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.

Coxiella burnetii is a human pathogen that causes the serious zoonotic disease Q fever. It is ubiquitous in the environment and due to its wide host range, long-range dispersal potential and classification as a bioterrorism agent, this microorganism is considered an HHS Select Agent. In the event of an outbreak or intentional release, laboratory strain typing methods can contribute to epidemiological investigations, law enforcement investigation and the public health response by providing critical information about the relatedness between C. burnetii isolates collected from different sources. Laboratory cultivation of C. burnetii is both time-consuming and challenging. Availability of strain collections is often limited and while several strain typing methods have been described over the years, a true gold-standard method is still elusive. Building upon epidemiological knowledge from limited, historical strain collections and typing data is essential to more accurately infer C. burnetii phylogeny. Harmonization of auspicious high-resolution laboratory typing techniques is critical to support epidemiological and law enforcement investigation. The single nucleotide polymorphism (SNP) -based genotyping approach offers simplicity, rapidity and robustness. Herein, we demonstrate SNPs identified within 16S rRNA gene sequences can differentiate C. burnetii strains. Using this method, 55 isolates were assigned to six groups based on six polymorphisms. These 16S rRNA SNP-based genotyping results were largely congruent with those obtained by analyzing restriction-endonuclease (RE)-digested DNA separated by SDS-PAGE and by the high-resolution approach based on SNPs within multispacer sequence typing (MST) loci. The SNPs identified within the 16S rRNA gene can be used as targets for the development of additional SNP-based genotyping assays for C. burnetii.

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.

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis including beta-lactams such as penicillins (amoxicillin/clavulanic acid), cephalosporins (ceftazidime), carbapenems (imipenem and meropenem), as well as tetracyclines and sulfonamides. We sequenced, assembled, and annotated the Bp1651 genome, and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A beta-lactamase and was previously implicated in resistance to beta-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance, and T147A contributed to amoxicillin/clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely due to a frame shift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

During high-impact events involving Bacillus anthracis, such as the Amerithrax incident of 2001 or the anthrax outbreaks in Russia and Sweden in 2016, critical decisions to reduce morbidity and mortality include rapid selection and distribution of effective antimicrobial agents for treatment and post-exposure prophylaxis. Detection of antimicrobial resistance currently relies on a conventional broth microdilution (BMD) method that requires a 16 - 20 hour incubation time for B. anthracis Advances in high-resolution optical screening offer a new technology to more rapidly evaluate antimicrobial susceptibility and to simultaneously assess growth characteristics of an isolate. Herein, we describe a new method developed and evaluated as a rapid antimicrobial susceptibility test for B. anthracis This method is based on automated, digital, time-lapse microscopy to observe growth and morphological effects of relevant antibiotics using an optical screening instrument, the oCelloScopeTM B. anthracis strains were monitored over time in the presence and absence of penicillin, ciprofloxacin, and doxycycline. Susceptibility to each antibiotic was determined in ≤ 4 hours, a 75-80% decrease in the time required for conventional methods. Time-lapse video imaging compiled from the optical screening images revealed unexpected differences in growth characteristics among strains of B. anthracis, which is considered to be a clonal organism. This technology provides a new approach for rapidly detecting phenotypic antimicrobial resistance and for documenting growth attributes that may be beneficial in further characterization of individual strains. IMPORTANCE: Early treatment of bacterial infections such as anthrax can dramatically improve survival rates and outcomes for affected populations. Conventional, gold-standard methods to detect drug resistance, such as broth microdilution, functionally assess the ability of bacteria to grow in the presence of drug, but are time intensive and rely on the subjective interpretation of results. Here, we describe the application of automated, time-lapsed optical imaging to rapidly and accurately detect single- and multi-drug resistant strains of Bacillus anthracis based on growth in microtiter cultures. Drug resistance was determined up to 16 hours faster than the conventional BMD method and the ability to visualize growth of B. anthracis in real-time revealed novel growth morphologies. Detailed growth characteristics of strains and rapid time-to-susceptibility results can assist in critical decision-making about clinical treatment or post-exposure prophylaxis regimes during public health emergency events involving infections such as anthrax.

The emergence and spread of colistin resistance among multidrug-resistant (MDR) Klebsiella pneumoniae represent a critical threat to global health. Here, we report the complete genome sequences of 10 MDR, colistin-susceptible and -resistant K. pneumoniae clinical isolates obtained in Pakistan between 2010 and 2013.

We report here the complete annotated genome sequence of the Burkholderia stabilis type strain ATCC BAA-67. There were three circular chromosomes with a combined size of 8,527,947 bp and G+C composition of 66.4%. These characteristics closely resemble the genomes of other sequenced members of the Burkholderia cepacia complex.

Analysis of 16S ribosomal RNA (rRNA) genes is important in phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data is generally a consensus sequence. We describe here valuable data that is missing from consensus sequences, variable effects on sequence data generated from non-identical 16S rRNA amplicons, and the appearance of data displayed by different sequence software. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of the Bacillus cereus group: Bacillus anthracis, B. cereus, B. mycoides, and B. thuringiensis These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP), previously reported as specific to B. anthracis was detected in some B. cereus strains. However, a different SNP at position 1139 was identified as specific to B. anthracis, which is a biothreat agent with high rates of mortality. Compared with visual analysis of the electropherograms, base caller software frequently missed gene sequence variations or could not identify variant bases due to overlapping base calls. Accurate detection of 16S rRNA gene sequences that include intra-genomic variations can improve discrimination between closely-related species, improve the utility of 16S rRNA databases, and assist in rapid bacterial identification by targeted DNA sequence analysis or by whole genome sequencing performed by clinical or reference laboratories.

Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or post-exposure prophylaxis in the aftermath of the release of bacterial biothreat agents such as Bacillus anthracis, Yersinia pestis, or Burkholderia pseudomallei. Conventional susceptibility tests require 16 to 48 h incubation, depending on the bacterial species. We evaluated a method that is based on laser light scattering technology that measures cell density in real time. We determined that it has the ability to rapidly differentiate between growth (resistant) and no growth (susceptible) of several bacterial threat agents in the presence of clinically relevant antimicrobials. Results were available in <4 h for B. anthracis and <6 h for Y. pestis and B. pseudomallei. One exception was B. pseudomallei in the presence of ceftazidime, which required >10 h incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% - 75% for B. anthracis, Y. pestis, and B. pseudomallei when compared to conventional methods.

Burkholderia pseudomallei strain Bp1651, a human isolate, is resistant to all clinically relevant antibiotics. We report here on the finished genome sequence assembly and annotation of the two chromosomes of this strain. This genome sequence may assist in understanding the mechanisms of antimicrobial resistance for this pathogenic species.

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.