Clematoclethra and Actinidia) in the Actinidiaceae via genome skimming technique. Comparative analyses revealed that the genome structure and content were rather conservative in three cp genomes in spite of different inheritance pattern, i.e.paternal in Actinidia and maternal in Clematoclethra. The clpP gene was lacked in all the three sequenced cp genomes examined here indicating that the clpP gene loss is likely a conspicuous synapomorphic characteristic during the cp genome evolution of Actinidiaceae. Comprehensive sequence comparisons in Actinidiaceae cp genomes uncovered that there were apparently heterogenous divergence patterns among the cpDNA regions, suggesting a preferred data-partitioned analysis for cp phylogenomics. Twenty non-coding cpDNA loci with fast evolutionary rates are further identified as potential molecular markers for systematics studies of Actinidiaceae. Moreover, the cp phylogenomic analyses including 31 angiosperm plastomes strongly supported the monophyly of Actinidia, being sister to Clematoclethra in Actinidiaceae which locates in the basal asterids, Ericales.Actinidiaceae is a well-known economically important plant family in asterids. To elucidate the chloroplast (cp) genome evolution within this family, here we present complete genomes of three species from two sister genera ( Actinidia chinensis Planch. and A. chinensis var. deliciosa H.L.Li that can be eaten as fresh fruit or made into juice, jelly and dried fruit. There are c. 357 species included in three genera, i.e. Actinidia Lindl., Clematoclethra Maxim. and Saurauia Willd., in the kiwifruit family [Clematoclethra species have bisexual flowers in one individual i.e. hermaphrodites, while in Actinidia species are dioecious or functionally dioecious [Clematoclethra and Actinidia descend differently, that is with maternal and paternal transmission in Clematoclethra and Actinidia respectively [Actinidiaceae as an economically important plant family includes the well-known kiwifruit species, i.e. t family . This faioecious –3. The cectively . HoweverThe size of cp genomes in flowering plants ranges from 19  to 218 kActinidia, remains largely elusive and controversial [Actinidia).Rates of nucleotide change between non-coding (intergenic regions and introns) and coding (exons of protein-coding genes) sequences can vary tremendously within a given taxonomic group –16. As loversial –21. TherA. chinensis) been sequenced the cp genomes [Clematoclethra (C. lanosa) and two species of Actinidia (A. polygama and A. tetramera) from Actinidiaceae using genome skimming approach. By applying comparative genomic analyses we aimed to 1) test whether the cp genomes of Clematoclethra and Actinidia evolved congruently in spite of their contrasting genetic patterns; 2) identify the most variable cp genome-wide markers for subsequent evolutionary studies of the kiwifruit family; and 3) uncover the potentiality of cp phylogenomic practice at varying taxonomic levels in asterids.Genome skimming technique is so far one of the simplest and the most economical methodologies to obtain high-copy sequences –23, for  genomes . Undoubt genomes . In thisActinidia polygama (ZXZ15021), Actinidia tetramera (ZXZ15014) and Clematoclethra lanosa (ZXZ15007) were collected from Huoditang forestry farm of Qinling Moutains in Shaanxi Province, China, in July 2015. Field studies were permitted and supported by Northwest A&F University to whom the Huoditang forestry farm belongs as an education and research experimental base. The voucher specimens of these three species were all deposited at the Trees Herbarium of Northwest A&F University.Fresh leaves of c. 500 bp) for each species were constructed from fragmented genomic DNA following standard Illumina protocols . DNAs from different species were indexed by barcodes and pooled together. Prepared libraries for all three species were finally sequenced in one lane on the Illumina HiSeq 2000 platform in Beijing Genomics Institute (BGI) in Shenzhen, China.Total genomic DNA was extracted from fresh leaf tissues of single individual per species using CTAB method . Paired-de novo assembled using CLC Genomics Workbench v7.5 software . Contigs with length <300 bp and sequence coverage <50 were discarded [http://blast.ncbi.nlm.nih.gov/) against the cp genome of A. chinensis [Raw reads of each species were quality-filtered and iscarded . The remhinensis  which wahinensis  and Zhanhinensis .A. chinensis cp genome [Initial cp genome annotation was performed using DOGMA software . Start/sp genome . tRNA gep genome . OGDRAW p genome  was applhttp://pgrc.ipk-gatersleben.de/misa/). Minimum repeat unit was defined as 10 for mono-, 6 for di-, and 5 for tri-, tetra-, penta-, and hexanucleotide SSRs [Tandem and palindromic repeats were determined by applying program REPuter . Minimalide SSRs .A. chinensis (NC_026690) and A. chinensis var. deliciosa (NC_026691), were downloaded from GenBank. Together with our own sequenced cp genomes of A. polygama, A. tetramera and C. lanosa, we aligned these five cp genomes using MAFFT program [A. chinensis [Chloroplast genome sequences of two additional Actinidiaceae species, i.e.  program  and manu program  by referhinensis . Multiplhinensis . The numhinensis .Five Actinidiaceae cp genomes were analysed to identify rapidly evolving molecular markers which can be used in subsequent phylogenetic studies. Both the coding and non-coding DNA fragments >200 bp in each cp genome were extracted separately by applying “Extract Sequences” option of DOGMA . Then thArabidopsis thaliana (L.) Heynh. and Vitis vinifera L. from superrosids were defined as outgroups according to previous studies [Thirty-one complete cp genomes representing eight major lineages of the superasterids were included for phylogenomic analyses . Arabido studies , 48. Giv studies , we anal studies  in Genei studies , and thePhylogenetic analyses of the concatenated datasets were carried out by maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). MP analyses were implemented in PAUP* v4.0b10 . ParsimoML and BI trees were inferred using RAxML v.8.2.8  and MrBaA. polygama, A. tetramera and C. lanosa, repsetively   . The seqA. polygama, A. tetramera and C. lanosa was 156,583, 157,659 and 156,731 bp respectively , only rps12 and ycf3 contained two intervening introns, while others contained one intron each. The boundaries between IRs and single copy regions were conserved in Clematoclethra and Actinidia cp genomes, with ycf1 and psbA extending into the IRb  than that in two Actinidia species (34 and 41 respectively)  and A. tetramera , but only two types in C. lanosa (mono- and tri-nucleotide)  were investigated in our sequenced cp genomes. Tandem repeats that ranged from 10 to 20 bp were the most abundant, that had their sizes between 20 and 30 bp were secondarily abundant, while that with lengths greater than 30 bp were much less in all three species . Palindrctively) . Analysictively) . Not allleotide) .A. chinensis, A. chinensis var. deliciosa, A. polygama, A. tetramera and C. lanosa) were visually quantified by “percent identity plots” and estimated by the percentage of nucleotide substitutions (SNPs) (Sequence divergences among the five Actinidiaceae species (s (SNPs) . Global s (SNPs) . Across s (SNPs) . The ests (SNPs) . Coding s (SNPs) .rpl20, accD and ycf1 had their variations exceeded 5%. In contrast, there were 32 non-coding loci showed relative high degree of divergence  differed largely in coding and non-coding loci (>200 bp) in these five Actinidiaceae cp genomes . It showThe final length of the aligned matrix for the “data-incomplete” and “data-complete” data sets contained 91,222 and 58,876 unambiguously aligned nucleotide characters respectively. Because these two data sets generated congruent phylogenetic trees, we here only reported the results based on the 56 common protein-coding plastid genes (“data-complete” data set) as shown in Actinidia taxa clustered in a clade with high statistic supports (100/100/1.00), being sister to Clematoclethra. A. tetramera diverged firstly (95/97/1.00), followed by A. polygama (100/100/1.00) in the Actinidia lineage. As expected, A. chinensis and A. chinensis var. deliciosa were closely related (100/100/1.00).The Actinidiaceae were recovered as a monophyly in Ericales with high statistic supports (100/100/1.00). The phylogenomic tree indicated a branching order of Actinidiaceae, Ericaceae, Primulaceae and Theaceae as , Theaceae), Primulaceae) in Ericales, each node getting high support values (100/100/1.00). The sampled four Epipogium aphyllum and Epipogium roseum [Orobanche [Petrosavia stellaris [Clematoclethra species (C. lanosa) bears a highly conserved cp genome in terms of its architecture and linear sequence order with either the two newly sequenced Actinidia (A. polygama and A. tetramera) in this study or the published A. chinese cp genome [C. lanosa (156.7 kb) is similar to that of A. polygama (156.6 kb) and A. tetramera (157.7 kb)  , showing lanosa 1.7 kb is oclethra , no signclpP gene was completely lost in the sequenced cp genomes of Actinidia [this study, Clematoclethra (C. lanosa). The lack of certain plastid genes is not unusual since previous studies illustrated that several cp genes have been lost in certain groups, such like, the clpP gene in Primula poissonii [Camellia oleifera [accD gene in Trifolium [rpl32 gene in Populus [infA gene in rosids [clpP gene loss detected in both Clematoclethra and Actinidia is most likely a conspicuous common feature during their cp genomes evolution, suggesting that the clpP gene loss event might happen in their common ancestor. More species from the kiwifruit family, especially representatives of the third genus Saurauia, will be sequenced to determine whether clpP gene loss is synapomorphy of the Actinidiaceae.oissonii  and Cameoleifera , the accrifolium , the rpl Populus , and then rosids . The clpC. lanosa were more than that in two Actinidia species  [Alignment of our three cp genomes and two other published cp genomes of Actinidiaceae (liciosa)  uncovereliciosa) , which rliciosa) . Differeliciosa) , by codiliciosa)  or by geliciosa) –28, may Silene [accD (in the LSC region), rpl20 (in the LSC region) and ycf1 (in the SSC region). These exceptions display more SNPs than all other coding loci particularly with rpl20 as one which varies the most quickly. There have been some observations of accelerated variation rate of the plastid genes, such as psb in Poaceae [rps in Saxifragales [rps and clpP genes in Caryophyllales [Sequence variation analyses of the coding and non-coding loci (longer than 200 bp) across the five examined cp genomes indicate that each locus shows independent/distinct evolutionary patterns  as reveaSilene . In gene Poaceae , rps in fragales , and rpshyllales . Neverthhyllales , 70. It matK, psbC-trnS, rbcL, and trnL-trnF have been used most commonly in Actinidia phylogenetic studies [Actinidia, which will shed light on our understanding of the evolutionary history of this valuable economical plant group. The average proportion of variability in non-coding loci is higher (3.36%) than that in the coding loci (2.01%) -trnL(UAA) and rpl32-trnL(UAG) has