Analysis of in-patient evolution of Escherichia coli reveals potential links to relapse of bone and joint infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

Multicenter evaluation of the BIOFIRE Joint Infection Panel for the detection of bacteria, yeast, and AMR genes in synovial fluid samples.

The bioMérieux BIOFIRE Joint Infection (JI) Panel is a multiplex in vitro diagnostic test for the simultaneous and rapid (~1 h) detection of 39 potential pathogens and antimicrobial resistance (AMR) genes directly from synovial fluid (SF) samples. Thirty-one species or groups of microorganisms are included in the kit, as well as several AMR genes. This study, performed to evaluate the BIOFIRE JI Panel for regulatory clearance, provides data from a multicenter evaluation of 1,544 prospectively collected residual SF samples with performance compared to standard-of-care (SOC) culture for organisms or polymerase chain reaction (PCR) and sequencing for AMR genes. The BIOFIRE JI Panel demonstrated a sensitivity of 90.9% or greater for all but six organisms and a positive percent agreement (PPA) of 100% for all AMR genes. The BIOFIRE JI Panel demonstrated a specificity of 98.5% or greater for detection of all organisms and a negative percent agreement (NPA) of 95.7% or greater for all AMR genes. The BIOFIRE JI Panel provides an improvement over SOC culture, with a substantially shorter time to result for both organisms and AMR genes with excellent sensitivity/PPA and specificity/NPA, and is anticipated to provide timely and actionable diagnostic information for joint infections in a variety of clinical scenarios.

Lysosomal alkalization to potentiate eradication of intra-osteoblastic Staphylococcus aureus in the bone and joint infection setting.

OBJECTIVES: Beyond intracellular penetration, acidic lysosomal pH might affect the intracellular activity of some antimicrobials. This study evaluated the ability of lysosomotropic alkalizing agents to potentiate the antimicrobial eradication of an intra-osteoblastic Staphylococcus aureus reservoir in the setting of bone and joint infection (BJI). METHODS: MICs of 16 anti-staphylococcal molecules active against methicillin-sensitive S. aureus (MSSA) were evaluated at pH 5 and pH 7. Additionally, the lysosomal alkalizing potential (spectrofluorometry) and cytotoxicity (MTT assay) of hydroxychloroquine, amantadine and ammonium chloride were assessed. The results led to further investigation of clindamycin, cotrimoxazole, daptomycin and levofloxacin-alone or in combination with hydroxychloroquine-in an in vitro model of osteoblast infection. The impact of hydroxychloroquine on autophagy was finally investigated using Western blot detection of two autophagic flux indicators, the LC3 membrane protein and the SQSTM1 cargo protein. RESULTS: Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone significantly decreased the intracellular staphylococcal reservoir (5.12 log10 CFU/100 000 cells) by 0.14 (95%CI 0.01-0.34), 0.25 (95%CI 0.12-0.43), 0.16 (95%CI 0.004-0.39) and 1.18 (95%CI 1.04-1.38) log10 CFU/100 000 cells, respectively (p < 10-3). Adding hydroxychloroquine (20 mg/L) increased intralysosomal pH from 4.8 to 7, and concomitantly the inoculum of each antimicrobial was reduced by 0.50 (95%CI 0.30-0.84), 0.73 (95%CI 0.59-0.96), 0.59 (95%CI 0.46-0.78) and 1.8 (95%CI 1.66-2.1) log10 CFU/100 000 cells, respectively (p < 10-4). Cellular levels of LC3II and SQSTM1 showed that hydroxychloroquine has direct activity on the autophagic flux, fostering the eradication of intracellular S. aureus by antimicrobials. CONCLUSION: At high concentrations, hydroxychloroquine used as an adjuvant to antimicrobials improves eradication of an S. aureus intra-osteoblastic reservoir in our in vitro cell infection model. These findings advocate further in vivo evaluation of alkalization efficacy and tolerance in S. aureus BJI.

In vitro antibiotic activity against intraosteoblastic Staphylococcus aureus: a narrative review of the literature.

Staphylococcus aureus - a major aetiological agent of bone and joint infection (BJI) - is associated with a high risk of relapse and chronicity, in part due to its ability to invade and persist in non-professional phagocytic bone cells such as osteoblasts. This intracellular reservoir protects S. aureus from the action of the immune system and most antibiotics. To date, the choice of antimicrobial strategies for BJI treatment mostly relies on standard susceptibility testing, bone penetration of antibiotics and their 'antibiofilm' activity. Despite the role of intracellular persistent S. aureus in the development of chronic infection, the ability of antibiotics to target the S. aureus intraosteoblastic reservoir is not considered in therapeutic choices but might represent a key determinant of treatment outcome. This review provides an overview of the intracellular pharmacokinetics of antistaphylococcal drugs used in the treatment of BJI and of their ability to target intraosteoblastic S. aureus. Thirteen studies focusing on the intraosteoblastic activity of antibiotics against S. aureus were reviewed, all relying on in vitro models of osteoblast infection. Despite varying incubation times, multiplicities of infection, bacterial strains, and the types of infected cell lines, rifamycins and fluoroquinolones remain the two most potent antimicrobial classes for intraosteoblastic S. aureus eradication, consistent with clinical data showing a superiority of this combination therapy in S. aureus orthopaedic device-related infections.

