Klebsiella pneumoniae: adaptive immune landscapes and vaccine horizons.

Klebsiella pneumoniae is among the most common antibiotic-resistant pathogens causing nosocomial infections. Additionally, it is a leading cause of neonatal sepsis and childhood mortality across the globe. Despite its clinical importance, we are only beginning to understand how the mammalian adaptive immune system responds to this pathogen. Further, many studies investigating potential K. pneumoniae vaccine candidates or alternative therapies have been launched in recent years. Here, we review the current state of knowledge on the adaptive immune response to K. pneumoniae infections and progress towards developing vaccines and other therapies to combat these infections.

Replicative Acinetobacter baumannii strains interfere with phagosomal maturation by modulating the vacuolar pH.

Bacterial pneumonia is a common infection of the lower respiratory tract that can afflict patients of all ages. Multidrug-resistant strains of Acinetobacter baumannii are increasingly responsible for causing nosocomial pneumonias, thus posing an urgent threat. Alveolar macrophages play a critical role in overcoming respiratory infections caused by this pathogen. Recently, we and others have shown that new clinical isolates of A. baumannii, but not the common lab strain ATCC 19606 (19606), can persist and replicate in macrophages within spacious vacuoles that we called Acinetobacter Containing Vacuoles (ACV). In this work, we demonstrate that the modern A. baumannii clinical isolate 398, but not the lab strain 19606, can infect alveolar macrophages and produce ACVs in vivo in a murine pneumonia model. Both strains initially interact with the macrophage endocytic pathway, as indicated by EEA1 and LAMP1 markers; however, the fate of these strains diverges at a later stage. While 19606 is eliminated in an autophagy pathway, 398 replicates in ACVs and are not degraded. We show that 398 reverts the natural acidification of the phagosome by secreting large amounts of ammonia, a by-product of amino acid catabolism. We propose that this ability to survive within macrophages may be critical for the persistence of clinical A. baumannii isolates in the lung during a respiratory infection.

Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae.

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.

A heptavalent O-antigen bioconjugate vaccine exhibits differential functional antibody responses against diverse Klebsiella pneumoniae isolates.

Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat due to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting seven predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Further, sera from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with O-antigen antibodies' ability to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates.

Discovery and characterization of a new class of O-linking oligosaccharyltransferases from the Moraxellaceae family.

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

Maintaining control: metabolism of molting Arctic seals in water and when hauled out.

Seals haul out of water for extended periods during the annual molt, when they shed and regrow their pelage. This behavior is believed to limit heat loss to the environment given increased peripheral blood flow to support tissue regeneration. The degree to which time in water, particularly during the molt, may affect thermoregulatory costs is poorly understood. We measured the resting metabolism of three spotted seals (Phoca largha), one ringed seal (Pusa hispida) and one bearded seal (Erignathus barbatus) during and outside the molting period, while resting in water and when hauled out. Metabolic rates were elevated in spotted and ringed seals during molt, but comparable in water and air for individuals of all species, regardless of molt status. Our data indicate that elevated metabolism during molt primarily reflects the cost of tissue regeneration, while increased haul out behavior is driven by the need to maintain elevated skin temperatures to support tissue regeneration.

Murine Respiratory Tract Infection with Classical Klebsiella pneumoniae Induces Bronchus-Associated Lymphoid Tissue.

Klebsiella pneumoniae is a Gram-negative, opportunistic pathogen that commonly causes nosocomial pneumonia, urinary tract infection, and septicemia. Our recent work utilizing a murine model of respiratory tract infection with classical K. pneumoniae demonstrated leukocyte aggregates in the lungs of mice at 28 days postinfection. Here, we sought to characterize the composition and development of these structures. Histopathological analyses of murine lungs revealed immune cell clusters surrounding the pulmonary vasculature and airways by 14 days postinfection, resembling inducible bronchus-associated lymphoid tissue (iBALT). Further investigation of these structures demonstrated central B cell aggregates with concomitant dispersed T cells. At day 28 postinfection, these lymphoid clusters expressed germinal center markers and CXCL12, qualifying these structures as iBALT with nonclassical B cell follicles. Investigations in mutant mice revealed that those lacking B and/or T cells were not able to form fully defined iBALT structures, although some rudimentary B cell clusters were identified in mice lacking T cells. The longevity of K. pneumoniae-induced BALT was assessed for up to 120 days postinfection. Lymphoid aggregates significantly decreased in size and quantity by 90 days after K. pneumoniae infection; however, aggregates persisted in mice that were restimulated with K. pneumoniae every 30 days. Finally, infections of mice with an array of classical K. pneumoniae clinical isolates demonstrated that the development of these structures is a common feature of K. pneumoniae lung infection. Together, these data confirm that murine lungs infected with K. pneumoniae develop iBALT, which may play a role in pulmonary immunity to this troublesome pathogen.

