Showing papers by "Frank R. DeLeo published in 2012"

Neutrophils in innate host defense against Staphylococcus aureus infections

Abstract: Staphylococcus aureus has been an important human pathogen throughout history and is currently a leading cause of bacterial infections worldwide. S. aureus has the unique ability to cause a continuum of diseases, ranging from minor skin infections to fatal necrotizing pneumonia. Moreover, the emergence of highly virulent, drug-resistant strains such as methicillin-resistant S. aureus in both healthcare and community settings is a major therapeutic concern. Neutrophils are the most prominent cellular component of the innate immune system and provide an essential primary defense against bacterial pathogens such as S. aureus. Neutrophils are rapidly recruited to sites of infection where they bind and ingest invading S. aureus, and this process triggers potent oxidative and non-oxidative antimicrobial killing mechanisms that serve to limit pathogen survival and dissemination. S. aureus has evolved numerous mechanisms to evade host defense strategies employed by neutrophils, including the ability to modulate normal neutrophil turnover, a process critical to the resolution of acute inflammation. Here we provide an overview of the role of neutrophils in host defense against bacterial pathogens and discuss strategies employed by S. aureus to circumvent neutrophil function.

Identification of a Highly Transmissible Animal-Independent Staphylococcus aureus ST398 Clone with Distinct Genomic and Cell Adhesion Properties

Abstract: A methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In con- trast, human ST398 MSSA isolates harbored the prophage 3 and the human-specific immune evasion cluster (IEC) genes chp andscn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with signifi- cantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans. IMPORTANCE Staphylococcus aureus strains have generally been considered to be species specific. However, cross-species trans- fers of S. aureus clones, such as ST398 methicillin-resistant S. aureus (MRSA), from swine to humans have been reported. Re- cently, we observed the emergence of ST398 methicillin-susceptible S. aureus (MSSA) as a colonizing strain of humans in north- ern Manhattan. Here we report that ST398 is a frequent cause of MSSA infections in this urban setting. The ST398 MSSA clone was readily transmitted within households, independent of animal contact. We discovered that human ST398 MSSA genomes were smaller than that of the LA-ST398 strain S0385 due to fewer mobile genetic elements. Human and LA-ST398 strains also differed in their composition of adhesion genes and their ability to bind to human skin keratinocytes, providing a potential mechanism of S. aureus host adaptation. Our findings illustrate the importance of implementing molecular surveillance of MSSA given the evidence for the rapid and clinically undetected spread of ST398 MSSA.

Staphylococcus aureus Leukotoxin GH Promotes Inflammation

Abstract: Staphylococcus aureus is a prominent cause of bacterial infections worldwide. The pathogen produces a variety of molecules that presumably facilitate survival in or on the human host (reviewed in [1]). Bicomponent, pore-forming leukotoxins are among the secreted molecules produced by S. aureus. These molecules have cytolytic activity toward leukocytes, most notably polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes. Some of these leukotoxins, such as Panton-Valentine leukocidin (PVL), are encoded on mobile genetic elements such as prophage, whereas others are encoded by genes located in the core genome and are thus present in the majority of strains [2–4]. Bicomponent leukotoxins are composed of S and F subunits that oligomerize and assemble into a β-barrel pore in the plasma membrane of host cells [2, 5]. Recently we and others described a new member of the leukotoxin family—LukGH (named LukAB by DuMont et al [6])—that is an abundant surface-associated and freely secreted protein of S. aureus [3]. Although these studies demonstrated that LukGH causes lysis of phagocytes in vitro and promotes S. aureus colonization and survival in the mouse, our understanding of the role of this molecule during S. aureus infection remains incomplete [3, 6]. To address this deficiency in knowledge, we tested the ability of purified LukGH to elicit an inflammatory response in the skin of mammals (monkey, rabbit, and mouse) and evaluated contribution of the toxin to the severity of S. aureus infection in mice and rabbits.

A NET Outcome.

Abstract: Neutrophils constitute a critical part of innate immunity and are well known for their ability to phagocytose and kill invading microorganisms. The microbicidal processes employed by neutrophils are highly effective at killing most ingested bacteria and fungi. However, an alternative non-phagocytic antimicrobial mechanism of neutrophils has been proposed whereby microorganisms are eliminated by neutrophil extracellular traps (NETs). NETs are comprised of DNA, histones, and antimicrobial proteins extruded by neutrophils during NETosis, a cell death pathway reported to be distinct from apoptosis, phagocytosis-induced cell death, and necrosis. Although multiple laboratories have reported NETs using various stimuli in vitro, the molecular mechanisms involved in this process have yet to be definitively elucidated, and many questions regarding the formation and putative role or function of NETs in innate host defense remain unanswered. It is with these questions in mind that we provide some reflection and perspective on NETs and NETosis.

