Clinical presentation, antimicrobial resistance, and treatment outcomes of Aeromonas human infections: A 14-year retrospective study and comparative genomics of two isolates from fatal cases.

BACKGROUND: Aeromonas virulence may not be entirely dependent on the host immune status. Pathophysiologic determinants of disease progression and severity remain unclear. METHODS: One hundred five patients with Aeromonas infections and 112 isolates were identified, their clinical presentations and outcomes analyzed, and their antimicrobial resistance (AMR) patterns assessed. Two isolates (A and B) from fatal cases of Aeromonas dhakensis bacteremia were characterized using whole genome sequence analysis. Virulence factor- and AMR-encoding genes from these isolates were compared with a well-characterized diarrheal isolate A. dhakensis SSU, and environmental isolate A. hydrophila ATCC_7966T. RESULTS: Skin and soft tissue infections, traumatic wound infections, sepsis, burns, and intraabdominal infections were common. Diabetes, malignancy, and cirrhosis were frequent comorbidities. Male sex, age ≥ 65 years, hospitalization, burns, and intensive care were associated with complicated disease. High rates of AMR to carbapenems and piperacillin-tazobactam were found. Treatment failure was observed in 25.7% of cases. Septic shock and hospital-acquired infections were predictors of treatment failure. All four isolates harbored assorted broad-spectrum AMR genes including blaOXA, ampC, cphA, and efflux pumps. Only clinical isolates possessed both polar and lateral flagellar genes, genes for various surface adhesion proteins, type 3- and -6 secretion systems and their effectors, and toxin genes, including exotoxin A. Both isolates A and B were resistant to colistin and harbored the mobile colistin resistance-3 (mcr-3) gene. CONCLUSIONS: Empirical therapy tailored to local Aeromonas antibiograms may facilitate more favorable outcomes, while advanced diagnostic methods may aid in identifying correct Aeromonas spp. of significant clinical importance.

Antimicrobial resistance in aeromonads and new therapies targeting quorum sensing.

Aeromonas species (spp.) are well-known fish pathogens, several of which have been recognized as emerging human pathogens. The organism is capable of causing a wide spectrum of diseases in humans, ranging from gastroenteritis, wound infections, and septicemia to devastating necrotizing fasciitis. The systemic form of infection is often fatal, particularly in patients with underlying chronic diseases. Indeed, recent trends demonstrate rising numbers of hospital-acquired Aeromonas infections, especially in immuno-compromised individuals. Additionally, Aeromonas-associated antibiotic resistance is an increasing challenge in combating both fish and human infections. The acquisition of antibiotic resistance is related to Aeromonas' innate transformative properties including its ability to share plasmids and integron-related gene cassettes between species and with the environment. As a result, alternatives to antibiotic treatments are desperately needed. In that vein, many treatments have been proposed and studied extensively in the fish-farming industry, including treatments that target Aeromonas quorum sensing. In this review, we discuss current strategies targeting quorum sensing inhibition and propose that such studies empower the development of novel chemotherapeutic approaches to combat drug-resistant Aeromonas spp. infections in humans. KEY POINTS: • Aeromonas notoriously acquires and maintains antimicrobial resistance, making treatment options limited. • Quorum sensing is an essential virulence mechanism in Aeromonas infections. • Inhibiting quorum sensing can be an effective strategy in combating Aeromonas infections in animals and humans.

Glycopyrrolate as an Adjunct in the Management of Anastomotic Leak Following Repair of Esophageal Atresia: A Clinicoradiological Perspective.

