Identification of Pathogenic Bacteria from Public Libraries via Proteomics Analysis.

Hazardous organisms may thrive on surfaces that are often exposed to human contact, including children's library books. In this study, swab samples were taken from 42 children's books collected from four public libraries in Texas and California. Samples were then cultivated in brain⁻heart infusion (BHI) medium and then in Luria broth (LB) medium containing either ampicillin or kanamycin. All 42 samples (100%) were positive for bacterial growth in normal BHI medium. Furthermore, 35 samples (83.3%) and 20 samples (47.6%) in total were positive in LB medium containing ampicillin or kanamycin, respectively. Bacterial populations were then identified in samples using an Orbitrap Fusion™ Tribrid ™ mass spectrometer, a state-of-the-art proteomic analysis tool. Identified bacterial species grown in ampicillin included Bacillus, Acinetobacter, Pseudomonas, Staphylococcus, Enterobacter, Klebsiella, Serratia, Streptococcus, Escherichia, Salmonella, and Enterococcus. In contrast, identified bacteria grown in kanamycin included Staphylococcus, Streptococcus, Enterococcus, and Bacillus. The presences of pathogenic bacteria species were also confirmed. The results of this study warrant follow up studies to assess the potential health risks of identified pathogens. This study demonstrates the utility of proteomics in identifying environmental pathogenic bacteria for specific public health risk evaluations.

The fibronectin-binding protein Fnm contributes to adherence to extracellular matrix components and virulence of Enterococcus faecium.

The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P < 0.05) in the adhesion of E. faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P < 0.001) in the ability of the mutant, TX6128, to bind fibronectin relative to that of the wild-type strain; in situ reconstitution of fnm in the deletion mutant strain restored adherence. In addition, the Δfnm mutant was highly attenuated relative to TX82 (P ≤ 0.0001) in a mixed-inoculum rat endocarditis model. Taken together, these results demonstrate that Fnm affects the adherence of E. faecium to fibronectin and is important in the pathogenesis of experimental endocarditis.

Surface-Associated Lipoproteins Link Enterococcus faecalis Virulence to Colitogenic Activity in IL-10-Deficient Mice Independent of Their Expression Levels.

The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2±1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (ΔepaB score: 4.7±2.3; p<0.05) and was almost completely abrogated in E. faecalis deficient for lipoproteins (Δlgt score: 2.3±2.3; p<0.0001). Consistently both E. faecalis mutants showed significantly impaired virulence in Galleria mellonella and Caenorhabditis elegans. Loss of E-cadherin in the epithelium was shown for all bacterial strains in inflamed IL-10-/- but not wild type mice. Inactivation of epaB in E. faecalis reduced microcolony and biofilm formation in vitro, altered bacterial adhesion to intestinal epithelium of germ-free Manduca sexta larvae and impaired penetration into the colonic mucus layer of IL-10-/- mice. Lipoprotein-deficient E. faecalis exhibited an impaired TLR2-mediated activation of BMDCs in vitro despite their ability to fully reactivate MLN cells as well as MLN-derived colitogenic T cells ex vivo. E. faecalis virulence factors accounting for bacterial adhesion to mucosal surfaces as well as intestinal barrier disruption partially contribute to colitogenic activity of E. faecalis. Beyond their well-known role in infections, cell surface-associated lipoproteins are essential structures for colitogenic activity of E. faecalis by mediating innate immune cell activation.

CcpA is important for growth and virulence of Enterococcus faecium.

