Metabolic and spatio-taxonomic response of uncultivated seafloor bacteria following the Deepwater Horizon oil spill.

The release of 700 million liters of oil into the Gulf of Mexico over a few months in 2010 produced dramatic changes in the microbial ecology of the water and sediment. Here, we reconstructed the genomes of 57 widespread uncultivated bacteria from post-spill deep-sea sediments, and recovered their gene expression pattern across the seafloor. These genomes comprised a common collection of bacteria that were enriched in heavily affected sediments around the wellhead. Although rare in distal sediments, some members were still detectable at sites up to 60 km away. Many of these genomes exhibited phylogenetic clustering indicative of common trait selection by the environment, and within half we identified 264 genes associated with hydrocarbon degradation. Alkane degradation ability was near ubiquitous among candidate hydrocarbon degraders, whereas just three harbored elaborate gene inventories for the degradation of alkanes and aromatic and polycyclic aromatic hydrocarbons (PAHs). Differential gene expression profiles revealed a spill-promoted microbial sulfur cycle alongside gene upregulation associated with PAH degradation. Gene expression associated with alkane degradation was widespread, although active alkane degrader identities changed along the pollution gradient. Analyses suggest that a broad metabolic capacity to respond to oil inputs exists across a large array of usually rare indigenous deep-sea bacteria.

Association of placental weight with cerebral palsy: population-based cohort study in Norway.

OBJECTIVE: To study the risk of cerebral palsy (CP) associated with placental weight, and also with placental weight/birthweight ratio and placental weight/birth length ratio. DESIGN: Population-based cohort study. SETTING: Perinatal data in the Medical Birth Registry of Norway were linked with clinical data in the CP Register of Norway. POPULATION: A total of 533 743 singleton liveborn children in Norway during 1999-2008. Of these, 779 children were diagnosed with CP. METHODS: Placental weight, placental weight/birthweight ratio, and placental weight/birth length ratio were grouped into gestational age-specific quartiles. Odds ratios (OR) with 95% confidence intervals (95% CI) for CP were calculated for children with exposure variables in the lowest or in the highest quartile, using the second to third quartile as the reference. MAIN OUTCOME MEASURES: CP and CP subtypes. RESULTS: Overall, children with low placental weight had increased risk for CP (OR 1.5, 95% CI 1.2-1.7). Low placental weight/birthweight ratio (OR 1.2, 95% CI 1.0-1.4) and low placental weight/birth length ratio (OR 1.5, 95% CI 1.2-1.8) were also associated with increased risk for CP. In children born at term, low placental weight was associated with a twofold increase in risk for spastic bilateral CP (including both quadriplegia and diplegia) (OR 2.1, 95% CI 1.5-2.9). In children born preterm, high placental ratios were associated with increased risk for spastic quadriplegia. CONCLUSIONS: Our results suggest that placental dysfunction may be involved in causal pathways leading to the more severe subtypes of CP. TWEETABLE ABSTRACT: Low placental weight increases the risk for cerebral palsy, especially for the spastic bilateral subtype.

Bone mineral density and vitamin D status in ambulatory and non-ambulatory children with cerebral palsy.

UNLABELLED: This study assessed distal femur and lumbar spine bone mineral density (BMD) Z-scores in children with cerebral palsy. BMD z-score was lower in non-ambulatory than in ambulatory children. Somewhat surprisingly, among ambulatory children, those with better walking abilities had higher BMD z-score than those with more impaired walking ability. INTRODUCTION: Children with cerebral palsy (CP) have increased risk for low bone mineral density (BMD). The aim was to explore the difference in BMD at the distal femur and lumbar spine between ambulatory and non-ambulatory children with CP and the relationship between vitamin D status and BMD. METHODS: Fifty-one children (age range 8-18 years; 20 girls) with CP participated. Their BMD Z-scores were measured in the lumbar spine and the distal femur using dual X-ray absorptiometry, and 25-hydroxy-vitamin D (25-OHD) concentrations were measured in serum. Children with GMFCS level I-III were defined as 'walkers' while children with level IV-V were defined as 'non-walkers. RESULTS: Non-walkers had lower mean BMD Z-scores (range -1.7 to -5.4) than walkers at all sites (range -0.8 to -1.5). Among walkers, BMD Z-scores at the distal femur were lower in those with GMFCS level II than with level I (p values < 0.004). A similar difference was found between the affected and unaffected limb in children with hemiplegia. Mean 25-OHD concentration was 45 nmol/L (SD = 18); lower in walkers (mean = 41 nmol/L; SD = 18) than in non-walkers (mean = 53 nmol/L; SD = 19; p = 0.041). There were no correlations between 25-OHD and BMD z-scores. CONCLUSIONS: The main predictor of low BMD Z-scores in the distal femur was the inability to walk, but the results suggest that the degree of the neuromotor impairment may also be a significant predictor. Vitamin D status did not correlate with BMD z-scores.

