Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

Genomic profile of Brazilian methicillin-resistant Staphylococcus aureus resembles clones dispersed worldwide.

PURPOSE: Comparative genomic analysis of strains may help us to better understand the wide diversity of their genetic profiles. The aim of this study was to analyse the genomic features of the resistome and virulome of Brazilian first methicillin-resistant Staphylococcus aureus (MRSA) isolates and their relationship to other Brazilian and international MRSA strains. METHODOLOGY: The whole genomes of three MRSA strains previously isolated in Vitória da Conquista were sequenced, assembled, annotated and compared with other MRSA genomes. A phylogenetic tree was constructed and the pan-genome and accessory and core genomes were constructed. The resistomes and virulomes of all strains were identified.Results/Key findings. Phylogenetic analysis of all 49 strains indicated different clones showing high similarity. The pan-genome of the analysed strains consisted of 4484 genes, with 31 % comprising the gene portion of the core genome, 47 % comprising the accessory genome and 22 % being singletons. Most strains showed at least one gene related to virulence factors associated with immune system evasion, followed by enterotoxins. The strains showed multiresistance, with the most recurrent genes conferring resistance to beta-lactams, fluoroquinolones, aminoglycosides and macrolides. CONCLUSIONS: Our comparative genomic analysis showed that there is no pattern of virulence gene distribution among the clones analysed in the different regions. The Brazilian strains showed similarity with clones from several continents.

RNA-Seq based transcriptome of whole blood from immunocompetent pigs (Sus scrofa) experimentally infected with Mycoplasma suis strain Illinois.

Pigs are popular animal models in biomedical research. RNA-Seq is becoming the predominant tool to investigate transcriptional changes of the pig's response to infection. The high sensitivity of this tool requires a strict control of the study design beginning with the selection of healthy animals to provide accurate interpretation of research data. Pigs chronically infected with Mycoplasma suis often show no obvious clinical signs, however the infection may affect the validity of animal research. The goal of this study was to investigate whether or not this silent infection is also silent at the host transcriptional level. Therefore, immunocompetent pigs were experimentally infected with M. suis and transcriptional profiles of whole blood, generated by RNA-Seq, were analyzed and compared to non-infected animals. RNA-Seq showed 55 differentially expressed (DE) genes in the M. suis infected pigs. Down-regulation of genes related to innate immunity (tlr8, chemokines, chemokines receptors) and genes containing IFN gamma-activated sequence (gbp1, gbp2, il15, cxcl10, casp1, cd274) suggests a general suppression of the immune response in the infected animals. Sixteen (29.09%) of the DE genes were involved in two protein interaction networks: one involving chemokines, chemokine receptors and interleukin-15 and another involving the complement cascade. Genes related to vascular permeability, blood coagulation, and endothelium integrity were also DE in infected pigs. These findings suggest that M. suis subclinical infection causes significant alterations in blood mRNA levels, which could impact data interpretation of research using pigs. Screening of pigs for M. suis infection before initiating animal studies is strongly recommended.

Genome Sequence of Listeria monocytogenes Strain F4244, a 4b Serotype.

Listeria monocytogenes is an opportunistic invasive foodborne pathogen. Here, we performed whole-genome sequencing of L. monocytogenes strain F4244 (serotype 4b) using Illumina sequencing. The sequence showed 94.5% identity with strain F2365, serotype 4b, and 90.6% with EGD-e, serotype 1/2a.

Draft Genome Sequence of Methicillin-Resistant Staphylococcus aureus Strain LC33 Isolated from Human Breast Milk.

Here, we report the draft genome sequence of Staphylococcus aureus strain LC33, isolated from human breast milk in Brazil. This microorganism has been typed as ST1/t127/sccmecV. To our knowledge, this is the first draft genome sequence of a methicillin-resistant S. aureus strain isolated from human breast milk.

Draft Genome Sequences of Two Clinical Methicillin-Resistant Staphylococcus aureus Strains Isolated from Healthy Children in Brazil.

