Mariana Neri Lucas Kurihara

Aim: Cutibacterium acnes remains susceptible to a broad range of antibiotics, but resistant strains have emerged. This study evaluated the therapeutic activity of ceftriaxone (CTX), vancomycin (VA), and gentamicin (GEN) on C. acnes using the Galleria mellonella infection model, and the interactions of these compounds with bioactive glass S53P4 (BAG) supernatant on biofilm formation. We also assessed the in vitro elution efficacy of CTX, VA and GEN released from antibioticloaded polymethylmethacrylate (PMMA) and calcium sulfate (CaSO4) on C. acnes strains. Method: Sequenced C. acnes strains 30.3 (phylotype IB, ST42, CC5) and 64.1 (phylotype IB, ST5, CC5) were selected. Antimicrobial interactions were evaluated using a checkerboard assay, and combinations exhibiting synergism or partial synergism were analyzed for their effects on biofilm formation, alone and in combination with BAG supernatant. Biofilm formation was quantified. Therapeutic potential was assessed in the G. mellonella infection model by infecting larvae with C. acnes (OD600 = 3.0) and administering 10 µL of antimicrobials, their combinations, or BAG supernatant under prophylactic and therapeutic regimens. Zone of inhibition (ZOI) testing assessed in vitro elution efficacy. Results: Partial synergism we identified between VA (0.25 µL/mL) and CTX (0.01 µL/mL) for C. acnes ATCC 6919. Strain 30.3 exhibited partial synergism between GEN (0.06 µL/mL) and CTX (1 µL/mL), while strain 64.1 showed synergism with GEN (0.06 µL/mL) and CTX (0.5 µL/mL). Biofilm reduction was observed across all tested strains, with CTX and GEN combinations achieving a threefold reduction. The addition of BAG supernatant reduced the efficacy of certain antimicrobial combinations, though notable biofilm reductions were still observed in specific strain-treatment pairs (e.g., ATCC 6919 + CTX; 30.3 + CTX; 64.1 + CTX; 64.1 + BG supernatant; 30.3 + BG supernatant + CTX + GEN; and ATCC + BG supernatant). The untreated G. mellonella larvae exhibited 100% mortality within 24 hours, while prophylactic and immediate post-infection treatments significantly improved survival, with some larvae surviving the full five-day experimental period. ZOI of VA and VA+CTX persisted for over 40 days, with PMMA exhibiting longer elution. GEN elution was completed within 25 days for both biomaterials. Conclusions: We identified potential antimicrobial combinations, particularly CTX and GEN in reducing C. acnes biofilm formation, enabling the use of lower therapeutic concentrations. While BAG supernatant exhibited limited synergistic effects, the larvae model emphasized the importance of early antimicrobial interventions for improved survival. VA and VA+CTX beads demonstrated prolonged elution in both CaSO4 and PMMA.

Specific virulence factors that likely influence C. acnes invasion into deep tissues remain to be elucidated. Herein, we describe the frequency of C. acnes identification in deep tissue specimens of patients undergoing clean shoulder surgery and assess its phenotypic and genetic traits associated with virulence and antibiotic resistance patterns, compared with isolates from the skin of healthy volunteers. Multiple deep tissue specimens from the bone fragments, tendons, and bursa of 84 otherwise healthy patients undergoing primary clean-open and arthroscopic shoulder surgeries were aseptically collected. The overall yield of tissue sample cultures was 21.5% (55/255), with 11.8% (30/255) identified as C. acnes in 27.3% (23/84) of patients. Antibiotic resistance rates were low, with most strains expressing susceptibility to first-line antibiotics, while a few were resistant to penicillin and rifampicin. Phylotypes IB (73.3%) and II (23.3%) were predominant in deep tissue samples. Genomic analysis demonstrated differences in the pangenome of the isolates from the same clade. Even though strains displayed a range of pathogenic markers, such as biofilm formation, patients did not evolve to infection during the 1-year follow-up. This suggests that the presence of polyclonal C. acnes in multiple deep tissue samples does not necessarily indicate infection.

Resumo: Objetivo Pesquisa e identificação de Cutibacterium acnes (C. acnes) e de outros microrganismos em amostras de tecidos profundos coletados em cirurgias limpas de ombro em pacientes que não foram submetidos a nenhum procedimento invasivo articular prévio e que não possuíam antecedentes clínicos de infecção. Métodos Foram analisados os resultados das culturas de amostras de tecidos profundos intraoperatórias de 84 pacientes submetidos à cirurgia limpa primária do ombro. Foram utilizados tubos contendo meio de cultivo para armazenamento e transporte de agentes anaeróbicos, tempo prolongado de incubação e espectrômetro de massa para diagnósticos de agentes bacterianos. Resultados Foi evidenciado o crescimento de bactérias em 34 pacientes (40,4%) dos 84 incluídos no estudo. Desses, 23 apresentavam crescimento de C. acnes em pelo menos uma amostra de tecido profundo coletada, correspondendo a 27,3% do total de pacientes. O segundo agente mais encontrado foi o Staphylococcus epidermidis, presente em 7,2% do total de indivíduos incluídos. Evidenciamos maior relação da positividade de amostras com o gênero masculino, uma média de idade inferior, a ausência de diabetes mellitus, o escore ASA I e a profilaxia antibiótica na indução anestésica com cefuroxima. Conclusões Verificou-se um elevado percentual de isolados de diferentes bactérias em amostras de tecidos de ombros de pacientes submetidos a cirurgias limpas e primárias e sem histórico de infecção anterior. A identificação de C. acnes foi elevada (27,6%) e o Staphylococcus epidermidis foi o segundo agente mais frequente (7,2%).

