Meredith S. Wright

Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities

The acquisition of new genetic material via horizontal gene transfer allows bacteria to rapidly evolve. One key to estimating the contribution of horizontal gene transfer to bacterial evolution is to quantify the abundance of mobile genetic elements (MGEs) in bacterial communities under varying degrees of selective pressure. We quantified class 1 integrase (intI1) gene abundance in total community DNA extracted from contaminated and reference riverine and estuarine microhabitats, and in metal- or antibiotic-amended freshwater microcosms. The intI1 gene was more abundant in all contaminant-exposed communities indicating that relative gene transfer potential is higher in these communities. A second key to assessing the contributions of MGEs to bacterial evolution is to examine the structure and function of the MGE-associated gene pool. We determined that the gene cassette pool is a novel and diverse resource available for bacterial acquisition, but that contamination has no discernible effect on cassette richness. Gene cassette profiles were more similar within sites than among sites, yet bacterial community profiles were not, suggesting that selective pressures can shape the structure of the gene cassette pool. Of the 46 sequenced gene cassette products, 37 were novel sequences, while the 9 gene cassettes with similarity to database sequences were primarily to hypothetical proteins. That class 1 integrons are ubiquitous and abundant in environmental bacterial communities indicates that this group of MGEs can play a substantial role in the acquisition of a diverse array of gene cassettes beyond their demonstrated impact in mediating multidrug resistance in clinical bacteria.

New Insights into Dissemination and Variation of the Health Care-Associated Pathogen Acinetobacter baumannii from Genomic Analysis

ABSTRACT Acinetobacter baumannii is a globally important nosocomial pathogen characterized by an increasing incidence of multidrug resistance. Routes of dissemination and gene flow among health care facilities are poorly resolved and are important for understanding the epidemiology of A. baumannii, minimizing disease transmission, and improving patient outcomes. We used whole-genome sequencing to assess diversity and genome dynamics in 49 isolates from one United States hospital system during one year from 2007 to 2008. Core single-nucleotide-variant-based phylogenetic analysis revealed multiple founder strains and multiple independent strains recovered from the same patient yet was insufficient to fully resolve strain relationships, where gene content and insertion sequence patterns added additional discriminatory power. Gene content comparisons illustrated extensive and redundant antibiotic resistance gene carriage and direct evidence of gene transfer, recombination, gene loss, and mutation. Evidence of barriers to gene flow among hospital components was not found, suggesting complex mixing of strains and a large reservoir of A. baumannii strains capable of colonizing patients. IMPORTANCE Genome sequencing was used to characterize multidrug-resistant Acinetobacter baumannii strains from one United States hospital system during a 1-year period to better understand how A. baumannii strains that cause infection are related to one another. Extensive variation in gene content was found, even among strains that were very closely related phylogenetically and epidemiologically. Several mechanisms contributed to this diversity, including transfer of mobile genetic elements, mobilization of insertion sequences, insertion sequence-mediated deletions, and genome-wide homologous recombination. Variation in gene content, however, lacked clear spatial or temporal patterns, suggesting a diverse pool of circulating strains with considerable interaction between strains and hospital locations. Widespread genetic variation among strains from the same hospital and even the same patient, particularly involving antibiotic resistance genes, reinforces the need for molecular diagnostic testing and genomic analysis to determine resistance profiles, rather than a reliance primarily on strain typing and antimicrobial resistance phenotypes for epidemiological studies. Genome sequencing was used to characterize multidrug-resistant Acinetobacter baumannii strains from one United States hospital system during a 1-year period to better understand how A. baumannii strains that cause infection are related to one another. Extensive variation in gene content was found, even among strains that were very closely related phylogenetically and epidemiologically. Several mechanisms contributed to this diversity, including transfer of mobile genetic elements, mobilization of insertion sequences, insertion sequence-mediated deletions, and genome-wide homologous recombination. Variation in gene content, however, lacked clear spatial or temporal patterns, suggesting a diverse pool of circulating strains with considerable interaction between strains and hospital locations. Widespread genetic variation among strains from the same hospital and even the same patient, particularly involving antibiotic resistance genes, reinforces the need for molecular diagnostic testing and genomic analysis to determine resistance profiles, rather than a reliance primarily on strain typing and antimicrobial resistance phenotypes for epidemiological studies.

