A review of pulmonary neutrophilia and insights into the key role of neutrophils in particle-induced pathogenesis in the lung from animal studies of lunar dusts and other poorly soluble dust particles.

The mechanisms of particle-induced pathogenesis in the lung remain poorly understood. Neutrophilic inflammation and oxidative stress in the lung are hallmarks of toxicity. Some investigators have postulated that oxidative stress from particle surface reactive oxygen species (psROS) on the dust produces the toxicopathology in the lungs of dust-exposed animals. This postulate was tested concurrently with the studies to elucidate the toxicity of lunar dust (LD), which is believed to contain psROS due to high-speed micrometeoroid bombardment that fractured and pulverized lunar surface regolith. Results from studies of rats intratracheally instilled (ITI) with three LDs (prepared from an Apollo-14 lunar regolith), which differed 14-fold in levels of psROS, and two toxicity reference dusts (TiO2 and quartz) indicated that psROS had no significant contribution to the dusts' toxicity in the lung. Reported here are results of further investigations by the LD toxicity study team on the toxicological role of oxidants in alveolar neutrophils that were harvested from rats in the 5-dust ITI study and from rats that were exposed to airborne LD for 4 weeks. The oxidants per neutrophils and all neutrophils increased with dose, exposure time and dust's cytotoxicity. The results suggest that alveolar neutrophils play a critical role in particle-induced injury and toxicity in the lung of dust-exposed animals. Based on these results, we propose an adverse outcome pathway (AOP) for particle-associated lung disease that centers on the crucial role of alveolar neutrophil-derived oxidant species. A critical review of the toxicology literature on particle exposure and lung disease further supports a neutrophil-centric mechanism in the pathogenesis of lung disease and may explain previously reported animal species differences in responses to poorly soluble particles. Key findings from the toxicology literature indicate that (1) after exposures to the same dust at the same amount, rats have more alveolar neutrophils than hamsters; hamsters clear more particles from their lungs, consequently contributing to fewer neutrophils and less severe lung lesions; (2) rats exposed to nano-sized TiO2 have more neutrophils and more severe lesions in their lungs than rats exposed to the same mass-concentration of micron-sized TiO2; nano-sized dust has a greater number of particles and a larger total particle-cell contact surface area than the same mass of micron-sized dust, which triggers more alveolar epithelial cells (AECs) to synthesize and release more cytokines that recruit a greater number of neutrophils leading to more severe lesions. Thus, we postulate that, during chronic dust exposure, particle-inflicted AECs persistently release cytokines, which recruit neutrophils and activate them to produce oxidants resulting in a prolonged continuous source of endogenous oxidative stress that leads to lung toxicity. This neutrophil-driven lung pathogenesis explains why dust exposure induces more severe lesions in rats than hamsters; why, on a mass-dose basis, nano-sized dusts are more toxic than the micron-sized dusts; why lung lesions progress with time; and why dose-response curves of particle toxicity exhibit a hockey stick like shape with a threshold. The neutrophil centric AOP for particle-induced lung disease has implications for risk assessment of human exposures to dust particles and environmental particulate matter.

Comparative pulmonary toxicities of lunar dusts and terrestrial dusts (TiO2 & SiO2) in rats and an assessment of the impact of particle-generated oxidants on the dusts' toxicities.

Humans will set foot on the Moon again soon. The lunar dust (LD) is potentially reactive and could pose an inhalation hazard to lunar explorers. We elucidated LD toxicity and investigated the toxicological impact of particle surface reactivity (SR) using three LDs, quartz, and TiO2. We first isolated the respirable-size-fraction of an Apollo-14 regolith and ground two coarser samples to produce fine LDs with increased SR. SR measurements of these five respirable-sized dusts, determined by their in-vitro ability to generate hydroxyl radicals (•OH), showed that ground LDs > unground LD ≥ TiO2 ≥ quartz. Rats were each intratracheally instilled with 0, 1, 2.5, or 7.5 mg of a test dust. Toxicity biomarkers and histopathology were assessed up to 13 weeks after the bolus instillation. All dusts caused dose-dependent-increases in pulmonary lesions and toxicity biomarkers. The three LDs, which possessed mineral compositions/properties similar to Arizona volcanic ash, were moderately toxic. Despite a 14-fold •OH difference among these three LDs, their toxicities were indistinguishable. Quartz produced the lowest •OH amount but showed the greatest toxicity. Our results showed no correlation between the toxicity of mineral dusts and their ability to generate free radicals. We also showed that the amounts of oxidants per neutrophil increased with doses, time and the cytotoxicity of the dusts in the lung, which supports our postulation that dust-elicited neutrophilia is the major persistent source of oxidative stress. These results and the discussion of the crucial roles of the short-lived, continuously replenished neutrophils in dust-induced pathogenesis are presented.

