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Background While the treatment of ESBL-producing Enterobacterales osteomyelitis relies on carbapenems, the optimal regimen for OXA48 types remains unclear. We evaluated the efficacy of ceftazidime/avibactam in different combinations in an experimental model of OXA-48-/ESBL-producing Escherichia coli osteomyelitis. Methods E. coli pACYC184 is a clinical strain harbouring blaOXA-48 and blaCTX-M-15 inserts, with ‘increased exposure susceptibility’ to imipenem (MIC, 2 mg/L), gentamicin (MIC, 0.5 mg/L), colistin (MIC, 0.25 mg/L), ceftazidime/avibactam (MIC, 0.094 mg/L) and fosfomycin (MIC, 1 mg/L), and resistance to ceftazidime (MIC, 16 mg/L). Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu of OXA-48/ESBL E. coli. Treatment started 14 days later for 7 days in six groups: (1) control, (2) colistin 150.000 IU/kg subcutaneously (SC) q8h, (3) ceftazidime/avibactam 100/25 mg/kg SC q8h, (4) ceftazidime/avibactam + colistin, (5) ceftazidime/avibactam + fosfomycin 150 mg/kg SC q12h, (6) ceftazidime/avibactam + gentamicin 15 mg/kg intramuscularly (IM) q24h. Treatment was evaluated at Day 24 according to bone cultures. Results In vitro, time–kill curves of ceftazidime/avibactam in combination showed a synergistic effect. In vivo, compared with controls, rabbits treated with colistin alone had similar bone bacterial density (P = 0.50), whereas ceftazidime/avibactam alone or in combinations significantly decreased bone bacterial densities (P = 0.004 and P Conclusions In our model of E. coli OXA-48/ESBL osteomyelitis, ceftazidime/avibactam in combination was more effective than any single therapy, whatever the companion drug used (gentamicin or colistin or fosfomycin).

Background Resistance to linezolid has become a worldwide concern since it is one of the last-resort antibiotics to treat multidrug-resistant staphylococcal and enterococcal infections. Objectives We investigated staphylococcal infections caused by 16 cfr-positive linezolid-resistant Staphylococcus epidermidis and Staphylococcus aureus isolates in a French university hospital from 2015 to 2018. Methods Antimicrobial susceptibility of isolates was tested by broth microdilution and gradient strips. Genetic determinants of linezolid resistance (including cfr gene and 23S rRNA mutations) were assessed by PCR and WGS; the latter was also used to characterize the cfr-carrying plasmids in S. epidermidis and S. aureus, and to explore the clonal relationship of isolates. Results All linezolid-resistant staphylococcal isolates harboured the same cfr-carrying plasmid, sharing 99% identity with the previously described pSA737. The three S. aureus isolates belonged to different STs (ST8, ST72, ST2416); the 13 methicillin-resistant S. epidermidis (MRSE) belonged to ST2 and harboured both cfr and mutations in genes encoding 23S rRNA and ribosomal proteins. Phylogenetic analysis grouped the MRSE isolates into two clusters, one of which (n = 12 isolates) belonged to the recently reported multidrug-resistant worldwide-disseminated S. epidermidis lineages. Conclusions The results presented herein highlight the persistence and efficient spread of a cfr-carrying plasmid in a hospital related both to the dissemination of a multidrug-resistant S. epidermidis clone and the in vivo interspecies transfer of cfr between S. epidermidis and S. aureus. The emergence of linezolid-resistant strains should be closely monitored, and the mechanisms involved systematically explored in order to limit the spread of plasmid-mediated resistance.

Background It is unclear whether Staphylococcus aureus with heterogeneous intermediate vancomycin resistance (hVISA) can develop vancomycin resistance faster than vancomycin-susceptible S. aureus (VSSA) strains. Methods We compared the kinetics of vancomycin MIC increase for 15 days of sustained in vitro vancomycin exposure for clinical hVISA (n = 12) and VSSA (n = 24) isolates, as well as for reference strains Mu3 (hVISA) and ATCC 29213 (VSSA). Clinical isolates were categorized as hVISA using the population analysis profile method. MICs were monitored for 15 days and the rate of MIC increase under exposure, for each strain, was evaluated in a linear regression model relative to time. Results All isolates acquired vancomycin resistance upon exposure. Vancomycin MICs increased faster for VSSA compared with hVISA isolates (P < 0.01). Conclusions The hVISA phenotype does not correspond to an enhanced adaptation potential to in vitro vancomycin pressure.