been used in phylogenetic studies of intractable plant groups, e.g. in woody bamboos  with relatively robust solutions [Chloroplast DNA fragments such like  studies –21, 71,  (2.01%) . In addiequences –19. We tolutions . These 2The presence of missing data is not unusual in phylogenomic analyses, though its impact on phylogenetic evaluation has been widely debated –76. UsinActinidia taxa clustering in a clade as sister to Clematoclethra , are highly supported as stated previously , 46, 77.oclethra . The sis studies , 71. MorTo date, the genome skimming method has been thought as an economical and advanced technique with great potential not only in the plastomes but also for the mitochondrial and nuclear genomes studies –83. ChloClematoclethra and two species of Actinidia, using genome skimming approach. These finished cp genomes will facilitate the development of plastid genetic engineering for these valuable fruit species and provide additional information about the evolutionary history of cp genomes in the whole kiwifruit family. The cp genomes of Clematoclethra and Actinidia are highly conserved in the structure and content in spite of their contrasting genetic processes of plastomes. The clpP gene loss likely provides a prominent synapomorphic characteristic of Actinidiaceae. Obviously heterogenous divergence patterns in different regions of the Actinidiaceae cp genomes are revealed, according to which 20 rapidly evolving intergenic loci are identified as potential cp genome-wide markers for subsequent phylogenetic studies of this family. The Actinidiaceae and its subordinate genera are both strongly supported as monophyletic located in the Ericales in basal asterids. The phylogenetic relationships within the large asterids clade, even within the enigmatical genus Actinidia, have been robustly resolved based on the cp genomes data, suggesting the bright probability of cp phylogenomics for tackling difficult phylogenies in flowering plants.In summary, in the present study we generated cp genomes of three species from the family Actinidiaceae, including one species of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Clostridium difficile colonization to infection develops in large intestine. However, the real colonization pattern of C. difficile in preoperative colorectal cancer patients has not been studied. In this study, 33 C. difficile strains (16.1%) were isolated from stool samples of 205 preoperative colorectal cancer patients. C. difficile colonization rates in lymph node metastasis patients (22.3%) were significantly higher than lymph node negative patients (10.8%) . Meanwhile, patients positive for stool occult blood had lower C. difficile colonization rates than negative patients . A total of 16 sequence types were revealed by multilocus sequence typing. Minimum spanning tree and time-space cluster analysis indicated that all C. difficile isolates were epidemiologically unrelated. Antibiotic susceptibility testing showed all isolates were susceptible to vancomycin and metronidazole. The results suggested that the prevalence of C. difficile colonization is high in preoperative colorectal cancer patients, and the colonization is not acquired in the hospital. Since lymph node metastasis colorectal cancer patients inevitably require adjuvant chemotherapy and C. difficile infection may halt the ongoing treatment, the call for sustained monitoring of C. difficile in those patients is apparently urgent.The entire process of  Clostridium difficile infection (CDI) is one of the leading causes of antibiotic-associated diarrhea. World-widely, the incidence of CDI has increased significantly since ribotype 027 strains appeared at the beginning of the century. There have been a lot of outbreaks with severe cases reported in the United States and Europe. A recent study indicated that approximately 453,000 cases of CDI and 29,000 deaths associated with CDI were identified each year in the United States [d States . The risd States , 7.C. difficile mainly colonizes the large bowel as a part of normal intestinal flora. There are toxigenic and non-toxigenic strains. Toxigenic strains release exotoxins TcdA and TcdB to result in colitis and other diseases. Prevalence of C. difficile colonization for ICU patients [C. difficile colonization in adult patients are different in different regions, but significantly lower than those in children [C. difficile colonization have been demonstrated to serve as reservoirs for CDI [C. difficile strains might prevent CDI in humans, toxigenic C. difficile colonization is regarded as an independent risk factor prone to developing CDI subsequently [C. difficile colonization include previous hospitalization [patients , cancer patients , 10, patpatients , 12, respatients , and heapatients  has beenchildren . A subst for CDI . Althougequently . The rislization , 15, 17,lization , and hoslization –15.C. difficile colonization in preoperative colorectal cancer (CRC) patients has not been previously reported. The main transmission pattern and specific risk factors of C. difficile colonization in hospitalized CRC patients remains unclear.The intestinal microbiota is also believed to be directly involved in colorectal carcinogenesis . HoweverC. difficile colonization in preoperative CRC patients admitted in a tertiary teaching hospital in China. The aim of this study is to reveal the rate of C. difficile colonization and its correlation to clinical characteristics in preoperative CRC patients. In addition, genotypes and antibiotic resistance profiles of C. difficile strains in those patients were also analyzed.Here we performed a preliminary study to investigate the pattern of C. difficile. Among these thirty-three C. difficile isolates, twenty-eight (84.8%) were positive for both tcdA and tcdB (A+B+), four (12.1%) were negative for tcdA and positive for tcdB (A-B+), and one isolate (3.0%) was non-toxigenic with neither tcdA nor tcdB (A-B-). Toxigenic isolates were dominant at a rate of 97.0%. There was only one type of C. difficile isolate detected in each culture-positive stool specimen.A total of 205 preoperative CRC patients were included in this study. Among them, Thirty-three (16.1%) were positive for C. difficile colonization rate in male patients was 15.7%, whereas in female patients was 16.9% (P = 0.820). The mean age is 63.3 years old. The mean ages of the toxigenic C. difficile positive and C. difficile negative patients were 64.1 and 62.2 years old, respectively. As shown in Table C. difficile colonization rate in patients over 60 years old (P = 0.249). Among all CRC patients, 29 of the 173 (16.8%) left-sided CRC patients were positive for C. difficile, and 4 of 32 (12.5%) right-sided CRC patients were positive for C. difficile (P = 0.547), no colonization preference was found between different sides.There were 134 (65.4%) male and 71 (34.6%) female patients. C. difficile colonization. However, as shown in Table C. difficile colonization is also inconclusive (data not shown).Pathological features including cancer sites of origin (colon to rectum), macroscopic morphology (exophytic to ulcerative), and differentiation (well to poor) of the CRC patients were then analyzed for correlation with C. difficile colonization in CRC patients at different stages, a comparative analysis of rate of colonization between T4 and non-T4, LN metastasis and LN negative patients was performed. Interestingly, CRC patents with high risk factors are likely to have higher C. difficile colonization rate than others, as shown in Table C. difficile colonization than those of negative LN . Multivariate logistic regression analysis also showed that LN metastasis is an independent risk factor for C. difficile colonization in CRC patients (P = 0.044).Generally speaking, T4  and lymph node (LN) metastasis are of great importance for CRC patients as poor prognostic factors and indicators for adjuvant chemotherapy. While in terms of C. difficile colonization. Among all 205 CRC patients studied, OB negative patients had significantly higher C. difficile positive rate than OB positive patients . Multivariate logistic regression analysis also revealed OB negativity as an independent predictor for C. difficile colonization (P =0.004). No relationship was found between blood hemoglobin level and C. difficile colonization. Nutritional indexes and metabolic profiles of CRC patients, include albumin, fast blood glucose, triglyceride, total cholesterol, high density lipoprotein, and body mass index, were collected and analyzed for possible relation to C. difficile colonization. However, as shown in Table C. difficile colonization in CRC patients.CRC frequently causes chronic bleeding, inducing anemia and malnutrition. Therefore, stool OB and blood hemoglobin of the CRC patients were used as differential tools in correlation with C. difficile colonization has effected the duration of hospitalization was also studied. For the 172 C. difficile negative patients, the average days in hospital were 15.4 ± 0.34 days (mean ± s.e.m.), while the average days in hospital were 15.0 ± 0.98 days (mean ± s.e.m.) for the C. difficile positive patients, the result was of no statistical difference (P >0.05).In addition, whether C. difficile isolates into 16 STs, indicating a great diversity of the genotype of C. difficile colonization. As previously described, three toxin gene profiles were included labeled as A+B+, A-B+, and A-B-. ST35 (21.2%) was predominant which consists of 7 isolates. A minimum spanning tree was constructed . In addition, CRC patients over 60 years old carried more moxifloxacin susceptible isolates than patients less than 60 years old . Antibiotic resistance profiles were also correlated with MLST relationships. All ST35 isolates and all but one ST37 isolates were resistant to tetracycline. All ST 37 isolates were resistant to clindamycin. All but one ST35 isolates were resistant to erythromycin (data not shown). Notably, the only one non-toxigenic isolate, ST15, was resistant to tetracycline only, but susceptible to all other antibiotics. It implies that the non-toxigenic C. difficile isolate had a lower level of antibiotics resistance than toxigenic C. difficile isolate.Antimicrobial resistance of the 33 F. nucleatum, enterotoxigenic B. fragilis and adherent-invasive E. coli are all found to promote the adenoma-carcinoma sequence [CRC is among the most commonly diagnosed malignancy all over the world . Apart fsequence –24.C. difficile in CRC patients has not been determined yet. In a prospective study, 19% of patients were colonized with toxigenic C. difficile on admission to oncology [C. difficile colonization in admitted children in hematologic ward was reported to be 25.6%, with a 92.6% of toxigenic strains [C. difficile colonization in cancer patients [C. difficile colonization in preoperative CRC patients was high in China from our clinical experience. We speculated that antiemetic pharmaceuticals might inhibit patients’ diarrhea symptoms to some extent in the course of ongoing treatment. Furthermore, CDI clinical severity is generally mild to moderate in Chinese patients in our other studies [C. difficile colonization and CDI in cancer patients