Torque Teno Virus Load Predicts Opportunistic Infections after Kidney Transplantation but Is Not Associated with Maintenance Immunosuppression Exposure.

Measuring the non-pathogenic Torque Teno Virus (TTV) load allows assessing the net immunosuppressive state after kidney transplantation (KTx). Currently, it is not known how exposure to maintenance immunosuppression affects TTV load. We hypothesized that TTV load is associated with the exposure to mycophenolic acid (MPA) and tacrolimus. We performed a prospective study including 54 consecutive KTx. Blood TTV load was measured by an in-house PCR at months 1 and 3. Together with doses and trough blood levels of tacrolimus and MPA, we calculated the coefficient of variability (CV), time in therapeutic range (TTR) and concentration/dose ratio (C/D) of tacrolimus, and the MPA-area under the curve (AUC-MPA) at the third month. TTV load at the first and third month discriminated those patients at risk of developing opportunistic infections between months 1 and 3 (AUC-ROC 0.723, 95%CI 0.559-0.905, p = 0.023) and between months 3 and 6 (AUC-ROC 0.778, 95%CI 0.599-0.957, p = 0.028), respectively, but not those at risk of acute rejection. TTV load did not relate to mean tacrolimus blood level, CV, TTR, C/D and AUC-MPA. To conclude, although TTV is a useful marker of net immunosuppressive status after KTx, it is not related to exposure to maintenance immunosuppression.

Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases.

Plasmid pAD1 is a 60-kb conjugative element commonly found in clinical isolates of Enterococcus faecalis. The relaxase TraX and the primary origin of transfer oriT2 are located close to each other and have been shown to be essential for conjugation. The oriT2 site contains a large inverted repeat (where the nic site is located) adjacent to a series of short direct repeats. TraX does not show any of the typical relaxase sequence motifs but is the prototype of a unique family of relaxases (MOBC). The present study focuses on the genetic, biochemical, and structural analysis of TraX, whose 3D structure could be predicted by protein threading. The structure consists of two domains: (i) an N-terminal domain sharing the topology of the DNA binding domain of the MarR family of transcriptional regulators and (ii) a C-terminal catalytic domain related to the PD-(D/E)XK family of restriction endonucleases. Alignment of MOBC relaxase amino acid sequences pointed to several conserved polar amino acid residues (E28, D152, E170, E172, K176, R180, Y181, and Y203) that were mutated to alanine. Functional analysis of these mutants (in vivo DNA transfer and cleavage assays) revealed the importance of these residues for relaxase activity and suggests Y181 as a potential catalytic residue similarly to His-hydrophobe-His relaxases. We also show that TraX binds specifically to dsDNA containing the oriT2 direct repeat sequences, confirming their role in transfer specificity. The results provide insights into the catalytic mechanism of MOBC relaxases, which differs radically from that of His-hydrophobe-His relaxases.

Microevolutionary events involving narrow host plasmids influences local fixation of vancomycin-resistance in Enterococcus populations.

Vancomycin-resistance in enterococci (VRE) is associated with isolates within ST18, ST17, ST78 Enterococcus faecium (Efm) and ST6 Enterococcus faecalis (Efs) human adapted lineages. Despite of its global spread, vancomycin resistance rates in enterococcal populations greatly vary temporally and geographically. Portugal is one of the European countries where Tn1546 (vanA) is consistently found in a variety of environments. A comprehensive multi-hierarchical analysis of VRE isolates (75 Efm and 29 Efs) from Portuguese hospitals and aquatic surroundings (1996-2008) was performed to clarify the local dynamics of VRE. Clonal relatedness was established by PFGE and MLST while plasmid characterization comprised the analysis of known relaxases, rep initiator proteins and toxin-antitoxin systems (TA) by PCR-based typing schemes, RFLP comparison, hybridization and sequencing. Tn1546 variants were characterized by PCR overlapping/sequencing. Intra- and inter-hospital dissemination of Efm ST18, ST132 and ST280 and Efs ST6 clones, carrying rolling-circle (pEFNP1/pRI1) and theta-replicating (pCIZ2-like, Inc18, pHTß-like, two pRUM-variants, pLG1-like, and pheromone-responsive) plasmids was documented. Tn1546 variants, mostly containing ISEf1 or IS1216, were located on plasmids (30-150 kb) with a high degree of mosaicism and heterogeneous RFLP patterns that seem to have resulted from the interplay between broad host Inc18 plasmids (pIP501, pRE25, pEF1), and narrow host RepA_N plasmids (pRUM, pAD1-like). TAs of Inc18 (ω-ε-ζ) and pRUM (Axe-Txe) plasmids were infrequently detected. Some plasmid chimeras were persistently recovered over years from different clonal lineages. This work represents the first multi-hierarchical analysis of VRE, revealing a frequent recombinatorial diversification of a limited number of interacting clonal backgrounds, plasmids and transposons at local scale. These interactions provide a continuous process of parapatric clonalization driving a full exploration of the local adaptive landscape, which might assure long-term maintenance of resistant clones and eventually fixation of Tn1546 in particular geographic areas.

