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.].

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.

Identification and characterization of variable-number tandem repeats in the Yersinia pestis genome.

Yersinia pestis, the infamous plague-causing pathogen, appears to have emerged in relatively recent history. Evidence of this fact comes from several studies that document a lack of nucleotide diversity in the Y. pestis genome. In contrast, we report that variable-number tandem repeat (VNTR) sequences are common in the Y. pestis genome and occur frequently in gene coding regions. Larger tandem repeat arrays, most useful for phylogenetic analysis, are present at an average of 2.18 arrays per 10 kbp and are distributed evenly throughout the genome and the two virulence plasmids, pCD1 and pMT1. We examined allelic diversity at 42 chromosomal VNTR loci in 24 selected isolates (12 globally distributed and 12 from Siskiyou County, Calif.). Vast differences in diversity were observed among the 42 VNTR loci, ranging from 2 to 11 alleles. We found that the maximum copy number of repeats in an array was highly correlated with diversity (R = 0.86). VNTR-based phylogenetic analysis of the 24 strains successfully grouped isolates from biovar orientalis and most of the antiqua and mediaevalis strains. Hence, multiple-locus VNTR analysis (MLVA) appears capable of both distinguishing closely related strains and successfully classifying more distant relationships. Harnessing the power of MLVA to establish standardized databases will enable researchers to better understand plague ecology and evolution around the world.

Identification of nucleotide sequences for the specific and rapid detection of Yersinia pestis.

Suppression subtractive hybridization, a cost-effective approach for targeting unique DNA, was used to identify a 41.7-kb Yersinia pestis-specific region. One primer pair designed from this region amplified PCR products from natural isolates of Y. pestis and produced no false positives for near neighbors, an important criterion for unambiguous bacterial identification.

Diversity in a variable-number tandem repeat from Yersinia pestis.

We have identified a tetranucleotide repeat sequence, (CAAA)(N), in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague.

Isolation of an asporogenic (spoOA) protective antigen-producing strain of Bacillus anthracis.

We found that Congo red agar allows identification of sporulation-deficient Bacillus anthracis. Using Congo red agar, we isolated an asporogenic derivative of the protective antigen-producing strain B. anthracis delta Sterne-1(pPA102). Polymerase chain reaction and Southern hybridization analyses of DNA from the asporogenic mutant revealed that a deletion was present in spoOA, an essential gene for the initiation of sporulation. The deletion also encompassed the spoIVB homologue and a portion of the recN homologue. The avirulent spoOA strain delta Sterne-1(pPA102)CR4 is suitable for the safe production of protective antigen without endospore contamination of the vaccine production facility.

Protection of mice from fatal bubonic and pneumonic plague by passive immunization with monoclonal antibodies against the F1 protein of Yersinia pestis.

Monoclonal antibodies (MAbs) to the fraction 1 (F1) protein of Yersinia pestis protected mice against fatal pneumonic as well as bubonic plague from wild-type F1+ organisms. The rare isolation of a virulent F1- isolate from surviving animals supports earlier studies suggesting that improved vaccines should consist of immunogens to protect against F1- variants. The high degree of protection with IgG MAb suggests that secretory IgA is not required for protection from pneumonic plague.

Recombinant V antigen protects mice against pneumonic and bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis.

The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.

Pathology of experimental pneumonic plague produced by fraction 1-positive and fraction 1-negative Yersinia pestis in African green monkeys (Cercopithecus aethiops).

OBJECTIVE: The protein capsule of Yersinia pestis, known as Fraction 1 or F1, is a protective immunogen and is an assumed, but not proven, virulence factor. Our objectives were to determine if inhaled F1-negative and/or F1-positive strains of Y pestis were virulent in the African green monkey and, if so, to differentiate F1-negative from F1-positive monkeys. Because F1-negative strains have been isolated from natural sources and have caused experimental fatal disease, we felt that this information was crucial to the development of future vaccines and diagnostic tests. MATERIALS AND METHODS: Adult African green monkeys were exposed by aerosol to F1-positive (CO92, n=15) or F1-negative (CO92-C12, n=6; Java-9, n=2) Y pestis strains. RESULTS: All monkeys died 4 to 10 days postexposure and had lesions consistent with primary pneumonic plague. Antibodies to F1 antigen and other Y pestis antigens allowed us to differentiate F1-positive from F1-negative Y pestis strains in fixed tissues. CONCLUSIONS: In this study, F1 antigen was not a required virulence factor. Therefore, there may be a need for vaccines and diagnostic assays that are not solely based on the F1 antigen.

Relationship between virulence and immunity as revealed in recent studies of the F1 capsule of Yersinia pestis.

Yersinia pestis, the causative agent of plague, possesses multiple virulence determinants encoded on its three plasmids and on its chromosome. We evaluated the role of the protein capsule F1 in virulence an immunity against plague. Strains lacking F1, either those that are naturally occurring or those with genetically defined nonpolar mutations in the structural gene, retained their virulence for mice and nonhuman primates. However, both active immunization with F1, from either a recombinant vector or Y. pestis, and passive immunization with F1 monoclonal antibody protected mice from experimental infection with wild-type F1-positive organisms. These results suggest that protective immunogens like F1 need not be essential for virulence. The rare isolation of virulent F1-negative organisms from F1-immunized animals infected with F1-positive strains supports this conclusion and also suggests that, in addition to F1, an optimal vaccine against plague should include essential virulence factors as immunogens.

Postexposure prophylaxis against experimental inhalation anthrax.