We report here the draft genome sequences of two community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains, C18 and C80, isolated from healthy children from day care centers. To our knowledge, these are the first draft genome sequences of CA-MRSA ST398/CC398/SccmecV and CA-MRSA ST5/CC5/SccmecIVa isolated from healthy children in Brazil.

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host.

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)-Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.

Detection of hemoplasma infection of goats by use of a quantitative polymerase chain reaction assay and risk factor analysis for infection.

OBJECTIVE To develop and validate a real-time quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma ovis in goats and investigate the prevalence and risk factors for hemoplasma infection of goats located in Indiana. ANIMALS 362 adult female goats on 61 farms. PROCEDURES Primers were designed for amplification of a fragment of the dnaK gene of M ovis by use of a qPCR assay. Blood samples were collected into EDTA-containing tubes for use in total DNA extraction, blood film evaluation, and determination of PCV. Limit of detection, intra-assay variability, interassay variability, and specificity of the assay were determined. RESULTS Reaction efficiency of the qPCR assay was 94.45% (R(2), 0.99; slope, -3.4623), and the assay consistently detected as few as 10 copies of plasmid/reaction. Prevalence of infection in goats on the basis of results for the qPCR assay was 18.0% (95% confidence interval, 14% to 22%), with infected goats ranging from 1 to 14 years old, whereby 61% (95% confidence interval, 47% to 73%) of the farms had at least 1 infected goat. Bacterial load in goats infected with M ovis ranged from 1.05 × 10(3) target copies/mL of blood to 1.85 × 10(5) target copies/mL of blood; however, no bacteria were observed on blood films. Production use of a goat was the only risk factor significantly associated with hemoplasma infection. CONCLUSIONS AND CLINICAL RELEVANCE The qPCR assay was more sensitive for detecting hemoplasma infection than was evaluation of a blood film, and production use of a goat was a risk factor for infection.

Cattle experimentally infected by Anaplasma marginale: Influence of splenectomy on disease pathogenesis, oxidative profile, and antioxidant status.

Bovine anaplasmosis is caused by the obligate intraerythrocytic bacteria Anaplasma marginale. These bacteria are transmitted by tick species such as Rhipicephalus (Boophilus) microplus, blood-sucking insects, and fomites (needles, clippers, and other blood contaminated equipment). During the acute phase of infection, animals may develop fever, anemia, jaundice, and hepatosplenomegaly. The aims of this study are to quantify the bacteremia by quantitative PCR in eight naïve calves experimentally infected by A. marginale [splenectomized (n = 4), and intact/non-splenectomized (n = 4)], and to correlate these findings with markers of oxidative stress on days 0, 8, 15, 21 and 23 post-infection. Complete blood counts (CBC) were performed in both groups. Lipid peroxidation was estimated by quantifying thiobarbituric acid reactive substances (TBARS); and non-enzymatic antioxidants were assessed by erythrocyte content of non-protein thiols (NPSH). There were no significant differences in complete blood counts (CBC) between the two groups. However, both groups had a slight decrease on packet cell volume (PCV), erythrocytes and hemoglobin concentration, as well as an increase in total leukocyte counts due to elevated lymphocytes when comparing pre and post-infection with A. marginale. Progressive increase on TBARS levels and concomitant decrease on NPSH content were observed in all animals, without significant differences between splenectomized and intact animals. A positive correlation between bacteremia and TBARS, and a negative correlation between bacteremia and NPSH were observed in both groups with higher correlation for NPSH in splenectomized animals. A negative correlation between TBARS and NPSH levels was observed in both groups indicating lipid peroxidation without a non-enzymatic antioxidant response. The results of experimental infection by A. marginale in cattle showed that bacteremia has an impact on lipid peroxidation regardless of the splenectomy.

Genome Sequence of Ureaplasma diversum Strain ATCC 49782.

Here, we report the complete genome sequence of Ureaplasma diversum strain ATCC 49782. This species is of bovine origin, having an association with reproductive disorders in cattle, including placentitis, fetal alveolitis, abortion, and birth of weak calves. It has a small circular chromosome of 975,425 bp.

Microscopy and genomic analysis of Mycoplasma parvum strain Indiana.

