The molecular detection of carbapenem markers with a two-levels amplification screening protocol: epidemiological and resistome insights.

Objectives: Carbapenem-resistance is a challenging healthcare concern and require specific stewardship programs. Monitoring workflows include the identification from surveillance samples, such as rectal swabs. Although culture assays represent the gold standard, data report a significant effectiveness in detecting carbapenemases genes directly from rectal swabs. The aim of this study was to evaluate the REALQUALITY Carba-Screen kit (AB ANALITICA, Padova, Italy) in detecting carbapenemases genes directly from rectal swabs, also comparing its effectiveness to culture assays results. A next-generation sequencing (NGS) was performed to investigate the positive samples about resistance markers and sequence type (ST). Methods: A number of 136 rectal swabs were collected from the University Hospital Policlinico of Catania critical wards. The samples simultaneously underwent culture and molecular assays (REALQUALITY Carba-Screen kit). The molecular method included two-steps. The first step (1 h and 6 min) rapidly excluded negative samples, while the second one (1 h and 6 min) included only positive samples for a resistance confirmation. All the positive culture samples underwent NGS analysis. Results: Statistical evaluations demonstrated high sensitivity (100%) and detection rates (92.6%) for the REALQUALITY Carba-Screen kit, which mostly correlated to the standard workflow. All the culture positive results matched the positive molecular results, which were mainly confirmed by the NGS resistome analysis. The identified ST appeared to be diversified and different from the clinically significative strains of the same setting, furnishing interesting epidemiological evidence. Conclusion: The molecular detection allowed a coordinate approach in a high-prevalence multi-drug-resistance area. The rapid identification with a multi-step procedure accelerated the infection control procedures, while the preliminary negative results reduced the overtreatment episodes. The molecular method efficacy was confirmed through the NGS. In conclusion, the molecular screening could initially lead to a more conservative approach, which may be reevaluated after a culture result about the microorganisms' identification and susceptibility profile.

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages.

Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.

Interactions of Gram-Positive Bacterial Membrane Vesicles and Hosts: Updates and Future Directions.

Extracellular vesicles (EVs) are lipid bilayers derived from cell membranes, released by both eukaryotic cells and bacteria into the extracellular environment. During production, EVs carry proteins, nucleic acids, and various compounds, which are then released. While Gram-positive bacteria were traditionally thought incapable of producing EVs due to their thick peptidoglycan cell walls, recent studies on membrane vesicles (MVs) in Gram-positive bacteria have revealed their significant role in bacterial physiology and disease progression. This review explores the current understanding of MVs in Gram-positive bacteria, including the characterization of their content and functions, as well as their interactions with host and bacterial cells. It offers a fresh perspective to enhance our comprehension of Gram-positive bacterial EVs.

Role of transcriptomic and genomic analyses in improving the comprehension of cefiderocol activity in Acinetobacter baumannii.

The mechanisms of action and resistance of cefiderocol (FDC) in Acinetobacter baumannii are still not fully elucidated, but iron transport systems have been evoked in its entry into the cell to reach the penicillin-binding proteins (PBPs). To capture the dynamics of gene expression related to FDC action in various conditions, we report on the genomic and transcriptomic features of seven A. baumannii strains with different FDC susceptibility, focusing on the variants in genes associated with ß-lactam resistance and the expression of the siderophore biosynthesis and transport systems acinetobactin and baumannoferrin. We also investigated the expression of the TonB energy transduction system (ETS) and siderophore receptors piuA and pirA. The four clinical samples belonged to the same clonal complex (CC2), and the two strains with the highest FDC MICs showed peculiar variants in PBP2 and ampC. Similarly, the two clinical strains with the lowest MICs shared variants in an outer membrane protein as well as ampC. Gene expression analyses highlighted the up-regulation of the acinetobactin and baumannoferrin genes in response to iron depletion and a down-regulation in the presence of high iron concentrations. In response to FDC, gene expression seemed strain-dependent, probably due to the different metabolic features of each strain. Overall, FDC activates the ETS, confirming the active import of the drug; baumannoferrin, more than acinetobactin, appeared stimulated by FDC in an iron-depleted medium. In conclusion, iron transport systems play a clear role in the FDC uptake, and their expression likely contributes to MIC variation together with ß-lactam resistance determinants.IMPORTANCEAcinetobacter baumannii poses a threat to healthcare due to its ability to give difficult-to-treat infections as a consequence of our shortage of antibiotic molecules active on this multidrug-resistant bacterium. Cefiderocol (FDC) represents one of the few drugs active on A. baumannii, and to preserve its activity, this study explored the transcriptomic and genomic features of seven strains with varying susceptibility to FDC. Transcriptomic analyses revealed the different effects of FDC on iron transport systems, promoting mainly baumannoferrin expression-thus more likely related to FDC entry-and the energy transduction systems. These findings suggest that not all iron transport systems are equally involved in FDC entry into A. baumannii cells. Finally, mutations in PBPs and ß-lactamases may contribute to the resistance onset. Overall, the study sheds light on the importance of iron availability and metabolic differences in FDC resistance, offering insights into understanding the evolution of resistance in A. baumannii strains.