remains unclear. Further investigations are needed to clarify the risk factors triggering the transformation from C. difficile colonization to CDI in cancer patients.So far the exact colonization pattern of oncology . The rat strains . Our pre studies . However studies , the relC. difficile colonization. It is accepted that the colonization of C. difficile in large intestine is prevented by the barrier of the gut microbiota. Weakening of this resistance by cancer is the major risky condition leading to infection. Differences in colon microbiota between individuals with a normal colonoscopy and CRC have been reported [C. difficile colonization. From the pathological perspective, changes in the composition of the gut microbiota may lead to the instability of homeostasis, resulting inflammation, dysplasia, and carcinogenesis [C. difficile in CRC patients, especially during the adjuvant chemotherapy, might develop CDI and result in severe diarrhea, which in turn halts the ongoing chemotherapy [C. difficile colonization should be examined for all preoperativeCRC patients as a part of risk stratification for further cancer therapy.Stage T4 and LN metastasis are both high risk factors for recurrence and indications for adjuvant chemotherapy in CRC patients. Our results indicated that CRC patients with more advanced disease (T4 or LN metastasis) who definitely need adjuvant chemotherapy after surgery tend to have higher rate of reported , 29. In ogenesis –32. Presotherapy . TherefoC. difficile colonization and the sites of origin, histological morphology, and metabolic factors of the cancer. Although a previous study demonstrated a predictable role of albumin in CDI [C. difficile colonization in CRC patients. However, we notably found OB negative patients had a higher rate of C. difficile colonization. Previous studies also disclosed that there are extremely few CDI patients with positive fecal OB [C. difficile colonization. Moreover, blood components might also hinder C. difficile colonization or CDI. More studies are needed in order to clarify the relationship between OB and C. difficile colonizationThere were no clear correlation between the rate of n in CDI , none offecal OB . The posThere was a specific correlation between antibiotic resistance and clinical characteristics, indicating that the individualized therapeutic scheme should be considered according to clinical characteristics of the patients. All the isolates in this study were susceptible to metronidazole and vancomycin with low minimal inhibitory concentration. Besides, the resistant rate to piperacillin-tazobactam is relatively low, which suggests it can serve as an alternative option for treatment of CDI in CRC patients. The non-toxigenic isolate was resistant only to tetracycline, indicating a significant narrower antibiotic resistance in the non-toxigenic isolates than that in the toxigenic isolates.C. difficile colonization are lower [C. difficile from same patients 48 hours after admission. The results showed the molecular spectrum of C. difficile isolates was still wide (data not shown), suggesting these patients were community-acquired according to the 2010 SHEA/IDSA guideline [C. difficile transmission pattern in China.Compared with hospital-acquired infection, morbidity and mortality associated with community-acquired re lower . All theuideline . The finC. difficile colonization in CRC patients in China. CRC patients harboring high risk factors such as T4 or LN metastasis tended to have higher C. difficile colonization rate. Therefore, peri-operative screening and monitoring for C. difficile is of great importance in these patients in order to avoid discontinuation of chemotherapy due to severe diarrhea and postoperative complications. Besides, OB negativity increases risk of C. difficile colonization in CRC patient. As high as 97.0% of colonized C. difficile strains were toxigenic with multiple antibiotics resistance. C. difficile colonization may not be mainly acquired in hospitals due to the absence of epidemiologic relatedness in preoperative CRC patients. A larger-population-based study and mechanism-specific research is needed in the future in order to clarify the exact role of C. difficile colonization in CRC patients.In conclusion, this is a preliminary study in determining prevalence and revealing risk factors of Stool samples were collected from consecutive preoperative CRC patients who were scheduled to accept radical cancer resection in Department of Colorectal Surgery, the First Affiliated Hospital of Zhejiang University. The study protocol was approved by the Institutional Ethics Committees of the hospital and the study was performed in accordance with the guidelines recommended by National Institute of Health involving human subjects and animal care and 1975 Declaration of Helsinki. All patients provided written consent. Stool samples within 24 hours of admission were collected from each patient, and delivered to Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention for culture, identification of toxin gene and antibiotic susceptibility testing.The study was performed between October, 2014 and August, 2015. All CRC patients were to undergo surgical resection after stool sample collection, with no preoperative chemotherapy received. No notable long-term diarrhea was reported by the patients. No antibiotics were used prior to the stool sample collection. Re-admitted patients were excluded. Demographic information and pathological findings collected from the patients include age, gender, body mass index, cancer stages , cancer differentiation, and sites of origin. Laboratory results such as blood hemoglobin level, stool occult blood (OB), fasting blood glucose, serum albumin, triglyceride, total cholesterol, and high density lipoprotein were also included.C. difficile isolation from the stool collected within 24 hours of admission was processed as the follows. Specimens were firstly treated with alcohol. The mixture was then inoculated into cefoxitin-cycloserine fructose agar (CCFA) plates . After incubation for 48 hours at 37°C in a GENbag anaerobic chamber , up to ten suspected colonies for each stool specimen were analyzed and confirmed to be C. difficile as described by Loo et al [C. difficile isolates were stored within the blood broth in a preservation kit  at -80°C.tcdA and tcdB) in order to determine between non-toxigenic and toxigenic isolates. Seven housekeeping genes  selected based on a previous study by Griffiths et al. were used for multilocus sequence typing (MLST) [C. difficile MLST database, accessible at http://pubmlst.org/cdifficile, for determining sequence types (ST). The eBURST was used to identify clonal complexes, each of those are defined by one of the seven typed genes, which bear different features from one another. Minimum spanning tree that reveals the allelic difference between isolates of the seven housekeeping genes was constructed by application of Bionumerics software (Applied Math). The Simpson's index of diversity  was calculated with an in-house program, MLEECOMP package.All bacterial colonies for each stool specimen were identified. Bacterial genomic DNAs were extracted using DNeasy Blood & Tissue kit  according to the manufacturer's instructions. A multiplex real time PCR assay provided by the UK Bio company in Hangzhou was used for detection of the two toxin genes (g (MLST) . After tIn vitro testing for susceptibilities of antibiotics including fusidic acid, ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, piperacillin-tazobactam, rifampicin, clindamycin, erythromycin, tetracycline, metronidazole and vancomycin was carried out using an E-test assay according to the supplement from Clinical and Laboratory Standards Institute (CLSI)[te (CLSI). Six E-tte (CLSI)–41.P < 0.05.Statistical analysis was performed by applying SPSS 20.0 . Categorized data were analyzed with the chi-squared test. The F-test and T-test were conducted for hetero or equal variance analysis. Significance of characteristics in individual patients was determined by multivariate logistic regression analysis. Statistical significance for all tests was defined as


Tuber indicum) on the diversity of microbial communities associated with an indigenous tree, Pinus armandii, and the microbial communities in the surrounding ectomycorhizosphere soil. High-throughput sequencing was used to analyze the richness of microbial communities in the roots or rhizosphere of treatments with or without ectomycorrhizae. The results indicated that the bacterial diversity of ectomycorhizosphere soil was significantly lower compared with the control soil. Presumably, the dominance of truffle mycelia in ectomycorhizosphere soil (80.91%) and ectomycorrhizae (97.64%) was the main factor that resulted in lower diversity and abundance of endophytic pathogenic fungi, including Fusarium, Monographella, Ustilago and Rhizopus and other competitive mycorrhizal fungi, such as Amanita, Lactarius and Boletus. Bacterial genera Reyranena, Rhizomicrobium, Nordella, Pseudomonas and fungal genera, Cuphophyllus, Leucangium, Histoplasma were significantly more abundant in ectomycorrhizosphere soil and ectomycorrhizae. Hierarchical cluster analysis of the similarities between rhizosphere and ectomycorrhizosphere soil based on the soil properties differed significantly, indicating the mycorrhizal synthesis may have a feedback effect on soil properties. Meanwhile, some soil properties were significantly correlated with bacterial and fungal diversity in the rhizosphere or root tips. Overall, this work illustrates the interactive network that exists among ectomycorrhizal fungi, soil properties and microbial communities associated with the host plant and furthers our understanding of the ecology and cultivation of T. indicum.The aim of this study was to investigate the effect of an ectomycorrhizal fungus ( Tuber genus , are ectomycorrhizal fungi (ECMF) that produce hypogeous fruiting bodies [Quercus, Corylus, Pinus and Abies [Tuber melanosporum) and the white truffle (T. magnatum) are highly valued on the European market because of their unique flavors [T. indicum, commonly known as the Chinese black truffle and one of China’s major commercial species [T. melanosporum. Polysaccharides and ribonuclease isolated from its fruiting bodies and fermentation system showed a strong antitumor and antiproliferative activity [T. indicum forms typical ectomycorrhizae structures in Chinese indigenous plants, such as Castanea mollissima and Pinus armandii, and ectomycorrhizae has been successfully synthesized in China [Truffles, belonging to the g bodies , 2 that nd Abies –6. The b flavors –9. T. in species , 11. shaactivity , 13. T. in China . The synTuber spp. may exert different competitive effects on other mycobionts. Ectomycorrhizae can be beneficial to plant productivity by enhancing plant growth or resistance to abiotic stress [Ectomycorrhizal fungi play an important role in ecosystems. Truffle species often form brûlé (an area devoid of herbaceous cover) around a host tree, and reduce biodiversity of bacteria and fungi in the brûlé area , 16. Truc stress –26. Ectoc stress –29. The c stress . NeverthT. indicum