Adeline R. Porter

Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae

Significance Carbapenem-resistant Klebsiella pneumoniae has emerged globally as a multidrug-resistant hospital pathogen for which there are few treatment options. Clinical isolates classified by multilocus sequence typing (ST) as ST258 are the most widespread. The basis for the success of ST258 organisms above and beyond antibiotic resistance is not known, nor is it clear whether infections are caused by a single clone. We used genome sequencing to reveal unexpected genetic diversity among ST258 organisms (thus disproving the single-clone hypothesis) and identified a recombination hotspot that accounts for the majority of divergence—and presumably for serologic variation—among ST258 clinical isolates. Our findings will facilitate the development of new clinical strategies designed to prevent or treat infections caused by multidrug-resistant K. pneumoniae. Infections caused by drug-resistant bacteria are a major problem worldwide. Carbapenem-resistant Klebsiella pneumoniae, most notably isolates classified as multilocus sequence type (ST) 258, have emerged as an important cause of hospital deaths. ST258 isolates are predominantly multidrug resistant, and therefore infections caused by them are difficult to treat. It is not known why the ST258 lineage is the most prevalent cause of multidrug-resistant K. pneumoniae infections in the United States and other countries. Here we tested the hypothesis that carbapenem-resistant ST258 K. pneumoniae is a single genetic clone that has disseminated worldwide. We sequenced to closure the genomes of two ST258 clinical isolates and used these genomes as references for comparative genome sequencing of 83 additional clinical isolates recovered from patients at diverse geographic locations worldwide. Phylogenetic analysis of the SNPs in the core genome of these isolates revealed that ST258 K. pneumoniae organisms are two distinct genetic clades. This unexpected finding disproves the single-clone hypothesis. Notably, genetic differentiation between the two clades results from an ∼215-kb region of divergence that includes genes involved in capsule polysaccharide biosynthesis. The region of divergence appears to be a hotspot for DNA recombination events, and we suggest that this region has contributed to the success of ST258 K. pneumoniae. Our findings will accelerate research on novel diagnostic, therapeutic, and vaccine strategies designed to prevent and/or treat infections caused by multidrug resistant K. pneumoniae.

Phagocytosis and Killing of Staphylococcus aureus by Human Neutrophils

Neutrophils are essential for host defense against Staphylococcus aureus infections. Although significant progress has been made, our understanding of neutrophil interactions with S. aureus remains incomplete. To provide a more comprehensive view of this process, we investigated phagocytosis and killing of S. aureus by human neutrophils using varied assay conditions in vitro. A greater percentage of bacteria were internalized by adherent neutrophils compared to those in suspension, and, unexpectedly, uptake of S. aureus by adherent neutrophils occurred efficiently in the absence of opsonins. An antibody specific for S. aureus promoted uptake of unopsonized bacteria in suspension, but had little or no capacity to enhance phagocytosis of S. aureus opsonized with normal human serum or by adherent neutrophils. Collectively, these results indicate that assay conditions can have a significant influence on the phagocytosis and killing of S. aureus by neutrophils. More importantly, the results suggest a vaccine approach directed to enhance opsonophagocytosis alone is not sufficient to promote increased killing of S. aureus by human neutrophils. With the emergence and reemergence of antibiotic-resistant microorganisms, establishing parameters that are optimal for studying neutrophil-S. aureus interactions will pave the way towards developing immune-directed strategies for anti-staphylococcal therapies.

Insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (PSM) α3 peptide

Phenol‐soluble modulins (PSMs) are a family of peptides with multiple functions in staphylococcal pathogenesis. To gain insight into the structural features affecting PSM functions, we analyzed an alanine substitution library of PSMα3, a strongly cytolytic and proinflammatory PSM of Staphylococcus aureus with a significant contribution to S. aureus virulence. Lysine residues were essential for both receptor‐dependent proinflammatory and receptor‐independent cytolytic activities. Both phenotypes also required additional structural features, with the C terminus being crucial for receptor activation. Biofilm formation was affected mostly by hydrophobic amino acid positions, suggesting that the capacity to disrupt hydrophobic interactions is responsible for the effect of PSMs on biofilm structure. Antimicrobial activity, absent from natural PSMα3, could be created by the exchange of large hydrophobic side chains, indicating that PSMα3 has evolved to exhibit cytolytic rather than antimicrobial activity. In addition to gaining insight into the structure‐function relationship in PSMs, our study identifies nontoxic PSMα3 derivatives for active vaccination strategies and lays the foundation for future efforts aimed to understand the biological role of PSM recognition by innate host defense.—Cheung, G. Y., Kretschmer, D., Queck, S. Y., Joo, H.‐S., Wang, R., Duong, A. C., Nguyen, T. H., Bach, T.‐H., Porter, A. R., DeLeo, F. R., Peschel, A., Otto, M. Insight into structure‐function relationship in phenol‐soluble modulins using an alanine screen of the phenol‐soluble modulin (PSM) α3 peptide. FASEB J. 28, 153–161 (2014). www.fasebj.org