Stat1-Deficient Mice Are Not an Appropriate Model for Efficacy Testing of Recombinant Vesicular Stomatitis Virus-Based Filovirus Vaccines.

Stat1(-/-) mice lack a response to interferon α, ß, and γ, allowing for replication of nonadapted wild-type (wt) Ebolavirus and Marburgvirus. We sought to establish a mouse model for efficacy testing of live attenuated recombinant vesicular stomatitis virus (rVSV)-based filovirus vaccine vectors using wt Ebolavirus and Marburgvirus challenge strains. While infection of immunocompetent mice with different rVSV-based filovirus vectors did not cause disease, infection of Stat1(-/-) mice with the same vectors resulted in systemic infection and lethal outcome for the majority of tested rVSVs. Despite differences in viral loads, organ tropism was remarkably similar between rVSV filovirus vaccine vectors and rVSVwt, with the exception of the brain. In conclusion, Stat1(-/-) mice are not an appropriate immunocompromised mouse model for efficacy testing of live attenuated, replication-competent rVSV vaccine vectors.

African green monkeys recapitulate the clinical experience with replication of live attenuated pandemic influenza virus vaccine candidates.

Live attenuated cold-adapted (ca) H5N1, H7N3, H6N1, and H9N2 influenza vaccine viruses replicated in the respiratory tract of mice and ferrets, and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild-type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in phase I clinical trials, there were inconsistencies between the observations in animal models and in humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative with respect to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a nonhuman primate model. The distribution of sialic acid (SA) receptors in the respiratory tract of AGMs was similar to that in humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) virus subtypes H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently, while replication of the ca vaccine viruses was restricted to the upper respiratory tract. Interestingly, the patterns and sites of virus replication differed among the different subtypes. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3, and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than when it was delivered intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects and that AGMs may be a useful model to evaluate the replication of ca vaccine candidates. Importance: Ferrets and mice are commonly used for preclinical evaluation of influenza vaccines. However, we observed significant inconsistencies between observations in humans and in these animal models. We used African green monkeys (AGMs) as a nonhuman primate (NHP) model for a comprehensive and comparative evaluation of pairs of wild-type and pandemic live attenuated influenza virus vaccines (pLAIV) representing four subtypes of avian influenza viruses and found that pLAIVs replicate similarly in AGMs and humans and that AGMs can be useful for evaluation of the protective efficacy of pLAIV.

A Syrian golden hamster model recapitulating ebola hemorrhagic fever.

Ebola hemorrhagic fever (EHF) is a severe viral infection for which no effective treatment or vaccine is currently available. While the nonhuman primate (NHP) model is used for final evaluation of experimental vaccines and therapeutic efficacy, rodent models have been widely used in ebolavirus research because of their convenience. However, the validity of rodent models has been questioned given their low predictive value for efficacy testing of vaccines and therapeutics, a result of the inconsistent manifestation of coagulopathy seen in EHF. Here, we describe a lethal Syrian hamster model of EHF using mouse-adapted Ebola virus. Infected hamsters displayed most clinical hallmarks of EHF, including severe coagulopathy and uncontrolled host immune responses. Thus, the hamster seems to be superior to the existing rodent models, offering a better tool for understanding the critical processes in pathogenesis and providing a new model for evaluating prophylactic and postexposure interventions prior to testing in NHPs.

Pathogenesis and host response in Syrian hamsters following intranasal infection with Andes virus.

Hantavirus pulmonary syndrome (HPS), also referred to as hantavirus cardiopulmonary syndrome (HCPS), is a rare but frequently fatal disease caused by New World hantaviruses. In humans HPS is associated with severe pulmonary edema and cardiogenic shock; however, the pathogenesis of this disease remains unclear largely due to a lack of suitable animal models for the study of disease progression. In this study we monitored clinical, virological, pathophysiological parameters and host immunological responses to decipher pathological factors and events in the lethal Syrian hamster model of HPS following intranasal inoculation of Andes virus. Transcriptional profiling of the host gene responses demonstrated a suppression of innate immune responses in most organs analyzed during the early stage of infection, except for in the lung which had low level activation of several pro-inflammatory genes. During this phase Andes virus established a systemic infection in hamsters, with viral antigen readily detectable in the endothelium of the majority of tissues analyzed by 7-8 days post-inoculation. Despite wide-spread infection, histological analysis confirmed pathological abnormalities were almost exclusively found in the lungs. Immediately preceding clinical signs of disease, intense activation of pro-inflammatory and Th1/Th2 responses were observed in the lungs as well as the heart, but not in peripheral organs, suggesting that localized immune-modulations by infection is paramount to pathogenesis. Throughout the course of infection a strong suppression of regulatory T-cell responses was noted and is hypothesized to be the basis of the aberrant immune activations. The unique and comprehensive monitoring of host immune responses to hantavirus infection increases our understanding of the immuno-pathogenesis of HPS and will facilitate the development of treatment strategies targeting deleterious host immunological responses.

