Genomics of Aminoglycoside Resistance in Pseudomonas aeruginosa Bloodstream Infections at a United States Academic Hospital.

Pseudomonas aeruginosa frequently becomes resistant to aminoglycosides by the acquisition of aminoglycoside modifying enzyme (AME) genes and the occurrence of mutations in the mexZ, fusA1, parRS, and armZ genes. We examined resistance to aminoglycosides in a collection of 227 P. aeruginosa bloodstream isolates collected over 2 decades from a single United States academic medical institution. Resistance rates of tobramycin and amikacin were relatively stable over this time, while the resistance rates of gentamicin were somewhat more variable. For comparison, we examined resistance rates to piperacillin-tazobactam, cefepime, meropenem, ciprofloxacin, and colistin. Resistance rates to the first four antibiotics were also stable, although uniformly higher for ciprofloxacin. Colistin resistance rates were initially quite low, rose substantially, and then began to decrease at the end of the study. Clinically relevant AME genes were identified in 14% of isolates, and mutations predicted to cause resistance were relatively common in the mexZ and armZ genes. In a regression analysis, resistance to gentamicin was associated with the presence of at least one gentamicin-active AME gene and significant mutations in mexZ, parS, and fusA1. Resistance to tobramycin was associated with the presence of at least one tobramycin-active AME gene. An extensively drug-resistant strain, PS1871, was examined further and found to contain five AME genes, most of which were within clusters of antibiotic resistance genes embedded in transposable elements. These findings demonstrate the relative contributions of aminoglycoside resistance determinants to P. aeruginosa susceptibilities at a United States medical center. IMPORTANCE Pseudomonas aeruginosa is frequently resistant to multiple antibiotics, including aminoglycosides. The rates of resistance to aminoglycosides in bloodstream isolates collected over 2 decades at a United States hospital remained constant, suggesting that antibiotic stewardship programs may be effective in countering an increase in resistance. Mutations in the mexZ, fusA1, parR, pasS, and armZ genes were more common than acquisition of genes encoding aminoglycoside modifying enzymes. The whole-genome sequence of an extensively drug resistant isolate indicates that resistance mechanisms can accumulate in a single strain. Together, these results suggest that aminoglycoside resistance in P. aeruginosa remains problematic and confirm known resistance mechanisms that can be targeted for the development of novel therapeutics.

Skimming for barcodes: rapid production of mitochondrial genome and nuclear ribosomal repeat reference markers through shallow shotgun sequencing.

DNA barcoding is critical to conservation and biodiversity research, yet public reference databases are incomplete. Existing barcode databases are biased toward cytochrome oxidase subunit I (COI) and frequently lack associated voucher specimens or geospatial metadata, which can hinder reliable species assignments. The emergence of metabarcoding approaches such as environmental DNA (eDNA) has necessitated multiple marker techniques combined with barcode reference databases backed by voucher specimens. Reference barcodes have traditionally been generated by Sanger sequencing, however sequencing multiple markers is costly for large numbers of specimens, requires multiple separate PCR reactions, and limits resulting sequences to targeted regions. High-throughput sequencing techniques such as genome skimming enable assembly of complete mitogenomes, which contain the most commonly used barcoding loci (e.g., COI, 12S, 16S), as well as nuclear ribosomal repeat regions (e.g., ITS1&2, 18S). We evaluated the feasibility of genome skimming to generate barcode references databases for marine fishes by assembling complete mitogenomes and nuclear ribosomal repeats. We tested genome skimming across a taxonomically diverse selection of 12 marine fish species from the collections of the National Museum of Natural History, Smithsonian Institution. We generated two sequencing libraries per species to test the impact of shearing method (enzymatic or mechanical), extraction method (kit-based or automated), and input DNA concentration. We produced complete mitogenomes for all non-chondrichthyans (11/12 species) and assembled nuclear ribosomal repeats (18S-ITS1-5.8S-ITS2-28S) for all taxa. The quality and completeness of mitogenome assemblies was not impacted by shearing method, extraction method or input DNA concentration. Our results reaffirm that genome skimming is an efficient and (at scale) cost-effective method to generate all mitochondrial and common nuclear DNA barcoding loci for multiple species simultaneously, which has great potential to scale for future projects and facilitate completing barcode reference databases for marine fishes.

Environmental DNA from Marine Waters and Substrates: Protocols for Sampling and eDNA Extraction.

Environmental DNA (eDNA) analysis has emerged in recent years as a powerful tool for the detection, monitoring, and characterization of aquatic metazoan communities, including vulnerable species. The rapid rate of adopting the eDNA approach across diverse habitats and taxonomic groups attests to its value for a wide array of investigative goals, from understanding natural or changing biodiversity to informing on conservation efforts at local and global scales. Regardless of research objectives, eDNA workflows commonly include the following essential steps: environmental sample acquisition, processing and preservation of samples, and eDNA extraction, followed by eDNA sequencing library preparation, high-capacity sequencing and sequence data analysis, or other methods of genetic detection. In this chapter, we supply instructional details for the early steps in the workflow to facilitate researchers considering adopting eDNA analysis to address questions in marine environments. Specifically, we detail sampling, preservation, extraction, and quantification protocols for eDNA originating from marine water, shallow substrates, and deeper sediments. eDNA is prone to degradation and loss, and to contamination through improper handling; these factors crucially influence the outcome and validity of an eDNA study. Thus, we also provide guidance on avoiding these pitfalls. Following extraction, purified eDNA is often sequenced on massively parallel sequencing platforms for comprehensive faunal diversity assessment using a metabarcoding or metagenomic approach, or for the detection and quantification of specific taxa by qPCR methods. These components of the workflow are project-specific and thus not included in this chapter. Instead, we briefly touch on the preparation of eDNA libraries and discuss comparisons between sequencing approaches to aid considerations in project design.

Genomic Features Associated with the Degree of Phenotypic Resistance to Carbapenems in Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae strains cause severe infections that are difficult to treat. The production of carbapenemases such as the K. pneumoniae carbapenemase (KPC) is a common mechanism by which these strains resist killing by the carbapenems. However, the degree of phenotypic carbapenem resistance (MIC) may differ markedly between isolates with similar carbapenemase genes, suggesting that our understanding of the underlying mechanisms of carbapenem resistance remains incomplete. To address this problem, we determined the whole-genome sequences of 166 K. pneumoniae clinical isolates resistant to meropenem, imipenem, or ertapenem. Multiple linear regression analysis of this collection of largely blaKPC-3-containing sequence type 258 (ST258) isolates indicated that blaKPC copy number and some outer membrane porin gene mutations were associated with higher MICs to carbapenems. A trend toward higher MICs was also observed with those blaKPC genes carried by the d isoform of Tn4401. In contrast, ompK37 mutations were associated with lower carbapenem MICs, and extended spectrum ß-lactamase genes were not associated with higher or lower MICs in carbapenem-resistant K. pneumoniae. A machine learning approach based on the whole-genome sequences of these isolates did not result in a substantial improvement in prediction of isolates with high or low MICs. These results build upon previous findings suggesting that multiple factors influence the overall carbapenem resistance levels in carbapenem-resistant K. pneumoniae isolates. IMPORTANCE Klebsiella pneumoniae can cause severe infections in the blood, urinary tract, and lungs. Resistance to carbapenems in K. pneumoniae is an urgent public health threat, since it can make these isolates difficult to treat. While individual contributors to carbapenem resistance in K. pneumoniae have been studied, few reports explore their combined effects in clinical isolates. We sequenced 166 clinical carbapenem-resistant K. pneumoniae isolates to evaluate the contribution of known genes to carbapenem MICs and to try to identify novel genes associated with higher carbapenem MICs. The blaKPC copy number and some outer membrane porin gene mutations were associated with higher carbapenem MICs. In contrast, mutations in one specific porin, ompK37, were associated with lower carbapenem MICs. Machine learning did not result in a substantial improvement in the prediction of carbapenem resistance nor did it identify novel genes associated with carbapenem resistance. These findings enhance our understanding of the many contributors to carbapenem resistance in K. pneumoniae.