and Pinus armandii with the goal of testing how the ectomycorrhizal symbiotic relationship affects microbial diversity and the communities associated with ectomycorrhizae and the soil surrounding the host plant during the early symbiotic stage. We also analyzed changes in soil properties and their correlation with microbial communities to lay a foundation for artificial cultivation of T. indicum and reveal the microbial interactions among ectomycorrhizal fungi, surrounding soil communities and the host plant.Although cultivation of truffles has been attempted for decades, the frequent failure to develop productive plantations indicates that the reproduction, cultivation and conditions stimulating ascocarp formation remain mysterious. Endophytes and soil microbes play an important role in the growth and development of the plant host –34. TheyTuber indicum fruiting bodies were found most frequently in the soil surrounding the indigenous plant, Pinus armandii. To reveal the effects of ectomycorrhizal fungi on rhizosphere soil and the microbial community associated with host plant, the ectomycorrhizae of T. indicum and P. armandii were artificially synthesized in greenhouses. P. armandii seeds were purchased from a commercial company. Seeds were surface sterilized with 30% H2O2 for 4 h and washed three times with distilled water [P. armandii forest and identified by morphological and molecular analysis) that had been surface sterilized with 75% alcohol, and soaked with sterile water to incite the spores to be released and germinate. We inoculated 2 g of this spore powder into the substrates. Three P. armandii seedlings that were not inoculated with truffle spores served as controls. All pots (each treatment had three replicates) were maintained in the greenhouse under the same conditions. Plants were watered every three days. No fertilizers were added to the plants [P. armandii roots, mycorrhized with truffle mycelia (ECM) or not colonized (CK), were surface sterilized, and separately with their surrounding soil, were extracted for their DNA for high throughput sequencing. The properties of soil samples around the roots were analyzed according to our previously described method [P. armandii mycorrhized with T. indicum were assigned to ECM (ectomycorrhiza) and the surrounding soils were assigned to ECM.S. Roots from the control trees lacking the T. indicum partner were assigned to CK and soils were assigned to CK.S. All experiments were conducted in triplicate.In southwest China, ed water . Surfaceed water  . PCR was conducted according to Li et al [® DNA PCR-Free Sample Preparation Kit  following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina HiSeq 2500 platform and 250 bp paired-end reads were generated [All samples 16S V4 and ITS1 genes were amplified using the universal primers 515F-806R and ITS5-1737F with the barcode as a marker for distinguishing samples respectively . All PCRLi et al . PCR proenerated .http://www.mothur.org/wiki/Chao), Shannon, Simpson, ACE (http://www.mothur.org/wiki/Ace), Good-coverage. All this indices and beta diversity in our samples were calculated with QIIME (Version 1.7.0). Raw reads were submitted to Sequence Read Archive (SRA) database (accession number: SRR5278654-SRR5278665).Paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Reads that overlapped, which were generated from the opposite end of the same DNA fragment, were merged using FLASH . QualityData of this study are presented as means ± standard deviation (SD) of three biological triplicates for each treatment. Statistical analysis was carried out by one-way analysis of variance (ANOVA) using SPSS 19.0. Least significant difference (LSD) was performed to test if the ANOVA result between different treated groups was significant at P < 0.05.To determine the composition of bacterial communities in the ectomycorrhizae and in the surrounding soil, we carried out 16S V4 rRNA barcoded pyrosequencing. 45,077–79,441 reads were obtained per sample after quality control procedures . AltogetAbout 38,052–73,338 qualified reads were obtained per sample , represeP. armandii root tips differed between treatments with or without T. indicum , Bacteroidetes (6.45%–13.87%), Actinobacteria (4.00%–14.88%), Acidobacteria (1.45%–9.14%) and Verrucomicrobia (1.90%–8.26%) occupied the dominant position in all samples. Verrucomicrobia were found in greatest abundance in ECM and ECM.S than in other samples (P <0.05). The relative abundance of Gemmatimonadetes was significantly lower in ECM.S than in CK.S (P < 0.05). ECM contained more Proteobacteria than CK (P < 0.05).Each bacterial 16S rRNA gene sequence was taxonomically assigned from the phylum level to the species level based on RDP 3 classifier. A total of 41 bacterial phyla were identified, out of which 23 were identified in all 12 samples . Two arcAmong the 81 bacterial classes detected, Alphaproteobacteria (21.28%– 31.65%), Betaproteobacteria (10.46%– 27.88%), Deltaproteobacteria (4.54%– 9.79%) and Gammaproteobacteria (5.24%– 7.84%) were the most abundant . ECM andStreptomyces (average 3.45%), Pseudolabrys (2.08%), Opitutus (1.65%), Pseudomonas (1.55%), Ohtaekwangia (1.44%), Dactylosporangium (1.44%) and Mesorhizobium (1.29%). Mesorhizobium, Reyranena, Rhizomicrobium and Nordella were significantly more abundant in ECM.S than in CK.S (P < 0.05). Pseudomonas was more abundant in ECM compared with CK (P < 0.05).190 of the 439 genera were identified in all samples . Among tA total of six phyla were observed in soil and root samples . The relAt the class level, Pezizomycetes (average 46.82%), Sordariomycetes (26.79%), Eurotiomycetes (10.03%), Agaricomycetes (5.61%) and Incertaesedis Zygomycota (1.13%) were the dominant taxa in all samples, of the 21 classes detected . PezizomTuber (average relative abundance of 89.28% in ECM and ECM.S), Monographella (average 7.48%), Cuphophyllus (2.04%) and Melanconiella (1.34%). Tuber was only detected in soil and P. armandii roots inoculated with T. indicum. Cuphophyllus, Melanconiella and Gonapodya were not observed in the ECM treatment.A total of 122 genera were observed, and 27 of these were detected in all samples . The mosT. indicum in association with P. armandii, the associating bacterial community structure was significantly different compared with the control treatments, implying that the ectomycorrhizal fungi (ECMF) can affect the structure of the microbial community on its host and in the surrounding rhizosphere soil.The differences in bacterial and fungal communities between the samples were estimated using UniFrac analysis . In the There were significant correlations between some soil properties and the diversity of microbial communities . Clay coEffective nitrogen and available copper were negatively correlated with soil fungi according to the Shannon index, observed fungal species and the Shannon index . Fungal T. indicum ectomycorrhizal association on the microbial communities of Pinus armandii and the surrounding ectomycorrhizosphere soil during early symbiotic stage. When T. indicum was present it was dominant (80.91% in ectomycorrhizosphere soil and 97.64% in ectomycorrhizae) reducing bacterial and fungal diversity in the root and in the surrounding soil , which indicated that the truffle mycelia exert specific effects on the surrounding microbial communities. Previous study[Sinorhizobium, Rhizobium and Bradyrhizobium spp and γ-Proteobacteria, mostly fluorescent pseudomonads, were predominant components of truffle bacterial communities. Our results also indicated a more abundant Bradyrhizobium, Rhizobium and Pseudomonas communities existing in ectomycorrhizosphere soil and ectomycorrhizae compared with the CK and CK.S, indicating that these species are closely related to the presence of truffle mycelia and may play an important role in the growth and mycorrhizal synthesis of truffles. Previous study indicated that T. melanosporum ascocarps selected specific bacterial communities from the surrounding soil which may contribute to the development, maturation and even aroma of the Black truffle [T. indicum shaped the microbial communities of ectomycorhizosphere soil and ectomycorrhizae during the colonization of P. armandii even in the symbiotic stage.Previous work has shown evidence for reduced biodiversity of bacteria and fungi in the brûlé area in cultivated or wild conditions , 19, 50.ous study found th truffle , 51. OurReyranena, Rhizomicrobium, Nordella and Pseudomonas and some fungal genera, such as Cuphophyllus, Leucangium and Histoplasma were significantly enriched in ectomycorrhizosphere soil and ectomycorrhizae compared with CK.S and CK. Previous studies showed that combination of Pseudomonas fluorescens and T. melanosporum improved the establishment and functioning of ectomycorrhizal symbiosis [Pseudomonas was more abundant in ectomycorrhizosphere soil. However, it was clear that the dominance of truffle mycelia did reduce the diversity and composition of endophytic pathogenic fungi, such as Fusarium, Monographella, Ustilago and Rhizopus and other competitive mycorrhizal fungi, such as Amanita, Lactarius and Boletus, supporting previous work that suggests ectomycorrhizal fungi can have a protective effect on the growth of plant host [Despite the reduced diversity of bacteria and fungi in ectomycorrhizosphere soil and ectomycorrhizae compared to the control samples, some bacterial genera, such as ymbiosis . Pseudomant host –54, for T. indicum mycelia were dominant (80.91%– 97.64%) among the fungal communities in the ectomycorrhizosphere and ectomycorrhizae, which was consistent with other studies. Whereas in another study, T. magnatum ectomycorrhiza seem to be absent or very rare when its ascomata formed and other mycorrhizal fungi were co-localized in the same root tips [oot tips , 55. Thioot tips , revealeTuber melanosporum) production are well known. Compared with the black truffle T. melanosporum and the Italian white truffle T. magnatum, which are more suitable for alkalescence soil [T. indicum is often found in slightly acidic soil [Hierarchical cluster analysis of the similarities between rhizosphere and ectomycorrhizosphere soil based on the soil properties differentiated significantly , indicatnce soil , 57, T. dic soil . MeanwhiSuccessful ectomycorrhizal synthesis is the basis of efficient cultivation , 4, 5. IS1 FigPinus armandii mycorrhized with Tuber indicum) and ectomycorrhizosphere soil. CK and CK.S, roots and soils from cultivated P. armandii without T. indicum partner.In the rarefaction curves, the number of OTUs increased with sequencing reads. ECM and ECM.S, ectomycorrhizae ((DOCX)Click here for additional data file.S2 FigPinus armandii in association with Tuber indicum) and ectomycorrhizosphere soil. CK and CK.S, roots and soils from cultivated P. armandii without T. indicum partner.ECM and ECM.S, ectomycorrhizae ((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Mesoplodon densirostris and the Cuvier’s beaked whale Ziphius cavirostris, confounded expectations of a simple allometric relationship, with exceptionally long (mean: 46.1 min & 65.4 min) and deep dives (mean: 1129 m & 1179 m), and comparatively small body masses (med.: 