Animal models of ricin toxicosis.

Animal models of ricin toxicosis are necessary for testing the efficacy of therapeutic measures, as well studying the mechanisms by which ricin exerts its toxicity in intact animals. Because ricin can serve as a particularly well-characterized model of tissue damage, and the host response to that damage, studies of the mechanisms of ricin toxicity may have more general applicability. For example, our studies of the molecular mechanisms underlying the development of ricin-induced hypoglycemia may help elucidate the relationship of type II diabetes, insulin resistance, and inflammation. Studies in non-human primates are most relevant for testing and developing agents having clinical utility. But these animals are expensive and limited in quantity, and so rodents are used for most mechanistic studies.

Toxicity of lunar dust assessed in inhalation-exposed rats.

Humans will again set foot on the moon. The moon is covered by a layer of fine dust, which can pose a respiratory hazard. We investigated the pulmonary toxicity of lunar dust in rats exposed to 0, 2.1, 6.8, 20.8 and 60.6 mg/m(3) of respirable-size lunar dust for 4 weeks (6 h/day, 5 days/week); the aerosols in the nose-only exposure chambers were generated from a jet-mill ground preparation of a lunar soil collected during the Apollo 14 mission. After 4 weeks of exposure to air or lunar dust, groups of five rats were euthanized 1 day, 1 week, 4 weeks or 13 weeks after the last exposure for assessment of pulmonary toxicity. Biomarkers of toxicity assessed in bronchoalveolar fluids showed concentration-dependent changes; biomarkers that showed treatment effects were total cell and neutrophil counts, total protein concentrations and cellular enzymes (lactate dehydrogenase, glutamyl transferase and aspartate transaminase). No statistically significant differences in these biomarkers were detected between rats exposed to air and those exposed to the two low concentrations of lunar dust. Dose-dependent histopathology, including inflammation, septal thickening, fibrosis and granulomas, in the lung was observed at the two higher exposure concentrations. No lesions were detected in rats exposed to ≤6.8 mg/m(3). This 4-week exposure study in rats showed that 6.8 mg/m(3) was the highest no-observable-adverse-effect level (NOAEL). These results will be useful for assessing the health risk to humans of exposure to lunar dust, establishing human exposure limits and guiding the design of dust mitigation systems in lunar landers or habitats.

Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDS in its public health impact. The pandemic clone USA300, like other CA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), a pore-forming toxin that targets polymorphonuclear leukocytes (PMNs). PVL is thought to play a key role in the pathogenesis of necrotizing pneumonia, but data from rodent infection models are inconclusive. Rodent PMNs are less susceptible than human PMNs to PVL-induced cytolysis, whereas rabbit PMNs, like those of humans, are highly susceptible to PVL-induced cytolysis. This difference in target cell susceptibility could affect results of experimental models. Therefore, we developed a rabbit model of necrotizing pneumonia to compare the virulence of a USA300 wild-type strain with that of isogenic PVL-deletion mutant and -complemented strains. PVL enhanced the capacity of USA300 to cause severe lung necrosis, pulmonary edema, alveolar hemorrhage, hemoptysis, and death, hallmark clinical features of fatal human necrotizing pneumonia. Purified PVL instilled directly into the lung caused lung inflammation and injury by recruiting and lysing PMNs, which damage the lung by releasing cytotoxic granule contents. These findings provide insights into the mechanism of PVL-induced lung injury and inflammation and demonstrate the utility of the rabbit for studying PVL-mediated pathogenesis.

Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus.

Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.

Staphylococcus aureus leukotoxin GH promotes inflammation.