The collagen adhesin Acm was the first virulence determinant reported to be important for the pathogenesis of Enterococcus faecium in a rat infective endocarditis model. We had previously reported that there was a slight growth delay associated with acm allelic replacement (cat) mutant strain TX6051 used in that study. Recently, we generated a nonpolar markerless acm deletion mutant and did not observe a delay in growth. We therefore performed comparative genome sequence analysis of wild-type strain TX82 and TX6051 and found a single mutation, a nonsense mutation in the ccpA gene of TX6051. After correcting this mutation, the growth defect of TX6051 was abolished, implicating a role for CcpA in the growth of E. faecium. To confirm this, we created a ccpA deletion mutant of TX82, which also exhibited a slight delay in growth. Furthermore, the ccpA deletion mutant was attenuated (P = 0.0024) in a mixed-inoculum (TX82 plus TX82 ΔccpA) rat endocarditis model and also in an in vitro competitive growth assay; a ccpA-complemented strain showed neither reduced growth nor reduced virulence. We also found attenuation in the endocarditis model with the new acm deletion mutant although not as great as that previously observed with TX6051 carrying the ccpA mutation. Taken together, our data confirm the role of Acm in the pathogenesis of endocarditis. We also show that CcpA affects the growth of E. faecium, that an intact ccpA gene is important for full virulence, and that a ccpA mutation was partly responsible for the highly attenuated phenotype of TX6051.

Whole-genome analysis of a daptomycin-susceptible enterococcus faecium strain and its daptomycin-resistant variant arising during therapy.

Development of daptomycin (DAP) resistance in Enterococcus faecalis has recently been associated with mutations in genes encoding proteins with two main functions: (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase [cls]). However, the genetic bases for DAP resistance in Enterococcus faecium are unclear. We performed whole-genome comparative analysis of a clinical strain pair, DAP-susceptible E. faecium S447 and its DAP-resistant derivative R446, which was recovered from a single patient during DAP therapy. By comparative whole-genome sequencing, DAP resistance in R446 was associated with changes in 8 genes. Two of these genes encoded proteins involved in phospholipid metabolism: (i) an R218Q substitution in Cls and (ii) an A292G reversion in a putative cyclopropane fatty acid synthase enzyme. The DAP-resistant derivative R446 also exhibited an S333L substitution in the putative histidine kinase YycG, a member of the YycFG system, which, similar to LiaFSR, has been involved in cell envelope homeostasis and DAP resistance in other Gram-positive cocci. Additional changes identified in E. faecium R446 (DAP resistant) included two putative proteins involved in transport (one for carbohydrate and one for sulfate) and three enzymes predicted to play a role in general metabolism. Exchange of the "susceptible" cls allele from S447 for the "resistant" one belonging to R446 did not affect DAP susceptibility. Our results suggest that, apart from the LiaFSR system, the essential YycFG system is likely to be an important mediator of DAP resistance in some E. faecium strains.

Functional importance of a pair of conserved glutamic acid residues and of Ca(2+) binding in the cbb(3)-type oxygen reductases from Rhodobacter sphaeroides and Vibrio cholerae.

The cbb(3)-type cytochrome c oxidases are members of the family of heme-copper proton pumping respiratory oxygen reductases. The structure of the cbb(3)-type oxidase from Pseudomonas stutzeri reveals that, in addition to the six redox-active metal centers (two b-type hemes, three c-type hemes, and Cu(B)), the enzyme also contains at least one Ca(2+). The calcium bridges two propionate carboxyls at the interface between the low-spin heme b and the active-site heme b(3) and, in addition, is ligated to a serine in subunit CcoO and by a glutamate in subunit CcoN. The glutamate that is ligated to Ca(2+) is one of a pair of glutamic acid residues that has previously been suggested to be part of a proton exit pathway for pumped protons. In this work, mutations of these glutamates are investigated in the cbb(3)-type oxidases from Vibrio cholerae and Rhodobacter sphaeroides. Metal analysis shows that each of these wild-type enzymes contains Ca(2+). Mutations of the glutamate expected to ligate the Ca(2+) in each of these enzymes (E126 in V. cholerae and E180 in R. sphaeroides) result in a loss of activity as well as a loss of Ca(2+). Mutations of the nearby glutamate (E129 in V. cholerae and E183 in R. sphaeroides) also resulted in a loss of oxidase activity and a loss of Ca(2+). It is concluded that the Ca(2+) is essential for assembly of the fully functional enzyme and that neither of the glutamates is likely to be part of a pathway for pumped protons within the cbb(3)-type oxygen reductases. A more likely role for these glutamates is the maintenance of the structural integrity of the active conformation of the enzyme.

