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Lipopolysaccharide-Deficient Mutants of Salmonella enterica Have Increased Sensitivity to Catechins  [PDF]
Miho Yoshii, Akira Okamoto, Michio Ohta
Advances in Microbiology (AiM) , 2013, DOI: 10.4236/aim.2013.35054
Abstract:

Antimicrobial activity is one of the well-known biological characteristics of catechins, the main extract of green tea leaves. It is thought that catechins intercalate into the bacterial cell membrane and damage the lipid bilayer. However, the association between catechins and lipopolysaccharides, which consist of an O side chain, core oligosaccharide, and lipid A, has not been previously investigated. In this study, we evaluated the catechin sensitivity of Salmonella enterica mutants that lack the O side chain and have core oligosaccharides of different lengths. These rough mutants were more sensitive to catechins than a bacterial strain with intact lipopolysaccharide. We conclude that the O side chain and core oligosaccharide play an important role in protecting Gram-negative bacteria against the antimicrobial activity of catechins.

Phage-resistance of Salmonella enterica serovar Enteritidis and pathogenesis in Caenorhabditis elegans is mediated by the lipopolysaccharide
Santander,Javier; Robeson,James;
Electronic Journal of Biotechnology , 2007,
Abstract: phage therapy has been used in the past as an alternative therapy against bacterial pathogens. however, phage-resistant bacterial strains can emerge. some studies show that these phage-resistant strains are avirulent. in this study, we report that phage-resistant strains of salmonella enterica serovar enteritidis (hereafter s. enteritidis) were avirulent in the caenorhabditis elegans animal model. we isolated phage-resistant strains of s. enteritidis atcc 13076 by using three lytic phages (f2αse, f3αse and f18αse). in these mutants, we explored different virulence factors like lipopolysaccharide (lps), virulence plasmid (pla), motility and type i fimbriae, all of which may have effects on virulence and could furthermore be related to phage resistance. the phage-resistant strains of s. enteritidis showed loss of o-polysaccharide (o-ps) and auto-agglutination, present a rough phenotype and consequently they are avirulent in the c. elegans animal model. we speculate that the o-ps is necessary for phage attachment to the s. enteritidis cell surface.
Adaptation and Preadaptation of Salmonella enterica to Bile  [PDF]
Sara B. Hernández,Ignacio Cota,Adrien Ducret,Laurent Aussel,Josep Casadesús
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002459
Abstract: Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS? mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10?6 and 10?7 per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations.
O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells  [PDF]
Claudia U. Duerr,Sebastian F. Zenk,Cécilia Chassin,Johanna Pott,Dominique Gütle,Michael Hensel,Mathias W. Hornef
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000567
Abstract: Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.
Analysis of Triclosan-Selected Salmonella enterica Mutants of Eight Serovars Revealed Increased Aminoglycoside Susceptibility and Reduced Growth Rates  [PDF]
Ulrike Rensch, Guenter Klein, Corinna Kehrenberg
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0078310
Abstract: The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPCTRC values were 8 - 64-fold higher than MIC values and ranged between 1 - 16 μg/ml. The frequencies at which mutants were selected varied between 1.3 x 10-10 - 9.9 x 10-11. Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly93→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 μg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly93→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased susceptibility to triclosan.
Exposure of Salmonella enterica Serovar Typhimurium to High Level Biocide Challenge Can Select Multidrug Resistant Mutants in a Single Step  [PDF]
Rebekah N. Whitehead, Tim W. Overton, Caroline L. Kemp, Mark A. Webber
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0022833
Abstract: Background Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux. Methodology/Principal Findings Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide. Conclusions/Significance These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.
Production and Kinetics of Salmonella enterica serovar enteritidis in Vibrofermentor
A. Nalbantsoy,O. Akpolat,I. Karaboz,S.I. Deliloglu-Gurhan
Biotechnology , 2007,
Abstract: Food-borne human Salmonellosis caused by Salmonella enterica serovar enteritidis has given a rise to many of economic losses in terms of both poultry and food industries. Salmonella has a great importance among the major bacterial pathogens of poultry. In Turkey as well as all over the world prevention of Salmonella infection can be achieved by good monitoring and screening programs. More recently immunological test systems are used for diagnosis based on the detection of surface antigens such as lipopolysaccharide (LPS), flagellin etc. As to its production some different methods are used practically. In this work, the lab-scale production of Salmonella enterica serovar enteritidis was achieved both in shake-flask and vibrofermentor cultures and a comparative optimization of the process yield and production kinetics was carried out. The microorganisms were cultivated in brain heart infusion broth, at 37°C for 5 h. Specific growth rates and doubling times for both vibroferment r and shake-flask were estimated as 0.509 and 0.709 h-1 and 75.0 and 58.6 min, respectively. LPS and flagella were extracted from lyophilized cultures.
Lipopolysaccharide-Deficient Brucella Variants Arise Spontaneously during Infection  [PDF]
Joshua E. Turse,Jianwu Pei,Thomas A. Ficht
Frontiers in Microbiology , 2011, DOI: 10.3389/fmicb.2011.00054
Abstract: Lipopolysaccharide-deficient mutants of smooth Brucella species (rough mutants) have been shown to arise spontaneously in culture. However, in situ analysis of Brucella infected macrophages using antibody directed against O-polysaccharide suggested a loss of reactivity of Brucella consistent with the appearance of rough organisms, and a potential contribution to infection. The experiments reported describe the direct recovery of Brucella from macrophages infected in vitro and from the spleens of infected mice at a frequency similar to that described in vitro, suggesting that Brucella dissociation is not simply an in vitro artifact. The frequency of appearance of spontaneous rough organisms deficient in O-polysaccharide expression measured in vitro is approximately 2–3 logs higher than the appearance of mutation to antibiotic resistance, purine auxotrophy, or reversion of erythritol sensitive ΔeryC mutants to tolerance. Genetic trans-complementation using a plasmid-based expression of Brucella manBA successfully restored O-polysaccharide expression in only one-third of O-polysaccharide deficient spontaneous mutants. Suggesting that the appearance of rough mutants is caused by mutation at more than one locus. In addition, Sanger sequencing of the manBA structural genes detected multiple sequence changes that may explain the observed phenotypic differences. The presence of O-polysaccharide resulted in macrophage and neutrophil infiltration into the peritoneal cavity and systemic distribution of the organism. In contrast, rough organisms are controlled by resident macrophages or by extracellular killing mechanisms and rapidly cleared from this compartment consistent with the inability to cause disease. Loss of O-polysaccharide expression appears to be stochastic giving rise to organisms with biological properties distinct from the parental smooth organism during the course of infection.
An Altered Immune Response, but Not Individual Cationic Antimicrobial Peptides, Is Associated with the Oral Attenuation of Ara4N-Deficient Salmonella enterica Serovar Typhimurium in Mice  [PDF]
Kristi L. Strandberg, Susan M. Richards, Rita Tamayo, Linh T. Reeves, John S. Gunn
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0049588
Abstract: Salmonella enterica serovar Typhimurium (S. Typhimurium) uses two-component regulatory systems (TCRS) to respond to stimuli in the local microenvironment. Upon infection, the Salmonella TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals in the intestinal lumen and within host cells. TCRS-mediated gene expression results in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide resistance. The PmrA-regulated pmrHFIJKLM operon mediates 4-amino-4-deoxy-L-arabinose (Ara4N) production and attachment to the lipid A of LPS. A ΔpmrF S. Typhimurium strain cannot produce Ara4N, exhibits increased sensitivity to cationic antimicrobial peptide (CAMP)-mediated killing, and attenuated virulence in mice upon oral infection. CAMPs are predicted to play a role in elimination of Salmonella, and may activate PhoPQ and PmrAB in vivo, which could increase bacterial resistance to host defenses. Competition experiments between wild type (WT) and ΔpmrF mutant strains of S. Typhimurium indicated that selection against this mutant first occurs within the intestinal lumen early during infection. However, CRAMP and active cryptdins alone are not responsible for elimination of Ara4N-deficient bacteria in vivo. Investigation into the early immune response to ΔpmrF showed that it differed slightly from the early immune response to WT S. Typhimurium. Further investigation into the early immune response to infection of Peyer’s patches suggests a role for IL-13 in the attenution of the ΔpmrF mutant strain. Thus, prominent CAMPs present in the mouse intestine are not responsible for the selection against the ΔpmrF strain in this location, but limited alterations in innate immune induction were observed that affect bacterial survival and virulence.
Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export  [PDF]
David González, María-Jesús Grilló, María-Jesús De Miguel, Tara Ali, Vilma Arce-Gorvel, Rose-May Delrue, Raquel Conde-álvarez, Pilar Mu?oz, Ignacio López-Go?i, Maite Iriarte, Clara-M. Marín, Andrej Weintraub, G?ran Widmalm, Michel Zygmunt, Jean-Jacques Letesson, Jean-Pierre Gorvel, José-María Blasco, Ignacio Moriyón
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002760
Abstract: Background The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.
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