Evaluation of the Effects of Heteroaryl Ethylene Molecules in Combination with Antibiotics: A Preliminary Study on Control Strains.

The discovery of compounds with antibacterial activity is crucial in the ongoing battle against antibiotic resistance. We developed two QSAR models to design six novel heteroaryl drug candidates and assessed their antibacterial properties against nine ATCC strains, including Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and also Salmonella enterica and Escherichia coli, many of which belong to the ESKAPE group. We combined PB4, a previously tested compound from published studies, with GC-VI-70, a newly discovered compound, with the best cytotoxicity/MIC profile. By testing sub-MIC concentrations of PB4 with five antibiotics (linezolid, gentamycin, ampicillin, erythromycin, rifampin, and imipenem), we evaluated the combination's efficacy against the ATCC strains. To assess the compounds' cytotoxicity, we conducted a 24 h and 48 h 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on colorectal adenocarcinoma (CaCo-2) cells. We tested the antibiotics alone and in combination with PB4. Encouragingly, PB4 reduced the MIC values for GC-VI-70 and for the various clinically used antibiotics. However, it is essential to note that all the compounds studied in this research exhibited cytotoxic activity against cells. These findings highlight the potential of using these compounds in combination with antibiotics to enhance their effectiveness at lower concentrations while minimizing cytotoxic effects.

The relationship between the gut microbiota, benign prostatic hyperplasia, and erectile dysfunction.

Microbiota is defined as the group of commensal microorganisms that inhabit a specific human body site. The composition of each individual's gastrointestinal microbiota is influenced by several factors such as age, diet, lifestyle, and drug intake, but an increasing number of studies have shown that the differences between a healthy microbiota and a dysbiotic one can be related to different diseases such as benign prostatic hyperplasia (BPH) and erectile dysfunction (ED). The aim of this review is to give an overview of the role of the gut microbiota on BPH and ED. Gut microbiota modifications can influence prostate health indirectly by the activation of the immune system and the production of proinflammatory cytokines such as IL-17, IL-23, TNF-alpha, and IFN-gamma, which are able to promote an inflammatory state. Gut dysbiosis may lead to the onset of ED by the alteration of hormone levels and metabolic profiles, the modulation of stress/anxiety-mediated sexual dysfunction, the development of altered metabolic conditions such as obesity and diabetes mellitus, and the development of hypertension. In conclusion, much evidence suggests that the intestinal microbiota has an influence on various pathologies including BPH and ED.

Acinetobacter baumannii and Cefiderocol, between Cidality and Adaptability.

Among the bacterial species included in the ESKAPE group, Acinetobacter baumannii is of great interest due to its intrinsic and acquired resistance to many antibiotics and its ability to infect different body regions. Cefiderocol (FDC) is a novel cephalosporin that is active against Gram-negative bacteria, with promising efficacy for A. baumannii infections, but some studies have reported therapeutic failures even in the presence of susceptible strains. This study aims to investigate the interactions between FDC and 10 A. baumannii strains with different susceptibilities to this drug. We confirmed diverse susceptibility profiles, with resistance values close to the EUCAST-proposed breakpoints. The minimal bactericidal concentration (MBC)/MIC ratios demonstrated bactericidal activity of the drug, with ratio values of ≤4 for all of the strains except ATCC 19606; however, bacterial regrowth was evident after exposure to FDC, as were changes in the shapes of colonies and bacterial cells. A switch to a nonsusceptible phenotype in the presence of high FDC concentrations was found in 1 strain as an adaptation mechanism implemented to overcome the cidal activity of this antibiotic, which was confirmed by the presence of heteroresistant, unstable subpopulations in 8/10 samples. Genomic analyses revealed the presence of mutations in penicillin-binding protein 3 (PBP3) and TonB3 that were shared by all of the strains regardless of their resistance phenotype. Because our isolates harbored ß-lactamase genes, ß-lactamase inhibitors showed the ability to restore the antimicrobial activity of FDC despite the different nonsusceptibility levels of the tested strains. These in vitro results support the concept of using combination therapy to eliminate drug-adapted subpopulations and regain full FDC activity in this difficult-to-treat species. IMPORTANCE This work demonstrates the underrated presence of Acinetobacter baumannii heteroresistant subpopulations after exposure of A. baumannii strains to FDC and its instability. Both A. baumannii and FDC are of great interest for the scientific community, as well as for clinicians; the former represents a major threat to public health due to its resistance to antibiotics, with related costs of prolonged hospitalization, and the latter is a novel, promising cephalosporin currently under the magnifying glass.