Inhalation anthrax is a rare disease that is almost invariably fatal. This study determined whether a prolonged course of postexposure antibiotics with or without vaccination would protect monkeys exposed to a lethal aerosol dose of Bacillus anthracis when the antibiotic was discontinued. Beginning 1 day after exposure, groups of 10 animals were given penicillin, ciprofloxacin, doxycycline, doxycycline plus vaccination, vaccination alone, or saline. Antibiotics were administered for 30 days and then discontinued. Vaccine was given on days 1 and 15. Two animals died of causes other than anthrax and were not included in the statistical analysis. Nine of 10 controls and 8 of 10 animals given only vaccine died. Each antibiotic regimen completely protected animals while on therapy and provided significant long-term protection upon discontinuance of the drug (penicillin, 7 of 10 survived, P < .02; ciprofloxacin, 8 of 9 survived, P < .002; doxycycline, 9 of 10 survived, P < .002; doxycycline plus vaccination, 9 of 9 survived, P < .0002). Protection against rechallenge was provided by combining postexposure antibiotic treatment with vaccination.

Killer toxin production in Pichia acaciae is associated with linear DNA plasmids.

We have identified a strain of the yeast Pichia acaciae which produces a "killer" toxin active against the yeast Debaryomyces tamarii. The killer phenotype was associated with the presence of two DNA plasmids, pPacl-1 (13.6 kilobase pairs) and pPacl-2 (7.3 kilobase pairs). P. acaciae strains, cured of these plasmids by irradiation with ultraviolet light, lacked killer activity and were sensitive to toxin produced by the parental strain. A partially cured strain, GS-1215, missing only the smaller plasmid, pPacl-2, also exhibited loss of both toxin activity and immunity. Exonuclease studies revealed that both plasmids were linear double-stranded DNA molecules with 5' protected ends. The P. acaciae system differs from that of the well-studied Kluyveromyces lactis "killer" system both in the range of susceptible strains and in the sizes of the plasmids involved. Our studies contradict previous reports that Pichia killer systems are invariably chromosomal.

Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy.

We have developed a simple and efficient transformation system for the dimorphic fungus Histoplasma capsulatum. Mutants of H. capsulatum defective in orotidine-5'-monophosphate pyrophosphorylase were transformed to prototrophy by the cloned URA5 gene of the filamentous fungus Podospora anserina. Abortive and mitotically stable transformants were obtained. The stable transformants had integrated copies of the plasmid, some in tandem head-to-tail orientation. Free plasmid identical to the transforming plasmid was present in some of the transformants. We obtained a transformation efficiency of up to 30 transformants/micrograms DNA for plasmid pPAura5-1 (9.2 kb). pPW2001, a smaller plasmid (4.7 kb) derived from pPAura5-1, transformed H. capsulatum more efficiently (up to 155 transformants/micrograms DNA).

Selection and characterization of ura5 mutants of Histoplasma capsulatum.

The combined use of non-aggregating Histoplasma capsulatum strains and a defined medium which allows quantitative plating of the yeast phase has allowed us to select 5-fluoroorotic acid (5-FOA)-resistant mutants of this dimorphic fungus. Approximately two-thirds of the 5-FOA-resistant strains were auxotrophic for uracil; all were deficient in orotidine-5'-monophosphate pyrophosphorylase (OMPpase) activity. One class of OMPpase mutant (alpha), which retained a low level of OMPpase activity, was auxotrophic in the yeast phase (37 degrees C) but grew slowly in the mycelial phase (25 degrees C) without exogenous uracil. This phenotype was not due to a temperature-sensitive OMPpase activity. Both wild-type and alpha mutants had a higher OMPpase activity in the mycelial phase than the yeast phase; this increased activity may be sufficient to allow mycelial growth of alpha mutants.

Quantitative plating of Histoplasma capsulatum without addition of conditioned medium or siderophores.

We have formulated two defined media which yield excellent plating efficiency of the yeast phase of Histoplasma capsulatum. Neither requires the addition of conditioned medium or purified siderophores, although the simpler of the two media includes hemin as a source of solubilized iron. Agarose, rather than conventional agar, is the solidifying agent for both media. Plating efficiency usually exceeds 90% with both North American and Central American isolates of H. capsulatum.

Locus affecting regulation of the colicin I receptor by iron.

Using a strain containing a cir-lac operon fusion and a selective medium, we isolated a regulatory mutant of the colicin I receptor, which we have designated cirR. Cells carrying the cirR mutation were defective in the transcriptional regulation of cir by iron, but synthesis of other iron-regulated proteins was unaffected. cirR was found to be cis dominant. This is in contrast to previously described mutations in iron regulation which are pleiomorphic and trans dominant. Temperature regulation of colicin I receptor production was unaffected by cirR.

Effect of growth temperature on the acquisition of iron by Salmonella typhimurium and Escherichia coli.

We have examined the effect of growth temperature on three systems normally induced under conditions of iron limitation: synthesis of the siderophore enterochelin (enterobactin), transport of ferric enterochelin, and production of the outer membrane protein which serves as the colicin I receptor. We found that although Salmonella typhimurium produces less enterochelin when grown at 42 degrees C, synthesis of this siderophore was not diminished in Escherichia coli grown under the same conditions. Growth at 42 degrees C under a condition of iron stress led to a reduction in the ability of cells to transport ferric enterochelin in both organisms. A two- to threefold decrease in the number of colicin I receptors was observed in cells of E. coli or S. typhimurium grown at 42 degrees C as compared with the number of receptors observed in cells grown at 37 degrees C. The colicin I receptor was shown not to be inherently unstable at 42 degrees C. By using a cir-lacZ operon fusion, it was shown that at least part of the decrease in receptor levels found in cells grown at high temperature was the result of decreased transcription of cir, the receptor structural gene. The effect of growth temperature on these systems was shown to be independent of fur, a regulatory element which mediates their enhanced production in response to iron stress. We suggest that a second regulatory element common to gene products involved in iron sequestration may be responsible for temperature regulation of these systems.