The continuing emergence of antibiotic-resistant pathogens is a concern to human health and highlights the urgent need for the development of alternative therapeutic strategies. Quorum sensing (QS) regulates virulence in many bacterial pathogens, and thus, is a promising target for antivirulence therapy which may inhibit virulence instead of cell growth and division. This means that there is little selective pressure for the evolution of resistance. Many natural quorum quenching (QQ) agents have been identified. Moreover, it has been shown that many microorganisms are capable of producing small molecular QS inhibitors and/or macromolecular QQ enzymes, which could be regarded as a strategy for bacteria to gain benefits in competitive environments. More than 30 species of marine QQ bacteria have been identified thus far, but only a few of them have been intensively studied. Recent studies indicate that an enormous number of QQ microorganisms are undiscovered in the highly diverse marine environments, and these marine microorganism-derived QQ agents may be valuable resources for antivirulence therapy. Antibiotics are recognized as effective antimicrobial agents for curing diseases caused by pathogenic bacteria. Traditional antibiotics are bactericidal or bacteriostatic by targeting essential processes for bacterial growth including cell wall synthesis, DNA replication, RNA transcription and protein synthesis . However Pseudomonas aeruginosa, Erwinia carotovora, Vibrio spp. and Burkholderia spp. [Quorum sensing (QS) is a process for bacteria to communicate, regulate gene expression and synchronize social behaviors, such as biofilm formation, bioluminescence and secretion of virulence factors ,3. Hitheria spp. . In addiria spp. .i.e., small molecular QS inhibitors and macromolecular QQ enzymes, are discussed. The current status and trends of antivirulence therapy utilizing QQ resources from the marine environment are considered. Based on this knowledge, a critical appraisal and perspective of the resource for antivirulence in the environment is presented.Here, we provide an overall summary of the uniformity and diversity of QS in microorganisms, as well as the QS circuits in some representative species. Two main QQ agents, i.e., V. fischeri and V. harveyi, use signaling molecules  as sensors of cell density so that the population as a whole may coordinate the social behavior of bioluminescence [et al. [The term QS was first proposed to describe the phenomenon that marine bacteria, nescence . Howevernescence ,6,7,8,9.nescence  proposednescence ,11, whicnescence . In addinescence . However [et al.  argued t [et al. .Despite the presence of distinct QS systems in different microorganisms, the fundamental processes are similar. Initially, AIs are synthesized by AI synthases and diffuse away. When the concentrations of AIs increase to a threshold, AIs are detected by receptors. Subsequently, the AI-bound receptors activate the expression of relevant genes, including AI synthase-encoding genes. This results in a positive feed-back loop for biosynthesis of AIs, which may be able to promote the synchrony among a population.N-acylhomoserine lactones (AHLs) and autoinducing peptides (AIPs) are mainly used by Gram-negative and Gram-positive bacteria for intraspecies communication, respectively. Autoinducter-2 (AI-2) signals are hypothesized to be used for interspecies communication because AI-2 production and the synthase LuxS homologues are widespread among Gram-negative and Gram-positive bacteria [Exiguobacterium sp. MPO, as a QS signaling molecule to regulate biofilm formation and extracellular polymeric substance production [Thermotoga maritima [Cryptococcus neoformans [V. cholerae autoinducer-1 (CAI-1) family and other particular signals, such as Pseudomonas quinolone signal (PQS), integrating QS signal (IQS) and pyrone signal are also employed by some Gram-negative bacteria  [V. fischeri possesses a multichannel system (C8-HSL- and AI-2-dependent channels) and an additionally LuxI/R circuit (3OC6-HSL) [P. aeruginosa employs various QS systems, including two complete AHL-dependent QS circuits (RhlI/R and LasI/R circuits), a 3OC12-HSL-responsive orphan receptor QscR, a Pseudomonas quinolone signal (PQS)-dependent QS, and a recently identified IQS (integrating the QS network) signal, which together compose a hierarchical QS network to regulate virulence expression and respond to environmental stress [In most cases, one species usually employs a single type of QS. However, some species may harbor multiple QS systems. For example, hannels) ,38, wherOC6-HSL) . P. aerul stress ,39.p-coumaroyl-HSL and cinnamoyl-HSL) [N-carboxyl-acyl-homoserine lactone [AHL-dependent QS exists in many pathogenic bacteria. A typical AHL molecule consists of a homoserine lactone and an acyl chain with an even number of carbons (C4-C18) together with an occasional modification at the C3 position (hydroxy or olefinic double bond) . The shooyl-HSL) ,29, branoyl-HSL)  and N-ca lactone  , LuxS/PQ (AI-2-dependent), CqsA/S (CAI-1-dependent) [R-THMF and S-THMF  or low NO concentration, phosphates from unliganded receptors are transduced to a single phosphotransfer protein LuxU, and subsequently to LuxO. Phospho-LuxO, together with a sigma factor σ54, activates the transcriptions of five small regulatory RNAs. Small RNAs repress the translation of LuxR and activate the translation of AphA [The LuxI/R circuit in marine bioluminescent fischeri a and theilar way b. V. harpendent) . In AHL-d S-THMF . R-THMF directly , whereas by CqsA . The cog by CqsA . LuxR an of AphA ,44. At h of AphA . The two of AphA .S-adenosylmethionine (SAM) and the recycling of methionine by detoxification of S-adenosyl-L-homocysteine (SAH) (S-ribosylhomocysteine (SRH) [Intriguingly, the biosynthesis pathways of AHLs, AI-2 and CAI-1 are related to each other and involved in the AMC, which is an important metabolic pathway responsible for the generation of the cell major methyl donor ne (SAH) c. AHL isne (SRH) . LuxS cane (SRH) . Unlike All of the discovered QS circuits share three critical steps: (1) production of signal molecules,(2) release of signal molecules, and(3) recognition of signal molecules by receptors.Pantoea stewartii functions as a transcriptional repressor. Unliganded EsaR binds DNA and represses transcription. The binding of AI to EsaR promotes DNA release and thereby promotes gene expression [In most cases, the LuxR-type transcriptional regulators are able to activate target genes, whereas the EsaR in pression . Also, Qpression .i.e., small molecular and macromolecular QQ agents, which are also referred to as QS inhibitors and QQ enzymes, respectively.Quorum quenching is now referred to as the interference with QS, and may reverse the QS regulated phenotypes, such as virulence. Because QS is not essential for the growth of bacteria, quenching QS has been recommended as a promising strategy for antivirulence therapy. In particular, diverse QQ agents have been identified from various sources and organisms. All of the QQ agents may be classified into two groups according to their molecular weight, The words “quorum quenching” and “QS inhibitors” were first used to describe the enzymatic degradation of AHL signals  and inhilacZ, gfp or luxCDABEG) in response to specific QS signals, are potent and indispensable tools for identifying QS inhibitors.There is abundant literature about the identification of novel and effective QS inhibitors from natural products and synthetic molecules. Multiple methods have been applied to identify QS inhibitors: purification from crude extracts of candidate organisms, random and high-throughput screening of QS inhibitors from compound libraries, and computer-aided screening from 3D structure libraries of known compounds. However, QS biosensors, which are genetically manufactured strains that express reporter genes  [Lyngbya majuscula produces four different compounds, including malyngolide (MAL), 8-epi-malyngamide C, lyngbic acid and lyngbyoic acid, which inhibit LasR of P. aeruginosa responding to exogenous 3OC12-HSL [Leptolyngbya crossbyana, possess dual-inhibitory activity against both QS and inflammation, which may represent a new strategy for developing multi-functional drugs. It is noteworthy that marine cyanobacteria produce many different kinds of AHL-dependent QS inhibitors, and most likely have evolved mechanisms to control the associated microbial communities by interfering with their cell-to-cell communication.An increasing number of studies indicate that marine cyanobacteria have become one of the richest resources for biologically active and structurally unique natural products. Many AHL-dependent QS inhibitors with divergent structures have been discovered in several species of marine cyanobacteria . The tumf 62 μM) . MoreoveOC12-HSL . InteresOC12-HSL , isolateHalobacillus salinus, produces two compounds, namely N-(2-phenylethyl)-isobutyramide and 3-methyl-N-(2-phenylethyl)-butyramide, which are capable of inhibiting violacein biosynthesis of Chromobacterium violaceum CV026 and GFP production of Escherichia coli JB525 in the presence of exogenous AHLs [Bacillus cereus and Gram-negative Marinobacter sp. SK-3 produce diketopiperazines (DKPs) which inhibit AHL-dependent QS [C. violaceum CV026 [Other marine microorganisms also produce QS inhibitors . The Graous AHLs . Both Grndent QS ,61. Addium CV026 ,108.Delisea pulchra produces a number of halogenated furanones with antifouling and antimicrobial properties [V. fischeri [V. harveyi [Vibrio species [P. aeruginosa lung infection [Ahnfeltiopsis flabelliformes, may also inhibit the reporter strain A. tumefaciens NTL4 (pCF218) (pCF372) responding to 3OC6-HSL [E. coli JB523 and V. harveyi JMH612 [Another source for QS inhibitors is marine algae. The red alga operties ,109. Furoperties ,110. It fischeri  and atte harveyi . Moreove harveyi . The pro species ,115,116 nfection  have bee3OC6-HSL . In addii JMH612 .Combretum albiflorum bark, is the first identified flavonoid compound and is capable of reducing the production of virulence factors in P. aeruginosa PAO1 by interfering with RhlR [C. violaceum and P. aeruginosa, respectively, which may be attributed to the high abundance of flavonoids in honey and propolis [C. violaceum or P. aeruginosa [Various natural products from a wide range of terrestrial organisms have been demonstrated to show inhibitory activity against AHL-dependent QS. Most of these bioactive substances are derived from plants, although some originate from bacteria, fungi and insects . Many heith RhlR . Severalith RhlR ,106,120.propolis . Hydrolyruginosa ,91.agr-dependent QS of Staphylococcus spp. [C. albicans as a QS signal, inhibits the PQS circuit of P. aeruginosa by promoting non-productive interaction between PqsR and the pqsA promoter [Drimys winteri and sesquiterpene lactones from Centratherum punctatum (Argentine herb), were identified as AHL-dependent QS inhibitors [V. harveyi [V. harveyi. CA and its analogs could increase significantly the survival of the nematode Caenorhabditis elegans, brine shrimp and giant freshwater prawn, Macrobrachium rosenbergii, infected with pathogenic Vibrio spp. [Compared with the plentiful natural AHL-dependent QS inhibitors, natural inhibitors against other QS systems are rarely reported . Peptidecus spp. ,106,107.promoter . It was promoter . Besideshibitors ,87. Refehibitors . Low con harveyi . Subsequrio spp. ,102,121.D. pulchra furanone compounds are unable to inhibit the QS of P. aeruginosa, but modified furanone analogs enhance the inhibitory effectiveness against this organism [Leptolyngbya was recently identified as an inhibitor against QS of V. harveyi and V. fischeri [Over the last decade, many natural substances have been evaluated for their ability to interfere with QS. QS inhibitors isolated from natural products are excellent resources for developing potent antivirulence drugs insofar as they may provide novel scaffolds for drug design. The natural organism . Honaucifischeri , whereasfischeri . Despiteet al. [V. harveyiluxN mutant without affecting growth in a complex medium and therefore claimed as an AI-2 QS inhibitor [Firstly, there is an increasing requirement for standardization to verify the true QS inhibitory activity. In some cases, the QS inhibitory activities of bioactive substances have been challenged because of the lack of suitable methodologies. Several QS reporter strains are widely used to identify QS inhibitors. Recently, Defoirdt et al.  detailednhibitor . Howevernhibitor . The appnhibitor . Hence, nhibitor .Secondly, further studies on natural crude extracts with identified QS inhibitory activity are required. In many cases, the precise compounds of the bioactive molecules have not been elucidated. As mentioned above, natural inhibitors are resources for drug design to develop more potent antivirulence drugs, and purification of individual compounds is necessary to improve the likelihood of understanding the mechanisms of inhibition .Moreover, the active mechanisms of these compounds are poorly understood. Successful drug design relies much on the knowledge of molecular mechanism of the connection between native signals or inhibitors and synthase or receptor, such as binding sites, conformational change and affinity change. Currently, the X-ray crystal structures of some AHL-bound LuxR-type receptors and computational protein docking methods provide powerful tools to determine the molecular mechanisms of interaction. However, the molecular mechanisms of only a few effective natural QS inhibitors have been studied in depth. The lack of information of binding interactions has thwarted the rational design of a more potent QS inhibitor.Bacillus, Staphylococcus and Pseudomonas possess DSF inactivation activity [Arthrobacter nitroguajacolicus is able to cleave PQS [in vitro, the extracellular phosphor-AI-2 molecules cannot be transported into cells and are degraded overnight. LsrK-mediated degradation of AI-2 attenuates the QS response in S. enterica serovar Typhimurium and V. harveyi [Various macromolecular agents have been found to possess the capability to quench QS. Unlike small molecular QS inhibitors, macromolecular QQ agents interfere with QS mostly through degrading signals rather than competitively binding to signal receptors. Most of the identified macromolecular QQ agents target the AHL-dependent QS, although enzymatic degradations of DSF, PQS and AI-2 have also been reported. activity . The 2,4eave PQS . In the eave PQS . When Ls harveyi .To date, many macromolecular QQ agents against AHL-dependent QS have been reported. Generally QQ enzymes are a major portion of the macromolecular agents, although a few antibodies have been generated to interfere with AHL-dependent QS through sequestration or hydrolyzation of AHLs . EnzymatN-acyl-homoserine. This hydrolyzation may also occur spontaneously at alkaline pH, and may be reversed under acid pH [AHL-degrading enzymes may be classified into three major types according to their enzymatic mechanisms: AHL lactonase (lactone hydrolysis), AHL acylase (amidohydrolysis) and AHL oxidase and reductase (oxidoreduction). AHL lactonase hydrolyzes the ester bond of AHL yielding the corresponding  acid pH . AHL acyi.e., AiiA, AidC and a novel marine AHL lactonase cluster [To date, approximately 30 types of AHL lactonases have been identified (except for predicted AHL lactonases or highly similar enzymes in one genus). According to the amino acid sequences, these lactonases belong to the metallo-β-lactamase superfamily, the phosphotriesterase (PTE) family and other particular families a. Among  cluster   from ide bond . The aiiactivity . AiiA isactivity . Crystal His 235 . These tf Asp191 . Additiof Asp191 ,134. Aiif Asp191 , howeverf Asp191 ,134. Recter ones . Althougter ones . Heterol therapy .240B1, were found in the plant pathogen A. tumefaciens C58, and their corresponding coding genes attm and aiib  located on the pAt plasmid and the pTi plasmid [A. tumefaciens infects a broad range of dicotyledonous plants and may transfer an oncogenic DNA fragment, the T-DNA, from its tumor-inducing plasmid to the nuclear genome of the plant hosts. These processes are regulated by TraI/R QS circuit, lactonases AttM and AiiB [A. tumefaciens infection in wounded plant hosts, the expression of attm is promoted by γ-aminobutyric acid, a molecule synthesized for defense by plants, and thereby the 3OC8-HSL-dependent virulence expression is attenuated. However, in the growing tumor, the high synthesis rate of 3OC8-HSL and the moderated expression of attm caused by plant produced l-proline permit the expression of QS-regulated functions including the transfer of Ti plasmid by conjugation [Two members of AiiA cluster, AttM and AiiB, showing 32% and 28% identity to AiiA plasmid , respect plasmid . A. tumeand AiiB ,141. DurChryseobacterium sp. StRB126 and Sinorhizobium fredii NGR234, respectively, and additionally includes YtnP, another AHL lactonase from Bacillus species [ytnP in B. subtilis may be induced by streptomycin, and YtnP may conversely inhibit the production of streptomycin and the development of aerial mycelium in Streptomyces griseus. B. subtilis may activate ytnP expression in response to the presence of certain antimicrobial compounds as a defensive strategy against threatening bacteria since it would allow B. subtilis to selectively inhibit the QS-regulated behaviors of harmful microbial communities [Muricauda olearia Th120, possesses a gene encoding a novel AHL lactonase which represents a new cluster of AHL lactonase in the metallo-β-lactamase family [The AidC cluster contains AidC and QsdR1 encoded by  species ,143,144.munities , and onee family   for their best substrate paraoxon, which is a widely used pesticide introduced in the 20th century. PTEs could have therefore evolved from a member of the PLL family, utilizing its latent promiscuous paraoxonase activity as an essential starting point [Apart from members of the metallo-β-lactamase family, another group of AHL lactonases shares key sequence and active site features with phosphotriesterases (PTEs), and these lactonases are termed PTE-like lactonases (PLLs). PLLs exist in both bacteria and archaea. Afriat et al.  discovertivities . Crystaltivities . Signifiembranes . The theembranes , as welltophilus  and SisLlandicus , is an alandicus ,148,149.landicus ,148,149.ng point . Additiong point ,151. Theng point . They alng point .Pseudomonas, Ochrobactrum, Arthrobacter, Streptomyces, Nostoc and Brucella  hydrolase superfamily, and shares 22%–24% identities with several cephalosporin and penicillin acylases [Streptomyces sp. which shows 35% identity with AiiD in the deduced amino acid sequence, was capable of degrading penicillin G and long-chain AHLs [V. paradoxus was not identified until whole genomic sequencing of V. paradoxus revealed recently a putative AHL acylase gene (Vapar_3883 of V. paradoxus S110) [aiiD in P. aeruginosa PAO1 weakened its ability to swarm, to produce elastase and pyocyanin, and to paralyse nematodes [P. aeruginosa also reduced the accumulation of AHLs, and decreased the production of virulence factors including elastase, total protease and LasA [AHL acylases have been found in bacteria including Brucella b. The Ai acylase , althougd energy . AiiD beacylases . Actuallain AHLs . The genus S110) . Howeverematodes . The addand LasA .P. aeruginosa PAO1 was previously found to utilize AHLs for growth, so far three proteins  belonging to the Ntn hydrolase have been characterized biochemically [quiP (pa1032) rather than pvdQ that is responsible for the ability of P. aeruginosa PAO1 to utilize AHLs as a sole carbon and energy source for growth [P. aeruginosa PAO1 is intriguing. PvdQ was found to be expressed only when iron is present at very low concentrations [pvdQ did not affect the growth of P. aeruginosa but abrogated pyoverdine production and greatly affected swarming motility and biofilm formation at low iron concentrations [pvdQ mutant against C. elegans was reduced. All of these data indicate that PvdQ plays an essential role in siderophore biosynthesis, on which P. aeruginosa depends for growth in iron-limited environments [emically . Among temically . The matemically . Howeverr growth ,157. Addr growth . The phytrations . Mutatiotrations . Moreoveronments . Thereforonments . NeverthTo date, most of the identified AHL acylases belong to the Ntn hydrolase superfamily, except QsdB and AiiO which belong to the amidase family and α/β hydrolase fold family, respectively ,162. AHLRhodococcus erythropolis in which AHLs with 3-oxo substituents were rapidly degraded by reduction of the keto group at the β position, yielding the corresponding 3-hydroxy derivative AHLs [Bacillus megaterium, a widely studied cytochrome P450, is the second identified AHL oxidoreductases that oxidizes AHL at the ω-1, ω-2, and ω-3 carbons of the acyl chain [bpiB09 in P. aeruginosa reduced its swimming motilities, pyocyanin production, biofilm formation and thereby the pathogenicity to C. elegans [Burkholderia sp. GG4, isolated from ginger rhizosphere, was previously found to possess a unique AHL-modifying activity that reduces 3-oxo-AHLs to 3-hydroxy-AHLs [D. pulchra [Laminaria digitata [Nitzschia cf pellucida [2O2-dependent mechanism.Compared to the abundant data of AHL lactonases and acylases, there are fewer reports about inactivation of AHLs by the modification of chemical structure of AHLs; only a few AHL oxidoreductases have been identified thus far. Because signal receptors usually respond to specific AHLs, the modification might affect the signal recognition, and thereby interfere with QS-regulated functions. Bacteria-derived AHL reduction activity was first discovered in ive AHLs . Howeveryl chain . Furtheryl chain . The thi elegans . Moreoveoxy-AHLs  althoughoxy-AHLs . AHL can pulchra , Laminardigitata  and Nitzellucida  via a H2Microorganisms exist in a multi-species and competitive environment, and have developed many survival strategies to gain benefits and compete for space, nutrition and ecological niches. QS is possibly one critical strategy used for competition by microorganisms to synchronize and coordinate social behaviors. Many of these behaviors  are primarily advantageous when expressed by a group of bacteria but seemingly futile if performed by a single bacterium , though P. aeruginosa, is toxic to S. delphini [P. aeruginosa via suppressing the production of pyocyanin [C. albicans [C. albicans blocks the PQS circuit and thus the pyocyanin biosynthesis of P. aeruginosa [P. aeruginosa virulence [The discovery of co-existence of QS and QQ bacteria in various environments might provide supports for this hypothesis ,172,173.delphini  that proalbicans . With thruginosa . Farnesoirulence . It seemC. violaceum ATCC 31532 is C6-HSL but the QS-regulated violacein production can be inhibited by long-chain AHLs produced by C. violaceum ATCC 12472 or other bacteria [S. aureus utilizes four different groups of AIPs. Each can specifically activate its cognate AgrC receptor, but inhibit all others by competitive binding to the non-cognate receptors [The interference of QS exists not only between different species but also between different strains of the same species.In Gram-negative bacteria, the native AHL utilized by bacteria ,177. Graeceptors ,180,181.P. aeruginosa participates in siderophore biosynthesis [A. tumefaciens is for the degradation of γ-butyrolactone rather than regulation of AHL accumulation, and the AHL-degrading activity might be only a side effect [V. paradoxus and Arthrobacter sp. utilize AHLs as a source of nitrogen or/and carbon for growth depending on their AHL-degrading enzymes [B. thuringensis [et al. [B. subtilis was induced by streptomycin, an antibiotic produced by the Streptomyces species. Conversely, YtnP inhibits the production of streptomycin in S. griseus probably by degrading its QS signaling molecule γ-butyrolactone [. The streptomycin-induced expression of YtnP may allow B. subtilis to response to certain antimicrobial