BACKGROUND: Staphylococcus aureus produces numerous molecules that facilitate survival in the host. We recently identified a novel S. aureus leukotoxin (leukotoxin GH [LukGH]) using proteomics, but its role in virulence remains unclear. Here we investigated the role of LukGH in vivo. METHODS: We tested cytotoxicity of LukGH toward polymorphonuclear leukocytes (PMNs) from mice, rabbits, monkeys, and humans. LukGH was administered to mice, rabbits, and a cynomolgus monkey by subcutaneous or intradermal injection to assess cytotoxicity or host response in vivo. The effects of LukGH in vivo were compared with those of Panton-Valentine leukocidin (PVL), a well-characterized S. aureus leukotoxin. The contribution of LukGH to S. aureus infection was tested using mouse and rabbit infection models. RESULTS: Susceptibility of PMNs to LukGH was similar between humans and cynomolgus monkeys, and was greater than that of rabbits, which in turn was greater than that of mice. LukGH or PVL caused skin inflammation in rabbits and a monkey, but deletion of neither lukGH nor lukGH and lukS/F-PV reduced severity of USA300 infections in rabbits or mice. Rather, some disease parameters (eg, rabbit abscess size) were increased following infection with a lukGH and lukS/F-PV deletion strain. CONCLUSIONS: Our findings indicate that S. aureus leukotoxins enhance the host inflammatory response and influence the outcome of infection.

Kinetics of innate immune response to Yersinia pestis after intradermal infection in a mouse model.

A hallmark of Yersinia pestis infection is a delayed inflammatory response early in infection. In this study, we use an intradermal model of infection to study early innate immune cell recruitment. Mice were injected intradermally in the ear with wild-type (WT) or attenuated Y. pestis lacking the pYV virulence plasmid (pYV(-)). The inflammatory responses in ear and draining lymph node samples were evaluated by flow cytometry and immunohistochemistry. As measured by flow cytometry, total neutrophil and macrophage recruitment to the ear in WT-infected mice did not differ from phosphate-buffered saline (PBS) controls or mice infected with pYV(-), except for a transient increase in macrophages at 6 h compared to the PBS control. Limited inflammation was apparent even in animals with high bacterial loads (10(5) to 10(6) CFU). In addition, activation of inflammatory cells was significantly reduced in WT-infected mice as measured by CD11b and major histocompatibility complex class II (MHC-II) expression. When mice infected with WT were injected 12 h later at the same intradermal site with purified LPS, Y. pestis did not prevent recruitment of neutrophils. However, significant reduction in neutrophil activation remained compared to that of PBS and pYV(-) controls. Immunohistochemistry revealed qualitative differences in neutrophil recruitment to the skin and draining lymph node, with WT-infected mice producing a diffuse inflammatory response. In contrast, focal sites of neutrophil recruitment were sustained through 48 h postinfection in pYV(-)-infected mice. Thus, an important feature of Y. pestis infection is reduced activation and organization of inflammatory cells that is at least partially dependent on the pYV virulence plasmid.

Comparative analysis of USA300 virulence determinants in a rabbit model of skin and soft tissue infection.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are frequently associated with strains harboring genes encoding Panton-Valentine leukocidin (PVL). The role of PVL in the success of the epidemic CA-MRSA strain USA300 remains unknown. Here we developed a skin and soft tissue infection model in rabbits to test the hypothesis that PVL contributes to USA300 pathogenesis and compare it with well-established virulence determinants: alpha-hemolysin (Hla), phenol-soluble modulin-alpha peptides (PSMα), and accessory gene regulator (Agr). The data indicate that Hla, PSMα, and Agr contribute to the pathogenesis of USA300 skin infections in rabbits, whereas a role for PVL could not be detected.

Role of the Yersinia pestis Ail protein in preventing a protective polymorphonuclear leukocyte response during bubonic plague.

The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node.

Lack of a major role of Staphylococcus aureus Panton-Valentine leukocidin in lower respiratory tract infection in nonhuman primates.

Panton-Valentine leukocidin (PVL) is a two-component cytolytic toxin epidemiologically linked to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, including serious invasive infections caused by the epidemic clone referred to as strain USA300. Although PVL has long been known to be a S. aureus virulence molecule in vitro, the relative contribution of this leukotoxin to invasive CA-MRSA infections such as pneumonia remains controversial. We developed a nonhuman primate model of CA-MRSA pneumonia and used it to test the hypothesis that PVL contributes to lower respiratory tract infections caused by S. aureus strain USA300. The lower respiratory tract disease observed in this monkey model mimicked the clinical and pathological features of early mild to moderate S. aureus pneumonia in humans, including fine-structure histopathology. In this experiment using a large sample of monkeys and multiple time points of examination, no involvement of PVL in virulence could be detected. Compared with the wild-type parental USA300 strain, the isogenic PVL deletion-mutant strain caused equivalent lower respiratory tract pathology. We conclude that PVL does not contribute to lower respiratory tract infection in this nonhuman primate model of human CA-MRSA pneumonia.