Evaluation of intraosteoblastic activity of dalbavancin against Staphylococcus aureus in an ex vivo model of bone cell infection.

OBJECTIVES: Long-acting lipoglycopeptides are promising therapeutic options in Staphylococcus aureus bone and joint infections (BJIs). This study evaluated the ability of dalbavancin to eradicate the intraosteoblastic reservoir of S. aureus, associated with BJI chronicity. METHODS: Osteoblastic cells were infected with a standardized inoculum of the S. aureus reference strain HG001 and incubated for 24 h with dalbavancin, vancomycin or rifampicin using the MIC, 10×MIC, 100×MIC and/or the intraosseous concentrations reached using standard therapeutic doses (i.e. vancomycin, 10 mg/L; rifampicin, 2 mg/L; and dalbavancin, 6 mg/L). The remaining intracellular bacteria were quantified by plating cell lysates. RESULTS: MICs of dalbavancin, vancomycin and rifampicin were 0.125, 1 and 0.004 mg/L, respectively. Dalbavancin significantly reduced the intracellular inoculum of S. aureus starting at a concentration equal to the MIC, with a significant dose effect, ranging from a reduction of 31.4% (95% CI = 17.6%-45.2%) at MIC to 51.6% (95% CI = 39.8%-63.4%) at 100×MIC compared with untreated cells. Of note, dalbavancin was the only molecule to significantly reduce the intraosteoblastic inoculum at low concentration (MIC). At intraosseous concentrations, dalbavancin reduced the intracellular inoculum by 49.6% (95% CI = 45.1%-54.1%) compared with untreated cells (P < 0.001), with no significant difference compared with vancomycin (38.1%; 95% CI = 19.2%-57.0%; P = 0.646), and was less efficient than rifampicin (69.0%; 95% CI = 63.2-74.8; P < 0.001). CONCLUSIONS: Dalbavancin was able to decrease the intraosteoblastic S. aureus inoculum by 50% at intraosseous concentrations reached during standard human therapeutic dosing, with no difference compared with vancomycin, and remained less efficient than rifampicin. However, it was the only molecule significantly active at low concentration.

Performance of the Hologic Panther Fusion® MRSA Assay for the nasal screening of methicillin-sensitive and methicillin-resistant Staphylococcus aureus carriage.

Staphylococcus aureus (SA) nasal carriage screening is usually based on either culture or molecular biology. The aim of the study was to evaluate the performance of the Panther Fusion® MRSA Assay (PF) that proposes a complete automation of the molecular screening for MSSA and MRSA carriage. Four hundred thirty-four nasal samples collected on ESwab™ were screened using PF. Results were compared with standard culture on BBL™ CHROMagar™ Staph aureus and chromID® MRSA agar. Discordant results were analyzed with additional techniques: Xpert SA Nasal Complete on GeneXpert (GX), culture on selective agar after 24 h in broth enrichment, and, if necessary, characterization of mec gene and SCCmec cassette using DNA microarray. The PF presented an overall agreement of 97.5% for SA detection and 97.9% for MRSA detection. Furthermore, 7.1% (31/434) of the samples were SA-negative in primary culture but SA-positive using PF and GX, confirming the greater sensitivity of molecular tests compared with culture. Of note, 4 out of 30 MRSA-positive samples were not detected due to an atypical SCCmec cassette, while 2 samples were falsely detected as MRSA due to co-colonization with a MSSA drop-out strain and a methicillin-resistant coagulase-negative staphylococcal strain. Considering all results, the PF instrument appears as a reliable and rapid (< 3 h) package for MSSA/MRSA nasal screening. This technology using random access capability and direct sampling of the primary container is innovative and corresponds therefore to a new step in complete molecular biology automation in bacteriology.

Antibiofilm and intraosteoblastic activities of rifamycins against Staphylococcus aureus: promising in vitro profile of rifabutin.