compounds and selectively inhibit the QS of harmful microorganisms before being killed. It seems likely that QQ enzyme could allow its producers to obtain competitive advantages over competitors in natural ecosystems.The physiological function of QQ enzyme has been discussed repeatedly but is still unclear ,183,184.ynthesis . The maje effect . V. para enzymes ,186. Aiiingensis . However [et al.  reportedolactone . The strThe hypothesis that microorganisms produce QQ agents in order to gain competitive advantage is not sufficient to explain all of these discoveries. Although several examples showed that the QQ agent producers could survive or even gain benefit through inhibiting QS-regulated harmful behaviors of their competitors, the possibility of “accidental” QQ activity of some QQ agents still exists.et al. [Alphaproteobacteria, Gammaproteobacteria Actinobacteria, Flavobacteriia and Firmicutes have been identified thus far  to known lactonases are below 30%, and the top eight strains (with identity higher than 39%) using BLASTP against the NR protein database are also typical marine bacteria. Therefore, MomL was believed to represent a new class of AHL lactonase, which may be widespread in the marine environment. Likewise, AHL acylase MomA (Muricauda olearia marine AHL acylase) may represent a marine-derived AHL acylase. It is even more astounding that the ethyl acetate extracts of Th120 culture showed inhibitory activity in A. tumefaciens A136 plate assay [The high diversity and abundance of marine QQ bacteria may lead to the discovery of new QQ enzymes and AHL-degrading mechanisms. However, few responsible genes in these bacteria have been identified . One of  acylase  (Figure te assay . All of in vitro and in vivo. However, both have advantages and drawbacks due to their entirely distinct molecular structures and functional mechanisms.The antivirulence activities of small molecular QS inhibitors and QQ enzymes have been demonstrated C. violaceum and LuxN of V. harveyi. However, only mBTL is capable of inhibiting the pyocyanin production of P. aeruginosa PA14 whereas CL and CTL show non-inhibitory activity [QS inhibitors may target one specific signal receptor or some homogenous receptors, such as the LuxR-like family. For example, compounds 4606–4327, CTL, CL and mBTL are synthetic AHL analogs with similar structures . Each isactivity . This maactivity .Compared with QQ enzymes, the structures of QS inhibitors are relatively simple and can be easily modified through synthetic methods. Moreover, small QS inhibitors allow for temporal control of a biological system, and this control is often rapid, depending on the diffusibility of compounds. For example, the low molecular weight of QS inhibitors facilitates their absorption by animals; they may be administered orally or intravenously like other drugs. Furthermore, the nonproteinaceous nature as well as low molecular weight of QS inhibitors can effectively prevent an antibody-based immune response unlike that of QQ enzymes.et al. [Stability is another important issue that should be taken into account for both of these two agents. QQ enzymes could be easily proteolyzed and most of them are sensitive to heat. Likewise, QS inhibitors may be degraded by abiotic or biotic elements. This is of concern especially for some QS inhibitors possessing similar structures with native AHLs. This type of QS inhibitor is suspected to be degraded by AHL-degrading enzymes because AiiA can degrade AHL analogs ,137, andet al.  for the et al. [P. aeruginosa PA14 on minimal medium using adenosine as the sole carbon source and with the simultaneous exposure to the synthetic QS inhibitor furanone C-30 [mexR and nalC genes, both of which encode negative regulators of the MexAB-OprM multidrug resistance efflux pump [mexR and nalC mutations were found in several clinical P. aeruginosa isolates. However, unlike QS inhibitors, application of QQ lactonase would be less likely to induce QQ resistance [The emergence of resistance to QQ compounds has raised doubts about whether QS is an ideal target for antivirulence therapy. Defoirdt et al.  proposedone C-30 . Furtherlux pump . Moreovesistance ,199. Theet al. [The potential for the development of antivirulence drugs has been emphasized for years, and a number of QQ agents have been discovered or synthesized, but none have been marketed. Certainly, the lack of appropriate delivery systems is one challenge. The corresponding QQ agents should be introduced into the hosts against different pathogens by controlling the rate, time, and place of release. The novel biological nanofactories engineered by Fernandes and colleagues would provide a promising specific delivery of QQ agents . Anotheret al.  argued tet al. . Thus, tet al. .in vitro and in vivo. The natural QQ agents, especially those derived from marine microorganisms, are great resources for developing antivirulence therapy. Recently, several studies have revealed a wide spread of QQ activities in marine microorganisms, however, these QQ resources need to be explored more deeply. Therefore, further research on QQ resources and mechanisms would provide more alternatives for developing antivirulence therapy.In summary, the utilization of quorum quenching as a promising strategy of antivirulence therapy has been demonstrated


Bordetella bronchiseptica causes acute and chronic respiratory infection in a variety of animals. Currently, there is no vaccine to prevent these infections. To identify useful candidate antigens for such a vaccine, five B. bronchiseptica genes including amino acid ATP-binding cassette transporter substrate-binding protein (ABC), lipoprotein (PL), outer membrane porin protein (PPP), leu/ile/val-binding protein (BPP), and conserved hypothetical protein (CHP) were cloned and the recombinant proteins were expressed. The immune responses of mice to vaccination with individual recombinant proteins were measured.The Gram-negative pathogen B. bronchiseptica. The protection ratios were 62.5 and 50 %, respectively, compared with 12.5 % for control vaccinations. The protection ratios of ABC, BPP, and CHP were not significantly different from the controls. IgG-subtype and cytokine analysis demonstrated that PPP and PL can induce two immune responses: a humoral immune response and a cell-mediated immune response. The humoral immunity-mediated, Th2-type response dominated.Each of the tested recombinant proteins induced a high antibody titer. PPP and PL showed protective indices against challenges with B. bronchiseptica.The identification of PPP and PL, which offer immune-protective potential, identifies them as candidates for the development of a diagnostic test or a vaccine for  Bordetella bronchiseptica is an important bacterial pathogen that causes a number of respiratory diseases in livestock and poultry. B. bronchiseptica can colonize the host for its entire lifetime, resulting in great economic losses worldwide. Examples of diseases caused by B. bronchiseptica include atrophic rhinitis in swine, kennel cough in dogs, and snuffles in rabbits  compared with PBS alone [protection ratio of 12.5 % (1/8); Fig. B. bronchiseptica was recovered from all the mice that died. For the acquired immune-protective rPPP and rPL, further tests were conducted to determine antibody subtypes. Mice immunized with rPPP produced high titers of specific IgG1 and IgG2a antibodies. The specific IgG1 titer was higher than the specific IgG2a titer (IgG1:IgG2a = 1.38)  Fig. . The mic38) Fig. . ExaminaLymphocyte proliferation assays showed that rPPP and rPL effectively stimulated lymphocyte proliferation Table . Levels B. bronchiseptica is an important pathogen responsible for infectious rhinitis in rabbits, as well as diseases in other animals. The protective effectiveness of the B. bronchiseptica vaccine currently used is not satisfactory. Therefore, the identification of antigenic proteins for a new vaccine is urgently needed. Subunit vaccines are attractive and useful tools for the development of protective immunity against pathogenic microorganisms [B. bronchiseptica outer membrane proteins have been identified, including filamentous hemagglutinin, pertussis adhesin, adenylyl cyclase hemolysin, tracheal colonization factor, iron-regulating outer membrane protein, outer membrane protein A, lipoprotein, and others. Among these proteins, filamentous hemagglutinin and the outer membrane protein pertactin are factors important for attachment during infection. They also serve as protective immunogens and are among the most important components of acellular vaccines for whooping cough [B. bronchiseptica proteins [B. bronchiseptica HB in mice.rganisms , 8. A nung cough . Previouproteins . Five ofPseudomonas aeruginosa [Salmonella enterica ssp. Typhi were immunogenic [Aeromonas hydrophila was also reported to have potential as a vaccine candidate, because it could induce a Th1-type immune response. Our results show that rPPP, a novel target antigen derived from an outer membrane porin in B. bronchiseptica, can stimulate splenocytes in vitro and elicits humoral and cellular immune responses to confer significant protection in mice against challenge by a virulent strain of B. bronchiseptica.PPP is an outer membrane porin protein, also known as a channel protein or protein matrix, found in Gram-negative bacteria . Outer mruginosa . Researcunogenic –15. In cunogenic . Recombiet al. screened 71 Pasteurella multocida proteins and found that only the outer membrane lipoprotein plpE could produce a protective immune response in a denatured state [P. multocida is a putative vaccine candidate [B. bronchiseptica. IgG-subtype and cytokine analysis demonstrated that rPL induced humoral and cell-mediated immune responses in mice and that Th2-type immunity dominates response profile. Th2- type responses have higher levels of IL-10 and produce more IgG1 [PL was identified as a lipoprotein in our previous study. Lipoproteins abound in the outer membranes of Gram-negative bacteria and have long been considered potential target antigens for vaccine development. Hatfaludi ed state . Anotherandidate . Our resore IgG1 –21. In tBordetella pertussis, Shigella flexneri, and Brucella abortus have been shown to be highly immunogenic [Leishmania HSP20 is antigenic during natural infections, but as DNA vaccine, it does not protect BALB/c mice against experimental L. amazonensis infection [Leu/ile/val-binding protein is a component of the leucine, isoleucine, valine, threonine transport system. This system is one of two periplasmic binding-protein-dependent transport systems used for the high-affinity transport of the branched-chain amino acids. Leu/ile/val-binding proteins isolated from unogenic –24. Putaunogenic , 26. ABCunogenic . We clonnfection . Furthernfection , 29, 30.B. bronchiseptica. These data suggest that rPPP and rPL are potential immunomodulators or vaccine candidates if different vaccination approaches such as prime-and-boost vaccination or multivalent subunit vaccines are followed.In conclusion, this study shows that rPPP and rPL can induce protective humoral and cellular immune responses against


