Multidrug resistance plasmids commonly reprogram the expression of metabolic genes in Escherichia coli.

Multidrug-resistant Escherichia coli is a leading cause of global mortality. Transfer of plasmids carrying genes encoding beta-lactamases, carbapenamases, and colistin resistance between lineages is driving the rising rates of hard-to-treat nosocomial and community infections. Multidrug resistance (MDR) plasmid acquisition commonly causes transcriptional disruption, and while a number of studies have shown strain-specific fitness and transcriptional effects of an MDR plasmid across diverse bacterial lineages, fewer studies have compared the impacts of different MDR plasmids in a common bacterial host. As such, our ability to predict which MDR plasmids are the most likely to be maintained and spread in bacterial populations is limited. Here, we introduced eight diverse MDR plasmids encoding resistances against a range of clinically important antibiotics into E. coli K-12 MG1655 and measured their fitness costs and transcriptional impacts. The scale of the transcriptional responses varied substantially between plasmids, ranging from >650 to <20 chromosomal genes being differentially expressed. However, the scale of regulatory disruption did not correlate significantly with the magnitude of the plasmid fitness cost, which also varied between plasmids. The identities of differentially expressed genes differed between transconjugants, although the expression of certain metabolic genes and functions were convergently affected by multiple plasmids, including the downregulation of genes involved in L-methionine transport and metabolism. Our data show the complexity of the interaction between host genetic background and plasmid genetic background in determining the impact of MDR plasmid acquisition on E. coli. IMPORTANCE: The increase in infections that are resistant to multiple classes of antibiotics, including those isolates that carry carbapenamases, beta-lactamases, and colistin resistance genes, is of global concern. Many of these resistances are spread by conjugative plasmids. Understanding more about how an isolate responds to an incoming plasmid that encodes antibiotic resistance will provide information that could be used to predict the emergence of MDR lineages. Here, the identification of metabolic networks as being particularly sensitive to incoming plasmids suggests the possible targets for reducing plasmid transfer.

Parallel loss of type VI secretion systems in two multi-drug-resistant Escherichia coli lineages.

The repeated emergence of multi-drug-resistant (MDR) Escherichia coli clones is a threat to public health globally. In recent work, drug-resistant E. coli were shown to be capable of displacing commensal E. coli in the human gut. Given the rapid colonization observed in travel studies, it is possible that the presence of a type VI secretion system (T6SS) may be responsible for the rapid competitive advantage of drug-resistant E. coli clones. We employed large-scale genomic approaches to investigate this hypothesis. First, we searched for T6SS genes across a curated dataset of over 20 000 genomes representing the full phylogenetic diversity of E. coli. This revealed large, non-phylogenetic variation in the presence of T6SS genes. No association was found between T6SS gene carriage and MDR lineages. However, multiple clades containing MDR clones have lost essential structural T6SS genes. We characterized the T6SS loci of ST410 and ST131 and identified specific recombination and insertion events responsible for the parallel loss of essential T6SS genes in two MDR clones.

Growth in a biofilm promotes conjugation of a bla NDM-1-bearing plasmid between Klebsiella pneumoniae strains.

