Genomewide Screening for Novel Genetic Variations Associated with Ciprofloxacin Resistance in Bacillus anthracis

Selection and isolation of CIP-resistant mutants.Japanese isolates of B. anthracis BA103 or BA104 were used as the parental strains. The BA103 strain was isolated from beef cattle in 1991, while BA104 was isolated from pigs in a sporadic incident in 1982. CIP-resistant mutants were isolated by serial passage on Muller-Hinton (MH) agar supplemented with 2-fold stepwise increasing concentrations of ciprofloxacin (CIP) (Wako, Osaka, Japan), from 0.0625 to 16 mg/liter CIP at 37°C. The MIC to antibiotics was determined using Etest (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions.

The handling of live B. anthracis was performed at biosafety level 3 (BSL-3) according to the Regulations on the Safety Control of Laboratories Handling Pathogenic Agents (SCLHPA) authorized by the biorisk management committee of the National Institute of Infectious Diseases (NIID), Japan. The management of group 2 agents, such as B. anthracis, was also regulated and required obligatory special permission, special biosafety facilities, and other measures for its possession and use as stated in the Law Concerning the Prevention of Infectious Diseases and Medical Care for Patients of Infections (the Infectious Diseases Control Law) by the Ministry of Health, Labor, and Welfare.

Short sequencing reads using GA II.Library preparation was performed using a genomic DNA sample prep kit (Illumina, San Diego, CA), and DNA clusters were generated on a slide using the cluster generation kit (version 2) on an Illumina cluster station (Illumina), according to the manufacturer's instructions. To obtain ∼10 million clusters for one lane, the general procedure as described in the standard recipe (Illumina) was performed as follows: template hybridization, isothermal amplification, linearization, blocking, denaturation, and hybridization of the sequencing primer (Illumina). All sequencing runs were performed with the Illumina genome analyzer II (GA II) using the Illumina sequencing kit (version 3). Fluorescent images were analyzed with the Illumina base-calling pipeline 1.3.2 to obtain FASTQ formatted sequence data.

Identification of single nucleotide variations (SNVs) and indels specific to in vitro-selected CIP-resistant B. anthracis mutants.A schematic flow chart of the data processing procedure is shown in Fig. S2 in the supplemental material. To identify specific SNVs/indels compared with the reference sequence of B. anthracis Ames 0581 (GenBank accession number NC_007530), Maq software (version 0.7.1) (12), a mapping assembler for short reads generated by the next-generation sequencer, was used with the easyrun Perl command as a default parameter. Read alignment for the validation of SNVs/indels was performed using the MapView graphical alignment viewer (1). Strain-specific SNVs/indels were extracted from the “cns.final.snp” or “cns.indelse” file, respectively, by comparison of the parental or CIP-resistant strains to Ames 0581. SNVs located in repetitive sequence regions (e.g., variable-number tandem repeat [VNTR], rRNA, and insertion sequence [IS]) were excluded from the analysis. Regarding the reliable detection of indels, the cns.indelse file, which includes potential indels showing abnormal alignment patterns, was processed by the recommended filtering technique to reduce the number of false positives, as described in the manual. Furthermore, we analyzed the 100 nucleotides surrounding the indels with a BlastN search against all sequenced short reads (parameter −F F −e 1.0E−10 −m 3). Putative SNVs/indels were finally verified by Sanger sequencing using the BigDye terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) (see Fig. S6 in the supplemental material). PCR and sequence primers are listed in Table S2 in the supplemental material.

qRT-PCR analysis.For the preparation of total RNA from B. anthracis, bacterial cells were cultured in brain heart infusion broth at 37°C to the mid-log phase, and then total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Total RNA was treated with the turbo DNA-free kit (Ambion, TX). Quantitative reverse transcription-PCR (qRT-PCR) was performed using 100 ng of total RNA, gene-specific primers designed by PrimerQuest (see Table S3 in the supplemental material), and the SuperScript III Platinum SYBR green one-step qRT-PCR kit with ROX (Invitrogen) and analyzed using the ABI Prism 7900HT real-time PCR system (Applied Biosystems). We used the following qRT-PCR program: RT reaction, 50°C for 3 min; initial denaturation, 95°C for 5 min; and 2 steps of amplification (40 cycles), 95°C for 15 s and 60°C for 30 s. The rplB gene encoding the 50S ribosomal protein L2 was used as the internal control.

Inhibition assay of multidrug resistance efflux system with reserpine.The MIC was determined using a CIP Etest (AB Biodisk) on MH agar containing 10 mg/liter reserpine (Sigma-Aldrich, St. Louis, MO) (14). Dimethyl sulfoxide was used as the control solvent.

