Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocases with E. coli Tat components.The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Streptomyces coelicolor were able to form heterologous translocases with substantive Tat transport activity. However, only the TatA and TatB proteins of Pseudomonas syringae were able to functionally interact with the E. coli Tat system even though the two organisms are closely related. Of the Tat components from the phylogenetically distant hyperthermophillic bacterium Aquifex aeolicus only the TatA proteins showed any detectable level of heterologous functionality. The heterologously expressed TatA proteins of S. coelicolor and A. aeolicus were found exclusively in the membrane fraction.All three subunits of the Tat system from the Gram positive organism S. coelicolor TatA protein was functional and found only in the membrane fraction, it suggests that soluble forms of Streptomyces TatA reported by others do not play a role in protein export.Our results show that of the three Tat proteins, TatA is most likely to show cross-species complementation. By contrast, TatB and TatC do not always show cross-complementation, probably because they must recognise heterologous signal peptides. Since heterologously-expressed  There are two general pathways by which proteins are translocated across the cytoplasmic membranes of bacteria. The Sec pathway, which is ubiquitous, uses a threading mechanism to transport unfolded polypeptides across the membrane, driven by the energy of ATP hydrolysis and the transmembrane proton gradient . By contEscherichia coli is comprised of the three major components, TatA, TatB and TatC, along with the minor component TatE which is a poorly expressed TatA orthologue . How. Howtat n strain .tat genes from three different bacterial species to compensate for the absence of the cognate E. coli tat gene. The organisms we selected for this study are Aquifex aeolicus, a thermophilic bacterium which forms the deepest branch in bacterial phylogeny, Streptomyces coelicolor, a Gram positive actinomycete and Pseudomonas syringae pv maculicola ES4326, which, like E. coli, is a gamma Proteobacterium. E. coli diverged from A. aeolicus approximately 4 billion years ago, from S. coelicolor approximately 3.2 billion years ago and from P. syringae approximately 1.3 billion years ago  was rep/or AmiC . HoweverE. coli partner subunit, recognition of non-native signal peptides and associated conformational changes to promote assembly of the active translocase. It is notable that the A. aeolicus TatA proteins, which would normally be operating at temperatures of 80°C, retain function when expressed in E. coli cells growing at 37°C. Thus either the TatA protein does not change conformation during the transport cycle or the protein retains sufficient flexibility for function even at temperatures considerably lower than physiological.It is striking to note that wherever cross-species complementation has been tested, TatA proteins always seem to retain some level of function in the heterologous host ,47. ThisS. coelicolor and A. aeolicus are found only in the membrane fraction. It is not clear why extra-membraneous forms of TatA/TatB are present in some organisms, but clearly since the S. coelicolor TatA protein  supportE. coli strains and plasmids employed in this study are shown in Table E. coli strains were grown aerobically in Luria-Bertani (LB) medium [The ) medium . Concent) medium . The gro) medium  suppleme) medium . For TMA) medium , supplemP. syringae pv maculicola ES4326 tatA, tatB and tatC genes, respectively, under the control of the E. coli tat promoter. They were cloned following amplification with primers GCGGCCGGATCCATGGGTATTTTTGACTGG and GCGCTCTAGATTAAACC TGGTCTTTGCGG to amplify tatA, GCGCGGGGATCCATGTTCGGTATCAGC and GCGCGCTCTAGATCATGGGGCTCGCGGTGGC to amplify tatB, and GCGC GCGGATCCATGAGCGCTGATATCCCG and GCGCGCTCTAGATCACGGTGT GGTGGCGGGCGG to amplify tatC (restriction sites shown in bold) and plasmid pKS-PSMtatABC [BamHI and XbaI and cloned into pUniprom [S. coelicolor tatA, tatB and tatC genes, respectively, under the control of the E. coli tat promoter. They were cloned following amplification with primers GCGCGCGGATCCATGTTCGGAAGGCTCG GC and GCGCGCTCTAGATCAGCGCTTGGTCGTGTC to amplify tatA, GCGCG CGGATTCGTGTTCAATGACATAGGC and GCGCGCTCTAGATCAGGTGGCG TCCATGTC to amplify tatB, and GCGCGCGGATCCATGCCGCTCGCGGAACA C and GCGCGCTCTAGATCAGGTCACGTCGTCG to amplify tatC with S. coelicolor chromosomal DNA as template. Each product was digested with BamHI and XbaI and cloned into pUniprom that had been similarly digested.Plasmids pUniprom-PA, pUniprom-PB and pUniprom-PC carry the SMtatABC  as templpUniprom  that hadtatA1 and tatA2 genes of A. aeolicus, under control of the lac promoter. The tatA1 and tatA2 genes, whose reading frames overlap, were amplified with the following primers: GCGCGCGAATTCCCCTTAAATTATTCTC TAAGGAGGC and GCGCGCGGATCCGCTGAGTTAAGCCTCTACCTTTTCC, with A. aeolicus chromosomal DNA (a kind gift of R. Huber) as template. The product was digested with EcoRI and BamHI and cloned into pBluescript that had been digested with the same enzymes. The A. aeolicus tatB gene is not annotated on the genome [E. coli TatB is found immediately upstream of the tatC gene (see Table E. coli protein), and we herein refer to it as tatB. Plasmid pFATAQ2 contains the tatB and tatC genes of A. aeolicus, under control of the lac promoter. The genes, whose reading frames also overlap, were amplified with primers GCGCGGATCCGGAAA GCAATCCCTATTAACGGAAG and GCGCTCTAGAGCTTATGCCTTTTGAATT TCCTTC, digested with BamHI and XbaI and cloned into pBluescript that had been pre-digested with the same enzymes. Plasmid pFATAQ3 contains an artificial operon encoding the tatA1, -A2, -B and -C genes of A. aeolicus and was generated by subcloning the tatBC coding region from pFATAQ2 by digestion with BamHI and XbaI and ligation into pFATAQ1 that had been similarly digested. For overproduction of the A. aeolicus Tat proteins with C-terminal hexahistidine affinity tags, the encoding tat genes were re-amplified and cloned into the overexpression vector pQE70. Plasmid pQEAQ1 contains the gene encoding the TatA1 protein with a C-terminal histag and was amplified using primers GCGCGCATGCACTTTCCTCTGC CGTGGC and GCGCAGATCTTTTCACCCTCCTTTTTAACTTCC, and pFATAQ1 as template. The product was digested with SphI and BglII, and cloned into similarly digested pQE70. Plasmid pQEAQ2 is analogous to pQEAQ1 but encodes a tagged version of the TatA2 protein. It was cloned in a similar manner, using primers GCGC ACATGTTTTCCCGGCGGAATATCTATG and GCGCAGATCTAGCCTCTACC TTTTCCTTCTC to amplify the encoding gene. Plasmid pQEAQ12 carries both of the A. aeolicus tatA genes, with just TatA2 supplied with the C-terminal histag. The genes were amplified using primers GCGCGCATGCACTTTCCTCTGCCGTGGC and GC GCAGATCTAGCCTCTACCTTTTCCTTCTC, the product digested with SphI and BglII, and cloned into similarly digested pQE70.Plasmids pFATAQ1 contains the e genome , howeverAll clones obtained from PCR amplified DNA were sequenced to ensure that no mutations (other than site-specific mutations) had been introduced.S. coelicolor TatA protein were raised in rabbits, using a peptide of amino acid sequence CTSSRPVTEPTDTTKR, by Davids biotechnology . Antibodies to E. coli TatA have been described previously [et al. [SDS-PAGE and immunoblotting were carried out as described ,54 and ieviously , the anteviously . TMAO:beeviously . Protein [et al. . Prepara [et al. .N-oxide, TorA – TMAO reductase DMSO – dimethylsulphoxide, DmsABC – membrane-bound DMSO reductase.Sec -general secretory pathway, Tat – Twin arginine translocation pathway, TMAO -trimethylamine-MGH, DG, GB, IC and DW undertook experiments, BCB and TP wrote the manuscript.