Toxoplasma gondii, a parasite with at least three distinct clonal lineages. This article reports the whole genome sequencing and de novo assembly of T. gondii RH (type I representative strain), as well as genome-wide comparison across major T. gondii lineages. Genomic DNA was extracted from tachyzoites of T. gondii RH strain and its identity was verified by PCR and LAMP. Subsequently, whole genome sequencing was performed, followed by sequence filtering, genome assembly, gene annotation assignments, clustering of gene orthologs and phylogenetic tree construction. Genome comparison was done with the already archived genomes of T. gondii. From this study, the genome size of T. gondii RH strain was found to be 69.35Mb, with a mean GC content of 52%. The genome shares high similarity to the archived genomes of T. gondii GT1, ME49 and VEG strains. Nevertheless, 111 genes were found to be unique to T. gondii RH strain. Importantly, unique genes annotated to functions that are potentially critical for T. gondii virulence were found, which may explain the unique phenotypes of this particular strain. This report complements the genomic archive of T. gondii. Data obtained from this study contribute to better understanding of T. gondii and serve as a reference for future studies on this parasite.Toxoplasmosis is a widespread parasitic infection by  Toxoplasma gondii is a globally widespread zoonotic parasite that infects approximately one-third of the world's population [T. gondii infections are subclinical, infection by this parasite in immuno-compromised groups and pregnant women can result in severe outcomes [T. gondii can persist within the host via latent infection, it may cause catastrophic consequences to those infected with HIV following reactivation of the parasite. This parasite is incriminated as one of the most fatal foodborne pathogens in USA [pulation , 2. Althoutcomes , 4. As Ts in USA .T. gondii shows a complex life cycle, during which sexual reproduction happens within its definite host (cat). Asexual reproduction occurs within its wide range of homoeothermic intermediate hosts, including humans. Since sexual reproduction is part of its life cycle, T. gondii was expected to show high variation across the world. However, the majority of the T. gondii strains from most parts of the world belong (but are not restricted) to three distinct clonal lineages  with minimal genetic differences among them [T. gondii lineages show varied virulence [T. gondii is the most virulent lineage in murine models [Similar to many other members of the Apicomplexa phylum, ong them –8. Howevirulence . For inse models . Furthere models . Type I e models , 12.T. gondii type I lineage is represented by GT1  and RH  strains [in vitro cultivation and commonly used in laboratory work [The  strains , 14. RH ory work . It showory work . On the ory work , with tyory work , 19 whilory work , 20. WitT. gondii can be understood better by performing in-depth genome analyses and comparisons on all major lineages of T. gondii. To date, whole genome sequencing has been completed on 62 T. gondii strains, including type I (GT1 and RH strains), type II (ME49 strain) and type III (VEG strain), as well as recombinant strains [T. gondii lineages will contribute towards better understanding on different virulence attributed to these lineages. Here, we performed whole genome de novo sequencing and assembly on T. gondii RH strain and compared its genome with those of T. gondii GT1, ME49 and VEG strains.The mechanisms behind the virulence and pathogenesis as well as other biological aspects of  strains –27. A clExperiments involving mouse model were carried out in compliance with the animal ethics approved by Institutional Animal Care and Use Committee (IACUC) of the University of Malaya, Faculty of Medicine (2014-06-03/PARA/R/CXT).T. gondii RH strain were maintained by serial intraperitoneal passage in BALB/c mice. The tachyzoites were harvested from peritoneal fluids three to four days-post- infection. The parasites were washed and re-suspended in sterile phosphate buffered saline (1x PBS) prior to usage. High molecular weight genomic DNA was extracted using DNeasy Blood and Tissue Kit . The specific identity of the extracted DNA was verified by conventional nested PCR directed against the B1 gene of T. gondii  and Loop-Mediated Isothermal Amplification Method (LAMP) [Tachyzoites of d (LAMP) , 29. TheT. gondii were used for the constructions of five libraries with various insert sizes of 200 bp, 500 bp, 800 bp, 5 kb and 10 kb. Paired-end sequencing was performed on these five libraries through Illumina HiSeq 2000 which allows users to determine the length of the insert and to sequence both ends of the insert, generating high quality sequence data. This process generated a total of 30.89 Gb of raw sequence data  were filtered to ensure high accuracy in scaffold construction. Besides, removal of low quality sequences such as those with an excess of “N” bases (> 10%) was done. Different bacterial and viral genomes were used for similarity mapping with Burrows-Wheeler Aligner (BWA). Reads mapped to the bacterial and viral genomes were removed. From these steps, the data size of all five libraries (post-filtered) was reduced significantly  to construct de Bruijn graph. To avoid chimeric reads, which are responsible for misassembles by generating incorrect sequence overlap, only short insert-size (<1 kb) of single- and paired-end reads were used in this assembly. Further corrections performed during de Bruijn graph simplification encompassed errors removals  and tiny repeats resolving. The graph was transformed to a contig graph by transforming linearly connected k-mers into a pre-contig node. Dijkstra’s algorithm (Skiena) was used to detect bubbles, which were then merged into a single pathway when the branches sequences were identical. Using this method, repetitive sequences were collapsed and consensus sequences were obtained.The corrected reads were assembled using SOAPdenovo, a software that assembles short oligonucleotide into contigs and scaffolds through de Bruijn graph algorithm . During k-mers in an unambiguous path. These contigs were broken into fragments at the boundaries of repeat ambiguous connections. All reads were realigned onto the contig sequences to obtain aligned paired-end reads. Contigs were linked to a scaffolding graph with paired-end reads. Connections between contigs comprised the edges in this graph, and the branch length demonstrated the gap size, which was calculated from the insert size of the paired-end reads. Subsequently, sub-graph linearization was applied to transform interleaving contigs into a linear structure. Repeat masking was used to mask complicated connections for repeat contigs. Following this, the amount of shared paired-end relationships between contig pairs were calculated, the ratio of consistent and conflicting paired-ends were determined, and scaffolds construction was done from the shortest to the longest insert size paired-ends in a step by step manner.The contig sequences were obtained by conjoining the Gaps, which composed mainly of masked repeats during scaffold construction, were found. Through the paired-end information, we retrieved read pairs that had one read well-aligned on the contigs and another read located in the gap region. Local assembly was performed on these reads to fill in small gaps within the scaffolds. We constructed a 27-mers de Bruijn graph with reads in the gaps and the contig ends on both sides using approach similar to that of contig construction. Gaps were filled with path sequences if an unambiguous path was found between those two contig ends. Each read in the gaps was checked to find one that had unambiguously mapped ends on both sides of the contigs and filled the gaps with read sequences. Most of the small tandem-repeat gaps were correctly filled by this way. However, longer tandem-repeat gaps could not be resolved by read sequences. Hence, we filled them with two repeat units along with a string of “N”. The GC-depth content difference is a primary factor for non-random sequencing-depth distribution . Therefode novo approach was employed in this study to identify the interspersed repeated sequences. Known transposable elements (TEs) were identified using RepeatMasker and RepeatProteinMask against the Repbase library of TEs  were used in homology-based approach [Neospora caninum, T. gondii GT1, T. gondii VEG and T. gondii ME49, whereas de novo approach involved usage of AUGUSTUS, Genscan and SNAP softwares to filter partial and small genes with coding length shorter than 150 bp in order to reduce false positives. The predictions were aligned to a TE protein database using BlastP with E-value of 10−5 and filtered TE-derived genes that had more than 50% aligning rate. Different gene sources were then integrated to form a consensus gene set using GLEAN [T. gondii genes has revealed key data that can further our understanding of differences between different strains. Uniprot database [Both homology-based and approach ; Neosporng GLEAN  by combidatabase  was refeT. gondii GT1 strain, contigs reordering was done on the assembled scaffolds of T. gondii RH strain, where all the 14 chromosomes were reordered and reassembled with Mauve Aligner [T. gondii were studied using BLASTn . Synteny blocks were retrived from the blast results to constitute the synteny plot with Circos v0.69 [Besides, whole genome synteny analysis was performed. Based on the chromosomes of  Aligner . Similaros v0.69 .T. gondii RH, T. gondii GT1, T. gondii ME49, T. gondii VEG, Neospora caninum) (Table H in −7), and con-joined fragment alignments for every gene were used through program Solar (Skiena). A connection (edge) was assigned between two nodes (genes) if more than one third of the region aligned to both genes. A h-score (0 to 100) was used to weight the similarity (edge). For two genes, G1 and G2, the h-score was defined as score (G1xG2) / max score (G1G1), score (G2G2), whereby the score was actually the BLAST raw score. Extracting gene families using clustering by Hcluster_sg, the average distance were used for the hierarchical clustering algorithm, requiring the minimum edge weight (h-score) to be larger than five, and minimum edge density  to be larger than one third. The clustering of gene families was terminated if out-group genes were identified.All protein sequences were compared against a database containing a protein dataset of all the species studied , consists of sequences LLKL01000001-LLKL01000441.T. gondii RH strain (TOXRH) was sequenced at 171-fold coverage , T. gondii ME49 (Type II) and T. gondii VEG (Type III)  and almost all have a sequencing depth above 60X  were analyzed. Of the predicted T. gondii RH gene families, 97.8% (5246 gene families) have orthologs (BLASTp cut-off: 10−5) in T. gondii GT1 compared to that in T. gondii ME49  and T. gondii VEG   . From ph; 97.5%) , close eH genome . From thny level .T. gondii RH except chromosomes 11 and 12  screening against genomes of other T. gondii strains available from ToxoDB. From this local alignment, 12 of the 111 genes showed good BLAST matches with genes of other T. gondii strains, validating the presence of similar homologs within the genomes of other T. gondii strains  that is different from other T. gondii clonal lineages.Most of the unique genes were annotated to unknown functions. Nevertheless, 17 unique genes were annotated to proteins with vital functions , mitotic cell cycle regulation , and meiosis regulation (TOXaeaD_GLEAN_10004604). It is noteworthy that the unique genes responsible for replication regulation were found to be annotated to functions that are closely associated with gene expression regulation as well. After all, the speed of replication is linked to the tachyzoite-bradyzoite stage conversion [Four nversion , which iT. gondii RH genome with PCR and sequencing as well . As indicated by the name, this protein is involved in calcium ion binding. Another unique gene (TOXaeaD_GLEAN_10003157) is annotated to calmodulin-like protein 3 (CALML3), which is also known as protein NB-1. CALML3 is an exosomal protein involved in calcium binding and release . We beliT. gondii RH strain to stay “active” as tachyzoites instead of facing “starvation” that drives bradyzoite stage conversion [Two genes were found to be annotated to enzymatic digestion. Gene TOXaeaD_GLEAN_10006826 was annotated to pepsin II-4 (pepsin A), whereas gene TOXaeaD_GLEAN_10004048 was annotated to a putative peroxisomal acyl-coenzyme A oxidase 1.2, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs. It regulates beta oxidation of fatty acids, which transforms lipids into acetyl CoA. The transformed acetyl CoA will then be led into Kreb’s cycle for energy production. Efficient metabolism and energy generation may enable nversion .T. gondii RH (which showed larger plaque formation) were significantly over-expressed