Bacterial communities in commercial aircraft high-efficiency particulate air (HEPA) filters assessed by PhyloChip analysis.

UNLABELLED: Air travel can rapidly transport infectious diseases globally. To facilitate the design of biosensors for infectious organisms in commercial aircraft, we characterized bacterial diversity in aircraft air. Samples from 61 aircraft high-efficiency particulate air (HEPA) filters were analyzed with a custom microarray of 16S rRNA gene sequences (PhyloChip), representing bacterial lineages. A total of 606 subfamilies from 41 phyla were detected. The most abundant bacterial subfamilies included bacteria associated with humans, especially skin, gastrointestinal and respiratory tracts, and with water and soil habitats. Operational taxonomic units that contain important human pathogens as well as their close, more benign relatives were detected. When compared to 43 samples of urban outdoor air, aircraft samples differed in composition, with higher relative abundance of Firmicutes and Gammaproteobacteria lineages in aircraft samples, and higher relative abundance of Actinobacteria and Betaproteobacteria lineages in outdoor air samples. In addition, aircraft and outdoor air samples differed in the incidence of taxa containing human pathogens. Overall, these results demonstrate that HEPA filter samples can be used to deeply characterize bacterial diversity in aircraft air and suggest that the presence of close relatives of certain pathogens must be taken into account in probe design for aircraft biosensors. PRACTICAL IMPLICATIONS: A biosensor that could be deployed in commercial aircraft would be required to function at an extremely low false alarm rate, making an understanding of microbial background important. This study reveals a diverse bacterial background present on aircraft, including bacteria closely related to pathogens of public health concern. Furthermore, this aircraft background is different from outdoor air, suggesting different probes may be needed to detect airborne contaminants to achieve minimal false alarm rates. This study also indicates that aircraft HEPA filters could be used with other molecular techniques to further characterize background bacteria and in investigations in the wake of a disease outbreak.

PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity.

BACKGROUND: Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease. METHODS: Using a novel comprehensive, high-density DNA microarray (PhyloChip) we performed a phylogenetic analysis of the microbial community of the large bowel in a rat model in which intracolonic acetic acid in neonates was used to induce long lasting colonic hypersensitivity and decreased stool water content and frequency, representing the equivalent of human constipation-predominant IBS. KEY RESULTS: Our results revealed a significantly increased compositional difference in the microbial communities in rats with neonatal irritation as compared with controls. Even more striking was the dramatic change in the ratio of Firmicutes relative to Bacteroidetes, where neonatally irritated rats were enriched more with Bacteroidetes and also contained a different composition of species within this phylum. Our study also revealed differences at the level of bacterial families and species. CONCLUSIONS & INFERENCES: The PhyloChip is a useful and convenient method to study enteric microflora. Further, this rat model system may be a useful experimental platform to study the causes and consequences of changes in microbial community composition associated with IBS.

Role of the rapA gene in controlling antibiotic resistance of Escherichia coli biofilms.