TCA cycle inactivation in Staphylococcus aureus alters nitric oxide production in RAW 264.7 cells.

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts of nitric oxide (NO(•)) and an inducible nitric oxide synthase as compared to those cells exposed to wild-type bacteria. Despite the decrease in NO(•) synthesis, the expression of antigen-presentation and costimulatory molecules was similar in cells cultured with wild-type and those cultured with aconitase mutant bacteria. The data suggest that staphylococci can evade innate immune responses and potentially enhance their ability to survive in infected hosts by altering their metabolism. This may also explain the occurrence of TCA cycle mutants in clinical S. aureus isolates.

Targeting of alpha-hemolysin by active or passive immunization decreases severity of USA300 skin infection in a mouse model.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are predominantly those affecting skin and soft tissues. Although progress has been made, our knowledge of the molecules that contribute to the pathogenesis of CA-MRSA skin infections is incomplete. We tested the hypothesis that alpha-hemolysin (Hla) contributes to the severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Isogenic hla-negative (Deltahla) strains caused skin lesions in a mouse infection model that were significantly smaller than those caused by wild-type USA300 and Newman strains. Moreover, infection due to wild-type strains produced dermonecrotic skin lesions, whereas there was little or no dermonecrosis in mice infected with Deltahla strains. Passive immunization with Hla-specific antisera or active immunization with a nontoxigenic form of Hla significantly reduced the size of skin lesions caused by USA300 and prevented dermonecrosis. We conclude that Hla is a potential target for therapeutics or vaccines designed to moderate severe S. aureus skin infections.

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.

Transcriptomic and innate immune responses to Yersinia pestis in the lymph node during bubonic plague.

A delayed inflammatory response is a prominent feature of infection with Yersinia pestis, the agent of bubonic and pneumonic plague. Using a rat model of bubonic plague, we examined lymph node histopathology, transcriptome, and extracellular cytokine levels to broadly characterize the kinetics and extent of the host response to Y. pestis and how it is influenced by the Yersinia virulence plasmid (pYV). Remarkably, dissemination and multiplication of wild-type Y. pestis during the bubonic stage of disease did not induce any detectable gene expression or cytokine response by host lymph node cells in the developing bubo. Only after systemic spread had led to terminal septicemic plague was a transcriptomic response detected, which included upregulation of several cytokine, chemokine, and other immune response genes. Although an initial intracellular phase of Y. pestis infection has been postulated, a Th1-type cytokine response associated with classical activation of macrophages was not observed during the bubonic stage of disease. However, elevated levels of interleukin-17 (IL-17) were present in infected lymph nodes. In the absence of pYV, sustained recruitment to the lymph node of polymorphonuclear leukocytes (PMN, or neutrophils), the major IL-17 effector cells, correlated with clearance of infection. Thus, the ability to counteract a PMN response in the lymph node appears to be a major in vivo function of the Y. pestis virulence plasmid.

Frameshift mutations in a single novel virulence factor alter the in vivo pathogenicity of Chlamydia trachomatis for the female murine genital tract.

Chlamydia trachomatis is a human pathogen of global importance. An obstacle to studying the pathophysiology of human chlamydial disease is the lack of a suitable murine model of C. trachomatis infection. Mice are less susceptible to infection with human isolates due in part to innate mouse-specific host defense mechanisms to which human strains are sensitive. Another possible factor that influences the susceptibility of mice to infection is that human isolates are commonly cultivated in vitro prior to infection of mice; therefore, virulence genes could be lost as a consequence of negative selective pressure. We tested this hypothesis by infecting innate immunity-deficient C3H/HeJ female mice intravaginally with a human serovar D urogenital isolate that had undergone multiple in vitro passages. We observed early and late infection clearance phenotypes. Strains of each phenotype were isolated and then used to reinfect naïve mice. Following infection, the late-clearance strain was significantly more virulent. It caused unvarying infections of much longer durations with greater infectious burdens that naturally ascended to the upper genital tract, causing salpingitis. Despite contrasting in vivo virulence characteristics, the strains exhibited no differences in the results of in vitro infectivity assays or sensitivities to gamma interferon. Genome sequencing of the strains revealed mutations that localized to a single gene (CT135), implicating it as a critical virulence factor. Mutations in CT135 were not unique to serovar D but were also found in multiple oculogenital reference strains. Our findings provide new information about the pathogenomics of chlamydial infection and insights for improving murine models of infection using human strains.