Convergent Molecular Evolution of Genomic Cores in Salmonella enterica and Escherichia coli

ABSTRACTOne of the strongest signals of adaptive molecular evolution of proteins is the occurrence of convergent hot spot mutations: repeated changes in the same amino acid positions. We performed a comparative genome-wide analysis of mutation-driven evolution of core (omnipresent) genes in 17 strains ofSalmonella entericasubspecies I and 22 strains ofEscherichia coli. More than 20% of core genes in bothSalmonellaandE. coliaccumulated hot spot mutations, with a predominance of identical changes having recent evolutionary origin. There is a significant overlap in the functional categories of the adaptively evolving genes in both species, although mostly via separate molecular mechanisms. As a strong evidence of the link between adaptive mutations and virulence inSalmonella, two human-restricted serovars, Typhi and Paratyphi A, shared the highest number of genes with serovar-specific hot spot mutations. Many of the core genes affected by Typhi/Paratyphi A-specific mutations have known virulence functions. For each species, a list of nonrecombinant core genes (and the hot spot mutations therein) under positive selection is provided.

Download Full-text

Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

Applied and Environmental Microbiology ◽

10.1128/aem.03079-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 713-725 ◽

Cited By ~ 44

Author(s):

John W. Schmidt ◽

Getahun E. Agga ◽

Joseph M. Bosilevac ◽

Dayna M. Brichta-Harhay ◽

Steven D. Shackelford ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Generation Cephalosporin ◽

Resistant Bacteria ◽

Processing Plant ◽

Cattle Production ◽

Content Type ◽

E Coli ◽

Beef Processing ◽

Tract Infections

ABSTRACTSpecific concerns have been raised that third-generation cephalosporin-resistant (3GCr)Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COTr)E. coli, 3GCrSalmonella enterica, and nalidixic acid-resistant (NALr)S. entericamay be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n= 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GCrSalmonellawas detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NALrS. entericawas detected on only one hide. 3GCrE. coliand COTrE. coliwere detected on 100.0% of hides during processing. Concentrations of 3GCrE. coliand COTrE. colion hides were correlated with pre-evisceration carcass contamination. 3GCrE. coliand COTrE. coliwere each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenicE. coli(ExPEC) virulence-associated markers. Only two COTrE. coliisolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria.

Download Full-text

Characterization of Epidemic IncI1-Iγ Plasmids Harboring Ambler Class A and C Genes in Escherichia coli and Salmonella enterica from Animals and Humans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05006-14 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5357-5365 ◽

Cited By ~ 36

Author(s):

Hilde Smith ◽

Alex Bossers ◽

Frank Harders ◽

Guanghui Wu ◽

Neil Woodford ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Phylogenetic Trees ◽

Functional Study ◽

Content Type ◽

Gene Associations ◽

Genes Encoding ◽

Marked Resistance

ABSTRACTThe aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained fromEscherichia coliandSalmonella entericaisolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation intraYandexcAgenes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

Download Full-text

Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

Applied and Environmental Microbiology ◽

10.1128/aem.02567-17 ◽

2018 ◽

Vol 84 (8) ◽

pp. e02567-17 ◽

Cited By ~ 12

Author(s):

H. Bart van den Berg van Saparoea ◽

Diane Houben ◽

Marien I. de Jonge ◽

Wouter S. P. Jong ◽

Joen Luirink

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Salmonella Enterica ◽

Membrane Vesicles ◽

Therapeutic Agents ◽

Outer Membrane Vesicles ◽

High Density ◽

Content Type ◽

Protein Ligation ◽

Serovar Typhimurium

ABSTRACT The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

Download Full-text

Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073

Journal of Bacteriology ◽

10.1128/jb.00168-17 ◽

2017 ◽

Vol 199 (24) ◽

Cited By ~ 7

Author(s):

Sébastien Crépin ◽

Gaëlle Porcheron ◽

Sébastien Houle ◽

Josée Harel ◽

Charles M. Dozois

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Molecular Mechanisms ◽

Diguanylate Cyclase ◽

Altered Expression ◽

Content Type ◽

Type 1 Fimbriae ◽

Tract Infections ◽

Pst System

ABSTRACT The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC (adrA in Salmonella) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC. This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA, affecting at the same time the inversion of the fim promoter switch (fimS). In the pst mutant, inactivation of yaiC restored fim-dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC, which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.

