Publications by authors named "Eva Heinz"

54 Publications

Stable high-density and maternally inherited Wolbachia infections in Anopheles moucheti and Anopheles demeilloni mosquitoes.

Curr Biol 2021 Apr 8. Epub 2021 Apr 8.

Departments of Vector Biology and Tropical Disease Biology, Centre for Neglected Tropical Diseases, Liverpool School of Tropical Medicine, Liverpool, UK. Electronic address:

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An.moucheti, and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria.
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http://dx.doi.org/10.1016/j.cub.2021.03.056DOI Listing
April 2021

Spatiotemporal persistence of multiple, diverse clades and toxins of Corynebacterium diphtheriae.

Nat Commun 2021 03 8;12(1):1500. Epub 2021 Mar 8.

Department of Medicine, Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), University of Cambridge, Cambridge, UK.

Diphtheria is a respiratory disease caused by the bacterium Corynebacterium diphtheriae. Although the development of a toxin-based vaccine in the 1930s has allowed a high level of control over the disease, cases have increased in recent years. Here, we describe the genomic variation of 502 C. diphtheriae isolates across 16 countries and territories over 122 years. We generate a core gene phylogeny and determine the presence of antimicrobial resistance genes and variation within the tox gene of 291 tox isolates. Numerous, highly diverse clusters of C. diphtheriae are observed across the phylogeny, each containing isolates from multiple countries, regions and time of isolation. The number of antimicrobial resistance genes, as well as the breadth of antibiotic resistance, is substantially greater in the last decade than ever before. We identified and analysed 18 tox gene variants, with mutations estimated to be of medium to high structural impact.
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http://dx.doi.org/10.1038/s41467-021-21870-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940655PMC
March 2021

A comprehensive and high-quality collection of genomes and their genes.

Microb Genom 2021 02;7(2)

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.

is a highly diverse organism that includes a range of commensal and pathogenic variants found across a range of niches and worldwide. In addition to causing severe intestinal and extraintestinal disease, is considered a priority pathogen due to high levels of observed drug resistance. The diversity in the population is driven by high genome plasticity and a very large gene pool. All these have made one of the most well-studied organisms, as well as a commonly used laboratory strain. Today, there are thousands of sequenced genomes stored in public databases. While data is widely available, accessing the information in order to perform analyses can still be a challenge. Collecting relevant available data requires accessing different sources, where data may be stored in a range of formats, and often requires further manipulation and processing to apply various analyses and extract useful information. In this study, we collated and intensely curated a collection of over 10 000 and genomes to provide a single, uniform, high-quality dataset. were included as they are considered specialized pathovars of . We provide these data in a number of easily accessible formats that can be used as the foundation for future studies addressing the biological differences between lineages and the distribution and flow of genes in the population at a high resolution. The analysis we present emphasizes our lack of understanding of the true diversity of the species, and the biased nature of our current understanding of the genetic diversity of such a key pathogen.
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http://dx.doi.org/10.1099/mgen.0.000499DOI Listing
February 2021

Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation.

Elife 2020 12 14;9. Epub 2020 Dec 14.

Institute of Microbiology and Infection, University of Birmingham, Edgbaston, United Kingdom.

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.
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http://dx.doi.org/10.7554/eLife.62614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806268PMC
December 2020

Microbial interactions in the mosquito gut determine Serratia colonization and blood-feeding propensity.

ISME J 2021 Jan 7;15(1):93-108. Epub 2020 Sep 7.

Departments of Vector Biology and Tropical Disease Biology, Centre for Neglected Tropical Disease, Liverpool School of Tropical Medicine, Liverpool, UK.

How microbe-microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host-microbe interactions and provides evidence that microbial interactions influence mosquito behavior.
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http://dx.doi.org/10.1038/s41396-020-00763-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852612PMC
January 2021

Genomic and Phenotypic Analyses of Isolates From Three Tertiary Care Hospitals in Thailand.

Front Microbiol 2020 6;11:548. Epub 2020 Apr 6.

School of Pharmacy, University College London, London, United Kingdom.