Phagocytosis and Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils

Carbapenem-resistant Klebsiella pneumoniae strains classified as multilocus sequence type 258 (ST258) are among the most widespread multidrug-resistant hospital-acquired pathogens. Treatment of infections caused by these organisms is difficult, and mortality is high. The basis for the success of ST258, outside of antibiotic resistance, remains incompletely determined. Here we tested the hypothesis that ST258K. pneumoniae has enhanced capacity to circumvent killing by human neutrophils, the primary cellular defense against bacterial infections. There was limited binding and uptake of ST258 by human neutrophils, and correspondingly, there was limited killing of bacteria. On the other hand, transmission electron microscopy revealed that any ingested organisms were degraded readily within neutrophil phagosomes, thus indicating that survival in the neutrophil assays is due to limited phagocytosis, rather than to microbicide resistance after uptake. Our findings suggest that enhancing neutrophil phagocytosis is a potential therapeutic approach for treatment of infection caused by carbapenem-resistant ST258K. pneumoniae.

Seasonal H3N2 influenza A virus fails to enhance Staphylococcus aureus co-infection in a non-human primate respiratory tract infection model

Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.

Contribution of Staphylococcus aureus Coagulases and Clumping Factor A to Abscess Formation in a Rabbit Model of Skin and Soft Tissue Infection

Staphylococcus aureus produces numerous factors that facilitate survival in the human host. S. aureus coagulase (Coa) and von Willebrand factor-binding protein (vWbp) are known to clot plasma through activation of prothrombin and conversion of fibrinogen to fibrin. In addition, S. aureus clumping factor A (ClfA) binds fibrinogen and contributes to platelet aggregation via a fibrinogen- or complement-dependent mechanism. Here, we evaluated the contribution of Coa, vWbp and ClfA to S. aureus pathogenesis in a rabbit model of skin and soft tissue infection. Compared to skin abscesses caused by the Newman wild-type strain, those caused by isogenic coa, vwb, or clfA deletion strains, or a strain deficient in coa and vwb, were significantly smaller following subcutaneous inoculation in rabbits. Unexpectedly, we found that fibrin deposition and abscess capsule formation appear to be independent of S. aureus coagulase activity in the rabbit infection model. Similarities notwithstanding, S. aureus strains deficient in coa and vwb elicited reduced levels of several proinflammatory molecules in human blood in vitro. Although a specific mechanism remains to be determined, we conclude that S. aureus Coa, vWbp and ClfA contribute to abscess formation in rabbits.

Contribution of Secreted NADase and Streptolysin O to the Pathogenesis of Epidemic Serotype M1 Streptococcus pyogenes Infections

Streptococcus pyogenes secretes many toxins that facilitate human colonization, invasion, and dissemination. NADase (SPN) and streptolysin O (SLO) are two toxins that play important roles in pathogenesis. We previously showed that increased production of SPN and SLO in epidemic serotype M1 and M89 S. pyogenes strains is associated with rapid intercontinental spread and enhanced virulence. The biological functions of SPN and SLO have been extensively studied using eukaryotic cell lines, but the relative contribution of each of these two toxins to pathogenesis of epidemic M1 or M89 strains remains unexplored. Herein, using a genetically representative epidemic M1 strain and a panel of isogenic mutant derivative strains, we evaluated the relative contributions of SPN and SLO toxins to virulence in mouse models of necrotizing myositis, bacteremia, and skin and soft tissue infection. We found that isogenic mutants lacking SPN, SLO, and both toxins are equally impaired in ability to cause necrotizing myositis. In addition, mutants lacking either SPN or SLO are significantly attenuated in the bacteremia and soft tissue infection models, and the mutant strain lacking production of both toxins is further attenuated. The mutant strain lacking both SPN and SLO production is severely attenuated in ability to resist killing by human polymorphonuclear leukocytes. We conclude that both SPN and SLO contribute significantly to S. pyogenes pathogenesis in these virulence assays.