Antimicrobial resistance (AMR) is a growing problem, especially in Gram-negative Enterobacteriaceae such as Klebsiella pneumoniae. Horizontal transfer of conjugative plasmids contributes to AMR gene dissemination. Bacteria such as K. pneumoniae commonly exist in biofilms, yet most studies focus on planktonic cultures. Here we studied the transfer of a multi-drug resistance plasmid in planktonic and biofilm populations of K. pneumoniae. We determined plasmid transfer from a clinical isolate, CPE16, which carried four plasmids, including the 119-kbp blaNDM-1-bearing F-type plasmid pCPE16_3, in planktonic and biofilm conditions. We found that transfer frequency of pCPE16_3 in a biofilm was orders-of-magnitude higher than between planktonic cells. In 5/7 sequenced transconjugants (TCs) multiple plasmids had transferred. Plasmid acquisition had no detectable growth impact on TCs. Gene expression of the recipient and a transconjugant was investigated by RNA-sequencing in three lifestyles: planktonic exponential growth, planktonic stationary phase, and biofilm. We found that lifestyle had a substantial impact on chromosomal gene expression, and plasmid carriage affected chromosomal gene expression most in stationary planktonic and biofilm lifestyles. Furthermore, expression of plasmid genes was lifestyle-dependent, with distinct signatures across the three conditions. Our study shows that growth in biofilm greatly increased the risk of conjugative transfer of a carbapenem resistance plasmid in K. pneumoniae without fitness costs and minimal transcriptional rearrangements, thus highlighting the importance of biofilms in the spread of AMR in this opportunistic pathogen. IMPORTANCE Carbapenem-resistant K. pneumoniae is particularly problematic in hospital settings. Carbapenem resistance genes can transfer between bacteria via plasmid conjugation. Alongside drug resistance, K. pneumoniae can form biofilms on hospital surfaces, at infection sites and on implanted devices. Biofilms are naturally protected and can be inherently more tolerant to antimicrobials than their free-floating counterparts. There have been indications that plasmid transfer may be more likely in biofilm populations, thus creating a conjugation "hotspot". However, there is no clear consensus on the effect of the biofilm lifestyle on plasmid transfer. Therefore, we aimed to explore the transfer of a plasmid in planktonic and biofilm conditions, and the impact of plasmid acquisition on a new bacterial host. Our data show transfer of a resistance plasmid is increased in a biofilm, which may be a significant contributing factor to the rapid dissemination of resistance plasmids in K. pneumoniae.

Updated Acoustic Normative Data through the Lifespan: A Scoping Review.

OBJECTIVE: To assess the recent literature for voice acoustic data values reported for individuals without voice disorder through the lifespan as a means to develop an updated normative acoustic data resource for children and adults. METHODS: A scoping review was conducted using the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist. English language, full-text publications were identified through Medline (EBSCO & OVID), PubMed, APA PsycINFO, Web of Science, Google Scholar, and ProQuest Theses and Dissertations Global. RESULTS: A total of 903 sources were retrieved; of these 510 were duplicates. Abstracts of 393 were screened, with 68 full-text review. From the eligible studies, citation review yielded 51 additional resources. Twenty-eight sources were included for data extraction. For the normative acoustic data extracted for males and females across the lifespan, lower fundamental frequency for adult females was observed and few studies collected semitone range, sound level range, or frequency range. Data extraction also indicated a predominately gender binary reporting of acoustic measures with few studies reporting gender identity, race, or ethnicity as variables of interest. CONCLUSIONS: The scoping review yielded updated acoustic normative data that is of value for clinicians and researchers who rely on this normative data to make determinations about vocal function. The limited availability of acoustic data by gender, race, and ethnicity creates barriers for generalization of these normative values across all patients, clients, and research volunteers.

Prokaryote pangenomes are dynamic entities.

Prokaryote pangenomes are influenced heavily by environmental factors and the opportunity for gene gain and loss events. As the field of pangenome analysis has expanded, so has the need to fully understand the complexity of how eco-evolutionary dynamics shape pangenomes. Here, we describe current models of pangenome evolution and discuss their suitability and accuracy. We suggest that pangenomes are dynamic entities under constant flux, highlighting the influence of two-way interactions between pangenome and environment. New classifications of core and accessory genes are also considered, underscoring the need for continuous evaluation of nomenclature in a fast-moving field. We conclude that future models of pangenome evolution should incorporate eco-evolutionary dynamics to fully encompass their dynamic, changeable nature.

The role of potentiating mutations in the evolution of pandemic Escherichia coli clones.

The Escherichia coli species exhibits a vast array of variable lifestyles, including environmental, commensal, and pathogenic organisms. Many of these E. coli contribute significantly to the global threat of antimicrobial resistance (AMR). Multidrug-resistant (MDR) clones of E. coli have arisen multiple times over varying timescales. The repeated emergence of successful pandemic clones, including the notorious ST131 lineage, highlights a desperate need to further study the evolutionary processes underlying their emergence and success. Here, we review the evolutionary emergence of E. coli ST131 pandemic clones and draw parallels between their evolutionary trajectories and those of other lineages. From colonization and expansion to the acquisition of multidrug resistance plasmids, potentiating mutations are present at each stage, leading to a proposed sequence of events that may result in the formation of an antimicrobial-resistant pandemic clone.

Gene-gene relationships in an Escherichia coli accessory genome are linked to function and mobility.