A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) is globally disseminating as a community-acquired pathogen causing life-threatening infections in healthy individuals. The fact that a dose as little as 50 bacteria is lethal to mice illustrates the dramatic increase of virulence associated with hvKp strains compared with classical K. pneumoniae (cKp) strains, which require lethal doses greater than 107 bacteria. Until recently, these virulent strains were mostly antibiotic-susceptible. However, multidrug-resistant (MDR) hvKp strains have been emerging, spawning a new generation of hypervirulent "superbugs." The mechanisms of hypervirulence are not fully defined, but overproduction of capsular polysaccharide significantly impedes host clearance, resulting in increased pathogenicity of hvKp strains. While there are more than 80 serotypes of K. pneumoniae, the K1 and K2 serotypes cause the vast majority of hypervirulent infections. Therefore, a glycoconjugate vaccine targeting these 2 serotypes could significantly reduce hvKp infection. Conventionally, glycoconjugate vaccines are manufactured using intricate chemical methodologies to covalently attach purified polysaccharides to carrier proteins, which is widely considered to be technically challenging. Here we report on the recombinant production and analytical characterization of bioconjugate vaccines, enzymatically produced in glycoengineered Escherichia coli cells, against the 2 predominant hypervirulent K. pneumoniae serotypes, K1 and K2. The K. pneumoniae bioconjugates are immunogenic and efficacious, protecting mice against lethal infection from 2 hvKp strains, NTUH K-2044 and ATCC 43816. This preclinical study constitutes a key step toward preventing further global dissemination of hypervirulent MDR hvKp strains.

CARDIAC EXAMINATIONS OF ANESTHETIZED STELLER SEA LIONS (EUMETOPIAS JUBATUS), NORTHERN FUR SEALS (CALLORHINUS URSINUS), AND A WALRUS (ODOBENUS ROSMARUS).

Pinniped hearts have been well described via dissection, but in vivo measurements of cardiac structure, function, and electrophysiology are lacking. Electrocardiograms (ECGs) were recorded under anesthesia from eight Steller sea lions (Eumetopias jubatus), five northern fur seals (Callorhinus ursinus), and one walrus (Odobenus rosmarus) to investigate cardiac electrophysiology in pinnipeds. In addition, echocardiograms were performed on all eight anesthetized Steller sea lions to evaluate in vivo cardiac structure and function. Measured and calculated ECG parameters included P-wave, PQ, QRS, and QT interval durations, P-, R-, and T-wave amplitudes, P- and T-wave polarities, and the mean electrical axis (MEA). Measured and calculated echocardiographic parameters included left ventricular internal diameter, interventricular septum thickness, and left ventricular posterior wall thickness in systole and diastole (using M-mode), left atrium and aortic root dimensions (using 2D), and maximum aortic and pulmonary flow velocities (using pulsed-wave spectral Doppler). ECG measurements were similar to those reported for other pinniped species, but there was considerable variation in the MEAs of Steller sea lions and northern fur seals. Echocardiographic measurements were similar to those reported for southern sea lions (Otaria flavenscens), including five out of eight Steller sea lions having a left atrial to aortic root ratio <1, which may indicate that they have an enlarged aortic root compared to awake terrestrial mammals. Isoflurane anesthesia likely affected some of the measurements as evidenced by the reduced fractional shortening found in Steller sea lions compared to awake terrestrial mammals. The values reported are useful reference points for assessing cardiac health in pinnipeds under human care.

In silico discovery of small molecules that inhibit RfaH recruitment to RNA polymerase.

RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus biosynthesis operons by reducing transcription termination and activating translation. While E. coli RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of E. coli and K. pneumoniae RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP ß subunit gate loop. We focused on high-affinity RfaH interactions with the RNAP ß' subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by in silico docking using ZINC database, 3 ligands were able to inhibit E. coli RfaH recruitment in vitro. The most potent lead was active against both E. coli and K. pneumoniae RfaHs in vitro. Our results demonstrate the feasibility of identifying RfaH inhibitors using in silico docking and pave the way for rational design of antivirulence therapeutics against antibiotic-resistant pathogens.