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.

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 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.

The heterogeneity of bacterial growth and replicative rates within a population was proposed a century ago notably to explain the presence of bacterial persisters. The term “growth rate” at the single-cell level corresponds to the increase in size or mass of an individual bacterium while the “replicative rate” refers to its division capacity within a defined temporality. After a decades long hiatus, recent technical innovative approaches allow population growth and replicative rates heterogeneity monitoring at the single-cell level resuming in earnest. Among these techniques, the oldest and widely used is time-lapse microscopy, most recently combined with microfluidics. We also discuss recent fluorescence dilution methods informing only on replicative rates and best suited. Some new elegant single cell methods so far only sporadically used such as buoyant mass measurement and stable isotope probing have emerged. Overall, such tools are widely used to investigate and compare the growth and replicative rates of bacteria displaying drug-persistent behaviors to that of bacteria growing in specific ecological niches or collected from patients. In this review, we describe the current methods available, discussing both the type of queries these have been used to answer and the specific strengths and limitations of each method.

ObjectivesIn a new experimental model of carbapenemase-producing Klebsiella pneumoniae osteomyelitis we evaluated the efficacy of colistin alone and in various combinations and examined the emergence of colistin-resistant strains and cross-resistance to host defence peptides (HDPs).MethodsKPC-99YC is a clinical strain with intermediate susceptibility to meropenem (MIC = 4 mg/L) and full susceptibility to gentamicin, colistin and tigecycline (MICs = 1 mg/L) and fosfomycin (MIC = 32 mg/L). Time–kill curves were performed at 4× MIC. Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu. Treatment started 14 days later for 7 days in seven groups: (i) control; (ii) colistin; (iii) colistin + gentamicin; (iv) colistin + tigecycline; (v) colistin + meropenem; (vi) colistin + meropenem + gentamicin; and (vii) colistin + fosfomycin.ResultsIn vitro, colistin was rapidly bactericidal, but regrowth occurred after 9 h. Combinations of colistin with meropenem or fosfomycin were synergistic, whereas combination with tigecycline was antagonistic. In vivo, colistin alone was not effective. Combinations of colistin with meropenem or fosfomycin were bactericidal (P ConclusionsIn this model, combinations of colistin plus meropenem, with or without gentamicin, or colistin plus fosfomycin were the only effective therapies. The combination of colistin and tigecycline should be administered with caution, as it may be antagonistic in vitro and in vivo.

ObjectiveTo identify the role of Staphylococcus aureus (SA) or its intracellular small-colony variant phenotype (SCV) with co-trimoxazole (CTX) or ANCA-associated vasculitis (AAV) activity.MethodsAll consecutive AAV patients with granulomatosis with polyangiitis (GPA), eosinophilic GPA or microscopic polyangiitis, followed at the French National Vasculitis Referral Center (09/2012–05/2013), and hospitalized non-AAV controls, exclusively for SA/SCV carriage comparisons, were enrolled. All had bilateral anterior nasal swab cultures. Nasal SA or SCV carriage was determined and associations with relapse(s), BVAS, ANCA-positivity, anti-staphylococcal and immunosuppressant use, were analysed ⩾4 years post-inclusion.ResultsNasal SA carriage rates did not differ among AAVs (P = 0.53): GPA (24/80; 30%), EGPA (7/28; 25%) and microscopic polyangiitis (3/11; 27.3%); and the rate was less frequent in controls than in GPA patients not taking CTX (P = 0.04). AAV patients taking CTX prophylaxis had less nasal SA carriage (8.7% vs 36.2%; P = 0.02). Nasal SA carriage or CTX use did not modify relapse rates, BVAS or ANCA-positivity at inclusion or during follow-up. Nasal SCV carriage, found in 15/207 (7.2%) patients, was similar for GPA (10/24; 41.7%), microscopic polyangiitis (2/7; 28.6%) and eosinophilic GPA (2/3; 66.7%), but higher (P = 0.02) than controls (1/14; 7.1%). SCV carriage by AAV groups did not modify relapse rates or ANCA positivity at inclusion or during follow-up; a trend towards higher BVAS was observed only for anti-PR3 ANCA patients.ConclusionNasal SA or SCV carriage was comparable among AAVs but more frequent than in controls. Nasal SA or SCV carriage and CTX use did not modify AAV relapse rates.