Coxiella burnetii, the causative agent of Q fever, may cause culture-negative vascular graft infections. Very few cases of C. burnetii infection of a vascular graft have been reported. All were diagnosed by serology.Coxiella burnetii vascular graft infection diagnosed by broad-range PCR and discuss the diagnostic approaches and treatment strategies of chronic C. burnetii infection.We report the first case of C. burnetii should be considered as etiological agent in patients with a vascular graft and fever, abdominal pain, and laboratory signs of inflammation, with or without exposure history. Broad-range PCR should be performed on culture-negative surgical samples in patients with suspected infection of vascular graft. Staphylococcus aureus (30%), Enterobacteriaceae (25%), coagulase-negative Staphylococci (12%), Enterococci (9%), Pseudomonas aeruginosa (7%) and Streptococci (5%)[C. burnetii account for some of these culture-negative vascular graft infections. Very few cases of C. burnetii infection of a vascular graft have been reported [C. burnetii infection was obtained after the serological diagnosis of chronic Q fever by culture [C. burnetii from vascular graft samples [C. burnetii vascular graft infection diagnosed by broad-range PCR from a surgical sample of a para-prosthetic abscess which was confirmed by serology. To our knowledge, ours is the first case where the diagnosis was made by broad-range PCR and suggests that broad-range PCR should be considered in all cases of culture-negative vascular graft infections.Infection of synthetic abdominal aortic grafts occurs in ≤1% of patients, with a higher risk 1.5–2%) for grafts that extend to the femoral location. Vascular graft infection may result from intra-operative contamination, local extension from infected adjacent tissue or by hematogenous seeding. The most commonly involved pathogens are % for gras aureus 0%, Enters aureus 0%, EnterA 63-year-old man presented to a regional hospital on September 8, 2003 with a 2-week history of diffuse abdominal pain and mild diarrhea, without fever. In 1988, he had received a Dacron aorto-bifemoral graft for an infra-renal aortic aneurysm. A computerized tomography (CT) of the abdomen revealed a para-prosthetic fluid collection. Blood cultures were sterile in the absence of any recent antibiotic therapy. Laboratory results showed a white blood cell count of 5.8 G/l, a CRP of 48 mg/l, no increase of liver enzymes and a normal serum creatinine level. Empirical ciprofloxacin and metronidazole therapy was initiated and abdominal pain improved.After two months of antibiotic therapy, the patient was admitted to the University Hospital in Lausanne for removal of the vascular prosthesis because of presumed persistent infection, despite two months of antibiotic treatment. On admission, the patient was afebrile. Clinical examination was normal except for mild periumbilical tenderness on deep palpation. Laboratory results showed a normal WBC count (4.9 G/l), a normal CRP (<2 mg/l), and normal renal and liver functions. At laparotomy, extensive adhesions and a right para-iliac purulent mass were found. The prosthetic graft was partially removed, and replaced by a homograft. Multiple intra-operative specimens did not grow any microorganisms in culture. Histopathology showed a chronic inflammatory infiltrate, ill-formed non-necrotizing granulomas, and degenerative changes such as calcifications and fibrosis Figure  &1B. No Coxiella burnetii, using the BAK11w forward and the PC3mod reverse primers [C. burnetii chronic infection was confirmed by a positive serology performed at Unité des Rickettsies, Marseille, France: phase I antibody titer (IgG): 1600, phase II antibody titer (IgG): 3200. Antibiotic therapy with doxycycline  and chloroquine  was started. The dose of doxycycline was increased to 300 mg daily to reach a concentration of at least 5 μg/mL [16S rRNA PCR amplification plus sequencing performed on a fragment of the para-iliac mass was positive for  5 μg/mL . EighteeC. burnetii is a strict intracellular bacterium. It is the causative agent of Q fever, a worldwide zoonosis mainly transmitted by inhalation of infected particles. There is a wide animal reservoir, and sheep and cattle are probably the main source of human infections. Our patient had no environmental exposure to C. burnetii. Chronic Q fever is mainly seen in patients with underlying risk factors such as valvulopathy, pregnancy, and immunosuppression. Endocarditis accounts for 73% of chronic Q fever cases, followed by vascular infection (8%), including infections of aneurysms and vascular grafts [C. burnetii infection is crucial to a successful therapeutic outcome. We report here a rare case of C. burnetii vascular graft infection. This unexpected diagnosis was based on broad-range PCR. Q fever is probably underdiagnosed since the diagnosis will be missed if it is not systematically looked for in patients with vascular graft infections of unknown etiology.r grafts ,8. GivenC. burnetii presents a variation of phase (phases I and II). Antiphase I IgG at titers of ≥1:800 by microimmunofluorescence are indicative of chronic Q fever [C. burnetii PCR. To shorten the diagnostic delay, Fenollar et al. developed a nested-PCR assay with serum as a template which showed a sensitivity of 64% and a specificity of 100% [C. burnetii [et al. observed a granulomatous inflammation in one third of cases with Q fever endocarditis, but well formed granuloma were not identified. In these cases, degenerative changes such as valvular calcifications or foreign body reaction prevented the establishment of a specific association between C. burnetii infection and granulomatous inflammation [Coxiella infection. Definite histological diagnosis relies upon immunohistology, that may demonstrate the presence of C. burnetii within the cytoplasm of macrophages [C. burnetii vascular graft infection. Cell culture is not widely available as a diagnostic technique as it requires trained technicians and a biosafety level 3 laboratory.The diagnosis of Q fever is most frequently made by serology.  Q fever ,10. The  of 100% . In the burnetii . The hisammation . The prerophages . Howeverrophages . Thus, iCoxiella-containing vacuole is acidic. Therefore, addition of chloroquine that acts as an alkalinizing agent of the vacuole is essential to improve the efficacy of doxycycline therapy [C. burnetii vascular graft infection. Since serum doxycycline concentration is correlated with decrease in levels of phase I antibodies, it is recommended to adjust the dosage of doxycycline [C. burnetii and emphasizes the need for prolonged antibiotic treatment course. Serological follow-up will guide our decision regarding the treatment duration.Tetracyclines are the antibiotics of choice for acute Q fever . The bac therapy . Combina therapy . This coycycline . Patientycycline . In thisC. burnetii infection should be considered in patients with a vascular graft and unexplained low-grade fever, abdominal discomfort, laboratory signs of inflammation, and/or a history of environmental exposure. Broad-range PCR should be performed on surgical samples in patients with suspected infection of aneurysm or vascular graft. C. burnetii serology and/or specific C. burnetii PCR should also systematically be performed in cases of culture-negative vascular graft infection.The author(s) declare that they have no competing interests.LS, MF, DR, LVS, TC and GG were involved in patient care. LS wrote the first draft of the manuscript. AM did the histology and provided the images. All authors improved the manuscript and approved its final version.The pre-publication history for this paper can be accessed here:


Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success.The highly successful human pathogen  Mycobacterium tuberculosis, the agent of tuberculosis, is a highly successful human pathogen and kills nearly 3 million persons each year. This pathogen and its close relatives sum up in a single and compact clonal group dating back only a few tens of thousands of years. Using genetic data, the researchers have discovered that human tubercle bacilli from East Africa represent extant bacteria of a much broader progenitor species from which the M. tuberculosis clonal group evolved. They estimate that this progenitor species is as old as 3 million years. This suggests that our remote hominid ancestors may well have already suffered from tuberculosis. In addition, the researchers show that tubercle bacilli are able to exchange parts of their genome with other strains, a process that is known to play a crucial role in adaptation of pathogens to their hosts. Thus, the M. tuberculosis genome appears to be a composite assembly, resulting from ancient horizontal DNA exchanges before its clonal expansion. These findings open novel perspectives for unraveling the origin and the molecular bases of M. tuberculosis evolutionary success, and lead to reconsideration of the impact of tuberculosis on human natural selection. Salmonella enterica serotype Typhi [Yersinia pestis [Mycobacterium tuberculosis complex (MTBC), the agents responsible for tuberculosis, are among the most successful human pathogens. The MTBC as defined here comprises the so-called M. tuberculosis, M. bovis, M. microti, M. africanum, M. pinnipedii, and M. caprae species. Although the members of the MTBC display different phenotypic characteristics and mammalian host ranges, they represent one of the most extreme examples of genetic homogeneity, with about 0.01%–0.03% synonymous nucleotide variation [Most bacterial species consist of a wide spectrum of distinct clones or clonal complexes –3 that dpe Typhi  and Yersa pestis  essentiaariation –12 and nariation ,13–15. Tariation ,8,11,16.M. canettii, a rare tubercle bacillus with an unusual smooth colony phenotype [M. canettii and other smooth tubercle bacilli actually correspond to pre-bottleneck lineages, belonging to a much broader progenitor species from which the MTBC emerged.However, the nature and the boundaries of the bacterial pool that existed prior to the putative bottleneck, as well as the time of the transition to pathogenicity for mammalian hosts, have not yet been identified. A preliminary report suggested that henotype , could rhenotype . HoweverM. canettii isolates, as defined by van Soolingen et al. [M. canettii but differed by the presence of can,RD12 characteristically deleted in M. canettii, and by the absence of IS1081 insertion sequence. The five other groups of smooth tubercle bacilli were remarkably distinctive from M. canettii and the thousands of MTBC strains globally investigated up to now, notably by lacking IS1081 and/or the direct repeat (DR) locus [We extensively characterized 37 pulmonary and extra-pulmonary isolates of smooth tubercle bacilli see  from Eurn et al.  and Brosn et al. . Group BR) locus .Mycobacterium genus, we classically sequenced portions of six housekeeping genes  and the complete 16S rRNA gene of all isolates of groups A, B, E, F, G, H, and I, of representative isolates of group C/D, and of representative strains of the MTBC members  and nonsynonymous sites (Ka). The distances between the alleles of the MTBC strains were always much smaller than those between the alleles of the smooth strains . Furtherh groups .Staphylococcus aureus (0.023–0.037) [M. tuberculosis CDC1551 and H37Rv strains [The mean synonymous distance among distinct alleles in the tubercle bacilli (0.0083–0.039) was similar to that observed in many bacterial species known to be diverse, such as 3–0.037) ,5,20. Mo3–0.037)  showed t3–0.037) ,23, but  strains . This hi strains ,10.Y. pestis or S. enterica serotype Typhi [M. canettii and the other smooth groups. This supports the bottleneck hypothesis [M. prototuberculosis, to reflect its status as the M. tuberculosis progenitor  and Sawyer's (p < 0.05) statistical tests. In contrast, the rare minor allele differences among the smooth strains, such as those between gyrB alleles 9 and 10, are probably due to point mutations rather than recombination. Altogether, these observations provide evidence that both mutations and DNA recombination have occurred during the evolution of smooth tubercle bacilli.To investigate the contribution of horizontal DNA exchanges to the genetic diversity of equences  and the equences . The netM. prototuberculosis, the concatenated sequences of the six housekeeping genes of the MTBC strains appear to be constituted of a mosaic of patches identical or nearly identical to sequence patches from different smooth groups . Trees were drawn using TreeView version 1.5 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Bootstrap analysis was performed with 1,000 replicates. Numbers of synonymous substitutions per synonymous site (Ks) and nonsynonymous substitutions per nonsynonymous site (Ka) were estimated using DNASP version 4.00, using the Nei and Gojobori method after Jukes-Cantor correction for multiple substitutions [g-scale parameter of one and using both sequence triplets or sequence pairs scanning methods. p-Values were obtained with the KA method. The chi-square method was implemented using the MaxChi algorithm of RDP. Given an alignment, MaxChi examines sequence pairs and seeks recombination breakpoints by comparing the number of variable and nonvariable sites on both sides of the breakpoint. Split decomposition analysis was performed using SplitsTree version 4b06 [Neighbor-joining trees were constructed using PAUP* version 4.0b10 with Jukes-Cantor distance correction  DR region analysis by spoligotyping.1081, (D) IS6110, and (E) ISMyca1, a 1.8-kb insertion sequence related to the IS4 family (see (B–E) Southern blot analysis with DNA probes against (B) the DR region, (C) ISmily see .can.RD12 PCR using primers targeting the regions flanking canRD12 and further sequencing of these amplification products demonstrated an identical deletion in groups A, C/D, E, and H, whereas deletion in group F overlapped can.RD12(F) Southern blot analysis with a DNA probe directed against region (373 KB DOC)Click here for additional data file.Figure S2The unrooted trees were obtained using Megalign version 5.53 .(343 KB DOC)Click here for additional data file.Protocol S1(25 KB DOC)Click here for additional data file.Protocol S2(27 KB DOC)Click here for additional data file.Table S1(57 KB DOC)Click here for additional data file.Table S2(361 KB DOC)Click here for additional data file.Table S3(26 KB DOC)Click here for additional data file.http://www.ebi.ac.uk/embl/) accession numbers for the sequenced portions of katG, gyrB, gyrA, rpoB, and hsp65 genes of the smooth tubercle bacilli are AJ749904–AJ749948. The M. canettii ISMyca1 sequence has been deposited in the EMBL database under accession number AJ619854.The EMBL (