Antibiotic resistant strains of are responsible for a large and increasing burden of nosocomial infections in Thailand and other countries of Southeast Asia. New approaches to their control and treatment are urgently needed and an attractive strategy is to remove the bacterial polysaccharide capsule, and thus the protection from the host's immune system. To examine phylogenetic relationships, distribution of capsule chemotypes, acquired antibiotic resistance determinants, susceptibility to complement and other traits associated with systemic infection, we sequenced 191 isolates from three tertiary referral hospitals in Thailand and used phenotypic assays to characterize key aspects of infectivity. Several distinct lineages were circulating in three hospitals and the majority belonged to global clonal group 2 (GC2). Very high levels of resistance to carbapenems and other front-line antibiotics were found, as were a number of widespread plasmid replicons. A high diversity of capsule genotypes was encountered, with only three of these (KL6, KL10, and KL47) showing more than 10% frequency. Almost 90% of GC2 isolates belonged to the most common capsule genotypes and were fully resistant to the bactericidal action of human serum complement, most likely protected by their polysaccharide capsule, which represents a key determinant of virulence for systemic infection. Our study further highlights the importance to develop therapeutic strategies to remove the polysaccharide capsule from extensively drug-resistant during the course of systemic infection.
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http://dx.doi.org/10.3389/fmicb.2020.00548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153491PMC
April 2020

Type II and type IV toxin-antitoxin systems show different evolutionary patterns in the global Klebsiella pneumoniae population.

Nucleic Acids Res 2020 05;48(8):4357-4370

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1RQ, UK.

The Klebsiella pneumoniae species complex includes important opportunistic pathogens which have become public health priorities linked to major hospital outbreaks and the recent emergence of multidrug-resistant hypervirulent strains. Bacterial virulence and the spread of multidrug resistance have previously been linked to toxin-antitoxin (TA) systems. TA systems encode a toxin that disrupts essential cellular processes, and a cognate antitoxin which counteracts this activity. Whilst associated with the maintenance of plasmids, they also act in bacterial immunity and antibiotic tolerance. However, the evolutionary dynamics and distribution of TA systems in clinical pathogens are not well understood. Here, we present a comprehensive survey and description of the diversity of TA systems in 259 clinically relevant genomes of K. pneumoniae. We show that TA systems are highly prevalent with a median of 20 loci per strain. Importantly, these toxins differ substantially in their distribution patterns and in their range of cognate antitoxins. Classification along these properties suggests different roles of TA systems and highlights the association and co-evolution of toxins and antitoxins.
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http://dx.doi.org/10.1093/nar/gkaa198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192599PMC
May 2020

Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2.

J Exp Med 2020 02;217(2)

Translational Gastroenterology Unit, Experimental Medicine Division, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.
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http://dx.doi.org/10.1084/jem.20180649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041704PMC
February 2020

CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes.

PLoS Negl Trop Dis 2019 12 2;13(12):e0007883. Epub 2019 Dec 2.

Departments of Vector Biology and Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Background: Symbiotic bacteria are pervasive in mosquitoes and their presence can influence many host phenotypes that affect vectoral capacity. While it is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. The CRISPR/Cas9 gene editing system is a powerful tool to alter bacterial genomes facilitating investigations into host-microbe interactions but has yet to be applied to insect symbionts.

Methodology/principal Findings: To investigate the role of bacterial genetic factors in mosquito biology and in colonization of mosquitoes we used CRISPR/Cas9 gene editing system to mutate the outer membrane protein A (ompA) gene of a Cedecea neteri symbiont isolated from Aedes mosquitoes. The ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adult mosquitoes, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting genetic factors are particularly important for adult gut colonization. We also used the CRISPR/Cas9 system to integrate genes (antibiotic resistance and fluorescent markers) into the symbionts genome and demonstrated that these genes were functional in vitro and in vivo.

Conclusions/significance: Our results shed insights into the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. The ability to use this technology for site-specific integration of genes into the symbiont will facilitate the development of paratransgenic control strategies to interfere with arboviral pathogens such Chikungunya, dengue, Zika and Yellow fever viruses transmitted by Aedes mosquitoes.
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http://dx.doi.org/10.1371/journal.pntd.0007883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907859PMC
December 2019

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Nat Commun 2019 09 19;10(1):4280. Epub 2019 Sep 19.

Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000, Antwerp, Belgium.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.
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http://dx.doi.org/10.1038/s41467-019-11844-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753159PMC
September 2019

Contrasting patterns of longitudinal population dynamics and antimicrobial resistance mechanisms in two priority bacterial pathogens over 7 years in a single center.

