Correction: Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

[This corrects the article DOI: 10.1371/journal.pone.0171363.].

Incorporating germination-induction into decontamination strategies for bacterial spores.

Bacterial spores resist environmental extremes and protect key spore macromolecules until more supportive conditions arise. Spores germinate upon sensing specific molecules, such as nutrients. Germination is regulated by specialized mechanisms or structural features of the spore that limit contact with germinants and enzymes that regulate germination. Importantly, germination renders spores more susceptible to inactivating processes such as heat, desiccation, and ultraviolet radiation, to which they are normally refractory. Thus, germination can be intentionally induced through a process called germination-induction and subsequent treatment of these germinated spores with common disinfectants or gentle heat will inactivate them. However, while the principle of germination-induction has been shown effective in the laboratory, this strategy has not yet been fully implemented in real-word scenarios. Here, we briefly review the mechanisms of bacterial spore germination and discuss the evolution of germination-induction as a decontamination strategy. Finally, we examine progress towards implementing germination-induction in three contexts: biodefense, hospital settings and food manufacture. SIGNIFICANCE AND IMPACT: This article reviews implementation of germination-induction as part of a decontamination strategy for the cleanup of bacterial spores. To our knowledge this is the first time that germination-induction studies have been reviewed in this context. This article will provide a resource which summarizes the mechanisms of germination in Clostridia and Bacillus species, challenges and successes in germination-induction, and potential areas where this strategy may be implemented.

Comparison of sampling methods to recover germinated Bacillus anthracis and Bacillus thuringiensis endospores from surface coupons.

AIMS: In an attempt to devise decontamination methods that are both effective and minimally detrimental to the environment, we evaluated germination induction as an enhancement to strategies for Bacillus anthracis spore decontamination. To determine an optimal method for the recovery of germinating spores from different matrices, it was critical to ensure that the sampling procedures did not negatively impact the viability of the germinating spores possibly confounding the results and downstream analyses of field trial data. METHODS AND RESULTS: Therefore, the two main objectives of this study were the following: (i) development of an effective processing protocol capable of recovering the maximum number of viable germinating or germinated spores from different surface materials; and (ii) using a model system of spore contamination, employ this protocol to evaluate the potential applicability of germination induction to wide-area decontamination of B. anthracis spores. We examined parameters affecting the sampling efficiencies of B. anthracis and the surrogate species Bacillus thuringiensis on nonporous and porous materials. CONCLUSIONS: The most efficient extraction from all matrices was observed using PBS with 0·01% Tween 80 extraction buffer. The addition of a sonication and/or extended vortex treatment did not yield significant increases in spore or germinated spore recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data demonstrate that previous germination-induction experiments performed in suspension can be reproduced when Bacillus spores are deposited onto reference surfaces materials. Our proof of concept experiment illustrated that a germination pretreatment step significantly improves conventional secondary decontamination strategies and remediation plans.

Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated "Smooth" and "Rough", under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants' genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a Bp strain will be leveraged to better understand the mechanism of Bp phenotypic variability and to possibly identify in vitro markers of infection.

Characterization of in vitro phenotypes of Burkholderia pseudomallei and Burkholderia mallei strains potentially associated with persistent infection in mice.

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the agents of melioidosis and glanders, respectively, are Tier 1 biothreats. They infect humans and animals, causing disease ranging from acute and fatal to protracted and chronic. Chronic infections are especially challenging to treat, and the identification of in vitro phenotypic markers which signal progression from acute to persistent infection would be extremely valuable. First, a phenotyping strategy was developed employing colony morphotyping, chemical sensitivity testing, macrophage infection, and lipopolysaccharide fingerprint analyses to distinguish Burkholderia strains. Then mouse spleen isolates collected 3-180 days after infection were characterized phenotypically. Isolates from long-term infections often exhibited increased colony morphology differences and altered patterns of antimicrobial sensitivity and macrophage infection. Some of the Bp and Bm persistent infection isolates clearly displayed enhanced virulence in mice. Future studies will evaluate the potential role and significance of these phenotypic markers in signaling the establishment of a chronic infection.

