Symbiosis in insects is accumulating significant amount of studies: the description of a wide array of mutualistic associations across the evolutionary history of insects suggests that resident microbiota acts as a driving force by affecting several aspects of hosts biology.Among arthropods, mosquito midgut microbiota has been largely investigated, providing crucial insights on the role and implications of host-symbiont relationships. However, limited amount of studies addressed their efforts on the investigation of microbiota colonizing salivary glands and reproductive tracts, crucial organs for pathogen invasion and vertical transmission of symbiotic microorganisms. Using 16S rRNA gene sequencing-based approach, we analysed the microbiota of gut, salivary glands and reproductive tracts of several mosquito species, representing some of the main vectors of diseases, aiming at describing the dynamics of bacterial communities within the individual.We identified a shared core microbiota between different mosquito species, although interesting inter- and intra-species differences were detected. Additionally, our results showed deep divergences between genera, underlining microbiota specificity and adaptation to their host.The comprehensive landscape of the bacterial microbiota components may ultimately provide crucial insights and novel targets for possible application of symbionts in innovative strategies for the control of vector borne diseases, globally named Symbiotic Control (SC), and suggesting that the holobiont of different mosquito species may significantly vary. Moreover, mosquito species are characterized by distinctive microbiota in different organs, likely reflecting different functions and/or adaptation processes.The online version of this article (10.1186/s12866-018-1266-9) contains supplementary material, which is available to authorized users. Insects represent the most diverse and abundant clade of metazoans, accounting for more than 90% species and dominating a variety of terrestrial habitats. Diverse insect habits are founded on associations with microorganisms, whose diversity reflects the variety of their hosts . These cDrosophila melanogaster capable of affecting mating preferences by changing the levels of cuticular hydrocarbons and sex pheromones , two belonging to the Aedes genus  and Cx. quinquefasciatus. Herein, data of an extensive study aimed to characterize bacterial microbiota associated to gut, salivary glands and reproductive organs are reported. The importance of describing the microbiota of these organs lies in possibility to develop innovative paratransgenic approaches for vector control. The selection of suitable symbiont candidates primarily requires a specific tissue tropism in the gut, the salivary glands and the reproductive organs [We investigated nine species of mosquitoes belonging to the main representative mosquito genera, in terms of geographical diffusion and public health interest. This includes six anopheline species [e organs , since iAe. aegypti harbours the highest number of bacteria species compared to others, followed by An. coluzzii and An. quadriannulatus. An. gambiae G3 and An. stephensi contain the lowest number of bacteria species, explained by the long adaptation in laboratory conditions  salivary glands of all species apart from An. gambiae G3 and An. coluzzii; iv) the reproductive organs of both males and females of An. arabiensis, An. merus, An. stephensi, Ae. albopictus and Ae. aegypti, the ovaries of An. gambiae G3 and organs of Cx. quinquefasciatus males. However, Bacteroidetes (> 68%) are present in male guts and salivary glands of both An. gambiae G3 and An. coluzzii, and in the reproductive organs of An. gambiae G3 males. The guts of An. gambiae Kisumu males harbour more than 28% of Bacteroidetes, which also encompasses 10% of taxa in Ae. aegypti organs. Firmicutes and Actinobacteria strongly colonize the reproductive organs of both males and females of An. coluzzii, An. quadriannulatus and An. gambiae Kisumu, whereas they are found only in ovaries of Cx. quinquefasciatus.A total of 951.930 reads were generated after removal of short reads < 250 bp), chimeras and the discard of spurious OTUs  from all50 bp, chAn. stephensi males and An. gambiae G3 females are divergent from other samples  plot was generated, revealing that the reproductive organs of les Fig. . This shzii Fig.  b; an adles Fig. . The resles Fig.  . A globles Fig. .Fig. 2PrAe. albopictus  and Cx. quinquefasciatus (♂: 99%); whereas in An. gambiae G3, An. coluzzii males, An. gambiae Kisumu females, it accounts for less than 1% of the OTUs. Lower abundance is detected in An. arabiensis , An. stephensi (♀: 48%), Ae. aegypti (♀ and ♂ 32%), Cx. quinquefasciatus (♀: 13%) and between 1 and 6% in the remaining samples  and for Betaproteobacteria in Cx. quinquefasciatus males and females, An. stephensi and An. gambiae Kisumu females  in females and Flavobacteria (74%) in males dominate the overall microbiota  followed by Escherichia-Shigella (0.5%), Sphingomonas (0.2%), Cupriavidus (0.2%) and Elizabethkingia (0.2%). The latter, on the contrary, represents the dominant taxon (73%) in male organs, co-habiting with Burkholderia (18%) and, in much lower amount, with Serratia (0.3%). Similarly, in An. stephensi, Serratia predominantly inhabits male reproductive organs, while in ovaries it is exceeded by Asaia, whose presence in males is reduced. Finally, Cx. quinquefasciatus offered a divergent scenario: male reproductive organs are mainly colonized by Phyllobacterium (91%), although its contribution in ovaries microbiota was minimal (0.8%) ; in male reproductive organs, it coexists with Sphingomonas, Cupriavidus and Serratia.Despite a few exceptions, the analysis of taxa at genus level showed the persistence of a shared core of bacteria. 8%) Fig. . DiffereAe. aegypti show an interestingly high richness of taxa: Ochrobactrum, Pseudomonas, Sphingomonas, Stenothropomonas, Acinetobacter and Elizabethkinghia are the most abundant genera detected in female and male individuals  and in Ae. albopictus, and, although less abundant, in An. merus, in Ae. aegypti and in Cx. quinquefasciatus. Alphaproteobacteria, followed by Betaproteobacteria, constitutes the remaining portion , Asaia (14%) and in lower proportion Serratia and Cupriavidus. A completely divergent pattern is visible in An. gambiae G3 and An. coluzzii mosquitoes, where 69% of OTUs are addressed to the Elizabethkingia sp. genus, whose abundance reaches almost undetectable levels in the salivary glands of other species (apart from 1.4% in Ae. aegypti). In addition, salivary glands of An. gambiae G3 and An. coluzzii are inhabited by Betaproteobacteria , whose colonization is found in lower amount (7.6%) in An. quadriannulatus samples.Ae. albopictus is mainly colonized by Serratia (88%) like An. stephensi. Similarly to other organs, a conserved core of communities is found in salivary glands, consisting of Asaia, Pseudomonas, Sphingomonas, Cupriavidus, Serratia and Escherichia-Shigella. Moreover, other taxa are identified, including Acetobacter and Ochrobactrum (Alphaproteobacteria) and Elizabethkingia (Bacteroidetes)  Fig. .Serratia, apart from An. gambiae G3 and An. coluzzii males, where Flavobacteria (represented by Elizabethkingia) reaches 78% and 95% of OTUs, respectively  are dominant in females, while Gammaproteobacteria (Yersinia) largely colonize male guts  in some samples. Overall, the incidence of bacteria contaminations in our data appears minimal; moreover, the taxa described above have been previously presented in metagenomics studies of insects, and for this reason they have been included in the data analysis.We are aware that bacterial DNA contamination in extraction kits and laboratory reagents can significantly affect the results of microbiota studies, particularly when samples contain low microbial biomass and when highly sensitive techniques are used. Previous studies described the list of contaminants detected in sequenced negative blank controls derived from reagents contamination, users and extraction kits . The proAn. culicifacies [Elizabethkingia: it largely colonizes guts and reproductive organs of An. gambiae G3 males, while in An. coluzzii, it is prevalent in guts of males and salivary glands of females. Its association with guts of An. gambiae was already reported in mosquitoes reared in standard insectary conditions, suggesting the establishment of an own thriving niche, and possibly explained with the ability of Bacteroidetes to degrade sugar [Serratia, although presenting variable abundances in each sample, is found to be dominant in the gut of female anophelines, being