Draft Whole-Genome Sequences of Ciprofloxacin-Resistant Derivatives of a Bacillus anthracis ΔANR Strain Lacking pXO1 and pXO2 Plasmids

Mutants of an attenuated Bacillus anthracis (ΔANR) strain conferring increasing levels of ciprofloxacin resistance have been described. Here, we report the draft genome sequences of the parent strain (ΔANR pXO1⁻, pXO2⁻) and its derivatives conferring low (step 1; 0.5 μg/ml), medium (step 2; 8 to 16 μg/ml), and high (step 3; 32 to 64 μg/ml) levels of ciprofloxacin resistance.

ABSTRACT

Mutants of an attenuated Bacillus anthracis (ΔANR) strain conferring increasing levels of ciprofloxacin resistance have been described. Here, we report the draft genome sequences of the parent strain (ΔANR pXO1⁻, pXO2⁻) and its derivatives conferring low (step 1; 0.5 μg/ml), medium (step 2; 8 to 16 μg/ml), and high (step 3; 32 to 64 μg/ml) levels of ciprofloxacin resistance.

ANNOUNCEMENT

Bacillus anthracis is a spore-forming, Gram-positive bacterium that causes anthrax disease. Natural resistance to the two commonly used postexposure prophylactic antibiotics, doxycycline (DOX) and ciprofloxacin (CIP), is rare. To understand the mechanism of ciprofloxacin resistance, mutants of an attenuated Bacillus anthracis strain with high-level CIP resistance (>32 μg/ml) were isolated using a three-step in vitro selection protocol (1). Step 1 mutants were resistant to 0.5 μg/ml of CIP and had one of two gyrA quinolone resistance-determining region (QRDR) mutations. Step 2 mutants, derived from step 1 isolates, were resistant to 8 to 16 μg/ml of CIP and had one of three parC QRDR mutations. Step 3 mutants, derived from step 2 isolates, were resistant to 32 to 64 μg/ml of CIP. Mutants with an MIC of 64 μg/ml had one of two additional mutations within the gyrA QRDR or one of two mutations within the gyrB QRDR. Mutants for which the MIC was 32 μg/ml had no additional target modifications but showed evidence of enhanced ciprofloxacin efflux. Indeed, mutations in a TetR-type transcriptional regulator upstream of a gene encoding an efflux pump protein were shown to be present in some of these mutants (2). To establish the link between the high-level CIP resistance phenotype and the unknown non-QRDR genetic changes, we sequenced the genomes of these mutants. Here, we report the whole-genome sequences of the parent and its derivatives.

For DNA preparation, all strains were plated from glycerol stocks onto tryptic soy agar plates and incubated overnight at 37°C. One colony was transferred to 15 ml of tryptic soy broth and grown overnight at 37°C with shaking at 250 rpm. The culture tube was centrifuged, and the pellet was used for DNA extraction using the Epicentre MasterPure Gram-positive DNA purification kit (catalog no. MGP04100) according to the manufacturer’s instructions. DNA samples were quantified using the Invitrogen Quant-iT PicoGreen double-stranded DNA (dsDNA) kit and a Lambda DNA standard. The concentration and purity of 2-μl DNA samples were determined from absorption ratios at both 260/280 and 230/260 nm using the NanoDrop 2000 instrument.

Sequencing libraries were prepared using the Illumina Nextera XT kit and sequenced on an Illumina MiSeq (v2 mid-output 2 × 151-bp) instrument. MiSeq quality filters were used on quality-controlled (QC) reads, and the average Phred score was Q30. Adapter sequences were removed with Cutadapt (3) and assembled using Velvet v1.2.10 (4). The genome was annotated with the NCBI Prokaryotic Genome Annotation Pipeline v4.10 (5), and reference mapping was done with Bowtie 2 v2.3.5.1 (6). Default parameters were used for all software with the exception of Velvet (k-mer size, 2/3 of the average read length after adapter removal; -exp_cov auto) and Bowtie (-n-ceil “0.0.30”; –very-sensitive-local).

