Recent reports have established the escalating threat of carbapenem-resistant Enterobacter cloacae complex (CREC). Here, we demonstrate that CREC has evolved as a highly antibiotic-resistant rather than highly virulent nosocomial pathogen. Applying genomics and Bayesian phylogenetic analyses to a 7-year collection of CREC isolates from a northern Manhattan hospital system and to a large set of publicly available, geographically diverse genomes, we demonstrate clonal spread of a single clone, ST171. We estimate that two major clades of epidemic ST171 diverged prior to 1962, subsequently spreading in parallel from the Northeastern to the Mid-Atlantic and Midwestern United States and demonstrating links to international sites. Acquisition of carbapenem and fluoroquinolone resistance determinants by both clades preceded widespread use of these drugs in the mid-1980s, suggesting that antibiotic pressure contributed substantially to its spread. Despite a unique mobile repertoire, ST171 isolates showed decreased virulence in vitro. While a second clone, ST78, substantially contributed to the emergence of CREC, it encompasses diverse carbapenemase-harboring plasmids, including a potentially hypertransmissible IncN plasmid, also present in other sequence types. Rather than heightened virulence, CREC demonstrates lineage-specific, multifactorial adaptations to nosocomial environments coupled with a unique potential to acquire and disseminate carbapenem resistance genes. These findings indicate a need for robust surveillance efforts that are attentive to the potential for local and international spread of high-risk CREC clones.

Highlights d S. aureus leukocidins target DARC on endothelial cells to cause vascular dysfunction d Targeting DARC leads to organ damage during S. aureus bloodstream infection d Primary human endothelial cells are susceptible to S. aureus leukocidins d Leukocidins target host leukocytes, red blood cells, and endothelial cells

A key aspect underlying the severity of infections caused by Staphylococcus aureus is the abundance of virulence factors that the pathogen uses to thwart critical components of the human immune response. One such mechanism involves the destruction of host immune cells by cytolytic toxins secreted by S. aureus, including five bicomponent leukocidins: PVL, HlgAB, HlgCB, LukED, and LukAB. Purified leukocidins can lyse immune cells ex vivo, and systemic injections of purified LukED or HlgAB can acutely kill mice. Here, we describe the generation and characterization of centyrins that bind S. aureus leukocidins with high affinity and protect primary human immune cells from toxin-mediated cytolysis. Centyrins are small protein scaffolds derived from the fibronectin type III–binding domain of the human protein tenascin-C. Although centyrins are potent in tissue culture assays, their short serum half-lives limit their efficacies in vivo. By extending the serum half-lives of centyrins through their fusion to an albumin-binding consensus domain, we demonstrate the in vivo efficacy of these biologics in a murine intoxication model and in models of both prophylactic and therapeutic treatment of live S. aureus systemic infections. These biologics that target S. aureus virulence factors have potential for treating and preventing serious staphylococcal infections.

Staphylococcus aureus is responsible for various diseases in humans, and recurrent infections are commonly observed. S. aureus produces an array of bicomponent pore-forming toxins that target and kill leukocytes, known collectively as the leukocidins. The contribution of these leukocidins to impair the development of anti–S. aureus adaptive immunity and facilitate reinfection is unclear. Using a murine model of recurrent bacteremia, we demonstrate that infection with a leukocidin mutant results in increased levels of anti–S. aureus antibodies compared with mice infected with the WT parental strain, indicating that leukocidins negatively impact the generation of anti–S. aureus antibodies in vivo. We hypothesized that neutralizing leukocidin-mediated immune subversion by vaccination may shift this host-pathogen interaction in favor of the host. Leukocidin-immunized mice produce potent leukocidin-neutralizing antibodies and robust Th1 and Th17 responses, which collectively protect against bloodstream infections. Altogether, these results demonstrate that blocking leukocidin-mediated immune evasion can promote host protection against S. aureus bloodstream infection.

Neutrophils, polymorphonuclear leukocytes (PMN), play a critical role in the innate immune response to Staphylococcus aureus, a pathogen that continues to be associated with significant morbidity and mortality. Neutrophil extracellular trap (NET) formation is involved in ensnaring and killing of S. aureus, but this hostpathogen interaction also leads to host tissue damage. Importantly, NET components including neutrophil proteases are under consideration as therapeutic targets in a variety of disease processes. Although S. aureus lipoproteins are recognized to activate cells via TLRs, specific mechanisms of interaction with neutrophils are poorly delineated. We hypothesized that a lipoprotein-containing cell membrane preparation from methicillin-resistant S. aureus (MRSA-CMP) would elicit PMN activation, including NET formation. We investigated MRSA-CMPelicited NET formation, regulated elastase release, and IL-8 production in human neutrophils. We studied PMN from healthy donors with or without a common single-nucleotide polymorphism in TLR1, previously demonstrated to impact TLR2/1 signaling, and used cell membrane preparation from both wild-type methicillin-resistant S. aureus and a mutant lacking palmitoylated lipoproteins (lgt). MRSA-CMP elicited NET formation, elastase release, and IL-8 production in a lipoprotein-dependent manner. TLR2/1 signaling was involved in NET formation and IL-8 production, but not elastase release, suggesting that MRSA-CMPelicited elastase release is not mediated by triacylated lipoproteins. MRSA-CMP also primed neutrophils for enhanced NET formation in response to a subsequent stimulus. MRSA-CMPelicited NET formation did not require Nox2-derived reactive oxygen species and was partially dependent on the activity of peptidyl arginine deiminase (PAD). In conclusion, lipoproteins from S. aureus mediate NET formation via TLR2/1 with clear implications for patients with sepsis.