Ureaplasma urealyticum pyelonephritis presenting with progressive dysuria, renal failure, and neurologic symptoms in an immunocompromised patient.

Ureaplasma urealyticum is a bacterial species correlated with urethritis in healthy individuals and invasive infections in immunocompromised patients. We describe a 20-year-old female with a history of remote heart transplant on everolimus, mycophenolate, and rituximab presenting with progressive urinary tract symptoms, renal failure, and neurologic symptoms. An extensive workup ultimately identified U urealyticum infection, and the patient successfully recovered after a course of azithromycin and doxycycline.

High Levels of Cyclic Di-GMP in Klebsiella pneumoniae Attenuate Virulence in the Lung.

The bacterial second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) has been shown to influence the expression of virulence factors in certain pathogenic bacteria, but little is known about its activity in the increasingly antibiotic-resistant pathogen Klebsiella pneumoniae Here, the expression in K. pneumoniae of a heterologous diguanylate cyclase increased the bacterial c-di-GMP concentration and attenuated pathogenesis in murine pneumonia. This attenuation remained evident in mice lacking the c-di-GMP sensor STING, indicating that the high c-di-GMP concentration exerted its influence not on host responses but on bacterial physiology. While serum resistance and capsule expression were unaffected by the increased c-di-GMP concentration, both type 3 and type 1 pili were strongly upregulated. Importantly, attenuation of K. pneumoniae virulence by high c-di-GMP levels was abrogated when type 1 pilus expression was silenced. We conclude that increased type 1 piliation may hamper K. pneumoniae virulence in the respiratory tract and that c-di-GMP signaling represents a potential therapeutic target for antibiotic-resistant K. pneumoniae in this niche.

REFERENCE RANGES AND AGE-RELATED AND DIVING EXERCISE EFFECTS ON HEMATOLOGY AND SERUM CHEMISTRY OF FEMALE STELLER SEA LIONS ( EUMETOPIAS JUBATUS).

Decreased health may have lowered the birth and survival rates of Steller sea lions ( Eumetopias jubatus) in the Gulf of Alaska and Aleutian Islands over the past 30 yr. Reference ranges for clinical hematology and serum chemistry parameters needed to assess the health of wild sea lion populations are limited. Here, blood parameters were serially measured in 12 captive female Steller sea lions ranging in age from 3 wk to 16 yr to establish baseline values and investigate age-related changes. Whether diving activity affects hematology parameters in animals swimming in the ocean compared with animals in a traditional aquarium setting was also examined. Almost all blood parameters measured exhibited significant changes with age. Many of the age-related changes reflected developmental life history changes, including a change in diet during weaning, an improvement of diving capacity, and the maturity of the immune system. Mean corpuscular hemoglobin and mean corpuscular volume were also higher in the ocean diving group compared with the aquarium group, likely reflecting responses to increased exercise regimes. These data provide ranges of hematology and serum chemistry values needed to evaluate and compare the health and nutritional status of captive and wild Steller sea lions.

Klebsiella pneumoniae FimK Promotes Virulence in Murine Pneumonia.

Klebsiella pneumoniae, a chief cause of nosocomial pneumonia, is a versatile and commonly multidrug-resistant human pathogen for which further insight into pathogenesis is needed. We show that the pilus regulatory gene fimK promotes the virulence of K. pneumoniae strain TOP52 in murine pneumonia. This contrasts with the attenuating effect of fimK on urinary tract virulence, illustrating that a single factor may exert opposing effects on pathogenesis in distinct host niches. Loss of fimK in TOP52 pneumonia was associated with diminished lung bacterial burden, limited innate responses within the lung, and improved host survival. FimK expression was shown to promote serum resistance, capsule production, and protection from phagocytosis by host immune cells. Finally, while the widely used K. pneumoniae model strain 43816 produces rapid dissemination and death in mice, TOP52 caused largely localized pneumonia with limited lethality, thereby providing an alternative tool for studying K. pneumoniae pathogenesis and control within the lung.

Dive, food, and exercise effects on blood microparticles in Steller sea lions (Eumetopias jubatus): exploring a biomarker for decompression sickness.