Nucleotide sequence accession numbers.The short-read archives have been deposited in the DNA Data Bank of Japan (DDBJ; accession numbers DRA000067, DRA000068, DRA000069 and DRA000070 for BA103, BA104, BA103-CIPr, and BA104-CIPr, respectively).

Susceptibility testing for in vitro-selected CIP-resistant mutants of B. anthracis.Both BA103 and BA104 were susceptible to CIP at 0.064 mg/liter, the MIC as determined using Etest (Fig. 1 D). Resistant mutants were identified by repeating the selection procedure until a final concentration of 16.0 μg/ml CIP. Two CIP-resistant mutants, BA103-CIPr (MIC, 1.5 mg/liter) and BA104-CIPr (MIC, 6 mg/liter), were isolated from BA103 and BA104, respectively. Their CIP resistance levels increased by 23- and 94-fold, respectively (Fig. 1D). The two selected mutants also showed reduced susceptibility to another quinolone antibiotic, levofloxacin (see Fig. S1 in the supplemental material): for BA103 and BA104, the MIC was 0.047 mg/liter; for BA103-CIPr, 0.125 mg/liter; and for BA104-CIPr, 0.50 mg/liter. Cross-resistance to other antibiotics was not observed with the selected mutants: for penicillin, the MIC was 0.023 mg/liter; for tetracycline, <0.016 mg/liter; and for amikacin, 0.25 mg/liter (see Fig. S1 in the supplemental material).

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FIG. 1.

(A) Schematic gene organization around the GBAA0834 locus. Vertical arrowheads indicate positions of the mutations (black, deletion mutation; white, insertion mutation; gray, SNV). The detailed sequence is shown in Fig. S4 in the supplemental material. Numbers indicate the genomic position of the Ames 0581 strain. Relative transcriptional expression around GBAA0834 in BA103 (B) and BA104 (C) derivatives. (D) Testing of susceptibility to CIP on Muller-Hinton agar containing 10 mg/liter reserpine, which acts by blocking the multidrug efflux protein. Dimethyl sulfoxide was used as the control solvent (−). Arrows indicate MICs for CIP.

Summary of sequence reads and coverage.The Illumina GA II sequencer produced 6.85 to 10.31 million 50-base-long reads per strain after applying the quality filter of the Illumina base-calling pipeline 1.32 (see Table S1 in the supplemental material). More than 94.4% of the filter-passed reads were aligned to the reference sequences of the Ames 0581 strain using Maq software, resulting in more than a 56-fold coverage depth on average and a 92.7 to 99.34% coverage of the sequence, excluding ambiguous repetitive regions (Table S1 in the supplemental material). Since genomic analysis of laboratory strains of B. subtilis using Illumina GA II was performed at a maximum coverage of 51.7-fold (26), the coverage in the present study would be sufficient for the identification of SNVs and indels.

Detection of specific SNVs associated with quinolone resistance.At the first screening step, with reads mapping to Ames 0581, 93 possible SNVs were found in BA103 and 85 in BA103-CIPr (see Fig. S3A in the supplemental material). The Venn diagram in Fig. S3A indicates that 16, 6, and 75 positions could be SNV candidates for BA103-specific, BA103-CIPr-specific, and common SNVs, respectively. Incidentally, these SNV candidates appeared to be wrongly identified due to a misalignment with Maq, because 5 of the 6 BA103-CIPr-specific SNV candidates were found to be incorrect by Sanger sequencing methods. In most cases of such incorrect extractions, mapped reads showed a lower coverage depth at a 50% coefficient of variation (CV) at SNV candidates than those around the SNV position. Thus, the original filtering method was performed by calculating the CV with a coverage depth value around 100 bp at each SNV candidate. This filtering enabled us to refine the potential SNVs and led to the identification of one BA103-CIPr-specific SNV (Fig. S3A).

This BA103-CIPr-specific SNV was at genomic position 842518 and was a C-to-T base substitution in the GBAA0834 gene, encoding the TetR family transcriptional regulator. This substitution resulted in an amino acid substitution of threonine to isoleucine at the 38th amino acid (Thr₃₈Ile) (Table 1; Fig. 1A). SNV was not detected in the pXO1 and pXO2 plasmids between BA103 derivatives.

