Unveiling the Relationship between Ceftobiprole and High-Molecular-Mass (HMM) Penicillin-Binding Proteins (PBPs) in Enterococcus faecalis.

Low-affinity PBP4, historically linked to penicillin resistance in Enterococcus faecalis, may still have affinity for novel cephalosporins. Ceftobiprole (BPR) is a common therapeutic choice, even with PBP4-related overexpression and amino acid substitution due to mutations. Our study aims to explore the interaction between BPR and High-Molecular-Mass (HMM) low-reactive PBPs in Penicillin-Resistant-Ampicillin-Susceptible/Ceftobiprole Non-Susceptible (PRAS/BPR-NS) E. faecalis clinical isolates. We conducted competition assays examining class A and B HMM PBPs from four PRAS/BPR-NS E. faecalis strains using purified membrane proteins and fluorescent penicillin (Bocillin FL), in treated and untreated conditions. Interaction strength was assessed calculating the 50% inhibitory concentration (IC50) values for ceftobiprole, by analyzing fluorescence intensity trends. Due to its low affinity, PBP4 did not display significant acylation among all strains. Moreover, both PBP1a and PBP1b showed a similar insensitivity trend. Conversely, other PBPs showed IC50 values ranging from 1/2-fold to 4-fold MICs. Upon higher BPR concentrations, increased percentages of PBP4 inhibition were observed in all strains. Our results support the hypothesis that PBP4 is necessary but not sufficient for BPR resistance, changing the paradigm for enterococcal cephalosporin resistance. We hypothesize that cooperation between class B PBP4 and at least one bifunctional class A PBP could be required to synthesize peptidoglycan and promote growth.

Staphylococcus aureus ST228 and ST239 as models for expression studies of diverse markers during osteoblast infection and persistence.

The ability of S. aureus to infect bone and osteoblasts is correlated with its incredible virulence armamentarium that can mediate the invasion/internalization process, cytotoxicity, membrane damage, and intracellular persistence. We comparatively analyzed the interaction, persistence, and modulation of expression of selected genes and cell viability in an ex vivo model using human MG-63 osteoblasts of two previously studied and well-characterized S. aureus clinical strains belonging to the ST239-SCCmecIII-t037 and ST228-SCCmecI-t041 clones at 3 h and 24 h post-infection (p.i). S. aureus ATCC12598 ST30-t076 was used as a control strain. Using imaging flow cytometry (IFC), we found that these strains invaded and persisted in MG-63 osteoblasts to different extents. The invasion was evaluated at 3 h p.i and persistence at 24 h p.i., in particular: ATCC12598 internalized in 70% and persisted in 50% of MG-63 cells; ST239-SCCmecIII internalized in 50% and persisted in 45% of MG-63 cells; and ST228-SCCmecI internalized in 30% and persisted in 20% of MG-63 cells. During the infection period, ST239-III exerted significant cytotoxic activity resulting from overexpression of hla and psmA and increased expression of the genes involved in adhesion, probably due to the release and re-entry of bacteria inside MG-63 cells at 24 h p.i. The lower invasiveness of ST228-I was also associated with non-cytotoxic activity inside osteoblasts. This clone was unable to activate sufficient cellular reaction and succumbed inside MG-63 cells. Our findings support the idea of considering new strategies, based on a translational approach-eukaryotic host-pathogen interaction (EHPI)-and to be applied on a large scale, to predict S. aureus /osteoblast interaction and treat bone infections. Such strategies rely on the study of the genetic and biochemical basis of both pathogen and host.

Staphylococcus aureus Internalization in Osteoblast Cells: Mechanisms, Interactions and Biochemical Processes. What Did We Learn from Experimental Models?

Bacterial internalization is a strategy that non-intracellular microorganisms use to escape the host immune system and survive inside the human body. Among bacterial species, Staphylococcus aureus showed the ability to interact with and infect osteoblasts, causing osteomyelitis as well as bone and joint infection, while also becoming increasingly resistant to antibiotic therapy and a reservoir of bacteria that can make the infection difficult to cure. Despite being a serious issue in orthopedic surgery, little is known about the mechanisms that allow bacteria to enter and survive inside the osteoblasts, due to the lack of consistent experimental models. In this review, we describe the current knowledge about S. aureus internalization mechanisms and various aspects of the interaction between bacteria and osteoblasts (e.g., best experimental conditions, bacteria-induced damages and immune system response), focusing on studies performed using the MG-63 osteoblastic cell line, the best traditional (2D) model for the study of this phenomenon to date. At the same time, as it has been widely demonstrated that 2D culture systems are not completely indicative of the dynamic environment in vivo, and more recent 3D models-representative of bone infection-have also been investigated.

In Vitro Activity of Dalbavancin against Refractory Multidrug-Resistant (MDR) Staphylococcus aureus Isolates.

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections, always treated with vancomycin and daptomycin, has led to the emergence of vancomycin-intermediate (VISA), heteroresistant vancomycin-intermediate (hVISA) and daptomycin non-susceptible (DNS) S. aureus. Even if glycopeptides and daptomycin remain the keystone for treatment of resistant S. aureus, the need for alternative therapies that target MRSA has now become imperative. The in vitro antibacterial and bactericidal activity of dalbavancin was evaluated against clinically relevant S. aureus showing raised antibiotic resistance levels, from methicillin-susceptible to Multidrug-Resistant (MDR) MRSA, including hVISA, DNS and rifampicin-resistant (RIF-R) strains. A total of 124 S. aureus strains were tested for dalbavancin susceptibility, by the broth microdilution method. Two VISA and 2 hVISA reference strains, as well as a vancomycin-resistant (VRSA) reference strain and a methicillin-susceptible Staphylococcus aureus (MSSA) reference strain, were included as controls. Time-kill curves were assayed to assess bactericidal activity. Dalbavancin demonstrated excellent in vitro antibacterial and bactericidal activity against all S. aureus resistance classes, including hVISA and DNS isolates. The RIF-R strains showed the highest percentage of isolates with non-susceptibility, reflecting the correlation between rpoB mutations and VISA/hVISA emergence. Our observations suggest that dalbavancin can be considered as an effective alternative for the management of severe MRSA infections also sustained by refractory phenotypes.

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

Methicillin-resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well-characterized Staphylococcus aureus invasive strains belonging to the main ST-MRSA-SCCmec clones. To overcome the limitations of the cell culture method, we comparatively analyzed the ability of internalization within human MG-63 osteoblasts with imaging flow cytometry (IFC). After evaluation by cell culture assay, the MRSA clones in the study were all able to readily internalize at 3h postinfection, the persistence of intracellular bacteria was evaluated at 24h both by routine cell culture and IFC assay, after vancomycin-BODIPY staining. A statistical difference of persistence was found in ST5-SCCmecII (26.59%), ST228-SCCmecI (20.25%), ST8-SCCmecIV (19.52%), ST239-SCCmecIII (47.82%), and ST22-SCCmecIVh (50.55%) showing the same ability to internalize as ATCC12598 (51%), the invasive isolate used as control strain for invasion and persistence assays. We demonstrated that the intracellular persistence process depends on the total number of infected cells. Comparing our data obtained by IFC with those of the cell culture assay, we obtained greater reproducibility rates and a number of intracellular bacteria, with the advantage of analyzing live host cells. Moreover, with some limitations related to the lack of whole-genome sequencing analysis, we validated the different proclivities to persist in the main Italian HA-MRSA invasive isolates and our results highlighted the heterogeneity of the different clones to persist during cell infection.