Context: Anastomotic leak after primary repair of esophageal atresia (EA) with tracheoesophageal fistula (TEF) is a well-known complication and can represent a challenging clinical scenario. Aims: The present study aimed to evaluate the role of glycopyrrolate as an adjunct in the treatment of anastomotic leak after primary repair of EA Vogt type 3b. Settings and Design: A retrospective study was carried out in our tertiary care teaching institute from January 2015 to December 2022. Materials and Methods: Neonates with EA with distal TEF with primary repair who had developed anastomotic leak, managed by the author(s), were studied. The study included patients with major, minor, and radiological leaks. Glycopyrrolate was administered in the dose of 4 µg/kg 8 hourly. The outcomes of the study were either resolution or progression of the leak. Results: There were 21 patients who were managed with glycopyrrolate in addition to the classical treatment of the anastomotic leak following repair of EA with distal TEF. The male: female ratio was 1:1.1. All the cases had anastomotic leaks with either clinically detectable in the chest tube (15) or radiological leak (6). The leaks were detected early in patients with major leak (mean = 3.2 ± 0.84 days) compared to minor leak (mean =4.9 ± 1.29 days). Radiological leaks were detected in all the neonates on postoperative day 7. In five patients with major leak, there was a negligible reduction in the amount of chest tube output, and were subjected to diversion procedures. There were a total of three deaths out of five in this group. In 10 patients with minor leak, there was complete resolution of anastomotic leak in eight patients (80%); there was one patient each with mortality and diversion procedure. The patients with a radiological leak (6) did not show any deterioration, and they were fed 1-5 days after the esophagogram. Conclusions: Glycopyrrolate may be an advantageous postoperative adjunct in the management of minor and radiological leak after tracheoesophageal repair.

T6SS and ExoA of flesh-eating Aeromonas hydrophila in peritonitis and necrotizing fasciitis during mono- and polymicrobial infections.

An earlier report described a human case of necrotizing fasciitis (NF) caused by mixed infection with 4 Aeromonas hydrophila strains (NF1-NF4). While the NF2, NF3, and NF4 strains were clonal and possessed exotoxin A (ExoA), the NF1 strain was determined to be phylogenetically distinct, harboring a unique type 6 secretion system (T6SS) effector (TseC). During NF1 and NF2 mixed infection, only NF1 disseminated, while NF2 was rapidly killed by a contact-dependent mechanism and macrophage phagocytosis, as was demonstrated by using in vitro models. To confirm these findings, we developed 2 NF1 mutants (NF1ΔtseC and NF1ΔvasK); vasK encodes an essential T6SS structural component. NF1 VasK and TseC were proven to be involved in contact-dependent killing of NF2 in vitro, as well as in its elimination at the intramuscular injection site in vivo during mixed infection, with overall reduced mouse mortality. ExoA was shown to have an important role in NF by both NF1-exoA (with cis exoA) and NF2 during monomicrobial infection. However, the contribution of ExoA was more important for NF2 than NF1 in the murine peritonitis model. The NF2∆exoA mutant did not significantly alter animal mortality or NF1 dissemination during mixed infection in the NF model, suggesting that the ExoA activity was significant at the injection site. Immunization of mice to ExoA protected animals from NF2 monomicrobial challenge, but not from polymicrobial infection because of NF2 clearance. This study clarified the roles of T6SS and ExoA in pathogenesis caused by A. hydrophila NF strains in both mouse peritonitis and NF models in monomicrobial and polymicrobial infections.

Plague vaccines: new developments in an ongoing search.

As the reality of pandemic threats challenges humanity, exemplified during the ongoing SARS-CoV-2 infections, the development of vaccines targeting these etiological agents of disease has become increasingly critical. Of paramount concern are novel and reemerging pathogens that could trigger such events, including the plague bacterium Yersinia pestis. Y. pestis is responsible for more human deaths than any other known pathogen and exists globally in endemic regions of the world, including the four corners region and Northern California in the USA. Recent cases have been scattered throughout the world, including China and the USA, with serious outbreaks in Madagascar during 2008, 2013-2014, and, most recently, 2017-2018. This review will focus on recent advances in plague vaccine development, a seemingly necessary endeavor, as there is no Food and Drug Administration-licensed vaccine available for human distribution in western nations, and that antibiotic-resistant strains are recovered clinically or intentionally developed. Progress and recent development involving subunit, live-attenuated, and nucleic acid-based plague vaccine candidates will be discussed in this review. KEY POINTS: • Plague vaccine development remains elusive yet critical. • DNA, animal, and live-attenuated vaccine candidates gain traction.

Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

Dissemination of non-typhoidal Salmonella during Plasmodium chabaudi infection affects anti-malarial immunity.