The pangenome contains all genes encoded by a species, with the core genome present in all strains and the accessory genome in only a subset. Coincident gene relationships are expected within the accessory genome, where the presence or absence of one gene is influenced by the presence or absence of another. Here, we analysed the accessory genome of an Escherichia coli pangenome consisting of 400 genomes from 20 sequence types to identify genes that display significant co-occurrence or avoidance patterns with one another. We present a complex network of genes that are either found together or that avoid one another more often than would be expected by chance, and show that these relationships vary by lineage. We demonstrate that genes co-occur by function, and that several highly connected gene relationships are linked to mobile genetic elements. We find that genes are more likely to co-occur with, rather than avoid, another gene in the accessory genome. This work furthers our understanding of the dynamic nature of prokaryote pangenomes and implicates both function and mobility as drivers of gene relationships.

Evidence for Selection in the Abundant Accessory Gene Content of a Prokaryote Pangenome.

A pangenome is the complete set of genes (core and accessory) present in a phylogenetic clade. We hypothesize that a pangenome's accessory gene content is structured and maintained by selection. To test this hypothesis, we interrogated the genomes of 40 Pseudomonas species for statistically significant coincident (i.e., co-occurring/avoiding) gene patterns. We found that 86.7% of common accessory genes are involved in ≥1 coincident relationship. Further, genes that co-occur and/or avoid each other-but are not vertically inherited-are more likely to share functional categories, are more likely to be simultaneously transcribed, and are more likely to produce interacting proteins, than would be expected by chance. These results are not due to coincident genes being adjacent to one another on the chromosome. Together, these findings suggest that the accessory genome is structured into sets of genes that function together within a given strain. Given the similarity of the Pseudomonas pangenome with open pangenomes of other prokaryotic species, we speculate that these results are generalizable.

Horizontal Gene Transfer as a Source of Conflict and Cooperation in Prokaryotes.

Horizontal gene transfer (HGT) is one of the most important processes in prokaryote evolution. The sharing of DNA can spread neutral or beneficial genes, as well as genetic parasites across populations and communities, creating a large proportion of the variability acted on by natural selection. Here, we highlight the role of HGT in enhancing the opportunities for conflict and cooperation within and between prokaryote genomes. We discuss how horizontally acquired genes can cooperate or conflict both with each other and with a recipient genome, resulting in signature patterns of gene co-occurrence, avoidance, and dependence. We then describe how interactions involving horizontally transferred genes may influence cooperation and conflict at higher levels (populations, communities, and symbioses). Finally, we consider the benefits and drawbacks of HGT for prokaryotes and its fundamental role in understanding conflict and cooperation from the gene-gene to the microbiome level.

Simulating the evolutionary trajectories of metabolic pathways for insect symbionts in the genus Sodalis.

Insect-bacterial symbioses are ubiquitous, but there is still much to uncover about how these relationships establish, persist and evolve. The tsetse endosymbiont Sodalis glossinidius displays intriguing metabolic adaptations to its microenvironment, but the process by which this relationship evolved remains to be elucidated. The recent chance discovery of the free-living species of the genus Sodalis, Sodalis praecaptivus, provides a serendipitous starting point from which to investigate the evolution of this symbiosis. Here, we present a flux balance model for S. praecaptivus and empirically verify its predictions. Metabolic modelling is used in combination with a multi-objective evolutionary algorithm to explore the trajectories that S. glossinidius may have undertaken from this starting point after becoming internalized. The order in which key genes are lost is shown to influence the evolved populations, providing possible targets for future in vitro genetic manipulation. This method provides a detailed perspective on possible evolutionary trajectories for S. glossinidius in this fundamental process of evolutionary and ecological change.

A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission.

Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters.

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.

The relationship of 3-D translabial ultrasound anal sphincter complex measurements to postpartum anal and fecal incontinence.