Download Full-text

Evaluation of Aerated Steam Treatment of Alfalfa and Mung Bean Seeds To Eliminate High Levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica, and Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00443-13 ◽

2013 ◽

Vol 79 (15) ◽

pp. 4613-4619 ◽

Cited By ~ 14

Author(s):

Patrick Studer ◽

Werner E. Heller ◽

Jörg Hummerjohann ◽

David Drissner

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Salmonella Enterica ◽

Mung Bean ◽

Germination Rate ◽

Steam Treatment ◽

Heat Sensitivity ◽

Human Pathogens ◽

Content Type ◽

E Coli

ABSTRACTSprouts contaminated with human pathogens are able to cause food-borne diseases due to the favorable growth conditions for bacteria during germination and because of minimal processing steps prior to consumption. We have investigated the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung bean seeds which have been artificially contaminated withEscherichia coliO157:H7,Salmonella entericasubsp.entericaserovar Weltevreden, andListeria monocytogenesScott A. In addition, a recently collectedE. coliO178:H12 isolate, characterized by a reduced heat sensitivity, was exposed to the treatment described. Populations ofE. coliO157:H7 andS. entericaon alfalfa and mung bean seeds could be completely eliminated by a 300-s treatment with steam at 70 ± 1°C as revealed by enrichment studies.L. monocytogenesandE. coliO178:H12 could not be completely eliminated from artificially inoculated seeds. However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung beans was not affected by the 300-s treatment compared to the germination rate of untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%. This chemical-free method is an effective alternative to the 20,000-ppm hypochlorite treatment presently recommended by the U.S. Food and Drug Administration (FDA).

Download Full-text

Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

Applied and Environmental Microbiology ◽

10.1128/aem.00599-11 ◽

2011 ◽

Vol 77 (20) ◽

pp. 7104-7112 ◽

Cited By ~ 59

Author(s):

Maria Karczmarczyk ◽

Yvonne Abbott ◽

Ciara Walsh ◽

Nola Leonard ◽

Séamus Fanning

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Gene Array ◽

Multidrug Resistant ◽

Genetic Determinants ◽

Gene Encoding ◽

Content Type ◽

E Coli ◽

Class 1

ABSTRACTIn this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection ofEscherichia coliisolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1,dfrA1-aadA1,dfrA17-aadA5,dfrA12-orfF-aadA2,blaOXA-30-aadA1,aacC1-orf1-orf2-aadA1,dfr7). Class 2 integrons (13.5%) contained thedfrA1-sat1-aadA1gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected includedblaTEM,cat,floR,aadB,aphA1,strA-strB,sul2, andtet(B), respectively. TheblaCTX-M-2gene, encoding an extended-spectrum β-lactamase (ESβL), andblaCMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensalE. coliisolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, theblaCTX-M-2gene has not previously been reported in Ireland.

Download Full-text

Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves

Applied and Environmental Microbiology ◽

10.1128/aem.07454-11 ◽

2012 ◽

Vol 78 (6) ◽

pp. 1752-1764 ◽

Cited By ~ 64

Author(s):

Ryan C. Fink ◽

Elaine P. Black ◽

Zhe Hou ◽

Masayuki Sugawara ◽

Michael J. Sadowsky ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Leaf Surface ◽

Short Term ◽

Transcriptional Responses ◽

Content Type ◽

E Coli ◽

Leaf Surfaces ◽

K 12

ABSTRACTAn increasing number of outbreaks of gastroenteritis recently caused byEscherichia coliO157:H7 have been linked to the consumption of leafy green vegetables. Although it is known thatE. colisurvives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identifyE. coligenes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparingE. coliK-12, a model system, andE. coliO157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, includingtnaA(33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsAandybiM) and curli production (csgAandcsgB) were significantly upregulated inE. coliK-12 and O157:H7. BothcsgAandbhsA(ycfR) mutants were impaired in the long-term colonization of the leaf surface, but onlycsgAmutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction ofE. coliK-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation.