Several studies have reported higher rates of antimicrobial resistance among isolates from intensive care units than among isolates from general patient-care areas. The aims of this study were to review the pathogens associated with nosocomial infections in a surgical intensive care unit of a university hospital in Turkey and to summarize rates of antimicrobial resistance in the most common pathogens. The survey was conducted over a period of twelve months in a tertiary-care teaching hospital located in the south-eastern part of Turkey, Gaziantep. A total of 871 clinical specimens from 615 adult patients were collected. From 871 clinical specimens 771 bacterial and fungal isolates were identified.Pseudomonas aeruginosa (20.3%), Candida species (15%) and Staphylococcus aureus (12.9%). Among the Gram-negative microorganisms P. aeruginosa were mostly resistant to third-generation cephalosporins (71.3–98.1%), while Acinetobacter baumannii were resistant in all cases to piperacillin, ceftazidime and ceftriaxone. Isolates of S. aureus were mostly resistant to penicillin, ampicillin, and methicillin (82–95%), whereas coagulase-negative staphylococci were 98.6% resistant to methicillin and in all cases resistant to ampicillin and tetracycline.Most commonly isolated microorganisms were: In order to reduce the emergence and spread of antimicrobial-resistant pathogens in ICUs, monitoring and optimization of antimicrobial use in hospitals are strictly recommended. Therefore local resistance surveillance programs are of most value in developing appropriate therapeutic guidelines for specific infections and patient types. Antimicrobial resistance among intensive care unit (ICU) pathogens is generally increasing, but variations do exist among different countries, probably due to individual antimicrobial use patterns. When new medical practices and alternative antimicrobials are introduced, changes in the dominant microbial etiologies may emerge prompting novel empiric selections. Appropriate therapy of ICU infections directed by local resistance data can have significant consequences for both patient and the healthcare system.Data from National Nosocomial Infections Surveillance (NNIS) shows that from 1988 to 1995 the number of intensive care unit (ICU) beds at the hospitals has increased 17%, whereas total hospital bed capacity decreased slightly [This study reviews the pathogens associated with nosocomial infections among ICU patients, summarizes rates of antimicrobial resistance in the most common pathogens and provides an overview of strategies to prevent the proliferation of antimicrobial-resistant microorganisms.During the study period a total of 871 clinical specimens from 615 adult patients were collected. None of the patients were receiving immunosuppressive therapy. In 771 (88.5%) of cases the isolate recovered by culture was confirmed to be the etiologic agent of a nosocomial infection. The distribution of infections was examined by the major site of infection. Four major infection sites represented 91.8% of all reported infections; lower respiratory tract infections were most frequent (31.5%), followed by urinary tract infections (27.8%), bloodstream infections (23.1%) and surgical site infections (9.3%).P. aeruginosa 20.3%, Candida spp. 15%, S. aureus 12.9%, A. baumannii 9.6%, and coagulase-negative staphylococci 8.9%. In lower respiratory tract infections, P. aeruginosa (27.9%) was the most commonly isolated pathogen, which was followed by S. aureus (13.6%) and A. baumannii (13.2%). Fifty-seven percent of reported isolates from the urinary tract were aerobic Gram-negative bacilli, and in 16.3% of cases Candida spp. were isolated. Bloodstream infections were mostly caused S. aureus and coagulase-negative staphylococci with 16.8% and 16.3%, respectively, whereas P. aeruginosa species (14.6%) were also frequently isolated from blood. In surgical site infections S. aureus (23.6%) and P. aeruginosa (22.2%) were the most commonly isolated infectious agents  and methicillin-resistant coagulase-negative staphylococci were 82% and 98.6%, respectively (Table S. aureus) isolated from infections in ICU were shown in Table Each of the pathogens listed on Table Pseudomonas aeruginosa showed high proportion of resistance against antipseudomonal penicillins  and third-generation cephalosporins . Isolates of A. baumannii were in all cases resistant to ceftriaxone and in most cases to aztreonam (94.6%) and ticarcillin (93.2%). Although Enterobacter species showed multiple resistance to most antimicrobials tested, they were uniformly susceptible to imipenem and ciprofloxacin. Klebsiella pneumoniae were usually resistant to multiple antimicrobials and hydrolized third-generation cephalosporins and aztreonam (72–88%)  oxacillin-resistant S. aureus and vancomycin-resistant enterococci. All of these resistance profiles have been documented in other regional and global ICU surveillance studies including Project Intensive Care Antimicrobial Resistance Epidemiology , the Meropenem Yearly Susceptibility Test Information Collection Program , the ICU Surveillance Study  and the SENTRY Program  [The types of organisms that have emerged as most problematic for patients within the ICU include the members of the family S. aureus (24.1%), P. aeruginosa (12.2%), E. coli (10.1%), Klebsiella spp. (8.9%), Enterococcus spp. (7.2%), coagulase-negative staphylococci (7%) and Enterobacter spp. (7%) (5). In the MYSTIC Study, P. aeruginosa (33%) was the most frequent isolate, followed by A. baumannii (17.1%), K. pneumoniae (12.1%), E. coli (10.5%) and E. cloacae (7.9%) . In this study P. aeruginosa (20.3%) was the most commonly isolated pathogen in ICU, which was followed by Candida spp. (15%), S. aureus (12.9%), A. baumannii (9.6%), and coagulase-negative staphylococci (8.9%).The rank order of pathogens recovered in SENTRY Antimicrobial Surveillance Program was Patients in the ICU are more likely than others to be colonized or infected with an antimicrobial-resistant pathogen, therefore the rates of resistance are significantly higher in patients cared for in the ICU than in non-ICU patients -14.S. aureus and coagulase-negative staphylococci shows that rates of methicillin-resistant isolates in ICU have increased steadily over the past decade [S. aureus strains and 98.6% of coagulase-negative staphylococci were resistant to methicillin.Examination of Gram-positive microorganisms, such as t decade -15. In oP. aeruginosa, especially fluoroquinolones, was increasing [of P. aeruginosa in this study were resistant 71.4 to 98% to third generation cephalosporins, and 69.4 to 93% to antipseudomonal penicillins. The relatively high susceptibility of Gram-negative bacilli to fluoroquinolones in this study can be attributed to antimicrobial prescribing protocol used in our ICU, which is preferably directed to broad-spectrum antibiotics.Gram-negative bacilli are frequently associated with nosocomial infections in ICUs. Data from a multicenter Intensive Care Unit Surveillance Study (ISS) in the United States demonstrated that resistance to antipseudomonal agents among ICU isolates of creasing . The isoAcinetobacter spp. have emerged as important pathogens of ICUs, most of them being resistant to ampicillin, carbenicillin, cefotaxime, chloramphenicol, and gentamicin [A. baumannii isolates were resistant to third generation cephalosporins, and most of them were resistant to aztreonam, ticarcillin, and gentamicin with 94.6%, 93.2%, and 85.1%, respectively.In recent years ntamicin ,18. In oEnterobacter species are resistant to first-generation cephalosporins and develop antibiotic resistance readily to second- and third-generation cephalosporins owing to an inducible chromosomally encoded cephalosporinase. Emergence of this form of resistance is seen frequently, when infection due to these organisms, particularly Enterobacter cloacae, are treated with broad-spectrum cephalosporins [Enterobacter isolates 35–50% were resistant to ceftazidime and piperacillin. In this study the resistance rates of Enterobacter strains to third generation cephalosporins were between 59.4–75%, whereas higher rates were recorded to aztreonam and gentamicin (both 81.2%).osporins . Pfallerosporins  reportedKlebsiella pneumoniae, which is producing extended-spectrum beta-lactamases (ESBLs). Our isolates of K. pneumoniae showed high resistance to broad-spectrum cephalosporins and aztreonam .Another type of commonly seen antimicrobial-resistant pathogen encountered among ICU patients is Many studies have demonstrated that the development of antimicrobial resistance at hospital level is strongly correlated with the use of the relevant antimicrobial . Data frIn order to prevent the emergence and spread of antimicrobial resistant pathogens in ICU, the pattern of antimicrobial use has to be determined. A multidisciplinary approach is required to succeed in combating the problem. Hospitals should monitor antimicrobial use to determine whether specific ICUs or the entire hospital is overusing antimicrobials. These data could be used, in conjunction with other related studies, to properly interpret significant resistance patterns and choose the most appropriate antimicrobial regimens for empirical therapy.Data were collected from adult patients hospitalized in the surgical ICU of the Medical Faculty Hospital during one year period between January and December in 2001. Standard Centers for Disease Control and Prevention/NNIS definitions of infection were used . All pat2 for 18–24 hours. Methods used for confirmation of identification included examination of colonial morphology and haemolytic characteristics on appropriate agar media, Gram stain, rapid tests  and use of an automated identification system, VITEK , which is a microbroth dilution method with two wells across pre-determined breakpoints.Lower respiratory tract specimens included bronchoalveolar lavage, transtracheal aspiration, and pleural fluid. Urinary tract specimens included urine and aspiration from the urinary catheter. Other types of specimens obtained from the patients were; swabs from surgical wounds, cerebrospinal fluid, pericardial fluid, peritonal fluid, synovial fluid, biopsy material, oral swab, rectal and anal swabs. All specimens were collected at the bed site, transferred to the laboratory immediately and were inoculated on proper culture media within two hours. Clinical specimens were inoculated onto 5% sheep blood agar, eosine-methylene-blue agar, Sabouraud's dextrose agar, and except the urine specimens, onto chocolate agar . Plates were incubated at 37°C in 5% COS. aureus ATCC 29213, P. aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212) were performed according to the instructions of CLSI (previously NCCLS) [Penicillin G, ampicillin, amoxicillin/clavulanate, methicillin, vancomycin, clindamycin, erythromycin, trimethoprim/sulfamethoxazol, ciprofloxacin, and tetracycline were used for antimicrobnial susceptibility