamination was suspected to occur through direct transmission from an animal reservoir, either by ingestion of undercooked meat prepared from an infected animal or contact with infected carcasses (cases 2, 3, 4, 7, and 8); possibly by an arthropod bite, as in case 8, in which the patient noted a tick bite 6 months before his initial knee arthroplasty; or by inhalation of contaminated aerosols or dust (cases 1, 5, and 6). For some patients, infectious symptoms were absent or mild, suggesting that the bacteria might be able to survive in a quiescent form with low virulence levels after seeding on the prosthesis. Mechanisms similar to those involved in the long-term persistence of F. tularensis subsp. holarctica in soil and water might occur, such as switching to a viable but noncultivable state or biofilm formation (4; C.D. Brunet et al., unpub. data, https://www.biorxiv.org/content/10.1101/2022.02.18.480867v3). Biofilm formation has been described for the Francisella species in vitro and might be a key mechanism in human pathogenesis and infection, especially PJIs (28,31).

According to the World Health Organization definition of tularemia, presumptive cases are defined as cases in persons with clinical symptoms compatible with tularemia and either positive DNA detection in 1 clinical sample or a single positive serum sample. An infection is confirmed when an F. tularensis strain is isolated and identified in culture or by a 4-fold increase in IgM or IgG titers in paired serum specimens (17). Although tularemia is primarily identified by serologic assays, the proportion of diagnoses assessed by molecular methods on tissue samples is increasing because of the development of specific PCRs or the availability of 16S rDNA sequencing. Positive bacterial cultures are usually obtained in <10% of cases (7,16) because F. tularensis is a fastidious bacterium that requires cysteine-supplemented agar (17). Growth occurs in 2–4 days of incubation at 37°C in aerobic or CO₂-enriched atmosphere, and colonies are gray-white, round, and smooth with a small halo (32,33). Surprisingly, in these 8 cases, the diagnosis was made by positive culture on joint aspiration or perioperative tissues. This finding highlights the major need for rapid seeding of fresh tissues on an agar media to enable the growth of this fastidious bacterium from human samples (34). Serologic testing, when performed, identified mostly high titers in MAT, ELISA, or IFA, confirming Francisella infection, but was performed after the initial diagnosis. The identification of 1 strain was suspected on the basis of results of the Vitek 2 GN assay. MALDI-TOF mass spectrometry enabled identification of 2 strains but did not provide subspecies distinction (35–37). F. tularensis has been identified using the in vitro diagnosis database of the Biotyper mass spectrometer since 2017 (partial integration of 5 species of the security-relevant library as a library extension) only if the extension was added to the database by the user. In contrast, the Vitek MS database does not contain any F. tularensis spectra. In 7 of 8 cases, the strain was identified or confirmed by molecular methods (Table 1). Because of the high level of 16S rDNA similarity among the Francisella species (98.5%–99.9%), 16S rDNA amplification and sequencing enables identifying the bacterium only to the genus level (38). Species or subspecies determination requires PCR targeting specific genes, such as ISFtu2, 23kDa, tul4, or fopA (39). Some of those targets, however, might cross-react with F. novicida, F. philomiragia, or Francisella-like endosymbionts. Additional techniques are necessary to identify F. tularensis subspecies, such as PCR targeting a junction between ISFtu2 and a flanking 3′ region (18), identification on the basis of size differences of amplified DNA products (Ft-M19, ISFtu2, RD1, and pdpD-2 assay) (17,40), and amplification and sequencing of the 16S-23S rRNA intergenic spacer region (16).