The complete mitochondrial genome of Costapex baldwinae (Gastropoda: Neogastropoda: Turbinelloidea: Costellariidae) from the Caribbean Deep-Sea.

We report the complete mitochondrial genome sequence of Costapex baldwinae, a Caribbean representative of a predominantly Indo-Pacific genus of gastropods that occurs on sunken wood at bathyal depths. The mitogenome is 15,321 bp in length and has a base composition of 29.2% A, 41.8% T, 12.0% C and 17.0% G. It contains 13 protein-coding, two ribosomal RNA, and 22 tRNA genes with the same gene order and strand orientation as other non-toxoglossan neogastropods. Phylogenetic analyses indicate that the superfamily Turbinelloidea, represented by this species, diverged early within the Neogastropod radiation, forming the sister group to a clade that includes five of the seven presently recognized superfamilies.

Comparison of genomic diversity and structure of sable antelope (Hippotragus niger) in zoos, conservation centers, and private ranches in North America.

As we enter the sixth mass extinction, many species that are no longer self-sustaining in their natural habitat will require ex situ management. Zoos have finite resources for ex situ management, and there is a need for holistic conservation programs between the public and private sector. Ex situ populations of sable antelope, Hippotragus niger, have existed in zoos and privately owned ranches in North America since the 1910s. Unknown founder representation and relatedness has made the genetic management of this species challenging within zoos, while populations on privately owned ranches are managed independently and retain minimal-to-no pedigree history. Consequences of such challenges include an increased risk of inbreeding and a loss of genetic diversity. Here, we developed and applied a customized targeted sequence capture panel based on 5,000 genomewide single-nucleotide polymorphisms to investigate the genomic diversity present in these uniquely managed populations. We genotyped 111 sable antelope: 23 from zoos, 43 from a single conservation center, and 45 from ranches. We found significantly higher genetic diversity and significantly lower inbreeding in herds housed in zoos and conservation centers, when compared to those in privately owned ranches, likely due to genetic-based breeding recommendations implemented in the former populations. Genetic clustering was strong among all three populations, possibly as a result of genetic drift. We propose that the North American ex situ population of sable antelope would benefit from a metapopulation management system, to halt genetic drift, reduce the occurrence of inbreeding, and enable sustainable population sizes to be managed ex situ.

Characterization of the complete mitochondrial genome of Amblyomma ovale, comparative analyses and phylogenetic considerations.

In this study, we sequenced two complete mitochondrial genomes of Amblyomma ovale, a tick of public health importance. Sequencing two distinct individuals, the resulting mitochondrial genomes were 14,756 and 14,760 bp in length and maintained the same gene order previously reported in Amblyomma. These were combined with RNA-seq derived mitochondrial sequences from three additional species, Amblyomma aureolatum, Amblyomma maculatum, and Amblyomma moreliae, to carry out mitogenome comparative and evolutionary analyses against all previously published tick mitochondrial genomes. We described a derivative genome rearrangement that isolates Ixodes from the remaining Ixodidae and consists of both a reverse translocation as well as an event of Tandem Duplication Random Loss. Genetic distance analyses indicated that cox2, nd1, nd5, and 16S are good candidates for future population studies in A. ovale. The phylogenetic analyses corroborated the utility of complete mitochondrial genomes as phylogenetic markers within the group. This study further supplements the genome information available for Amblyomma and facilitates future evolutionary and population genetic studies within the genus.

A tale of two genes: divergent evolutionary fate of haptoglobin and hemopexin in hemoglobinless Antarctic icefishes.