By using a high-throughput screening method, a mutant of a uropathogenic Escherichia coli strain affected in the rapA gene was isolated. The mutant formed normal-architecture biofilms but showed decreased penicillin G resistance, although the mutation did not affect planktonic cell resistance. Transcriptome analysis showed that 22 genes were down-regulated in the mutant biofilm. One of these genes was yhcQ, which encodes a putative multidrug resistance pump. Mutants with mutations in this gene also formed biofilms with decreased resistance, although the effect was less pronounced than that of the rapA mutation. Thus, an additional mechanism(s) controlled by a rapA-regulated gene(s) was involved in wild-type biofilm resistance. The search for this mechanism was guided by the fact that another down-regulated gene in rapA biofilms, yeeZ, is suspected to be involved in extra cell wall-related functions. A comparison of the biofilm matrix of the wild-type and rapA strains revealed decreased polysaccharide quantities and coverage in the mutant biofilms. Furthermore, the (fluorescent) functional penicillin G homologue Bocillin FL penetrated the mutant biofilms more readily. The results strongly suggest a dual mechanism for the wild-type biofilm penicillin G resistance, retarded penetration, and effective efflux. The results of studies with an E. coli K-12 strain pointed to the same conclusion. Since efflux and penetration can be general resistance mechanisms, tests were conducted with other antibiotics. The rapA biofilm was also more sensitive to norfloxacin, chloramphenicol, and gentamicin.

Loss of bacterial diversity during antibiotic treatment of intubated patients colonized with Pseudomonas aeruginosa.

Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic treatment. Bacterial 16S rRNA genes were absent from aspirates obtained from patients briefly intubated for elective surgery but were detected by PCR in samples from all patients intubated for longer periods. Sequencing of 16S rRNA clone libraries demonstrated the presence of many orally, nasally, and gastrointestinally associated bacteria, including known pathogens, in the lungs of patients colonized with P. aeruginosa. PhyloChip analysis detected the same organisms and many additional bacterial groups present at low abundance that were not detected in clone libraries. For each patient, both culture-independent methods showed that bacterial diversity decreased following the administration of antibiotics, and communities became dominated by a pulmonary pathogen. P. aeruginosa became the dominant species in six of seven patients studied, despite treatment of five of these six with antibiotics to which it was sensitive in vitro. Our data demonstrate that the loss of bacterial diversity under antibiotic selection is highly associated with the development of pneumonia in ventilated patients colonized with P. aeruginosa. Interestingly, PhyloChip analysis demonstrated reciprocal changes in abundance between P. aeruginosa and the class Bacilli, suggesting that these groups may compete for a similar ecological niche and suggesting possible mechanisms through which the loss of microbial diversity may directly contribute to pathogen selection and persistence.

NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes.

Microbiologists conducting surveys of bacterial and archaeal diversity often require comparative alignments of thousands of 16S rRNA genes collected from a sample. The computational resources and bioinformatics expertise required to construct such an alignment has inhibited high-throughput analysis. It was hypothesized that an online tool could be developed to efficiently align thousands of 16S rRNA genes via the NAST (Nearest Alignment Space Termination) algorithm for creating multiple sequence alignments (MSA). The tool was implemented with a web-interface at http://greengenes.lbl.gov/NAST. Each user-submitted sequence is compared with Greengenes' 'Core Set', comprising approximately 10,000 aligned non-chimeric sequences representative of the currently recognized diversity among bacteria and archaea. User sequences are oriented and paired with their closest match in the Core Set to serve as a template for inserting gap characters. Non-16S data (sequence from vector or surrounding genomic regions) are conveniently removed in the returned alignment. From the resulting MSA, distance matrices can be calculated for diversity estimates and organisms can be classified by taxonomy. The ability to align and categorize large sequence sets using a simple interface has enabled researchers with various experience levels to obtain bacterial and archaeal community profiles.

Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.

A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.

Comprehensive aligned sequence construction for automated design of effective probes (CASCADE-P) using 16S rDNA.

