RESUMEN
Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony forming unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.
Asunto(s)
Osteomielitis , Osteomielitis/microbiología , Humanos , Carga Bacteriana , Técnicas de Cocultivo , Recuento de Colonia Microbiana , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/clasificaciónRESUMEN
Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.
Asunto(s)
Biopelículas , ConsensoRESUMEN
The intracellular infection of osteocytes represents a clinically important aspect of osteomyelitis. However, few human osteocyte in vitro models exist and the differentiation of immature osteoblasts to an osteocyte stage typically takes at least 4-weeks of culture, making the study of this process challenging and time consuming. The osteosarcoma cell line Saos-2 has proved to be a useful model of human osteoblast to mature osteocyte differentiation. Culture under osteogenic conditions in a standard normoxic (21% O2 ) atmosphere results in reproducible mineralization and acquisition of mature osteocyte markers over the expected 28-35 day culture period. In order to expedite experimental assays, we tested whether reducing available oxygen to mimic concentrations experienced by osteocytes in vivo would increase the rate of differentiation. Cells cultured under 1% O2 exhibited maximal mineral deposition by 14 days. Early (COLA1, MEPE) and mature (PHEX, DMP1, GJA1, SOST) osteocyte markers were upregulated earlier under hypoxia compared to normoxia. Cells differentiated under 1% O2 for 14 days displayed a similar ability to internalize Staphylococcus aureus as day 28 cells grown under normoxic conditions. Thus, low oxygen accelerates Saos-2 osteocyte differentiation, resulting in a useful human osteocyte-like cell model within 14 days.
Asunto(s)
Osteocitos , Staphylococcus aureus , Humanos , Osteocitos/metabolismo , Regulación de la Expresión Génica , Osteoblastos/metabolismo , Diferenciación Celular , Hipoxia/metabolismo , Oxígeno/metabolismo , Células CultivadasRESUMEN
Approximately 40% of treatments of chronic and recurrent osteomyelitis fail in part due to bacterial persistence. Staphylococcus aureus, the predominant pathogen in human osteomyelitis, is known to persist by phenotypic adaptation as small-colony variants (SCVs) and by formation of intracellular reservoirs, including those in major bone cell types, reducing susceptibility to antibiotics. Intracellular infections with S. aureus are difficult to treat; however, there are no evidence-based clinical guidelines addressing these infections in osteomyelitis. We conducted a systematic review of the literature to determine the demonstrated efficacy of all antibiotics against intracellular S. aureus relevant to osteomyelitis, including protein biosynthesis inhibitors (lincosamides, streptogramins, macrolides, oxazolidines, tetracyclines, fusidic acid, and aminoglycosides), enzyme inhibitors (fluoroquinolones and ansamycines), and cell wall inhibitors (beta-lactam inhibitors, glycopeptides, fosfomycin, and lipopeptides). The PubMed and Embase databases were screened for articles related to intracellular S. aureus infections that compared the effectiveness of multiple antibiotics or a single antibiotic together with another treatment, which resulted in 34 full-text articles fitting the inclusion criteria. The combined findings of these studies were largely inconclusive, most likely due to the plethora of methodologies utilized. Therefore, the reported findings in the context of the models employed and possible solutions for improved understanding are explored here. While rifampicin, oritavancin, linezolid, moxifloxacin and oxacillin were identified as the most effective potential intracellular treatments, the scientific evidence for these is still relatively weak. We advocate for more standardized research on determining the intracellular effectiveness of antibiotics in S. aureus osteomyelitis to improve treatments and patient outcomes.
RESUMEN
Infectious osteomyelitis associated with periprosthetic joint infections is often recalcitrant to treatment and has a high rate of recurrence. In the case of Staphylococcus aureus, the most common pathogen in all forms of osteomyelitis, this may be attributed in part to residual intracellular infection of host cells, yet this is not generally considered in the treatment strategy. Osteocytes represent a unique cell type in this context due to their abundance, their formation of a syncytium throughout the bone that could facilitate bacterial spread and their relative inaccessibility to professional immune cells. As such, there is potential value in studying the host-pathogen interactions in the context of this cell type in a replicable and scalable in vitro model. Here, we examined the utility of the human osteosarcoma cell line SaOS2 differentiated to an osteocyte-like stage (SaOS2-OY) as an intracellular infection model for S. aureus. We demonstrate that S. aureus is capable of generating stable intracellular infections in SaOS2-OY cells but not in undifferentiated, osteoblast-like SaOS2 cells (SaOS2-OB). In SaOS2-OY cells, S. aureus transitioned towards a quasi-dormant small colony variant (SCV) growth phenotype over a 15-day post-infection period. The infected cells exhibited changes in the expression of key immunomodulatory mediators that are consistent with the infection response of primary osteocytes. Thus, SaOS2-OY is an appropriate cell line model that may be predictive of the interactions between S. aureus and human osteocytes, and this will be useful for studying mechanisms of persistence and for testing the efficacy of potential antimicrobial strategies.