The impact of inducing germination of Bacillus anthracis and Bacillus thuringiensis spores on potential secondary decontamination strategies.

AIMS: Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. METHODS AND RESULTS: Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germination-induction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. CONCLUSIONS: These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. SIGNIFICANCE AND IMPACT OF THE STUDY: By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more efficient and safe decontamination measures and may obviate the need for more stringent methods that are currently in place.

D-cycloserine or similar physiochemical compounds may be uniquely suited for use in Bacillus anthracis spore decontamination strategies.

AIMS: As observed in the aftermath of the anthrax attacks of 2001, decontamination and remediation of a site contaminated by the accidental or intentional release of Bacillus anthracis spores is difficult, costly and potentially damaging to the environment. The identification of novel strategies that neutralize the threat of spores while minimizing environmental damage remains a high priority. We investigated the efficacy of d-cycloserine (DCS), an antibiotic and inhibitor of the spore-associated enzyme (alanine racemase) responsible for converting l-alanine to d-alanine, as a spore germination enhancer and antimicrobial agent. METHODS AND RESULTS: We characterized the impact of DCS exposure on both germinating spores and vegetative cells of fully virulent B. anthracis by evaluating spore germination kinetics, determining the minimum inhibitory concentrations (MICs) required to affect growth of the bacteria and performing macrophage viability assays. DCS enhanced germination induced by l-alanine and also efficiently killed the newly germinated spores. Furthermore, DCS proved nontoxic to macrophages at concentrations that provided protection from the killing effects of spores. Similar tests were conducted with Bacillus thuringiensis (subspecies kurstaki and Al Hakam) to determine its potential as a possible surrogate for B. anthracis field trials. Bacillus thuringiensis spores responded in a similar manner to B. anthracis spores when exposed to DCS. CONCLUSIONS: These results further support that DCS augments the germination response of spores in the presence of l-alanine but also reveal that DCS is bactericidal towards germinating spores. SIGNIFICANCE AND IMPACT OF THE STUDY: DCS (or similar compounds) may be uniquely suited for use as part of decontamination strategies by augmenting the induction of spore germination and then rendering the germinated spores nonviable.

Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia.

Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ΔAmes strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.

Humanized theta-defensins (retrocyclins) enhance macrophage performance and protect mice from experimental anthrax infections.

Retrocyclins are humanized versions of the -defensin peptides expressed by the leukocytes of several nonhuman primates. Previous studies, performed in serum-free media, determined that retrocyclins 1 (RC1) and RC2 could prevent successful germination of Bacillus anthracis spores, kill vegetative B. anthracis cells, and inactivate anthrax lethal factor. We now report that retrocyclins are extensively bound by components of native mouse, human, and fetal calf sera, that heat-inactivated sera show greatly enhanced retrocyclin binding, and that native and (especially) heat-inactivated sera greatly reduce the direct activities of retrocyclins against spores and vegetative cells of B. anthracis. Nevertheless, we also found that retrocyclins protected mice challenged in vivo by subcutaneous, intraperitoneal, or intranasal instillation of B. anthracis spores. Retrocyclin 1 bound extensively to B. anthracis spores and enhanced their phagocytosis and killing by murine RAW264.7 cells. Based on the assumption that spore-bound RC1 enters phagosomes by "piggyback phagocytosis," model calculations showed that the intraphagosomal concentration of RC1 would greatly exceed its extracellular concentration. Murine alveolar macrophages took up fluorescently labeled retrocyclin, suggesting that macrophages may also acquire extracellular RC1 directly. Overall, these data demonstrate that retrocyclins are effective in vivo against experimental murine anthrax infections and suggest that enhanced macrophage function contributes to this property.

Effects of altering the germination potential of Bacillus anthracis spores by exogenous means in a mouse model.