Polymethylmethacrylate (PMMA) remains the gold standard antibiotic carrier in the management of osteomyelitis. However, biodegradable ceramic carriers may exhibit more efficient antibiotic elution properties. Through zone of inhibition (ZOI) testing and biofilm killing assays, we assessed the in vitro elution efficacy of vancomycin released from calcium sulfate (PG‐CSH) and PMMA beads as carriers on clinical strains of Staphylococcus aureus and Staphylococcus epidermidis, which were isolated from sonication fluid of orthopedic implant‐associated infections. Overall, vancomycin‐loaded PMMA and PG‐CSH beads showed potency (ZOI above 4 cm²) for up to 14 days against ATCC and clinical strains. Vancomycin‐loaded PG‐CSH beads displayed higher rates, exhibited a more stable antibiotic elution, had greater impacts on bacterial colony‐forming unit counts and produced higher ZOIs; additionally, statistically significant differences (Student's t test) were observed in different time sets during the experiment. In the biofilm killing assay, PG‐CSH loaded with vancomycin resulted in more bacterial deaths. In conclusion, in the present study, both PG‐CSH and PMMA beads acted as good carriers, but greater antimicrobial elution and biofilm bacterial killing were observed with PG‐CSH than PMMA. Future in vitro research should focus on testing other difficult‐to‐treat clinical strains, including multidrug resistant coagulase‐negative staphylococci and Gram‐negative bacilli.

Fosfomycin disodium is a potential therapeutic option to manage difficult-to-treat infections, especially when combined with other antimicrobials. In this study, we evaluated the activity of fosfomycin in combination with meropenem or polymyxin B against contemporaneous KPC-2-producing K. pneumoniae clinical isolates (KPC-KPN). Synergistic activity was assessed by checkerboard (CKA) and time–kill (TKA) assays. TKA was performed using serum peak and trough concentrations. The activity of these combinations was also assessed in the Galleria mellonella model. Biofilm disruption was assessed by the microtiter plate technique. CKA resulted in an 8- to 2048-fold decrease in meropenem MIC, restoring meropenem activity for 82.4% of the isolates when combined with fosfomycin. For the fosfomycin + polymyxin B combination, a 2- to 128-fold reduction in polymyxin B MIC was achieved, restoring polymyxin B activity for 47% of the isolates. TKA resulted in the synergism of fosfomycin + meropenem (3.0–6.7 log₁₀ CFU/mL decrease) and fosfomycin + polymyxin B (6.0–6.2 log₁₀ CFU/mL decrease) at peak concentrations. All larvae treated with fosfomycin + meropenem survived. Larvae survival rate was higher with fosfomycin monotherapy (95%) than that observed for fosfomycin + polymyxin B (75%) (p-value < 0.0001). Finally, a higher biofilm disruption was observed under exposure to fosfomycin + polymyxin B (2.4–3.4-fold reduction). In summary, we observed a synergistic effect of fosfomycin + meropenem and fosfomycin + polymyxin B combinations, in vitro and in vivo, against KPC-KPN, as well as biofilm disruption.

Staphylococcus spp. remain the leading biofilm-forming agents causing orthopedic implant-associated infections (OIAI). This is a descriptive study of phenotypic and genomic features identified in clinical isolates of S. aureus and coagulase-negative Staphylococcus (CoNS) recovered from OIAIs patients that progressed to treatment failure. Ten isolates were identified by matrix-time-of-flight laser-assisted desorption mass spectrometry (MALDI-TOF-MS) and tested for antibiotic susceptibility and biofilm formation. Genotypic characteristics, including, MLST (Multi Locus Sequence Typing), SCCmec typing, virulence and resistance genes were assessed by whole-genome sequencing (WGS). All S. aureus harbored mecA, blaZ, and multiple resistance genes for aminoglycosides and quinolones. All MRSA were strong biofilm producers harboring the complete icaADBC and icaR operon. Seven CoNS isolates comprising five species (S. epidermidis, S. haemolyticus, S. sciuri, S. capitis and S. lugdunensis) were analyzed, with mecA gene detected in five isolates. S. haemolitycus (isolate 95), and S. lugdunensis were unable to form biofilm and did not harbor the complete icaADBCR operon. High variability of adhesion genes was detected, with atl, ebp, icaADBC operon, and IS256 being the most common. In conclusion, MRSA and CoNS isolates carrying genes for biofilm production, and resistance to β-lactam and aminoglycosides are associated with treatment failure in OIAIs.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a growing threat to public health. A 3-year retrospective study was conducted to evaluate the prevalence and lethality of multidrug-resistant (MDR) A. baumannii isolated from Brazilian patients. In this study, 219 Acinetobacter baumannii isolates were identified, of which 70.8% (155/219) were isolated from patients hospitalized in intensive care units. Of these, 57.4% (n = 89/155) were assessed, of which 92.1% (82/89) were carbapenem-resistant, and 49 were classified as infected. The lethality rate was 79.6% (39/49). We highlight the need of an effective epidemiological surveillance measure to contain the dissemination of CRAB in the hospital environment.