BACKGROUND: Targeting biofilm-embedded and intraosteoblastic Staphylococcus aureus, rifampicin gained a pivotal role in bone and joint infection (BJI) treatment. Two other rifamycins, rifabutin and rifapentine, may represent better-tolerated alternatives, but their activity against bacterial reservoirs associated with BJI chronicity has never been evaluated. OBJECTIVES: To evaluate the activities of rifampicin, rifabutin and rifapentine in osteoblast infection models. METHODS: Using three S. aureus isolates, rifamycins were compared regarding: (i) their intracellular activity in 'acute' (24 h) and 'chronic' (7 days) osteoblast infection models at 0.1× MIC, 1× MIC, 10× MIC and 100× MIC, while impacting infection-induced cytotoxicity (MTT assay), intracellular phenol-soluble modulin (PSM) secretion (RT-PCR), resistance selection and small colony variant (SCV) emergence; and (ii) their minimal biofilm eradication concentration (MBEC) and their MIC to prevent biofilm formation (bMIC). RESULTS: At 0.1× MIC, only rifabutin significantly reduced intracellular inoculum and PSM secretion. All rifamycins allowed a 50% reduction of intraosteoblastic inoculum at higher concentrations, with no difference between acute and chronic infection models, while reducing infection-induced cytotoxicity and PSM secretion. Dose-dependent emergence of intracellular SCVs was observed for all molecules. No intracellular emergence of resistance was detected. bMICs were equivalent for all molecules, but MBEC90s of rifapentine and rifabutin were 10- to 100-fold lower than those of rifampicin, respectively. CONCLUSIONS: All rifamycins are efficient in reducing the S. aureus intraosteoblastic reservoir while limiting infection-induced cytotoxicity, with a higher activity of rifabutin at low concentrations. All molecules prevent biofilm formation, but only rifapentine and rifabutin consistently reduce formed biofilm-embedded bacteria for all isolates. The activity of rifabutin at lower doses highlights its therapeutic potential.

Evaluation of the ability of linezolid and tedizolid to eradicate intraosteoblastic and biofilm-embedded Staphylococcus aureus in the bone and joint infection setting.

OBJECTIVES: Prolonged use of linezolid for bone and joint infection (BJI) is limited by its long-term toxicity. The better safety profile of tedizolid, a recently developed oxazolidinone, could offer an alternative. However, its efficacy against biofilm-embedded and intracellular Staphylococcus aureus, the two main bacterial reservoirs associated with BJI chronicity, is unknown. METHODS: Using three S. aureus strains (6850 and two clinical BJI isolates), linezolid and tedizolid were compared regarding their ability: (i) to target the S. aureus intracellular reservoir in an in vitro model of osteoblast infection, using three concentrations increasing from the bone concentration reached with standard therapeutic doses (Cbone = 2.5 × MIC; Cplasm = 10 × MIC; Cmax = 40 × MIC); (ii) to eradicate mature biofilm [minimal biofilm eradication concentration (MBEC)]; and (iii) to prevent biofilm formation [biofilm MIC (bMIC) and confocal microscopy]. RESULTS: Linezolid and tedizolid weakly reduced the intracellular inoculum of S. aureus in a strain-dependent manner despite the similar MICs for the tested strains, but improved cell viability even in the absence of an intracellular bactericidal effect. Conversely, linezolid and tedizolid were ineffective in eradicating mature biofilm formed in vitro, with MBEC >2000 and >675 mg/L, respectively. bMICs of tedizolid were 4-fold lower than those of linezolid for all strains. CONCLUSIONS: Linezolid and tedizolid alone are not optimal candidates to target bacterial phenotypes associated with chronic forms of BJI. Despite weak intracellular activity, they both reduce infection-related cytotoxicity, suggesting a role in modulating intracellular expression of staphylococcal virulence factors. Although inactive against biofilm-embedded S. aureus, both-but particularly tedizolid-are able to prevent biofilm formation.

Intraosteoblastic activity of daptomycin in combination with oxacillin and ceftaroline against MSSA and MRSA.

BACKGROUND: The Staphylococcus aureus intracellular reservoir is associated with bone and joint infection (BJI) chronicity. As daptomycin is increasingly prescribed in BJI, strategies for improving its reduced intracellular activity should be promoted. OBJECTIVES: Based on the known in vitro synergy of daptomycin with ß-lactams, the aim of the present study was to evaluate the intracellular activity of these combinations in an ex vivo osteoblast infection model. METHODS: Osteoblastic cells infected with an MRSA strain or its isogenic MSSA counterpart were incubated for 24 h with daptomycin, oxacillin or ceftaroline alone or in combination using usual intraosseous therapeutic concentrations. Intracellular bacteria were quantified by plating cell lysates. MICs for MSSA and MRSA were determined using the chequerboard method at pH 5 to mimic conditions expected within lysosomes, the foremost S. aureus intracellular location. RESULTS: Daptomycin failed to reduce the intracellular MSSA inoculum, and was weakly active against MRSA compared with untreated cells (-27.6%; P < 10-3). Oxacillin and ceftaroline revealed significant intracellular activity, including oxacillin against MRSA-infected cells (-43.2%; P < 10-3). The daptomycin/oxacillin combination was significantly more active against intracellular MSSA and MRSA compared with daptomycin and oxacillin alone (-44.4% and -57.2%, respectively; P < 0.05). In contrast, daptomycin/ceftaroline was not more efficient than ceftaroline alone. Interestingly, oxacillin MICs for MRSA decreased in vitro from >256 to 0.023 mg/L when the pH decreased from 7 to 5, and chequerboards showed an additive effect of the daptomycin/oxacillin combination against MRSA at pH 5. CONCLUSIONS: In acidic intracellular conditions, oxacillin enhances daptomycin activity against the intraosteoblastic reservoir of S. aureus, including MRSA.