when compared to T. gondii GT1, suggestive of association between the ABC transporters and the parasite’s higher growth rate [T. gondii is different from those of non-type I T. gondii parasites [T. gondii has been shown to be unrelated to ABC transporters [T. gondii RH-unique ABC transporter may play important roles in detox machinery of the parasite. This protein may modulate accumulation and removal of other toxic substances or xenobiotics, which enables longer extracellular survival for the parasite.One gene (TOXaeaD_GLEAN_10002720) was annotated to a transporter known as multidrug resistance-associated protein 1 (MRP1), which is also known as ATP-binding cassette sub-family C member 1 protein (ABC.C1). The ABC proteins have been linked to drug resistance in protozoan parasites . This “dwth rate . Besidesarasites . Althougsporters , we beliWhole genome alignments represent the fundamental basis for comparative analyses aimed at identifying and characterising functional related stretches of genes that are clustered. For instance, similarity across a wide range of evolutionary distance can be detected by a multiple alignments of homologous sequences from several species, whereby conserved and important biological similarities are usually revealed. Similarly, estimation of local rates of evolution on the basis of multiple alignments provides quantitative assessment of the strength of evolutionary constraints and the importance of functional elements. Collectively, these contribute to better understanding of the cellular biology of a parasite.T. gondii RH strain that was slightly larger than those reported by a number of studies [T. gondii RH strain used in this study may contribute to the larger genome size as well. Interestingly, the number of coding genes in T. gondii RH draft genome of this study happened to be of the lowest under comparison with the genomes of T. gondii GT1, ME49 and VEG strains  was not found in T. gondii RH draft genome  than draft genomes of other strains. Such differences may be associated with certain phenotypic variations among the different strains of T. gondii. On the whole, T. gondii RH shared the closest evolutionary relationship with T. gondii GT1, as compared with the ME49 and VEG strains.By referring to a list of pathogenesis-associated, parasite gene families presented by a recently published work , we consT. gondii RH genome shared high level of synteny with the genomes of three strains of T. gondii   and type I (likely the second generation offspring) parasites [Our genome synteny analysis revealed that and VEG) . The levarasites . It is lT. gondii show varied biological properties. For instance, type I strain was reported to show higher trans-epithelial migration rate than type II and type III strains [T. gondii strains of the same clonal lineage have been reported to demonstrate different phenotypes. The laboratory-adapted RH strains are known to be incapable of forming bradyzoites that are responsible for chronic infection [T. gondii clonal lineages show high similarity at the genome level, with the RH strain having only 111 unique genes. Therefore, these unique genes may be responsible for the different characteristics of T. gondii RH strain compared to other strains. We managed to locate these genes at the chromosomes of the parasite  found in this studyFigure D. Sequence depth distribution curve. Figure E. Primer design, PCR and sequencing of a randomly selected .(DOC)Click here for additional data file.S2 FileT. gondii RH genome. Table F. General statistics of predicted protein-coding genes. Table G. Summary of evidence for the GLEAN gene models. Table H. Statistical analysis of gene families. Table I. Statistics of the assembled sequence length. Table J. Chromosome assembly. Table K. Non-coding RNA genes in the genome. Table L. The 111 genes were predicted to be unique to the T. gondii RH genome based on Clustering Method. Table M. Chromosomal loci of the 111 T. gondii RH-unique genes. Table N. BLASTp result of 111 unique genes to the latest release from Toxodb.org. Table O. BLASTx of T. gondii ME49 unique genes vs T.gondii RH  domains. . Table  al. 2016).(XLS)Click here for additional data file.


S. aureus. Recent studies identified their presence in animal coagulase-negative staphylococci (CNS). The emergence of human-associated SAg+ CNS would mark a prodigious shift in virulence capabilities. We examined CNS isolates from healthy human nares and diseased individuals, and determined that no known SAgs were present.Superantigens (SAgs) are important virulence factors in  The SAg family consists of 23 members which includes toxic shock syndrome toxin-1 (TSST-1), the cause of nearly all cases of menstrual TSS and 50% of non-menstrual cases, staphylococcal enterotoxins (SEs), common causes of food poisoning and non-menstrual TSS, and SE-like . Fourteen nasal S. aureus isolates were cultured from these healthy individuals. Additionally, 48 nasal CNS isolates were obtained from these same individuals. CNS typing of the 48 nasal organisms was performed using the bioMérieux api Staph identification system according to manufacturer’s instructions. Briefly, bacterial strains were plated on TSA blood agar plates and grown at 37°C overnight for 20 hrs. A homogenous bacterial suspension was prepared in API Staph medium to a turbidity of 0.5 McFarland and used to inoculate the api strip. Strips were read after incubation 37°C for 24 hrs. All 48 organisms were determined to be S. epidermidis.Fifty healthy adult volunteers were analyzed for staphylococcal nasal colonization. Sterile cotton swabs were used to obtain individual samples from both nares of the volunteers. Staphylococcal isolates were identified by colony color on mannitol salt agar plates and morphology  and catalase activity. S. epidermidis as determined by the clinical microbiology laboratories of submitting medical centers. These isolates were tested for TSST-1, SEB, and SEC by antibody-based assays [S. epidermidis isolates from TSS patients were available for SAg gene testing by PCR.Fifty-seven coagulase negative staphylococci from human patients with TSS as defined by the treating physician were submitted for SAg testing to the Schlievert laboratory between 1980 and 2007. We have previously shown that physicians have high accuracy in identifying TSS cases based on patient symptoms and signs of disease . All of d assays . In multd assays –16. ThisS. aureus isolates from the nares of healthy individuals; these also served as controls for SAg detection. Group 2 included the 48 nasal S. epidermidis isolates from healthy individuals. Group 3  included 60 total S. epidermidis isolates recovered from the bloodstream of patients with TSS, and an additional 10 clinical S. lugdunensis isolates from the University of Iowa hospitals and clinics. Thus, we tested a total of 118 CNS for SAgs by antibody tests or their genes by PCR.Collectively, three groups of isolates were compared for the presence of SAgs or their genes. Group 1 included the 14 S. aureus strains, TSST-1 is typically produced at 3.5 ug/ml, whereas SEB and SEC are produced at 25–100 ug/ml.CNS isolates from TSS patients (57) were tested by an antibody-based test for the three major SAg causes of TSS, notably TSST-1, SEB, and SEC. Briefly, the isolates were cultured at 37°C with shaking (200 rpm) overnight in 50 ml Todd Hewitt broth and then treated with 4 volumes of absolute ethanol to precipitate SAgs. Subsequently, the precipitates were resuspended in 0.1 ml of distilled water (500 times concentrated) and clarified by centrifugation . The clarified supernates were tested by double immunodiffusion for reaction with hyperimmune rabbit antisera specific for each toxin. The lower limit of detection of SAgs by this method was 0.01 ug/ml original culture fluid. In tests of thousands of S. epidermidis) isolates from TSS patients, the organisms were cultured in 10 mL of Todd Hewitt broth and then subjected to DNA extraction for use in PCR [For all other isolates, including the three more recent CNS  carried staphylococci in their anterior nares. Among the volunteers, 48 of 48 (100%) individuals were positive for CNS, and 14 out of 48 (29%) individuals were positive for both CNS and sea-seg, tstH, and selh-selx in three groups of staphylococcal isolates: (Group 1) nasal S. aureus isolates from 14 healthy individuals, (Group 2) nasal CNS isolates from healthy individuals (48 S. epidermidis isolates), and (Group 3) clinical CNS isolates (60 S. epidermidis and 10 S. lugdunensis) . There was no detection of SEB, SED, SEl-J, SEl-K, SEl-Q, SEl-R, SEl-S, and SEl-T  isolates in both Groups 2 and 3 regardless of their origin  SAgs known to cause most cases of TSS.Conventional PCR was used to screen for the presence of canonical SAg genes unensis) . As expe isolate . Among tnd SEl-T . Data for origin . None ofS. epidermidis) [S. aureus strains, noting that TSST-1 positive S. aureus may give weak coagulase test reactions and appearing instead to be CNS [S. aureus. The Schlievert and Bergdoll (now deceased) laboratories both tested a sampling of S. epidermidis strains from unused tampons that had been isolated by the CDC to test their hypothesis. Both the Schlievert and Bergdoll laboratories showed that none of the S. epidermidis strains were positive for TSST-1 [When TSST-1 was first described in 1981 ,18, one o be CNS . Howeverr TSST-1 . It was r TSST-1 .6-fold in rabbits [S. epidermidis, 10 times concentrated culture fluids, for ability to induce fever and enhance endotoxin shock in rabbits; all 22 strains were negative, suggesting these CNS did not produce known or yet to be described SAgs that caused the TSS symptoms [The original defining immunobiological activities of SAgs  are ability to cause fever and enhance host susceptibility to lethal endotoxin shock by up to 10 rabbits . Subsequ rabbits . BecauseS. aureus can be occasionally present in CNS [S. epidermidis, do not encode S. aureus SAgs [S. epidermidis strain originally provided by the Bergdoll laboratory, the organism has been clearly established as both CNS and verified to produce S. aureus SAgs [S. epidermidis and none of 12 S. lugenunensis strains in that study produced known SAgs, in agreement with the findings from the 1980s [S. epidermidis and S. lugdunensis strains produce any of the known SAgs. Thus, with one exception strain (FRI909) [S. epidermidis and S. lugdunensis) that infect humans do not produce known or unknown SAgs, despite their abilities to cause human diseases.Recent studies indicate that known SAgs present in nearly all t in CNS ,10. Theseus SAgs . In one eus SAgs . There heus SAgs ,11. Addihe 1980s  and our (FRI909) , the majS. hominis and S. capitis, and showed none produced TSST-1, SEB, and/or SEC [S. hominis and capitis strains from humans produce one or more of these same three SAgs [In a prior study, we tested a small number of CNS that only occasionally cause human infections, for example d/or SEC . In contree SAgs . These dS. aureus and CNS can be isolated from anterior nares of humans. While only 24% of tested individuals were positive for S. aureus, 96% were positive for S. epidermidis CNS. Interestingly, individuals colonized with S. aureus were also co-colonized with CNS, but when dually present, S. aureus dominated. This is in agreement with a previous study where we reported that S. aureus and CNS are inversely correlated in colonization of mucosal surfaces [S. aureus isolates encoded an average of 5 SAgs per strain. To date, this is the only study to evaluate a large collection of S. epidermidis strains for the presence of any of the 23 known staphylococcal SAg genes.Our study indicates, as expected, that both surfaces . When weS1 DatasetS. aureus strains and 51 S. epidermidis strains tested by PCR, 10 S. lugdunensis strains tested by PCR and 57 S. epidermidis isolates from TSS patients tested by antibody reaction against hyperimmune serum raised specifically against TSST-1, SEB, or SEC.These include the 14 (XLS)Click here for additional data file.


