Interaction of multidrug-resistant hypervirulent Klebsiella pneumoniae with components of human innate host defense.

IMPORTANCE: Klebsiella pneumoniae strains with a combination of multidrug resistance and hypervirulence genotypes (MDR hvKp) have emerged as a cause of human infections. The ability of these microbes to avoid killing by the innate immune system remains to be tested fully. To that end, we compared the ability of a global collection of hvKp and MDR hvKp clinical isolates to survive in human blood and resist phagocytic killing by human neutrophils. The two MDR hvKp clinical isolates tested (ST11 and ST147) were killed in human blood and by human neutrophils in vitro, whereas phagocytic killing of hvKp clinical isolates (ST23 and ST86) required specific antisera. Although the data were varied and often isolate specific, they are an important first step toward gaining an enhanced understanding of host defense against MDR hvKp.

Microbes and the fate of neutrophils.

Neutrophils or polymorphonuclear neutrophils (PMNs) are an important component of innate host defense. These phagocytic leukocytes are recruited to infected tissues and kill invading microbes. There are several general characteristics of neutrophils that make them highly effective as antimicrobial cells. First, there is tremendous daily production and turnover of granulocytes in healthy adults-typically 1011 per day. The vast majority (~95%) of these cells are neutrophils. In addition, neutrophils are mobilized rapidly in response to chemotactic factors and are among the first leukocytes recruited to infected tissues. Most notably, neutrophils contain and/or produce an abundance of antimicrobial molecules. Many of these antimicrobial molecules are toxic to host cells and can destroy host tissues. Thus, neutrophil activation and turnover are highly regulated processes. To that end, aged neutrophils undergo apoptosis constitutively, a process that contains antimicrobial function and proinflammatory capacity. Importantly, apoptosis facilitates nonphlogistic turnover of neutrophils and removal by macrophages. This homeostatic process is altered by interaction with microbes and their products, as well as host proinflammatory molecules. Microbial pathogens can delay neutrophil apoptosis, accelerate apoptosis following phagocytosis, or cause neutrophil cytolysis. Here, we review these processes and provide perspective on recent studies that have potential to impact this paradigm.

Subinhibitory Concentrations of Antibiotics Alter the Response of Klebsiella pneumoniae to Components of Innate Host Defense.

Carbapenem-resistant Klebsiella pneumoniae isolates classified as multilocus sequence type 258 (ST258) are a problem in health care settings in many countries globally. ST258 isolates are resistant to multiple classes of antibiotics and can cause life-threatening infections, such as pneumonia and sepsis, in susceptible individuals. Treatment strategies for such infections are limited. Understanding the response of K. pneumoniae to host factors in the presence of antibiotics could reveal mechanisms employed by the pathogen to evade killing in the susceptible host, as well as inform treatment of infections. Here, we investigated the ability of antibiotics at subinhibitory concentrations to alter K. pneumoniae capsular polysaccharide (CPS) production and survival in normal human serum (NHS). Unexpectedly, pretreatment with some of the antibiotics tested enhanced ST258 survival in NHS. For example, a subinhibitory concentration of mupirocin increased survival for 7 of 10 clinical isolates evaluated and there was increased cell-associated CPS for 3 of these isolates compared with untreated controls. Additionally, mupirocin pretreatment caused concomitant reduction in the deposition of the serum complement protein C5b-9 on the surface of these three isolates. Transcriptome analyses with a selected ST258 isolate (34446) indicated that genes implicated in the stringent response and/or serum resistance were upregulated following mupirocin treatment and/or culture in NHS. In conclusion, mupirocin and/or human serum causes changes in the K. pneumoniae transcriptome that likely contribute to the observed decrease in serum susceptibility via a multifactorial process. Whether these responses can be extended more broadly and thus impact clinical outcome in the human host merits further investigation. IMPORTANCE The extent to which commensal bacteria are altered by exposure to subinhibitory concentrations of antibiotics (outside resistance) remains incompletely determined. To gain a better understanding of this phenomenon, we tested the ability of selected antibiotics (at subinhibitory concentrations) to alter survival of ST258 clinical isolates in normal human serum. We found that exposure of ST258 to antibiotics at low concentrations differentially altered gene expression, capsule production, serum complement deposition, and bacterial survival. The findings were isolate and antibiotic dependent but provide insight into a potential confounding issue associated with ST258 infections.

