Benjamin J. Perry

Emergence of mmpT5 variants during bedaquiline treatment of mycobacterium intracellulare lung disease

ABSTRACT Bedaquiline (BDQ), a diarylquinoline antibiotic that targets ATP synthase, is effective for the treatment of Mycobacterium tuberculosis infections that no longer respond to conventional drugs. While investigating the off-label use of BDQ as salvage therapy, seven of 13 patients with Mycobacterium intracellulare lung disease had an initial microbiological response and then relapsed. Whole-genome comparison of pretreatment and relapse isolates of M. intracellulare uncovered mutations in a previously uncharacterized locus, mmpT5. Preliminary analysis suggested similarities between mmpT5 and the mmpR5 locus, which is associated with low-level BDQ resistance in M. tuberculosis. Both genes encode transcriptional regulators and are adjacent to orthologs of the mmpS5-mmpL5 drug efflux operon. However, MmpT5 belongs to the TetR superfamily, whereas MmpR5 is a MarR family protein. Targeted sequencing uncovered nonsynonymous mmpT5 mutations in isolates from all seven relapse cases, including two pretreatment isolates. In contrast, only two relapse patient isolates had nonsynonymous changes in ATP synthase subunit c (atpE), the primary target of BDQ. Susceptibility testing indicated that mmpT5 mutations are associated with modest 2- to 8-fold increases in MICs for BDQ and clofazimine, whereas one atpE mutant exhibited a 50-fold increase in MIC for BDQ. Bedaquiline shows potential for the treatment of M. intracellulare lung disease, but optimization of treatment regimens is required to prevent the emergence of mmpT5 variants and microbiological relapse.

Construction of a mariner-based transposon vector for use in insertion sequence mutagenesis in selected members of the Rhizobiaceae

BackgroundThe Rhizobiaceae family of Gram-negative bacteria often engage in symbiosis with plants of economic importance. Historically, genetic studies to identify the function of individual genes, and characterize the biology of these bacteria have relied on the use of classical transposon mutagenesis. To increase the rate of scientific discovery in the Rhizobiaceae there is a need to adapt high-throughput genetic screens like insertion sequencing for use in this family of bacteria. Here we describe a Rhizobiaceae compatible MmeI-adapted mariner transposon that can be used with insertion sequencing for high-throughput genetic screening.ResultsThe newly constructed mariner transposon pSAM_Rl mutagenized R. leguminosarum, S. meliloti, and A. tumefaciens at a high frequency. In R. leguminosarum, mutant pools were generated that saturated 88% of potential mariner insertions sites in the genome. Analysis of the R. leguminosarum transposon insertion sequencing data with a previously described hidden Markov model-based method resulted in assignment of the contribution of all annotated genes in the R. leguminosarum 3841 genome for growth on a complex medium. Good concordance was observed between genes observed to be required for growth on the complex medium, and previous studies.ConclusionsThe newly described Rhizobiaceaee compatible mariner transposon insertion sequencing vector pSAM_Rl has been shown to mutagenize at a high frequency and to be an effective tool for use in high-throughput genetic screening. The construction and validation of this transposon insertion sequencing tool for use in the Rhizobiziaceae will provide an opportunity for researchers in the Rhizobiaceae community to use high-throughput genetic screening, allowing for significant increase in the rate of genetic discovery, particularly given the recent release of genome sequences from many Rhizobiaceae strains.

The Use of Transposon Insertion Sequencing to Interrogate the Core Functional Genome of the Legume Symbiont Rhizobium leguminosarum

The free-living legume symbiont Rhizobium leguminosarum is of significant economic value because of its ability to provide fixed nitrogen to globally important leguminous food crops, such as peas and lentils. Discovery based research into the genetics and physiology of R. leguminosarum provides the foundational knowledge necessary for understanding the bacteriums complex lifestyle, necessary for augmenting its use in an agricultural setting. Transposon insertion sequencing (INSeq) facilitates high-throughput forward genetic screening at a genomic scale to identify individual genes required for growth in a specific environment. In this study we applied INSeq to screen the genome of R. leguminosarum bv. viciae strain 3841 (RLV3841) for genes required for growth on minimal mannitol containing medium. Results from this study were contrasted with a prior INSeq experiment screened on peptide rich media to identify a common set of functional genes necessary for basic physiology. Contrasting the two growth conditions indicated that approximately 10% of the chromosome was required for growth, under both growth conditions. Specific genes that were essential to singular growth conditions were also identified. Data from INSeq screening on mannitol as a sole carbon source were used to reconstruct a metabolic map summarizing growth impaired phenotypes observed in the Embden-Meyerhof-Parnas pathway, Entner-Doudoroff pathway, pentose phosphate pathway, and tricarboxylic acid cycle. This revealed the presence of mannitol dependent and independent metabolic pathways required for growth, along with identifying metabolic steps with isozymes or possible carbon flux by-passes. Additionally, genes were identified on plasmids pRL11 and pRL12 that are likely to encode functional activities important to the central physiology of RLV3841.