Salmonella from chicken and pig slaughterhouses in Henan, China and antimicrobial susceptibility of these isolates to antibiotics was determined. From 283 chicken samples and 240 pig samples collected, 128 and 70 Salmonella isolates were recovered with an isolation rate of 45.2 and 29.2% respectively. The predominant serovars in chicken samples were S. enterica serovar Enteritidis, S. enterica serovar Hadar and S. enterica serovar Indiana, while those in pig samples were S. enterica serovar Typhimurium, S. enterica serovar Derby and S. enterica serovar Enteritidis. Resistance to ciprofloxacin was 8.6 and 10.0% for isolates from chickens and pigs respectively, whereas resistance to cefotaxime was 5.5 and 8.6%, respectively. Multidrug resistance  was markedly higher in pig isolates (57.1%) than in chicken isolates (39.8%). Of particular concern was the detection of ciprofloxacin and cefotaxime co-resistant S. enterica serovar Indiana isolates, which pose risk to public health. All 16 S. enterica serovar Indiana isolates detected were resistant to ciprofloxacin, among which 11 were co-resistant to cefotaxime. The S. enterica serovar Indiana isolates accumulated point mutations in quinolone resistance determination regions of gyrA (S83F/D87G or S83F/D87N) and parC (T57S/S80R). Two plasmid mediated quinolone resistant determinants were found with aac (6')-Ib-cr and oqxAB in 16 and 12 S. enterica serovar Indiana isolates respectively. Cefotaxime-resistance of S. enterica serovar Indiana was associated with the acquisition of a blaCTX-M-65 gene. The potential risk of ciprofloxacin and cefotaxime co-resistant S. enterica serovar Indiana infection is a significant concern due to limited alternative treatment options. Reduction of Salmonella in chicken and pig slaughterhouses, in particular, ciprofloxacin and cefotaxime co-resistant S. enterica serovar Indiana will be an important measure to reduce the public health burden of Salmonella infections.The prevalence of  Salmonella infections with 155,000 deaths and 85% of these cases are foodborne  resulted in high-level resistance to ciprofloxacin .Two PMQR determinants were identified in our ndrogram  suggestsm , different from those of the isolates in cluster A, lending further support to its independent origin. Therefore both clonal spread and independent acquisition of cefotaxime resistance may have acted to contribute to the emergence and spread of ciprofloxacin and cefotaxime co-resistant S. enterica serovar Indiana in Henan province.The genetic relationship of the S. enterica serovar Indiana has already been reported in human infections in China [S. enterica serovar Indiana isolates from human infections in Henan were resistant to ciprofloxacin but susceptible to cefotaxime [S. enterica serovar Indiana emerged in Henan will transmit to humans, posing a significant threat to public health. The co-resistance limits the optimal treatment alternatives of salmonellosis in humans.Multidrug resistant in China ,27. All Salmonella in the chicken and pig slaughterhouses in Henan was found to be high, and represented by a small number of different serovars such as S. enterica serovar Enteritidis, S. enterica serovar Hadar and S. enterica serovar Indiana in chickens and S. enterica serovar Typhimurium, S. enterica serovar Derby and S. enterica serovar Enteritidis in pigs. This finding highlights the fact that food-producing animals at processing plants are a significant reservoir of antimicrobial-resistant Salmonella. Our data may contribute to the development of interventions at the processing level to interrupt the transmission of Salmonella to humans. Ciprofloxacin and cefotaxime co-resistant S. enterica serovar Indiana possibly emerged only recently through acquisition of blaCTX-M-65. The potential risk of ciprofloxacin and cefotaxime co-resistant S. enterica serovar Indiana infections in humans requires urgent attention. The emergence of ciprofloxacin and cefotaxime co-resistance also underscores the importance of strict control on the use of antibiotics in animal production to prevent the emergence of antibiotic resistance in Salmonella.The prevalence of


Dicathais orbita is a mollusc of the Muricidae family and is well known for the production of the expensive dye Tyrian purple and its brominated precursors that have anticancer properties, in addition to choline esters with muscle-relaxing properties. However, the biosynthetic pathways that produce these secondary metabolites in D. orbita are not known. Illumina HiSeq 2000 transcriptome sequencing of hypobranchial glands, prostate glands, albumen glands, capsule glands, and mantle and foot tissues of D. orbita generated over 201 million high quality reads that were de novo assembled into 219,437 contigs. Annotation with reference to the Nr, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases identified candidate-coding regions in 76,152 of these contigs, with transcripts for many enzymes in various metabolic pathways associated with secondary metabolite biosynthesis represented. This study revealed that D. orbita expresses a number of genes associated with indole, sulfur and histidine metabolism pathways that are relevant to Tyrian purple precursor biosynthesis, and many of which were not found in the fully annotated genomes of three other molluscs in the KEGG database. However, there were no matches to known bromoperoxidase enzymes within the D. orbita transcripts. These transcriptome data provide a significant molecular resource for gastropod research in general and Tyrian purple producing Muricidae in particular. Dicathais orbita is well known for the production of the dye Tyrian purple, which is a historically important colourant exclusively produced by the Muricidae family of marine molluscs [molluscs ,2. Tyriamolluscs ,4. Once molluscs ,6,7,8,9.molluscs ; howeverReishia (Thais) clavigera [Concholepas concholepas [Colubraia reticulata [Conus consors [C. tribblei, C. lenavati [Lophiotoma olangoensis, a Turrid snail [The “post-genomics era” has seen an expansion in the application of bioinformatics to the fields of transcriptomics, proteomics and metabolomics . Transcrlavigera , the mancholepas , the aliticulata , the ven consors  and the lenavati  and Lophid snail .D. orbita is an ideal model species for gene expression studies of biosynthetic pathways that may be involved in the synthesis of biologically active secondary metabolites [D. orbita used suppressive subtractive hybridisation to identify genes that were upregulated in the hypobranchial gland, the biosynthetic organ where Tyrian purple is produced [D. orbita (Muricidae Neogastropoda) and then search these transcriptomes for potential metabolic pathways that could contribute to indole and choline ester biosynthesis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAYS database.Transcriptomics can identify genes involved in the biosynthesis of secondary metabolites ,32. The produced . This stproduced . Howeverhttp://sourceforge.net/projects/bbmap), which resulted in 201 million high quality reads. Transcripome sequencing across the six different tissue types  resulted in approximately 221 million sequencing reads . Raw seqhttp://transdecoder.github.io/) identified 76,152 contigs that contained candidate-coding regions that were used for annotation and further downstream analysis.High quality reads were de novo assembled into 219,437 contigs using CLC Genomics server  . Transde−5). The D. orbita contig BLAST hit rate was in a similar range to other whole mollusc genome/transcriptome studies with hits ranging from 25% to 40% [D. orbita tissues [k-mers generated in the quality control phase of RNAseq reads and confirmed that these are mostly mitochondria RNA rather than bacterial (<0.1%).Basic Local Alignment Search Tool (BLAST) analysis was performed using 76,152 contigs with Open Reading Frames (ORF)s against non-redundant protein database National Center for Biotechnology Information (NCBI) Nr (Mollusc specific proteins), Swiss-Prot and KEGG protein databases. Overall 28,364 contigs ~37%) had significant BLAST hits  involved in the production of serotonin. As the other three molluscs all contain matches to tryptophan 5-monoxygenase in their genomes  compared to L. gigantea (five genes), O. bimaculoides (five genes) and C. gigas (five genes)  .D. orbita transcripts with reference to the sulfur metabolism pathway revealed that most of the genes involved in D. orbita sulfur metabolism were found to be similar to the other three molluscs. Enzymes like dimethyl-sulfide monooxygenase , O. bimaculoides (12 genes) and C. gigas (14 genes) (Tyrindoxyl sulphate contains a methane thiol group at the 2′ position of the indole ring A. Examin4 genes) .D. orbita has the ability to produce 3-methylthioproprionate, 3-mercaptopyruvate and thiosulfate, but no match was found to methionine-gamma-lyase  compared to L. gigantea (32 genes), O. bimaculoides (30 genes) and C. gigas (33 genes)  . This abD. orbita [D. orbita transcripts against known bromoperoxidase genes using BLAST . However, no evidence of matches to bromoperoxidase genes was found using these sequences. This is consistent with a previous study that examined the transcripts that were up-regulated in the D. orbita hypobranchial glands relative to other D. orbita tissues, which also found no matches to bromoperoxidase genes [D. orbita hypobranchial glands revealed the presence of bacterial symbionts known to produce bromoperoxidase enzymes [D. orbita transcriptome data therefore supports the suggestion that symbiotic bacteria play a role in the biosynthesis of Tyrian purple.Tyridoxyl sulfate is a 6-brominated indole derivative A, and br. orbita  and othe. orbita . Consequse genes . There a enzymes . Our D. D. orbita transcriptome compared to the annotated genomes of other molluscs   . The D. D. orbita transcriptome  when compared to L. gigantea (10 genes), O. bimaculoides (11 genes) and C. gigas (13 genes)  . Specifi3 genes)  was onlyD. orbita . The stabilized tissue was incubated at 4 °C overnight and stored at −80 °C, prior to extracting the total RNA. The total RNA was extracted from the RNAlater stabilized tissue using the E.Z.N.A. Mollusc RNA Kit  following the manufacturer’s instructions. The concentration and quality (purity and integrity) of total RNA was assessed by NanoDrop and the Agilent Bioanalyzer 2100 System . The total RNA extracted from three biological replicates of each tissue type  was pooled within the same tube for each tissue in equal masses. The pooled extracted RNA was stored at −80 °C until further used. The RNA samples were shipped to Macrogen Inc.  for high throughput sequencing. Prior to shipping, each RNA sample was