Mycoplasma parvum [Eperythrozoon parvum] is the second hemotrophic mycoplasma (hemoplasma) described in pigs. Unlike M. suis, its closest phylogenetic relative, M. parvum, is considered a non-pathogenic bacterium in this host species. Natural infection of a domestic, 6-month-old splenectomized pig with M. parvum strain Indiana is described herein. Light and scanning electron microscopy of the bacteria were performed in addition to whole genome sequencing, analysis, and comparison to the genome of M. suis strain Illinois. Neither clinical signs nor anemia were observed during the infection. Microscopy analyses revealed coccoid to rod- shaped organisms varying from 0.2 to 0.5 µm; they were observed individually or in short chains by both light and electron microscopy, however less than 30% of the red blood cells were infected at peak bacteremia. The single circular chromosome of M. parvum was only 564 395 bp, smaller than M. genitalium, previously considered the tiniest member of the Mollicutes. Its general genomic features were similar to others in this class and species circumscription was verified by phylogenomic analysis. A gene-by-gene comparison between M. suis and M. parvum revealed all protein coding sequences (CDS) with assigned functions were shared, including metabolic functions, transporters and putative virulence factors. However, the number of CDS in paralogous gene families was remarkably different with about half as many paralogs in M. parvum. The differences in paralogous genes may be implicated in the different pathogenic potential of these two species, however variable gene expression may also play a role. Both are areas of ongoing investigation.

Comparative genomics and phylogenomics of hemotrophic mycoplasmas.

Hemotrophic mycoplasmas (hemoplasmas) are a group of animal pathogens of the Mollicutes class. Recently, the genomes of 8 hemoplasmas have been completely sequenced. The aim of this study was to gain a better understanding of their genomic features and relationship to other Mycoplasma species. The genome structure and dynamics of hemoplasmas were analyzed by evaluating gene synteny, adaptive evolution of paralogous gene families (PGF) and horizontal gene transfer (HGT). The Mollicutes class was then phylogenetically analyzed by constructing a distance matrix of the 16S rRNA genes and a phylogenetic tree with 32 conserved, concatenated proteins. Our results suggest that the hemoplasmas have dynamic genomes. The genome size variation (from 547 to 1,545 genes) indicates substantial gene gain/loss throughout evolution. Poorly conserved gene syntenies among hemoplasmas, positional shuffling of paralogous genes between strains, HGT, and codons under positive selection in PGFs were also observed. When compared to other Mollicutes species, the hemoplasmas experienced further metabolic reduction, and the 16S rRNA gene distance matrix of the available mollicutes suggests that these organisms presently constitute the most divergent clade within its class. Our phylogenetic tree of concatenated proteins showed some differences when compared to the 16S rRNA gene tree, but non-mycoplasma organisms, such as Ureaplasma spp. and Mesoplasma spp., continue to branch within Mycoplasma clades. In conclusion, while the hemoplasmas experienced further metabolic shrinkage through gene loss, PGFs with positively selected codons are likely beneficial to these species. Phylogeny of the mollicutes based on 16S rRNA genes or concatenated proteins do not obey the current taxonomy. The metabolism and genetic diversity of the mollicutes, the presence of HGT, and lack of standard for genus circumscription are likely to hinder attempts to classify these organisms based on phylogenetic analyses.

Complete Genome Sequence of Mycoplasma ovis Strain Michigan, a Hemoplasma of Sheep with Two Distinct 16S rRNA Genes.

We report the complete genome sequence of Mycoplasma ovis strain Michigan. Its single circular chromosome has 702,511 bp and contains 2 copies of the 16S rRNA gene, one corresponding to M. ovis and the other to "Candidatus Mycoplasma haemovis." All housekeeping genes and the 5S-23S rRNA genes are present in single copies.

Genome Sequence of Mycoplasma parvum (Formerly Eperythrozoon parvum), a Diminutive Hemoplasma of the Pig.

We report the complete genome sequence of Mycoplasma parvum strain Indiana. Its circular chromosome is 564,395 bp, which is smaller than that of Mycoplasma genitalium, which was previously considered the smallest member of the Mollicutes. Comparative analyses of the genomes of M. parvum and Mycoplasma suis will provide novel insights into the molecular basis of their virulence.