Genome Biol 2019 09 2;20(1):184. Epub 2019 Sep 2.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Background: Two of the most important pathogens contributing to the global rise in antimicrobial resistance (AMR) are Klebsiella pneumoniae and Enterobacter cloacae. Despite this, most of our knowledge about the changing patterns of disease caused by these two pathogens is based on studies with limited timeframes that provide few insights into their population dynamics or the dynamics in AMR elements that they can carry.

Results: We investigate the population dynamics of two priority AMR pathogens over 7 years between 2007 and 2012 in a major UK hospital, spanning changes made to UK national antimicrobial prescribing policy in 2007. Between 2006 and 2012, K. pneumoniae showed epidemiological cycles of multi-drug-resistant (MDR) lineages being replaced approximately every 2 years. This contrasted E. cloacae where there was no temporally changing pattern, but a continuous presence of the mixed population.

Conclusions: The differing patterns of clonal replacement and acquisition of mobile elements shows that the flux in the K. pneumoniae population was linked to the introduction of globally recognized MDR clones carrying drug resistance markers on mobile elements. However, E. cloacae carries a chromosomally encoded ampC conferring resistance to front-line treatments and shows that MDR plasmid acquisition in E. cloacae was not indicative of success in the hospital. This led to markedly different dynamics in the AMR populations of these two pathogens and shows that the mechanism of the resistance and its location in the genome or mobile elements is crucial to predict population dynamics of opportunistic pathogens in clinical settings.
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http://dx.doi.org/10.1186/s13059-019-1785-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717969PMC
September 2019

Investigation of the role of typhoid toxin in acute typhoid fever in a human challenge model.

Nat Med 2019 07 3;25(7):1082-1088. Epub 2019 Jul 3.

Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, UK.

Salmonella Typhi is a human host-restricted pathogen that is responsible for typhoid fever in approximately 10.9 million people annually. The typhoid toxin is postulated to have a central role in disease pathogenesis, the establishment of chronic infection and human host restriction. However, its precise role in typhoid disease in humans is not fully defined. We studied the role of typhoid toxin in acute infection using a randomized, double-blind S. Typhi human challenge model. Forty healthy volunteers were randomized (1:1) to oral challenge with 10 colony-forming units of wild-type or an isogenic typhoid toxin deletion mutant (TN) of S. Typhi. We observed no significant difference in the rate of typhoid infection (fever ≥38 °C for ≥12 h and/or S. Typhi bacteremia) between participants challenged with wild-type or TN S. Typhi (15 out of 21 (71%) versus 15 out of 19 (79%); P = 0.58). The duration of bacteremia was significantly longer in participants challenged with the TN strain compared with wild-type (47.6 hours (28.9-97.0) versus 30.3(3.6-49.4); P ≤ 0.001). The clinical syndrome was otherwise indistinguishable between wild-type and TN groups. These data suggest that the typhoid toxin is not required for infection and the development of early typhoid fever symptoms within the context of a human challenge model. Further clinical data are required to assess the role of typhoid toxin in severe disease or the establishment of bacterial carriage.
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http://dx.doi.org/10.1038/s41591-019-0505-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892374PMC
July 2019

Caribbean multi-centre study of Klebsiella pneumoniae: whole-genome sequencing, antimicrobial resistance and virulence factors.

Microb Genom 2019 05 29;5(5). Epub 2019 Apr 29.

1​Wellcome Trust Sanger Institute, Hinxton, UK.

The surveillance of antimicrobial-resistant isolates has proven to be one of the most valuable tools to understand the global rise of multidrug-resistant bacterial pathogens. We report the first insights into the current situation in the Caribbean, where a pilot project to monitor antimicrobial resistance (AMR) through phenotypic resistance measurements combined with whole-genome sequencing was set up in collaboration with the Caribbean Public Health Agency (CARPHA). Our first study focused on Klebsiella pneumoniae, a highly relevant organism amongst the Gram-negative opportunistic pathogens worldwide causing hospital- and community-acquired infections. Our results show that not only carbapenem resistance, but also hypervirulent strains, are circulating in patients in the Caribbean. Our current data does not allow us to infer their prevalence in the population. We argue for the urgent need to further support AMR surveillance and stewardship in this almost uncharted territory, which can make a significant impact on the reduction of antimicrobial usage. This article contains data hosted by Microreact (https://microreact.org).
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http://dx.doi.org/10.1099/mgen.0.000266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562249PMC
May 2019

rPinecone: Define sub-lineages of a clonal expansion via a phylogenetic tree.

Microb Genom 2019 04 28;5(4). Epub 2019 Mar 28.

