Rapid Nanopore Whole-Genome Sequencing for Anthrax Emergency Preparedness.

Human anthrax cases necessitate rapid response. We completed Bacillus anthracis nanopore whole-genome sequencing in our high-containment laboratory from a human anthrax isolate hours after receipt. The de novo assembled genome showed no evidence of known antimicrobial resistance genes or introduced plasmid(s). Same-day genomic characterization enhances public health emergency response.

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis.

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ß-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). 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 ß-lactamase and was previously implicated in resistance to ß-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 because of a frameshift 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 the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle.

Infections with tick-transmitted Borreliella (Borrelia) burgdorferi, the cause of Lyme disease, represent an increasingly large public health problem in North America and Europe. The ability of these spirochetes to maintain themselves for extended periods of time in their tick vectors and vertebrate reservoirs is crucial for continuance of the enzootic cycle as well as for the increasing exposure of humans to them. The stringent response mediated by the alarmone (p)ppGpp has been determined to be a master regulator in B. burgdorferi. It modulates the expression of identified and unidentified open reading frames needed to deal with and overcome the many nutritional stresses and other challenges faced by the spirochete in ticks and animal reservoirs. The metabolic and morphologic changes resulting from activation of the stringent response in B. burgdorferi may also be involved in the recently described non-genetic phenotypic phenomenon of tolerance to otherwise lethal doses of antimicrobials and to other antimicrobial activities. It may thus constitute a linchpin in multiple aspects of infections with Lyme disease borrelia, providing a link between the micro-ecological challenges of its enzootic life-cycle and long-term residence in the tissues of its animal reservoirs, with the evolutionary side effect of potential persistence in incidental human hosts.

Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis, Yersinia pestis, and Burkholderia pseudomallei by Use of Laser Light Scattering Technology.

Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or postexposure 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 of 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 of incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% to 75% for B. anthracis, Y. pestis, and B. pseudomallei compared to conventional methods.

The ribonucleases J1 and J2 are essential for growth and have independent roles in mRNA decay in Streptococcus pyogenes.

The paralogous ribonucleases J1 and J2, recently identified in Bacillus subtilis, have both endoribonucleolytic and 5'-to-3' exoribonucleolytic activities and participate in degradation and regulatory processing of mRNA. RNases J1 and J2 have partially overlapping target specificities, but only RNase J1 is essential for B. subtilis growth. Because mRNA decay is important in regulation of virulence factors of Streptococcus pyogenes (the group A streptococcus, GAS), we investigated the role of these newly described RNases in GAS. We found that conditional mutants for both RNases J1 and J2 require induction for growth, so we conclude that, unlike the case in B. subtilis, both of these RNases are essential for GAS growth, and therefore their functions are not redundant. We compared decay of representatives of the two classes of messages we had previously identified: Class I, which decay rapidly in exponential and stationary phase of growth (hasA and gyrA), and Class II, which are stable in stationary phase and exhibit a biphasic decay curve in exponential phase (sagA and sda). We report that RNases J1 and J2 affect the rate of decay of Class I messages and the length of the first phase in decay of Class II messages.

The histone-like protein Hlp is essential for growth of Streptococcus pyogenes: comparison of genetic approaches to study essential genes.

Selection of possible targets for vaccine and drug development requires an understanding of the physiology of bacterial pathogens, for which the ability to manipulate expression of essential genes is critical. For Streptococcus pyogenes (the group A streptococcus [GAS]), an important human pathogen, the lack of genetic tools for such studies has seriously hampered research. To address this problem, we characterized variants of the inducible Ptet cassette, in both sense and antisense contexts, as tools to regulate transcription from GAS genes. We found that although the three-operator Ptet construct [Ptet(O)3] had low uninduced expression, its induction level was low, while the two-operator construct [Ptet(O)2] was inducible to a high level but showed significant constitutive expression. Use of Ptet(O)3 in the chromosome allowed us to demonstrate previously that RNases J1 and J2 are required for growth of GAS. Here we report that the uninduced level from the chromosomally inserted Ptet(O)2 construct was too high for us to observe differential growth. For the highly expressed histone-like protein (Hlp) of GAS, neither chromosomal insertion of Ptet(O)2 or Ptet(O)3 nor their use on a high-copy-number plasmid to produce antisense RNA specific to hlp resulted in adequate differential expression. However, by replacing the ribosome binding site of hlp with an engineered riboswitch to control translation of Hlp, we demonstrated for the first time that this protein is essential for GAS growth.