Inhalational anthrax is the most severe form of anthrax. It has been shown in small-animal and non-human primate models that relatively large pools of ungerminated Bacillus anthracis spores can remain within the alveolar spaces for days to weeks post-inhalation or until transported to areas more favourable for germination and bacillary outgrowth. In this study, spores of the Ames strain that were exposed to germination-inducing media prior to intranasal delivery were significantly less infectious than spores delivered in either water or germination-inhibitory medium. The effect of manipulating the germination potential of these spores within the lungs of infected mice by exogenous germination-altering media was examined. The data suggested that neither inducing germination nor inhibiting germination of spores within the lungs protected mice from the ensuing infection. Germination-altering strategies could, instead, significantly increase the severity of disease in a mouse model of inhalational anthrax when implemented in vivo. It was shown that germination-altering strategies, in this study, were not beneficial to the infected host and are impractical as in vivo countermeasures.

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

Analysis of a novel spore antigen in Bacillus anthracis that contributes to spore opsonization.

The significance of Bacillus anthracis as an agent of bioterrorism has been well established. An understanding of both the pathogenesis and the host response is required to elucidate approaches to more rapidly detect and effectively prevent or treat anthrax. Current vaccine strategies are focused primarily on production of antibodies against the protective antigen components of the anthrax toxins, which are secreted by the bacilli. A better understanding of the dynamic morphology of the dormant and germinating spore and its interaction with the host immune system could be important in developing an optimally efficacious anthrax vaccine. A spore-associated protein was identified that was specific to the Bacillus cereus group of bacteria and referred to as spore opsonization-associated antigen A (SoaA). Immuno-electron microscopy localized this protein to the area of the cortex beneath the coat of the dormant spore. Although our data suggested that SoaA was found below the coat layers of the ungerminated spore, SoaA was involved in the interaction of spores with macrophages shortly after infection. To investigate further the specific properties of the SoaA protein, the soaA gene was inactivated in the B. anthracis Ames strain. The SoaA protein in the Ames strain of B. anthracis increased the phagocytic uptake of the spores in the presence of anti-spore antibodies. Unlike the wild-type strain, the mutant soaA : : Kan strain was not readily opsonized by anti-spore antibodies. While the mutant spores retained characteristic resistance properties in vitro and virulence in vivo, the soaA : : Kan mutant strain was significantly less suited for survival in vivo when competed against the wild-type Ames strain.

Development of in vitro correlate assays of immunity to infection with Yersinia pestis.

Pneumonic plague is a severe, rapidly progressing disease for which there is no effective vaccine. Since the efficacy of new vaccines cannot be tested in humans, it is essential to develop in vitro surrogate assays that are valid predictors of immunity. The F1 capsule antigen stimulates a protective immune response to most strains of Yersinia pestis. However, strains of Y. pestis that are F1- but still virulent have been isolated, and an in vitro assay, the results which can predict protection against both F1+ and F1- strains, is needed. The virulence antigen (V) is an essential virulence factor of Y. pestis and stimulates protective antibodies. We investigated potential correlates of plague immunity that are based on anti-V antibody-mediated neutralization of Yersinia-induced macrophage cytotoxicity. The neutralizing activity of sera from mice vaccinated with an F1-V fusion candidate vaccine was determined. The decrease in the level of the apoptosis-specific enzyme caspase-3 significantly predicted survival in one- and two-dose vaccination experiments. Sera from F1-V-vaccinated nonhuman primates were evaluated with macrophage assays based on caspase-3 and on other markers manifested at the different stages in cell death. Using murine- and human-derived macrophages in microscopic and fluorescence-activated-cell-sorting-based live/dead staining assays of terminal necrosis, we demonstrated a strong association between in vitro neutralization of macrophage cytotoxicity induced by serum-treated Yersinia and in vivo protection against lethal infection. These results provide a strong base for the development of reliable in vitro correlate bioassays that are predictive of protective immunity to plague.

Fully virulent Bacillus anthracis does not require the immunodominant protein BclA for pathogenesis.

The BclA protein is the immunodominant epitope on the surface of Bacillus anthracis spores; however, its roles in pathogenesis are unclear. We constructed a BclA deletion mutant (bclA) of the fully virulent Ames strain. This derivative retained full virulence in several small-animal models of infection despite the bclA deletion.

The detection of protective antigen (PA) associated with spores of Bacillus anthracis and the effects of anti-PA antibodies on spore germination and macrophage interactions.

