Lessons Learned from An Implant-Related Infection with Bacillus Spp of the Proximal Femur: A Rare and Insidious Complication After Internal Fixation of Closed Fractures

Background: Bacillus are Gram-positive, primarily aerobic, rod-shaped and sporeforming bacteria. Ubiquitary in environment, infections with Bacillus are known to occur after open fractures but are rarely described after internal fixation of closed fractures. Based on a case of implant-related infection of the proximal femur with Bacillus spp after internal fixation, we report on the associated diagnostic and therapeutic challenges.

were sampled for microbiological workup (aerobic and anaerobic cultures, including enrichment broth, with 14 days incubation). All the hardware was retained. Postoperatively, amoxicillin/clavulanate was administered parenterally. Due to persistent wound secretion and persistent elevation of the inflammatory parameters, wound revision was repeated five and nine days later. Three, respectively two microbiological tissue biopsies were sampled on these occasions. Bacillus spp was detected for the first time in two of two samples from the third debridement, whereas all the previous samples failed to detect any microorganisms at all. However, these positive samples had been discarded as contamination and the antibiotic treatment was discontinued. The patient was discharged nine days after the last operation, despite persisting minor secretion from the former suction drain port.
The wound from the surgical approach presented dry without any signs of wound healing disturbances. At discharge, leucocytes had normalized, but the CRP remained elevated at 77 mg/l. However, three days after discharge, the patient had to be readmitted with a breakdown of the wound with serous discharge. The WBC was elevated again, at 10.2 G/l, and the CRP had increased to 101 mg/l, but there was no elevated temperature. Another revision was performed the same day, with debridement, removal of the cerclage, and drainage. Intraoperatively, three biopsies had been sampled for microbiological workup, but this time before any interfering antibiotic treatment. Intravenous antibiotic treatment with amoxicillin/clavulanate was re-established postoperatively.
One tissue sample showed growth of Bacillus spp. Now the infection was diagnosed, integrating the results from the previous revisions, with Bacillus spp being identified in three separate samples, fulfilling even the more rigorous historical diagnostic criteria for low-virulence implant-associated infections [1]. MALDI- However no recurrence of infection was seen ( Figure 2C & 2D).
Informed consent for publication of anonymised health-related data was obtained from the patient.

Discussion
We present a case of an implant-related infection with Bacillus spp after internal fixation of a closed trochanteric fracture with a cephalomedullary nail. To the best of our knowledge, this is only the second case reported in the literature showing a bacillus infection after surgical treatment of a closed fracture [6]. Infections with low virulence microorganisms, such as Bacillus spp, may be difficult to identify and may also be misinterpreted as contaminants [1,2]. Therefore, it is crucial to have an adequate and well-established diagnostic approach if implant-related infection is to be suspected.
The procedure was not optimal in this case, and this hampered proper diagnosis and treatment. The current recommendations are reviewed, focusing on elementary steps in diagnostics that would permit to perform better. Bacillus spp are Gram positive, primarily aerobic, rod-shaped and spore-forming bacteria, which are widely spread in the environment and mostly found in the soil [7]. The spores are formed within the mother cell compartment, and they are extremely resistant to most environmental stress factors [8]. They are released when the mother cell lyses [8]. Spore resistance and dormancy is due to particular structure and components, such as an exosporium, an interspace and a coat layer, which are not found in growing cells [8][9][10][11]. Most Bacillus spp produce necrotizing toxins (cereolysin) and mimic the effect of staphylococcal and clostridial toxins [12]. Therefore, they can induce food poisoning, but are identified more frequently in osteomyelitis, bacteremia, pneumonia, endocarditis and meningitis, especially in immunocompromised patients [12]. Osteomyelitis caused by Bacillus spp is known to occur after open fractures, as this microorganism is ubiquitously present in the soil [7,12,13]. Most Bacillus spp however are of low virulence and are mostly discarded as non-pathogenic contaminants in clinical samples [2]. In presence of an implant, a detailed analysis of results is necessary before such conclusions can properly be drawn. An exception is B. anthracis, which might cause necrotizing and fulminant infections, particularly when airborne. In our case, both samples taken at the third revision showed growth of Bacillus spp. This was misinterpreted as contamination by the laboratory, considering only each sample individually. The patient was immunocompromised to a certain degree due to chronic alcohol abuse, which represents a known risk factor [12]. Even if the clinical signs were inconspicuous, postoperative wound oozing after implant-surgery should already alert the treating physician [2]. To ensure a better interpretation of the microbiological findings, proper sampling is crucial. Four to five tissue biopsies should be sampled in immediate proximity to the implant for microbiological analysis [14][15][16].
Tissue biopsies provide a higher sensitivity and specificity than swab cultures, particularly for detecting microorganisms of low virulence [16,17]. Prolonged incubation on various media under aerobic and anaerobic conditions is crucial to identify fastidious microorganisms, which might be present in implantrelated infections [14,18,19]. Whenever possible, antimicrobial therapy should be withheld for at least 2 weeks before sampling [2,20,21]. In our case, empirical antibiotic treatment interfered with microbiological workup and should not have been started preoperatively before sampling. Identification of phenotypically identical microorganisms in two or more samples is sufficient to diagnose infection [1,14,21]. Histology is very useful for a better differentiation between contamination and infection [22,23].

Granulocyte quantification thresholds have been defined for both
periprosthetic joint infections and fracture-related infection, but technical specificities have to be considered for appropriate counts [22,23]. As surgery induces acute granulocytic inflammation, sampling has to be performed at the first revision, to avoid false positive results.
A fracture-related infection of a non-consolidated fracture corresponds to an osteomyelitis Cierny-Mader stage IV [4], where fracture stabilization is part of the management to treat the infection. As the infection already had to be considered chronified when the diagnosis was made, removal of the implant was indicated [2].
However, Bacillus spp are microorganisms with a low virulence, allowing a one-stage exchange [2,21]. If a fracture already shows enough signs of consolidation, providing sufficient stability, simple implant removal alone may be considered. Suppressive therapy of the infection with hardware retention until fracture healing occurs represents another alternative approach [24]. Local application of vancomycin-loaded calcium sulphate is an adjunct that allows delivery of high and prolonged antibiotic concentrations at the site of infection, at concentrations even high enough to be active against staphylococcal biofilm [3]. Bacillus spp commonly produce beta-lactamases, but usually remain susceptible to a systemic antibiotic therapy with penicillin combined with a beta-lactamase inhibitor [12].
Fluorochinolones, aminoglycosides, clindamycin or vancomycin are alternative systemic antibiotic treatment options [12]. In our case we started postoperatively an intravenous antibiotic treatment with an aminopenicillin combined with a beta-lactamase inhibitor (amoxicillin/clavulanate), followed by an oral treatment with a fluorochinolone (levofloxacin). As sporulating bacterias are difficult to treat, an attempt was made to optimize the efficacy of the antibiotic treatment by adding rifampin, because of a particularly high sensitivity of the concerned bacteria antibiotic drug, expressed as a very low MIC. The addition of rifampin is otherwise only necessary to treat implant-related infections with Staphylococcus spp [21]. The antibiotic treatment was maintained for six months total in our case, to reduce the risk of late relapse of spore-forming bacteria, but no sufficient data is available in the literature regarding optimal duration of antibiotic treatment for such cases.