F53D23000800006

Staphylococcus aureus responsible for hospital acquired bloodstream infections: toxin production quantification and its relationship with clinical outcomes (STABITOX)

Descrizione

Staphylococcus aureus is one the most important bacteria responsible for bloodstream infections (BSI) attributable mortality. The population incidence of S. aureus bacteremia ranges from 10 to 30 per 100,000 person-year.. In the preantibiotic era, the case fatality rate for S. aureus bacteremia was 80%. The high mortality observed is due to several factors: antibiotic resistance properties, invasivity of S. aureus, strong cytokine release and bacterial pathogenicity in terms of toxins’ production, both in a qualitative (which toxins) and quantitative (amount) manner.

1. aureus carries more than 30 toxin genes. The different toxins have different potentials in determining tissue and systemic damage. Their pathogenic activity involve antiphagocytosis, hemolysis, coagulation system alterations, neurotoxic effects, immunomodulation. Actually, S. aureus bacteremia is a stable problem both in community and hospital settings, and the 30-day all-cause mortality of S. aureus bacteremia is 20% and has not changed since the 1990s. The standard of care toward S. aureus bloodstream infection is still antibiotic therapy alone (± source control). However, similarly to what we have witnessed with bezlotoxumab for Clostridioides difficile, the field of bacterial infections is preparing to be investigated for a targeted therapy with monoclonal antibodies, usually to be administered as adjuvants to antibiotics. In support of this, Liu et al. recently (2021) identified a human monoclonal antibody against S. aureus alpha-toxin. From this perspective, in the near future, it will become significantly important to know “which toxins” are produced and “how much” they are produced. This would help to select who will benefit from adjuvant anti-toxin therapies. Existing studies on the role of S. aureus toxins and mortality focused on the presence or absence of toxins and the outcome (survivor/non-survivor). These studies analyzed infections caused by strains with disrupted toxin genes. It is likely that the quantitative assessment of toxins (detection sensitivity improvement) will overcome the “qualitative” studies to identify those at higher risk of fatal infections.

Recently, Prof. di Masi (Roma Tre University) studied the relationship between toxemia levels and clinical outcome in C. difficile infection (CDI), demonstrating that high serum levels of toxin A correlate with disease severity in patients with CDI. Remarkably, a similar investigation for S. aureus in humans is lacking.

 

Finalità

The aims of the proposal are:

1. To investigate the relationship between the amount of toxins and the clinical outcomes of patients with S. aureus BSI.
2. To assess if human serum albumin (HSA) could prevent S.aureus toxin-associated damage in human cells. This aim relies on previous experiments from our group demonstrating that HSA can bind toxins produced by Gram positive bacteria, representing a component of the human innate immunity.

 

Risultati attesi

• We expect to find a correlation between the type and the amount of toxins produced by S. aureus strains and the clinical outcome of patients with S. aureus BSI.
• We expect to demonstrate in vitro a neutralizing effect of human serum albumin towards cellular intoxication with S. aureus toxins.

We are heading toward the so-called “targeted medicine”. This means that a tailored therapy will be administered according to analytic dosages of substances that correlates with the clinical course/severity of the diseases.

In this context, monoclonal antibodies are already being used for bacterial infections (e.g. bezlotoxumab for C. difficile infection) and they will be even more used as adjuvants to antibiotics. Monoclonal antibodies against S. aureus toxins (alpha-toxin), namely ASN100, suvratoxumab and tosatoxumab are currently in Phase II/III trials. It is likely that identifying patients who could benefit from these drugs will be important. In this regard the quantification of S. aureus produced toxins could provide a threshold/cutoff to determine who will benefit from monoclonal antibodies.

So our results could have at least 4 potential benefits in terms of advancement of knowledge:

1. to demonstrate a relationship between the type and the amount of toxins and the clinical outcomes in S. aureus BSI in humans.
2. to provide a threshold/cutoff to determine who will benefit from anti-toxin adjuvants drugs (e.g. monoclonal antibodies).
3. to provide insights into the potential of HSA against S. aureus toxins.

All the aforementioned potential benefits would have significant translational implications.

