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.

Draft Genomes for Eight Burkholderia mallei Isolates from Turkey.

Burkholderia mallei, the etiologic agent of glanders, is a Gram-negative, nonmotile, facultative intracellular pathogen. Although glanders has been eradicated from many parts of the world, the threat of B. mallei being used as a weapon is very real. Here we present draft genome assemblies of 8 Burkholderia mallei strains that were isolated in Turkey.

Identification and typing of Francisella tularensis with a highly automated genotyping assay.

A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay.

Avian cholera in a southern giant petrel (Macronectes giganteus) from Antarctica.

A southern giant petrel (Macronectes giganteus) was found dead at Potter Peninsula, King George Island, South Shetland, Antarctica. The adult male was discovered approximately 48 hr after death. Macroscopic and microscopic lesions were compatible with avian cholera and the bacterium Pasteurella multocida subsp. gallicida, serotype A1 was isolated from lung, heart, liver, pericardial sac, and air sacs. In addition, Escherichia coli was isolated from pericardial sac and air sacs. This is the first known report of avian cholera in a southern giant petrel in Antarctica.

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces.

AIMS: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. METHODS AND RESULTS: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek GNI+) and enterococci (API rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at >/=85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API(R) profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. CONCLUSIONS: Gull faeces could be a major contributor of E. coli (10(5)-10(9) CFU g(-1)) and enterococci (10(4)-10(8) CFU g(-)1) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. SIGNIFICANCE AND IMPACT OF THE STUDY: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.

Septicemic pasteurellosis in free-ranging neonatal pronghorn in Oregon.

As part of a study to determine the cause(s) of population decline and low survival of pronghorn (Antilocapra americana) neonates on Hart Mountain National Antelope Refuge (HMNAR), Oregon (USA), 55 of 104 neonates captured during May 1996 and 1997 were necropsied (n = 28, 1996; n = 27, 1997) to determine cause of death. Necropsies were conducted on fawns that died during May, June, or July of each year. The objectives of this study were to report the occurrence and pathology of pasteurellosis in neonates and determine if the isolated strain of Pasteurella multocida was unique. Septicemic pasteurellosis, caused by P. multocida, was diagnosed as the cause of death for two neonates in May and June 1997. Necropsy findings included widely scattered petechial and ecchymotic hemorrhages found over a large portion of the subcutaneous tissue, meninges of the brain, epicardium, skeletal muscle, and serosal surface of the thorasic and abdominal cavities. Histological examination of lung tissues revealed diffuse congestion and edema and moderate to marked multifocal infiltrate of macrophages, neutrophils, and numerous bacteria within many terminal bronchioles and alveoli. Pasteurella multocida serotypes A:3,4, and B:1 were isolated from several tissues including lung, intestinal, thorasic fluid, and heart blood. Each B:1 isolate had DNA restriction endonuclease fingerprint profiles distinct from isolates previously characterized from domestic cattle, swan (Olor spp.), moose (Alces alces), and pronghorn from Montana (USA). This is the first report of pasteurellosis in pronghorn from Oregon and the B:1 isolates appear to be unique in comparison to DNA fingerprint profiles from selected domestic and wild species.

Advances in nucleic acid-based detection methods.

Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids.