Penicillin (PEN) is a low-cost option for anthrax treatment, but naturally occurring resistance has been reported. beta-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 beta-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.

Clinical outcomes of melioidosis patients improve when the infecting agent, Burkholderia pseudomallei, is rapidly detected and identified by laboratory testing. Detection of B. pseudomallei DNA or recovery of the pathogen by culture from urine can support a diagnosis of melioidosis and guide patient care. Two new methods designated as Filter-Capture DNA Isolation (FCDI) and Filter Cellular Recovery (FCR) were developed to increase the sensitivity of detection and recovery of viable B. pseudomallei cells from small volumes (0.45 ml) of urine. DNA from eight strains of B. pseudomallei that were spiked into synthetic urine at low concentrations (1 x 102 CFU/ml) was detected in FCDI cell lysates using real-time PCR with greater consistency when compared with preparations from the QIAamp DNA Blood Mini kit. The FCR method showed greater B. pseudomallei detection sensitivity than conventional urine culture methods and resulted in typical colony growth at 24 h from as few as 1 x 102 CFU/ml. In addition, the FCR method does not rely on precipitation of a urine pellet by centrifugation and requires a smaller volume of urine. The FCDI and FCR methods described here could improve time to results and decrease the number of negative B. pseudomallei reports that are currently observed from urine culture as a consequence of samples containing low or variable bacterial cell concentrations.

In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis.

An effective public health response to a deliberate release of Bacillus anthracis will require rapid distribution of antimicrobial agents for post-exposure prophylaxis and treatment. However, conventional antimicrobial susceptibility testing for B. anthracis requires a 16-20 hr incubation period. To reduce this time, we have combined a modified broth microdilution (BMD) susceptibility testing method with real-time quantitative PCR (qPCR). Growth or inhibition of growth of B. anthracis cells incubated in two-fold dilutions of ciprofloxacin (CIP, 0.015-16 mug/ml) or doxycycline (DOX, 0.06-64 mug/ml) was determined by comparing the fluorescence threshold cycle (Ct) generated by target amplification from cells incubated in each drug concentration with the Ct of the no-drug (positive growth) control. This DeltaCt readily differentiated susceptible and non-susceptible strains. Among susceptible strains, the median DeltaCt was ≥7.51 cycles for CIP and ≥7.08 cycles for DOX when drug concentrations were at or above the CLSI breakpoint for susceptibility. For CIP- and DOX-nonsusceptible strains, the DeltaCt was <1.0 cycle at the breakpoint for susceptibility. When evaluated with 14 genetically and geographically diverse strains of B. anthracis, the rapid method provided the same susceptibility results as conventional methods but required less than 6 hr, significantly decreasing the time required for selection and distribution of appropriate medical countermeasures.