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.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. Unlike bone marrow-derived stromal cells (also known as bone marrow-derived mesenchymal stromal/stem cells), adipose-derived stromal cells (ASC) have so far failed to form a bone organ by ECO. The goal of the present study was to assess whether priming human ASC to a defined stage of chondrogenesis in vitro allows their autonomous ECO upon ectopic implantation. ASC were cultured either as micromass pellets or into collagen sponges in chondrogenic medium containing transforming growth factor-β3 and bone morphogenetic protein-6 for 4 weeks (early hypertrophic templates) or for two additional weeks in medium supplemented with β-glycerophosphate, l-thyroxin, and interleukin1-β to induce hypertrophic maturation (late hypertrophic templates). Constructs were implanted in vivo and analyzed after 8 weeks. In vitro, ASC deposited cartilaginous matrix positive for glycosaminoglycans, type II collagen, and Indian hedgehog. Hypertrophic maturation induced upregulation of type X collagen, bone sialoprotein, and matrix metalloproteinase13 (MMP13). In vivo, both early and late hypertrophic templates underwent cartilage remodeling, as assessed by MMP13- and tartrate-resistant acid phosphatase-positive staining, and developed bone ossicles, including bone marrow elements, although to variable degrees of efficiency. In situ hybridization for human-specific sequences and staining with a human specific anti-CD146 antibody demonstrated the direct contribution of ASC to bone and stromal tissue formation. In conclusion, despite their debated skeletal progenitor nature, human ASC can generate bone organs through ECO when suitably primed in vitro. Significance Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. This study demonstrated that expanded, human adult adipose-derived stromal cells can generate ectopic bone through ECO, as previously reported for bone marrow stromal cells. This system can be used as a model in a variety of settings for mimicking ECO during development, physiology, or pathology (e.g., to investigate the role of BMPs, their receptors, and signaling pathways). The findings have also translational relevance in the field of bone regeneration, which, despite several advances in the domains of materials and surgical techniques, still faces various limitations before being introduced in the routine clinical practice.

OBJECTIVES Mupirocin is the cornerstone of decolonization regimens, a successful strategy to prevent healthcare-associated staphylococcal infections. Several recent studies have reported alarming results: (i) an extending reservoir of mupA, the ancestral mobile resistance gene, among coagulase-negative staphylococci (CoNS); (ii) the emergence of a new resistance gene (mupB); and (iii) a growing number of mupirocin-resistant methicillin-resistant Staphylococcus aureus (MRSA), including highly pathogenic clones. We performed a nationwide prospective study in France to detect such trends among invasive staphylococci. METHODS Between October 2011 and February 2012, 367 MRSA and 708 CoNS invasive isolates were collected from 37 hospitals and analysed centrally. Mupirocin MICs were determined using the broth microdilution method. mupA/B PCR was performed for resistant isolates (MIC >1 mg/L). Genetic relatedness between mupirocin-resistant MRSA isolates was determined by PFGE analysis and related isolates were tested by microarray. RESULTS Among MRSA isolates 2.2% (n = 8) were classified as mupirocin resistant; 1.4% (n = 5) showing low-level resistance (MIC ≤256 mg/L) and 0.8% (n = 3) high-level resistance (MIC >256 mg/L). Only the latter isolates carried mupA. A clonal relationship was identified between two mupA-negative MRSA from the same hospital and three mupA-positive MRSA from three distant towns; these three isolates belonged to the Lyon clone. Mupirocin resistance was identified in 10.3% of CoNS, mainly highly resistant mupA-positive isolates (5.6%). The mupB gene was not detected in mupirocin-resistant MRSA or CoNS. CONCLUSIONS This first large national study indicates the need for thorough epidemiological monitoring and a stewardship programme to prevent and detect mupirocin resistance in staphylococci.

Background The recent discovery of a mecA homologue (mecA(LGA251)) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecA(LGA251) could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing. Methods In this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecA(LGA251), alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the Panton-Valentine leucocidin (PVL) toxin]. Results and discussion The assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecA(LGA251) from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecA(LGA251), including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecA(LGA251) in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.