Insights into the Environmental Resistance Gene Pool from the Genome Sequence of the Multidrug-Resistant Environmental Isolate Escherichia coli SMS-3-5

The increasing occurrence of multidrug-resistant pathogens of clinical and agricultural importance is a global public health concern. While antimicrobial use in human and veterinary medicine is known to contribute to the dissemination of antimicrobial resistance, the impact of microbial communities and mobile resistance genes from the environment in this process is not well understood. Isolated from an industrially polluted aquatic environment, Escherichia coli SMS-3-5 is resistant to a record number of antimicrobial compounds from all major classes, including two front-line fluoroquinolones (ciprofloxacin and moxifloxacin), and in many cases at record-high concentrations. To gain insights into antimicrobial resistance in environmental bacterial populations, the genome of E. coli SMS-3-5 was sequenced and compared to the genome sequences of other E. coli strains. In addition, selected genetic loci from E. coli SMS-3-5 predicted to be involved in antimicrobial resistance were phenotypically characterized. Using recombinant vector clones from shotgun sequencing libraries, resistance to tetracycline, streptomycin, and sulfonamide/trimethoprim was assigned to a single mosaic region on a 130-kb plasmid (pSMS35_130). The remaining plasmid backbone showed similarity to virulence plasmids from avian-pathogenic E. coli (APEC) strains. Individual resistance gene cassettes from pSMS35_130 are conserved among resistant bacterial isolates from multiple phylogenetic and geographic sources. Resistance to quinolones was assigned to several chromosomal loci, mostly encoding transport systems that are also present in susceptible E. coli isolates. Antimicrobial resistance in E. coli SMS-3-5 is therefore dependent both on determinants acquired from a mobile gene pool that is likely available to clinical and agricultural pathogens, as well, and on specifically adapted multidrug efflux systems. The association of antimicrobial resistance with APEC virulence genes on pSMS35_130 highlights the risk of promoting the spread of virulence through the extensive use of antibiotics.

Multi-site Analysis Reveals Widespread Antibiotic Resistance in the Marine Pathogen Vibrio vulnificus

Vibrio vulnificus is a serious opportunistic human pathogen commonly found in subtropical coastal waters, and is the leading cause of seafood-borne mortality in the USA. This taxon does not sustain prolonged presence in clinical or agricultural settings, where it would undergo human-induced selection for antibiotic resistance. Therefore, few studies have verified the effectiveness of commonly prescribed antibiotics in V. vulnificus treatment. Here we screened 151 coastal isolates and 10 primary septicaemia isolates against 26 antimicrobial agents representing diverse modes of action. The frequency of multiple resistances to antibiotics from all sources was unexpectedly high, particularly during summer months, and a substantial proportion of isolates (17.3%) were resistant to eight or more antimicrobial agents. Numerous isolates demonstrated resistance to antibiotics routinely prescribed for V. vulnificus infections, such as doxycycline, tetracycline, aminoglycosides and cephalosporins. These resistances were detected at similar frequencies in virulent and non-virulent strains (PCR-based virulence typing) and were present in septicaemia isolates, underlying the public health implications of our findings. Among environmental isolates, there were no consistent differences in the frequency of resistance between pristine and anthropogenically impacted estuaries, suggesting natural rather than human-derived sources of resistance traits. This report is the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the importance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resistance factors.

Genomic and Transcriptomic Analyses of Colistin-Resistant Clinical Isolates of Klebsiella pneumoniae Reveal Multiple Pathways of Resistance

ABSTRACT The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Colr) is increasingly reported from clinical settings. The genetic mechanisms that lead to Colr in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Colr clinical isolates. Colr was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-l-arabinose (Ara4N) modification of lipid A was found in all Colr strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Colr mechanisms may be dependent on the genetic background.

Population Structure of KPC-Producing Klebsiella pneumoniae Isolates from Midwestern U.S. Hospitals

ABSTRACT Genome sequencing of carbapenem-resistant Klebsiella pneumoniae isolates from regional U.S. hospitals was used to characterize strain diversity and the blaKPC genetic context. A phylogeny based on core single-nucleotide variants (SNVs) supports a division of sequence type 258 (ST258) into two distinct groups. The primary differences between the groups are in the capsular polysaccharide locus (cps) and their plasmid contents. A strict association between clade and KPC variant was found. The blaKPC gene was found on variants of two plasmid backbones. This study indicates that highly similar K. pneumoniae subpopulations coexist within the same hospitals over time.

Genetic Dissection of the Type VI Secretion System in Acinetobacter and Identification of a Novel Peptidoglycan Hydrolase, TagX, Required for Its Biogenesis

ABSTRACT The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi. By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l,d-endopeptidase activity. TagX shows homology to known bacteriophage l,d-endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium. IMPORTANCE Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter, and our results provide genetic and functional insights into the Acinetobacter T6SS. Through this analysis, we identified a novel peptidoglycan hydrolase, TagX, that is required for biogenesis of the T6SS apparatus. This is the first peptidoglycanase specialized in T6SS function identified in any species. We propose that this enzyme is required for the spatially and temporally regulated digestion of peptidoglycan to allow assembly of the T6SS machinery. Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter, and our results provide genetic and functional insights into the Acinetobacter T6SS. Through this analysis, we identified a novel peptidoglycan hydrolase, TagX, that is required for biogenesis of the T6SS apparatus. This is the first peptidoglycanase specialized in T6SS function identified in any species. We propose that this enzyme is required for the spatially and temporally regulated digestion of peptidoglycan to allow assembly of the T6SS machinery.