The protective antigen (PA) component of the anthrax toxins is an essential virulence factor of Bacillus anthracis and is the major protective immunogen. The kinetics of PA production during growth of B. anthracis, and the roles of anti-PA antibody in host immunity are not clearly defined. Production of PA by the vegetative organisms peaks during the shift from exponential to stationary phase of growth. Recently, PA was also found to be associated with spores. In our study, PA-specific mRNA was detected in spores by RT-PCR within 15-min of exposure to germinant. PA protein was detected by immunomagnetic electrochemiluminescence (ECL) on spores within 1 h of exposure to a germination medium and was rapidly released into the supernatant. PA was not demonstrated on ungerminated spores by RNA analysis, ECL, or spore-based anti-PA ELISA; however, it was detected on ungerminated spores by immunoelectron microscopy (immunoem). In rabbits, PA induces polyclonal antibodies (Abs) that, in addition to their anti-toxin neutralizing activities, exhibit anti-spore activities. In this study, the anti-spore effects of a human monoclonal Ab specific for PA (AVP-hPA mAb, Avanir Pharmaceuticals) were characterized. AVP-hPA mAb retarded germination in vitro, and enhanced the phagocytic and sporicidal activities of macrophages. The activities were comparable to those of the polyclonal rabbit anti-rPA Ab. Assays to detect germination inhibitory activity (GIA) in serum from vaccinated mice and guinea pigs suggested a possible role for anti-PA Abs in protection. Thus, anti-PA Ab-mediated, anti-spore activities may play a role in protection during the early stages of an anthrax infection.

Mu dI1(Ap lac) mutagenesis of Yersinia pestis plasmid pFra and identification of temperature-regulated loci associated with virulence.

The F1 capsule of Yersinia pestis, encoded by the 100 kb plasmid pFra, is often assumed to be essential for full virulence of Y. pestis. However, virulent strains of Y. pestis that are F1- and either pFra+ or pFra- have been reported. To assess the role of pFra-encoded factors in virulence, mutants in pFra with insertions of the defective transposing bacteriophage Mu dl(Ap lac) were obtained, by using the wild type (wt) and the pLcr-cured derivative of strain C092. Mutants that exhibited temperature regulation of lactose fermentation and retarded electrophoretic mobility of pFra were selected. A total of 15 insertion mutants were isolated in the wt strain (12 of which had a single insertion in the genome, in pFra); and 24 mutants in the isogenic pLcr- derivative. Four of the pLcr+ mutants, and none of the pLcr- mutants, were F1-. All F1- mutants were decreased in virulence for mice compared to the wt parent; and five of the F1+ mutants also were significantly attenuated in mice. Fusion end-joints of insert DNA were cloned into Escherichia coli by using pMLB524, a vector for rescuing operon fusions of lacZ. Recombinants were obtained which contained pFra inserts ranging from < 2kb to approximately 36 kb, and the insertions occurred at several sites on pFra. All of the four F1- mutants tested mapped within the F1 capsule operon (caf1). The remaining five attenuated mutants sequenced were F1+ and mapped outside of but near the operon. Sequencing and complete analysis of the pFra insertions mutants could facilitate identification of new potential virulence factors.

In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody.

Antibodies (Abs) to the protective antigen (PA) component of the anthrax toxins have anti-spore as well as anti-toxin activities. Anti-PA antisera and purified anti-PA Abs enhance the phagocytosis by murine-derived macrophages (MQs) of spores of the Ames and Sterne strains and retard the germination of extracellular spores in vitro. The fate after phagocytosis of untreated and anti-PA-treated spores was further studied in culture medium that supported phagocytosis without stimulating spore germination (Dulbecco's minimal essential medium with horse serum 10%). The spores germinated within cells of primary peritoneal murine MQs (C3H/HeN) and MQs of the RAW264.7 MQ-like cell line; germination was associated with a rapid decline in spore viability. Exposure of MQs to inhibitors of phago-endosomal acidification (bafilomycin A and chloroquine) reduced the efficiency of MQ killing and allowed outgrowth and replication of the organisms. Treatment of spores with anti-PA Abs stimulated their phagocytosis and was associated with enhanced MQ killing of the spores. The enhanced killing of spores correlated with the greater extent of germination of anti-PA-treated spores after phagocytosis. A PA null mutant of the Ames strain exhibited none of the effects associated with anti-PA Ab treatment ofthe parental strain. Thus, the anti-PA Ab-specific immunity induced by vaccines has anti-spore activities and its role in impeding the early stages of infection with Bacillusanthracis needs to be assessed.