Patterns and regulation of ribosomal RNA transcription in Borrelia burgdorferi.

BACKGROUND: Borrelia burgdorferi contains one 16S and two tandem sets of 23S-5S ribosomal (r) RNA genes whose patterns of transcription and regulation are unknown but are likely to be critical for survival and persistence in its hosts. RESULTS: RT-PCR of B. burgdorferi N40 and B31 revealed three rRNA region transcripts: 16S rRNA-alanine transfer RNA (tRNA Ala); tRNA Ile; and both sets of 23S-5S rRNA. At 34°C, there were no differences in growth rate or in accumulation of total protein, DNA and RNA in B31 cultured in Barbour-Stoenner-Kelly (BSK)-H whether rabbit serum was present or not. At 23°C, B31 grew more slowly in serum-containing BSK-H than at 34°C. DNA per cell was higher in cells in exponential as compared to stationary phase at either temperature; protein per cell was similar at both temperatures in both phases. Similar amounts of rRNA were produced in exponential phase at both temperatures, and rRNA was down-regulated in stationary phase at either temperature. Interestingly, a rel Bbu deletion mutant unable to generate (p)ppGpp did not down-regulate rRNA at transition to stationary phase in serum-containing BSK-H at 34°C, similar to the relaxed phenotype of E. coli relA mutants. CONCLUSIONS: We conclude that rRNA transcription in B. burgdorferi is complex and regulated both by growth phase and by the stringent response but not by temperature-modulated growth rate.

Antimicrobial Susceptibility of Western Hemisphere Isolates of Burkholderia pseudomallei: Phenotypic and Genomic Analyses.

Current antimicrobial treatment recommendations for melioidosis, the disease caused by Burkholderia pseudomallei, are largely based on studies of strains isolated from the Eastern Hemisphere (EH), where most human cases are identified and reported. In this study, we evaluated the antimicrobial susceptibility of 26 strains in the CDC (Centers for Diseases Control and Prevention) collection from the Western Hemisphere (WH) isolated from 1960 to 2015. Minimal inhibitory concentration (MIC) values were measured by standard broth microdilution for 16 antimicrobials following Clinical and Laboratory Standards Institute (CLSI) guidelines. Twenty-four of the 26 WH strains were susceptible to the six antimicrobials with CLSI-defined MIC susceptibility interpretive criteria for B. pseudomallei: amoxicillin/clavulanate, ceftazidime, imipenem, doxycycline, tetracycline, and trimethoprim/sulfamethoxazole. One WH strain demonstrated intermediate amoxicillin/clavulanate resistance and another strain had intermediate resistance to tetracycline. For all antimicrobials tested, the susceptibility profiles of WH isolates were comparable with previously reported MIC results of EH strains. The overall similarities suggest that the same antimicrobials are useful for melioidosis treatment in both the WH and EH. Using in silico analyses of WH genomes, we identified a novel amino acid substitution P258S in the beta-lactamase PenA, which may contribute to decreased susceptibility to amoxicillin/clavulanate in B. pseudomallei.

Regulation of virulence gene expression in Streptococcus pyogenes: determinants of differential mRNA decay.

Differential mRNA stability is an important mechanism for regulation of virulence factors in Streptococcus pyogenes (group A streptococcus, GAS), a serious and prevalent human pathogen. We have described 2 Classes of mRNA in GAS that are distinguishable by 1) stability in the stationary phase of growth, 2) kinetics of decay in exponential phase, and 3) effect of depletion of RNases J1 and J2 and polynucleotide phosphorylase (PNPase) on decay in exponential phase. We discuss features of the structure of an mRNA that appear to be important for determining the Class to which it belongs and present a model to explain differential mRNA decay.

