Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Abstract

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.

Fig. 1

Positions of subgroup–specific sequence differences in the 16S rRNA (A) and 23S rRNA (B) genes of seven B. cereus subgroups and corresponding sequences of reference microorganisms. The 16S rRNA and 23S rRNA sequences of B. anthracis Sterne (GenBank accession no. AF 176321 and AF 267877, respectively) in 5′ to 3′ orientation have been used as the consensus sequences. Vertical lines denote nucleotides identical to the consensus sequence. Numbers indicate distance from 5′-end of consensus rRNA sequence. Principle of pair probe's design is shown below sequences on Fig. 1A. R =G, or A; Y = T, or C.

Fig. 1

Positions of subgroup–specific sequence differences in the 16S rRNA (A) and 23S rRNA (B) genes of seven B. cereus subgroups and corresponding sequences of reference microorganisms. The 16S rRNA and 23S rRNA sequences of B. anthracis Sterne (GenBank accession no. AF 176321 and AF 267877, respectively) in 5′ to 3′ orientation have been used as the consensus sequences. Vertical lines denote nucleotides identical to the consensus sequence. Numbers indicate distance from 5′-end of consensus rRNA sequence. Principle of pair probe's design is shown below sequences on Fig. 1A. R =G, or A; Y = T, or C.

Fig. 2

Identification of B. cereus subgroup reference microorganisms with 16S rRNA oligonucleotide microarray. Bulk RNA from reference microorganisms was isolated, fluorescently labeled with LissRhod, and hybridized with a microarray bearing 20 b oligonucleotides. Positions of the probes and targeted subgroups (in rectangles) are shown in the upper right corner. Reference organisms and their subgroups (in parentheses) are indicated under each hybridization image captured with stationary microscope. For more details about probe design and probe abbreviations, see Table 1.

Fig. 3

Discrimination of subgroups Anthracis and Cereus A (A), or Mycoides A and Mycoides B (B) isolates with 23S rRNA probes. Image capture was carried out with stationary microscope for B. anthracis Sterne, B. thuringiensis B8 and B. cereus HER1414, and with portable reader for all other isolates (A, B). Probe signal ratio is shown on the right site of the images. For more details about probe design and probe abbreviations, see Table 2.

Fig. 3

Discrimination of subgroups Anthracis and Cereus A (A), or Mycoides A and Mycoides B (B) isolates with 23S rRNA probes. Image capture was carried out with stationary microscope for B. anthracis Sterne, B. thuringiensis B8 and B. cereus HER1414, and with portable reader for all other isolates (A, B). Probe signal ratio is shown on the right site of the images. For more details about probe design and probe abbreviations, see Table 2.

Fig. 4

Identification of single-base polymorphisms in reference microorganisms using hybridization of fluorescently labeled total RNA from B. cereus group bacteria to probes targeting the 23S rRNA. Images were captured with stationary microscope. R = G or A.

Fig. 5

Identification of “polymorphic” sites imitated in mixtures. Samples of B. thuringiensis B8 and B. thuringiensis 4Q281 labeled total RNA were hybridized with microarray separately (A, B) or mixed in proportions 1 to 2 (C), or 1 to 5 (D). Positions of the probes and targeted subgroups (in rectangles) are shown on scheme (E). Images were captured with stationary microscope. For probe signal ratio in pairs selected for SNP quantization see Table 3. Correlation between actual and predicted data is indicated on chart (F). Values of theoretical (x) versus experimental (y) signal ratios for four selected pairs obtained for two different mixtures, 1 to 2 and 1 to 5 (Table 3) determine each data point (blue rhombuses) position on the chart. Approximation line is described by equation y = Ax +k, where A and k are regression coefficient and error, respectively. One of the data point obtained for pair ps3/ps4 (rhombus in upper right corner) was not representative due to low hybridization signals. Calculations performed without these data revealed volumes of A to be equal to 0.96. The error values for all other pairs, where differences between experimental and theoretical ratios did not exceed 10% of the experimental ratio (Table 3), were 0.04 or less.

Fig. 6

Identification of microbial groups. Microarray containing probes ps25 and ps26 targeting 16S rRNA of the B. cereus group and the B. subtilis group, respectively, was hybridized with labeled bulk RNA of the reference microorganisms. Probe signal ratios are shown in the far right column. Images were captured with stationary microscope.

Fig. 7

Two strategies for microbial identification. Hierarchically nested probe design and analysis strategy (C and D) and endpoint probe design and analysis strategy (A and B). The hierarchical strategy reduces false positives due to concordance of positive microarray signals (red dots) at predecessor nodes in the phylogenetic tree, resulting in a “branch” analysis (D) instead of an “endpoint” analysis (B) for data interpretation and reporting.