consistent with recently reported data [An. stephensi, Serratia represents more than 90% of detected taxa in all organs, except for female ovaries where its abundance is reduced.Previous studies proved that commensal bacteria are mainly acquired from the environment or transmitted directly between hosts to their offspring. Often, these contributions are correlated, meaning that bacteria with complete transmission among hosts will evolve specialization for a particular niche –28. Althcifacies . A leadide sugar . Similarted data , 30. ForAsaia occurrence is in line with its role among symbiotic bacterial communities: it rarely showed, in fact, dominant behaviour, like Serratia or Elizabethkingia, but its persistence is found in guts and salivary glands of all mosquito genera and its relevant occurrence in reproductive organs of males and females of An. stephensi and An. arabiensis provides evidences of its widespread colonization ability.Notably, Asaia, Pantoea, Pseudomonas, Entrobacter, Serratia are gaining momentum as promising candidates for paratransgenic modifications for vector control strategies. Our findings confirm their abundance in key tissues of various mosquito species, corroborating and widening their role as broad-spectrum tools against mosquito borne diseases. Cupriavidus and Ochrobactrum could be also included in this bacterial arsenal: their association with mosquitoes was never reported before, but their recent engineering for biomedical purposes could be also translated in the field of mosquito control [Serratia and Wolbachia in different mosquito species are well-known to provide protective effects from pathogens infections, as reported in several studies [Bacteria such as  control , 32. In  studies , 34.In any case, before any field application of bacterial strains, a detailed characterization and analysis of the potential risks for human and animal health is an imperative.Our study describes the composition of microbial communities harboured in different tissues of nine mosquito species, transmitting devastating human diseases. The results highlight the importance of a comprehensive understanding of organisms, intended as holobionts, and thus composed by innumerable interacting communities, whose impact is increasingly apparent. The identification of interesting inter- and intra-species differences, coexisting with a shared core microbiota, suggest a singular adaptation and a tissue-specific tropism. The contextual findings of a deep degree of divergences between genera, underlines microbiota specificity and its adaptation to the host. Moving forward, our data lay the basis for the design of effective vector control strategies, relying on the use of mosquito-associated bacteria.The following laboratory strains were used:An. gambiae G3  established in Perugia insectary in 2013; An. gambiae Kisumu  established in Perugia insectary in 2013; An. coluzzii  established in Perugia insectary in 2013 ; An. arabiensis  established in Perugia insectary in 2013; An. merus  established in Perugia insectary in 2013; An. quadriannulatus  established in Perugia insectary in 2013; An. stephensi (SD-500) established in Perugia insectary in 2011; Ae. albopictus  established in Perugia insectary in 2013; Ae. aegypti  established in Perugia insectary in 2013; Cx. quinquefasciatus  established in Camerino insectary in 2011.Anopheles, Aedes and Culex larvae were reared in deionized water with 0.3 g/liter of artificial sea salts and fed daily with a diet composed by a slurry of 2:2:1 bovine liver powder, tuna meal and Vanderzant vitamin mix [Analyzed mosquitoes were all cyclic laboratory-strain colonies. Mosquitoes were reared with a 12 h day/night cycle at 27 °C and relative humidity of 70%. amin mix . Adults Prior dissecting, mosquitoes were surface sterilized in 70% ethanol for 5 min and rinsed twice in sterile PBS. Foreguts and midguts, salivary glands lobes and reproductive organs, consisting on testes and male accessory glands (MAGs) for males, and ovaries for females, were dissected with special care to reduce contaminations: needles and dissecting slides were sterilized and treated with 70% ethanol between each sample, while storing vials were UV treated before use. Pooled guts (10 whole guts), reproductive organs (10 sets) and salivary glands (20 pairs) were homogenized with sterile 