Surgical infections are one of the most common types of infections encountered in a hospital. Staphylococcus aureus is the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate, as the bacteria form biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria that are planktonic, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate that mazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allows S. aureus to transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host.

Staphylococcus aureus biofilms have a high tolerance to antibiotics, making the treatment of periprosthetic joint infection (PJI) challenging. From a clinical perspective, bacteria from surgical specimens are cultured in a planktonic state to determine antibiotic sensitivity. However, S. aureus exists primarily as established biofilms in PJI. To address this dichotomy, we developed a prospective registry of total knee and hip arthroplasty PJI S. aureus isolates to quantify the activity of clinically important antibiotics against isolates grown as biofilms. S. aureus planktonic minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed using clinical laboratory standard index assays for 10 antibiotics (cefazolin, clindamycin, vancomycin, rifampin, linezolid, nafcillin, gentamicin, trimethoprim/sulfamethoxazole, doxycycline, and daptomycin). Mature biofilms of each strain were grown in vitro, after which biofilm MIC (MBIC) and biofilm MBC (MBBC) were determined. Overall, isolates grown as biofilms displayed larger variations in antibiotic MICs as compared to planktonic MIC values. Only rifampin, doxycycline, and daptomycin had measurable biofilm MIC values across all S. aureus isolates tested. Biofilm MBC observations complemented biofilm MIC observations; rifampin, doxycycline, and daptomycin were the only antibiotics with measurable biofilm MBC values. 90% of S. aureus biofilms could be killed by rifampin, 50% by doxycycline, and only 15% by daptomycin. Biofilm formation increased bacterial antibiotic tolerance nonspecifically across all antibiotics, in both MSSA and MRSA samples. Rifampin and doxycycline were the most effective antibiotics at killing established S. aureus biofilms. ß

Antibiotics are unable to remove biofilms from surgical implants. This high antibiotic tolerance is related to bacterial persisters, a sub-population of bacteria phenotypically tolerant to antibiotics secondary to a reduced metabolic state. WLBU2 is an engineered cationic amphipathic peptide designed to maximize antimicrobial activity with minimal mammalian cell toxicity. The objective of this study was to test the ability of WLBU2 to remove Staphylococcus aureus surgical implant biofilms. WLBU2 effectively treated S. aureus biofilms formed by a variety of clinical MSSA and MRSA strains and created culture-negative implants in the in vitro biofilm model. Blocking bacterial metabolism by inhibiting oxidative phosphorylation did not affect WLBU2 killing compared to decreased killing by cefazolin. In the surgical implant infection animal model, WLBU2 decreased biofilm mass as compared to control, untreated samples. WLBU2 could rapidly eliminate implants in vitro and had sufficient efficacy in vivo with minimal systemic toxicity.

BackgroundOsteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined.MethodsThree human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment.ResultsLung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining.ConclusionsLung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.

Both Staphylococcus aureus and Staphylococcus epidermidis are commonly associated with periprosthetic joint infections (PJIs). The treatment of PJI can be challenging because biofilms are assumed to have an increased intolerance to antibiotics. This makes the treatment of PJI challenging from a clinical perspective. Although S. aureus has been previously demonstrated to have increased biofilm antibiotic tolerance, this has not been well established with Staphylococcus epidermidis. A prospective registry of PJI S. epidermidis isolates was developed. The efficacy of clinically relevant antibiotics was quantified against these isolates. S. epidermidis planktonic minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were collected using clinical laboratory standard index (CLSI) assays for eight antibiotics (doxycycline, vancomycin, daptomycin, clindamycin, rifampin, nafcillin, and trimethoprim/sulfamethoxazole). Mature biofilms were grown in vitro, after which minimum biofilm inhibitory concentration (MBIC) and minimum biofilm bactericidal concentration (MBBC) were quantified. Only rifampin and doxycycline had a measurable MBIC across all tested isolates. Based on MBBC, 64% of S. epidermidis biofilms could be eliminated by rifampin, whereas only 18% by doxycycline. S. epidermidis biofilm was observed to have a high tolerance to antibiotics as compared to planktonic culture. Isolate biofilm antibiotic tolerance varied to a larger degree than was seen in planktonic cultures.