Sublytic concentrations of Staphylococcus aureus Panton-Valentine leukocidin alter human PMN gene expression and enhance bactericidal capacity

Abstract: CA-MRSA infections are often caused by strains encoding PVL, which can cause lysis of PMNs and other myeloid cells in vitro, a function considered widely as the primary means by which PVL might contribute to disease. However, at sublytic concentrations, PVL can function as a PMN agonist. To better understand this phenomenon, we investigated the ability of PVL to alter human PMN function. PMNs exposed to PVL had enhanced capacity to produce O2− in response to fMLF, but unlike priming by LPS, this response did not require TLR signal transduction. On the other hand, there was subcellular redistribution of NADPH oxidase components in PMNs following exposure of these cells to PVL—a finding consistent with priming. Importantly, PMNs primed with PVL had an enhanced ability to bind/ingest and kill Staphylococcus aureus. Priming of PMNs with other agonists, such as IL-8 or GM-CSF, altered the ability of PVL to cause formation of pores in the plasma membranes of these cells. Microarray analysis revealed significant changes in the human PMN transcriptome following exposure to PVL, including up-regulation of molecules that regulate the inflammatory response. Consistent with the microarray data, mediators of the inflammatory response were released from PMNs after stimulation with PVL. We conclude that exposure of human PMNs to sublytic concentrations of PVL elicits a proinflammatory response that is regulated in part at the level of gene expression. We propose that PVL-mediated priming of PMNs enhances the host innate immune response.

Presence of genes encoding panton-valentine leukocidin is not the primary determinant of outcome in patients with hospital-acquired pneumonia due to Staphylococcus aureus.

Abstract: The impact of Panton-Valentine leukocidin (PVL) on the outcome in Staphylococcus aureus pneumonia is controversial. We genotyped S. aureus isolates from patients with hospital-acquired pneumonia (HAP) enrolled in two registrational multinational clinical trials for the genetic elements carrying pvl and 30 other virulence genes. A total of 287 isolates (173 methicillin-resistant S. aureus [MRSA] and 114 methicillin-susceptible S. aureus [MSSA] isolates) from patients from 127 centers in 34 countries for whom clinical outcomes of cure or failure were available underwent genotyping. Of these, pvl was detected by PCR and its product confirmed in 23 isolates (8.0%) (MRSA, 18/173 isolates [10.4%]; MSSA, 5/114 isolates [4.4%]). The presence of pvl was not associated with a higher risk for clinical failure (4/23 [17.4%] versus 48/264 [18.2%]; P = 1.00) or mortality. These findings persisted after adjustment for multiple potential confounding variables. No significant associations between clinical outcome and (i) presence of any of the 30 other virulence genes tested, (ii) presence of specific bacterial clone, (iii) levels of alpha-hemolysin, or (iv) delta-hemolysin production were identified. This study suggests that neither pvl presence nor in vitro level of alpha-hemolysin production is the primary determinant of outcome among patients with HAP caused by S. aureus.

Genomic Analysis of the Emergence of Vancomycin-Resistant Staphylococcus aureus

Abstract: Staphylococcus aureus is a human commensal bacterium and a prominent cause of infections globally. The high inci- dence ofS.aureusinfections is compounded by the ability of the microbe to readily acquire resistance to antibiotics. In the United States, methicillin-resistant S.aureus(MRSA) is a leading cause of morbidity and mortality by a single infectious agent. Therapeutic options for severe MRSA infections are limited to a few antibiotics to which the organism is typically susceptible, including vancomycin. Acquisition of high-level vancomycin resistance by MRSA is a major concern, but to date, there have been only 12 vancomycin-resistant S.aureus(VRSA) isolates reported in the United States and all belong to a phylogenetic lineage known as clonal complex 5. To gain enhanced understanding of the genetic characteristics conducive to the acquisition of vanco- mycin resistance byS.aureus, V. N. Kos et al. performed whole-genome sequencing of all 12 VRSA isolates and compared the DNA sequences to the genomes of other S.aureusstrains. Thefindings provide new information about the evolutionary history of VRSA and identify genetic features that may bear on the relationship between S.aureusclonal complex 5 strains and the ac- quisition of vancomycin resistance genes from enterococci.

Toward an understanding of the evolution of Staphylococcus aureus strain USA300 during colonization in community households.