Genetic basis for in vivo daptomycin resistance in enterococci.

BACKGROUND: Daptomycin is a lipopeptide with bactericidal activity that acts on the cell membrane of enterococci and is often used off-label to treat patients infected with vancomycin-resistant enterococci. However, the emergence of resistance to daptomycin during therapy threatens its usefulness. METHODS: We performed whole-genome sequencing and characterization of the cell envelope of a clinical pair of vancomycin-resistant Enterococcus faecalis isolates from the blood of a patient with fatal bacteremia; one isolate (S613) was from blood drawn before treatment and the other isolate (R712) was from blood drawn after treatment with daptomycin. The minimal inhibitory concentrations (MICs) of these two isolates were 1 and 12 µg per milliliter, respectively. Gene replacements were made to exchange the alleles found in isolate S613 with those in isolate R712. RESULTS: Isolate R712 had in-frame deletions in three genes. Two genes encoded putative enzymes involved in phospholipid metabolism, GdpD (which denotes glycerophosphoryl diester phosphodiesterase) and Cls (which denotes cardiolipin synthetase), and one gene encoded a putative membrane protein, LiaF (which denotes lipid II cycle-interfering antibiotics protein but whose exact function is not known). LiaF is predicted to be a member of a three-component regulatory system (LiaFSR) involved in the stress-sensing response of the cell envelope to antibiotics. Replacement of the liaF allele of isolate S613 with the liaF allele from isolate R712 quadrupled the MIC of daptomycin, whereas replacement of the gdpD allele had no effect on MIC. Replacement of both the liaF and gdpD alleles of isolate S613 with the liaF and gdpD alleles of isolate R712 raised the daptomycin MIC for isolate S613 to 12 µg per milliliter. As compared with isolate S613, isolate R712--the daptomycin-resistant isolate--had changes in the structure of the cell envelope and alterations in membrane permeability and membrane potential. CONCLUSIONS: Mutations in genes encoding LiaF and a GdpD-family protein were necessary and sufficient for the development of resistance to daptomycin during the treatment of vancomycin-resistant enterococci. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.).

Combining microarray and genomic data to predict DNA binding motifs.

The ability to detect regulatory elements within genome sequences is important in understanding how gene expression is controlled in biological systems. In this work, microarray data analysis is combined with genome sequence analysis to predict DNA sequences in the photosynthetic bacterium Rhodobacter sphaeroides that bind the regulators PrrA, PpsR and FnrL. These predictions were made by using hierarchical clustering to detect genes that share similar expression patterns. The DNA sequences upstream of these genes were then searched for possible transcription factor recognition motifs that may be involved in their co-regulation. The approach used promises to be widely applicable for the prediction of cis-acting DNA binding elements. Using this method the authors were independently able to detect and extend the previously described consensus sequences that have been suggested to bind FnrL and PpsR. In addition, sequences that may be recognized by the global regulator PrrA were predicted. The results support the earlier suggestions that the DNA binding sequence of PrrA may have a variable-sized gap between its conserved block elements. Using the predicted DNA binding sequences, a whole-genome-scale analysis was performed to determine the relative importance of the interplay between the three regulators PpsR, FnrL and PrrA. Results of this analysis showed that, compared to the regulation by PpsR and FnrL, a much larger number of genes are candidates to be regulated by PrrA. The study demonstrates by example that integration of multiple data types can be a powerful approach for inferring transcriptional regulatory patterns in microbial systems, and it allowed the detection of photosynthesis-related regulatory patterns in R. sphaeroides.