Toward Optimization of a Rabbit Model of Staphylococcus aureus (USA300) Skin and Soft Tissue Infection.

Staphylococcus aureus remains a leading cause of skin and soft tissue infections (SSTIs) globally. In the United States, many of these infections are caused by isolates classified as USA300. Our understanding of the success of USA300 as a human pathogen is due in part to data obtained from animal infection models, including rabbit SSTI models. These animal models have been used to study S. aureus virulence and pathogenesis and to gain an enhanced understanding of the host response to infection. Although significant knowledge has been gained, the need to use a relatively high inoculum of USA300 (1 × 108 to 5 × 108 CFU) is a caveat of these infection models. As a step toward addressing this issue, we created mutations in USA300 that mimic those found in S. aureus strains with naturally occurring rabbit tropism-namely, single nucleotide polymorphisms in dltB and/or deletion of rot. We then developed a rabbit SSTI model that utilizes an inoculum of 106 USA300 CFU to cause reproducible disease and tested whether primary SSTI protects rabbits against severe reinfection caused by the same strain. Although there was modest protection against severe reinfection, primary infection and reinfection with rabbit-tropic USA300 strains failed to increase the overall level of circulating anti-S. aureus antibodies significantly. These findings provide additional insight into the host response to S. aureus. More work is needed to further develop a low-inoculum infection model that can be used to better test the potential of new therapeutics or vaccine target antigens. IMPORTANCE Animal models of S. aureus infection are important for evaluating bacterial pathogenesis and host immune responses. These animal infection models are often used as an initial step in the testing of vaccine antigens and new therapeutics. The extent to which animal models of S. aureus infection approximate human infections remains a significant consideration for translation of results to human clinical trials. Although significant progress has been made with rabbit models of S. aureus infection, one concern is the high inoculum needed to cause reproducible disease. Here, we generated USA300 strains that have tropism for rabbits and developed a rabbit SSTI model that uses fewer CFU than previous models.

Innate Host Defense against Klebsiella pneumoniae and the Outlook for Development of Immunotherapies.

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative commensal bacterium and opportunistic pathogen. In healthy individuals, the innate immune system is adept at protecting against K. pneumoniae infection. Notably, the serum complement system and phagocytic leukocytes (e.g., neutrophils) are highly effective at eliminating K. pneumoniae and thereby preventing severe disease. On the other hand, the microbe is a major cause of healthcare-associated infections, especially in individuals with underlying susceptibility factors, such as pre-existing severe illness or immune suppression. The burden of K. pneumoniae infections in hospitals is compounded by antibiotic resistance. Treatment of these infections is often difficult largely because the microbes are usually resistant to multiple antibiotics (multidrug resistant [MDR]). There are a limited number of treatment options for these infections and new therapies, and preventative measures are needed. Here, we review host defense against K. pneumoniae and discuss recent therapeutic measures and vaccine approaches directed to treat and prevent severe disease caused by MDR K. pneumoniae.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