Survival of carbapenem-resistant ST258 Klebsiella pneumoniae in human blood

ABSTRACT Klebsiella pneumoniae is a prominent cause of nosocomial infections worldwide. Bloodstream infections caused by carbapenem-resistant K. pneumoniae, including the epidemic lineage known as multilocus sequence type 258 (ST258), are difficult to treat, and the rate of mortality from such infections is high. Thus, it is imperative that we gain a better understanding of host defense against this pathogen as a step toward developing novel therapies. Here we tested the hypothesis that the resistance of ST258 to bactericidal components of human blood, such as serum complement, is linked to virulence capacity in the context of bacteremia. There was significant variance in the survival of ST258 clinical isolates in heparinized human blood or normal human serum. The rate of survival of ST258 isolates in human blood was, in general, similar to that in normal human serum, suggesting a prominent role for complement (rather than leukocytes) in the healthy host defense against ST258 isolates and related organisms. Indeed, deposition of serum complement—the C5b to C9 (C5b-C9) membrane attack complex—onto the surface of ST258 isolates accompanied serum bactericidal activity. Human serum treated with pharmacological inhibitors of complement, depleted of antibody, or heated at 56°C for 30 min had significantly reduced or absent bactericidal activity. In contrast to heparinized blood from humans, that from BALB/c mice lacked bactericidal activity toward the ST258 isolates tested, but the virulence of these ST258 isolates in a mouse bacteremia model was inexplicably limited. Our data highlight the importance of the complement system in host defense against ST258 bacteremia, and we propose that there is the potential to enhance complement-mediated bactericidal activity using an antibody-based approach.

Genomic landscape of intrahost variation in Group A Streptococcus: repeated and abundant mutational inactivation of the fabT gene encoding a regulator of fatty acid synthesis.

ABSTRACT To obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here.

Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae is a problem worldwide. A carbapenem-resistant K. pneumoniae lineage classified as multilocus sequence type 258 (ST258) is prominent in the health care setting in many regions of the world, including the United States. ST258 strains can be resistant to virtually all clinically useful antibiotics; treatment of infections caused by these organisms is difficult, and mortality is high. As a step toward promoting development of new therapeutics for ST258 infections, we tested the ability of rabbit antibodies specific for ST258 capsule polysaccharide to enhance human serum bactericidal activity and promote phagocytosis and killing of these bacteria by human neutrophils. We first demonstrated that an isogenic wzy deletion strain is significantly more susceptible to killing by human heparinized blood, serum, and neutrophils than a wild-type ST258 strain. Consistent with the importance of capsule as an immune evasion molecule, rabbit immune serum and purified IgG specific for ST258 capsule polysaccharide type 2 (CPS2) enhanced killing by human blood and serum in vitro. Moreover, antibodies specific for CPS2 promoted phagocytosis and killing of ST258 by human neutrophils. Collectively, our findings suggest that ST258 CPS2 is a viable target for immunoprophylactics and/or therapeutics. IMPORTANCE Infections caused by carbapenem-resistant K. pneumoniae are difficult to treat, and mortality is high. New prophylactic approaches and/or therapeutic measures are needed to prevent or treat infections caused by these multidrug-resistant bacteria. A strain of carbapenem-resistant K. pneumoniae, classified by multilocus sequence typing as ST258, is present in many regions of the world and is the most prominent carbapenem-resistant K. pneumoniae lineage in the United States. Here we show that rabbit antibodies specific for capsule polysaccharide of ST258 significantly enhance human serum bactericidal activity and promote phagocytosis and killing of this pathogen by human neutrophils. These studies have provided strong support for the idea that development of an immunotherapy (vaccine) for carbapenem-resistant K. pneumoniae infections is feasible and has merit. Infections caused by carbapenem-resistant K. pneumoniae are difficult to treat, and mortality is high. New prophylactic approaches and/or therapeutic measures are needed to prevent or treat infections caused by these multidrug-resistant bacteria. A strain of carbapenem-resistant K. pneumoniae, classified by multilocus sequence typing as ST258, is present in many regions of the world and is the most prominent carbapenem-resistant K. pneumoniae lineage in the United States. Here we show that rabbit antibodies specific for capsule polysaccharide of ST258 significantly enhance human serum bactericidal activity and promote phagocytosis and killing of this pathogen by human neutrophils. These studies have provided strong support for the idea that development of an immunotherapy (vaccine) for carbapenem-resistant K. pneumoniae infections is feasible and has merit.