INTRODUCTION AND HYPOTHESIS: We aimed to determine whether anal sphincter complex (ASC) measurements on translabial ultrasound (TL-US) were related to anal incontinence (AI) or fecal incontinence (FI) symptoms 6 months postpartum. METHODS: A prospective cohort of primiparous women underwent TL-US 6 months after a vaginal birth (VB) or cesarean delivery (CD). Muscle thickness was measured at 3, 6, 9, and 12 o'clock positions of the external anal sphincter (EAS), the same four quadrants of the internal anal sphincter (IAS) at proximal, mid, and distal levels, and at the bilateral pubovisceralis muscle (PVM). Measurements were correlated to AI and FI on the Wexner Fecal Incontinence Scale, with sub-analyses by mode of delivery. The odds ratio (OR) of symptoms was calculated for every 1 mm increase in muscle thickness (E1MIT). RESULTS: A total of 423 women (299 VB, 124 CD) had TL-US 6 months postpartum. Decreased AI risk was associated with thicker measurements at the 6 o'clock (OR 0.74 E1MIT) and 9 o'clock proximal IAS (OR 0.71 E1MIT) in the entire cohort. For CD women, thicker measurements of the 9 o'clock proximal IAS were associated with decreased risk of AI (OR 0.56 E1MIT) and thicker distal 6 o'clock IAS measurements were related to a decreased risk of FI (OR 0.37 E1MIT). For VB women, no sphincter measurements were significantly related to symptoms, but thicker PVM measurements were associated with increased risk of AI (right side OR 1.32 E1MIT; left side OR 1.21 E1MIT). CONCLUSIONS: ASC anatomy is associated with AI and FI in certain locations; these locations vary based on the patient's mode of delivery.

Epidermal keratinocytes initiate wound healing and pro-inflammatory immune responses following percutaneous schistosome infection.

Keratinocytes constitute the majority of cells in the skin's epidermis, the first line of defence against percutaneous pathogens. Schistosome larvae (cercariae) actively penetrate the epidermis to establish infection, however the response of keratinocytes to invading cercariae has not been investigated. Here we address the hypothesis that cercariae activate epidermal keratinocytes to promote the development of a pro-inflammatory immune response in the skin. C57BL/6 mice were exposed to Schistosoma mansoni cercariae via each pinna and non-haematopoietic cells isolated from epidermal tissue were characterised for the presence of different keratinocyte sub-sets at 6, 24 and 96 h p.i. We identified an expansion of epidermal keratinocyte precursors (CD45(-), CD326(-), CD34(+)) within 24 h of infection relative to naïve animals. Following infection, cells within the precursor population displayed a more differentiated phenotype (α6integrin(-)) than in uninfected skin. Parallel immunohistochemical analysis of pinnae cryosections showed that this expansion corresponded to an increase in the intensity of CD34 staining, specifically in the basal bulge region of hair follicles of infected mice, and a higher frequency of keratinocyte Ki67(+) nuclei in both the hair follicle and interfollicular epidermis. Expression of pro-inflammatory cytokine and stress-associated keratin 6b genes was also transiently upregulated in the epidermal tissue of infected mice. In vitro exposure of keratinocyte precursors isolated from neonatal mouse skin to excretory/secretory antigens released by penetrating cercariae elicited IL-1α and IL-1ß production, supporting a role for keratinocyte precursors in initiating cutaneous inflammatory immune responses. Together, these observations indicate that S.mansoni cercariae and their excretory/secretory products act directly upon epidermal keratinocytes, which respond by initiating barrier repair and pro-inflammatory mechanisms similar to those observed in epidermal wound healing.

Anal sphincter complex: 2D and 3D endoanal and translabial ultrasound measurement variation in normal postpartum measurements.

INTRODUCTION AND HYPOTHESIS: Women may experience anal sphincter anatomy changes after vaginal birth (VB) or Cesarean delivery (CD). Therefore, accurate and acceptable imaging options to evaluate the anal sphincter complex (ASC) are needed. ASC measurements may differ between translabial (TLUS) and endoanal (EAUS) ultrasound imaging and between 2D and 3D US. The objective of this analysis was to describe measurement variation between these modalities. METHODS: Primiparous women underwent 2D and 3D TLUS imaging of the ASC 6 months after VB or CD. A subset of women also underwent EAUS measurements. Measurements included internal anal sphincter (IAS) thickness at proximal, mid, and distal levels and the external anal sphincter (EAS) at 3, 6, 9, and 12 o'clock positions, as well as bilateral thickness of the pubovisceralis muscle (PVM). RESULTS: There were 433 women presenting for US: 423 had TLUS and 64 had both TLUS and EAUS of the ASC. All IAS measurements were significantly thicker on TLUS than EAUS (all p < 0.01), while EAS measurements were significantly thicker on EAUS (p < 0.01). PVM measurements with 3D or 2D imaging were similar (p > 0.20). On both TLUS and EAUS, there were multiple sites where significant asymmetry existed in left versus right measurements. CONCLUSIONS: US modality used to image the ASC introduces small but significant changes in measurements, and the direction of the bias depends on the muscle and location being imaged.