testing. Susceptibility tests were performed with a commercial broth microdilution method  according to the manufacturers' guideline recommendations and interpretative criteria. Colonies from 18–24 hours old culture medium were used to inoculate the microdilution plates. Quality control for testing by VITEK was performed before each new lot of cards was used. Antimicrobial susceptibilities of the control microorganisms  software .AB carried out the collection of clinical samples from patients, cultured and identified pathogenic microorganisms from those specimens and perfomed antimicrobial susceptibility tests of the study strains. She also designed the study and performed acquisition, analysis and interpretation of data. IB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Both authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Helicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea.H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017 – 0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago.We obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either.Our mitochondrial DNA sequence data supports the single species status of  Helicoverpa (Hardwick 1965) is a group of 18 species [H. armigera (Hübner) and H. zea (Boddie) being the dominant pest species in the Old World and New World respectively. A few other species in the genus Helicoverpa are pests of a range of crops but they are either limited in host plant range or are geographically restricted [H. assulta (Guenée) which feeds only on Solanaceae, and is endemic to Asia, Africa and Australia [H. punctigera  which is polyphagous and endemic to Australia [Helicoverpa species are oligophagous and are not considered as major agricultural pests.The genus Hardwick 965 is a Hardwick 965 is a ustralia , and H. ustralia . The majH. zea and H. armigera were considered conspecific [Heliothis Oschenheimer [Heliothis armigera. Hardwick [H. armigera and H. zea as separate species within the new Helicoverpa genus, Matthews [Helicoverpa genus, developing methods to inflate the helical bladder-like appendages in this genus. Morphological characters and allozyme-based phylogeny suggested that H. punctigera is basal to H. assulta [H. armigera and H. zea was uncertain and they were considered monophyletic, sharing a common ancestor with H. assulta [H. armigera and H. punctigera but with H. zea displaying 61% less mean heterozygosity, and Mallet et al. [H. zea evolved from a small founding population of H. armigera (or of their joint common ancestor). Accurate demarcation of species boundaries and their distributions is essential for understanding pest demography. Apart from the work of Matthews [Helicoverpa pest species such as H. armigera and H. zea. This represents a significant handicap for basic and applied research into these two important pest species.Until the work of Hardwick H. zea anspecific  within tenheimer  as HelioHardwick  incorporMatthews  confirmeMatthews  for the  assulta . The evo assulta . Allozymt et al.  suggesteMatthews  which waH. armigera and H. zea are highly polyphagous. They possess the ability to enter diapause as pupae and are known to develop high levels of insecticide resistance [H. armigera have been conducted in different regions of the world using different genetic marker systems [H. armigera. However, microsatellite studies of Australian H. armigera populations suggested monthly genetic shifts, highly variable gene flow between populations between years, and limited moth movements which varied between and within seasons [H. armigera. Low genetic differentiation in the related long-range migratory pest Heliothis virescens across the cotton belt within the United States of America has also been reported [Larvae of sistance -11. Adulsistance . Populat systems -18. Vary systems , mitocho systems  and the  systems  suggest  seasons ,16. Resu seasons , and in reported .H. armigera as a polyphagous pest, very little systematic research has been conducted to resolve the question of the existence of sub-species at the local or global level. Based on taxonomic data, Paterson [H. armigera constituted a single genetic species or formed a complex of cryptic species over its geographic range. Although the existence of cryptic H. armigera species has not been disproved, mating experiments that involved H. armigera from different regions of the Old World strongly supported its single species status [H. armigera could be categorized into races, based on their host-feeding preferences, and that these races do not interbreed freely [H. armigera populations being sampled at these times from different crops. Differential responses of H. armigera populations to pheromone [H. armigera have also been generated through laboratory selection of field-collected insects on cotton fruit buds (squares). These races were characterized by differing (though overlapping) cornutal spine number on the male aedeagus [Despite intense agricultural interest in Paterson  raised ds status . In Indid freely ,23. Krand freely  reportedheromone  and paraheromone  were alsheromone ,28. "Cotaedeagus .H. armigera. Analysis of mtDNA has provided valuable insights into understanding natural genetic diversity and population structures in other organisms [H. armigera globally. The genetic information collected is used to address the questions of single species status and of host races, and to infer phylogenetic relationships amongst the four pest Helicoverpa species.To date, no molecular studies have assessed phylogeographic patterns among worldwide populations of rganisms . It is crganisms . Neverthrganisms , and hasrganisms . This stH. armigera and H. zea population study. The 62 randomly sampled H. armigera and 11 H. zea haplotype representative samples all possessed identical tRNA-Leu gene sequence to the deposited H. armigera tRNA-Leu sequence (DQ059302). In the 62 H. armigera samples, two transitional mutations (G/A and C/T) were detected. The G/A mutation involved one individual each from Burkina Faso and Pakistan that belonged to the same mtDNA haplotype (Harm1). The C/T base change involved an Australian (previously Harm2) and a Pakistan (previously Harm3) sample. There were four C/T transitional mutations from nucleotide positions 512 to 809 between the 11 H. zea and the 62 H. armigera samples. These four mutations increased the number of steps by one between haplotype Hzea-1 and Hzea-7, and the number of steps by three in the haplotype network between H. armigera and H. zea  indicated the partial COI gene at nucleotide positions 1 to 511 as being the most informative region for our H. armigera, H. zea, H. punctigera, H. assulta and Heliothis virescens gave PCR products of the expected size. A final 511 base pair (bp) sequence (nucleotide position 1 to 511) was analysed, following trimming of invalid end sequences and sequence alignment. The COI sequences showed no ambiguity and no premature stop codons. A total of 32 SNPs were identified from the 511 bp partial COI region in 249 H. armigera samples resulting in 33 mtDNA haplotypes. A total of 29 synonymous and 3 non-synonymous substitutions were detected in these partial COI sequences. Multiple amino acid sequence alignments revealed that the three non-synonymous changes are in haplotype Harm-11 (Glycine to Serine); Harm-13  and Harm-26 (Tyrosine to Cysteine). Amino acid substitutions in Harm-13 and Harm-26 resulted in the replacement of one hydrophobic amino acid with another, while in Harm-11 the amino acid substitution occurred between hydrophobic and polar groups. Of the 32 variable sites identified, 15 sites were parsimony informative and 17 were non-informative.PCR amplification of the informative partial mtDNA COI fragments for all individuals of H. armigera haplotypes according to either host plants or geographical clade  and South America (Brazil). Haplotypes Hzea-1, 2, 6 and 7 were found in both North and South American continents, while the remaining haplotypes were unique to either the North or South American populations. The H. zea haplotype network is most parsimoniously linked to the H. armigera clade through Harm-14 found in Burkina Faso .The most prevalent haplotype found in all countries was designated Harm-1. Harm-4, found in Uganda and Australia, was the most diverged haplotype with seven mutation steps from the major haplotype Harm-1 Fig. . The hapF-statistics analysis which showed significant gene flow between these three regions (Fst = 0.07). Analysis of Molecular Variance detected no genetic structure at various hierarchical levels  were low in the study samples, and ranged from approximately 0 to 0.06. The observed nucleotide diversity between countries was also very low and ranged from 0.0017 – 0.0039 , North  and South  in ls Table , with 9639 Table .H. armigera within Africa (Burkina Faso and Uganda) was 0.0 – 0.02, within Australia 0.0 – 0.018, and within Asia  was 0 – 0.014. Genetic distances between the three continents ranged from 0.0 – 0.02. Genetic distances between all four species of the genus Helicoverpa and Heliothis virescens were also estimated  is 0.014 as compared to within both the African (0.020) and Australia (0.018) continents. The isozyme study of Australian H. armigera populations sampled across a 3,000 km study area by Daly and Gregg [Fst = 0.012). Non-significant isozyme allele frequency differences were also found between populations of H. armigera located on either side of the Sahara desert, thereby suggesting that geographical barriers such as the Sahara desert have not prevented long distant migration in H. armigera [H. armigera has been further suggested by the trapping of adult moths on Ascension Island, 2,000 km from the African coast [H. armigera constitutes a single species across its distribution range with demonstrated long distance migration ability is further supported by the intercontinental crossing experiments of Colvin et al. [H. armigera from the African, Asian and Australian continents did not lead to a reduction in fecundity or offspring viability.Generally, the pattern of genetic variation seen at the partial COI region of movement . Our mtDnd Gregg  also repralia 0.0 continenan coast  and Willn et al. , wherebyH. armigera and across the Old World. Genetic studies of generalist phytophagous insects often reveal complexes of genetically differentiated host races or cryptic species, but the extent of genetic difference correlated with host plant association in the H. armigera samples available in our study is inconclusive both at the global level and more specifically within the Indian populations due to small sample sizes. Bhattacherjee [H. armigera showed variability in host preferences, responded differentially to parasitoid attacks [H. armigera based on a survey of male internal morphological characters and their association with hosts. Laboratory experiments of Kranthi et al. [H. armigera possessed distinct internal morphological features . As an example of a study in which mtDNA COI sequence comparisons did reveal cryptic species, Crespi et al. [H. armigera sub-species or races in the Old World. Further work using nuclear DNA makers or genes associated with the detoxification of secondary plant compounds may yet reveal differentiation. For example, strong genetic differentiation in maize and mugwort races of the European corn borer  was reported by Thomas et al. [The mtDNA COI haplotype network (haplotype clustering patterns) and phylogenetic analysis failed to revealed specific host affiliations, both within Indian populations of acherjee  reported attacks  and when attacks , suggest attacks  failed ti et al.  showed ti et al.  found ths et al.  based ons et al. ,44.Helicoverpa species indicated that all H. armigera haplotypes formed a monophyletic clade which included the New World species H. zea. This phylogeny also revealed that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea  will be necessary for constructing a robust Helicoverpa phylogeny, especially that between the two closely related H. armigera and H. zea species. A monophyletic relationship between H. armigera and H. zea  was inferred based on the nuclear Elongation Factor 1-alpha (EF1-α) gene [H. zea and H. armigera with a higher bootstrap value [Phylogenetic analysis of the four important pest zea Fig. . The cur-α) gene  and the -α) gene , while aap value .H. armigera and H. zea (0.031 – 0.047) were intermediate between that expected for intra- and interspecific comparisons, thereby indicating high levels of genetic similarity between H. zea and H. armigera. Hebert et al. [Busseola fusca, Sezonlin et al. [b gene between different clades of the same species. The similar genetic distance reported in this study suggests that H. armigera and H. zea are more closely related to each other than the case with the Busseola fusca species, possibly due to a more rapid