Host Fate Is Rapidly Determined by Innate Effector-Microbial Interactions During Acinetobacter baumannii Bacteremia

BACKGROUND Acinetobacter baumannii is one of the most antibiotic-resistant pathogens. Defining mechanisms driving pathogenesis is critical to enable new therapeutic approaches. METHODS We studied virulence differences across a diverse panel of A. baumannii clinical isolates during murine bacteremia to elucidate host-microbe interactions that drive outcome. RESULTS We identified hypervirulent strains that were lethal at low intravenous inocula and achieved very high early, and persistent, blood bacterial densities. Virulent strains were nonlethal at low inocula but lethal at 2.5-fold higher inocula. Finally, relatively avirulent (hypovirulent) strains were nonlethal at 20-fold higher inocula and were efficiently cleared by early time points. In vivo virulence correlated with in vitro resistance to complement and macrophage uptake. Depletion of complement, macrophages, and neutrophils each independently increased bacterial density of the hypovirulent strain but insufficiently to change lethality. However, disruption of all 3 effector mechanisms enabled early bacterial densities similar to hypervirulent strains, rendering infection 100% fatal. CONCLUSIONS The lethality of A. baumannii strains depends on distinct stages. Strains resistant to early innate effectors are able to establish very high early bacterial blood density, and subsequent sustained bacteremia leads to Toll-like receptor 4-mediated hyperinflammation and lethality. These results have important implications for translational efforts to develop therapies that modulate host-microbe interactions.

Genome dynamics of multidrug-resistant Acinetobacter baumannii during infection and treatment.

BackgroundLimited treatment options are available for patients infected with multidrug (MDR)- or pan-drug (PDR)-resistant bacterial pathogens, resulting in infections that can persist for weeks or months. In order to better understand transmission and evolutionary dynamics of MDR Acinetobacter baumannii (Ab) during long-term infection, we analyzed genomes from a series of isolates from individual patients at isolate-specific, patient-specific, and population levels.MethodsWhole genome analysis of longitudinal isolates (range 2-10 isolates per patient spanning 0-829 days) from 40 patients included detection of single-nucleotide variants (SNVs), insertion sequence (IS) mapping, and gene content changes.ResultsPhylogenetic analysis revealed that a significant fraction of apparently persistent infections are in fact due to re-infection with new strains. SNVs primarily resulted in protein coding changes, and IS events primarily interrupted genes or were in an orientation such that the adjacent gene would be over-expressed. Mutations acquired during infection were over-represented in transcriptional regulators, notably pmrAB and adeRS, which can mediate resistance to the last line therapies colistin and tigecycline, respectively, as well as transporters, surface structures, and iron acquisition genes.ConclusionsMost SNVs and IS events were isolate-specific indicating these mutations did not become fixed on the time scale investigated, yet over-representation of independent mutations in some genes or functional categories suggests that they are under selective pressure. Genome analysis at the population-level suggests that gene transfer including recombination also contributes to Ab evolutionary dynamics. These findings provide important insight into the transmission dynamics of Ab and the identification of patients with repeat infections has implications for infection control programs targeted to this pathogen.

Dissolved organic carbon lability increases with water residence time in the alluvial aquifer of a river floodplain ecosystem

We assessed spatial and temporal patterns of dissolved organic carbon (DOC) lability and composition throughout the alluvial aquifer of the 16 km2 Nyack Floodplain in northwest Montana, USA. Water influx to the aquifer derives almost exclusively from the Middle Fork of the Flathead River, and water residence times within the aquifer range from days to months. Across seasons and channel discharge conditions, we measured DOC concentration, lability, and optical properties of aquifer water sampled from 12 wells, both near and ~3 m below the water table. Concentrations of DOC were typically low (542 ± 22.7 µg L−1; mean ± se), and the percentage of labile DOC averaged 18 ± 12% during 3 day laboratory assays. Parallel factor analysis of fluorescence excitation-emission matrices revealed two humic-like and two amino acid-like fluorescence groups. Total DOC, humic-like components, and specific UV absorbance decreased with water residence time, consistent with sorption to aquifer sediments. However, labile DOC (both concentration and fraction) increased with water residence time, suggesting a concurrent influx or production of labile DOC. Thus, although the carbon-poor, oxygen-rich aquifer is a net sink for DOC, recalcitrant DOC appears to be replaced with more labile DOC along aquifer flow paths. Our observation of DOC production in long flow paths contrasts with studies of hyporheic DOC consumption along short (centimeters to meters) flow paths and highlights the importance of understanding the role of labile organic matter production and/or influx in alluvial aquifer carbon cycling.