0.5-mm wide glass beads (Bertin) for 30s at 6800 rpm in the automatic tissue homogenizer . Genomic DNA was extracted using a JetFlex Genomic DNA Purification kit (Genomed) according to the manufacturer’s instructions.Bacterial 16S V4 regions rDNA were amplified using bacteria/archaeal degenerate primers 515F/806R . Amplifihttp://qiime.org, http://www.r-project.org/).Quality control of raw data was done using FastQC, then adapters sequences and low quality scores (< 20) were trimmed by Trimmomatic software package . CleanedAdditional file 1:Figure S1. Alpha diversity. Box plots of bacterial species richness associated with the nine mosquito species. The box plots indicate median (middle line), upper and lower quartiles (box top and bottom), minimum and maximum (whiskers). (JPG 3163 kb)Additional file 2:Figure S2. Rarefaction curves. Rarefaction curves calculated for each sample based on the OTU computations, reflect different diversities in different samples and help to estimate whether bacterial communities were sampled properly, i.e. enough sequence reads per sample where collected. Rarefaction curves are expected to reach a plateau if sampling has been exhaustive. ♀: females; ♂: males. (JPG 1442 kb)Additional file 3:Table S1. Percentage of phyla OTUs in the reproductive organs (A) salivary glands (B) and guts (C). (DOCX 47 kb)Additional file 4:Figure S3. Family level composition (% of OTUs) in different organs of nine mosquito species. Only OTUs representing > 1% of the total reads are represented here. The family color code is given for (A), (B) and (C). GA G3: An. gambiae G3; COL: An. coluzzii; QUAD: An. quadriannulatus; GA KIS: An. gambiae Kisumu; ARA: An. arabiensis; MER: An. merus; STEP: An. stephensi; ALB: Ae. albopictus; AEG: Ae. aegypti; QUINQ: Cx. quinquefasciatus; ♀: females; ♂: males. (JPG 3132 kb)


TMEM95) seriously reduced reproductive performance in male Fleckvieh cattle; therefore, this locus was considered a candidate genetic marker in bovine marker-assisted selection (MAS) breeding. Until now, no study has identified this mutation in Chinese cattle breeds. Herein, we detected this c.483C > A mutation in 13 Chinese cattle breeds. Importantly, we found that this mutation did not exist at this locus in our analyzed breeds. Interestingly, we first identified a frameshift insertion/deletion (indel) mutation (NC_037346.1: g.27056998_27057000delCT) in the bovine TMEM95 gene in 11 cattle breeds. Together, the results of this study suggest that the mutation c.483C > A cannot serve as a genetic marker for molecular breeding among Chinese indigenous cattle breeds.Chinese indigenous cattle are economically important cattle breeds for animal husbandry development. The promotion and development of Chinese cattle breeds is essential. A previous study found that a nonsense mutation  in bovine transmembrane protein 95 gene (TMEM95) seriously reduced the reproductive performance in bulls, but few studies have detected this mutation in Chinese indigenous cattle breeds. Since the mutation c.483C > A may serve as a potential genetic marker for selecting higher fertility bulls, in the present study, using tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR), forced PCR restriction fragment length polymorphism (forced PCR-RFLP), and DNA sequencing methods, the mutation c.483C > A was detected in 765 individuals from 13 Chinese cattle breeds. However, the results showed that this mutation did not exist at this locus in our analyzed breeds. Interestingly, we identified a newly frameshift insertion/deletion (indel) mutation (NC_037346.1: g.27056998_27057000delCT) in the bovine TMEM95 gene in 11 cattle breeds, which changed the location of the termination codon and changed the 16 amino acids in the C-terminal to 21 amino acids. Combined with previous studies, our study provides evidence that in Chinese cattle breeds the mutation c.483C > A cannot be used as a genetic marker in molecular breeding.Chinese indigenous cattle breeds have abundant genetic resources, which are valuable for the molecular breeding of cattle around the world. Thus, identifying important candidate genes and their genetic markers is very important for cattle molecular breeding. A previous study found that a nonsense mutation  in the bovine transmembrane protein 95 gene ( Cattle are among the most important domestic