842.9 kg & 1556.7 kg). These two species also exhibited exceptionally long recovery periods between successive deep dives, or inter-deep-dive intervals . We examined competing hypotheses to explain observed patterns of vertical habitat use based on body mass, oxygen binding protein concentrations, and inter-deep-dive intervals in an assemblage of five sympatric toothed whales species in the Bahamas. Hypotheses were evaluated using dive data from satellite tags attached to the two beaked whales , as well as melon-headed whales Peponocephala electra (n = 13), short-finned pilot whales Globicephala macrorhynchus (n = 15), and sperm whales Physeter macrocephalus (n = 27). Body mass and myoglobin concentration together explained only 36% of the variance in maximum dive durations. The inclusion of inter-deep-dive intervals, substantially improved model fits (R2 = 0.92). This finding supported a hypothesis that beaked whales extend foraging dives by exceeding aerobic dive limits, with the extension of inter-deep-dive intervals corresponding to metabolism of accumulated lactic acid. This inference points to intriguing tradeoffs between body size, access to prey in different depth strata, and time allocation within dive cycles. These tradeoffs and resulting differences in habitat use have important implications for spatial distribution patterns, and relative vulnerabilities to anthropogenic impacts.Dive capacity among toothed whales (suborder: Odontoceti) has been shown to generally increase with body mass in a relationship closely linked to the allometric scaling of metabolic rates. However, two odontocete species tagged in this study, the Blainville’s beaked whale  Eubaleana spp. ) in muscles represent dimensions over which tissue oxygen reservoirs, and hence dive durations, can vary ,28,29. N. (2013)  found a . (2013) , and on . (2013) ,31.Leptonychotes weddellii completed >92% of dives within ≤26 min, necessitating minimal recovery time (<10 min) between dives during which time gases were exchanged ) was an additional covariate considered in the subsequent modelling of dive durations and dive depths. Species-level mean myoglobin concentrations from epaxial muscles (longissimus dorsi) were obtained from a variety of published sources , as well as skin biopsy samples collected using a remote dart biopsy technique , see Tmax potentially unrealistically collapsed all of the variation within each individual’s dive behavior to a single number, we also developed log-log PGLMM models of dive duration (T) and dive depth (Z) at the level of individual dives in For comparison with previous modeling efforts, we developed models of esiduals . Villemeesiduals  further y (2009) , as wellet al. (2009) , and IDDI or were able to extract these values from supplemental materials. The additional species included: belugas Delphinapterus leucas . This improvement in model fit was demonstrated by: 1) the selection of Model 2b (IDDI and m) as the most parsimonious model fit , and ziphiids (65 min). In addition to the IDDI of the two ziphiids being comparatively long in absolute terms  compared to ratios of 0.39, 0.38, and 0.17 among the delphinids and physeterids .Including differences in IDDI shown in  Mod 2b,  explaineodel fit  and 2 thte terms , these Ite terms , with meTmax, in this case over a wider range of taxonomic diversity than the species tagged in the present study. This analysis included representative species from all six primarily marine  families of Odontoceti. m and Tmax space, while the Ziphioidea (Family: Ziphiidae) were clearly clustered apart from these two superfamilies.The comparison of PGLS models in M. densirostris and Z. cavirostris individuals in temperature/approximate depth ranges thought to be associated with foraging activity , P. macrocephalus spent an average of 2.05x more of their time engaged in foraging dives relative to M. densirostris. Averaged over an entire diel cycle, the two delphinid species (P. electra and G. macrorhynchus) spent an average of 27.2% and 31.6% of their respective time budgets within target foraging strata, only moderately higher than the 24.0% and 22.6% spent by the two ziphiids  in over 869 hours of daytime behavior log and TAT data. Foraging dive activity in this species appears to be exclusively confined to nighttime periods. G. macrorhynchus, with the second shallowest median dive depths, undertook daytime dives that were on average, 417 m deeper and 69.9% less frequent than nighttime dives and appear to be concentrated during hours of lower incident light angles (early morning and late afternoon). Unlike any other species tagged in this study, the distribution of nighttime dives in G. macrorhynchus lacked bi-modality, with a continuous distribution from the central mesopelagic zone to near the surface. However, the nighttime dive pattern of G. macrorhynchus did differ between the sexes of this highly sexually dimorphic species, with adult males (2.05x larger by mass) showing greater consistency between daytime and nighttime dive depths, in contrast to greater variation between daytime and nighttime foraging dive depths of the smaller-bodied females. Overall P. macrocephalus displayed small diurnal differences between median daytime (920 m) and nighttime (888 m) dive depths. This pattern also varied among sexes and age classes: females and juvenile males that were tagged within matrilineal social groups dove on average to 3.5% shallower depths and exhibited a 26.4% larger diurnal difference in dive depths when compared with sub-adult males that were tagged either solitarily or in bachelor groups. M. densirostris exhibited daytime dives that were on average 142.3 m or 11.6% shallower than nighttime dives. This diurnal difference in dive depths was not observed in all tagged M. densirostris, but was detected in 5 of 7 individuals. Z. cavirostris, the deepest diving species, showed little to no variability in foraging dive depths between day and night.The tagged species in this study showed divergent responses to light levels, both in terms of dive depth and dive frequency . The shaP. macrocephalus, M. densirostris, and Z. cavirostris) were more frequently localized over areas where the bottom was within reach during their dives (P. electra and G. macrorhynchus) were proportionally less frequently localized over habitats where the benthos fell within their respective dive depth ranges , while the remaining two individuals  ranged widely over a variety of benthic depths that were beyond the range of their dive capacities  were consistently localized along the northern slope of the Great Bahama Canyon in an area where the benthos fell within their dive range. The shape of this distribution pattern also appears to correspond closely with the gradual shoaling of bathymetric topography from east to west along this slope. By contrast, sub-adult males (n = 16) that were typically encountered solitarily or in small bachelor groups were found to range widely over a variety of bathymetric habitats that were both shallower and deeper than their maximum dive depth (1344 m). Finally, the dive depths of Z. cavirostris showed a relatively high correlation with bottom depths (ρ = 0.62), across a wide range of bathymetric depths occupied by this species . All other species  showed low overall correlations of dive depths with bottom depths (ρ = 0.16–0.27).There was also some intra-specific variation in these patterns. In particular, pacities . P. macrZ. cavirostris (med. 1557 kg) relative to M. densirostris (med. 843 kg) also pointed to the relevance of body mass as a predictor of dive capacities. However, the comparatively low proportion of the overall variance in observed maximum dive duration (Tmax) explained by body mass (m) alone, suggested a need for additional covariates, especially to explain the dive durations of the two beaked whale species.Similar to previous research examining the relationship of dive duration with body mass –21, thisMoreover, selection on odontocete body size is likely considerably more complex than a simple optimization of dive efficiency and duration, so it is important to consider alternative hypotheses for the evolution of this trait. For example, larger body size generally reduces the per-unit-mass metabolic rate and external surface-area-to-volume ratios, fostering greater overall efficiency of energy use and thermoregulation ,64. Greae.g., blood, muscle, lungs), relative to metabolic rate  among ziphiids did not appear to be exceptional relative to other specialist deep-diving odontocete taxa  than glycolytic Type II fibers [Mb = 4.80) but overall similarity of bulk [Mb] between the beaked whales and other deep diving species [Mb might allow the beaked whales to more tightly space myoglobin units within proportionally rare Type I fibers.Regular reliance on anaerobic respiration may also account for several intriguing findings in previous studies ,74,91 whI fibers –94, migh species ,21,60,74e.g., brain and visceral tissues) versus low-cost tissues  when compared to other deep-diving species [Mesoplodon spp. and Z. cavirostris exhibit large-diameter muscle fibres, which reduce cellular surface-area-to-volume ratios and may thus minimize the metabolic demand of the active ion pumps needed to maintain muscle fibre membrane potential [G. macrorhynchus [In addition to the likely extension of foraging dives beyond ADL, ziphiids also exhibit a range of other metabolically economical adaptations that may allow further extension of dive durations. These include differences in the proportion by weight of tissue that incur high metabolic maintenance costs  [P. electra  [M. densirostris, Z. cavirostris, and P. macrocephalus, to varying degrees, interacted with prey layers tied geographically to benthic habitats. A specific example was the close match between the wedge-shaped westward spreading of tracks belonging to female P. macrocephalus and the gradual shoaling of benthic topography into the dive range of female P. macrocephalus along the northern slope of the Great Bahama Canyon. Furthermore the correlation of Z. cavirostris dive depths with benthic depths at estimated dive locations also circumstantially supported a hypothesis of BBL prey use. Intriguingly M. densirostris exhibited some inter-individual variation in spatial association with the benthos, which may reflect the flexibility in foraging strategy  documented by Arranz et al. (2011) [M. densirostris also exhibited shallower daytime and deeper nighttime dives, which may indicate either the pursuit of prey that undertook a reverse diel vertical migration, or a diurnal switch between mid-water and lower meso- and benthopelagic resources as shown by Arranz et al. (2011) [Benthic echoes recorded by digital acoustic recording tags (DTAG) deployed on ng dives . Althoug. (2011) , it also. (2011) . As furt. (2011) .et al. (2006) [In conclusion, this study confirmed that body mass is an important correlate of dive durations in toothed whales and is integral to their ability to access prey at different depths. However, substantial evidence also supported the hypothesis of Tyack . (2006)  that theS1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.