Human and swine hosts share vancomycin-resistant Enterococcus faecium CC17 and CC5 and Enterococcus faecalis CC2 clonal clusters harboring Tn1546 on indistinguishable plasmids.

VRE isolates from pigs (n = 29) and healthy persons (n = 12) recovered during wide surveillance studies performed in Portugal, Denmark, Spain, Switzerland, and the United States (1995 to 2008) were compared with outbreak/prevalent VRE clinical strains (n = 190; 23 countries; 1986 to 2009). Thirty clonally related Enterococcus faecium clonal complex 5 (CC5) isolates (17 sequence type 6 [ST6], 6 ST5, 5 ST185, 1 ST147, and 1 ST493) were obtained from feces of swine and healthy humans. This collection included isolates widespread among pigs of European Union (EU) countries since the mid-1990s. Each ST comprised isolates showing similar pulsed-field gel electrophoresis (PFGE) patterns (≤6 bands difference; >82% similarity). Some CC5 PFGE subtype strains from swine were indistinguishable from hospital vancomycin-resistant enterococci (VRE) causing infections. A truncated variant of Tn1546 (encoding resistance to vancomycin) and tcrB (coding for resistance to copper) were consistently located on 150- to 190-kb plasmids (rep(pLG1)). E. faecium CC17 (ST132) isolates from pig manure and two clinical samples showed identical PFGE profiles and contained a 60-kb mosaic plasmid (rep(Inc18) plus rep(pRUM)) carrying diverse Tn1546-IS1216 variants. The only Enterococcus faecalis isolate obtained from pigs (CC2-ST6) corresponded to a multidrug-resistant clone widely disseminated in hospitals in Italy, Portugal, and Spain, and both animal and human isolates harbored an indistinguishable 100-kb mosaic plasmid (rep(pRE25) plus rep(pCF10)) containing the whole Tn1546 backbone. The results indicate a current intra- and international spread of E. faecium and E. faecalis clones and their plasmids among swine and humans.

A multiresistance megaplasmid pLG1 bearing a hylEfm genomic island in hospital Enterococcus faecium isolates.

Enterococcus faecium is considered to be a nosocomial pathogen with increasing medical importance. The putative virulence factor, hyl(Efm), encoding a putative hyaluronidase, is enriched among the hospital-associated polyclonal subpopulation of E. faecium.. The hyl(Efm) gene is described to be part of a genomic island and was recently identified to be plasmid-located. Here, we present a description of the structure, localization, and distribution of the putative pathogenicity factor hyl(Efm) and its putative island among 39 clinical isolates and elucidate the composition and host range of pLG1, a hyl(Efm) multiresistance plasmid of approximately 281.02kb. The hyl(Efm) gene was located within a 17,824-bp element highly similar to the putative genomic island (GI) structure that had been previously described. This genomic region was conserved among 39 hyl(Efm)-positive strains with variation in a specific region downstream of hyl(Efm) in 18 strains. The putative hyl(Efm) was located on large plasmids (150-350kb) in 37 strains. pLG1 could be horizontally transferred into four different E. faecium recipient strains (n=4) but not into E. faecalis (n=3). Sequencing of pLG1 resolved putative plasmid replication, conjugation, and maintenance determinants as well as a pilin gene cluster, carbon uptake and utilization genes, heavy metal and antibiotic resistance clusters. The hyl(Efm) transferable plasmid pLG1 bears additional putative pathogenicity factors and antibiotic resistance genes. These findings suggest horizontal gene transfer of virulence factors and antibiotic resistance gene clusters by a single genetic event (conjugative transfer) which might be triggered by heavy antibiotic use common in health care units where E. faecium is increasingly prevalent.

The diversity of conjugative relaxases and its application in plasmid classification.

Bacterial conjugation is an efficient and sophisticated mechanism of DNA transfer among bacteria. While mobilizable plasmids only encode a minimal MOB machinery that allows them to be transported by other plasmids, conjugative plasmids encode a complete set of transfer genes (MOB1T4SS). The only essential ingredient of the MOB machinery is the relaxase, the protein that initiates and terminates conjugative DNA processing. In this review we compared the sequences and properties of the relaxase proteins contained in gene sequence databases. Proteins were arranged in families and phylogenetic trees constructed from the family alignments. This allowed the classification of conjugative transfer systems in six MOB families:MOB(F), MOB(H), MOB(Q), MOB(C), MOB(P) and MOB(V). The main characteristics of each family were reviewed. The phylogenetic relationships of the coupling proteins were also analysed and resulted in phylogenies congruent to those of the cognate relaxases. We propose that the sequences of plasmid relaxases can be used for plasmid classification. We hope our effort will provide researchers with a useful tool for further mining and analysing the plasmid universe both experimentally and in silico.