animals. China, vast in size and diverse in topography, has abundant cattle breeding resources. East Asian cattle are mainly composed of three distinct ancestries: East Asian taurine ancestry, Eurasian taurine ancestry, and Chinese indicine ancestry . The earTMEM95) gene was associated with idiopathic male subfertility in Fleckvieh cattle, but did not affect semen quality [TMEM95 consists of 183 amino acids, encoding a quite conserved single-pass type I transmembrane protein  [TMEM95 (p.Cys161X), which truncates 22 amino acids of the TMEM95 C-terminal sequence [Artificial insemination (AI) is a routine breeding technique in cattle production. The application of AI in cattle began at the beginning of the 20th century, and improves the breeding efficiency of bulls ,5. In or quality . Subfert quality ,8. Bovinsequence ,11. ThesTMEM95 gene mutation on male reproductive performance, testing and verifying the candidate molecular genetic markers in Chinese cattle breeds is beneficial and necessary. Herein, a total of 765 individuals from 13 Chinese representative cattle breeds were selected for this study, so as to lay the foundation for assessing the genetic variations of the bovine TMEM95 gene and evaluating the possibility of applying the genetic markers in cattle MAS breeding.Until now, no study has detected the abovementioned nonsense mutation (c.483C > A) in Chinese cattle. Considering the genetic effect of bovine All animal experiments in the present study were performed in conformity to the applicable guidelines, animal welfare laws, and policies by the Ministry of Science and Technology of the People’s Republic of China . Moreover, the experiments were also approved by Northwest A&F University (NWAFU), Shaanxi, P.R. China.n = 135, Jilin Province), Qinchuan cattle , Nanyang cattle , Jinnan cattle , Luxi cattle , Xia’nan cattle , Jiaxian Red cattle , Pi’nan cattle , Jinjiang cattle , De’nan cattle , Yunling cattle , Zaosheng cattle , and Bohai Black cattle .A total of 765 cattle samples from 13 Chinese indigenous cattle breeds were analyzed: Red Steppe cattle , the primers of tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR), which include two allele-specific inner primers and a pair of outer primers, were designed using the Primer1 website (http://primer1.soton.ac.uk/primer1.html) [A previous study found a potential nonsense mutation  in bovine r1.html) ,15. The Hha I endonuclease were 228 bp and 25 bp for the CC genotype; 253 bp, 228 bp, and 25 bp bands for genotype AC; genotype AA showed one band of 253 bp. The genotypes of individuals were classified by analyzing the electrophoresis images.The PCR reaction was carried out in a 13 μL reaction volume, and the PCR programs was the same as in our previous studies . Using 3This study also verified the genotypes by sequencing; the PCR product for sequencing is 765 bp in length. Red Steppe cattle were sequenced one by one. Then, we constructed 21 DNA pools in the analyzed cattle breeds . In each DNA pool, 30 DNA samples were randomly selected, then the DNA pools or DNA were used for amplifications and sequencing. The PCR reaction was performed in a 25 μL volume, and touchdown PCR reaction was implemented according to six steps, as follows ,19: stepTMEM95 gene. In this study, we failed to detect the different genotypes of this locus via the T-ARMS-PCR and forced PCR-RFLP methods. In a previous study , the nonWe further used the DNA sequencing method to identify the genotypes of this locus . First, TMEM95 gene, named NC_037346.1: g.27056998_27057000delCT, was revealed in the detected cattle breeds. This indel was found in 11 studied cattle breeds . However, in Zaosheng cattle and Red Steppe cattle no mutation was revealed in this indel locus  in the bovine h et al. . In 2017h et al.  examinedIn the present study, we first detected the c.483C > A in 13 Chinese indigenous cattle breeds. Surprisingly, there was no mutation in the c.483C > A locus in the detected Chinese cattle breeds. So, the fact that the nonsense mutation c.483C > A only exists in Fleckvieh cattle but not in Chinese cattle breeds  suggests that this locus is not suitable to be used in MAS breeding in Chinese cattle. Still, in order to understand why Chinese cattle breeds did not contain this mutation, follow-up studies are necessary to clarify the breeding discrepancy and