Mycobacterium mucogenicum is a rapidly growing mycobacterium found ubiquitously in water sources. It has been reported to cause widespread infections with infection entry from wound or central venous catheters especially in immunocompromised patients. Diagnosis is made from blood cultures which may take at least a week. Management includes removal of the source or drainage of wound infections and combination antimicrobial therapy. Mycobacterium mucogenicum belongs to the group of rapidly growing NTM, which are environmental organisms that survive in water distribution systems and tap water [M. chelonae-like organism [M. mucogenicum, is derived from the mucoid cell surface noted upon growth on the solid media. This mucoid surface provides the ability to form a protective biofilm, which facilitates infections of central venous catheters (CVC) and posttraumatic wounds, clinically the most common sites for M. mucogenicum infections [Nontuberculous mycobacteria (NTM) were first recognized as pathogens in the mid-1950s . Many spap water , 4. The fections , 4. The fections .A 46-year-old man with a history of heroin use presented to the emergency room with right hand pain, redness, and swelling four days after attempting to inject heroin into his vein but missing. The wound had progressively worsened to the point of severely limiting hand motion. The patient also reported fevers and chills. He was actively injecting heroin till the day of presentation but denied sharing needles. The past medical history was relevant for uncontrolled diabetes mellitus, hypertension, chronic hepatitis C infection, and chronic kidney disease stage III.Upon presentation, the patient was afebrile (temperature: 100.3°F) with a pulse of 96 beats per minute (bpm), blood pressure of 177/96 mm Hg, respiratory rate of 13/min, and had 98% saturation on room air. Physical exam was unremarkable except for generalized erythema and swelling of the right hand with decreased gross sensation and power in the right hand.µL with 80.8% neutrophils. Lactate was 2.8 meq/l. Blood cultures were sent, and the patient was started on intravenous (IV) vancomycin, piperacillin-tazobactam, and fluids for sepsis management. X-ray of the right hand showed dorsal soft tissue swelling without any other acute abnormalities or radiopaque foreign body. Urine drug screen was positive for heroin, opiates, and benzodiazepines. HIV screen was negative. Plastic surgery performed 2 incisions on the dorsum of the right hand which drained clear fluid without pus. Counterincision was made on the distal part on the dorsal aspect of the hand (Staphylococcus aureus (MSSA). His wound culture was also positive for MSSA. Upon return, he was afebrile (temperature: 97.9°F) with a pulse of 100 bpm, blood pressure of 177/96 mm Hg, respiratory rate of 13/min, and had 99% saturation on room air. He had persistent pain but was able to move his fingers. He acknowledged inhaling heroin before coming in. He was started on intravenous ampicillin/sulbactam, and blood cultures were sent on day 1 of second admission. White count was 11,400/µL with 70.5% neutrophils. Transthoracic echocardiogram was negative for vegetation.White blood cell count was 10,800/the hand . CultureµL), chest X-ray (negative for consolidation), and HIV viral load (negative) were done. On day 10, M. phocaicum was identified preliminarily via best match . Unfortunately, no wound cultures were sent for AFB testing. IV amikacin and cefoxitin with probenecid were started, and intravenous ampicillin/sulbactam was discontinued. Questioning to determine a source of infection revealed that the patient had used tap water and phenol as a solvent for heroin. Repeat blood cultures were negative. Mycobacterium was later confirmed as M. mucogenicum. Amikacin was stopped due to acute kidney injury, and the patient was instead put on meropenem and cefoxitin for 2 weeks followed by doxycycline 100 mg twice daily and trimethoprim-sulfamethoxazole (TMP-SMX) daily for 6 weeks. On follow-up, he had completed full course of antibiotics and his hand had improved significantly . Repeat cultures from day 1 of second admission also came back positive for AFB. TB QuantiFERON (negative), CD4 count (610/ficantly .M. mucogenicum is ubiquitously found in public and hospital tap water, as well as in shower faucets and environmental surfaces. There have been several case reports linking M. mucogenicum infections to potable water sources [M. mucogenicum are central venous catheter and wound infections in immunocompromised persons [M. mucogenicum bacteremia in bone marrow transplant recipients and oncology patients caused by water contamination of central venous catheters during bathing [M. mucogenicum infections of the central nervous system in immunocompetent hosts, skin lesions in the presence of the tumor necrosis factor-α antagonist etanercept, peritonitis associated with peritoneal dialysis, bacteremia in patients with hepatitis B and D associated cirrhosis with negative ascites fluid cultures, bone marrow involvement with retinal lesions and acute respiratory distress in a patient with idiopathic CD4+ T lymphocytopenia, and wound infections [M. mucogenicum is the most common RGM implicated in central line-associated bloodstream infections [M. mucogenicum [ sources , 7, 8. T persons , 8. Equi persons , 2. Klinfections . Undertrogenicum .M. mucogenicum is an organism that resides in water bodies and soil, a complete environmental assessment needs to be performed. It is crucial to collect water samples from dialysis machine, RO, and incoming water supply [Due to slow growth of mycobacteria, diagnosis is often difficult and delayed. Appropriate blood cultures are critical and may need verification with repeat cultures . Differer supply .M. mucogenicum infections includes both removal of indwelling catheters, similar to management of other RGM, and combination antimicrobial therapy [M. mucogenicum is generally more susceptible to antimicrobials [M. mucogenicum most susceptible to antibiotics including 100% susceptibility with amikacin, clarithromycin, imipenem, and TMP-SMZ [M. mucogenicum but do state that most isolates are susceptible to multiple antimicrobial agents including aminoglycosides, cefoxitin, clarithromycin, minocycline, doxycycline, quinolones, TMP-SMX, and imipenem [RGM are difficult to eradicate from venous catheters since they survive by biofilm formation. Successful therapy requires removal of the catheter or other infected devices in addition to appropriate antimicrobial agents , 4, 9. M therapy . RGM arecrobials . Howevercrobials . El Helo TMP-SMZ . Acquire TMP-SMZ . The curimipenem , 4, 9. Aimipenem . Empiricimipenem .M. mucogenicum, a rapidly growing NTM present in contaminated water, should be recognized as a potential source of infection, especially in the immunocompromised population [M. mucogenicum infection should be considered early in the care of transplant patients or of otherwise immunocompromised patients with suspected catheter-related bloodstream infection or peritonitis.RGM are emerging pathogens in the immune-compromised host . M. mucopulation . M. muco


Pseudomonas gingeri LMG 5327 (NCPPB 3146), the causative agent of ginger blotch in Agaricus bisporus, is reported. Together with another mushroom pathogen, Pseudomonas agarici, it belongs to a distinct phylogenomic group.The draft genome sequence of  Pseudomonas gingeri is responsible for ginger blotch disease on Agaricus bisporus fruit bodies (–P. agarici (Pseudomonas gingeri type strain LMG 5327 (NCPPB 3146), determined by an Illumina HiSeq 2000 sequencing system. A total of 9,604,938 reads were used for de novo assembly with the Velvet assembler , were generated. The final assembled length comprises 7,643,850 bp, with a G+C content of 62.6%, and the longest contig size is 252,545 bp. Automated annotation using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP)  to support interference competition (P. gingeri. The presence of a luxR-luxI gene pair can be linked to the high-level production of 3-oxo-C4-homoserine lactone (Pseudomonas alkylphenolica (The characteristic secretion systems of proteobacteria are all present in petition . The cappetition . Besides lactone . Adding henolica , and dethenolica .P. gingeri LMG 5327 reported here is a valuable source of information for studying the bacterium’s interaction with its host and its pathogenicity.The draft genome sequence of POWE00000000. The version described in this paper is version POWE01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


Artemia salina. Feeding assays consisted in 1-h incubations of brine shrimps with the toxic Prorocentrum lima, Gambierdiscus excentricus and Ostreopsis cf. ovata and the non-toxic Tetraselmis sp. Brine shrimps fed unselectively on all toxic and non-toxic algal preys, without significant differences in clearance and ingestion rates. Acute toxicity assays were performed with dinoflagellate cells in two growth phases during 7-h to assess differences in cell toxicity to A. salina. Additionally, exposure to cell-free medium was performed to evaluate its effects on A. salina survival. The behavior of brine shrimps significantly changed during exposure to the toxic dinoflagellates, becoming immobile at the bottom by the end of the trials. Dinoflagellates significantly affected A. salina survival with 100% mortality after 7-h exposure to cells in exponential phase  and to P. lima in stationary phase. Mortality rates of brine shrimps exposed to O. cf. ovata and G. excentricus in stationary phase were 91% and 75%, respectively. However, incubations of the brine shrimps with cell-free medium did not affect A. salina survivorship. Significant differences in toxic effects between cell growth phases were only found in the survival rates of A. salina exposed to G. excentricus. Acute exposure to benthic toxic dinoflagellates induced harmful effects on behavior and survival of A. salina. Negative effects related to the toxicity of benthic dinoflagellates are thus expected on their primary grazers making them more vulnerable to predation and vectors of toxins through the marine food webs.Harmful algae may differently affect their primary grazers, causing sub-lethal effects and/or leading to their death. The present study aim to compare the effects of three toxic benthic dinoflagellates on clearance and grazing rates, behavioral changes, and survival of  Ostreopsis cf. ovata have been recorded with increasing frequency, intensity and distribution, particularly in the Mediterranean Sea [O. cf. ovata became a recurrent event since 1998 in Rio de Janeiro state [Echinometra lucunter reported during these HAB events [O. siamensis caused decline in the numbers of the sea urchin Evechinus chloroticus [Ostreopsis cf. ovata produces ovatoxins, and other PLTX analogues, one of the most toxic molecules occurring in nature that cause intoxication in humans [O. cf. ovata, ostreol A, was shown to have in vitro cytotoxicity against the brine shrimp Artemia salina [Marine benthic dinoflagellates are important primary producers and most of their representatives are potentially toxic . Recentlnean Sea –3, with nean Sea . In Brazro state  with higB