Using the same filtering methods described above, we identified 3 BA104-CIPr-specific SNVs, which were verified by Sanger sequencing (see Fig. S3A in the supplemental material). Among these 3 BA104-CIPr-specific SNVs, an SNV at genomic position 6850 was a G-to-C nucleotide substitution resulting in an amino acid substitution (Ala₈₆Pro) in the QRDR of the gyrA gene (GBAA0006) (Table 1). There are no previous reports of this SNV associated with CIP resistance in B. anthracis; however, the substitution has been found as Ala₈₄Pro in Escherichia coli (25) and Ser₈₅Pro in Staphylococcus aureus (10). The second SNV was located at genomic position 2138798 and was an A-to-G nucleotide substitution resulting in an amino acid substitution (Lys₈₀Glu) in GBAA2291 encoding the major sporulation sensor histidine kinase. The third SNV was located at genomic position 4595343 and was a substitution of T to C in the intergenic region between GBAA5072 and GBAA5074. SNV was not detected in the pXO1 and pXO2 plasmids between BA104 derivatives.

Detection of specific indels associated with quinolone resistance.At the first screening step, with reads mapping to B. anthracis Ames 0581, 7 possible short indels were identified in BA103 and 10 in BA103-CIPr (see Fig. S3B in the supplemental material). The Venn diagram in Fig. S3B shows that 4, 7, and 3 positions could be candidates for BA103-specific indels, BA103-CIPr-specific indels, and common indels, respectively. Further verification with a BlastN search around the positions of these indels refined 2 potential BA103-CIPr-specific indels (Table 2). These were validated by Sanger sequencing (see Fig. S6 in the supplemental material). The first short indel, located at genomic positions 842291 to 842295, was a deletion of 5 nucleotides (TAACA) in BA103-CIPr. This indel was located in the 132-bp intergenic region between GBAA0833 and GBAA0834 (Fig. 1A; see also Fig. S4A in the supplemental material). The second short indel was located at genomic positions 5215555 to 5215556 and was an insertion of 2 nucleotides (CA) within GBAA5724. This indel caused a frameshift in the coding sequence, resulting in the truncation of the amino acid sequence from amino acids (aa) 366 to 197 (see Fig. S5 in the supplemental material). Indel was not detected in the pXO1 and pXO2 plasmids between BA103 derivatives.

Using the filtering methods described above, we identified 9 BA104-CIPr-specific indels (Table 2). The first short indel was located at genomic positions 336869 to 336873 and was a deletion of 5 nucleotides (ACTTA) in GBAA0328, encoding a small multidrug resistance (SMR) family multidrug efflux pump. This indel caused a frameshift mutation leading to an increase in the coding sequence from aa 107 to aa 114 (see Fig. S5 in the supplemental material). The second short indel was located at genomic position 842291 and was a single nucleotide (T) deletion in the 132-bp intergenic region between GBAA0833 and GBAA0834 (Fig. 1A). Intriguingly, this deletion overlapped the first BA103-CIPr indel (Table 2; see also Fig. S4A in the supplemental material). Moreover, the third indel was located at genomic position 842715 and was a single nucleotide (C) insertion within the GBAA0834 TetR family regulator, the same as that mentioned above, leading to a truncation of the amino acid sequence from aa 191 to aa 117 (Fig. 1A; see also Fig. S4C in the supplemental material). The other 6 indels appeared to be generated by tandem duplications of 2 to 6 nucleotide bases, as summarized in Table 2. These indels resulted in a frameshift causing the truncation of the coding sequences of GBAA1077, GBAA2814, and GBAA4143 (see Fig. S5 in the supplemental material). On the other hand, the tandem duplications of 6 bases in GBAA3656 (parC) and GBAA5329 resulted in the incorporation of an additional 2 amino acids without frameshift mutation (Fig. S5).

Disruptive mutations of GBAA0834 are associated with overexpression of multidrug efflux pumps, leading to quinolone resistance.Three genes of the putative multidrug efflux system, GBAA0832, GBAA0833, and blt (GBAA0835), are located adjacent to GBAA0834 (Fig. 1A). We performed qRT-PCR to determine whether the TetR-type regulator (GBAA0834) is involved in the regulation of the multidrug efflux systems. All 4 genes were significantly upregulated in both BA103-CIPr and BA104-CIPr compared with their respective parental strains (Fig. 1B and C).

To evaluate whether multidrug efflux systems are involved in CIP resistance, the MIC was determined with reserpine, which acts by blocking efflux ability (14). Reserpine reduced the MIC by 4-fold in BA103-CIPr and 8-fold in BA104-CIPr (Fig. 1D), whereas it reduced the MIC by 1.3-fold in the parental strains.