Genome Sequences of Penicillin-Resistant Bacillus anthracis Strains

Bacillus anthracis, the etiologic agent of anthrax, is characteristically susceptible to penicillin despite containing two chromosomal β-lactamase genes. Few naturally occurring penicillin-resistant B. anthracis isolates have been reported.

ABSTRACT

Bacillus anthracis, the etiologic agent of anthrax, is characteristically susceptible to penicillin despite containing two chromosomal β-lactamase genes. Few naturally occurring penicillin-resistant B. anthracis isolates have been reported. Here, we report the draft genome sequences for three penicillin-resistant B. anthracis strains, strain 32, UT308, and SK57.

ANNOUNCEMENT

Penicillin (PEN) is an effective treatment for anthrax, the disease caused by Bacillus anthracis; however, surveys indicate that PEN resistance occurs in 2 to 16% of strains (1). Expression of the B. anthracis β-lactamase genes (bla1 and bla2) is not sufficient to confer resistance to β-lactam antibiotics (1, 2). An extracytoplasmic function sigma factor (SigP) and its cognate anti-sigma factor (RsiP) regulate β-lactamase expression in B. anthracis (3). β-Lactamase expression and PEN resistance in B. anthracis strain 32 and derivatives of this strain were described previously (1–6); however, the genomes for these strains were not reported previously. Strain 32 was isolated from a fatal anthrax case in Northampton, England, in 1974 (7, 8). A mutation resulting in a truncated RsiP was described as the basis of PEN resistance in strain 32 (3). Previously, novobiocin was used to create the attenuated strain, UT308, from strain 32 (3), with a PEN MIC of >32 µg/ml (9). Strain SK57, with a PEN MIC of >32 µg/ml (9), was also studied as a PEN-resistant isolate (9, 10). SK57 was isolated from England in November 1975; however, additional details about the source are not available. Mutations in the sigP-rsiP region of these strains and β-lactamase expression are described by Ross et al. (3) and Gargis et al. (11).

Genomic DNA was purified from colonies cultured overnight on Trypticase soy agar II with 5% sheep blood (BD BBL) at 35°C in ambient air from glycerol stocks. DNA was isolated following MasterPure complete DNA and RNA purification kit (Epicentre) instructions using a modified lysis buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1.2% Triton X-100) with 60 min of incubation at 37°C. Sequencing was performed on an Illumina MiSeq platform using the version 2 kit (2 × 250-bp paired ends) with TruSeq DNA LT libraries for UT308 and Nextera XT libraries for strains 32 and SK57. Sequencing adapters were removed, and reads were trimmed using Cutadapt (version 1.14, settings: -m 75 -u -4 -U -4 -a AGATCGGAAGAG -A AGATCGGAAGAG). De novo assembly was performed using the SPAdes genome assembler (version 3.10.0, settings: –k 71,81,91,101). Assembled genomes were submitted to the NCBI Prokaryotic Genome Annotation Pipeline for annotation. The results are summarized in Table 1. Single-nucleotide polymorphisms (SNPs) between the assembled genomes and the Ames Ancestor reference sequence (GenBank accession number AE017334) were identified using the Parsnp utility (version 1.2, with default parameters) (12), and large insertions and deletions were identified using the MUMmer utility (version 4.0.0beta2, with default parameters) (13).

TABLE 1.

Genome characteristics of three penicillin-resistant B. anthracis strains^a

Strain/alternative name	GenBank accession no.	Total no. of reads	Chromosome				pXO1				pXO2
			Size (bp)	No. of contigs	No. of CDSs	Coverage (×)	Size (bp)	No. of contigs	No. of CDSs	Coverage (×)	Size (bp)	No. of contigs	No. of CDSs	Coverage (×)
2000031103/strain 32	QPKO00000000	12,627,396	5,178,673	39	5,097	71	180,475	6	162	88	94,895	1	92	68
UT308	QPKP00000000	13,700,164	5,227,967	27	5,189	175	135,250	5	99	991	NA	NA	NA	NA
2007740863/SK57	QPKQ00000000	12,655,760	5,163,883	34	5,100	89	180,476	6	161	157	94,899	1	92	128

^aCDSs, coding sequences; NA, information is not available, as pXO2 is absent in UT308. The NCBI RefSeq accession numbers are AE017334 (chromosome), AE017336 (plasmid pXO1), and AE017335 (plasmid pXO2).

Sequence analysis revealed that strains 32 and SK57 are closely related, with 159 SNPs separating the two strains. However, they have diverged and, relative to the Ames Ancestor reference sequence, there is a 13,945-bp deletion (positions 2124811 to 2138755) that is not found in strain 32 or UT308. This deletion results in the loss of 11 genes from the SK57 genome, including several amino acid transporters.

The three assembled genomes were each approximately 5.2 Mb long and had an average G+C content of 35.5%. Strains 32 and SK57 both contained pXO1 and pXO2. Plasmid pXO2 was absent from UT308, but a 135,293-bp contig corresponding to pXO1 was identified. In UT308, pXO1 is missing approximately 47 kb that includes the 44.8-kb pathogenicity island containing the three genes (lef, pagA, and cya) encoding the anthrax toxin proteins (14).

Data availability.

This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers QPKO00000000, QPKP00000000, and QPKQ00000000, and the first versions are described here. The raw reads were deposited in the Sequence Read Archive under the accession number SRP155512.