events . Similaroroticus . Ostreopn humans . MoreoveProrocentrum lima is a cosmopolitan species and often constitutes a significant part of benthic dinoflagellate communities worldwide [P. lima tested to date has been found to produce OA and its analogues in varying quantities [P. lima is found all year round growing epiphytically on macroalgae and locally isolated strains demonstrated the production of high OA concentrations [P. lima can act as a vector for DSP toxins in shellfish and suspected cases of intoxication have been recorded in Argentina [orldwide . This diorldwide . Evidencantities . In Rio trations . There irgentina , Canada rgentina , the Unirgentina  and Japargentina .Gambierdiscus excentricus produces maitotoxins (MTXs) and ciguatoxins (CTX) responsible for ciguatera fish poisoning [Gambierdiscus colonize upon [oisoning , a diseaoisoning . Moreoveize upon .Despite some grazers may avoid certain toxic algae –22, mostMost information on interactions between toxic algae and their grazers comprises phytoplankton and zooplankton grazers, particularly copepods , 34–35. Artemia salina is widely used for toxicity tests due to its widespread distribution, short life cycle, non-selective grazing, and sensitivity to toxic substances [A. salina seems to be a suitable model species to assess the toxicity of marine benthic dinoflagellates. The purpose of the present study was to evaluate the effects of the benthic dinoflagellates P. lima, G. excentricus, and O. cf. ovata on clearance and grazing rates, behavioral changes, and survival of A. salina during acute exposure. Acute toxicity assays were performed with cells in different growth phases of a batch culture  to assess differences in cell toxicity to brine shrimps.The brine shrimp bstances . TherefoProrocentrum lima (strain UNR-01), Ostreopsis cf. ovata (strain UNR-05) and Gambierdiscus excentricus (strain UNR-08) used in this study were isolated from Armação dos Búzios , Rio de Janeiro state as described in [Tetraselmis sp. was isolated from Guanabara Bay, Rio de Janeiro state . Scientific research and collecting permit authorizing field studies were obtained from Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), Brazilian Ministry of the Environment (permit number: 35192–3). No protected or endangered species was caught through field studies or used in experimental trials.Clonal cultures of ribed in , 39. A nO. cf. ovata which was grown in L2/2 medium. Salinity was adjusted to 34 for all the cultures, except for G. excentricus that was cultivated at salinity 32. All stock cultures were kept in a temperature-controlled cabinet at 24 ± 2°C, with a 12:12 h dark-light cycle and photon flux density of 60 μmol m-2s-1 provided by cool-white fluorescent tubes. Photosynthetically active radiation was measured with a QSL-100 quantum sensor .Benthic dinoflagellates were maintained in filtered seawater  supplemented with L2 enrichment medium [Prorocentrum lima (UNR-01) used in the current study synthesizes mostly OA and small amounts of DTX-1 [Gambierdiscus excentricus strain (UNR-08) produces at least one MTX analog, while the evaluation of CTX production by this strain has not yet been completed . The strain of Ostreopsis cf. ovata (UNR-05) has not been analyzed for toxin production. However, other strains of O. cf. ovata isolated from the same sampling location produce PLTX analogues (ovatoxins) [The strain of of DTX-1 . The Gamata UNR-0 has not Lugol preserved cells from the cultured strains were observed using light microscopy  to measure the cells for the determination of cellular biovolume. Images were collected using an Axiocam Icc1digital camera , and cells (n = 40) were measured using the Axiovision software . Cellular biovolume was estimated using geometric shapes and mathematical equations suggested for each genus : prolateA. salina (7 mm ± 1.14) were acclimatized in experimental conditions in a temperature-controlled cabinet at 24 ± 2°C for 24 to 48 h and fed ad libitum with Tetraselmis sp. Feeding trials were performed in 6-well plates containing, each well, three individuals in 10 ml of filtered seawater  at salinity 35.Adult individuals of A. salina grazing were carried out at various abundances of the non-toxic prey Tetraselmis sp. (150–720 cells ml-1) during an incubation interval that ranged from 30 min to 6 h. The results indicated the optimum incubation time (1 h) and Tetraselmis sp. abundance  for feeding experiments in which the brine shrimps depleted 15–30% of cell abundances. Cell size of microalgae used in this study ranged from 13 μm (Tetraselmis sp.) to 78 μm (G. excentricus). Thus, the same carbon concentration was established in all treatments allowing the comparison of clearance and grazing rates of A. salina among different prey species. Cell abundances of microalgae that corresponded to the carbon concentration of 40 ng C ml-1, per well, were: Tetraselmis sp. , Prorocentrum lima , Ostreopsis cf. ovata , and Gambierdiscus excentricus . Microalgae species were harvested during exponential phase for feeding trials.Previous tests of Each treatment containing the brine shrimps and one algal species was performed in triplicate, and the control containing solely algal cells without the brine shrimps was performed in duplicate. Aliquots of each replicate (1 ml) were collected using an automatic pipette at the beginning and after 1-h incubation. Cells were preserved with neutral Lugol's iodine solution for later cell counts using a Sedgewick-rafter chamber and observation in an inverted microscope .Artemia salina clearance  and ingestion  rates were calculated by the ratio of natural log (ln) of initial and final carbon concentrations after 1-h incubation, and corrected by the carbon concentration in controls [controls . ClearanA. salina (7 ± 1.14 mm) were acclimatized in the same experimental conditions described above for feeding experiments. Intoxication assays were performed in 6-well plates containing, each well, 10 ml of filtered seawater  at salinity 35. Three individuals of Artemia salina were incubated for 7-h with each dinoflagellate species (G. excentricus or O. cf. ovata or P. lima) in abundances of 200 cells ml-1 [A. salina individuals to each toxic species was performed twice on independent samples, first with cells at exponential phase and in the second time with cells at stationary phase.Adult individuals of lls ml-1 . Acute eTetraselmis sp. (control) was kept and harvested in exponential growth for all trials. Toxic treatments were performed in triplicates and the non-toxic control in duplicate. In total, toxic treatments consisted of six different incubations: 1) P. lima in exponential phase, 2) P. lima in stationary phase, 3) O. cf. ovata in exponential phase, 4) O. cf. ovata in stationary phase, 5) G. excentricus in exponential phase, and 6) G. excentricus in stationary phase. Changes on swimming activity (actively swimming or motionless), position  and survival of individuals were verified after the first 30 min and after every hour of incubation.Dinoflagellate growth curves were previously determined , 11, andG. excentricus, O. cf. ovata and P. lima cultures in abundances of 200 cells ml-1. Filtrates were used immediately. Incubations were performed in 6-well plates containing, each well, 10 ml of cell-free medium with six individuals of A. salina during 7-h. Each treatment of cell-free medium was performed in six replicates and the control (only filtered seawater) was performed in triplicate.Additional experiments were carried out to evaluate the effects of cell-free medium on brine shrimps survival. Cell-free medium were obtained by the filtration  of P. lima, O. cf. ovata, G. excentricus, and Tetraselmis sp.). Normality and homogeneity of variances were assessed using Kolmogorov-Smirnov and Levene Test, respectively, and log-transformation was applied when necessary. Analyses were performed using the software Statistica 8.0 (StatSoft).One-way ANOVA was applied to evaluate the influence of four different preys on the ingestion and clearance rates of brine shrimps—categorical factor  as covariable. A Monte Carlo permutation was used to test the significance of ordination model. The multivariate analysis was performed in the software CANOCO [A redundancy analysis (RDA) was applied to data of e CANOCO .A. salina individuals from toxic treatments  with exposure time (7-h) as continuous predictor in a full factorial design. Tukey test was applied whenever significant differences were detected by GLMs. A Kruskal-Wallis test was applied to survival data of A. salina individuals exposed to cell-free medium of dinoflagellate cultures. Analyses were performed using the software Statistica 8.0 (StatSoft).Generalized linear models (GLM) were applied to survival, swimming and position data (as dependent variables) of 50) in each treatment was also estimated. A Kaplan-Meier log rank test (Mantel-Cox) was applied to test significant differences in the survivorship curve of A. salina among toxic treatments using the software GraphPad Prism 5.0 . The Kaplan-Meier test takes into account the censored survivorship data [Survival functions were estimated from the continuous survival time for 7-h brine shrimps exposure to the toxic dinoflagellates. The time when 50% of the individuals died  and ingestion rates  of the brine shrimps among the different treatments . Adults A. salina clearly distinguished toxic treatments (negative coordinates on the first axis) from the non-toxic one .Redundancy analysis (RDA) applied to survival and behavioral data of A. salina exposed to the toxic dinoflagellates P. lima, O. cf. ovata and G. excentricus showed abnormal behavior related to swimming activity and its position on the water column, independently of dinoflagellate growth phase , leading to the immobility of individuals at the bottom. Behavioral changes in the swimming activity and the position of the brine shrimps were noticed in the first 30-min incubations with dinoflagellates in stationary phase. After 1-h exposure to toxic cells, either in exponential or stationary phase, individuals have shown changes in swimming activity and in their position on the water column  and by the interaction between dinoflagellate species and time of exposure . The A. salina activity was overall more affected by the exposure to P. lima and O. cf. ovata than to G. excentricus  was detected on A. salina position on the water column.The swimming activity of entricus . No signA. salina was significantly affected by dinoflagellate species , by the exposure time , and by the interaction between dinoflagellate and exposure time . The incubation of the brine shrimps with cell-free medium did not affect A. salina survivorship. There was no significant difference among treatments and control , survival rates ranged from ~92–100%: control , and cell-free medium of G. excentricus , O. cf. ovata , and P. lima . In contrast, the exposure to all the toxic dinoflagellate cells in exponential phase severely affected A. salina survival, with 100% mortality during assays  P. lima in stationary phase x G. excentricus in exponential phase , (2) P. lima in stationary phase x O. cf. ovata in stationary phase . During