Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Comparative genomics of early-diverging Brucella strains reveals a novel lipopolysaccharide biosynthesis pathway.

UNLABELLED: Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1(T) and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1(T) and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1(T) and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1(T) maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE: This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.

Evaluation of the Abbott ARCHITECT i2000 sirolimus assay and comparison with the Abbott IMx sirolimus assay and an established liquid chromatography-tandem mass spectrometry method.

BACKGROUND: Sirolimus is indicated for prophylaxis treatment to prevent solid organ rejection. Due to intrapatient and interpatient variabilities in pharmacokinetics, risk of concentration-related toxicity, risk of noncompliance, and a high likelihood of drug-drug interactions, therapeutic drug monitoring of sirolimus is essential. There are several methodologies used clinically to monitor sirolimus, ranging from immunoassay to tandem mass spectrometry, each with potential strengths and limitations. The purpose of our study was to validate the Abbott ARCHITECT i2000 sirolimus assay. MATERIALS AND METHODS: The Abbott ARCHITECT i2000 sirolimus assay was evaluated for linearity, limit of detection, limit of quantification, imprecision, common interferences, and accuracy. Accuracy was compared with the Abbott IMx sirolimus assay and with an established liquid chromatography-tandem mass spectrometry method. Stability of sirolimus when specimens were stored frozen (-20°C) or refrigerated (2-8°C) and degree of crossreactivity of the i2000 with everolimus were also evaluated. RESULTS: The ARCHITECT i2000 assay demonstrated good linearity, low imprecision, and was free of common interferences. Results for both immunoassay methods were biased slightly high, compared with those of liquid chromatography-tandem mass spectrometry. Sirolimus was stable when samples were stored either refrigerated or frozen. However, the results generated with samples stored refrigerated had an increase in scatter on the regression plots compared with those generated for samples that were stored frozen, indicating that extraction of the drug may be incomplete, particularly with the Abbott IMx assay. In addition, statistical performance indices suggest that the agreement between the ARCHITECT i2000 and Abbott IMx was better for frozen patient samples. The ARCHITECT i2000 and the Abbott IMx methods both exhibited a >100% crossreactivity with everolimus. CONCLUSIONS: The Abbott ARCHITECT i2000 instrument demonstrates reasonable sensitivity, precision, and accuracy for supporting routine therapeutic drug monitoring of sirolimus with either refrigerated or frozen whole blood collected from patients who are not comedicated with everolimus.

Characterization of novel Brucella strains originating from wild native rodent species in North Queensland, Australia.

We report on the characterization of a group of seven novel Brucella strains isolated in 1964 from three native rodent species in North Queensland, Australia, during a survey of wild animals. The strains were initially reported to be Brucella suis biovar 3 on the basis of microbiological test results. Our results indicated that the rodent strains had microbiological traits distinct from those of B. suis biovar 3 and all other Brucella spp. To reinvestigate these rodent strains, we sequenced the 16S rRNA, recA, and rpoB genes and nine housekeeping genes and also performed multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA). The rodent strains have a unique 16S rRNA gene sequence compared to the sequences of the classical Brucella spp. Sequence analysis of the recA, rpoB, and nine housekeeping genes reveals that the rodent strains are genetically identical to each other at these loci and divergent from any of the currently described Brucella sequence types. However, all seven of the rodent strains do exhibit distinctive allelic MLVA profiles, although none demonstrated an amplicon for VNTR 07, whereas the other Brucella spp. did. Phylogenetic analysis of the MLVA data reveals that the rodent strains form a distinct clade separate from the classical Brucella spp. Furthermore, whole-genome sequence comparison using the maximal unique exact matches index (MUMi) demonstrated a high degree of relatedness of one of the seven rodent Brucella strains (strain NF 2653) to another Australian rodent Brucella strain (strain 83-13). Our findings strongly suggest that this group of Brucella strains isolated from wild Australian rodents defines a new species in the Brucella genus.

Identification of an unusual Brucella strain (BO2) from a lung biopsy in a 52 year-old patient with chronic destructive pneumonia.

BACKGROUND: Brucellosis is primarily a zoonotic disease caused by Brucella species. There are currently ten Brucella spp. including the recently identified novel B. inopinata sp. isolated from a wound associated with a breast implant infection. In this study we report on the identification of an unusual Brucella-like strain (BO2) isolated from a lung biopsy in a 52-year-old patient in Australia with a clinical history of chronic destructive pneumonia. RESULTS: Standard biochemical profiles confirmed that the unusual strain was a member of the Brucella genus and the full-length 16S rRNA gene sequence was 100% identical to the recently identified B. inopinata sp. nov. (type strain BO1(T)). Additional sequence analysis of the recA, omp2a and 2b genes; and multiple locus sequence analysis (MLSA) demonstrated that strain BO2 exhibited significant similarity to the B. inopinata sp. compared to any of the other Brucella or Ochrobactrum species. Genotyping based on multiple-locus variable-number tandem repeat analysis (MLVA) established that the BO2 and BO1(T) strains form a distinct phylogenetic cluster separate from the other Brucella spp. CONCLUSION: Based on these molecular and microbiological characterizations, we propose that the BO2 strain is a novel lineage of the newly described B. inopinata species.