Postpartum translabial 2D and 3D ultrasound measurements of the anal sphincter complex in primiparous women delivering by vaginal birth versus Cesarean delivery.

INTRODUCTION AND HYPOTHESIS: Consensus on normal translabial ultrasound (TL-US) anal sphincter complex measurements for postpartum women is lacking. We aimed to evaluate normative measurements in 2D and 3D TL-US for the anal sphincter complex (ASC) at 6 months postpartum and compare these measurements in women who had a vaginal birth (VB) and in those who had a Cesarean delivery (CD). METHODS: A large, prospective cohort of primiparous women underwent 2D and 3D TL-US 6 months after their first delivery. For normative sphincter measurements, we excluded women with third- or fourth-degree lacerations or with sphincter interruption on TL-US. Measurements included the sphincter thickness at the 3, 6, 9, and 12 o'clock positions of the external anal sphincter (EAS) and the internal anal sphincter (IAS) at proximal, mid, and distal levels. We also measured the mean coronal diameter of the pubovisceralis muscle (PVM). RESULTS: 696 women consented to participate, and 433 women presented for ultrasound imaging 6 months later. Women who sustained a third- or fourth-degree laceration had significantly thicker EAS measurements at 12 o'clock. Sphincter asymmetry was common (69 %), but was not related to mode of delivery. Only IAS measurements at the proximal and distal 12 o'clock position were significantly thicker for CD patients. There were no significant differences in the EAS or PVM measurements between VB and CD women. CONCLUSIONS: There appear to be few differences in normative sphincter ultrasound measurements between primiparous patients who had VB or CD.

Translabial ultrasound assessment of the anal sphincter complex: normal measurements of the internal and external anal sphincters at the proximal, mid-, and distal levels.

The purpose of this study was to measure the internal and external anal sphincters using translabial ultrasound (TLU) at the proximal, mid, and distal levels of the anal sphincter complex. The human review committee approval was obtained and all women gave written informed consent. Sixty women presenting for gynecologic ultrasound for symptoms other than pelvic organ prolapse or urinary or anal incontinence underwent TLU. Thirty-six (60%) were asymptomatic and intact, 13 symptomatic and intact, and 11 disrupted. Anterior-posterior diameters of the internal anal sphincter at all levels and the external anal sphincter at the distal level were measured in four quadrants. Mean sphincter measurements are given for symptomatic and asymptomatic intact women and are comparable to previously reported endoanal MRI and ultrasound measurements.

Primary repair of obstetric anal sphincter laceration: a randomized trial of two surgical techniques.

OBJECTIVE: This study was undertaken to compare surgical techniques for the primary repair of obstetric anal sphincter lacerations. STUDY DESIGN: Patients with complete third- or fourth-degree lacerations were recruited and randomly assigned to either an end-to-end or overlapping repair. Data collection included demographic data, obstetric history, and intrapartum events. Postpartum, women completed incontinence questionnaires and underwent physical and ultrasound examinations. To detect a 36% difference between groups with an alpha = .05 and beta = .20, 30 patients were required. Data were analyzed with Student t test and chi2 analysis. RESULTS: Forty-one women were randomly assigned; 23 to an end-to-end and 18 to an overlapping repair. Twenty-seven percent of women underwent episiotomy and 61% operative vaginal delivery. Follow-up was limited to 26 of 41 patients. On physical examination, 3 patients had a separated anal sphincter. On ultrasound, overall 85% of patients had intact sphincters, with no difference between groups (all P > .05). Forty-two percent of women complained of anorectal symptoms with no differences between groups (all P > .28). CONCLUSION: We found no difference in anal incontinence symptoms, physical examination, or translabial ultrasonography findings between the 2 groups. Incontinence symptoms were common in both groups.