MOTIVATION: Prokaryotic organisms have been identified utilizing the sequence variation of the 16S rRNA gene. Variations steer the design of DNA probes for the detection of taxonomic groups or specific organisms. The long-term goal of our project is to create probe arrays capable of identifying 16S rDNA sequences in unknown samples. This necessitated the authentication, categorization and alignment of the >75 000 publicly available '16S' sequences. Preferably, the entire process should be computationally administrated so the aligned collection could periodically absorb 16S rDNA sequences from the public records. A complete multiple sequence alignment would provide a foundation for computational probe selection and facilitates microbial taxonomy and phylogeny. RESULTS: Here we report the alignment and similarity clustering of 62 662 16S rDNA sequences and an approach for designing effective probes for each cluster. A novel alignment compression algorithm, NAST (Nearest Alignment Space Termination), was designed to produce the uniform multiple sequence alignment referred to as the prokMSA. From the prokMSA, 9020 Operational Taxonomic Units (OTUs) were found based on transitive sequence similarities. An automated approach to probe design was straightforward using the prokMSA clustered into OTUs. As a test case, multiple probes were computationally picked for each of the 27 OTUs that were identified within the Staphylococcus Group. The probes were incorporated into a customized microarray and were able to correctly categorize Staphylococcus aureus and Bacillus anthracis into their correct OTUs. Although a successful probe picking strategy is outlined, the main focus of creating the prokMSA was to provide a comprehensive, categorized, updateable 16S rDNA collection useful as a foundation for any probe selection algorithm.

Sequence-specific identification of 18 pathogenic microorganisms using microarray technology.

We have developed a Multi-Pathogen Identification (MPID) microarray for high confidence identification of eighteen pathogenic prokaryotes, eukaryotes and viruses. Analysis of amplified products from pathogen genomic DNA using microarray hybridization allows for highly specific and sensitive detection, and allows the discrimination between true amplification products and false positive amplification products that might be derived from primers annealing to non-target sequences. Species-specific primer sets were used to amplify multiple diagnostic regions unique to each individual pathogen. Amplified products were washed over the surface of the microarray, and labelled with phycoerythrin-streptavidin for fluorescence detection. A series of overlapping 20-mer oligonucleotide probes hybridize to the entire diagnostic region, while parallel hybridizations on the same surface allow simultaneous screening for all organisms. Comparison to probes that differ by a single mismatch at the central position reduced the contribution of non-specific hybridization. Samples containing individual pathogens were analyzed in separate experiments and the corresponding species-specific diagnostic regions were identified by fluorescence among their highly redundant probe sets. On average, 91% of the 53 660 pathogen probes on the MPID microarray performed as predicted. The limit of detection was found to be as little as 10 fg of B. anthracis DNA in samples that were amplified with six diagnostic primer-pairs. In contrast, PCR products were not observed at this concentration when identical samples were prepared and visualized by agarose gel electrophoresis.

Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar enteritidis.

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.

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.

[Children and HIV/AIDS--prevention, follow up and treatment]. / Barn og HIV/AIDS--forebygging, oppfølging og behandling.

At the global level, the HIV/AIDS epidemic has reached dramatic proportions. According to WHO, more than 1 million children are currently living with HIV infection. In Norway (population 4.4 million), a cumulative total of only 33 children with HIV infection have been reported, making HIV/AIDS a rare disease. In view of the limited experience with HIV/AIDS among Norwegian children, strategies for the clinical management of HIV infection are discussed in light of recent guidelines from the United States and Europe. Procedures for prophylactic treatment and follow-up of children born to HIV infected mothers, and procedures and follow-up of antiretroviral treatment of HIV infected children are proposed.

Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization.

The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization. Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH). In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium. Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E. coli genome, the differences were directly placed in the genome map. About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other. Additional differences in most cases represent sequences that have diverged considerably in the course of evolution. Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species.

Molecular characterization and sequence of a methionine biosynthetic locus from Pseudomonas syringae.

Two methionine biosynthetic genes in Pseudomonas syringae pv. syringae, metX and metW, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces. The metXW locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness. Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa. The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserine O-acetyltransferases. This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus Leptospira, and several gram-positive bacterial genera. Both metX and metW were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine. MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes. Several classes of methionine auxotrophs, including metX and metW mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness. This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates.

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.

Influence of immigration on epiphytic bacterial populations on navel orange leaves.