Complete Genome Sequences for Three Chromosomes of the Burkholderia stabilis Type Strain (ATCC BAA-67).

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.

Characterization of the RelBbu Regulon in Borrelia burgdorferi Reveals Modulation of Glycerol Metabolism by (p)ppGpp.

The bacterial stringent response is triggered by deficiencies of available nutrients and other environmental stresses. It is mediated by 5'-triphosphate-guanosine-3'-diphosphate and 5'-diphosphate-guanosine-3'-diphosphate (collectively (p)ppGpp) and generates global changes in gene expression and metabolism that enable bacteria to adapt to and survive these challenges. Borrelia burgdorferi encounters multiple stressors in its cycling between ticks and mammals that could trigger the stringent response. We have previously shown that the B. burgdorferi stringent response is mediated by a single enzyme, RelBbu, with both (p)ppGpp synthase and hydrolase activities, and that a B. burgdorferi 297 relBbu null deletion mutant was defective in adapting to stationary phase, incapable of down-regulating synthesis of rRNA and could not infect mice. We have now used this deletion mutant and microarray analysis to identify genes comprising the rel regulon in B. burgdorferi cultured at 34°C, and found that transcription of genes involved in glycerol metabolism is induced by relBbu. Culture of the wild type parental strain, the relBbu deletion mutant and its complemented derivative at 34°C and 25°C in media containing glucose or glycerol as principal carbon sources revealed a growth defect in the mutant, most evident at the lower temperature. Transcriptional analysis of the glp operon for glycerol uptake and metabolism in these three strains confirmed that relBbu was necessary and sufficient to increase transcription of this operon in the presence of glycerol at both temperatures. These results confirm and extend previous findings regarding the stringent response in B. burgdorferi. They also demonstrate that the stringent response regulates glycerol metabolism in this organism and is likely crucial for its optimal growth in ticks.

Finished Annotated Genome Sequence of Burkholderia pseudomallei Strain Bp1651, a Multidrug-Resistant Clinical Isolate.

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.

Novel antibiotic-resistance markers in pGK12-derived vectors for Borrelia burgdorferi.

Extension of molecular genetics studies in Borrelia burgdorferi has been hampered by a lack of a variety of antibiotic resistance selective markers. Such markers are critical for isolation of B. burgdorferi strains with multiple mutants, for complementation with different cloning vectors, and for methods such as negative selection and reporter genes. To remedy this lack, resistance to various antibiotics of non-infectious (B31, 297) and infectious (N40) B. burgdorferi strains was examined and vectors incorporating appropriate antibiotic resistance genes as selective markers were developed. Minimal inhibitory concentrations for growth of B. burgdorferi on plates and in liquid media for aminoglycosides (kanamycin, gentamycin, sisomycin, amikacin, spectinomycin, neomycin), macrolides-lincosamids (erythromycin, lincomycin), coumarin derivatives (coumermycin A(1), novobiocin), glycopeptides (vancomycin, ristocetin), peptides (bacitracin, cycloserine), and chloramphenicol were found to differ significantly. There were also striking differences in resistance to these antibiotics between non-infectious and infectious B. burgdorferi strains. Antibiotic-resistance genes aph(3')-IIIa from Streptococcus faecalis, aad9 from Staphylococcus aureus Tn554, linA' from Staphylococcus aureus, and aac(3)-VIa from Enterobacter cloacae (conferring resistance to kanamycin, spectinomycin, lincomycin, and gentamycin/sisomycin, respectively) were subcloned either with their own promoters or under the control of the B. burgdorferi flaB promoter into pGK12 or its derivative pED1 to develop new cloning vectors for B. burgdorferi with the rationale that the absence of homologous regions between derived recombinant plasmids lacking the flaB promoter and the B. burgdorferi genome would permit avoidance of possible recombination with target DNA. Resistance to the corresponding antibiotic was conferred by vectors containing aph(3')-IIIa, aad9, linA' or aac(3)-VIa whether under the control of their own promoters or under the control of the flaB promoter. We conclude that these markers can be used for genetic study of B. burgdorferi and suggest they will be an important addition to the previously used coumermycin A(1), erythromycin and kanamycin in these studies.

