Severe soft tissue infections (SSTIs), such as necrotizing fasciitis (NF), also called flesh-eating disease, are among the most lethal bacterial infections. Several bacterial pathogens, such as Aeromonas spp., Staphylococcus spp., and Vibrio spp., can cause NF. Even when treated with a cocktail of antibiotics, NF still has severe sequelae and causes substantial mortality. In recent years, increasing numbers of SSTIs caused by Aeromonas dhakensis (also known as A. hydrophila sub. dhakensis) have been reported in southern Taiwan and worldwide [1], [2]. Aeromonas spp. are ubiquitous in aquatic environments and can cause opportunistic infections through unexpected exposure to contaminated water, soils, or aquatic animal food sources [2], [3]. On the basis of our previous finding, the barA-uvrY two-component system is the major regulator of the virulence of A. dhakensis and controls an array of virulence factors, including the haemolysin Ahh1[4]. In addition, we discovered that clinically virulent species, such as A. dhakensis and A. hydrophila, carry genes encoding the haemolysin Ahh1 and demonstrate greater virulence than other Aeromonas species [5], [6]. Haemolysin Ahh1 is a β pore-forming toxin that causes host cell membrane perforation and leads to cell death [7]. However, evidence connecting haemolysin Ahh1 and clinically severe SSTIs is lacking.

The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is a well-known protein complex that is mainly expressed in macrophages and contains a sensor (NLRP3), adaptor (ASC, also called PYCARD), and effector (caspase-1) [8]. The activated NLRP3 inflammasome induces cell death (known as pyroptosis) by cleaving the pore-forming protein gasdermin D N-terminus (GSDMD-N), which allows the subsequent release of the proinflammatory cytokines interleukin-18 (IL-18) and interleukin-1β (IL-1β) [9]. Generally, bacterial toxins can activate the assembly of the NLRP3 inflammasome, which induces pyroptosis [10]. In general, pyroptosis is a self-protective mechanism that eliminates severely damaged cells. However, excessive pyroptosis due to bacterial infection can cause severe tissue damage (such as necrotizing fasciitis), cause irreversible sequelae and require extensive surgical debridement and subsequent tissue reconstruction [11]. A previous study demonstrated that Aeromonas hydrophila can secrete cytotoxins and activate the NLRP3 inflammasome pathway but not the NLR family or CARD domain containing 4 (NLRC4) in macrophages [12]. However, the connection between NLRP3 activation and necrotizing fasciitis remains unclear.

In this study, we verified that the haemolysin Ahh1 is the major virulence factor of A. dhakensis and contributes to the development of SSTIs through the overexpression of IL-1β which is activated by the NLRP3 inflammatory signalling pathway. In addition, we demonstrated that antibiotics combined with an IL-1β antibody significantly alleviated the severity of tissue damage caused by A. dhakensis infection. Our study provides potential insight into how to improve the clinical outcomes of patients with SSTIs caused by toxin-producing bacteria.