divergence rate.Pairwise genetic distances between t et al.  cautionen et al.  reportedH. armigera and H. zea, by applying a linear rate of substitution in short term evolution [H. zea populations were established via a founder event from H. armigera no more than 1.5 million years ago. This is further supported by highly similar morphology [piggyBac, Zimowska and Handler [H. zea and H. armigera inter-mating as a possible explanation for the propagation of these mobile elements, while arguing for conspecific status in H. zea and H. armigera should these elements share the same genomic insertion sites in these two species. The founder event hypothesis should be further tested through a better H. zea sampling regime across the northern and southern American continents, as strong geographic components to intraspecific polymorphisms and inadequate sampling can lead to inaccurate phylogenies [The estimates of divergence time between volution  of 2% pevolution , suggestrphology  and the rphology ,52,53, arphology . Based o Handler  proposedlogenies .H. armigera differently. The current study does not suggest that H. armigera and H. zea are a single species but does show their close relationship and indicates a fairly recent (<1.5mya) divergence of H. zea from a parental H. armigera stock. We should utilise their genetic similarity as the basis of formulating strategies for concerted future research efforts into areas such as complete genome sequencing, and the mapping and identification of economically important genes  through crossing experiments. The scientific community is now presented with a valuable opportunity, whereby the complete genome sequencing of either one of these species will significantly advance our understanding of genome organisation of both, and thus our efforts in fighting against two of the most destructive lepidopteran pests of the Old and New Worlds.This study suggests that for pest management purposes, there is currently no molecular basis for treating geographic or host specific populations of H. armigera individuals were collected from India, China, Pakistan, Burkina Faso and Uganda as larvae, adults or pupae. Larvae were collected directly from host plants or were laboratory reared first generation individuals  or the method of Zraket et al. [A total of 249 ls Table . Genomict et al. .H. armigera , representative haplotypes of 11 H. zea and two of each H. assulta and H. punctigera at the mtDNA region (809 bp) that spanned the partial COI gene (552 bp) at the 3' end, the complete intergenic region between COI and COII genes  and the 5' partial region of the COII gene (190 bp). Due to the general homogeneous nature of sequences at the terminal region of COI, the tRNA-Leu gene and the COII partial gene (sequence data available upon request), we restricted our population analysis to include only the informative partial COI (511 bp) region. For all remaining population samples to be analysed, a 680 bp fragment of the mtDNA COI gene was initially PCR amplified using the primers COI-F01 (5' TTATTTCACATCAGCTACTAT 3') and COI-R01 (5' CTTTATAAATGGGGTTTAAAT 3') and subsequently trimmed to the desirable 511 bp. Primers were designed based on H. armigera COI sequences deposited in GenBank  using the primer analysis software Oligo 6.4 . PCR conditions used the following profile: 94°C for 4 minutes (one cycle); one minute each of 94°C, 50°C and 72°C (35 cycles), followed by a final extension cycle of 72°C for 5 minutes. PCR amplification of individual DNA samples was carried out in a total reaction volume of 25 μL, and contained 25 ng of genomic DNA, 0.2μM each of forward and reverse primers, 0.2 mM of dNTP's, 1× PCR reaction buffer (Promega), 1.5 mM Mg2+ and one unit of Taq DNA polymerase (Promega).In a preliminary population survey we randomly sequenced 62 ® PCR purification Kit  prior to the sequencing reaction. The DNA sequencing reaction used the ABI BigDye® dideoxy chain termination sequencing system (Applied Biosystems) following the supplier's instructions, and was electrophoresed by the Australian Genome Research Facility . Double stranded sequences were obtained for all samples, for accuracy. In addition, 45 individuals including representatives of each haplotype were sequenced at least twice using amplicons from separate PCR reactions to ascertain the accuracy of the single nucleotid polymorphisms (SNPs) detected. Sequences were assembled using Pregap4 and Gap4 programs within the molecular biology software STADEN package [Amplicons were purified using the QIAquick package . Nucleot package  and wereH. armigera was constructed manually and verified using the program TCS, version 1.13 [Helicoverpa moth species. A heuristic tree search with the 'asis' stepwise addition and TBR branch swapping algorithm was used to find the best ML tree. To assess branch support in our ML trees we used non-parametric bootstrapping with heuristic searches of 1,000 replications. We implement the criteria of Huelsenbeck and Hillis [A mtDNA COI haplotype network for ion 1.13 . For theion 1.13  was usedion 1.13  to assesd Hillis  to assesd Hillis  was usedH. armigera populations were estimated using single locus F-statistics of Weir and Cockerman [Fst values in the ten Indian populations, all ten populations were pooled in AMOVA analysis. Levels of polymorphism between countries were estimated using haplotype diversity  and nucleotide diversity (π) [Genetic differentiation among all predefined ockerman  in GENEPockerman . Analysiockerman ) and divockerman  using pasity (π) .RAPD, random amplified polymorphic DNA; mtDNA, mitochondrial DNA; COI, cytochrome oxidase subunit I; COII, cytochrome oxidase subunit II; tRNA-Leu, transfer RNA-Leucin; SNPs, single nucleotide polymorphisms; ML, maximum likelihood; INDELs, insertions and/or deletions.H. armigera samples, DAR obtained African and Asian H. armigera samples and various American H. zea samples. Field work in Victorian was carried out by GTB. GTB carried out all the molecular genetic work and generated all sequence data. Analyses of sequence data, phylogenetic and population genetic analyses were performed by GBT and WTT. GTB and BRA worked on the mtDNA haplotype network. WTT, PB and DGH helped with interpretation of sequence data and results from population genetic analyses. GTB led the writing of this study. The original version was critically read and significant improved by WTT, DAR, DGH and PB.DAR, BRA and KRK designed the study. KRK also provided various Indian


Both the recently updated consensus guidelines published by the American College of Chest Physicians, and the International Union of Angiology recommend thromboprophylaxis with either low-molecular-weight heparin (LMWH) or unfractionated heparin (UFH) in medical patients at risk of VTE. However, no guidance is given regarding the appropriate dosing regimens that should be used for thromboprophylaxis in this patient group. LMWH  and UFH have been shown to be effective for thromboprophylaxis in at-risk hospitalized medical patients. Although LMWH once daily (o.d.) has been shown to be as effective as UFH three times daily (t.i.d.) for thromboprophylaxis in at-risk medical patients, there are no data to show that UFH twice daily (b.i.d) is as effective as either LMWH o.d. or UFH t.i.d. On the basis of currently available evidence, the LMWHs enoxaparin and dalteparin are more attractive alternatives to UFH for the prevention of VTE in hospitalized medical patients because of their convenient once-daily administration and better safety profile, demonstrated in terms of reduced bleeding, HIT, and other adverse events. In the absence of thromboprophylaxis, the incidence of venous thromboembolism (VTE) ranges from 10–20% in general medical patients to 80% in trauma patients, spinal cord injury patients, and patients in the critical care unit -5, thromConsensus guidelines published by the American College of Chest Physicians (ACCP) and the International Union of Angiology (IUA) recommend assessment of all hospitalized medical patients for the risk of VTE and the provision of appropriate thromboprophylaxis ,2. FurthThe recently updated ACCP consensus guidelines give a grade 1A recommendation for thromboprophylaxis using either low-molecular-weight heparin (LMWH) or low-dose unfractionated heparin (UFH) in medical patients with congestive heart failure (CHF) or severe respiratory disease, or in medical patients who are confined to bed and have one or more risk factors for VTE, such as active malignancy, acute neurological disease, inflammatory bowel disease, previous VTE, or sepsis ,16.The aim of this manuscript is to review recent advances in thromboprophylaxis in hospitalized medical patients, and discuss them in light of the recently updated ACCP consensus guidelines.Several placebo-controlled studies have investigated the efficacy of thromboprophylaxis with UFH or LMWH in medical patients Table  3-5,17--17-26. Ip < 0.001), but not when 20 mg enoxaparin was used (Table p < 0.001). Moreover, Kaplan-Meier plots of the probability of 3-month survival rates suggest that the overall mortality was lower in patients receiving 40 mg enoxaparin than those receiving placebo, although differences in mortality rates between the three groups did not reach statistical significance. A retrospective multihospital analysis of data from hospitalized acutely ill medical patients in the USA also showed that thromboprophylaxis with enoxaparin resulted in a significant reduction in the incidence of VTE compared with no thromboprophylaxis  .-34.30-34< 0.001) . This me< 0.001) . No signed above . This adAlthough the recently updated ACCP consensus guidelines recommend thromboprophylaxis with either LMWH or UFH for at-risk medical patients, no recommendations are given regarding appropriate dosing regimens . The ACCWhen comparing UFH and LMWH, similar efficacy in the prevention of VTE in medical patients has only been observed when UFH t.i.d. was compared with LMWH o.d. Table . One stuThese findings were confirmed in a recently published systematic review of published randomized clinical trials evaluating the efficacy of UFH 5000 IU b.i.d., compared with LMWH and UFH 5000 IU t.i.d. . AlthougIn light of this, it is noteworthy that there are data to suggest better efficacy with enoxaparin than with UFH given on a t.i.d. basis to patients with congestive heart failure (CHF). When comparing enoxaparin 40 mg o.d. with UFH in patients with CHF in THE-PRINCE study, enoxaparin 40 mg o.d. showed a trend towards better efficacy than UFH, t.i.d. Figure . Venous In summary, only the t.i.d. regimen of UFH is supported by similar clinical outcomes compared with LMWH o.d.. Therefore, only UFH t.i.d. amd LMWH o.d.  and can be recommended for use as thromboprophylaxis in hospitalized medical patients at risk of VTE.p = 0.029; relative risk reduction 46.7%; 95% CI 7.7%-69.3%) (Table p = 0.029). Patient inclusion criteria in ARTEMIS differed from those used in the MEDENOX and PREVENT studies: patients were older (>60 years compared to >40 years) and were immobilised for longer (>4 days compared with ≤ 3 days) . The. Thep = p < 0.001) .-34.30-34< 0.001) . Severalp = 0.049) [p = 0.0001).In the previously described meta-analysis of randomized studies that showed equivalent thromboprophylactic efficacy when directly comparing LMWH and UFH , use of = 0.049) . Another= 0.049) . While tThe ARTEMIS study  reportedThe risk of heparin-induced thrombocytopenia (HIT) should also be considered when providing patients with thromboprophylaxis. The risk of HIT has been shown to be significantly lower in surgical patients receiving LMWH prophylaxis than in those receiving UFH . Recent Several economic studies have examined the cost-effectiveness of thromboprophylaxis in medical patients -45. ThreEvidence from clinical studies has shown the benefits of thromboprophylaxis with LMWH  or UFH for the prevention of VTE in at-risk medical patients. Thromboprophylaxis with LMWH is as effective as UFH at preventing VTE in this group of patients, but has a significantly better safety profile. If UFH is used, physicians should choose a t.i.d. dose regimen, as there are no data to show that UFH b.i.d. is as effective as LMWH o.d. or UFH t.i.d. dosing. Recent data suggest that fondaparinux may be an effective and safe option in high-risk elderly medical patients, but further studies are awaited before general recommendations can be given regarding its use in all acutely ill medical patients. On the basis of currently available evidence, it appears that, while low-dose UFH is also recommended by the ACCP  althoughMM Samama has periodically been an independent consultant for several pharmaceutical companies.FX Kleber has no competing interests.Both authors had significant roles in conception, drafting and reviewing the manuscript, and they approved the final version.


Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Sα RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Sα rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups . All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient  was T. cruzi II and the mitochondrial donor was T. cruzi III.Acquisition of detailed knowledge of the structure and evolution of  Trypanosoma cruzi causes Chagas disease, a malady that afflicts almost 20 million people in South America and Central America. Although the genome sequencing of T. cruzi has been recently completed, little is known about its population structure and evolution. Since 1999, two major evolutionary lineages presenting distinct epidemiological characteristics have been recognized in the parasite: T. cruzi I and T. cruzi II, the latter being much more associated with severe chronic cases of the disease. We describe new and important aspects of the population structure of the parasite, especially the characterization of a third ancestral lineage that we propose to call T. cruzi III. Through careful dissection of the genetic constitution of blocks of genes that are stably transmitted from generation to generation of the parasite we deduced at least two occurrences of the formation of hybrid strains from the parental lineages T. cruzi II and T. cruzi III, including the strain CLBrener, whose genome was sequenced. We did not find any hybrids originating from T. cruzi I. A fascinating finding was that both hybrids studied had the same mitochondrial DNA type as the T. cruzi III ancestral lineage, which was quite different from T.cruzi II.The parasite protozoan  Trypanosoma cruzi causes Chagas disease, a malady that afflicts almost 20 million people in South America and Central America, with more than 20,000 deaths reported each year  and DogT. cruzi can be envisaged with different molecular markers, such as isozymes [T. cruzi, they seem to have been only occasional and to have been subsequently stabilized by strong clonal propagation .T. cruzi stocks (75) isolated from both domestic and sylvatic transmission cycles were analyzed  and D72 (5′-TTTTCAGAATGGCCGAACAGT-3′) primers following protocols described previously [COII gene [Amplification of the mitochondrial OII gene  was perfOII gene .COII sequences, the AluI restriction endonuclease was chosen to perform RFLP analyses in the mitochondrial COII gene. After PCR amplification, the amplicons were submitted to enzyme digestion for 16 hours according to instructions provided by the manufacturer . Digested products were analyzed on polyacrylamide gel electrophoresis and silver stained.Based on the restriction map of CYb gene [ND1 (37) were obtained from GenBank.Sequences for the mitochondrial CYb gene  and the Based on the microsatellite results, a distance matrix between the strains was constructed as described previously . In ordeCOII, CYb, and ND1 sequences with the MEGA v. 3.1 software [NJ trees were obtained separately for the software  using thT. cruzi populations by using a Bayesian coalescent theory-based method contained in PHASE software (Version 2.0.2 for Linux) [http://www.fluxus-engineering.com).Haplotypes were reconstructed from the 75 r Linux) . The typr Linux) , availabTable S1(105 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genes and gene products discussed in this paper are 24S rDNA (L19411), mini-exon gene (X62674), COII (AF359041 and DQ343715–DQ343753), CYb , and ND1 .The GenBank (


Candida spp. have been collected from literature and six of these patients died after treatment with amphotericin B.Fungal mural endocarditis is a rare entity in which the antemortem diagnosis is seldom made. Seven cases of mural endocarditis caused by Candida parapsilosis. Because blood cultures continued to yield C. parapsilosis despite caspofungin monotherapy, treatment with voriconazole was added.We report a case of mural endocarditis diagnosed by transesophageal echocardiogram and positive blood cultures to C. parapsilosis mural endocarditis with caspofungin and voriconazole.This is the first description of successful treatment of  Candida species were recovered in 94.1% of yeast infections and Aspergillus species in 71.8% of mold infections. Mural endocarditis is inflammation and disruption of the nonvalvular endocardial surface of the cardiac chambers. Its presentation is remarkably similar to that of infective valvular endocarditis. Risk factors for mural endocarditis include chronic debilitating disease, immunosuppression, intravenous drug use, central venous catheterization, prolonged treatment with broad-spectrum antibiotics, and repeated surgery. The pathogenesis of mural involvement has been ascribed to hematogenous seeding or direct extension of myocardial abscesses [Fungal endocarditis has increased in incidence during the last 2 decades. In a review of 152 cases of fungal endocarditis reported between 1995 and 2002 [Aspergillus mural endocarditis has been diagnosed in small series of patients [Candida spp. has been exceptionally reported [Candida parapsilosis mural endocarditis cured by combined treatment with caspofungin and voriconazole.Kearney et al.  collectepatients -7 but mureported ,9. We prStaphylococcus aureus and enterobacteriacea. The patient was treated empirically with piperacillin/tazobactam and received total parenteral nutrition via a subclavian central venous catheter. Surgical treatment consisted of cholecystectomy, sequestrectomy of necrotic pancreatic tissue, and extensive debridement. Antibiotic treatment was continued for 23 more days, although intravenous fluconazole was added and given for 14 days due to oropharyngeal candidiasis.A 59-year-old woman had a 13-year history of biopsy proven alcoholic cirrhosis. She was admitted to the hospital because of abdominal pain, nausea, vomiting, and fever. A diagnosis of biliary acute pancreatitis was established. Twenty days after admission, the patient's condition deteriorated. A new dynamic abdominal computed tomography (CT) scan showed an increase of peripancreatic fluid collections and necrosis of approximately 80% of the gland. A CT-guided fine needle biopsy yielded a positive culture for methicillin-sensitive Enterococcus faecalis, methicillin-sensitive S. aureus, methicillin-resistant S. epidermidis, and Candida parapsilosis were documented on intraoperative and drainage cultures, 3) peripheral blood cultures were positive for C. parapsilosis, 4) a subclavian catheter was removed and another central venous catheter was inserted into a different site, and 5) culture of the subclavian catheter tip was also positive for C. parapsilosis. The minimal inhibitory concentrations (MICs) for the C. parapsilosis isolate were as follows: flucytosine, 0.125 μg/mL; amphotericin B, 0.25 μg/mL; ketoconazole, 0.25 μg/mL; fluconazole, 4 μg/mL; and voriconazole, 0.06 μg/mL. On the 5th postoperative day, meropenem was discontinued and intravenous fluconazole, 400 mg/12 h, was initiated. The patient's clinical condition improved. Transthoracic echocardiograms performed 24 hours and 14 days after the onset of treatment with fluconazole were unrevealing. Eye funduscopy was repeatedly negative.The clinical course was complicated by a new episode of abdominal pain and a second operation to drain large fluid collections of the retroperitoneal space was performed. Events during the immediate postoperative period were as follows: 1) treatment with meropenem and teicoplanin was started but fever reappeared, 2) ampicillin-sensitive C. parapsilosis. Antifungal treatment with intravenous caspofungin was started (a 70-mg loading dose and then 50 mg daily). A transesophageal echocardiogram showed a mobile, echodense, filamentous image (35 × 10 mm) near the basal part of the interventricular septum, protruding into the left ventricular outflow tract . Heart surgery was not indicated due to the lack of valvular involvement and the patient's poor clinical condition related to severe pancreatitis, previous abdominal operations, renal dysfunction, and hemodynamic instability. The central venous catheter was replaced and culture of the tip was negative. However, 48 hours after the onset of treatment with caspofungin, serial blood cultures continued to yield C. parapsilosis. The serum creatinine clearance was 40 mL/min  and because of the persistence of fungemia, 72 hours after starting caspofungin therapy, intravenous voriconazole at a dosage of 6 mg/kg/12 h followed by 4 mg/kg/12 h was added. Remarkably, the patient's clinical condition improved and fever subsided. Three days after the combined administration of caspofungin and voriconazole, a negative blood culture was obtained for the first time. Subsequent blood cultures were consistently negative. Serial follow-up transesophageal and transthoracic echocardiograms showed complete resolution of the fungal vegetation without significant aortic valve regurgitation. Treatment with caspofungin was maintained for 60 days. The patient was discharged on oral voriconazole (400 mg/day), which was given for 9 additional months. Oral voriconazole was well tolerated. In November 2005, 16 months after discharge from the hospital, she was asymptomatic without any evidence of infection.On the 17th postoperative day, the temperature rose to >38.5°C, the serum C reactive protein concentration increased (>200 mg/L), and peripheral blood cultures grew C. parapsilosis mural endocarditis was cured by combined treatment with caspofungin and voriconazole. The outcomes for patients with fungal mural endocarditis have been extremely poor [Candida spp. previously reported in the literature, 6 patients died after treatment with amphotericin B [Candida albicans in 2 cases, and C. parapsilosis, Candida krusei and Candida tropicalis in one case each; in 2 patients, the species of Candida was not identified. Prolonged use of central venous catheter was probably the implicated cause of hematogenous candidiasis and seeding of the endocardium. Intracardiac catheters can produce endothelial damage and predispose usually right-sided endocarditis. The mechanism of direct traumatization of the endocardium by the catheter makes central venous catheter-induced endocarditis bear pathophysiologic similarity to the experimental model of endocarditis in animals [C. parapsilosis are more frequently the cause of catheter-related infections than all other Candida species combined [C. parapsilosis as a nosocomial pathogen associated with invasive devices and parenteral nutrition has been emphasized [C. parapsilosis that may relate to its increasing occurrence in nosocomial settings include frequent colonization of the subungual space and an ability to proliferate in glucose-containing solutions, with a resultant increase in adherence to synthetic materials.This patient with ely poor . In the ericin B -10 and tericin B . These p animals . In our combined . On the phasized . CharactC. parapsilosis and C. guilliermondii, although the clinical significance is unclear [Candida endocarditis treated with caspofungin have been reported [Our patient was not medically fit for heart surgery and was successfully treated with 8 weeks of intravenous caspofungin, although voriconazole was added due to persistence of positive blood cultures. Caspofungin has MICs somewhat higher for  unclear . This mireported ,17, fourreported .C. parapsilosis, voriconazole