precipitated in a mixture of 2× ethanol (96%) and 0.1× sodium acetate (3 M). mRNA isolation and library construction were performed by Macrogen. The libraries were sequenced using the Illumina HiSeq 2000 platform .Three independent replicate snails were used for each tissue sample summarised in http://www.bioinformatics.babraham.ac.uk/projects/fastqc) [http://sourceforge.net/projects/bbmap) using command “bbduk.sh in1 = in1.fastq in2 = in2.fastq out1 = out1.fastq out2 = out2.fastq ref = adapters.fa qtrim = rl trimq = 20 ktrim = rl k = 28 hdist = 1 minlength = 20”. BLAST search of over-represented k-mers against NCBI confirmed these were mostly mitochondrial and ribosomal Dicathais obita genes, and no evidence of bacterial contamination was found. CLC Genomics Workbench, version 4.9  with the option to map reads back to contigs, automatic word size and automatic bubble size was used to de novo assemble the high quality reads. All the contigs were clustered using CD-hit-est  [http://transdecoder.github.io/) was used to identify candidate coding regions within transcript sequences.FASTQ format raw sequencing reads were checked for quality using FASTQC  . The ada/cd-hit) , and Trahttps://code.google.com/archive/p/interproscan) [http://wego.genomics.org.cn/cgi-bin/wego/index.p) [D. orbita transcripts were searched for bromoperoxidase gene by aligning against known bromoperoxidase genes available in the NCBI GenBank using BLAST  , Swiss-Prot and KEGG protein databases. Gene ontology analysis was performed with Interproscan   using coindex.p) . D. orbi × 10−3) .All raw sequence data were deposited in the European nucleotide archive (ENA) with the accession numbers PRJEB12262. Assembled contigs are available from the authors upon request.D. orbita generated over 216 million high quality reads that were de novo assembled into 219,437 contigs, of which 76,152 contigs contained candidate-coding regions that were annotated with Nr, Swiss-Prot and KEGG databases. This provides a significant new molecular resource for neogastropod molluscs, and adds to pool of genomic data for molluscs in general. Several genes that are potentially associated with Tyrian purple precursor biosynthesis in D. orbita were identified. It appears the neogastropod D. orbita has evolved a complex suite of metabolic capabilities that are not represented in the more primitive orthogastropods or bivalves, for which complete genome sequences are available.This transcriptome study of


ChloroMitoSSRDB 2.00 allows the end user to perform multiple SSRs searches and easy browsing through the SSRs using two repeat algorithms and provide primer pair information for identified SSRs for evolutionary genomics.Organelle genomes evolve rapidly as compared with nuclear genomes and have been widely used for developing microsatellites or simple sequence repeats (SSRs) markers for delineating phylogenomics. In our previous reports, we have established the largest repository of organelle SSRs, ChloroMitoSSRDB, which provides access to 2161 organelle genomes  with a total of 5838 perfect chloroplast SSRs, 37 297 imperfect chloroplast SSRs, 5898 perfect mitochondrial SSRs and 50 355 imperfect mitochondrial SSRs across organelle genomes. In the present research, we have updated ChloroMitoSSRDB by systematically analyzing and adding additional 191 chloroplast and 2102 mitochondrial genomes. With the recent update, ChloroMitoSSRDB 2.00 provides access to a total of 4454 organelle genomes displaying a total of 40 653 IMEx Perfect SSRs (11 802 Chloroplast Perfect SSRs and 28 851 Mitochondria Perfect SSRs), 275 981 IMEx Imperfect SSRs (78 972 Chloroplast Imperfect SSRs and 197 009 Mitochondria Imperfect SSRs), 35 250 MISA (MIcroSAtellite identification tool) Perfect SSRs and 3211 MISA Compound SSRs and associated information such as location of the repeats (coding and non-coding), size of repeat, motif and length polymorphism, and primer pairs. Additionally, we have integrated and made available several Database URL:http://www.mcr.org.in/chloromitossrdb Nuclear and organelle  genome-based microsatellites or simple sequence repeats (SSRs) markers have been widely used to demonstrate the underpinning differences in genetic patterns and for assessing the phylogenomic and genetic associations between species of particular clade . In compPinus (forest species) (Oryza sativa (Monocots).   to Oryzanocots). , 11 Rece plants.  However, plants. , primer  plants. , GOBASE  plants.  and AT_C plants. , althoug plants.  and Chlo plants.  have beeIn the present research, we present ChloroMitoSSRDB 2.00, an update to the previously established microsatellite (SSRs) repository ChloroMitoSSRDB, by systematically analyzing and adding SSRs entries for additional 191 chloroplast and 2102 mitochondrial genomes thus displaying a total of 4454 organelle genomes. Additionally, ChloroMitoSSRDB 2.00 provides a web-accessible unified portal for the identification of the maximal/perfect/imperfect SSRs repeats using IMEx (Imperfect Microsatellite Extractor)  either fAvailability of the SSR mining tools under a common portal plus a systematically curated in-house database, we believe that ChloroMitoSSRDB 2.00 will serve as a portal for the identification and assessment of organelle repeat evolution, developing species-specific markers, identifying estimates of genetic diversity based on organelle marker abundances, phylogenomics and other organelle-based genotyping approaches. To the best of our knowledge, this is the first integrated portal, which catalogs the repeat search pattern for thousands of organelle genomes, across diverse phylogenetic clades in a systematic manner along with the ‘on-the-fly’ availability of the organelle repeat search tools for organelle genomics, which is accessible via web-interface.www.ncbi.nlm.nih.gov/). Additionally, each organelle genome was scanned for SSRs patterns using two different tools: IMEx (http://pgrc.ipk-gatersleben.de/misa/misa.html). IMEx  were systematically downloaded from NCBI RefSeq database release 63 (http://pgrc.ipk-gatersleben.de/misa/misa.html), which allows the detection of the perfect, imperfect and compound repeats. For a stretch of nucleotide to be classified as the SSRs using MISA, a minimum length of ≥12 bp for Mono-, ≥6 bp for Di-, ≥4 bp for Tri- and ≥3 bp for Tetra-, Penta- and Hexa-nucleotide repeats were used as length thresholds, respectively. For the identification of the compound SSRs, we kept the minimum distance between any two identified SSRs as 100 bp. Following the identification of the SSRs using MISA, primer pairs for the each set of the MISA identified SSRs, primer pairs were designed using Primer3 with settings PRIMER_PRODUCT_SIZE_RANGE=100-280 and PRIMER_MAX_END_STABILITY=250. In order to make SSRs markers readily available for downstream analysis, we integrated only those MISA SSRs markers, which have the corresponding primer pair information. The genome composition and the repeat occurrence graphs were generated dynamically using HighCharts, a JavaScript chart-drawing library (www.highcharts.com/products/highcharts). ChloroMitoSSRDB 2.00 is hosted on a 64-bit Linux server pre-installed with Apache (http://www.apache.org/) and PHP (http://www.php.net/).Additionally, each organelle genome has been simultaneously analyzed using MISA algorithm tool . In this update, identified SSRs from the IMEx and MISA algorithms for each of the organelle genomes were integrated for visualization through respective algorithm specific pages. The relational database system of ChloroMitoSSRDB 2.00 has been updated by adding primer-pair information corresponding to MISA identified SSRs, as a separate query field. Supplementary Figures S1 and S2.A sketch of the updated computational workflow of ChloroMitoSSRDB 2.00 is given in http://www.mcr.org.in/chloromitossrdb/chloro_browse.php; mitochondrial, http://www.mcr.org.in/chloromitossrdb/mito_browse.php) genomes. Each organelle genome has been hyperlinked to the corresponding taxonomy record as previously available in ChloroMitoSSRDB. Organelle  genome-specific pages offer options for the end users to systematically browse through the results of the IMEx (http://www.mcr.org.in/chloromitossrdb/chloro_browse.php) and mitochondrial (http://www.mcr.org.in/chloromitossrdb/mito_browse.php) genomes . In line with the previous version of the database, each organelle genome page displays the genome composition  (http://www.mcr.org.in/chloromitossrdb/get_repeats.php?id=NC_015820&size=1&type=0&org=0). Additionally, in case of repeats localized with in the genic regions  B and 4B  regions D, the cohttp://www.mcr.org.in/chloromitossrdb/misa_get_repeats.php?id=NC_009268&size=1&type=0&org=0), and each identified SSRs motif has been hyperlinked to the corresponding primer pair information (http://www.mcr.org.in/chloromitossrdb/primers.php?id=NC_009268&start=4251&end=4268)  E. Availand=4268)  and MISAnd=4268)  SSRs alihttp://www.mcr.org.in/chloromitossrdb/ngs_upload.php using PAL finder (http://www.mcr.org.in/chloromitossrdb/extract.php), where user can analyze the SSRs from their assembled genome using any of the repeat mining algorithm such as IMEx  and size and length of repeat motif, we present a new advanced search panel with an enhanced drop-down box which is now available showing several additional search patterns based on coding and non-coding classification patterns A. With tL finder , 24 fromL finder B. To fac as IMEx , REputer as IMEx  or MISA  as IMEx C.FigureIn conclusion, ChloroMitoSSRDB 2.00 provides an enhanced visualization and unified update to the previously developed integrated repository of the organelle genome by integrating SSRs patterns from two different tools IMEx and MISA. ChloroMitoSSRDB 2.00 also overcomes the limitations of the SSR search pattern for the user-defined gene-based or the whole-genome-based sequences by integrating three widely implemented tools for SSRs search pattern. In future work, we plan to establish a sequence-based retrieval for the complete NGS-based SSRs patterns for marker development across a wide range of organelle genomes. We believe that the enhanced version of the database portal along with the comparative integration of two repeats mining algorithms, and ‘on-the-fly’ repeat extraction will support a wide range of the organelle genomics community and will serve as a platform for wider organelle genome-wide SSR explorations.GS designed the study and carried out the analysis; GVPR, SBM updated the database in discussion with GS; RP and DPS helped in the database update; GS wrote the manuscript; VB, GY, PJR and NLP provided revisions to the manuscript.Supplementary data are available at Database Online.