Genome sequence of "Candidatus Mycoplasma haemolamae" strain purdue, a red blood cell pathogen of alpacas (Vicugna pacos) and llamas (Lama glama).

We report the complete genome sequence of "Candidatus Mycoplasma haemolamae," an endemic red-cell pathogen of camelids. The single, circular chromosome has 756,845 bp, a 39.3% G+C content, and 925 coding sequences (CDSs). A great proportion (49.1%) of these CDSs are organized into paralogous gene families, which can now be further explored with regard to antigenic variation.

Mycoplasma haemocanis--the canine hemoplasma and its feline counterpart in the genomic era.

Mycoplasma haemocanis is a hemotrophic mycoplasma (hemoplasma), blood pathogen that may cause acute disease in immunosuppressed or splenectomized dogs. The genome of the strain Illinois, isolated from blood of a naturally infected dog, has been entirely sequenced and annotated to gain a better understanding of the biology of M. haemocanis. Its single circular chromosome has 919 992 bp and a low G + C content (35%), representing a typical mycoplasmal genome. A gene-by-gene comparison against its feline counterpart, M. haemofelis, reveals a very similar composition and architecture with most of the genes having conserved synteny extending over their entire chromosomes and differing only by a small set of unique protein coding sequences. As in M. haemofelis, M. haemocanis metabolic pathways are reduced and apparently rely heavily on the nutrients afforded by its host environment. The presence of a major percentage of its genome dedicated to paralogous genes (63.7%) suggests that this bacterium might use antigenic variation as a mechanism to evade the host's immune system as also observed in M. haemofelis genome. Phylogenomic comparisons based on average nucleotide identity (ANI) and tetranucleotide signature suggest that these two pathogens are different species of mycoplasmas, with M. haemocanis infecting dogs and M. haemofelis infecting cats.

Complete genome sequence of Mycoplasma wenyonii strain Massachusetts.

Mycoplasma wenyonii is a hemotrophic mycoplasma that causes acute and chronic infections in cattle. Here, we announce the first complete genome sequence of this organism. The genome is a single circular chromosome with 650,228 bp and G+C% of 33.9. Analyses of M. wenyonii genome will provide insights into its biology.

Complete genome sequence of Mycoplasma haemocanis strain Illinois.

Mycoplasma haemocanis is a blood pathogen that may cause acute disease in immunosuppressed or splenectomized dogs. The genome of the strain Illinois is a single circular chromosome with 919,992 bp and a GC content of 35%. Analyses of the M. haemocanis genome will provide insights into its biology and in vitro cultivation requirements.

Genome of Mycoplasma haemofelis, unraveling its strategies for survival and persistence.

Mycoplasma haemofelis is a mycoplasmal pathogen (hemoplasma) that attaches to the host's erythrocytes. Distributed worldwide, it has a significant impact on the health of cats causing acute disease and, despite treatment, establishing chronic infection. It might also have a role as a zoonotic agent, especially in immunocompromised patients. Whole genome sequencing and analyses of M. haemofelis strain Ohio2 was undertaken as a step toward understanding its survival and persistence. Metabolic pathways are reduced, relying on the host to supply many of the nutrients and metabolites needed for survival. M. haemofelis must import glucose for ATP generation and ribose derivates for RNA/DNA synthesis. Hypoxanthine, adenine, guanine, uracil and CMP are scavenged from the environment to support purine and pyrimidine synthesis. In addition, nicotinamide, amino acids and any vitamins needed for growth, must be acquired from its environment. The core proteome of M. haemofelis contains an abundance of paralogous gene families, corresponding to 70.6% of all the CDSs. This "paralog pool" is a rich source of different antigenic epitopes that can be varied to elude the host's immune system and establish chronic infection. M. haemofelis also appears to be capable of phase variation, which is particularly relevant to the cyclic bacteremia and persistence, characteristics of the infection in the cat. The data generated herein should be of great use for understanding the mechanisms of M. haemofelis infection. Further, it will provide new insights into its pathogenicity and clues needed to formulate media to support the in vitro cultivation of M. haemofelis.