was added without waiting for a possible resolution of candidemia. Voriconazole is a new triazole antifungal, while caspofungin is the first echinocandin antifungal. Voriconazole acts by inhibiting the synthesis of ergosterol in the fungal cell membrane. Caspofungin inhibits beta-1,3-D-glucan synthesis in the cell wall, a target present in fungal cells, and has shown excellent activity against Candida biofilm [Aspergillus and Candida infections. The efficacy of the antifungal combination therapy with caspofungin and voriconazole has been documented in a few case reports of invasive aspergillosis in patients with hematologic malignancies [Candida endophthalmitis [C. parapsilosis mural endocarditis with caspofungin and voriconazole. Although relapsing endocarditis seems unlikely, the patient has been followed for less than 2 years and late relapse is still possible.In our patient, combination therapy with caspofungin and voriconazole was an alternative to caspofungin monotherapy because of the persistence of positive blood cultures and the presence of renal insufficiency. On the other hand, continued fungemia at 3 days after the onset of caspofungin therapy cannot be considered that caspofungin was a failure, but given the known effect of grater MIC for  biofilm  as oppos biofilm ,23. Bothgnancies ,25 and ihalmitis . To our C. parapsilosis cured with a combination of caspofungin and voriconazole. The combination of these potent antifungal agents opens new perspectives in the management of severely ill patients with invasive fungal infection.In this paper, we firstly reported a non-operated patient with mural endocarditis by CT: computed tomographyMIC: minimal inhibitory concentrationSPSS: Statistical Package for the Social SciencesThe author(s) declare that they have no competing interests.  C.S-R. is an employee of Merck Sharp &  Dohme de España, S.A.V. López-Ciudad, M.J. Castro-Orjales, M.J. de la Torre-Fernábdez, A. Pérez de Juan-Romero, M.D. Collell-Llach, and M.D. Díaz-López participated actively in the diagnosis, care, and follow-up of the patient.V. López-Ciudad and M.J. Castro-Orjales obtained consent from the patient for this care report, reviewed the literature, provided clinical details, and drafted the manuscript.V. León, reviewed the literature and drafted the manuscript.C. Sanz-Rodríguez, reviewed the literature and gave helpful comments regarding the scientific content of the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Arthropod-borne viruses (arboviruses) transmitted by mosquito vectors cause many important emerging or resurging infectious diseases in humans including dengue, chikungunya and Zika. Understanding the co-evolutionary processes among viruses and vectors is essential for the development of novel transmission-blocking strategies. Episomal viral DNA fragments are produced from arboviral RNA upon infection of mosquito cells and adults. Additionally, sequences from insect-specific viruses and arboviruses have been found integrated into mosquito genomes.Aedes aegypti and Ae. albopictus, in which they are approximately ~10-fold more abundant than in other mosquito species analysed. Additionally, viral integrations are enriched in piRNA clusters of both the Ae. aegypti and Ae. albopictus genomes and, accordingly, they express piRNAs, but not siRNAs.We used a bioinformatic approach to analyse the presence, abundance, distribution, and transcriptional activity of integrations from 425 non-retroviral viruses, including 133 arboviruses, across the presently available 22 mosquito genome sequences. Large differences in abundance and types of viral integrations were observed in mosquito species from the same region. Viral integrations are unexpectedly abundant in the arboviral vector species Ae. aegypti and Ae. albopictus, and represent a thus far underappreciated component of their genomes. Additionally, the genome locations of viral integrations and their production of piRNAs indicate a functional link between viral integrations and the piRNA pathway. These results greatly expand the breadth and complexity of small RNA-mediated regulation and suggest a role for viral integrations in antiviral defense in these two mosquito species.Differences in the number of viral integrations in the genomes of mosquito species from the same geographic area support the conclusion that integrations of viral sequences is not dependent on viral exposure, but that lineage-specific interactions exist. Viral integrations are abundant in The online version of this article (doi:10.1186/s12864-017-3903-3) contains supplementary material, which is available to authorized users. Culicidae family are the most serious vectors in terms of their worldwide geographic distribution and the public health impact of the pathogens they transmit. The Culicidae is a large family whose members separated between 180 to 257 million years ago (mya) into the Culicinae and Anophelinae subfamilies ), viruses of the Venezuelan equine encephalitis [VEE] and eastern equine encephalitis [EEE] complexes), Orthobunyavirus , Phlebovirus  and Orbivirus (e.g. Orungo Virus) [Culex pipiens complex, such as Cx. pipens pipiens and Cx. quinquefasciatus, are the most prominent Culex vectors because of their wide distribution and close association with humans [Anophelinae is the alphavirus O’nyong-nyong [Nearly one-quarter of emerging or resurging infectious diseases in humans are vector-borne . Hematop viruses . Collectn vector , 5. Thesng-nyong . Recentlng-nyong –11.Drosophila melanogaster and Aedes mosquitoes, have shown that RNA interference (RNAi) is the main antiviral mechanism in insects [Aedes mosquitoes [D. melanogaster [Ae. aegypti, virus-derived sequences [Mosquito competence for arboviruses is a complex and evolving phenotype because it depends on the interaction of genetic factors from both mutation-prone RNA viruses and mosquito vectors with environmental variables –15. Not  insects –20. In t insects , 19. Whisquitoes . Althougnogaster . These pequences .D. melanogaster, Culex tarsalis, Ae. aegypti, and Ae. albopictus, as well as in adult mosquitoes. The episomal vDNA forms produced by this mechanism reside in the nucleus and it has been proposed that they contribute to the establishment of persistent infections through the RNAi machinery [Culicinae, they also suggest the functional involvement of RNAi. If vDNAs fragments from arboviruses integrate into host genomes, the NIRVS landscape should be different across mosquito species depending both on their viral exposure and the activity of their RNAi pathways. To test this hypothesis, we used a bioinformatics approach to analyse the presence, abundance, distribution, and transcriptional activity of NIRVS across the currently available 22 mosquito genome sequences. We probed these genomes for integrations from 425 non-retroviral viruses, including 133 arboviruses. We observed a ten-fold difference in the number of NIRVS between Aedes and the other tested mosquitoes. NIRVS were not evenly distributed across Aedes genomes, but occurred preferentially in piRNA clusters and, accordingly, they produced piRNAs. Among the viral species tested, integrations had the highest similarities to rhabdoviruses, flaviviruses and bunyaviruses, viruses that share the same evolutionary origin [Recent studies have shown that the genomes of some eukaryotic species, including mosquitoes, carry integrations from non-retroviral RNA viruses –32. Viraachinery , 36, 37.y origin .Ae. aegypti and Ae. albopictus, their genome locations and their production of piRNAs show that in these species genomic integration of viral sequences is a more pervasive process than previously thought. Based on the interplay between viral integrations and the piRNA pathway, we propose that viral integrations represent heritable immune signals and contribute to shape vector competence.The larger number of NIRVS identified in Culicinae species, along with the genome of D. melanogaster, were searched bioinformatically for sequence integrations derived from all 424 non-retroviral RNA viruses for which a complete genome sequence is currently available. Additionally, we tested the genome of African swine fever virus, the only known DNA arbovirus [Ae. albopictus Foshan mosquitoes were sequenced to further validate NIRVS in this species. Retrieved sequences longer than 100 base pairs (bp) were filtered based on gene ontology and the presence of partial or complete open reading frames (ORFs) of viral proteins. This stringent pipeline led to the characterization of a total of 242 loci harboring NIRVS across the genome of 15 mosquitoes  had similarities to genes encoding Nucleoprotein (N), Glycoprotein (G) and the RNA-dependent RNA polymerase (L), the relative abundance of which differed across mosquito species. We did not detect integrations corresponding to the matrix (M) or phosphoprotein (P) genes, consistent with observations in other arthropods [Culicinae and Anophelinae formed separate clades in phylogenetic trees, supporting the conclusion that independent integrations occurred in the two mosquito lineages  with similarities to structural genes  were less frequent than integrations corresponding to non-structural genes . Thus, we cannot determine whether these identical NIRVS represent recent independent integration events or arose from duplication or ectopic recombination after integration.Twenty-five genome assemblies from 22 rbovirus , giving ged Fig. . A total viruses , 40 and ily Fig. ,b. Integto virus   in both Ae. aegypti and Ae. albopictus , which replicate exclusively in arthropods, but are phylogenetically-related to arboviruses , 42 Fig. HoweverAe. aegypti and Ae. albopictus, the mosquitoes with the largest number of identified NIRVS. Previously, uncharacterized viral sequences were identified as piRNA producing loci in Ae. aegypti [Ae. aegypti genome and 0.61% of Ae. albopictus genome [Ae. aegypti was driven by two regions that harbored one fourth of all NIRVS loci and span three piRNA clusters  and 11% (n = 6) had at least 10 piRNA reads per million genome-mapped reads in Ae. aegypti and Ae. albopictus, respectively. In Ae. aegypti, the highest piRNA counts were a few hundred reads per million genome-mapped reads. In Ae. albopictus, the maximum piRNA counts per NIRVS were about 10 fold lower, suggesting that NIRVS piRNA are less efficiently produced or retained in this species. In both mosquito species, R-NIRVS showed higher piRNA coverage than F-NIRVS  derived from R-NIRVS  was found embedded within a full-length TE of the Pao-Bel family.piRNA clusters in y of TEs , 37. Moride Fig. . While LFlavivirus-like sequence characterized in mosquitoes and includes a complete ORF for NS3 [Ae. albopictus genomes  identified, suggesting low abundance of mosquito-specific viruses among Togaviridae [Bunyaviridae, Reoviridae, Rhabdoviridae and Flaviviridae in which many ISVs have been identified [Aedes, the distribution of piRNAs across the viral genome is not comparable between these genera, suggesting that piRNA biogenesis might be different [Across all 425 viral species tested, viral integrations had similarities primarily to ISVs of the families iviruses . For insaviridae , unlike ifferent . Both alifferent , 36, 37.Bunyaviridae, Flaviviridae and Rhabdoviridae are ancient and diversified within their hosts, and