1​Parasites and Microbes, Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, UK.

The ability to distinguish different circulating pathogen clones from each other is a fundamental requirement to understand the epidemiology of infectious diseases. Phylogenetic analysis of genomic data can provide a powerful platform to identify lineages within bacterial populations, and thus inform outbreak investigation and transmission dynamics. However, resolving differences between pathogens associated with low-variant (LV) populations carrying low median pairwise single nucleotide variant (SNV) distances remains a major challenge. Here we present rPinecone, an R package designed to define sub-lineages within closely related LV populations. rPinecone uses a root-to-tip directional approach to define sub-lineages within a phylogenetic tree according to SNV distance from the ancestral node. The utility of this software was demonstrated using both simulated outbreaks and real genomic data of two LV populations: a hospital outbreak of methicillin-resistant Staphylococcus aureus and endemic Salmonella Typhi from rural Cambodia. rPinecone identified the transmission branches of the hospital outbreak and geographically confined lineages in Cambodia. Sub-lineages identified by rPinecone in both analyses were phylogenetically robust. It is anticipated that rPinecone can be used to discriminate between lineages of bacteria from LV populations where other methods fail, enabling a deeper understanding of infectious disease epidemiology for public health purposes.
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http://dx.doi.org/10.1099/mgen.0.000264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521585PMC
April 2019

Discovery of trehalose phospholipids reveals functional convergence with mycobacteria.

J Exp Med 2019 04 25;216(4):757-771. Epub 2019 Feb 25.

Department of Infectious Diseases and Immunology, School of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

species are among the world's most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Paratyphi and Typhi, we focused on lipids that are enriched in Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6'-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by Typhi, lower amounts by other pathogens, and variable amounts by strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6'-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.
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http://dx.doi.org/10.1084/jem.20181812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446866PMC
April 2019

Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1.

Sci Rep 2019 02 20;9(1):2392. Epub 2019 Feb 20.

Department of Microbiology and Immunology, The University of Melbourne, at Peter Doherty Institute for Infection and Immunity, Melbourne, Australia.

Klebsiella pneumoniae is a major threat to public health with the emergence of isolates resistant to most, if not all, useful antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae collected from patients resident in a region of Pakistan, during the period 2010-2012, when the now globally-distributed carbapenemase bla-NDM-1 was being acquired by Klebsiella. We observed two dominant lineages, but neither the overall resistance profile nor virulence-associated factors, explain their evolutionary success. Phenotypic analysis of resistance shows few differences between the acquisition of resistance genes and the phenotypic resistance profile, including beta-lactam antibiotics that were used to treat ESBL-positive strains. Resistance against these drugs could be explained by inhibitor-resistant beta-lactamase enzymes, carbapenemases or ampC type beta-lactamases, at least one of which was detected in most, but not all relevant strains analysed. Complete genomes for six selected strains are reported, these provide detailed insights into the mobile elements present in these isolates during the initial spread of NDM-1. The unexplained success of some lineages within this pool of highly resistant strains, and the discontinuity between phenotypic resistance and genotype at the macro level, indicate that intrinsic mechanisms contribute to competitive advantage and/or resistance.
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http://dx.doi.org/10.1038/s41598-019-38943-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382945PMC
February 2019

Complement Susceptibility in Relation to Genome Sequence of Recent Klebsiella pneumoniae Isolates from Thai Hospitals.

mSphere 2018 11 7;3(6). Epub 2018 Nov 7.