The evolution of Antarctic notothenioid fishes in the isolated freezing Southern Ocean has led to remarkable trait gains and losses. One of the most extraordinary was the loss of the major oxygen carrier hemoglobin (Hb) in the icefishes (family Channichthyidae). Although the mechanisms of this loss and the resulting compensatory changes have been well studied, the impact of Hb loss on the network of genes that once supported its recycling and disposal has remained unexplored. Here, we report the functional fate and underlying molecular changes of two such key Hb-supporting proteins across the icefish family - haptoglobin (Hp) and hemopexin (Hx), crucial in removing cytotoxic free Hb and heme, respectively. Hp plays a critical role in binding free Hb for intracellular recycling and absent its primary client, icefish Hp transcription is now vanishingly little, and translation into a functional protein is nearly silenced. Hp genotype degeneration has manifested in separate lineages of the icefish phylogeny with three distinct nonsense mutations and a deletion frame shift, as well as mutated polyadenylation signal sequences. Thus, Hb loss appears to have diminished selective constraint on Hp maintenance, resulting in its stochastic, co-evolutionary drift towards extinction. Hx binds free heme for iron recycling in hepatocytes. In contrast to Hp, Hx genotype integrity is preserved in the icefishes and transcription occurs at levels comparable to those in the red-blooded notothenioids. The persistence of Hx likely owes to continued selective pressure for its function from mitochondrial and non-Hb cellular hemoproteins.

Molecular mechanism and history of non-sense to sense evolution of antifreeze glycoprotein gene in northern gadids.

A fundamental question in evolutionary biology is how genetic novelty arises. De novo gene birth is a recently recognized mechanism, but the evolutionary process and function of putative de novo genes remain largely obscure. With a clear life-saving function, the diverse antifreeze proteins of polar fishes are exemplary adaptive innovations and models for investigating new gene evolution. Here, we report clear evidence and a detailed molecular mechanism for the de novo formation of the northern gadid (codfish) antifreeze glycoprotein (AFGP) gene from a minimal noncoding sequence. We constructed genomic DNA libraries for AFGP-bearing and AFGP-lacking species across the gadid phylogeny and performed fine-scale comparative analyses of the AFGP genomic loci and homologs. We identified the noncoding founder region and a nine-nucleotide (9-nt) element therein that supplied the codons for one Thr-Ala-Ala unit from which the extant repetitive AFGP-coding sequence (cds) arose through tandem duplications. The latent signal peptide (SP)-coding exons were fortuitous noncoding DNA sequence immediately upstream of the 9-nt element, which, when spliced, supplied a typical secretory signal. Through a 1-nt frameshift mutation, these two parts formed a single read-through open reading frame (ORF). It became functionalized when a putative translocation event conferred the essential cis promoter for transcriptional initiation. We experimentally proved that all genic components of the extant gadid AFGP originated from entirely nongenic DNA. The gadid AFGP evolutionary process also represents a rare example of the proto-ORF model of de novo gene birth where a fully formed ORF existed before the regulatory element to activate transcription was acquired.

The genomic basis for colonizing the freezing Southern Ocean revealed by Antarctic toothfish and Patagonian robalo genomes.