Recent studies of stranded marine mammals indicate that exposure to underwater military sonar may induce pathophysiological responses consistent with decompression sickness (DCS). However, DCS has been difficult to diagnose in marine mammals. We investigated whether blood microparticles (MPs, measured as number/µl plasma), which increase in response to decompression stress in terrestrial mammals, are a suitable biomarker for DCS in marine mammals. We obtained blood samples from trained Steller sea lions (Eumetopias jubatus, 4 adult females) wearing time-depth recorders that dove to predetermined depths (either 5 or 50 meters). We hypothesized that MPs would be positively related to decompression stress (depth and duration underwater). We also tested the effect of feeding and exercise in isolation on MPs using the same blood sampling protocol. We found that feeding and exercise had no effect on blood MP levels, but that diving caused MPs to increase. However, blood MP levels did not correlate with diving depth, relative time underwater, and presumed decompression stress, possibly indicating acclimation following repeated exposure to depth.

Feeding kinematics and performance of basal otariid pinnipeds, Steller sea lions and northern fur seals: implications for the evolution of mammalian feeding.

Feeding performance studies can address questions relevant to feeding ecology and evolution. Our current understanding of feeding mechanisms for aquatic mammals is poor. Therefore, we characterized the feeding kinematics and performance of five Steller sea lions (Eumetopias jubatus) and six northern fur seals (Callorhinus ursinus). We tested the hypotheses that both species use suction as their primary feeding mode, and that rapid jaw opening was related to suction generation. Steller sea lions used suction as their primary feeding mode, but also used a biting feeding mode. In contrast, northern fur seals only used a biting feeding mode. Kinematic profiles of Steller sea lions were all indicative of suction feeding (i.e. a small gape, small gape angle, large depression of the hyolingual apparatus and lip pursing). However, jaw opening as measured by gape angle opening velocity (GAOV) was relatively slow in Steller sea lions. In contrast to Steller sea lions, the GAOV of northern fur seals was extremely fast, but their kinematic profiles indicated a biting feeding mode (i.e. northern fur seals exhibited a greater gape, a greater gape angle and minimal depression of the hyolingual apparatus compared with Steller sea lions). Steller sea lions produced both subambient and suprambient pressures at 45 kPa. In contrast, northern fur seals produced no detectable pressure measurements. Steller sea lions have a broader feeding repertoire than northern fur seals, which likely enables them to feed on a greater variety of prey, in more diverse habitats. Based on the basal phylogenetic position of northern fur seals, craniodental morphological data of the Callorhinus lineage, and the performance data provided in this study, we suggest that northern fur seals may be exhibiting their ancestral feeding mode.

Safety and efficacy of sodium nitroprusside during prolonged infusion in pediatric patients.

OBJECTIVE: Sodium nitroprusside is a direct-acting vasodilator used to lower blood pressure in the operating room and ICU. The efficacy of sodium nitroprusside has been analyzed in few pediatric randomized trials. This study assesses the efficacy and safety of sodium nitroprusside following at least 12 hours of IV infusion in children. DESIGN: Randomized, double-blind withdrawal to placebo study. SETTING: ICUs. PATIENTS: Pediatric patients younger than 17 years. INTERVENTIONS: Following 12-24 hours of open-label sodium nitroprusside titration, a blinded infusion of sodium nitroprusside or placebo was administered (at the stable rate used at the end of the open-label phase) for up to 30 minutes. MEASUREMENTS AND MAIN RESULTS: The primary efficacy measure was whether control of mean arterial blood pressure was lost, that is, increased above ambient baseline for two consecutive minutes during the blinded phase. The proportion of patients who lost mean arterial blood pressure control in the placebo group (15/19; 79%) was significantly different than those in the sodium nitroprusside group (9/20; 45%) (p = 0.048). Three patients experienced rebound hypertension during the blinded phase, and all were in the placebo group. Serious adverse event rates were low (7/52; 13%), and in only one patient was the serious adverse event determined to be related to sodium nitroprusside by the site investigator. Fourteen patients (27%) had whole blood cyanide levels above 0.5 µg/mL, with high correlation (0.7) between infusion rate and cyanide levels, but there were few clinical signs of cyanide toxicity. CONCLUSIONS: Sodium nitroprusside is efficacious in maintaining mean arterial blood pressure control in children following a 12-hour infusion. Although a high proportion of patients were found to have elevated cyanide levels, toxicity was not observed.