Further Insight into the Mechanism of Human PMN Lysis following Phagocytosis of Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen that can cause a variety of diseases ranging from mild superficial skin infections to life-threatening conditions like necrotizing pneumonia, endocarditis, and septicemia. Polymorphonuclear leukocytes (PMNs; neutrophils in particular herein) are essential for host defense against S. aureus infections, and the microbe is phagocytosed readily. Most ingested bacteria are killed, but some S. aureus strains-such as the epidemic USA300 strain-have an enhanced ability to cause PMN lysis after phagocytosis. Although progress has been made, the mechanism for lysis after phagocytosis of S. aureus remains incompletely determined. Here, we tested the hypothesis that disruption of phagosome integrity and escape of S. aureus from the PMN phagosome into the cytoplasm precedes PMN lysis. We used USA300 wild-type and isogenic deletion strains to evaluate and/or verify the role of selected S. aureus molecules in this cytolytic process. Compared to the wild-type USA300 strain, Δagr, Δhla, ΔlukGH, and Δpsm strains each caused significantly less lysis of human PMNs 3 h and/or 6 h after phagocytosis, consistent with previous studies. Most notably, confocal microscopy coupled with selective permeabilization assays demonstrated that phagosome membrane integrity is largely maintained prior to PMN lysis after S. aureus phagocytosis. We conclude that PMN lysis does not require escape of S. aureus from the phagosome to the cytoplasm and that these are independent phenomena. The findings are consistent with the ability of S. aureus (via selected molecules) to trigger lysis of human PMNs by an undetermined signaling mechanism. IMPORTANCE S. aureus strain USA300 has the ability to cause rapid lysis of human neutrophils after phagocytosis. Although this phenomenon likely contributes to the success of USA300 as a human pathogen, our knowledge of the mechanism remains incomplete. Here, we used a selective permeabilization assay coupled with confocal microscopy to demonstrate that USA300 is contained within human neutrophil phagosomes until the point of host cell lysis. Thus, consistent with a process in macrophages, S. aureus fails to escape into the neutrophil cytoplasm prior to cytolysis.

Bacteriophage Treatment Rescues Mice Infected with Multidrug-Resistant Klebsiella pneumoniae ST258.

Severe infections caused by multidrug-resistant Klebsiella pneumoniae sequence type 258 (ST258) highlight the need for new therapeutics with activity against this pathogen. Bacteriophage (phage) therapy is an alternative treatment approach for multidrug-resistant bacterial infections that has shown efficacy in experimental animal models and promise in clinical case reports. In this study, we assessed microbiologic, histopathologic, and survival outcomes following systemic administration of phage in ST258-infected mice. We found that prompt treatment with two phages, either individually or in combination, rescued mice with K. pneumoniae ST258 bacteremia. Among the three treatment groups, mice that received combination phage therapy demonstrated the greatest increase in survival and the lowest frequency of phage resistance among bacteria recovered from mouse blood and tissue. Our findings support the utility of phage therapy as an approach for refractory ST258 infections and underscore the potential of this treatment modality to be enhanced through strategic phage selection.IMPORTANCE Infections caused by multidrug-resistant K. pneumoniae pose a serious threat to at-risk patients and present a therapeutic challenge for clinicians. Bacteriophage (phage) therapy is an alternative treatment approach that has been associated with positive clinical outcomes when administered experimentally to patients with refractory bacterial infections. Inasmuch as these experimental treatments are prepared for individual patients and authorized for compassionate use only, they lack the rigor of a clinical trial and therefore cannot provide proof of efficacy. Here, we demonstrate that administration of viable phage provides effective treatment for multidrug-resistant K. pneumoniae (sequence type 258 [ST258]) bacteremia in a murine infection model. Moreover, we compare outcomes among three distinct phage treatment groups and identify potential correlates of therapeutic phage efficacy. These findings constitute an important first step toward optimizing and assessing phage therapy's potential for the treatment of severe ST258 infection in humans.

Staphylococcus aureus, Antibiotic Resistance, and the Interaction with Human Neutrophils.

Significance:Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The high burden of S. aureus among human and animal hosts, which includes asymptomatic carriage and infection, is coupled with a notorious ability of the microbe to become resistant to antibiotics. Notably, S. aureus has the ability to produce molecules that promote evasion of host defense, including the ability to avoid killing by neutrophils. Recent Advances: Significant progress has been made to better understand S. aureus-host interactions. These discoveries include elucidation of the role played by numerous S. aureus virulence molecules during infection. Based on putative functions, a number of these virulence molecules, including S. aureus alpha-hemolysin and protein A, have been identified as therapeutic targets. Although it has not been possible to develop a vaccine that can prevent S. aureus infections, monoclonal antibodies specific for S. aureus virulence molecules have the potential to moderate the severity of disease. Critical Issues: Therapeutic options for treatment of methicillin-resistant S. aureus (MRSA) are limited, and the microbe typically develops resistance to new antibiotics. New prophylactics and/or therapeutics are needed. Future Directions: Research that promotes an enhanced understanding of S. aureus-host interaction is an important step toward developing new therapeutic approaches directed to moderate disease severity and facilitate treatment of infection. This research effort includes studies that enhance our view of the interaction of S. aureus with human neutrophils. Antioxid. Redox Signal. 34, 452-470.