they seem to be maintained in mosquito populations through transovarial transmission [ISVs of the families smission , 42. Addsmission , for whismission . VerticaBunyaviridae and Rhabdoviridae have been identified in insects other than mosquitoes, including different Drosophila species and the tick Ixodes scapularis [Ae. aegypti and Ae. albopictus [Anophelinae mosquitoes analysed here were sampled in the same geographic area as Ae. albopictus, but showed ten times fewer NIRVS than Ae. albopictus and Ae. aegypti. Overall, these data indicate that viral exposure is not a determinant of NIRVS, but that virus-host lineage-specific interactions play a crucial role in how their genomes co-evolve. Additionally, our comparative analysis shows that Aedes mosquitoes acquire and retain fragments of infecting non-retroviral RNA viruses of primarily the Flaviviridae and Rhabdoviridae families, more frequently than other tested arthropods and vertebrates. A deeper understanding of the evolution of viruses within these large and diverse families along with insights into the variability of the genomes of mosquito populations is warranted to elucidate the dynamic species-specific interactions between RNA viruses and Aedes mosquitoes.NIRVS from apularis –28. In cbopictus , 32. Intbopictus –27. FinaAe. aegypti and Ae. albopictus, which could be the result of positive selection favoring the retention of those NIRVS that integrated by chance in these genomic loci [Flamenco) in Drosophila [Flamenco PTS in neither Ae. aegypti nor Ae. albopictus. It remains to be established whether other PTS sequences exist that may explain piRNA production from non-cluster associated NIRVS.NIRVS are significantly enriched in piRNA clusters in both mic loci . Howevermic loci , 44 or tosophila . We analAe. aegypti and Ae. albopictus genomes [Ae. aegypti. Despite many remaining uncertainties due to the highly repetitive and complex structure of the regions in which NIRVS map, these data confirm a functional link among NIRVS, TEs, and the piRNA pathway.Analyses of the integration sites showed that NIRVS are primarily associated with LTR transposons of the Gypsy and Pao families, which are the most abundant TE families in both the  genomes . Additio genomes , 37. Thi genomes . EctopicAe. aegypti and Ae. albopictus do not encode proteins that interfere in trans with viral proteins as was observed for bornavirus-derived NIRVS in vertebrates [Ae. albopictus Foshan strain has been reared under standard laboratory conditions without infection challenges for more than 30 years [Our data indicate that NIRVS in tebrates . Rather 30 years . HoweverAe. aegypti and Ae. albopictus have a high number of NIRVS in their genome, which confers heritable immune signals.Our results show a basal expression of NIRVS-derived primary piRNAs that are antisense to viral mRNA. These piRNAs could block novel infections with cognate viruses or they could interact with RNAi mechanisms to contain replication of incoming viruses at a level that does not become detrimental to mosquitoes. Albeit leading to opposite effects on vector competence, both mechanisms display functional similarities to the CRISPR-Cas system of prokaryotic adaptive immunity. Even if further studies are essential to clarify the effect of NIRVS-derived piRNAs on mosquito immunity, our study clearly demonstrates that Aedine than in Anopheline mosquitoes correlates with competence for a larger number of arboviruses of Aedine mosquitoes. In this regard, Cx. quinquefasciatus shows an interesting intermediate phenotype because it is phylogenetically closer to Aedine mosquitoes, but vectors a smaller range of arboviruses, and, like Anophelinae, it can vector more protozoans and nematodes than Aedine [Cx. quinquefasciatus has a number of NIRVS and TE load comparable to Anopheline, but an expanded PIWI gene family like Ae. aegypti [The higher number of NIRVS in n Aedine . Additio aegypti , 55, 56.Ae. aegypti and Ae. albopictus and the observation that NIRVS produce piRNAs and reside in piRNA clusters support the intriguing hypothesis that the formation and maintenance of NIRVS is coupled with the evolution of the PIWI pathway in these two species. This may have led to functional specialization of the expanded PIWI gene family, PIWI expression in the soma, and a role for the piRNA pathway in antiviral immunity [Aedine mosquitoes than in Culex and Anophelinae because of the higher abundance of retrotransposons in the genome of Aedine mosquitoes [in trans with virus replication, thereby affecting vector competence [NIRVS are regarded as viral fossils, occurring as occasional events during the long co-evolutionary history of viruses and their hosts , 35. Theimmunity , 45. Thisquitoes . NIRVS tmpetence . Second,mpetence . Under tD. melanogaster and 22 currently available mosquito species were screened in silico using tBLASTx and a library consisting of genome sequences of 424 non-retroviral RNA viruses and one DNA arbovirus  and protozoan vectors , depending on whether they most efficiently transmit arboviruses or protozoans to humans, respectively  to viral queries were extracted from the respective insect genomes using custom scripts. When several queries mapped to the same genomic region, only the query with the highest score was retained. Blast hits were considered different when they mapped to genomic positions at least 100 bp apart from each other, otherwise they were included in the same NIRVS locus.2 [All putative viral integrations were subjected to a three-step filtering process before being retained for further analyses to reduce the chance of false positives and ensure that the identified sequences are from non-retroviral RNA viruses . Filteri2 .Ae. albopictus and Ae. aegypti [While our search expanded the range of viral integrations identified in  aegypti , 26, 28,Ae. albopictus Foshan strain were sequenced. The strain was received from Dr. Chen of the Southern Medical University of Guangzhou (China) in 2013. Since 2013, the Foshan strain has been reared in an insectary of the University of Pavia at 70–80% relative humidity, 28 °C and with a 12–12 h light–dark photoperiod. Larvae are fed on finely-ground fish food . A membrane feeding apparatus and commercially available mutton blood is used for blood-feeding females.The genomes of 16 individual mosquitoes of the Ae. albopictus Foshan reference genome  using the Burrows-Wheeler Aligner (BWA) [Ae. albopictus NIRVS using BEDTools [DNA was extracted from single mosquitoes using the DNeasy Blood and Tissue Kit  following the manufacturer’s protocol. DNA was shipped to the Polo D’Innovazione Genomica, Genetica e Biologia  for quality control, DNA-seq library preparation and sequencing on an Illumina HiSeq 2500. After quality control, retrieved sequences were aligned to the er (BWA)  and markBEDTools . AlignmeBEDTools .Flavivirus, Rhabdovirus, Reovirus and Bunyavirus genomes using MUSCLE. Maximum likelihood (ML) phylogenies were estimated in MEGA6 [http://tree.bio.ed.ac.uk/software/figtree/).Deduced NIRVS protein sequences were aligned with subsets of corresponding proteins from in MEGA6 , implemeP < 0.05 suggests a statistically significant enrichment of integration events in the corresponding genomic region  using A. aegypti TEs retrieved from TEfam (http://tefam.biochem.vt.edu/tefam/), which was manually annotated. Percent TE occupancy (percent of bases in the genomic sequence that match TEs) was used as a measure for possible enrichment of certain TEs. TE copy numbers were not used because it is likely that some TEs are broken into multiple fragments and counted multiple times. We retrieved sequences of the viral integration sites plus 5 kb sequences flanking each side of the integration for the analysis. In addition, to identify potentially full-length TEs, 10 kb sequences flanking each side of the viral integration were analysed by RepeatMasker (version open-4.0.3). The presence of full-length TEs was verified by comparing the length of masked sequences with the length of the annotated TEs.Analysis of TE enrichment in all NIRVS sites and region 1 and region 2 of Ae. aegypti  [Ae. albopictus mosquitoes  [Reovirus NIRVS and normalized to the corresponding library size. The size of the ring-graph was scaled to reflect the normalized read counts. piRNA counts on individual NIRVS was also determined from acetone treated female Ae. aegypti, male Ae. aegypti, blood-fed Ae. albopictus and male Ae. albopictus mosquitoes. The data were obtained from the same studies as described above.Small RNA deep-sequencing data of female X397102) , Ae. albX201600) , and PIWX201600)  were dowX201600) . After rX201600) . From thX201600) . piRNA cTo identify the PIWI dependency of NIRVS-derived piRNAs, we analysed libraries from Aag2 cells transfected with double stranded RNA (dsRNA) targeting the somatic PIWI genes  and a non-targeting control  . These dAe. albopictus and Ae. aegypti mosquitoes, including both DENV-infected and non-infected mosquitoes were downloaded from NCBI Sequence Read Archive. Libraries analysed correspond to data SRA438038 for Ae. albopictus, and SRA058076, SRX253218, SRX253219 and SRX253220 for Ae. aegypti. RNA-seq reads were mapped using BWA [RNA deep-sequencing data of sing BWA  to NIRVSAe. albopictus developmental stages, total RNA was extracted using Trizol reagent (Life Technologies) from 3 pools of 5 entities for each condition . From each pool, a total of 100 ng of RNA was used for reverse transcription using the qScript cDNA SuperMix following the manufacturer’s protocol (Quanta Biosciences). AlbFlavi34 expression was quantified in a 20 μL final reaction volume containing 10 μL of QuantiNova SYBR Green PCR Master Mix (Qiagen), 700 nM of each forward (5’-CTTGCGACCCATGGTCTTCT-3’) and reverse (5’-GTCCTCGGCGCTGAATCATA-3’) primers and 5.0 μL cDNA sample on an Eppendorf RealPlex Real-Time PCR Detection System (Eppendorf). We used a two-step amplification protocol consisting of 40 cycles of amplification  after an initial denaturation of 2 min at 95 °C. AlbFlavi34 expression values were normalized to mRNA abundance levels of the Ae. albopictus Ribosomal Protein L34 (RPL34) gene [Flavivirus infection was verified using a published protocol [To analyze AlbFlavi34 expression in different protocol  on all sAdditional file 1: Table S1.List of Dipteran species analysed. (XLSX 34 kb)Additional file 2: Table S2.List of viral genomes used for in silico screening of Dipteran genomes. The only DNA arbovirus is in yellow. (XLSX 38 kb)Additional file 3: Table S3.Description of all identified NIRVS (XLSX 132 kb)Additional file 4: Table S4.Ae. albopictus mosquitoes (Foshan strain). (DOCX 20 kb)DNA-seq library statistics. Total number of reads, percentage of mapped reads and percentage of properly paired reads from DNA-seq of 16 individual Additional file 5: Table S5.Ae. aegypti and Ae. albopictus genomes. Classification was based on their sequence identity. Integrations with sequence identity >90% were grouped together. (DOCX 20 kb)Viral integrations (NIRVS) in the Additional file 6:Details of AlbFlavi34. (DOCX 17 kb)Additional file 7: Table S6.(A) piRNA coverage at NIRVS loci. (B) NIRVS transcriptional activity. (XLSX 90 kb)Additional file 8: Figure S1.Ae. albopictus. (PDF 102 kb)qRT-PCR based expression of AlbFlavi34 in different developmental stages of