School of Pharmacy, University College London, London, United Kingdom

The capacity to resist the bactericidal action of complement (C') is a strong but poorly understood virulence trait in spp. Killing requires activation of one or more C' pathways, assembly of C5b-9 membrane attack complexes (MACs) on the surface of the outer membrane (OM), and penetration of MACs into the target bilayer. We interrogated whole-genome sequences of 164 isolates from three tertiary hospitals in Thailand for genes encoding surface-located macromolecules considered to play a role in determination of C' resistance. Most isolates (154/164) were identified as , and the collection conformed to previously established population structures and antibiotic resistance patterns. The distribution of sequence types (STs) and capsular (K) types were also typical of global populations. The majority (64%) of isolates were resistant to C', and the remainder were either rapidly or slowly killed. All isolates carried genes encoding capsular polysaccharides (K antigens), which have been strongly linked to C' resistance. In contrast to previous reports, there were no differences in the amount of capsule produced by C'-resistant isolates compared to C'-susceptible isolates, nor was there any correlation between serum reactivity and the presence of hypermucoviscous capsules. Similarly, there were no correlations between the presence of genes specifying lipopolysaccharide O-side chains or major OM proteins. Some virulence factors were found more frequently in C'-resistant isolates but were considered to reflect clonal ST expansion. Thus, no single gene accounts for the C' resistance of the isolates sequenced in this study. Multidrug-resistant is responsible for an increasing proportion of nosocomial infections, and emerging hypervirulent clones now cause severe community-acquired infections in otherwise healthy individuals. These bacteria are adept at circumventing immune defenses, and most survive and grow in serum; their capacity to avoid C'-mediated destruction is correlated with their invasive potential. Killing of Gram-negative bacteria occurs following activation of the C' cascades and stable deposition of C5b-9 MACs onto the OM. For , studies with mutants and conjugants have invoked capsules, lipopolysaccharide O-side chains, and OM proteins as determinants of C' resistance, although the precise roles of the macromolecules are unclear. In this study, we sequenced 164 isolates with different C' susceptibilities to identify genes involved in resistance. We conclude that no single OM constituent can account for resistance, which is likely to depend on biophysical properties of the target bilayer.
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http://dx.doi.org/10.1128/mSphere.00537-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222052PMC
November 2018

The return of Pfeiffer's bacillus: Rising incidence of ampicillin resistance in Haemophilus influenzae.

Authors:
Eva Heinz

Microb Genom 2018 09 12;4(9). Epub 2018 Sep 12.

Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK.

Haemophilus influenzae, originally named Pfeiffer's bacillus after its discoverer Richard Pfeiffer in 1892, was a major risk for global health at the beginning of the 20th century, causing childhood pneumonia and invasive disease as well as otitis media and other upper respiratory tract infections. The implementation of the Hib vaccine, targeting the major capsule type of H. influenzae, almost eradicated the disease in countries that adapted the vaccination scheme. However, a rising number of infections are caused by non-typeable H. influenzae (NTHi), which has no capsule and against which the vaccine therefore provides no protection, as well as other serotypes equally not recognised by the vaccine. The first line of treatment is ampicillin, but there is a steady rise in ampicillin resistance. This is both through acquired as well as intrinsic mechanisms, and is cause for serious concern and the need for more surveillance. There are also increasing reports of new modifications of the intrinsic ampicillin-resistance mechanism leading to resistance against cephalosporins and carbapenems, the last line of well-tolerated drugs, and ampicillin-resistant H. influenzae was included in the recently released priority list of antibiotic-resistant bacteria by the WHO. This review provides an overview of ampicillin resistance prevalence and mechanisms in the context of our current knowledge about population dynamics of H. influenzae.
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http://dx.doi.org/10.1099/mgen.0.000214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202453PMC
September 2018

SLING: a tool to search for linked genes in bacterial datasets.

Nucleic Acids Res 2018 11;46(21):e128

Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

Gene arrays and operons that encode functionally linked proteins form the most basic unit of transcriptional regulation in bacteria. Rules that govern the order and orientation of genes in these systems have been defined; however, these were based on a small set of genomes that may not be representative. The growing availability of large genomic datasets presents an opportunity to test these rules, to define the full range and diversity of these systems, and to understand their evolution. Here we present SLING, a tool to Search for LINked Genes by searching for a single functionally essential gene, along with its neighbours in a rule-defined proximity (https://github.com/ghoresh11/sling/wiki). Examining this subset of genes enables us to understand the basic diversity of these genetic systems in large datasets. We demonstrate the utility of SLING on a clinical collection of enteropathogenic Escherichia coli for two relevant operons: toxin antitoxin (TA) systems and RND efflux pumps. By examining the diversity of these systems, we gain insight on distinct classes of operons which present variable levels of prevalence and ability to be lost or gained. The importance of this analysis is not limited to TA systems and RND pumps, and can be expanded to understand the diversity of many other relevant gene arrays.
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http://dx.doi.org/10.1093/nar/gky738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265476PMC
November 2018

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Mol Microbiol 2018 09 19;109(5):584-599. Epub 2018 Sep 19.

Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, 3800, Australia.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.
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http://dx.doi.org/10.1111/mmi.13990DOI Listing
September 2018

Kaptive Web: User-Friendly Capsule and Lipopolysaccharide Serotype Prediction for Klebsiella Genomes.