Streptococcus pyogenes genes that promote pharyngitis in primates.

Streptococcus pyogenes (group A streptococcus; GAS) causes 600 million cases of pharyngitis annually worldwide. There is no licensed human GAS vaccine despite a century of research. Although the human oropharynx is the primary site of GAS infection, the pathogenic genes and molecular processes used to colonize, cause disease, and persist in the upper respiratory tract are poorly understood. Using dense transposon mutant libraries made with serotype M1 and M28 GAS strains and transposon-directed insertion sequencing, we performed genome-wide screens in the nonhuman primate (NHP) oropharynx. We identified many potentially novel GAS fitness genes, including a common set of 115 genes that contribute to fitness in both genetically distinct GAS strains during experimental NHP pharyngitis. Targeted deletion of 4 identified fitness genes/operons confirmed that our newly identified targets are critical for GAS virulence during experimental pharyngitis. Our screens discovered many surface-exposed or secreted proteins - substrates for vaccine research - that potentially contribute to GAS pharyngitis, including lipoprotein HitA. Pooled human immune globulin reacted with purified HitA, suggesting that humans produce antibodies against this lipoprotein. Our findings provide new information about GAS fitness in the upper respiratory tract that may assist in translational research, including developing novel vaccines.

Genetic heterogeneity of the Spy1336/R28-Spy1337 virulence axis in Streptococcus pyogenes and effect on gene transcript levels and pathogenesis.

Streptococcus pyogenes is a strict human pathogen responsible for more than 700 million infections annually worldwide. Strains of serotype M28 S. pyogenes are typically among the five more abundant types causing invasive infections and pharyngitis in adults and children. Type M28 strains also have an unusual propensity to cause puerperal sepsis and neonatal disease. We recently discovered that a one-nucleotide indel in an intergenic homopolymeric tract located between genes Spy1336/R28 and Spy1337 altered virulence in a mouse model of infection. In the present study, we analyzed size variation in this homopolymeric tract and determined the extent of heterogeneity in the number of tandemly-repeated 79-amino acid domains in the coding region of Spy1336/R28 in large samples of strains recovered from humans with invasive infections. Both repeat sequence elements are highly polymorphic in natural populations of M28 strains. Variation in the homopolymeric tract results in (i) changes in transcript levels of Spy1336/R28 and Spy1337 in vitro, (ii) differences in virulence in a mouse model of necrotizing myositis, and (iii) global transcriptome changes as shown by RNAseq analysis of isogenic mutant strains. Variation in the number of tandem repeats in the coding sequence of Spy1336/R28 is responsible for size variation of R28 protein in natural populations. Isogenic mutant strains in which genes encoding R28 or transcriptional regulator Spy1337 are inactivated are significantly less virulent in a nonhuman primate model of necrotizing myositis. Our findings provide impetus for additional studies addressing the role of R28 and Spy1337 variation in pathogen-host interactions.

New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates.

A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcriptome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro, with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 (Spy0281 [dahA]), ihk-irr, slr, isp, and ciaH, that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications.IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.

Phagocytosis and neutrophil extracellular traps.

Neutrophils are recruited rapidly to sites of infection in response to host- and/or microbe-derived proinflammatory molecules. At such sites, neutrophils phagocytose microbes and are activated to produce superoxide and other reactive oxygen species (ROS). In addition, neutrophils contain stores of antimicrobial peptides and enzymes that work in concert with ROS to kill ingested microbes. Neutrophils can also release chromosomal DNA bound with antimicrobial peptides and enzymes to form web-like structures known as extracellular traps. Neutrophil extracellular traps (NETs) have been reported to ensnare and kill microbes and are commonly considered to be an important component of innate host defense. Notably, the formation of NETs is most often reported as a cytolytic process. Whereas intraphagosomal killing of microbes sequesters cytotoxic antimicrobial molecules that would otherwise damage host tissues, the formation of NETs and associated extracellular release of these molecules can contribute to host tissue destruction and disease. Here we compare and contrast phagocytosis and NETs in host defense, with emphasis on recent studies of NETs that ultimately underscore the importance of phagocytosis as the primary means by which neutrophils eliminate microbes.