Malaria-associated bacteremia accounts for up to one-third of deaths from severe malaria, and non-typhoidal Salmonella (NTS) has been reported as a major complication of severe malarial infection. Patients who develop NTS bacteremia during Plasmodium infection show higher mortality rates than individuals with malaria alone. Systemic bacteremia can be caused by a wound or translocation from epithelial or endothelial sites. NTS is an intestinal pathogen, however the contribution of bacterial translocation from the intestinal tract during Plasmodium infection is not well studied. Here, we investigated the integrity of the intestinal barrier function of P. chabaudi-infected mice using large molecules and Salmonella infection. Intestinal histology and the adaptive immune response to malaria were also studied using light microscopy and flow cytometry. P. chabaudi infection compromised intestinal barrier function, which led to increased intestinal cellular infiltration. In addition, we observed increased serum lipopolysaccharide binding protein and leakage of soluble molecules from the intestine into the blood in infected mice. Plasmodium infection also increased intestinal translocation and dissemination of NTS to the liver. The adaptive immune response to P. chabaudi infection was also significantly impacted by NTS translocation. Reduced B and T cell activation were observed in co-infected animals, suggesting interference in the malaria-specific immune responses by bacteremia. These studies demonstrate that P. chabaudi infection induces failure of the barrier function of the intestinal wall and enhanced intestinal bacterial translocation, affecting anti-malarial immunity.

Combating Multidrug-Resistant Pathogens with Host-Directed Nonantibiotic Therapeutics.

Earlier, we reported that three Food and Drug Administration-approved drugs, trifluoperazine (TFP; an antipsychotic), amoxapine (AXPN; an antidepressant), and doxapram (DXP; a breathing stimulant), identified from an in vitro murine macrophage cytotoxicity screen, provided mice with 40 to 60% protection against pneumonic plague when administered at the time of infection for 1 to 3 days. In the present study, the therapeutic potential of these drugs against pneumonic plague in mice was further evaluated when they were administered at up to 48 h postinfection. While the efficacy of TFP was somewhat diminished as treatment was delayed to 24 h, the protection of mice with AXPN and DXP increased as treatment was progressively delayed to 24 h. At 48 h postinfection, these drugs provided the animals with significant protection (up to 100%) against challenge with the agent of pneumonic or bubonic plague when they were administered in combination with levofloxacin. Likewise, when they were used in combination with vancomycin, all three drugs provided mice with 80 to 100% protection from fatal oral Clostridium difficile infection when they were administered at 24 h postinfection. Furthermore, AXPN provided 40 to 60% protection against respiratory infection with Klebsiella pneumoniae when it was administered at the time of infection or at 24 h postinfection. Using the same in vitro cytotoxicity assay, we identified an additional 76/780 nonantibiotic drugs effective against K. pneumoniae For Acinetobacter baumannii, 121 nonantibiotic drugs were identified to inhibit bacterium-induced cytotoxicity in murine macrophages. Of these 121 drugs, 13 inhibited the macrophage cytotoxicity induced by two additional multiple-antibiotic-resistant strains. Six of these drugs decreased the intracellular survival of all three A. baumannii strains in macrophages. These results provided further evidence of the broad applicability and utilization of drug repurposing screening to identify new therapeutics to combat multidrug-resistant pathogens of public health concern.

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

New Role for FDA-Approved Drugs in Combating Antibiotic-Resistant Bacteria.

Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.7 murine macrophages resistant to cytotoxicity induced by the highly virulent Yersinia pestis CO92 strain. Of these compounds, we identified 94 not classified as antibiotics as being effective at preventing Y. pestis-induced cytotoxicity. A total of 17 prioritized drugs, based on efficacy in in vitro screens, were chosen for further evaluation in a murine model of pneumonic plague to delineate if in vitro efficacy could be translated in vivo Three drugs, doxapram (DXP), amoxapine (AXPN), and trifluoperazine (TFP), increased animal survivability despite not exhibiting any direct bacteriostatic or bactericidal effect on Y. pestis and having no modulating effect on crucial Y. pestis virulence factors. These findings suggested that DXP, AXPN, and TFP may modulate host cell pathways necessary for disease pathogenesis. Finally, to further assess the broad applicability of drugs identified from in vitro screens, the therapeutic potential of TFP, the most efficacious drug in vivo, was evaluated in murine models of Salmonella enterica serovar Typhimurium and Clostridium difficile infections. In both models, TFP treatment resulted in increased survivability of infected animals. Taken together, these results demonstrate the broad applicability and potential use of nonantibiotic FDA-approved drugs to combat respiratory and gastrointestinal bacterial pathogens.