Background Optimal surgical therapy could be sometimes non-feasible, especially in the elderly population. Therefore, a medical therapy with oral prolonged suppressive antibiotic therapy (PSAT) seems to be an option to prevent recurrence and prosthesis loosening. Subcutaneous (SC) administration of injectable intravenous antibiotics as PSAT could be a convenient way when oral treatment is not available to facilitate ambulatory care, even if this practice is considered as an “off-label” practice. Methods All patients receiving SC PSAT since 2010 were prospectively enrolled in a cohort study evaluating treatment modalities, efficacy, and safety. Success was defined by the absence of clinical signs of infection at the time of last follow-up. Results We included 10 patients (median age of 79 years): seven had PJI and three chronic osteomyelitis. Six had plurimicrobial infections and four had infections due to multidrug-resistant Gram-negative pathogens. Suboptimal surgery was performed in seven patients, and three received only antibiotics. All patients received an induction-phase therapy with conventional antibiotic treatment before SC PSAT. For nine patients, SC injections were delivered by a 50 mlml 30 minute gravity infusion of the antibiotic, using butterfly disposable needle. One patient received direct flash SC administration. The most frequent drug used was ertapenem (n = 7; 1–2 g/day), followed by ceftriaxone (n = 2; 1 g/day), and ceftazidime (n = 1; 2 g/day). The dose was adjusted depending on the results of trough residual blood concentration. Median duration of treatment was 6 months (from 1 to 58 months), corresponding to a total of about 5,000 SC injections. SC PSAT had to be discontinued for side effects in only two patients, including skin necrosis in the patient receiving direct SC infusion (lost to follow-up after treatment discontinuation) and epilepsy under ertapenem therapy (with relapse of the BJI after the treatment discontinuation). One other patient experienced a relapse despite the SC PSAT. Finally, SC PSAT was still ongoing in seven patients with a favorable outcome at the last follow-up. Conclusion SC PSAT appears to be a safe and effective alternative therapy when optimal surgical strategy is not feasible and when oral treatment is not available. Disclosures T. Ferry, HERAEUS: Consultant, Speaker honorarium. S. Lustig, Heraeus: Consultant, Consulting fee

There is significant diversity in methicillin-resistant Staphylococcus aureus (MRSA) clones arising in the community worldwide, with considerable geographical differences in typical antimicrobial resistance profiles. Many community clones of MRSA have a non-multidrug resistant antimicrobial profile, providing increased options for empirical and directed therapy of infections caused by these strains. However, the recent description of increasing non-β lactam resistance in community clones of MRSA, especially USA300, provides a timely warning for clinicians making decisions about therapy for patients potentially infected with these strains. Continued monitoring of global epidemiology and emerging drug resistance data is critical for the effective management of these infections.

Background The French National Reference Center for Staphylococci used whole genome sequencing (WGS) to investigate outbreaks due to a virulent MRSA clone containing the toxic shock syndrome toxin-1 (TSST-1+, sequence type 5, Geraldine clone) increasingly reported in neonatal intensive care units (ICUs). Methods We analyzed 48 isolates previously characterized by spa typing: 31 isolates from outbreak 1 (infected or colonized patients, healthcare workers carriage and environment), 12 isolates from four distinct outbreaks (2, 3, 4, and 5) that occurred in geographically independent neonatal ICUs, and five sporadic strains. We performed WGS using a de novo assembly approach to perform comparisons between isolates (EpiSeq®, bioMérieux). A phylogenetic analysis was constructed by comparing single nucleotide variations (SNVs) in 2020 core-genes using a cutoff of 40 SNVs for defining isolates belonging to the same transmission cluster. We detected in silico resistance and virulence markers using the same bioinformatic pipeline. Results For outbreak 1, 25/31 isolates with two distinct but related spa types t002 and t111 were highly related (80 SNVs) from the previous cluster of 25 isolates suggesting their origin from separate sources. Interestingly the three isolates of outbreak 2 with a spa t111 differed by less than 22 SNVs from the main cluster of the 25 isolates of outbreak 1. This suggested origin from the same transmission cluster. The other three outbreaks showing respectively a spa t002 for outbreak 3 and outbreak 4 and a spa t045 for outbreak 5 were not affiliated to the main cluster of outbreak 1. The isolates carry numerous virulence factors (including TSST-1) and resistance markers confering a peculiar antibiotic resistance profile to the Geraldine clone. Conclusion WGS provides the resolution power to reveal unsuspected transmission events not indicated by conventional methods (different spa type). Based on its high resolution WGS is an all in one tool for epidemiology, virulence and resistance analysis. It really transforms outbreak management and infection control practice for an early response and should replace conventional methods for detection of MRSA transmission. Disclosures G. Durand, bioMérieux: Employee, Salary. F. Javerliat, bioMérieux: Employee, Salary.