Group A streptococcus inhibitors by high-throughput virtual screening.

Group A streptococcus (GAS) is a Gram-positive bacterium, which can cause multiple types of disease from mild infections of skin and throat to invasive and life-threatening infections. Recently RNase J1 and J2 were found to be essential for the growth of GAS. In order to identify inhibitors against RNase J1/J2, homology models of both the ligand-free apo-form and the ligand-bound holo-form complexes were constructed as templates for high-throughput virtual screening (HTVS). A focused small molecule library and the commercially available Maybridge database were employed as sources for potential inhibitors. A cell-based biological assay identified two compounds with 10 µM MIC activity.

Role of mRNA stability in growth phase regulation of gene expression in the group A streptococcus.

The impressive disease spectrum of Streptococcus pyogenes (the group A streptococcus [GAS]) is believed to be determined by its ability to modify gene expression in response to environmental stimuli. Virulence gene expression is controlled tightly by several different transcriptional regulators in this organism. In addition, expression of most, if not all, GAS genes is determined by a global mechanism dependent on growth phase. To begin an analysis of growth-phase regulation, we compared the transcriptome 2 h into stationary phase to that in late exponential phase of a serotype M3 GAS strain. We identified the arc transcript as more abundant in stationary phase in addition to the sag and sda transcripts that had been previously identified. We found that in stationary phase, the stability of sagA, sda, and arcT transcripts increased dramatically. We found that polynucleotide phosphorylase (PNPase [encoded by pnpA]) is rate limiting for decay of sagA and sda transcripts in late exponential phase, since the stability of these mRNAs was greater in a pnpA mutant, while stability of control mRNAs was unaffected by this mutation. Complementation restored the wild-type decay rate. Furthermore, in a pnpA mutant, the sagA mRNA appeared to be full length, as determined by Northern hybridization. It seems likely that mRNAs abundant in stationary phase are insensitive to the normal decay enzyme(s) and instead require PNPase for this process. It is possible that PNPase activity is limited in stationary phase, allowing persistence of these important virulence factor transcripts at this phase of growth.

Borrelia burgdorferi rel is responsible for generation of guanosine-3'-diphosphate-5'-triphosphate and growth control.

The global transcriptional regulator (p)ppGpp (guanosine-3'-diphosphate-5'-triphosphate and guanosine-3',5'-bisphosphate, collectively) produced by the relA and spoT genes in Escherichia coli allows bacteria to adapt to different environmental stresses. The genome of Borrelia burgdorferi encodes a single chromosomal rel gene (BB0198) (B. burgdorferi rel [rel(Bbu)]) homologous to relA and spoT of E. coli. Its role in (p)ppGpp synthesis, bacterial growth, and modulation of gene expression has not been studied in detail. We constructed a rel(Bbu) deletion mutant in an infectious B. burgdorferi 297 strain and isolated an extrachromosomally complemented derivative of this mutant. The mutant did not synthesize rel(Bbu) mRNA, Rel(Bbu) protein, or (p)ppGpp. This synthesis was restored in the complemented derivative, confirming that rel(Bbu) is necessary and sufficient for (p)ppGpp synthesis and degradation in B. burgdorferi. The rel(Bbu) mutant grew well during log phase in complete BSK-H but reached lower cell concentrations in the stationary phase than the wild-type parent, suggesting that (p)ppGpp may be an important factor in the ability of B. burgdorferi to adapt to stationary phase. Deletion of rel(Bbu) did not eliminate the temperature-elicited OspC shift, nor did it alter bmp gene expression or B. burgdorferi antibiotic susceptibility. Although deletion of rel(Bbu) eliminated B. burgdorferi virulence for mice, which was not restored by complementation, we suggest that rel(Bbu)-dependent accumulation of (p)ppGpp may be important for in vivo survival of this pathogen.