Heteroaryl-Ethylenes as New Effective Agents for High Priority Gram-Positive and Gram-Negative Bacterial Clinical Isolates.

The World Health Organization has identified antimicrobial resistance as a public health emergency and developed a global priority pathogens list of antibiotic-resistant bacteria that can be summarized in the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales species), reminding us of their ability to escape the effect of antibacterial drugs. We previously tested new heteroaryl-ethylene compounds in order to define their spectrum of activity and antibacterial capability. Now, we focus our attention on PB4, a compound with promising MIC and MBC values in all conditions tested. In the present study, we evaluate the activity of PB4 on selected samples of ESKAPE isolates from nosocomial infections: 14 S. aureus, 6 E. faecalis, 7 E. faecium, 12 E. coli and 14 A. baumannii. Furthermore, an ATCC control strain was selected for all species tested. The MIC tests were performed according to the standard method. The PB4 MIC values were within very low ranges regardless of bacterial species and resistance profiles: from 0.12 to 2 mg/L for S. aureus, E. faecalis, E. faecium and A. baumannii. For E. coli, the MIC values obtained were slightly higher (4-64 mg/L) but still promising. The PB4 heteroaryl-ethylenic compound was able to counteract the bacterial growth of both high-priority Gram-positive and Gram-negative clinical strains. Our study contributes to the search for new molecules that can fight bacterial infections, in particular those caused by MDR bacteria in hospitals. In the future, it would be interesting to evaluate the activity of PB4 in animal models to test for its toxicity.

Elexacaftor-Tezacaftor-Ivacaftor as a Final Frontier in the Treatment of Cystic Fibrosis: Definition of the Clinical and Microbiological Implications in a Case-Control Study.

The use of modulator drugs that target the Cystic Fibrosis transmembrane conductance regulator (CFTR) is the final frontier in the treatment of Cystic Fibrosis (CF), a genetic multiorgan disease. F508del is the most common mutation causing defective formation and function of CFTR. Elexacaftor-tezacaftor-ivacaftor is the first triple combination of CFTR modulators. Herein, we report on a one-year case-control study that involved 26 patients with at least one F508del mutation. Patients were assigned to two similar groups, and patients with the worse clinical condition received treatment with the triple combination therapy. The study aimed to define the clinical and especially microbiological implications of treatment administration. The treatment provided significant clinical benefits in terms of respiratory, pancreatic, and sweat function. After one year of therapy, airway infection rates decreased and pulmonary exacerbations were dramatically reduced. Finally, treated patients reported a surprising improvement in their quality of life. The use of triple combination therapy has become essential in most CF people carrying the F508del mutation. Although the clinical and instrumental benefits of treatment are thoroughly known, further investigations are needed to properly define its microbiological respiratory implications and establish the real advantage of life-long treatment with elexacaftor-tezacaftor-ivacaftor.

SARS-CoV-2's high rate of genetic mutation under immune selective pressure: from oropharyngeal B.1.1.7 to intrapulmonary B.1.533 in a vaccinated patient.

This is the case report of an 84-year-old man affected by COVID-19 between the 2 doses of vaccination, with negative exitus. We analyzed nasopharyngeal samples of viral RNA collected during the disease and nasopharyngeal and lung samples collected postmortem by reverse transcription LAMP (RT-LAMP) PCR and Next Generation Sequencing (NGS). NGS results were analyzed with different bioinformatic tools to define virus lineages and the related single-nucleotide polymorphisms (SNPs). Both lung and nasopharyngeal samples tested positive for SARS-CoV-2 on RT-LAMP. Through bioinformatic analysis, 2 viral RNAs from the nasal swabs, which belonged to the B.1.1.7 lineage, and 1 viral RNA from the lung sample, which belonged to the B.1.533 lineage, were identified. This genetic observation suggested that SARS-CoV-2 tends to change under selective pressure. The high mutation rate of ORFa1b, containing a replicase gene, was a biological image of a complex viral survival system.