Data availability.

The accession numbers and sequence statistics of the 20 genomes are listed in Table 1.

TABLE 1.

Genome sequence details of B. anthracis ΔANR and its CIP-resistant derivatives^a

Strain	UCC no.b	CIPr levelc	SRA accession no.	GenBank accession no.	Total no. of reads	Assembly size (bp)	No. of contigs	No. of CDSsd	Depth of coveragee (×)	Breadth of coveragef (%)
ΔANR	BACI355	Sen	SRR10820631	WUDB00000000	7,704,385	5,180,208	103	5,329	210	99.93
S1-1	BACI356	I	SRR10820630	WXXJ00000000	5,935,864	5,114,359	56	5,271	167	99.93
S1-2	BACI357	I	SRR10820619	WXXI00000000	7,674,652	5,442,523	67	5,566	206	99.93
S2-1	BACI358	II	SRR10820618	WXXH00000000	12,594,028	4,990,635	286	5,180	352	99.93
S2-2	BACI359	II	SRR10820617	WXXG00000000	10,599,687	5,048,612	191	5,206	292	99.93
S2-3	BACI360	II	SRR10820616	WXXF00000000	9,362,926	4,962,434	279	5,177	230	99.93
S3-1	BACI361	III	SRR10820615	WUDA00000000	10,556,085	4,901,717	252	5,117	216	98.01
S3-3	BACI362	III	SRR10820614	WUCZ00000000	8,594,796	5,187,801	34	5,293	240	99.93
S3-4	BACI363	III	SRR10820613	WXXE00000000	8,252,056	4,867,450	230	5,036	232	99.93
S3-5	BACI364	III	SRR10820612	WXXD00000000	11,159,148	5,110,766	173	5,261	287	99.93
S3-6	BACI365	III	SRR10820629	WXXC00000000	8,698,057	4,942,892	59	5,064	221	99.93
S3-7	BACI366	III	SRR10820628	WUCY00000000	10,373,919	5,732,249	53	5,834	287	99.90
S3-8	BACI367	III	SRR10820627	WUCX00000000	10,340,752	5,003,507	98	5,128	289	99.93
S3-11	BACI368	III	SRR10820626	WXXB00000000	7,704,786	5,064,510	39	5,164	215	99.93
S3-12	BACI369	III	SRR10820625	WXXA00000000	8,613,155	5,179,602	46	5,275	243	99.93
S3-14	BACI370	III	SRR10820624	WUCW00000000	6,026,125	5,070,985	33	5,182	169	99.93
S3-15	BACI371	III	SRR10820623	WUCV00000000	7,543,074	5,185,778	29	5,295	202	99.93
S3-16	BACI372	III	SRR10820622	WXWZ00000000	8,911,392	5,422,954	43	5,570	240	99.93
S3-17	BACI373	III	SRR10820621	WUCU00000000	7,496,750	5,239,352	30	5,335	211	99.93
S3-18	BACI374	III	SRR10820620	WUCT00000000	8,075,762	5,330,389	41	5,437	223	99.93

^aThe differences in the genome sizes and gene numbers between the parent and derivatives could be due to the fact that these are draft and unfinished sequences with multiple contigs, as opposed to a single closed contig, and hence may have potential gaps in the assembly.

^bUCC, Unified Culture Collection of the U.S. Army Medical Research Institute of Infectious Diseases (https://www.usamriid.army.mil/ucc/).

^cCIP^r, ciprofloxacin resistance; Sen, sensitive at 0.06 μg/ml. Resistance levels, I, 0.5 μg/ml; II, 8 to 16 μg/ml; and III, 32 to 64 μg/ml.

^dCDSs, coding sequences.

^eReference sequence accession no. NZ_CP008752.

^fAgainst the reference sequence.