The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids.

The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

Characterization of an active partition system for the Enterococcus faecalis pheromone-responding plasmid pAD1.

Enterococcus faecalis plasmid pAD1 is a 60-kb conjugative, low-copy-number plasmid that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin that contributes to virulence. Although aspects of conjugation have been studied extensively, relatively little is known about the control of pAD1 maintenance. Previous work on pAD1 identified a 5-kb region of DNA sufficient to support replication, copy control, and stable inheritance (K. E. Weaver, D. B. Clewell, and F. An, J. Bacteriol. 175:1900-1909, 1993), and recently, the pAD1 replication initiator (RepA) and the origin of vegetative replication (oriV) were characterized (M. V. Francia, S. Fujimoto, P. Tille, K. E. Weaver, and D. B. Clewell, J. Bacteriol. 186:5003-5016, 2004). The present study focuses on the adjacent determinants repB and repC, as well as a group of 25 8-bp direct repeats (iterons with the consensus sequence TAGTARRR) located between the divergently transcribed repA and repB. Through mutagenesis and trans-complementation experiments, RepB (a 33-kDa protein, a member of the ParA superfamily of ATPases) and RepC (a protein of 14.4 kDa) were shown to be required for maximal stabilization. Both were active in trans. The iteron region was shown to act as the pAD1 centromere-like site. Purified RepC was shown by DNA mobility shift and DNase I footprinting analyses to interact in a sequence-specific manner with the iteron repeats upstream of the repBC locus. The binding of RepC to the iteron region was shown to be modified by RepB in the presence of ATP via a possible interaction with the RepC-iteron complex. RepB did not bind to the iteron region in the absence of RepC.

Characterization of defective beta-lactamase genes in Yersinia enterocolitica.

OBJECTIVES: To study at the molecular level the heterogeneity of expression of the two chromosomal beta-lactamases, BlaA and BlaB, in Yersinia enterocolitica strains isolated from clinical samples. METHODS: MIC determination by the agar dilution method and beta-lactamase assays was performed to determine the resistance level conferred by these enzymes. DNA cloning, PCR and direct sequencing were used to detect the presence of mutations. RESULTS: The blaA allele from strain IP97 (blaA97) was found to carry a deletion of 51 bp which entirely abolished its beta-lactamase activity. Both the ampR gene and the promoter region of strain Y56 were shown to be functional by a gene swapping experiment. The blaB allele from strain Y56 was found to carry two point mutations, only one of them resulting in a change in the amino acid sequence of the protein. This single amino acid change created a practically inactive BlaB or AmpC cephalosporinase in Y. enterocolitica Y56. CONCLUSIONS: The lack of activity observed in the beta-lactamases of some Y. enterocolitica isolates was due to the presence of point mutations or small deletions in the corresponding genes.

Enterococcus resistentes a glucopéptidos en Europa: un problema hospitalario creciente / Glycopeptide-resistant enterococcus in Europe: an increasing nosocomial problem

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Replication of Enterococcus faecalis pheromone-responding plasmid pAD1: location of the minimal replicon and oriV site and RepA involvement in initiation of replication.

The hemolysin-determining plasmid pAD1 is a member of a widely disseminated family of highly conjugative elements commonly present in clinical isolates of Enterococcus faecalis. The determinants repA, repB, and repC, as well as adjacent iteron sequences, are believed to play important roles in pAD1 replication and maintenance. The repA gene encodes an initiator protein, whereas repB and repC encode proteins related to stability and copy number. The present study focuses specifically on repA and identifies a replication origin (oriV) within a central region of the repA determinant. A small segment of repA carrying oriV was able to support replication in cis of a plasmid vector otherwise unable to replicate, if an intact RepA was supplied in trans. We demonstrate that under conditions in which RepA is expressed from an artificial promoter, a segment of DNA carrying only repA is sufficient for stable replication in E. faecalis. We also show that RepA binds specifically to oriV DNA at several sites containing inverted repeat sequences (i.e., IR-1) and nonspecifically to single-stranded DNA, and related genetic analyses confirm that these sequences play an important role in replication. Finally, we reveal a relationship between the internal structure of RepA and its ability to recognize oriV. An in-frame deletion within repA resulting in loss of 105 nucleotides, including at least part of oriV, did not eliminate the ability of the altered RepA protein to initiate replication using an intact origin provided in trans. The relationship of RepA to other known initiator proteins is also discussed.