Discriminatory Weight of SNPs in Spike SARS-CoV-2 Variants: A Technically Rapid, Unambiguous, and Bioinformatically Validated Laboratory Approach.

BACKGROUND: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. METHODS: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). RESULTS: A "weight" could be assigned to each mutation that may be present in the selected portion of the S gene. The method's robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. CONCLUSIONS: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

Omic insights into various ceftazidime-avibactam-resistant Klebsiella pneumoniae isolates from two southern Italian regions.

Ceftazidime-avibactam (CZA) is one of the best therapeutic options available for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. However, sporadic reports of CZA-resistant strains have been rapidly increasing in patients. Herein, we provide detailed case reports of the emergence of ceftazidime-avibactam resistance to identify their resistome and virulome using genomic molecular approaches. Sixteen isolates were collected from 13 patients at three hospitals in Catania and Catanzaro (Italy) between 2020-2021. Antimicrobial susceptibility was determined by broth microdiluition. The samples included in study were analyzed for resistome, virulome and Sequence Type (ST) using Whole Genome Sequencing (WGS). All strains were resistant to ceftazidime/avibactam, ciprofloxacin, extended-spectrum cephalosporins and aztreonam, 13/16 to meropenem, 8/16 to colistin and 7/16 to fosfomycin; 15/16 were susceptible to meropenem/vaborbactam; all strains were susceptible to cefiderocol. Molecular analysis showed circulation of three major clones: ST101, ST307 and ST512. In 10/16 strains, we found a bla KPC-3 gene; in 6/16 strains, four different bla KPC variants (bla KPC28-31-34-50) were detected. A plethora of other beta-lactam genes (bla SHV28-45-55-100-106-187-205-212, bla OXA1-9-48, bla TEM-181 and bla CTX-M-15) was observed; bla OXA-9 was found in ST307 and ST512, instead bla OXA48 in one out four ST101 strains. With regard to membrane permeability, ompK35 and ompK36 harbored frameshift mutations in 15/16 strains; analysis of ompK37 gene revealed that all strains harbored a non-functional protein and carry wild-type PBP3. There is an urgent need to characterize the mechanisms underlying carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance. Furthermore, it is becoming increasingly important to explore feasible methods for accurate detection of different KPC enzymes.

Low Represented Mutation Clustering in SARS-CoV-2 B.1.1.7 Sublineage Group with Synonymous Mutations in the E Gene.

Starting in 2019, the COVID-19 pandemic is a global threat that is difficult to monitor. SARS-CoV-2 is known to undergo frequent mutations, including SNPs and deletions, which seem to be transmitted together, forming clusters that define specific lineages. Reverse-Transcription quantitative PCR (RT-qPCR) has been used for SARS-CoV-2 diagnosis and is still considered the gold standard method. Our Eukaryotic Host Pathogens Interaction (EHPI) laboratory received six SARS-CoV-2-positive samples from a Sicilian private analysis laboratory, four of which showed a dropout of the E gene. Our sequencing data revealed the presence of a synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene of all four samples showing the dropout in RT-qPCR. Interestingly, these samples also harbored three other mutations (S137L-Orf1ab; N439K-S gene; A156S-N gene), which had a very low diffusion rate worldwide. This combination suggested that these mutations may be linked to each other and more common in a specific area than in the rest of the world. Thus, we decided to analyze the 103 sequences in our internal database in order to confirm or disprove our "mutation cluster hypothesis". Within our database, one sample showed the synonymous mutation (c.26415 C > T, TAC > TAT) in the E gene. This work underlines the importance of territorial epidemiological surveillance by means of NGS and the sequencing of samples with clinical and or technical particularities, e.g., post-vaccine infections or RT-qPCR amplification failures, to allow for the early identification of these SNPs. This approach may be an effective method to detect new mutational clusters and thus to predict new emerging SARS-CoV-2 lineages before they spread globally.

Heteroaryl-Ethylenes as New Lead Compounds in the Fight against High Priority Bacterial Strains.