Neutrophils in innate immunity and systems biology-level approaches.

The innate immune system is the first line of host defense against invading microorganisms. Polymorphonuclear leukocytes (PMNs or neutrophils) are the most abundant leukocyte in humans and essential to the innate immune response against invading pathogens. Compared to the acquired immune response, which requires time to develop and is dependent on previous interaction with specific microbes, the ability of neutrophils to kill microorganisms is immediate, nonspecific, and not dependent on previous exposure to microorganisms. Historically, studies of PMN-pathogen interaction focused on the events leading to killing of microorganisms, such as recruitment/chemotaxis, transmigration, phagocytosis, and activation, whereas postphagocytosis sequelae were infrequently considered. In addition, it was widely accepted that human neutrophils possessed limited capacity for new gene transcription and thus, relatively little biosynthetic capacity. This notion has changed dramatically within the past 20 years. Further, there is now more effort directed to understand the events occurring in PMNs after killing of microbes. Herein, we give an updated review of the systems biology-level approaches that have been used to gain an enhanced view of the role of neutrophils during host-pathogen interaction and neutrophil-mediated diseases. We anticipate that these and future systems-level studies will continue to provide information important for understanding, treatment, and control of diseases caused by pathogenic microorganisms. This article is categorized under: Physiology > Organismal Responses to Environment Physiology > Mammalian Physiology in Health and Disease Biological Mechanisms > Cell Fates.

The Role of Neutrophils in the Immune System: An Overview.

Neutrophils, also known as polymorphonuclear neutrophils (PMNs), have long been considered as the short-lived, nonspecific white cells that form pus-and also happen to kill invading microbes. Indeed, neutrophils were often neglected (and largely not considered) as immune cells. This historic view of neutrophils has changed considerably over the past several decades, and we now know that in addition to playing the predominant role in the clearance of bacteria and fungi, they have a major role in shaping the host response to infection and immune system homeostasis. The change in our view of the role of neutrophils in the immune system has been due in large part to the study of these cells in vitro. Such work has been made possible by new and/or improved methods and approaches used to investigate neutrophils. These methods are the focus of this volume.

Isolation of Neutrophils from Nonhuman Species.

The development of new advances in understanding the role of neutrophils in inflammation requires effective procedures for isolating and purifying neutrophils. Methods for isolating human neutrophils are fairly standard, and some are covered in other chapters of this volume and previous editions. However, procedures for isolating neutrophils from nonhuman species used to model human diseases vary from those used in isolating human neutrophils and are not as well developed. Since neutrophils are highly reactive and sensitive to small perturbations, the methods of isolation are important to avoid isolation technique-induced alterations in cell function. We present methods here for reproducibly isolating highly purified neutrophils from large animal models (bovine, equine, ovine), small animal models (murine and rabbit), and nonhuman primates (cynomolgus macaques) and describe optimized details for obtaining the highest cell purity, yield, and viability.

Genome-Scale Transcript Analyses of Human Neutrophils.

Transcriptome analyses of unicellular and multicellular organisms have changed fundamental understanding of biological and pathological processes across multiple scientific disciplines. Over the past 15 years, studies of polymorphonuclear leukocyte (PMN or neutrophil) gene expression on a global scale have provided new insight into the molecular processes that promote resolution of infections in humans. Herein we present methods to analyze gene expression in human neutrophils using Affymetrix oligonucleotide microarrays and next-generation sequencing. Notably, the procedures utilize commercially available reagents and materials and thus represent a standardized approach for evaluating PMN transcript levels.

Vaccine Protection against Multidrug-Resistant Klebsiella pneumoniae in a Nonhuman Primate Model of Severe Lower Respiratory Tract Infection.