Download Full-text

Modeling Stochastic Variability in the Numbers of Surviving Salmonella enterica, Enterohemorrhagic Escherichia coli, and Listeria monocytogenes Cells at the Single-Cell Level in a Desiccated Environment

Applied and Environmental Microbiology ◽

10.1128/aem.02974-16 ◽

2016 ◽

Vol 83 (4) ◽

Cited By ~ 10

Author(s):

Kento Koyama ◽

Hidekazu Hokunan ◽

Mayumi Hasegawa ◽

Shuso Kawamura ◽

Shigenobu Koseki

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Probability Distribution ◽

Poisson Process ◽

Poisson Distribution ◽

Salmonella Enterica ◽

Bacterial Cells ◽

Content Type ◽

Cell Numbers ◽

Inactivation Process

ABSTRACT Despite effective inactivation procedures, small numbers of bacterial cells may still remain in food samples. The risk that bacteria will survive these procedures has not been estimated precisely because deterministic models cannot be used to describe the uncertain behavior of bacterial populations. We used the Poisson distribution as a representative probability distribution to estimate the variability in bacterial numbers during the inactivation process. Strains of four serotypes of Salmonella enterica, three serotypes of enterohemorrhagic Escherichia coli, and one serotype of Listeria monocytogenes were evaluated for survival. We prepared bacterial cell numbers following a Poisson distribution (indicated by the parameter λ, which was equal to 2) and plated the cells in 96-well microplates, which were stored in a desiccated environment at 10% to 20% relative humidity and at 5, 15, and 25°C. The survival or death of the bacterial cells in each well was confirmed by adding tryptic soy broth as an enrichment culture. Changes in the Poisson distribution parameter during the inactivation process, which represent the variability in the numbers of surviving bacteria, were described by nonlinear regression with an exponential function based on a Weibull distribution. We also examined random changes in the number of surviving bacteria using a random number generator and computer simulations to determine whether the number of surviving bacteria followed a Poisson distribution during the bacterial death process by use of the Poisson process. For small initial cell numbers, more than 80% of the simulated distributions (λ = 2 or 10) followed a Poisson distribution. The results demonstrate that variability in the number of surviving bacteria can be described as a Poisson distribution by use of the model developed by use of the Poisson process. IMPORTANCE We developed a model to enable the quantitative assessment of bacterial survivors of inactivation procedures because the presence of even one bacterium can cause foodborne disease. The results demonstrate that the variability in the numbers of surviving bacteria was described as a Poisson distribution by use of the model developed by use of the Poisson process. Description of the number of surviving bacteria as a probability distribution rather than as the point estimates used in a deterministic approach can provide a more realistic estimation of risk. The probability model should be useful for estimating the quantitative risk of bacterial survival during inactivation.

Download Full-text

Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Buffered Peptone Water and Apple Juice

Applied and Environmental Microbiology ◽

10.1128/aem.01818-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7122-7129 ◽

Cited By ~ 72

Author(s):

Il-Kyu Park ◽

Dong-Hyun Kang

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Electric Field ◽

Salmonella Enterica ◽

Apple Juice ◽

Ohmic Heating ◽

Conventional Heating ◽

Content Type ◽

Serovar Typhimurium ◽

Peptone Water

ABSTRACTThe effect of electric field-induced ohmic heating for inactivation ofEscherichia coliO157:H7,Salmonella entericaserovar Typhimurium, andListeria monocytogenesin buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P< 0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P< 0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating.

Download Full-text

Survival of Escherichia coli Cells on Solid Copper Surfaces Is Increased by Glutathione

Applied and Environmental Microbiology ◽

10.1128/aem.02842-14 ◽

2014 ◽

Vol 80 (22) ◽

pp. 7071-7078 ◽

Cited By ~ 18

Author(s):

Cornelia Große ◽

Grit Schleuder ◽

Christin Schmole ◽

Dietrich H. Nies

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Copper Tolerance ◽

Anaerobic Growth ◽

Resistant Bacteria ◽

Nadh:Ubiquinone Oxidoreductase ◽

Content Type ◽

Copper Surfaces ◽

Dps Protein ◽

Solid Copper

ABSTRACTBacteria are rapidly killed on solid copper surfaces, so this material could be useful to limit the spread of multiple-drug-resistant bacteria in hospitals. InEscherichia coli, the DNA-protecting Dps protein and the NADH:ubiquinone oxidoreductase II Ndh were not involved in tolerance to copper ions or survival on solid copper surfaces. Decreased copper tolerance under anaerobic growth conditions in the presence of ascorbate and with melibiose as the carbon source indicated that sodium-dependent symport systems may provide an import route for CuIinto the cytoplasm. Glutathione-free ΔcopAΔgshAdouble mutants ofE. coliwere more rapidly inactivated on solid copper surfaces than glutathione-containing wild-type cells. Therefore, while DNA protection by Dps was not required, glutathione was needed to protect the cytoplasm and the DNA against damage mediated by solid copper surfaces, which may explain the differences in the molecular mechanisms of killing between glutathione-containing Gram-negative and glutathione-free Gram-positive bacteria.

Download Full-text