reproduction differences between foreign and Chinese cattle breeds. TMEM95 gene. This is the first study to detect the indel mutation in this gene. Multiple studies have suggested that indel mutations located in coding or non-coding regions have notable effects on gene expression and phenotypes [TMEM95, though that needs further verification. Furthermore, two alternative splicing variants of the TMEM95 gene have been identified in the brain and testicular tissue of fetal Qinchuan cattle [TMEM95 gene has abundant genetic polymorphisms, but further investigation is needed to better understand the function of this gene as well as enhance the male reproductive performance of bulls.In this study, an indel mutation (NC_037346.1: g.27056998_27057000delCT) was discovered in 11 cattle breeds by direct sequencing of the bovine enotypes ; thus, tn cattle . Alternan cattle . Taken tTMEM95 gene in 11 cattle breeds, which changed the location of the termination codon and changed the 16 amino acids in the C-terminal to 21 amino acids. These results suggest that the c.483C > A mutation cannot serve as a genetic marker in molecular breeding in Chinese indigenous cattle breeds.In this study, T-ARMS-PCR, forced PCR-RFLP, and DNA sequencing methods were used to detect the c.483C > A mutation in 765 individuals from 13 Chinese cattle breeds. The c.483C > A mutation was not found in our analyzed breeds. However, we identified a new frameshift indel mutation (NC_037346.1: g.27056998_27057000delCT) in the bovine


Stenotrophomonas maltophilia is an emerging global opportunistic pathogen, responsible for a wide range of human infections, including respiratory tract infections. Intrinsic multidrug resistance and propensity to form biofilms make S. maltophilia infections recalcitrant to treatment. Colistin is among the second-line options in case of difficult-to-treat S. maltophilia infections, with the advantage of being also administrable by nebulization. We investigated the potential synergism of colistin in combination with N-acetylcysteine (NAC) (a mucolytic agent with antioxidant and anti-inflammatory properties) against S. maltophilia grown in planktonic phase and biofilm. Eighteen S. maltophilia clinical isolates (comprising three isolates from cystic fibrosis (CF) and two trimethoprim-sulfamethoxazole (SXT)-resistant strains) were included. Checkerboard assays showed a synergism of colistin/NAC combinations against the strains with colistin Minimum Inhibitory Concentration (MIC) >2 µg/mL (n = 13), suggesting that NAC could antagonize the mechanisms involved in colistin resistance. Nonetheless, time–kill assays revealed that NAC might potentiate colistin activity also in case of lower colistin MICs. A dose-dependent potentiation of colistin activity by NAC was also clearly observed against S. maltophilia biofilms, also at sub-MIC concentrations. Colistin/NAC combinations, at concentrations likely achievable by topical administration, might represent a valid option for the treatment of S. maltophilia respiratory infections and should be examined further. Stenotrophomonas maltophilia is an emerging global opportunistic pathogen, responsible for a wide range of human infections, including chronic lung colonization and acute exacerbations in patients affected by chronic respiratory diseases, such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and bronchiectasis .,13.S. mauginosa) . Overalltibility .P. aeruginosa) was observed with 12 strains in the presence of NAC 4 mg/mL and with all strains in the presence of NAC >4 mg/mL  ≤0.5) . In part>4 mg/mL . The 5 c>4 mg/mL .S. maltophilia strains, namely Z131 , Z157 , and Z66 . Colistin and NAC concentrations potentially achievable by topical administration were tested. Results showed a relevant dose-dependent potentiation of colistin activity by NAC, with the three strains investigated /peg after 24 h of growth. Overall, a synergism of colistin/NAC combinations was observed with all the colistin-resistant strains, except for strain Z155 (colistin MIC = 4 µg/mL)  was extremely susceptible to NAC 16 mg/mL, which alone achieved complete eradication of the in vitro biofilm model  . In part4 µg/mL) . S. maltlm model . In addilm model .A statistically significant potentiation of colistin activity by NAC was also observed with one of the five colistin-susceptible strains  . For theS. maltophilia strains. Biofilm susceptibility to colistin/NAC combinations was anyway strain-dependent and not directly correlated to colistin or NAC MICs.Taken together, these results indicated a NAC-mediated dose-dependent potentiation of the antibiofilm activity of colistin against A. baumannii grown in planktonic and biofilm phase [Colistin/NAC combinations, at the high concentrations potentially achievable by topical administration, have been recently found to exert a relevant synergistic activity against lm phase . In partlm phase .S. maltophilia, an emerging global difficult-to-treat opportunistic pathogen, with a relevant role in respiratory tract infections, especially in CF. Our study demonstrated that the antimicrobial and antibiofilm synergism of colistin/NAC combinations is also exerted against Inhaled colistin has been increasingly used since late 1980s, especially for the treatment of individuals with CF, health care-associated pneumonia, and ventilator-associated pneumonia ,25,26,27Despite inhaled NAC has been used safely for decades as a mucus-dissolving treatment in respiratory diseases associated to abundant and/or thick mucus production , the actual NAC concentrations achievable in the ELF after topical administration have never been determined. Nonetheless, considering the multiple-dosage regimes of nebulized administration , the higher performance of last-generation nebulizers, and the possibility of direct instillation, topical NAC could reach the ELF concentrations needed for exerting the antimicrobial and antibiofilm potentiation of colistin activity ,29. In aColistin has gained a renewed interest only in the last years as salvage therapy for the treatment of infections caused by multidrug-resistant Gram-negative pathogens, and many aspects concerning the molecular mechanisms of colistin bactericidal activity and acquired resistance remain still scarcely known ,33. AparSimilarly, no solid data are available on the molecular mechanisms accounting for the intrinsic antimicrobial and antibiofilm activity of NAC, which most likely is multifactorial . NAC hasThe reasons accounting for the antimicrobial and antibiofilm synergism of colistin/NAC combinations are not easy to be hypothesized due to the relevant knowledge gaps on the mechanisms of action of both compounds. In this perspective, understanding the mechanisms of such a synergism would be relevant not only for optimization of clinical applications but also for drug discovery purposes .Based on the in vitro evidence of potentiation of colistin antimicrobial and antibiofilm activity by NAC, in vivo animal models to evaluate the potential clinical relevance of topical colistin/NAC combinations are warranted.S. maltophilia clinical isolates were investigated, including isolates from CF and SXT-resistant strains  [A total of 18  strains . The strtophilia . Colistitophilia . For thesistant) ,13.NAC stock solutions (100 mg/mL) were prepared immediately before use. NAC powder  was dissolved in sterile double-distilled water, pH was adjusted at 6.5–6.8 with NaOH, and the solution was filtered through a 0.22-μm membrane filter. All experiments were performed in cation-adjusted Mueller-Hinton broth , starting from an appropriately concentrated medium in order to avoid broth dilution when testing high NAC concentrations.The potential synergism of colistin/NAC combinations was investigated by checkerboard assay as described previously . The ranS. maltophilia strains: Z131 , Z157 , and Z66   . Two colThe potential antibiofilm synergism of colistin/NAC combinations was investigated using the Nunc-TSP lid system , as described previously . BrieflyStatistical analysis was performed using GraphPad Prism version 6.0 . D’Agostino-Pearson and Shapiro-Wilk normality tests were applied. Multiple comparison tests were performed by Kruskal-Wallis test with Dunn’s correction.S. maltophilia, an emerging global difficult-to-treat opportunistic pathogen, with an important role also in CF.In conclusion, we demonstrated a relevant in vitro antimicrobial and antibiofilm activity of colistin/NAC combinations  against Further studies are needed to understand the molecular bases of such a synergism and to evaluate the potential clinical relevance of colistin/NAC topical formulations.International patent application No. WO2018/154091.