Background Pubic osteomyelitis (PO) is a neglected bone and joint infections (BJI), as its management is still poorly codified. We aim to describe PO epidemiology and to look for factors associated with management failure. Methods We performed a retrospective cohort study in two French reference centers including patients with PO in 2010–2016. Treatment failure was defined by clinical (persistence or recurrence of clinical signs) and/or microbiological failure. Factors associated with treatment failure were determined by univariate Cox analysis (hazard ratio [HR] and 95% confidence interval calculation). Kaplan–Meier curve was compared between groups by log-rank test. Results Twenty-five patients were included over 13 years (median age 67 years; 19 men, median ASA score 3). Six (24 %) had a PO from haematogenous origin. Those were all monomicrobial infection, due to S.aureus, mostly identified in young patients without comorbidities, especially in athletes. No surgery was required if no abcess or bone sequestrum were found. Nineteen patients (76 %) had a post-operative chronic PO (developed from 1 month to 11 years after a pelvic surgery); 15 of them had history of pelvic cancer (60%); 12 received radiotherapy at the site of infection (48 %). Infection was polymicrobial in 68 % of cases, including 32 % of cases with multidrug-resistant pathogens. A clinical success was recorded in only 14 patients (56%). Treatment failure was always noticed in chronic post-operative forms. Potential risk factors associated with failure management were: pelvic cancer history (HR 3.8; P = 0,089); pelvic radiotherapy history (HR 2.9; P = 0.122); clinical sinus tract (HR 5.1; P = 0,011); infection with multidrug-resistant bacteria (HR 2.8; P = 0,116), and polymicrobial infection (HR 70.5; P = 0,090). Conclusion Our study highlights predominant chronic complex post-operative forms of PO. They are mostly plurimicrobial, sometimes associated with multi-drug resistance, occurring in fragile patients with pelvic cancer. It frequently leads to complex antibiotherapy, with important risk of relapse. Aggressive surgical procedure with large bone resection is frequently required in patients who underwent pelvic radiotherapy. Disclosures T. Ferry, HERAEUS: Consultant, Speaker honorarium. E. Senneville, 1959: Board Member and Consultant, Consulting fee and Speaker honorarium

Department of Biology and Infection Control, Centre Hospitalier de Dourdan, 91415 Dourdan, France; Department of Biology, Centre Hospitalier du Bassin de Thau, 34200 Sete, France; Centre National de Reference des Staphylocoques, Faculte Laennec, INSERM 851, 69372 Lyon, France; Department of Bacteriology, Hopital de la Croix Rousse, 69317 Lyon, France; Department of Microbiology and Infection Control, AP-HP, Centre HospitaloUniversitaire Antoine Beclere, 92140 Clamart, France; Department of Microbiology, EA 4065, Faculte des Sciences Pharmaceutiques et Biologiques, Universite Paris Descartes, Paris, France

CHU Limoges, Laboratoire de Bacteriologie-Virologie-Hygiene, 2 Avenue Martin Luther King, Limoges F-87042, France; INSERM, U1092, Limoges F-87000, France; Universite de Limoges, UMRS1092, Faculte de Medecine, 2 rue du Dr Marcland, Limoges F-87025, France; Hospices Civils de Lyon, Centre National de Reference des Staphylocoques, 59 Boulevard Pinel, Bron cedex F-69677, France; Centre International de Recherche en Infectiologie (CIRI)-Inserm U1111, CNRS UMR5308, UCBL, ENSL, Equipe ‘Pathogenese des infections a staphylocoques’, 7 rue Guillaume Paradin, Lyon F-69317, France; CHU Limoges, Service de Reanimation Polyvalente, 2 Avenue Martin Luther King, Limoges F-87042, France

