Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis

Abstract

Penicillin (PEN) is a low-cost option for anthrax treatment, but naturally occurring resistance has been reported. β-Lactamase expression (bla1, bla2) in Bacillus anthracis is regulated by a sigma factor (SigP) and its cognate anti-sigma factor (RsiP). Mutations leading to truncation of RsiP were previously described as a basis for PEN resistance. Here, we analyze whole-genome sequencing (WGS) data and compare the chromosomal sigP-bla1 regions from 374 B. anthracis strains to determine the frequency of mutations, identify mutations associated with PEN resistance, and evaluate the usefulness of WGS for predicting PEN resistance. Few (3.5%) strains contained at least 1 of 11 different mutations in sigP, rsiP, or bla1. Nine of these mutations have not been previously associated with PEN resistance. Four strains showed PEN resistance (PEN-R) by conventional broth microdilution, including 1 strain with a novel frameshift in rsiP. One strain that carries the same rsiP frameshift mutation as that found previously in a PEN-R strain showed a PEN-susceptible (PEN-S) phenotype and exhibited decreased bla1 and bla2 transcription. An unexpectedly small colony size, a reduced growth rate, and undetectable β-lactamase activity levels (culture supernatant and cell lysate) were observed in this PEN-S strain. Sequence analysis revealed mutations in genes associated with growth defects that may contribute to this phenotype. While B. anthracis rsiP mutations cannot be exclusively used to predict resistance, four of the five strains with rsiP mutations were PEN-R. Therefore, the B. anthracis sigP-bla1 region is a useful locus for WGS-based PEN resistance prediction, but phenotypic testing remains essential. IMPORTANCE Determination of antimicrobial susceptibility of B. anthracis is essential for the appropriate distribution of antimicrobial agents for postexposure prophylaxis (PEP) and treatment of anthrax. Analysis of WGS data allows for the rapid detection of mutations in antimicrobial resistance (AMR) genes in an isolate, but the presence of a mutation in an AMR gene does not always accurately predict resistance. As mutations in the anti-sigma factor RsiP have been previously associated with high-level penicillin resistance in a limited number of strains, we investigated WGS assemblies from 374 strains to determine the frequency of mutations and performed functional antimicrobial susceptibility testing. Of the five strains that contained mutations in rsiP, only four were PEN-R by functional antimicrobial susceptibility testing. We conclude that while sequence analysis of this region is useful for AMR prediction in B. anthracis, genetic analysis should not be used exclusively and phenotypic susceptibility testing remains essential.

Keywords: Bacillus anthracis; anthrax; penicillin resistance; whole-genome sequencing.

FIG 1

Diagram of the B. anthracis sigP-bla1 region. The numbers refer to ORFs in the B. anthracis Ames Ancestor reference sequence. The asterisk (*) indicates that pbp2 contains a frameshift that results in two predicted ORFs; the first contains 124 amino acids of the predicted PBP2, and the second contains the remaining 586 amino acids of the predicted PBP2. (Adapted from reference with permission).

FIG 2

Neighbor-joining tree of B. anthracis strains containing mutations within sigP, rsiP, and bla1 identified from the WGS screen. Strains from the three major B. anthracis clades (A, B, and C) were identified in the WGS screen (color coded) (24). PEN-R strains are colored in red. Asterisks (*) indicate strains containing an rsiP mutation. Control strains SK57, UT308, 2000031103 (strain 32), and Sterne, as well as the Ames Ancestor reference strain, were included for comparison.

FIG 3

Growth characteristics of Ba3027. (A) Growth kinetics of strains SK57 (PEN-R, rsiP 10 mutation), Ba3027 (PEN-S, rsiP 10 mutation), and Ba0878 (PEN-S, wild-type strain) were evaluated over a 12-h incubation at 35°C in broth. Growth was measured by the Segmentation and Extraction of Surface Area (SESA) algorithm. Graphs represent the average growth value ± standard deviations from three replicate wells. (B) Microscope images (×8) of single colonies were taken following an 18-h incubation at 35°C in ambient air on SBA (top); optical screen images represent bacterial growth in a 100-µl cell suspension after 7 h (bottom).

FIG 4

β-Lactamase production and semiquantitative RT-PCR analysis of bla1, bla2, and 16S transcripts in B. anthracis strains. (A) β-Lactamase activity of culture supernatants from strains SK57 (PEN-R, rsiP 10 mutation), UT308 (PEN-R, sigP 183 mutation, rsiP 10 mutation), Ba3027 (PEN-S, rsiP 10 mutation), Sterne (PEN-S), and Ba0878 (PEN-S, wild-type strain) was measured using nitrocefin. Error bars represent averages ± standard deviations. **, P = 0.001 to 0.01 (statistical significance compared to β-lactamase-producing strain UT308); ***, P < 0.001 (statistical significance of UT308 compared to SK57). (B) Expression of SK57, UT308, Ba3027, Sterne, and Ba0878 bla1, bla2, and 16S genes was analyzed by semiquantitative RT-PCR after 20 cycles. The molecular marker was run in lane M.