Abstract: Staphylococcus aureus is a frequent cause of serious infections and also a human commensal. The emergence of community-associated methicillin-resistant S. aureus led to a dramatic increase in skin and soft tissue infections worldwide. This epidemic has been driven by a limited number of clones, such as USA300 in the United States. To better understand the extent of USA300 evolution and diversification within communities, we performed comparative whole-genome sequencing of three clinical and five colonizing USA300 isolates collected longitudinally from three unrelated households over a 15-month period. Phylogenetic analysis that incorporated additional geographically diverse USA300 isolates indicated that all but one likely arose from a common recent ancestor. Although limited genetic adaptation occurred over the study period, the greatest genetic heterogeneity occurred between isolates from different households and within one heavily colonized household. This diversity allowed for a more accurate tracking of interpersonal USA300 transmission. Sequencing of persisting USA300 isolates revealed mutations in genes involved in major aspects of S. aureus function: adhesion, cell wall biosynthesis, virulence, and carbohydrate metabolism. Genetic variations also included accumulation of multiple polymorphisms within select genes of two multigene operons, suggestive of small genome rearrangements rather than de novo single point mutations. Such rearrangements have been underappreciated in S. aureus and may represent novel means of strain variation. Subtle genetic changes may contribute to USA300 fitness and persistence. Elucidation of small genome rearrangements reveals a potentially new and intriguing mechanism of directed S. aureus genome diversification in environmental niches and during pathogen–host interactions.

Innate immunity against Granulibacter bethesdensis, an emerging gram-negative bacterial pathogen.

Abstract: Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H2O2 but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O2-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.

Community-associated methicillin-resistant Staphylococcus aureus and athletes.

Abstract: The remarkable ability of Staphylococcus aureus to develop antibiotic resistance in conjunction with the emergence of highly virulent and/or transmissible strains has established the pathogen as a leading cause of human bacterial infections worldwide. Historically, methicillin-resistant S aureus (MRSA) was found almost exclusively in hospitals and/or health care-related facilities. However, in the late 1990s, community-associated MRSA strains emerged in the United States and rapidly became the leading cause of community-associated bacterial infections. An enhanced understanding of the pathogenesis and epidemiology of this bacterium is fundamental for the prevention and/or treatment of community-associated MRSA infections. This review highlights salient features of S aureus biology that contribute to the exceptional ability of this pathogen to cause human disease, as well as discusses, in brief, the established approaches for treatment and prevention of infection.

Presence of Genes Encoding the Panton Valentine Leukocidin (PVL) is Not The Primary

Abstract: 31 The impact of Panton Valentine Leukocidin (PVL) on the outcome in Staphylococcus 32 aureus pneumonia is controversial. We genotyped S. aureus isolates from patients with Hospital33 Acquired Pneumonia (HAP) enrolled in two registrational multinational clinical trials for the 34 genetic elements encoding pvl and 30 other virulence genes. A total of 287 isolates (173 35 methicillin resistant [MRSA] and 114 methicillin-susceptible S. aureus [MSSA]) from patients 36 from 127 centers in 34 countries for whom clinical outcomes of cure or failure were available 37 underwent genotyping. Of these, pvl was detected by PCR and its product confirmed in 23 38 isolates (8.0%) (MRSA 18/173 isolates [10.4%]; MSSA 5/114 isolates [4.4%]). Presence of pvl 39 was not associated with a higher risk for clinical failure (4/23 [17.4%] vs. 48/264 [18.2%]; p = 40 1.00) or mortality. These findings persisted after adjustment for multiple potential confounding 41 variables. No significant associations were identified between clinical outcome and a) presence 42 of any of the 30 other virulence genes tested; b) presence of specific bacterial clone; c) levels of 43 alpha hemolysin; or d) delta hemolysin production. This study suggests that neither pvl presence 44 nor in vitro level of alpha hemolysin production are the primary determinants of outcome among 45 patients with HAP caused by S. aureus. 46

Flexicate molecules as a potential new class of antibiotics.

Abstract: Evaluation of: Howson SE, Bolhuis A, Brabec V et al. Optically pure, water-stable metallo–helical ‘flexicate’ assemblies with antibiotic activity. Nat. Chem. 4(1), 31–36 (2011). Helicates are α-helical, nonpeptide complexes that bind to DNA and exhibit antimicrobial activity. In the past, enthusiasm for the use of helicates in biological applications was limited, at least in part, by the presence of a racemic mixture of enantiomers or the formation of complexes that are insoluble in aqueous solutions. Recently, Howson et al. overcame the barriers associated with helicate synthesis by generating helicate-like complexes that are soluble and stable in water, optically pure and synthetically flexible. The mechanism synthesizes nonpeptide mimetic α-helical ‘flexicates’ that bind to DNA and show broad-spectrum antimicrobial activity against representative Gram-positive and Gram-negative bacterial pathogens. Although the application of flexicates as an antimicrobial therapy remains to be determined, this study p...