Effect of carbon dioxide on broth microdilution susceptibility testing of Brucella spp.

Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO2 incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log2 dilution higher in CO2 than in AA; tetracycline-class MICs to be 1 log2 dilution lower in CO2.

Analytical performance characteristics of the Abbott Architect i2000 Tacrolimus assay; comparisons with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Abbott IMx methods.

BACKGROUND: Tacrolimus, an immunosuppressive drug discovered in 1984, is used to decrease the risk of organ rejection. The 2007 European Consensus Conference on Tacrolimus Optimization recommended the use of methods with a limit of quantitation at < or =1ng/ml for monitoring low dose tacrolimus therapy. METHODS: The performance characteristics of the Abbott i2000 Tacrolimus assay were evaluated and compared to LC-MS/MS and Abbott IMx methods. RESULTS: The limit of detection of the Abbott i2000 method was 0.21ng/ml. Total imprecision was 6.9, 5.8 and 4.2% at three target concentrations of tacrolimus (3.2, 8.5 and 15.9ng/ml) respectively. The method was linear up to 30ng/ml. The limit of quantitation (LOQ) was 0.5ng/ml. Correlation of patient specimen results between the Abbott i2000 method and LC-MS/MS yielded a Deming slope of 1.072ng/ml (CI 1.005 to 1.140), r=0.952 and an intercept of -0.491ng/ml (CI -1.387 to 0.405). The mean bias, as determined by Bland-Altman analysis, was 0.36ng/ml. Comparing results generated by the widely used Abbott IMx assay with LC-MS/MS and Architect i2000 methods yielded Deming slopes of 1.062 and 0.973, with intercepts of -0.214ng/ml and -0.01ng/ml respectively. For both comparisons, r=0.97. The corresponding mean bias of results generated by the Abbott IMx assay (Bland-Altman plots) was 0.53ng/ml vs LC-MS/MS and -0.38ng/ml vs the Architect i2000. CONCLUSIONS: The Architect i2000 method is a sensitive and highly precise method that achieves a LOQ of <1.0 ng/ml and demonstrated overall accuracy of tacrolimus measurements within 0.4ng/ml.

Performance characteristics of beta-2-microglobulin measurements on a Beckman Immage analyzer with the DakoCytomation reagent kit.

OBJECTIVE: Evaluation of serum beta-2-microglobulin (beta2M) in an automated analyzer. DESIGN AND METHODS: The DakoCytomation beta2M kit is an antibody based reagent intended for quantitative determination of beta2M in serum and plasma by rate nephelometry. RESULTS: The limit of blank is 0.16 mg/L. The method is linear up to 17.9 mg/L. The imprecision ranged from 2.1% to 7.9% at the concentrations of 1.77 and 7.19 mg/L, respectively. Method comparison yielded slope=1.009, r=0.998. No interference was observed from hemolytic or icteric specimens. Reference interval of a healthy population was 1.13 mg/L to 3.04 mg/L. CONCLUSION: The DakoCytomation reagent is acceptable to measure serum beta2M.

Comparison of two multiple-locus variable-number tandem-repeat analysis methods for molecular strain typing of human Brucella melitensis isolates from the Middle East.

Brucella species are highly monomorphic, with minimal genetic variation among species, hindering the development of reliable subtyping tools for epidemiologic and phylogenetic analyses. Our objective was to compare two distinct multiple-locus variable-number tandem-repeat analysis (MLVA) subtyping methods on a collection of 101 Brucella melitensis isolates from sporadic human cases of brucellosis in Egypt (n = 83), Qatar (n = 17), and Libya (n = 1). A gel-based MLVA technique, MLVA-15(IGM), was compared to an automated capillary electrophoresis-based method, MLVA-15(NAU), with each MLVA scheme examining a unique set of variable-number tandem repeats. Both the MLVA(IGM) and MLVA(NAU) methods were highly discriminatory, resolving 99 and 101 distinct genotypes, respectively, and were able to largely separate genotypes from Egypt and Qatar. The MLVA-15(NAU) scheme presented higher strain-to-strain diversity in our test population than that observed with the MLVA-15(IGM) assay. Both schemes were able to genetically correlate some strains originating from the same hospital or region within a country. In addition to comparing the genotyping abilities of these two schemes, we also compared the usability, limitations, and advantages of the two MLVA systems and their applications in the epidemiological genotyping of human B. melitensis strains.

Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades.

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

Novel Brucella strain (BO1) associated with a prosthetic breast implant infection.