In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine.

The bacteriophage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56-µm-long, 170-kb viral genome into 120 nm × 86 nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, highly antigenic outer capsid protein (Hoc) and small outer capsid protein (Soc). T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein in the T4 head elicited robust antibody and cellular immune responses. This "progene delivery" approach might lead to new types of vaccines and genetic therapies.

High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection.

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD50. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD50 were 90% protected upon subsequent challenge with 12 LD50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s).

Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection.

Mutated and bacteriophage T4 nanoparticle arrayed F1-V immunogens from Yersinia pestis as next generation plague vaccines.

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal ß-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the ß-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.

Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate.

Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.

Functional genomic characterization of virulence factors from necrotizing fasciitis-causing strains of Aeromonas hydrophila.

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF.

Low-shear force associated with modeled microgravity and spaceflight does not similarly impact the virulence of notable bacterial pathogens.

As their environments change, microbes experience various threats and stressors, and in the hypercompetitive microbial world, dynamism and the ability to rapidly respond to such changes allow microbes to outcompete their nutrient-seeking neighbors. Viewed in that light, the very difference between microbial life and death depends on effective stress response mechanisms. In addition to the more commonly studied temperature, nutritional, and chemical stressors, research has begun to characterize microbial responses to physical stress, namely low-shear stress. In fact, microbial responses to low-shear modeled microgravity (LSMMG), which emulates the microgravity experienced in space, have been studied quite widely in both prokaryotes and eukaryotes. Interestingly, LSMMG-induced changes in the virulence potential of several Gram-negative enteric bacteria, e.g., an increased enterotoxigenic Escherichia coli-mediated fluid secretion in ligated ileal loops of mice, an increased adherent invasive E. coli-mediated infectivity of Caco-2 cells, an increased Salmonella typhimurium-mediated invasion of both epithelial and macrophage cells, and S. typhimurium hypervirulence phenotype in BALB/c mice when infected by the intraperitoneal route. Although these were some examples where virulence of the bacteria was increased, there are instances where organisms became less virulent under LSMMG, e.g., hypovirulence of Yersinia pestis in cell culture infections and hypovirulence of methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, and Listeria monocytogenes in a Caenorhabditis elegans infection model. In general, a number of LSMMG-exposed bacteria (but not all) seemed better equipped to handle subsequent stressors such as osmotic shock, acid shock, heat shock, and exposure to chemotherapeutics. This mini-review primarily discusses both LSMMG-induced as well as bona fide spaceflight-specific alterations in bacterial virulence potential, demonstrating that pathogens' responses to low-shear forces vary dramatically. Ultimately, a careful characterization of numerous bacterial pathogens' responses to low-shear forces is necessary to evaluate a more complete picture of how this physical stress impacts bacterial virulence since a "one-size-fits-all" response is clearly not the case.

Bacteriophage T4 as a Protein-Based, Adjuvant- and Needle-Free, Mucosal Pandemic Vaccine Design Platform.

The COVID-19 pandemic has transformed vaccinology. Rapid deployment of mRNA vaccines has saved countless lives. However, these platforms have inherent limitations including lack of durability of immune responses and mucosal immunity, high cost, and thermal instability. These and uncertainties about the nature of future pandemics underscore the need for exploring next-generation vaccine platforms. Here, we present a novel protein-based, bacteriophage T4 platform for rapid design of efficacious vaccines against bacterial and viral pathogens. Full-length antigens can be displayed at high density on a 120 × 86 nm phage capsid through nonessential capsid binding proteins Soc and Hoc. Such nanoparticles, without any adjuvant, induce robust humoral, cellular, and mucosal responses when administered intranasally and confer sterilizing immunity. Combined with structural stability and ease of manufacture, T4 phage provides an excellent needle-free, mucosal pandemic vaccine platform and allows equitable vaccine access to low- and middle-income communities across the globe.