J Clin Microbiol 2018 06 25;56(6). Epub 2018 May 25.

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria, Australia.

As whole-genome sequencing becomes an established component of the microbiologist's toolbox, it is imperative that researchers, clinical microbiologists, and public health professionals have access to genomic analysis tools for the rapid extraction of epidemiologically and clinically relevant information. For the Gram-negative hospital pathogens such as , initial efforts have focused on the detection and surveillance of antimicrobial resistance genes and clones. However, with the resurgence of interest in alternative infection control strategies targeting surface polysaccharides, the ability to extract information about these antigens is increasingly important. Here we present Kaptive Web, an online tool for the rapid typing of K and O loci, which encode the polysaccharide capsule and lipopolysaccharide O antigen, respectively. Kaptive Web enables users to upload and analyze genome assemblies in a web browser. The results can be downloaded in tabular format or explored in detail via the graphical interface, making it accessible for users at all levels of computational expertise. We demonstrate Kaptive Web's utility by analyzing >500 genomes. We identify extensive K and O locus diversity among 201 genomes belonging to the carbapenemase-associated clonal group 258 (25 K and 6 O loci). The characterization of a further 309 genomes indicated that such diversity is common among the multidrug-resistant clones and that these loci represent useful epidemiological markers for strain subtyping. These findings reinforce the need for rapid, reliable, and accessible typing methods such as Kaptive Web. Kaptive Web is available for use at http://kaptive.holtlab.net/, and the source code is available at https://github.com/kelwyres/Kaptive-Web.
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http://dx.doi.org/10.1128/JCM.00197-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971559PMC
June 2018

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

PLoS Pathog 2018 03 30;14(3):e1006945. Epub 2018 Mar 30.

Biomedicine Discovery Institute and Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, Australia.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.
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http://dx.doi.org/10.1371/journal.ppat.1006945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877877PMC
March 2018

The great escape.

Authors:
Eva Heinz

Nat Rev Microbiol 2017 12;16(1)

Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

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http://dx.doi.org/10.1038/nrmicro.2017.156DOI Listing
December 2017

Constraints on lateral gene transfer in promoting fimbrial usher protein diversity and function.

Open Biol 2017 11;7(11)

Infection Genomics Program, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK

Fimbriae are long, adhesive structures widespread throughout members of the family Enterobacteriaceae. They are multimeric extrusions, which are moved out of the bacterial cell through an integral outer membrane protein called usher. The complex folding mechanics of the usher protein were recently revealed to be catalysed by the membrane-embedded translocation and assembly module (TAM). Here, we examine the diversity of usher proteins across a wide range of extraintestinal (ExPEC) and enteropathogenic (EPEC) , and further focus on a so far undescribed chaperone-usher system, with this usher referred to as UshC. The fimbrial system containing UshC is distributed across a discrete set of EPEC types, including model strains like E2348/67, as well as ExPEC ST131, currently the most prominent multi-drug-resistant uropathogenic strain worldwide. Deletion of the TAM from a naive strain of results in a drastic time delay in folding of UshC, which can be observed for a protein from EPEC as well as for two introduced proteins from related organisms, and We suggest that this models why the TAM machinery is essential for efficient folding of proteins acquired via lateral gene transfer.
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http://dx.doi.org/10.1098/rsob.170144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717340PMC
November 2017

Phylogenetic Analysis of Klebsiella pneumoniae from Hospitalized Children, Pakistan.

Emerg Infect Dis 2017 11;23(11):1872-1875

Klebsiella pneumoniae shows increasing emergence of multidrug-resistant lineages, including strains resistant to all available antimicrobial drugs. We conducted whole-genome sequencing of 178 highly drug-resistant isolates from a tertiary hospital in Lahore, Pakistan. Phylogenetic analyses to place these isolates into global context demonstrate the expansion of multiple independent lineages, including K. quasipneumoniae.
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http://dx.doi.org/10.3201/eid2311.170833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652443PMC
November 2017

Reshaping the tree of life.

Nat Rev Microbiol 2017 05;15(6):322

Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

This month's Genome Watch highlights how metagenomics is continuing to reveal the diversity of microorganisms in the environment and how it is challenging and expanding our understanding of how life evolved on Earth.
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http://dx.doi.org/10.1038/nrmicro.2017.51DOI Listing
May 2017

The diversity of surface polysaccharides.

Microb Genom 2016 08 25;2(8):e000073. Epub 2016 Aug 25.