Klebsiella pneumoniae is a human gut communal organism and notorious opportunistic pathogen. The relative high burden of asymptomatic colonization by K. pneumoniae is often compounded by multidrug resistance-a potential problem for individuals with significant comorbidities or other risk factors for infection. A carbapenem-resistant K. pneumoniae strain classified as multilocus sequence type 258 (ST258) is widespread in the United States and is usually multidrug resistant. Thus, treatment of ST258 infections is often difficult. Inasmuch as new preventive and/or therapeutic measures are needed for treatment of such infections, we developed an ST258 pneumonia model in cynomolgus macaques and tested the ability of an ST258 capsule polysaccharide type 2 (CPS2) vaccine to moderate disease severity. Compared with sham-vaccinated animals, those vaccinated with ST258 CPS2 had significantly less disease as assessed by radiography 24 h after intrabronchial installation of 108 CFU of ST258. All macaques vaccinated with CPS2 ultimately developed ST258-specific antibodies that significantly enhanced serum bactericidal activity and killing of ST258 by macaque neutrophils ex vivo Consistent with a protective immune response to CPS2, transcripts encoding inflammatory mediators were increased in infected lung tissues obtained from CPS-vaccinated animals compared with control, sham-vaccinated macaques. Taken together, our data provide support for the idea that vaccination with ST258 CPS can be used to prevent or moderate infections caused by ST258. As with studies performed decades earlier, we propose that this prime-boost vaccination approach can be extended to include multiple capsule types.IMPORTANCE Multidrug-resistant bacteria continue to be a major problem worldwide, especially among individuals with significant comorbidities and other risk factors for infection. K. pneumoniae is among the leading causes of health care-associated infections, and the organism is often resistant to multiple classes of antibiotics. A carbapenem-resistant K. pneumoniae strain known as multilocus sequence type 258 (ST258) is the predominant carbapenem-resistant Enterobacteriaceae in the health care setting in the United States. Infections caused by ST258 are often difficult to treat and new prophylactic measures and therapeutic approaches are needed. To that end, we developed a lower respiratory tract infection model in cynomolgus macaques in which to test the ability of ST258 CPS to protect against severe ST258 infection.

Klebsiella pneumoniae capsule polysaccharide as a target for therapeutics and vaccines.

Carbapenem-resistant (CR) Klebsiella pneumoniae has emerged as an urgent public health threat in many industrialized countries worldwide, including the United States. Infections caused by CR K. pneumoniae are difficult to treat because these organisms are typically resistant to multiple antibiotics, and the patients have significant comorbidities. Notably, there is high (∼50%) mortality among individuals with bacteremia caused by CR K. pneumoniae. Given the dearth of new antibiotics, and the recent convergence of multidrug resistance and hypervirulence, there is a critical need for alternative strategies for the treatment of CR K. pneumoniae infections. The capsule polysaccharide (CPS) of K. pneumoniae has long been viewed as an important virulence factor that promotes resistance to phagocytosis and serum bactericidal activity. Thus, the CPS has been targeted previously for the development of therapeutics and vaccines, although there is no licensed CPS-based vaccine or therapy for the treatment of CR K. pneumoniae infections. Here, we discuss immunoprophylactic and immunotherapeutic approaches that have been tested previously for the treatment of Klebsiella infections. We also suggest potential strategies to promote development of CPS-based vaccines and therapies for prevention and treatment of CR K. pneumoniae infections.

Mouse Model of Staphylococcus aureus Skin Infection.

Bacterial skin and soft tissue infections are abundant worldwide, and many are caused by Staphylococcus aureus. Indeed, S. aureus is the leading cause of skin and soft tissue infections in the USA. Here we describe a mouse model of skin and soft tissue infection induced by subcutaneous inoculation of S. aureus. This animal model can be used to investigate a number of factors related to the pathogenesis of skin and soft tissue infections, including strain virulence and the contribution of specific bacterial molecules to disease, and it can be employed to test the potential effectiveness of antibiotic therapies or vaccine candidates.