Michelle L. Kirtley

Pathogenesis of Y. enterocolitica and Y. pseudotuberculosis in Human Yersiniosis

Yersiniosis is a food-borne illness that has become more prevalent in recent years due to human transmission via the fecal-oral route and prevalence in farm animals. Yersiniosis is primarily caused by Yersinia enterocolitica and less frequently by Yersinia pseudotuberculosis. Infection is usually characterized by a self-limiting acute infection beginning in the intestine and spreading to the mesenteric lymph nodes. However, more serious infections and chronic conditions can also occur, particularly in immunocompromised individuals. Y. enterocolitica and Y. pseudotuberculosis are both heterogeneous organisms that vary considerably in their degrees of pathogenicity, although some generalizations can be ascribed to pathogenic variants. Adhesion molecules and a type III secretion system are critical for the establishment and progression of infection. Additionally, host innate and adaptive immune responses are both required for yersiniae clearance. Despite the ubiquity of enteric Yersinia species and their association as important causes of food poisoning world-wide, few national enteric pathogen surveillance programs include the yersiniae as notifiable pathogens. Moreover, no standard exists whereby identification and reporting systems can be effectively compared and global trends developed. This review discusses yersinial virulence factors, mechanisms of infection, and host responses in addition to the current state of surveillance, detection, and prevention of yersiniosis.

Characterization of Aeromonas hydrophila Wound Pathotypes by Comparative Genomic and Functional Analyses of Virulence Genes

ABSTRACT Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966T, and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966T. The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966T and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity. IMPORTANCE Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms. Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms.

Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection

ABSTRACT Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-κB activation. ATII cells pretreated with an NF-κB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis.

Progress on plague vaccine development

Yersinia pestis (YP), the gram-negative plague bacterium, has shaped human history unlike any other pathogen known to mankind. YP (transmitted by the bite of an infected flea) diverged only recently from the related enteric pathogen Yersinia pseudotuberculosis but causes radically different diseases. Three forms of plague exist in humans: bubonic (swollen lymph nodes or bubos), septicemic (spread of YP through the lymphatics or bloodstream from the bubos to other organs), and contagious, pneumonic plague which can be communicated via YP-charged respiratory droplets resulting in person–person transmission and rapid death if left untreated (50–90% mortality). Despite the potential threat of weaponized YP being employed in bioterrorism and YP infections remaining prevalent in endemic regions of the world where rodent populations are high (including the four corner regions of the USA), an efficacious vaccine that confers immunoprotection has yet to be developed. This review article will describe the current vaccine candidates being evaluated in various model systems and provide an overall summary on the progress of this important endeavor.

Levofloxacin Rescues Mice from Lethal Intra-nasal Infections with Virulent Francisella tularensis and Induces Immunity and Production of Protective Antibody

The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis.

Role of Hcp, a type 6 secretion system effector, of Aeromonas hydrophila in modulating activation of host immune cells

Recently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-β, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.

Characterization of an F1 Deletion Mutant of Yersinia pestis CO92, Pathogenic Role of F1 Antigen in Bubonic and Pneumonic Plague, and Evaluation of Sensitivity and Specificity of F1 Antigen Capture-Based Dipsticks

ABSTRACT We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37°C, although the mutants growth dropped slightly during the late phase at 37°C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 μg/ml of purified F1 antigen or 1 × 105 to 5 × 105 CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 108 CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis

Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

Immunomodulatory and Protective Roles of Quorum-Sensing Signaling Molecules N-Acyl Homoserine Lactones during Infection of Mice with Aeromonas hydrophila

ABSTRACT Aeromonas hydrophila leads to both intestinal and extraintestinal infections in animals and humans, and the underlying mechanisms leading to mortality are largely unknown. By using a septicemic mouse model of infection, we showed that animals challenged with A. hydrophila die because of kidney and liver damage, hypoglycemia, and thrombocytopenia. Pretreatment of animals with quorum-sensing-associated signaling molecules N-acyl homoserine lactones (AHLs), such as butanoyl and hexanoyl homoserine lactones (C4- and C6-HSLs), as well as N-3-oxododecanoyl (3-oxo-C12)-HSL, prevented clinical sequelae, resulting in increased survivability of mice. Since little is known as to how different AHLs modulate the immune response during infection, we treated mice with the above AHLs prior to lethal A. hydrophila infection. When we compared results in such animals to those in controls, the treated animals exhibited a significantly reduced bacterial load in the blood and other mouse organs, as well as various levels of cytokines/chemokines. Importantly, neutrophil numbers were significantly elevated in the blood of C6-HSL-treated mice compared to those in animals given phosphate-buffered saline and then infected with the bacteria. These findings coincided with the fact that neutropenic animals were more susceptible to A. hydrophila infection than normal mice. Our data suggested that neutrophils quickly cleared bacteria by either phagocytosis or possibly another mechanism(s) during infection. In a parallel study, we indeed showed that other predominant immune cells inflicted during A. hydrophila infections, such as murine macrophages, when they were pretreated with AHLs, rapidly phagocytosed bacteria, whereas untreated cells phagocytosed fewer bacteria. This study is the first to report that AHL pretreatment modulates the innate immune response in mice and enhances their survivability during A. hydrophila infection.

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

ABSTRACT 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.