Factors that influenced the increase in epiphytic bacterial population size on navel orange leaves during winter months were investigated to test the assumption that such populations were the result of multiplication on orange leaves. The population sizes of bacteria of different kinds, including ice nucleation-active (Ice(sup+)) bacteria, were from 6- to 30-fold larger on leaves of navel orange trees adjacent to other plant species than on trees growing near other citrus species. Total and Ice(sup+) bacterial population sizes on other plant species growing near navel orange trees were from 18- to 60-fold and 2- to 18,000-fold larger, respectively, than on navel orange trees. About twice the number of bacterial cells of a given type were deposited onto petri dishes opened simultaneously in navel orange orchards with other plant species nearby as in orchards surrounded by citrus trees. Epiphytic bacteria and airborne bacteria were more numerous near the upwind edge of orchards bordering on other plant species, but not in orchards adjacent to other citrus trees, and decreased with distance from other plant species. Navel orange leaves also exhibited progressive increases in the ability to supercool as a function of increasing distance from the upwind edge of orchards adjacent to other plant species but not in orchards adjacent to other citrus trees. While the population size of three different bacterial strains remained nearly constant for 60 days after inoculation, total bacterial populations increased more than 50-fold during this period. These results suggest that immigration of bacteria from plants having high epiphytic bacterial populations could account for most, if not all, of the seasonal increase in bacterial populations on navel orange leaves and have important implications for procedures to modify bacterial communities on leaves.

Identification of a region of genetic variability among Bacillus anthracis strains and related species.

The identification of a region of sequence variability among individual isolates of Bacillus anthracis as well as the two closely related species, Bacillus cereus and Bacillus mycoides, has made a sequence-based approach for the rapid differentiation among members of this group possible. We have identified this region of sequence divergence by comparison of arbitrarily primed (AP)-PCR "fingerprints" generated by an M13 bacteriophage-derived primer and sequencing the respective forms of the only polymorphic fragment observed. The 1,480-bp fragment derived from genomic DNA of the Sterne strain of B. anthracis contained four consecutive repeats of CAATATCAACAA. The same fragment from the Vollum strain was identical except that two of these repeats were deleted. The Ames strain of B. anthracis differed from the Sterne strain by a single-nucleotide deletion. More than 150 nucleotide differences separated B. cereus and B. mycoides from B. anthracis in pairwise comparisons. The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kDa protein located between the carboxy terminus of an upstream ORF (designated orf1) and the amino terminus of a downstream ORF (designated lytB). The sequence variation was primarily in vrrA, which was glutamine- and proline-rich (30% of total) and contained repetitive regions. A large proportion of the nucleotide substitutions between species were synonymous. vrrA has 35% identity with the microfilarial sheath protein shp2 of the parasitic worm Litomosoides carinii.

Genetic variability of Bacillus anthracis and related species.

We evaluated the abilities of pulsed-field gel electrophoresis (PFGE) and sequences of intergenic spacer regions (ISRs) between two highly conserved genes, 16S-23S rDNA and gyrB-gyrA ISRs, to detect variation in strains of Bacillus anthracis as well as two closely related species, B. cereus ATCC 14579 and B. mycoides ATCC 6462. For each restriction enzyme, (NotI, SfiI, and SmaI), the PFGE banding patterns for three B. anthracis strains (Ames, Vollum, and Sterne) were identical. However, closely related species could be differentiated from B. anthracis and from each other. PCR amplification of the 16S-23S rDNA ISR yielded a 143- to 144-bp fragment, showing identical sequences for B. anthracis strains, one nucleotide deletion between B. cerus and B. anthracis, and 13 nucleotide differences between B. mycoides and B. anthracis. The gyrase ISR sequences (121 bp) in B. anthracis strains were also identical, but those in B. cereus and B. mycoides differed from that in B. anthracis by 1 and 2 nucleotides, respectively, and from each other by only 1 nucleotide. Given the diverse geographic origins of these B. anthracis strains, this species is very homogenous. We conclude that methods such as PFGE and sequences of ISRs may be useful in separating B. anthracis from closely related species, but more sensitive methods are needed for strain identification of B. anthracis.