2​The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

is considered an urgent health concern due to the emergence of multi-drug-resistant strains for which vaccination offers a potential remedy. Vaccines based on surface polysaccharides are highly promising but need to address the high diversity of surface-exposed polysaccharides, synthesized as O-antigens (lipopolysaccharide, LPS) and K-antigens (capsule polysaccharide, CPS), present in . We present a comprehensive and clinically relevant study of the diversity of O- and K-antigen biosynthesis gene clusters across a global collection of over 500 whole-genome sequences and the seroepidemiology of human isolates from different infection types. Our study defines the genetic diversity of O- and K-antigen biosynthesis cluster sequences across this collection, identifying sequences for known serotypes as well as identifying novel LPS and CPS gene clusters found in circulating contemporary isolates. Serotypes O1, O2 and O3 were most prevalent in our sample set, accounting for approximately 80 % of all infections. In contrast, K serotypes showed an order of magnitude higher diversity and differ among infection types. In addition we investigated a potential association of O or K serotypes with phylogenetic lineage, infection type and the presence of known virulence genes. K1 and K2 serotypes, which are associated with hypervirulent , were associated with a higher abundance of virulence genes and more diverse O serotypes compared to other common K serotypes.
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http://dx.doi.org/10.1099/mgen.0.000073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320592PMC
August 2016

Evolutionary conservation and in vitro reconstitution of microsporidian iron-sulfur cluster biosynthesis.

Nat Commun 2017 01 4;8:13932. Epub 2017 Jan 4.

Institut für Zytobiologie und Zytopathologie, Philipps-Universität, Robert-Koch-Strasse 6, Marburg 35032, Germany.

Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron-sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe-2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron-sulfur protein assembly (CIA) pathway includes the essential Cfd1-Nbp35 scaffold complex that assembles a [4Fe-4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes.
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http://dx.doi.org/10.1038/ncomms13932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5216125PMC
January 2017

Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family.

Genome Biol Evol 2016 06 13;8(6):1690-705. Epub 2016 Jun 13.

Department of Microbiology, Infection & Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, Australia

The bacterial cell surface proteins intimin and invasin are virulence factors that share a common domain structure and bind selectively to host cell receptors in the course of bacterial pathogenesis. The β-barrel domains of intimin and invasin show significant sequence and structural similarities. Conversely, a variety of proteins with sometimes limited sequence similarity have also been annotated as "intimin-like" and "invasin" in genome datasets, while other recent work on apparently unrelated virulence-associated proteins ultimately revealed similarities to intimin and invasin. Here we characterize the sequence and structural relationships across this complex protein family. Surprisingly, intimins and invasins represent a very small minority of the sequence diversity in what has been previously the "intimin/invasin protein family". Analysis of the assembly pathway for expression of the classic intimin, EaeA, and a characteristic example of the most prevalent members of the group, FdeC, revealed a dependence on the translocation and assembly module as a common feature for both these proteins. While the majority of the sequences in the grouping are most similar to FdeC, a further and widespread group is two-partner secretion systems that use the β-barrel domain as the delivery device for secretion of a variety of virulence factors. This comprehensive analysis supports the adoption of the "inverse autotransporter protein family" as the most accurate nomenclature for the family and, in turn, has important consequences for our overall understanding of the Type V secretion systems of bacterial pathogens.
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http://dx.doi.org/10.1093/gbe/evw112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943183PMC
June 2016

Removal of hydrocarbons from synthetic road runoff through adsorptive filters.

Water Sci Technol 2015 ;72(5):802-9

Institute of Urban Water Management and Environmental Engineering, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany E-mail:

Compact filter systems, which are installed to significantly reduce the load of pollutants from road runoff, are very promising treatments for urban runoff. The objective of this research was to evaluate the effectiveness of activated carbon, activated lignite, zero valent iron, exfoliated graphite, amorphous ferric hydroxide, and activated alumina at removing petrol hydrocarbons from synthetic road runoff. Therefore, the kinetics and the equilibrium adsorption of petrol hydrocarbons onto these adsorbents were investigated using column adsorption experiments at levels ranging from 100 to 42 g L(-1). Of the tested adsorbents, exfoliated graphite is the most effective with a maximum adsorption capacity for petrol hydrocarbons of 3,850 mg g(-1). The experimental equilibrium data are fitted to the Freundlich and Langmuir models.
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http://dx.doi.org/10.2166/wst.2015.285DOI Listing
December 2015