We report the microbiological, biochemical, and molecular characterization of an unusual Brucella strain (BO1) isolated from a breast implant wound in a 71-year-old woman with clinical symptoms consistent with brucellosis. Initial phenotypic analysis, including biochemical and antimicrobial susceptibility testing, cellular fatty acid analysis, and molecular analysis based on DNA-DNA reassociation and the presence of multiple copies of IS711 element suggested that the isolate was a Brucella-like organism, but species determination using microbiological algorithms was unsuccessful. Furthermore, molecular data based on 16S rRNA gene sequencing and multilocus sequence analysis demonstrated that BO1 was an unusual Brucella strain and not closely related to any currently described Brucella species. However, comparison with equivalent sequences in Ochrobactrum spp. confirms that the isolate is much more closely related to Brucella than to Ochrobactrum spp., and thus the isolate likely represents an atypical and novel strain within the genus Brucella.

Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes.

Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-D-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.

Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates.

Members of the genus Brucella are categorized as biothreat agents and pose a hazard for both humans and animals. Current identification methods rely on biochemical tests that may require up to 7 days for results. We sequenced the 16S rRNA genes of 65 Brucella strains along with 17 related strains likely to present a differential diagnostic challenge. All Brucella 16S rRNA gene sequences were determined to be identical and were clearly different from the 17 related strains, suggesting that 16S rRNA gene sequencing is a reliable tool for rapid genus-level identification of Brucella spp. and their differentiation from closely related organisms.

Standardization and denoising algorithms for mass spectra to classify whole-organism bacterial specimens.

MOTIVATION: Application of mass spectrometry in proteomics is a breakthrough in high-throughput analyses. Early applications have focused on protein expression profiles to differentiate among various types of tissue samples (e.g. normal versus tumor). Here our goal is to use mass spectra to differentiate bacterial species using whole-organism samples. The raw spectra are similar to spectra of tissue samples, raising some of the same statistical issues (e.g. non-uniform baselines and higher noise associated with higher baseline), but are substantially noisier. As a result, new preprocessing procedures are required before these spectra can be used for statistical classification. RESULTS: In this study, we introduce novel preprocessing steps that can be used with any mass spectra. These comprise a standardization step and a denoising step. The noise level for each spectrum is determined using only data from that spectrum. Only spectral features that exceed a threshold defined by the noise level are subsequently used for classification. Using this approach, we trained the Random Forest program to classify 240 mass spectra into four bacterial types. The method resulted in zero prediction errors in the training samples and in two test datasets having 240 and 300 spectra, respectively.

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry.

Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) is a species-common, immunogenic surface lipoprotein. In this study, the psaA gene was expressed as a nonfusion acylated protein in an Escherichia coli expression system. Yields of pure recombinant PsaA (rPsaA) were 8-10 mg/liter of fermentation culture. Analysis of rPsaA tryptic digests by HPLC-electrospray mass spectrometry (MS) confirmed 98% of the expected protein sequence. GC/MS data demonstrated very similar acylation of native and rPsaA by C12:0-C22:0 fatty acids, with C16 and C18 predominating. Negative ion electrospray MS/MS analysis of the rPsaA lipid anchor released by Pronase-E confirmed that the structure was based on an N-terminal palmitoylcysteine (Pam(3)Cys). Electrospray MS heterogeneity analysis of intact rPsaA indicated that all of the observed heterogeneity could be accounted for by the fatty acid distributions. The availability of well-characterized rPsaA will facilitate the continued research and development of protein-based vaccines for the prevention of pneumococcal disease.

Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei.

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.

Performance characteristics of the Dimension RxL iron and total iron-binding capacity methods.

BACKGROUND: Measurements of serum iron and total iron-binding capacity (TIBC) can be used to aid the diagnosis of iron deficiency and iron overload states. A variety of different methods for these measurements are commercially available. METHODS: Linearity, imprecision and hemoglobin interference of homogeneous iron and TIBC methods on a Dimension RxL analyzer were assessed. Method comparison studies were performed with a Vitros 950 analyzer. RESULTS: The Dimension RxL iron method was linear from 40 to 1000 microg/dl. The coefficient of variation of the iron and TIBC methods were <4% and <2%, respectively, at iron concentrations of 68 and 228 microg/dl and TIBC concentrations of 206 and 384 microg/dl. Comparison of the Vitros 950 and Dimension RxL iron methods gave a slope of 0.97, an intercept of 6.0 and r=0.99. Corresponding results for the TIBC methods were 1.02, -6.6 and r=0.97, respectively. Simulation of acute iron overload in vitro did not produce the same effect on TIBC measurements as in vivo overload. Iron recovery by the Dimension RxL method was reduced 40-60% by the addition of deferoxamine at a concentration of 200 micromol/l. CONCLUSIONS: The Dimension RxL assay provides acceptable measurements of iron and TIBC in routine patient samples. Iron measurements are unreliable in the presence of deferoxamine.

A two-component direct fluorescent-antibody assay for rapid identification of Bacillus anthracis.

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non-B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 aging clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens.