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Melissa L. Madsen

Bio: Melissa L. Madsen is an academic researcher from Iowa State University. The author has contributed to research in topics: Mycoplasma hyopneumoniae & Mutant. The author has an hindex of 10, co-authored 12 publications receiving 657 citations.

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Journal ArticleDOI
TL;DR: The complete genome sequence of Mycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex, is presented, finding few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation and it is not clear how M. hyop pneumoniae evades the immune response and establishes a chronic infection.
Abstract: We present the complete genome sequence of Mycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex. The genome is composed of 892,758 bp and has an average G+C content of 28.6 mol%. There are 692 predicted protein coding sequences, the average protein size is 388 amino acids, and the mean coding density is 91%. Functions have been assigned to 304 (44%) of the predicted protein coding sequences, while 261 (38%) of the proteins are conserved hypothetical proteins and 127 (18%) are unique hypothetical proteins. There is a single 16S-23S rRNA operon, and there are 30 tRNA coding sequences. The cilium adhesin gene has six paralogs in the genome, only one of which contains the cilium binding site. The companion gene, P102, also has six paralogs. Gene families constitute 26.3% of the total coding sequences, and the largest family is the 34-member ABC transporter family. Protein secretion occurs through a truncated pathway consisting of SecA, SecY, SecD, PrsA, DnaK, Tig, and LepA. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The DnaK-DnaJ-GrpR complex is intact, providing the only control over protein folding. There are several proteases that might serve as virulence factors, and there are 53 coding sequences with prokaryotic lipoprotein lipid attachment sites. Unlike other mycoplasmas, M. hyopneumoniae contains few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation. Thus, it is not clear how M. hyopneumoniae evades the immune response and establishes a chronic infection.

279 citations

Journal ArticleDOI
TL;DR: Two new real-time PCR assays that are specific and capable of detecting all of the M. hyopneumoniae isolates used in this study were developed were developed.
Abstract: Mycoplasma hyopneumoniae is an important cause of pneumonia in pigs around the world, but confirming its presence in (or absence from) pigs can be difficult. Culture for diagnosis is impractical, and seroconversion is often delayed after natural infection, limiting the use of serology. Numerous PCR assays for the detection of M. hyopneumoniae have been developed, targeting several different genes. Recently, genetic diversity among strains of M. hyopneumoniae was demonstrated. The effect of this diversity on the accuracy and sensitivity of the M. hyopneumoniae PCR assays could result in false-negative results in current PCR tests. In this study, a panel of isolates of M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae were tested with a number of M. hyopneumoniae-specific PCR assays. Some M. hyopneumoniae PCR assays tested did not detect all isolates of M. hyopneumoniae. To increase the efficiency of PCR testing, two new real-time PCR assays that are specific and capable of detecting all of the M. hyopneumoniae isolates used in this study were developed.

87 citations

Journal ArticleDOI
TL;DR: 91 genes that had significant transcriptional differences in response to heat shock conditions were identified and many of the heat shock proteins previously characterized in other bacteria were identified as significant in this study as well.
Abstract: Bacterial pathogens undergo stress during host colonization and disease processes. These stresses result in changes in gene expression to compensate for potentially lethal environments developed in the host during disease. Mycoplasma hyopneumoniae colonizes the swine epithelium and causes a pneumonia that predisposes the host to enhanced disease from other pathogens. How M. hyopneumoniae responds to changing environments in the respiratory tract during disease progression is not known. In fact, little is known concerning the capabilities of mycoplasmas to respond to changing growth environments. With limited genes, mycoplasmas are thought to possess only a few mechanisms for gene regulation. A microarray consisting of 632 of the 698 open reading frames of M. hyopneumoniae was constructed and used to study gene expression differences during a temperature shift from 37°C to 42°C, a temperature swing that might be encountered during disease. To enhance sensitivity, a unique hexamer primer set was employed for generating cDNA from only mRNA species. Our analysis identified 91 genes that had significant transcriptional differences in response to heat shock conditions (P < 0.01) with an estimated false-discovery rate of 4 percent. Thirty-three genes had a change threshold of 1.5-fold or greater. Many of the heat shock proteins previously characterized in other bacteria were identified as significant in this study as well. A proportion of the identified genes (54 of 91) currently have no assigned function.

73 citations

Journal ArticleDOI
TL;DR: In response to norepinephrine, M. hyopneumoniae appears to upregulate protein expression while downregulating general metabolism, which correlated with the reduction in growth of the mycoplasma.
Abstract: Mycoplasma hyopneumoniae, a component of the porcine respiratory disease complex, colonizes the respiratory tract of swine by binding to the cilia of the bronchial epithelial cells. Mechanisms of pathogenesis are poorly understood for M. hyopneumoniae, but previous work has indicated that it responds to the environmental stressors heat shock, iron deprivation and oxidative compounds. For successful infection, M. hyopneumoniae must effectively resist host responses to the colonization of the respiratory tract. Among these are changes in hormonal levels in the mucosal secretions. Recent work in the stress responses of other bacteria has included the response to the catecholamine norepinephrine. The idea that M. hyopneumoniae can respond to a host hormone, however, is novel and has not previously been demonstrated. To test this, organisms in the early exponential phase of growth were exposed to 100 μM norepinephrine for 4 h, and RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. The M. hyopneumoniae response included slowed growth and changes in mRNA transcript levels of 84 genes, 53 of which were upregulated in response to norepinephrine. A larger proportion of the genes upregulated than those downregulated were involved with transcription and translation. The downregulated genes were mostly involved with metabolism, which correlated with the reduction in growth of the mycoplasma. Approximately 51 % of the genes were hypothetical with no known function. Thus, in response to norepinephrine, M. hyopneumoniae appears to upregulate protein expression while downregulating general metabolism.

58 citations

Journal ArticleDOI
TL;DR: During lung infection, the analysis indicated that 79 M. hyopneumoniae genes were differentially expressed, and 28 of 46 lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes.
Abstract: Mycoplasma hyopneumoniae causes swine pneumonia and contributes significantly to the porcine respiratory disease complex. The mechanisms of pathogenesis are difficult to address, since there is a lack of genetic tools, but microarrays are available and can be used to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Mycoplasmas were collected from bronchial alveolar lavage samples and compared to broth-grown cells using microarrays. Bronchial alveolar lavage was performed on pigs 28 days postinfection, and mycoplasmas were isolated by differential centrifugation. Mycoplasma RNA-enriched preparations were then obtained from total RNA by subtracting eucaryotic ribosomal and messenger RNAs. Labeled cDNAs were generated with mycoplasma open reading frame-specific primers. Nine biological replicates were analyzed. During lung infection, our analysis indicated that 79 M. hyopneumoniae genes were differentially expressed (P < 0.01), at a false-discovery rate of <2.7%. Of the down-regulated genes, 28 of 46 (61%) lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes. Four down-regulated genes and two up-regulated genes encoded putative lipoproteins. secA (mhp295) (P = 0.003) and two glycerol transport permease genes (potA [mhp380; P = 0.006] and ugpA [mhp381; P = 0.003]) were up-regulated in vivo. Elongation factor EF-G (fusA [mhp083]) (P = 0.002), RNA polymerase beta chain (rpoC [mhp635]) (P = 0.003), adenylate kinase (adk [mhp208]) (P = 0.001), prolyl aminoacyl tRNA synthetase (proS [mhp397]) (P = 0.009), and cysteinyl-tRNA synthetase (cysS [mhp661]) (P < 0.001) were down-regulated in vivo.

51 citations


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Journal ArticleDOI
TL;DR: The main effects of vaccination include less clinical symptoms, lung lesions and medication use, and improved performance, however, bacterins provide only partial protection and do not prevent colonization of the organism.

370 citations

Journal ArticleDOI
TL;DR: The results show that the PVK-GO nanocomposite presents higher antimicrobial effects than the pristine GO, and offers a great opportunity for potential applications in important biomedical and industrial fields.
Abstract: It is critical to develop highly effective antimicrobial agents that are not harmful to humans and do not present adverse effects on the environment. Although antimicrobial studies of graphene-based nanomaterials are still quite limited, some researchers have paid particular attention to such nanocomposites as promising candidates for the next generation of antimicrobial agents. The polyvinyl-N-carbazole (PVK)–graphene oxide (GO) nanocomposite (PVK–GO), which contains only 3 wt% of GO well-dispersed in a 97 wt% PVK matrix, presents excellent antibacterial properties without significant cytotoxicity to mammalian cells. The high polymer content in this nanocomposite makes future large-scale material manufacturing possible in a high-yield process of adiabatic bulk polymerization. In this study, the toxicity of PVK–GO was assessed with planktonic microbial cells, biofilms, and NIH 3T3 fibroblast cells. The antibacterial effects were evaluated against two Gram-negative bacteria: Escherichia coli and Cupriavidus metallidurans; and two Gram-positive bacteria: Bacillus subtilis and Rhodococcus opacus. The results show that the PVK–GO nanocomposite presents higher antimicrobial effects than the pristine GO. The effectiveness of the PVK–GO in solution was demonstrated as the nanocomposite “encapsulated” the bacterial cells, which led to reduced microbial metabolic activity and cell death. The fact that the PVK–GO did not present significant cytotoxicity to fibroblast cells offers a great opportunity for potential applications in important biomedical and industrial fields.

336 citations

Journal ArticleDOI
TL;DR: The applications of genotyping methods to the study of bacterial strain diversity are described and compared and the progresses allowed by the availability of genomic sequences are investigated.
Abstract: Bacterial strain typing, or identifying bacteria at the strain level, is particularly important for diagnosis, treatment, and epidemiological surveillance of bacterial infections. This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics. Over the last two decades, molecular methods have progressively replaced phenotypic assays to type bacterial strains. In this article, we review the current bacterial genotyping methods and classify them into three main categories: (1) DNA banding pattern-based methods, which classify bacteria according to the size of fragments generated by amplification and/or enzymatic digestion of genomic DNA, (2) DNA sequencing-based methods, which study the polymorphism of DNA sequences, and (3) DNA hybridization-based methods using nucleotidic probes. We described and compared the applications of genotyping methods to the study of bacterial strain diversity. We also discussed the selection of appropriate genotyping methods and the challenges of bacterial strain typing, described the current trends of genotyping methods, and investigated the progresses allowed by the availability of genomic sequences.

321 citations

Journal ArticleDOI
Ana Tereza Ribeiro de Vasconcelos, Henrique Bunselmeyer Ferreira1, Cristiano Valim Bizarro1, Sandro L. Bonatto2, Marcos Oliveira de Carvalho1, Paulo Marcos Pinto1, Darcy F. de Almeida3, Luiz Gonzaga Paula de Almeida, Almeida Rosana De4, Leonardo Alves-Filho1, Enedina Nogueira de Assunção5, Vasco Azevedo6, Maurício Reis Bogo2, Marcelo M. Brigido7, Marcelo Brocchi8, Marcelo Brocchi4, Hélio Almeida Burity9, Anamaria A. Camargo10, Sandro da Silva Camargo1, Marta S. P. Carepo11, Dirce Maria Carraro10, J.C.M. Cascardo12, Luiza Amaral de Castro1, Gisele Cavalcanti, Gustavo Chemale1, Rosane G. Collevatti13, Cristina W. Cunha14, Bruno Dallagiovanna, Bibiana Paula Dambrós15, Odir Antônio Dellagostin14, Clarissa Falcão13, Fabiana Fantinatti-Garboggini8, Maria Sueli Soares Felipe7, Laurimar Fiorentin16, Glória Regina Franco6, Nara Suzy Aguiar De Freitas17, Diego Frias12, Thalles B. Grangeiro18, Edmundo C. Grisard15, Claudia Teixeira Guimarães9, Mariangela Hungria9, Silvia Neto Jardim9, Marco Aurélio Krieger, Jomar Pereira Laurino2, Lucymara Fassarella Agnez Lima19, Maryellen I. Lopes20, Élgion Lúcio da Silva Loreto21, Humberto Maciel França Madeira22, Gilson P. Manfio8, Andrea Queiroz Maranhão7, Christyanne T. Martinkovics1, Silvia Regina Batistuzzo de Medeiros19, Miguel Angêlo Martins Moreira, Márcia Neiva5, Cicero Eduardo Ramalho-Neto23, Marisa Fabiana Nicolás9, Sergio C. Oliveira6, Roger Ferreira Cury Paixão, Fábio O. Pedrosa24, Sérgio D.J. Pena6, Maristela Pereira25, Lilian Pereira-Ferrari22, Itamar Antônio Piffer16, Luciano da Silva Pinto18, Deise Porto Potrich1, Anna Christina M. Salim10, Fabrício R. Santos6, Renata Schmitt20, Maria Paula Cruz Schneider11, Augusto Schrank1, Irene Silveira Schrank1, Adriana F. Schuck1, Héctor N. Seuánez, Denise Wanderlei Silva23, Rosane Silva3, Sergio Ceroni da Silva1, Célia Maria de Almeida Soares25, Kelly Rose Lobo de Souza, Rangel C. Souza, Charley Christian Staats1, Maria B. R. Steffens24, Santuza M. R. Teixeira6, Turán P. Ürményi3, Marilene Henning Vainstein1, Luciana W. Zuccherato6, Andrew J. G. Simpson10, Arnaldo Zaha1 
TL;DR: Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species, and indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.
Abstract: This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

314 citations

Journal ArticleDOI
TL;DR: A total of 223 complete bacterial genomes are analyzed for the presence of genes encoding modular polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) and, whenever known, the metabolites they synthesize.

299 citations