BACKGROUND: The Southern Ocean is the coldest ocean on Earth but a hot spot of evolution. The bottom-dwelling Eocene ancestor of Antarctic notothenioid fishes survived polar marine glaciation and underwent adaptive radiation, forming >120 species that fill all water column niches today. Genome-wide changes enabling physiological adaptations and the rapid expansion of the Antarctic notothenioids remain poorly understood. RESULTS: We sequenced and compared 2 notothenioid genomes-the cold-adapted and neutrally buoyant Antarctic toothfish Dissostichus mawsoni and the basal Patagonian robalo Eleginops maclovinus, representing the temperate ancestor. We detected >200 protein gene families that had expanded and thousands of genes that had evolved faster in the toothfish, with diverse cold-relevant functions including stress response, lipid metabolism, protein homeostasis, and freeze resistance. Besides antifreeze glycoprotein, an eggshell protein had functionally diversified to aid in cellular freezing resistance. Genomic and transcriptomic comparisons revealed proliferation of selcys-transfer RNA genes and broad transcriptional upregulation across anti-oxidative selenoproteins, signifying their prominent role in mitigating oxidative stress in the oxygen-rich Southern Ocean. We found expansion of transposable elements, temporally correlated to Antarctic notothenioid diversification. Additionally, the toothfish exhibited remarkable shifts in genetic programs towards enhanced fat cell differentiation and lipid storage, and promotion of chondrogenesis while inhibiting osteogenesis in bone development, collectively contributing to the achievement of neutral buoyancy and pelagicism. CONCLUSIONS: Our study revealed a comprehensive landscape of evolutionary changes essential for Antarctic notothenioid cold adaptation and ecological expansion. The 2 genomes are valuable resources for further exploration of mechanisms underlying the spectacular notothenioid radiation in the coldest marine environment.

The complete mitochondrial genome of the critically endangered Atlantic humpback dolphin, Sousa teuszii (Kükenthal, 1892).

The Atlantic humpback dolphin remains an understudied, critically endangered cetacean species. Here, we describe the first complete mitogenome of Sousa teuszii, derived from an animal stranded on Île des Oiseaux, Sine Saloum, Senegal. The S. teuszii mitogenome is composed of 16,384 base pairs and is 98.1% identical to its closest relative with a mitogenome, Sousa chinensis. Phylogenetic analysis confirms its placement with S. chinensis, as well as the placement of the genus Sousa within subfamily Delphininae.

Environmental reservoirs for exoS+ and exoU+ strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses its type III secretion system to inject the effector proteins ExoS and ExoU into eukaryotic cells, which subverts these cells to the bacterium's advantage and contributes to severe infections. We studied the environmental reservoirs of exoS+ and exoU+ strains of P. aeruginosa by collecting water, soil, moist substrates and plant samples from environments in the Chicago region and neighbouring states. Whole-genome sequencing was used to determine the phylogeny and type III secretion system genotypes of 120 environmental isolates. No correlation existed between geographic separation of isolates and their genetic relatedness, which confirmed previous findings of both high genetic diversity within a single site and the widespread distribution of P. aeruginosa clonal complexes. After excluding clonal isolates cultured from the same samples, 74 exoS+ isolates and 16 exoU+ isolates remained. Of the exoS+ isolates, 41 (55%) were from natural environmental sites and 33 (45%) were from man-made sites. Of the exoU+ isolates, only 3 (19%) were from natural environmental sites and 13 (81%) were from man-made sites (p < 0.05). These findings suggest that man-made water systems may be a reservoir from which patients acquire exoU+ P. aeruginosa strains.

Reconstruction of the repetitive antifreeze glycoprotein genomic loci in the cold-water gadids Boreogadus saida and Microgadus tomcod.