Role of O 2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate

Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547 [phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O2 (pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O2 and cell death. IMPORTANCE Rhizobium leguminosarum, a soil bacterium that forms N2-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O2 tensions during growth in soil and N2 fixation in symbiosis with legumes.

Draft Genome Sequence and Annotation of Phyllosphere-Persisting Salmonella enterica subsp. enterica Serovar Livingstone Strain CKY-S4, Isolated from an Urban Lake in Regina, Canada

ABSTRACT Here, we report the first draft genome sequence of Salmonella enterica subsp. enterica serovar Livingstone. This S. Livingstone strain CKY-S4 displayed biofilm formation and cellulose production and could persist on lettuce. This genome may help the study of mechanisms by which enteric pathogens colonize food crops.

Functional and genetic characterization of an incF-type multidrug resistance plasmid isolated from fresh spinach.

The presence of antibiotic-resistant bacteria and clinically-relevant antibiotic resistance genes within raw foods is an on-going food safety concern. It is particularly important to be aware of the microbial quality of fresh produce because foods such as leafy greens including lettuce and spinach are minimally processed and often consumed raw therefore they often lack a microbial inactivation step. This study characterizes the genetic and functional aspects of a mobile, multidrug resistance plasmid, pLGP4, isolated from fresh spinach bought from a farmers’ market. pLGP4 was isolated using a bacterial conjugation approach. The functional characteristics of the plasmid were determined using multidrug resistance profiling and plasmid stability assays. pLGP4 was resistant to six of the eight antibiotics tested and included ciprofloxacin and meropenem. The plasmid was stably maintained within host strains in the absence of an antibiotic selection. The plasmid DNA was sequenced using an Illumina MiSeq high throughput sequencing approach and assembled into contigs using SPAdes. PCR mapping and Sanger DNA sequencing of PCR amplicons was used to complete the plasmid DNA sequence. Comparative sequence analysis determined that the plasmid was similar to plasmids that have been frequently associated with multidrug resistant clinical isolates of Klebsiella spp. DNA sequence analysis showed pLGP4 harboured qnrB1 and several other antibiotic resistance genes including three β-lactamases: blaTEM-1, blaCTX-M-15 and blaOXA-1. The detection of a multidrug-resistant, clinically-relevant plasmid on fresh spinach emphasizes the importance for vegetable producers to implement evidence-based food safety approaches into their production practises to ensure the food safety of leafy greens.

Draft Whole-Genome Sequence of Deinococcus sp. UR1, a Putative Novel Species Isolated from an External Stainless Steel Surface in the Canadian Prairies

ABSTRACT Deinococcus sp. strain UR1, a resilient bacterium isolated from the surface of a stainless steel sign located on the University of Regina campus in Saskatchewan, Canada, was sequenced to 56-fold coverage to produce 73 contigs with a consensus length of 4,472,838 bp and a G+C content of 69.37%.

Whole-Genome Sequence and Annotation of Salmonella enterica subsp. enterica Serovar Enteritidis Phage Type 8 Strain EN1660

ABSTRACT The genome of Salmonella enterica subspecies enterica serovar Enteritidis phage type 8 strain EN1660, isolated from an outbreak in Thunder Bay, Canada, was sequenced to 46-fold coverage using an Illumina MiSeq with 300-bp paired-end sequencing chemistry to produce 28 contigs with an N50 value of 490,721 bp.

Draft Genome Sequence of Rheinheimera sp. KL1, Isolated from a Freshwater Lake in Southern Saskatchewan, Canada

ABSTRACT Rheinheimera sp. KL1 was isolated from an algal bloom in Katepwa Lake, Saskatchewan, Canada. The isolate shares genetic and physiological similarities with Rheinheimera tangshanensis. The genome is estimated to be 4,295,060 bp in length with a GC content of 46.37%. Sequence analysis suggests the strain carries a previously uncharacterized prophage.