exposure to P. lima in stationary phase, less than 25% of A. salina individuals died in the first 2-h and, after a sharp decline in survival, all individuals were dead within 6-h incubation. In contrast, survival rate of A. salina exposed to G. excentricus cells in exponential phase slightly declined with over 30% survival after 6-h exposure. During exposure to O. cf. ovata cells in stationary phase, there was a sharp decline in A. salina survival in the first hours of incubation followed by a trend to stabilization close to 10% survival in the last hour . The survival of brine shrimps exposed to G. excentricus cells in exponential phase showed a steady decline until the end of incubation, reaching 100% mortality. In contrast, the exposure to G. excentricus in stationary phase led to a sharp reduction in survival within the first 3-h, with a loss of 25% of individuals per hour, followed by a stabilization in survival rate until the end of incubation with ~25% survival  during exposure to Gambierdiscus excentricus in stationary phase  of A. salina was found in the same treatment after 7-h exposure  producer Alexandrium fundyense at rates similar to that displayed for the non-harmful cryptophyte Rhodomonas salina [A. fundyense decreased when dinoflagellate abundances increased, while an opposite trend was observed in feeding rates on the non-harmful species [A. salina were observed when grazing on toxic Alexandrium species  compared to grazing on a mixture of toxic and non-toxic dinoflagellates (A. tamarense + Prorocentrum donghaiense), and solely on the non-toxic P. donghaiense [In addition, neither preference nor avoidance of toxic prey has been shown by the brine shrimps. The clearance and ingestion rates of brine shrimps were not affected by the toxicity of the dinoflagellates during the short-term experiment of 1-h incubation. This is probably due to the low cell abundances offered to them ~4–19 cells ml pungens . In the s salina . However species . Lower fghaiense .A. salina tract can be transferred to secondary consumers as demonstrated for Gambierdiscus cells [A. salina exposed to PSTs [A. salina, responsible for detoxification of various toxic compounds [Artemia salina shown in the present study) can act as vectors of dinoflagellate toxins in marine food webs [The threat of natural marine toxins increases considerable when bioaccumulation is considered along the food chains . As the us cells . The tox to PSTs  and micr to PSTs . Phycotoompounds . Crustacood webs .Artemia salina [Prorocentrum lima, Ostreopsis cf. ovata and Gambierdiscus excentricus isolated from tropical marine systems caused harmful effects on behavior and affected the survival of A. salina at abundances of 200 cells ml-1. The harmful effects on behavior and survival of the brine shrimps were demonstrated by multivariate analysis (RDA) in which toxic treatments were clearly distinguished from the non-toxic control that was positively correlated with swimming activity, water column position and survival of A. salina.The current intoxication trials contribute to increase the knowledge about direct toxic effects of benthic dinoflagellates on a marine invertebrate widely used as model organism in bioassays—a salina . The toxA. salina. Healthy Artemia individuals are active swimmers, thus, alterations in swimming activity are valid as behavioral endpoints to detect stress at sub-lethal concentrations of toxic compounds [A. salina individuals exposed to G. excentricus cells in stationary phase showed any movement after 7-h incubation.Immobility and mortality were used as endpoints to measure the toxicity of the three benthic dinoflagellates to adults of ompounds . The briompounds . Only ~2O. cf. ovata and P. lima induced the loss of movement or death of all the individuals until the end of incubation. Similarly, adult females of the copepod Tigriopus japonicus exposed to Fukuyoa sp. (as Gambierdiscus cf. yasumotoi) showed a decrease in activity, loss of motor control, and abnormal swimming [-1 of Fukuyoa sp. (as Gambierdiscus cf. yasumotoi) [Besides, brine shrimp exposure to swimming . Howeversumotoi) , the samsumotoi) –59. In asumotoi) .P. lima and O. cf. ovata were able to induce similar effects on behavior, particularly swimming activity, and survival of the brine shrimp A. salina. The exposure to these two dinoflagellates caused a sharp reduction on swimming activity followed by an increase in mortality of A. salina adults. The OA and DTX-1 toxins produced by P. lima probably lead to physiological disturbances in brine shrimps related to loss of body fluids and lack of physiological control of fluid dynamics, as previously observed in affected organisms [O. cf. ovata alter the mechanisms of ion homeostasis [A. salina induced by the exposure to P. lima and O. cf. ovata seem to be thus related to brine shrimp ion regulation.The present results showed that the toxic dinoflagellates rganisms , 61. Simeostasis , with dieostasis . In the A. salina during acute exposure. All the brine shrimps exposed to the three toxic dinoflagellates in exponential phase died, while the mortality of brine shrimps exposed to cells in stationary phase varied according to dinoflagellate species: 100% (P. lima), 91% (O. cf. ovata), and 75% (G. excentricus). However, the cell-free medium did not significantly affect A. salina survival during acute exposure. Cell-free medium from cultures with higher abundances of O. cf. ovata (~4000 cells ml-1) [A. salina. In the present study, the high survival of the brine shrimps exposed to cell-free medium of G. excentricus, O. cf. ovata and P. lima confirms that the concentration of diluted cell exudates was within A. salina tolerance and did not compromise their survival.The toxic benthic dinoflagellates significantly affected the survival rates of ls ml-1)  were harArtemia salina exhibited similar sensitivity to Prorocentrum lima and Ostreopsis cf. ovata acute exposure; the same pattern seen for behavioral responses. Adults of A. salina showed high sensitivity to P. lima either in exponential or stationary growth phases  and to O. cf. ovata, particularly in exponential growth phase (tD50 = 3h), with 100% mortality after 6-h exposure. Conversely, minimal mortalities of A. salina adults have been reported after exposure to P. lima and P. concavum in abundances up to 1000 cells individual-1[A. salina larvae have demonstrated high sensitivity of nauplii to both P. lima (tD50 = 1.7h) [O. cf. ovata cells [A. salina nauplii were reported to be extremely sensitive to O. cf. ovata cells with half maximum effective concentration (EC50) ranging from 6 to 24 cells ml-1 [O. cf. ovata cells revealed that A. salina larvae have the highest sensitivity among them, with a half lethal concentration (LC50) lower than 4 cells ml-1 [Artemia salina has also showed high sensitivity to other Ostreopsis species, the PTX-like producer O. siamensis [A. salina in the two stages of its life cycle (nauplius and adult) to these toxic dinoflagellates confirms this crustacean as a suitable model species to assess the toxicity of marine harmful algae.ividual-1. Previou = 1.7h)  and O. cta cells , 63. In lls ml-1 . Artemiaiamensis . The senGambierdiscus excentricus rapidly affected the swimming activity, water column position and survival of exposed-individuals of A. salina in the present assays. A short stabilization of effects was observed after 3-h exposure, followed by a reduction in the percentage of individuals swimming on the water column as well as in the survival of the brine shrimps between 5 to 7-h exposure. Maitotoxins (MTX) alter ion transport systems causing an increase in free intracellular Ca2+ [Alexandrium spp. that act on the voltage-gated Na+ channel of nerve cells, leading to loss of motor control and abnormal swimming on copepod [Gambierdiscus sp. (as Gambierdiscus toxicus) and Fukuyoa sp. (as Gambierdiscus cf. yasumotoi) was lethal to the adult brine shrimp Artemia spp. [Tigriopus japonicus [A. salina showed high sensitivity to G. excentricus in both growth phases  at 200 cells ml-1, with 100% mortality after 7-h exposure to cells in exponential phase. Similarly, strains of Gambierdiscus sp. (as G. toxicus) showed high toxicity to Artemia with median lethal dose (LD50) from 2.8 to 104.5 cells individual-1 [Fukuyoa sp. (as Gambierdiscus cf. yasumotoi) at 200 cells ml-1 [Fukuyoa sp. [G. excentricus (current study), as well as the feeding selectivity against harmful prey or sensitivity of the tested species .The dinoflagellate lar Ca2+  while cilar Ca2+ –67. The  copepod . Exposurmia spp.  and the aponicus . In the vidual-1 . Lower ells ml-1 , the samlls ml-1 . Differeuyoa sp.  and G. eO. cf. ovata [Gambierdiscus, MTX-related hemolytic activity increased from log phase  to late log-early stationary phase, but then declined in mid-stationary phase, although no significant differences in toxicity were observed among growth phases [Gambierdiscus species. In the current study, significant differences between the toxic effects of dinoflagellate growth phases was solely found in survival rates of A. salina exposed to G. excentricus. Exposure to cells in stationary phase induced an initial quick response on A. salina survival, with high mortality of 50% within the first 3-h incubation, and 75% mortality at the end of 7-h incubation. In contrast, G. excentricus cells in exponential phase induced a steady decrease in survival rates and caused 100% mortality of brine shrimps at the end of incubation. The difference in A. salina survival rates may be related to an effect of the production of different toxin congeners with varying toxicities for the brine shrimps in each growth phase. Moreover, G. excentricus is a large thecate (with cellulose plates) dinoflagellate and it is likely that aged cells (in stationary phase) are thicker than actively growing  cells. As cells enter the stationary phase, the growth rate diminishes, but cells may continue to expand in size. In many armored species, the edges of each thecal plate overlap, sliding apart as the cells increase in size, likely producing a more robust theca. The thicker and more robust aged cell is possibly more resistant to the digestion by A. salina, which will affect toxin assimilation rate and ultimately the survival of potential grazers.The toxin content or cell quota may vary along the growth phases of a dinoflagellate species in a batch culture and may be lower during the exponential phase relative to the stationary and the declining phase, as observed for f. ovata –70. In eh phases . FurtherProrocentrum lima, Ostreopsis cf. ovata and Gambierdiscus excentricus isolated from tropical marine systems induced severe effects on behavior and survival of adult individuals of Artemia salina. Further studies are necessary to identify the different modes of action of diverse toxic compounds produced by benthic dinoflagellates on their primary grazers. Negative effects related to the toxicity of benthic dinoflagellates are expected on diverse grazers making them more vulnerable to predation and vectors of toxins through the marine food webs.Finally, acute exposure to the toxic benthic dinoflagellates S1 File(XLS)Click here for additional data file.