Antifreeze glycoproteins (AFGPs) are a novel evolutionary innovation in members of the northern cod fish family (Gadidae), crucial in preventing death from inoculative freezing by environmental ice in their frigid Arctic and sub-Arctic habitats. However, the genomic origin and molecular mechanism of evolution of this novel life-saving adaptive genetic trait remained to be definitively determined. To this end, we constructed large insert genomic DNA BAC (bacterial artificial chromosome) libraries for two AFGP-bearing gadids, the high-Arctic polar cod Boreogadus saida and the cold-temperate Atlantic tomcod Microgadus tomcod, to isolate and sequence their AFGP genomic regions for fine resolution evolutionary analyses. The BAC library construction encountered poor cloning efficiency initially, which we resolved by pretreating the agarose-embedded erythrocyte DNA with a cationic detergent, a method that may be of general use to BAC cloning for teleost species and/or where erythrocytes are the source of input DNA. The polar cod BAC library encompassed 92,160 clones with an average insert size of 94.7 kbp, and the Atlantic tomcod library contained 73,728 clones with an average insert size of 89.6 kbp. The genome sizes of B. saida and M. tomcod were estimated by cell flow cytometry to be 836 Mbp and 645 Mbp respectively, thus their BAC libraries have approximately 10- and 9.7-fold genome coverage respectively. The inclusiveness and depth of coverage were empirically confirmed by screening the libraries with three housekeeping genes. The BAC clones that mapped to the AFGP genomic loci of the two gadids were then isolated by screening the BAC libraries with gadid AFGP gene probes. Eight minimal tiling path (MTP) clones were identified for B. saida, sequenced, and assembled. The B. saida AFGP locus reconstruction produced both haplotypes, and the locus comprises three distinct AFGP gene clusters, containing a total of 16 AFGP genes and spanning a combined distance of 512 kbp. The M. tomcod AFGP locus is much smaller at approximately 80 kbp, and contains only three AFGP genes. Fluorescent in situ hybridization with an AFGP gene probe showed the AFGP locus in both species occupies a single chromosomal location. The large AFGP locus with its high gene dosage in B. saida is consistent with its chronically freezing high Arctic habitats, while the small gene family in M. tomcod correlates with its milder habitats in lower latitudes. The results from this study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms and history of gadid AFGP gene evolution driven by northern hemisphere glaciation.

Metagenomic sequencing of environmental DNA reveals marine faunal assemblages from the West Antarctic Peninsula.

The West Antarctic Peninsula (WAP) is the fastest warming region in Antarctica where climate impact on the cold-adapted marine ecosystem is already visible. To monitor faunal changes in remote vast bodies of Antarctic waters, efficient and informative tools are essential. High-throughput sequencing of environmental DNA (eDNA) has emerged as one such tool for monitoring biodiversity and ecosystems, as it increases detection sensitivity of taxa, and sampling is often simpler and less costly than traditional collection methods. We collected water samples from four WAP shallow (≤300m) shelf regions, recovered the eDNA therein, and performed metagenomic shotgun sequencing and analyses to determine the effectiveness of this method to assess marine benthic faunal diversity; this includes the detection of deep-water predatory king crabs whose potential shoreward expansion to warming shelves has sparked much concern. Using a customized bioinformatics pipeline, we identified abundant signatures of common benthic invertebrate fauna, endemic notothenioid fishes, as well as lithodid king crabs. We also uncovered species richness and diversity comparable to biological inventories compiled by the use of traditional survey methods, supporting the efficacy of the eDNA shotgun sequencing approach. As the rate of eDNA degradation affects faunal detection sensitivity, we also quantified mitochondrial ND2 gene copies in eDNA derived from a WAP icefish and found ND2 copies persisted to at least 20days in the cold WAP water, much longer than values reported for temperate environments. We propose that eDNA metagenomic sequencing complements traditional sampling, and combining both will enable more inclusive biodiversity detection and faunal change monitoring in the vast Southern Ocean.

Impact of Type III Secretion Effectors and of Phenoxyacetamide Inhibitors of Type III Secretion on Abscess Formation in a Mouse Model of Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or microabscess formation. The rising incidence of multidrug resistance among P. aeruginosa isolates has increased both the economic burden and the morbidity and mortality associated with P. aeruginosa disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells in vitro In this study, we used a mouse model of P. aeruginosa abscess formation to test the in vivo efficacy of these compounds against the P. aeruginosa type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of the abscesses, as neutrophil depletion resulted in an increased abscess size, the formation of dermonecrotic lesions on the skin, and the dissemination of P. aeruginosa to internal organs. Consistent with the specificity of the T3SS-neutrophil interaction, P. aeruginosa bacteria lacking a functional T3SS were fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in the immune clearance of the bacteria. Finally, a P. aeruginosa strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together, our results further define the role of type III secretion in murine abscess formation and demonstrate the in vivo efficacy of phenoxyacetamide inhibitors in P. aeruginosa infection.