Sir, Denton et al. recently reported the results of the EPISA study, which compared the susceptibility of Staphylococcus aureus causing community-acquired skin and soft tissue infections (SSTIs) in three European countries. They highlighted in particular that the resistance rate to fusidic acid was significantly lower in French (6.3%) than in Irish (31.4%) or British (24.4%) isolates. This difference was attributed by the authors to the likely spread of a fusidic acid-resistant clone, named the epidemic European fusidic acid-resistant impetigo clone (EEFIC) in the latter two countries. This clone, initially described in Scandinavia, had so far been reported sporadically in the UK. It is characterized by: (i) agr allelic group 4, spa type t171 [or single-locus variants (t408, t659, t874, t875)], sequence type (ST) 123, clonal complex 121; (ii) the presence of the eta (exfoliative toxin A) gene with the majority of strains also carrying the etb (exfoliative toxin B) gene; (iii) resistance to fusidic acid (MIC of 2–8 mg/L) related to the expression of the fusB gene; and (iv) strong association with impetigo. In the same issue, Larsen et al. reported the molecular characterization of all these fusidic acid-resistant isolates (n 1⁄4 136) collected during the EPISA study. The results confirmed that the EEFIC represented 63% (34/54) of fusidic acid-resistant strains in the UK and 49% (34/69) in Ireland, but was not detected in France (0%, 0/13). These results seemed to indicate that the EEFIC was not present in France. We were puzzled by this unexpected and surprising French epidemiology and decided to screen a part of the collection of the French national reference centre for staphylococci (Lyon, France) for the presence of the EEFIC. First, between December 2003 and August 2004, corresponding to the appropriate period of the EPISA study, we identified 19 isolates harbouring agr allelic group 4 and the eta gene, characteristics matching the EEFIC. We then performed a fusidic acid susceptibility test using disc diffusion (using the breakpoint from the BSAC) and fusB PCR on these isolates. Four strains (21%, 4/19) appeared to be resistant and PCR-positive, strongly suggesting that they belonged to the EEFIC. Second, we applied the same screening to all S. aureus isolates collected from October 2000 to November 2003 (before the EPISA study) and from September 2004 to December 2007 (after the EPISA study). The agr allelic group 4 and eta positive strains represented a total of 256 isolates. Forty-four of these isolates were randomly selected to undergo the fusidic acid susceptibility test and fusB PCR. Six isolates (14%, 6/44) were detected as resistant to fusidic acid and positive for fusB PCR. Finally, the fusidic acid MICs (determined by Etest) and spa types were determined for the 10 isolates thus screened. All revealed MICs between 4 and 8 mg/L and belonged to spa type t171 or a related spa type (t171, n 1⁄4 8; t408, n 1⁄4 2) corresponding to ST123, which definitively confirmed that they belonged to the EEFIC. The 10 isolates were collected from eight different hospitals and over a long period (2000–07), confirming the geographical and temporal dissemination of the EEFIC in France. As expected, the clinical data for the 10 patients indicated that impetigo or its generalized form were the motives for consultation. Even if we are not presently able to establish the proportion of the EEFIC within fusidic acid-resistant S. aureus in our collection, because of the design of our screening procedure, these data definitively demonstrated that the EEFIC is present in France and likely represents 15% to 20% of all French S. aureus isolates harbouring exfoliative toxin A. In the EPISA study, the low rate of impetigo in the French patients (12.2%) compared with British patients (33.3%) could partly explain the underrepresentation of the EEFIC in the French data. The low number in the series is probably due to the French practice of consulting a specialist (paediatrician or dermatologist) or hospital emergency wards for impetigo, rather than a general practitioner as would tend to be the case in the UK and Ireland. So, the fact that the EPISA inclusion criterion was ‘patient attending a general practice clinic in the community with SSTIs’, probably introduced a recruitment bias in the French isolates implicated in impetigo and more generally in severe SSTIs. Conversely, the French national reference centre for staphylococci collects S. aureus strains of clinical interest with recruitment from hospitals as well as non-hospital community laboratories, thereby avoiding the bias found in the EPISA study and allowing us to detect the presence of the EEFIC in France in the 2000–07 period.