Megachile: Megachilidae) cut leaves from various trees, shrubs, wildflowers and grasses to partition and encase brood cells in hollow plant stems, decaying logs or in the ground. The identification of preferred plant species via morphological characters of the leaf fragments is challenging and direct observation of bees cutting leaves from certain plant species are difficult. As such, data are poor on leaf preference of leafcutting bees. In this study, I use DNA barcoding of the rcbL and ITS2 regions to identify and compare leaf preference of three Megachile bee species widespread in Toronto, Canada. Nests were opened and one leaf piece from one cell per nest of the native M. pugnata Say (N=45 leaf pieces), and the introduced M. rotundata Fabricius (N=64) and M. centuncularis (L.) (N=65) were analysed. From 174 individual DNA sequences, 54 plant species were identified. Preference by M. rotundata was most diverse =2.97), followed by M. centuncularis  then M. pugnata . Cluster analysis revealed significant overlap in leaf choice of M. rotundata and M. centuncularis. There was no significant preference for native leaves, and only M. centuncularis showed preference for leaves of woody plants over perennials. Interestingly, antimicrobial properties were present in all but six plants collected; all these were exotic plants and none were collected by the native bee, M. pugnata. These missing details in interpreting what bees need offers valuable information for conservation by accounting for necessary  nesting materials.Leafcutting bees ( TheseMegachile predominantly collect leaves by cutting pieces with their mandibles to construct their nests [M. montivaga Cresson) [M. sculpturalis Smith, M. campanulae Robertson), or even tile caulking and plastic bags [Megachilid bees and especially those in the genus ir nests –13. Not Cresson) ,15 and otic bags . Althougtic bags ,18, surpM. rotundata among 11 plant species. In enclosures, bees cut all plant species offered to them but showed a significant preference for leaves of buckwheat (Fagopyrum esculentum Moench), which were also significantly larger than the leaves of the other 10 species. Kim [M. apicalis Spinola visiting Wisteria sp. for leaf pieces in nature and subsequently used the plant in pots in enclosures to study relationships between female body size and fecundity. Finally, Nugent & Wagner [Megachile bee species on seven different Populus cultivars and found one cultivar was defoliated significantly less than all others. Mechanical properties of leaves are evidently important for leaf preference in leaf-collecting Megachile bees. However, studies of resin- and trichome (leaf hair)-collecting megachilid bees suggest antimicrobial factors are important to protect brood from diseases, moulds and parasites [Megachile bees.Nesting material choice may limit the geographical ranges and/or abundances of particular bee species. However, the leaf preferences of megachilid bee species have rarely been investigated. Identifying leaf preference could inform management and conservation , researcies. Kim  observed& Wagner  comparedarasites –26, and Hoplitis producta (Cresson) collecting Fragaria sp. leaf pieces [Hoplitis pilosifrons (Cresson) using Oenothera sp. leaves, M. centuncularis was observed cutting leaves from beech (Fagus) trees [M. integra Cresson using blackberry (Rubus sp.) [et al. [M. relativa Cresson to cutting plants in the genera Fragaria and Epilobium, and M. inermis Provancher to (among many others) Acer, Betula, Rubus and Amelanchier. The authors associated the size of the cuts on the leaves to the two different bees studied because the first bee species cut noticeably larger pieces than the second one.Studies that report the identities of leaves cut by megachilid bees do so based on the characteristic damage to leaves ,27–32 f. For exaf pieces , Michenef pieces  noted Hos) trees , and anobus sp.) . StricklDetermining leaf preference from observations of the bee in the act of cutting, or by surveying for the characteristic leaf cuts on plants, gives partial lists that can offer useful details about local megachilid bee nesting requirements. However, methods that more accurately link leaf–bee species interactions could provide details about inter- and intraspecific variation in leaf preference, contributing to gaps in knowledge on these essential pollinators. One technique that could ameliorate our understanding of leafcutting bee nesting preferences is DNA barcoding, which uses the diversity in short gene sequence regions to improve species-level identification –39. ThesM. pugnata Say) and two exotic bees that are naturalized in the study region, M. centuncularis (L.) and M. rotundata [M. pugnata) would prefer native leaves rather than exotic ones for nesting material. Lastly, I determine whether any bees exhibit specialization or affinities for native or exotic species, or particular kinds of vegetation, including trees, shrubs or flowering plants, which would have applications for conservation planning and management.In this study, I use DNA barcoding to identify leaf preference of three leafcutting bee species, one native , which are a preferred habitat for many cavity-nesting bee species ,17,44,45M. pugnata, and the introduced M. centuncularis and M. rotundata. M. pugnata uses mud and chewed leaves to line its brood cells, and makes partitions between adjacent cells using circular pieces of leaves laid one over the other , and the introduced M. rotundata  and M. centuncularis . A total of 62 sites were sampled over the 2012 and 2013 seasons. No sites were shared among all three bees, and only two sites were shared between M. pugnata and M. centuncularis, one between M. pugnata and M. rotundata, and four sites between M. centuncularis and M. rotundata.Thirty-six bee species (including five cleptoparasites) used the nest-boxes over the three seasons . From thhe other . M. rotud leaves . One lea2 square, washed in ethanol, air dried, and placed into an individual sampling vial in a 96-vial DNA extraction plate (AcroPrep) provided by the Canadian Centre for DNA Barcoding (CCDB). Two filled plates of leaf fragments were then sent to the CCDB for DNA extraction [To determine the identity of each leaf cut sample, using forceps I removed one leaf piece per brood cell, then each was cut into a 0.5 cmtraction  and barctraction ,49,50. Ttraction  for 100%traction . Lastly,traction –60.Tabl2.2H′) of leaf species preference for each of the three bee species. Rarefaction curves for leaf preference of all bee species were interpreted using iNEXT software [χ2-tests in the R package ‘MASS’. Some plant species used by the bees were biennials  or ‘non-woody’ .From the type and number of leaf species cut as determined by DNA barcoding, I determined the richness and Shannon diversity index . M. centuncularis collected 23 species in 20 genera and 10 families, and 23.1% of the total leaves analysed were Rosa multiflora Thunb. M. pugnata collected leaves from 18 species in 14 genera and 8 families, with Cornus racemosa Lam., Lythrum salicaria L., R. multiflora and R. virginiana P.Mill. all tied for the most identified leaf type (11.1% each). M. rotundata collected leaves from 36 species in 33 genera and 20 families. Cynanchum rossicum (Kleopow) Borhidi was the most often identified species for M. rotundata. Rarifying leaf species richness and sample completeness for all three bees to control for uneven sample size indicated that the estimate of total species richness was lower for M. pugnata and M. centuncularis than M. rotundata and that sampling more adequately characterized the leaf preference of M. pugnata than that of M. centuncularis and M. rotundata  then M. centuncularis (H′=2.38), and lowest in the native M. pugnata (H′=1.87). The pd in leaf preference was highest in M. rotundata (pd=2.97), followed by M. centuncularis (pd=1.51) and the native M. pugnata (pd=1.22) (M. centuncularis (0.64) and M. pugnata (0.65) were more constrained in pd than was M. rotundata (0.96).Diversity in leaf preference was greatest in the exotic pd=1.22) . ControlM. centuncularis and M. rotundata was more similar than that of M. pugnata . Four plant species were visited by all bee species and all were shrubs or trees: R. blanda Gray, R. glauca Pourret, R. multiflora and Prunus avium (L.) . Rosaceae was the most used family for both M. centuncularis  and M. pugnata . For M. pugnata, leaves in the family Rosaceae represented half of all species collected. Leaves of R. multiflora were the most abundantly collected species by all three species combined (N=25). Only one grass species was collected, Setaria italica (L.) P. Beauvois, by one M. rotundata individual  . Twelve dividual .Figure χ2=1.30, p=0.52) with 30 native plant species and 24 exotic species collected among the three bees , bird’s foot trefoil (Lotus corniculatus L.) and crown vetch (Securigera varia (L.) Lassen) as well as plants invasive to the Southern Ontario region, including purple loosestrife  and dog-strangling vine (C. rossicum) (Silene vulgaris (Moench) Garcke, L. corniculatus L., Stachys palustris L., Syringa vulgaris L., Rhamnus cathartica L. [Ampelopsis japonica (Thunb.) Makino [M. pugnata  significantly more than ‘non-woody’ plants (N=13)  . Interesossicum) . Only sirtica L.  and Ampe) Makino . All six pugnata . Lastly,4.χ2-tests none of the bee species exhibited a significant preference for native or exotic leaf species, indicating that the third hypothesis, e.g. that the native bee, M. pugnata, would prefer native plant species to exotic species, is not valid.Leafcutting bees are reported to be selective in leaf preference, and to forage on only a few plant species ,20. PastM. pugnata on nesting materials that are more phylogenetically related could make this species and other native bees more susceptible to environmental change [Dependence of the native l change . Ecologil change ,70. Somel change ,72 and tl change . Future l change ) among bl change .M. centuncularis exhibited a significant preference for woody plants (e.g. trees and shrubs) ) and invasive species ) (M. rotundata collected more exotic leaf types than the other two species (Cyananchum rossicum) [L. corniculatus L.) and sweet clover )  . None oficaria)) . The exo species , and amoossicum)  table 1ossicum) ,78. A nuossicum ,Bee diversity and abundance are strongly linked to characteristics of the local environment, including the presence and quantity of foraging and nesting materials, as well as the amount of, and distance between, habitat containing these resources –87. In uAnthidium manicatum, collects leaf trichomes to make their nests which have antimicrobial properties [M. rotundata, which preferred buckwheat unanimously over alfalfa and especially leaves greater than 1 cm2 [Almost all the leaves identified in the nests are known to contain antimicrobial properties . Many groperties  and theioperties . One stuan 1 cm2 . A combiMissing from this study is an examination of the continuity and variability in leaf choice within single nests, which limits interpretation of the diversity of leaf types used by individual females. Identifying all leaves comprising a complete nest of individual bees  would contribute to knowledge on how diverse leaf preference is among individual bees, or even how it changes over a season. Knowing the identities of plants used by leafcutting bees can inform ‘complete’ pollinator gardening and broader actions supporting wild pollinators that include both foraging and nesting requirements ,94–97.