Stato dell’arte

Staphylococcus aureus is one of the major “bacterial killer” in hospitalized patients with systemic infections. This microorganism isable to cause different clinical conditions, but one of the most impactful in terms of prognosis is bacteremia (bloodstream infection – BSI). In the industrialized world, the population incidence of S. aureus bacteremia ranges from 10 to 30 per 100,000 person-years. Interestingly, S. aureus bacteremia is a stable problem with a mortality of 20% that has not changed since the 1990s, despite the progress achieved in terms of antibacterial chemotherapy (outcome plateau).

Secreted toxins (exotoxins) produced by S. aureus contribute to pathogenicity and to the ability to colonize the host. These toxins can broadly be categorized as either superantigens or cytotoxic exotoxins. Although few studies directly examined the impact of exotoxin production on mortality in bacteremic patients, clues to the potential role that exotoxins play in mortality can be gathered from virulence studies and animal models. In 2021, researchers from the US demonstrated that mice infected with S. aureus strains harboring deletion in the gene for enterotoxin B experienced lower systemic inflammation and succumbed to infection significantly more slowly than mice infected with the wild-type strain.

1. aureus toxins can be grouped into three categories: 1) membrane-damaging toxins, which may work in a receptor-mediated or receptor-independent fashion, 2) toxins that interfere with receptor function but are not membrane-damaging, and 3) secreted enzymes, such as those that degrade host molecules or affect important host defense mechanisms.

It is likely that to overcome the plateau reached in mortality rates of S. aureus bacteremia, we would need to implement adjunctive therapy to antimicrobials. In this context, human monoclonal antibodies have been recognized against S. aureus alpha-toxin, and an increasing interest is now directed towards anti-toxin strategies to be combined to antimicrobials. New therapeutic strategies are on the horizon and monoclonal antibodies against S. aureus toxins are in Phase II/III trials. As it would be determinant to understand who will really benefit from this adjuvant therapy, a serum toxin quantification could be a key point to guide clinical decisions. To date, studies evaluating quantitative S. aureus toxins’ role in determining a poor prognosis are lacking.

In this context, the first aim of this project is to assess the relationship between the type and the amount of staphylococcal toxin produced in vivo and the clinical outcome of patients with S. aureus BSI. Within this framework, another molecule that has a great clinical potential by exploiting its ability to act as toxin scavenger is human serum albumin (HSA). HSA is the most abundant protein in human plasma and is responsible for several physiologic activities. Our research group demonstrated that HSA can act as part of the innate immunity by blocking toxins of gram positive bacteria (C. difficile and S. pyogenes). In detail the administration of HSA to human intestinal cells and zebrafish exposed to C. difficile toxin A or B determined a +200% survival of the cells and a +30% survival in animals. Similarly, HSA treatment was capable of reducing by more than 50% the hemolysis associated with exposure to S. pyogenes toxin SLO.

To evaluate if HSA can recognize also S. aureus virulence factors, molecular docking analyses were performed. Preliminary results highlight that the PFT α-hemolysin and γ-hemolysin possess the molecular determinants required for the recognition of HSA (i.e., low molecular interaction energy, high number of possible binding modes). These results have been compared to those obtained by the docking analysis between α-hemolysin and ADAM10, which is known to mediate α-hemolysin toxicity. The interaction energy values are very similar, supporting the potential capability of HSA to recognize this toxin. In contrast, staphylococcal enterotoxins B and C3 seem to be not recognized by HSA. These differences are possibly due to the different hydrophobic characteristics of the two protein families.

Moreover, HSA seems to be able to bind ClfA and ClfB, two fibrinogen binding proteins considered virulence factors in bloodstream infections and endocarditis. Furthermore, also Efb and Coa, two secreted fibrinogen binding protein which promotes clotting, seems to be recognized by are probably recognized by HSA.

To date, no evidence exists regarding the possible protective role of HSA towards toxins produced by bacteria other than C. difficile and S. pyogenes (both demonstrated by our group). Therefore, here we aim at evaluating HSA immunity potential also against S. aureus toxins.

 

Riferimento: PRIN 2022 – Codice progetto: 202229FKJF – CUP: F53D23000800006

Investimento totale del progetto: 202.215,00 €

 Partner/proponente: Prof. Stefano di Bella, Università degli Studi di Trieste (Trieste)

 Coordinatore dell’UdR Università degli Studi Roma Tre: Prof.ssa Alessandra di Masi