Biology subjects

McLaughlin, H. P.

Publications and source records attributed to McLaughlin, H. P..

3 recordsLinked to original sources

bioRxiv2021

In silico analyses of penicillin binding proteins in Burkholderia pseudomallei uncovers SNPs with utility for phylogeography, species differentiation, and sequence typing

BackgroundBurkholderia pseudomallei causes melioidosis. Sequence typing this pathogen can reveal geographical origin and uncover epidemiological associations. Here, we describe B. pseudomallei genes encoding putative penicillin binding proteins (PBPs) and investigate their utility for determining phylogeography and differentiating closely related species. Methodology & Principal FindingsWe performed in silico analysis to characterize 10 PBP homologs in B. pseudomallei 1026b. As PBP active site mutations can confer {beta}-lactam resistance in Gram-negative bacteria, PBP sequences in two resistant B. pseudomallei strains were examined for similar alterations. Sequence alignments revealed single amino acid polymorphisms (SAAPs) unique to the multidrug resistant strain Bp1651 in the transpeptidase domains of two PBPs, but not directly within the active sites. Using BLASTn analyses of complete assembled genomes in the NCBI database, we determined genes encoding PBPs were conserved among B. pseudomallei (n=101) and Burkholderia mallei (n=26) strains. Within these genes, single nucleotide polymorphisms (SNPs) useful for predicting geographic origin of B. pseudomallei were uncovered. SNPs unique to B. mallei were also identified. Based on 11 SNPs identified in two genes encoding predicted PBP-3s, a dual-locus sequence typing (DLST) scheme was developed. The robustness of this typing scheme was assessed using 1,523 RefSeq genomes from B. pseudomallei (n=1,442) and B. mallei (n=81) strains, resulting in 32 sequence types (STs). Compared to multi-locus sequence typing (MLST), the DLST scheme demonstrated less resolution to support the continental separation of Australian B. pseudomallei strains. However, several STs were unique to strains originating from a specific country or region. The phylogeography of Western Hemisphere B. pseudomallei strains was more highly resolved by DLST compared to internal transcribed spacer (ITS) typing, and all B. mallei strains formed a single ST. SignificanceConserved genes encoding PBPs in B. pseudomallei are useful for strain typing, can enhance predictions of geographic origin, and differentiate strains of closely related Burkholderia species. Author SummaryBurkholderia pseudomallei causes the life-threatening disease melioidosis and is considered a biological threat and select agent by the United States government. This soil-dwelling bacterium is commonly found in regions of southeast Asia and northern Australia, but it is also detected in other tropical and sub-tropical areas around the world. With a predicted global burden of 165,000 annual cases and mortality rate that can exceed 40% without prompt and appropriate antibiotic treatment, understanding the epidemiology of melioidosis and mechanisms of antibiotic resistance in B. pseudomallei can benefit public health and safety. Recently, we identified ten conserved genes encoding putative penicillin binding proteins (PBPs) in B. pseudomallei. Here, we examined B. pseudomallei PBP sequences for amino acid mutations that may contribute to {beta}-lactam resistance. We also uncovered nucleotide mutations with utility to predict the geographical origin of B. pseudomallei strains and to differentiate closely related Burkholderia species. Based on 11 informative single nucleotide polymorphisms in two genes each encoding a PBP-3, we developed a simple, targeted dual-locus typing approach.

microbiology↗

bioRxiv2020

Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during β-lactam antimicrobial susceptibility testing

BackgroundIn Gram-negative species, {beta}-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans. ResultsHere, we identified {beta}-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant {beta}-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin-clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis. B. pseudomallei morphology changes were more consistent in IPM. Spheroplast formation followed by cell lysis was observed for all strains in broth containing IPM at concentrations greater than or equal to MICs, however, the time to cell lysis was variable. Length of B. pseudomallei cells was strain-, drug- and drug concentration-dependent. ConclusionsBoth resistant and susceptible B. pseudomallei strains exhibited filamentation during early exposure to AMC and CAZ at concentrations used to interpret susceptibility (based on CLSI guidelines). While developing a rapid {beta}-lactam antimicrobial susceptibility test based on cell-shape alone requires more extensive analyses, optical microscopy detected B. pseudomallei growth attributes that lend insight into antibiotic response and antibacterial mechanisms of action.

microbiology↗

bioRxiv2019

Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing

Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore sequencer (MinION) for Y. pestis (6.5h) and B. anthracis (8.5h) and sequenced strains with different AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality metrics were defined for genome assembly and mutation analysis. AMR markers were correctly detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing reads. While chromosomes and large and small plasmids were accurately assembled, including novel multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line strategy for on-site pathogen characterization to speed the public health response during a biothreat emergency.

microbiology↗