The widespread use of antibiotics has led to a gradual increase in drug-resistant bacterial infections, which severely weakens the clinical efficacy of antibacterial therapies. In recent decades, stilbenes aroused great interest because of their high bioavailability, as well as their manifold biological activity. Our research efforts are focused on synthetic heteroaromatic stilbene derivatives as they represent a potentially new type of antibiotic with a wide antibacterial spectrum. Herein, a preliminary molecular modeling study and a versatile synthetic scheme allowed us to define eight heteroaromatic stilbene derivatives with potential antimicrobial activity. In order to evaluate our compound's activity spectrum and antibacterial ability, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests have been performed on Gram-positive and Gram-negative ATCC strains. Compounds PB4, PB5, PB7, and PB8 showed the best values in terms of MIC and were also evaluated for MBC, which was found to be greater than MIC, confirming a bacteriostatic activity. For all compounds, we evaluated toxicity on colon-rectal adenocarcinoma cells tumor cells (CaCo2), once it was established that the whole selected set was more active than 5-Fluorouracil in reducing CaCo-2 cells viability. To the best of our knowledge, the biological assays have shown for these derivatives an excellent bacteriostatic activity, compared to similar molecular structures previously reported, thus paving the way for a new class of antibiotic compounds.

Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples.

The Coronavirus Disease 19 (COVID-19) pandemic has caused an unexpected death toll worldwide. Even though several guidelines for the management of infectious corpses have been proposed, the limited number of post-mortem analyses during the pandemic has led to inaccuracies in the counting of COVID-19 deaths and contributed to a lack of important information about the pathophysiology of the SARS-CoV-2 infection. Due to the impossibility of carrying out autopsies on all corpses, the scientific community has raised the question of whether confirmatory analyses could be performed on exhumed bodies after a long period of burial to assess the presence of SARS-CoV-2 RNA. Post-mortem lung samples were collected from 16 patients who died from COVID-19 infection and were buried for a long period of time. A custom RNA extraction protocol was developed to enhance extraction of viral RNA from degraded samples and highly sensitive molecular methods, including RT-qPCR and droplet digital PCR (ddPCR), were used to detect the presence of SARS-CoV-2 RNA. The custom extraction protocol developed allowed us to extract total RNA effectively from all lung samples collected. SARS-CoV-2 viral RNA was effectively detected in all samples by both RT-qPCR and ddPCR, regardless of the length of burial. ddPCR results confirmed the persistence of the virus in this anatomical niche and revealed high viral loads in some lung samples, suggesting active infection at the time of death. To the best of our knowledge, this is the first study to demonstrate the persistence of SARS-CoV-2 viral RNA in the lung even after a long post-mortem interval (up to 78 days). The extraction protocol herein described, and the highly sensitive molecular analyses performed, could represent the standard procedures for SARS-CoV-2 detection in degraded lung specimens. Finally, the innovative results obtained encourage post-mortem confirmatory analyses even after a long post-mortem interval.

Resistance to Echinocandins Complicates a Case of Candida albicans Bloodstream Infection: A Case Report.

Invasive candidiasis is known to be one of the most common healthcare-associated complications and is caused by several Candida species. First-line drugs, particularly echinocandins, are effective, but there are increasing reports of resistance to these molecules, though rarely related to C. albicans. Even though the rate of echinocandins resistance remains low (<3%), sporadic cases are emerging. Here, we present a case of bloodstream infection by a pan-echinocandin-resistant Candida albicans affecting a critically ill patient, who died in an intensive care unit following therapeutic failure and multiple organ dysfunction syndrome. This case highlights the need to suspect pan-echinocandin resistance in patients with prolonged echinocandin exposure, particularly in the presence of urinary tract colonization. Our study shows the importance of sequencing to predict therapeutic failure in patients treated with echinocandins and persistent candidemia.

Combination of aztreonam, ceftazidime-avibactam and amikacin in the treatment of VIM-1 Pseudomonas aeruginosa ST235 osteomyelitis.

We describe a challenging case of patient with metallo-beta-lactamase-producing Pseudomonas aeruginosa sternal osteomyelitis following aortic valve replacement with biological prosthesis. The strain exhibited a multidrug-resistance phenotype carrying the blaVIM-1 gene and belonged to the high-risk clone sequence type ST235. The patient was successfully treated with surgical debridement plus antibiotic therapy with ceftazidime/avibactam, aztreonam, and amikacin. Time-kill curves showed that this triple antibiotic combination at 1 × MIC was strongly synergic after 8 h, achieving 99.9% killing and maintaining this until 48 h.

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.