Anthrax vaccines.

The only impetus for the development of new anthrax vaccines is to protect humans against the intentional use of Bacillus anthracis as a bioterrorist or warfare agent. Live attenuated vaccines against anthrax in domesticated animals were among the very first vaccines developed. This was followed by the development of nonliving component vaccines leading to the eventual licensure of protein-based vaccines for human use in the 1970s. This chapter will review the recent advances in developing protein, live attenuated, and genetic vaccines against anthrax.

Anti-V antigen antibody protects macrophages from Yersinia pestis -induced cell death and promotes phagocytosis.

The pathogenic Yersinia spp. harbor a common plasmid (pYV) essential for virulence. The plasmid encodes a type III secretion system that functions to translocate Yersinia outer proteins (Yops) into the host cytosol. Within the host cell, the Yops act to inhibit phagocytosis and induce apoptosis. One of the plasmid-encoded proteins, virulence antigen (V), is a major protective immunogen that is involved in Yop translocation. Yersinia pestis, like the enteric Yersinia spp., was both resistant to phagocytosis by and cytotoxic for J774.A1, a murine macrophage cell line. Both of these activities were dependent on culture of the bacteria at 37 degrees C for 1.5-2 h before infection. However, extending the preculture period at 37 degrees C to 24 h, which induced formation of a capsule, completely blocked cytotoxicity. Treating the bacteria with either rabbit polyclonal anti-V antibodies (R anti-V) or monoclonal antibody (MAb) 7.3, antibodies specific for V and protective against plague in vivo, protected J774.A1 cells from Y. pestis -induced cell death and also reversed the inhibition of phagocytosis. Whereas protection against cell cytotoxicity was afforded by the F(ab')(2) portion of R anti-V, the ability of anti-V to induce uptake of Y. pestis appeared to be dependent on the Fc portion of the Ab. The protective epitope(s) recognized by R anti-V was contained in the central region of Y. pestis V (aa 135-275) and were partially cross reactive with Y. pseudotuberculosis and Y. enterocolitica serotype 08 V antigens.

Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague.

The current human plague vaccine, a killed Yersinia pestis whole-cell preparation, does not protect against aerosol challenge and is reactogenic and antigenically undefined. Live attenuated Y. pestis, such as pigmentation-deficient (Pgm-) strains, have been used frequently as vaccines and are efficacious. They are used widely in plague research and assumed to be safe. However, they can cause serious adverse reactions, and their aerosol infectivity is not known. We tested the virulence of a defined Pgm- variant of the C092 strain of Y. pestis in mouse and non-human primate models of pneumonic plague. The ten-fold lower median lethal dose by the aerosol compared to the subcutaneous (s.c.) routes of the Pgm- strain in mice suggested that the Pgm- strain might be less attenuated by the former than by the latter route. After exposure of 16 African green monkeys to inhaled doses ranging from 1.1 x 10(4) to 8.1 x 10(7)cfu, eight died and eight survived. The terminal cultures collected from five of the non-survivors were all positive for Y. pestis. Two of the remaining three non-survivors were culture-negative but had pathologic and immunologic evidence of infection with Y. pestis, specimens could not be obtained nor the cause of death determined for the third one. The deaths were not dose-related, and there were some differences in the pathology associated with infection by the Pgm- strain compared to the wild-type (wt) strain. However, the Pgm- derivative was clearly virulent for monkeys by the aerosol route. A mutant of the Pgm- strain, which has a deletion in the plasminogen activator (Pla) virulence locus (pla), appeared to be more attenuated than was either the Pgm- single mutant (in NHPs and mice) or the Pla- single mutant strain (in mice) and has potential as a live vaccine.