Strategies for PJI treatment combine surgical interventions and antimicrobial therapy. Overall, in cases of early infection (<15 days after prosthesis placement), prosthesis retention with debridement is recommended. In chronic infection, prosthesis removal is the best option, performed as either a 1-stage or 2-stage replacement procedure, depending on the patient history, the bacterium identified, and susceptibility to antibiotics. Those surgical treatments are associated with a long duration (4–6 weeks) of antimicrobial therapy adapted to the antibiotic susceptibility of the bacteria identified (28,41,42). Several guidelines for tularemia treatment have been published (2,17,43,44). The antibiotic classes recommended for first-line treatment of tularemia are aminoglycosides, fluoroquinolones, and tetracycline because the bacterium is intrinsically resistant to many other antibiotic classes (all β-lactams, TMP/SXT, clindamycin, glycopeptides, and daptomycin) (19). For severe tularemia cases, parenteral gentamicin (5 mg/kg/d) treatment is recommended depending on the clinical response. In moderate cases, oral ciprofloxacin (800–1,000 mg/d) or doxycycline (200 mg/d) can be administered for a minimum duration of 10–14 days. However, those recommendations have not yet been established for BJIs. Of the 8 cases we reviewed, 5 patients underwent 1- or 2-stage revision joint replacement; 1 underwent debridement, antibiotic, and implant retention; and 2 underwent regular joint aspiration without prosthesis replacement. For 2 patients, aminoglycosides were administered during and after the surgery (120 mg amikacin for 5 days and 240 mg gentamicin for 10 days), followed by monotherapy with ciprofloxacin (500 mg 2×/d for 20 days). Three patients received monotherapy; 1 patient took ciprofloxacin (750 mg 2×/d) for 3 months, and 2 patients were prescribed doxycycline (100 mg 2×/d) for 6 weeks or 12 months. Combination therapy was administered in 2 other cases, consisting of ciprofloxacin with rifampin for >6 months or ciprofloxacin with doxycycline for 9 weeks. When information was available, the patients’ follow-up visits revealed favorable progress without joint infection relapse.

Strains have not been reported that are resistant to the recommended first-line antibiotic classes. A comprehensive review reported low MICs for ciprofloxacin (<0.002–0.125 mg/L), gentamicin (<0.016–2 mg/L), and doxycycline (0.064–4 mg/L) against F. tularensis strains (19). Aminoglycosides penetrate slowly intracellularly and are effective against extracellular bacteria. On the basis of our previous comprehensive review of antimicrobial susceptibility testing data in vitro, in cellular model, and in mice model of infection (19), we advise using aminoglycoside for only a short period during and after surgery, when a bacteremic phase can occur, to rapidly kill extracellular bacteria and prevent hematologic spread after surgery. Combining aminoglycosides with ciprofloxacin might confer the highest efficacy because of the rapid penetration of fluoroquinolones in bones and joint tissues and their efficient activity against the intracellular niche of Francisella. Thus, combining aminoglycosides and fluoroquinolones might be considered in these severe cases (44). Ciprofloxacin and doxycycline, alone or in combination, can be used for long-term treatment. Interest in combination therapy with rifampin has not been demonstrated, but in vitro activity of rifampin against Francisella has been observed (MICs range 0.094–3 mg/L), and its excellent bone diffusion might enhance successful outcomes, as demonstrated by case 1 (13,19,21,45). However, rifampin is not recommended for tularemia treatment because of insufficient in vivo data (19).

Because of the difficulty and delay involved in identifying Francisella strains, laboratory staff can be exposed to bacteria without recommended precautions. During surgical interventions, surgeons can be exposed through contact with infectious materials, accidental inoculation, or exposure to aerosols and infectious droplets. World Health Organization guidelines distinguish 3 situations: proven accidental laboratory exposure, potential exposure to F. tularensis aerosols, and unlikely exposure. Antibiotic prophylaxis (1,000 mg ciprofloxacin or 200 mg doxycycline for 14 days) or clinical follow-up was recommended according to exposure risk (17).

In conclusion, PJI is an unusual clinical manifestation of tularemia that might be underestimated because of the fastidious culture conditions and difficulty in strain identification. Infection might occur in tularemia-endemic areas or in the presence of risk factors. The combination of surgical and extended antibiotic treatment generally leads to favorable outcomes.