Pulmonary nontuberculous mycobacteria (NTM) disease is of increasing public health concern in China. Information is limited regarding risk factors associated with this disease in China. The objective of this study was to describe the epidemiology of pulmonary disease due to NTM in Southern China.We retrospectively reviewed the medical records of pulmonary NTM patients registered in the Guangzhou Chest Hospital with positive mycobacterial cultures during 2013–2016. We described sex, age, residence, treatment history, laboratory examination results and comorbidities of pulmonary NTM patients.Mycobacterium avium complex (44.5%), Mycobacterium abscessus complex (40.5%), Mycobacterium kansasii (10.0%) and Mycobacterium fortuitum (2.8%). The male:female ratio was significantly lower among patients infected with rapidly growing mycobacteria (RGM) than among those with slowly growing mycobacteria (SGM). The risk of developing SGM disease significantly increased with advancing age. In addition, pulmonary RGM diseases were more common in migrant population than resident population. Notably, patients with pulmonary RGM diseases were significantly more likely to have bronchiectasis underlying noted than those with SGM diseases. No significant difference was observed in in vitro drug susceptibility among NTM species.Among the 607 NTM cases, the most prevalent species were M. avium complex is the most predominant causative agent of pulmonary NTM disease in Southern China. Female, migrant population, the presence of bronchiectasis are independent risk factors for pulmonary diseases due to RGM. In addition, the prevalence of SGM increases significantly with advancing age.Our data illustrate that the  Mycobacterium tuberculosis complex and Mycobacterium leprae [M. tuberculosis, these environmental bacteria are associated with a wide array of clinical diseases, especially in HIV-infected patients or those with immunodeficiencies [Nontuberculous mycobacteria (NTM) are a heterogeneous group of species other than the m leprae . As the m leprae . Despiteciencies . Notablyciencies , while tciencies . The mosciencies , 6, whicciencies ; consequAlthough China has achieved impressive reductions in TB prevalence and mortality over the past 20 years , NTM infThis study was conducted at the Guangzhou Chest Hospital, an 800-bed regional tuberculosis clinical centre in Southern China. We retrospectively reviewed the medical records of pulmonary NTM patients registered in the hospital with positive mycobacterial cultures during 2013–2016. Factors that were assessed in this study included demographic and clinical characteristics, such as sex, age, residence, treatment history, laboratory examination results and comorbidities. The definition of NTM lung disease met the criteria established by the American Thoracic Society (ATS) in 2007 , includiMycobacterium tuberculosis (MTB) complex and NTM. The NTM strains identified by conventional biochemical method were subcultured on the Löwenstein-Jensen (L-J) medium [Mycobacterium chelonae-Mycobacterium abscessus group by Biochip were further divided into subspecies with the sequencing of multiple genes, including 16S rRNA, hsp65, rpoB, and 16S–23S rRNA internal transcribed spacer (ITS) sequence as previously reported [http://www.ncbi.nlm.nih.gov/BLAST).Media supplied with paranitrobenzoic acid was used for differential identification of ) medium . Colonie) medium . In addireported . The PCRM. abscessus complex was determined with a broth microdilution method based on the guidelines from the Clinical and Laboratory Standards Institute (CLSI) [M. avium and M. intracellulare isolates, three agents were selected for MIC assessment, including clarithromycin, moxifloxacin and linezolid. The in vitro drug susceptibility for these drugs were evaluated with a broth microdilution method, and their breakpoint values were followed the recommendation from CLSI [The in vitro drug susceptibility of e (CLSI) . Eight ae (CLSI) . For M. rom CLSI .P < 0.05. We conducted analyses by using SPSS version 20.0 .All collected data were entered using Epi Data version 3.1 . Each entry was cross checked independently to ensure the data quality. The predictor variables of age, sex, residence, previous history for tuberculosis, and comorbidity were tested for association with various NTM diseases using univariate and multivariate logistic analysis. The level of significance of univariate analysis was 0.05, and that for inclusion in the multivariate model was 0.15. Association between NTM diseases and predictor variables was calculated using adjusted odds ratio and 95% confidence interval. In addition, comparison of rate of drug resistance between different NTM species was evaluated by chi-square and Fisher’s exact tests. Differences were considered to be statistically significant at M. avium complex , M. abscessus complex , M. kansasii  and M. fortuitum . These four groups accounted for 97.9% of all mycobacteria identified. Of 270 M. avium complex isolates, there were 171 M. intracellulare  and 99 M. avium  isolates, respectively. In addition, 58.9% (145/246) of M. abscessus complex isolates were M. abscessus subspecies abscessus, and the remining 41.1% (101/246) belonged to M. abscessus subspecies massiliense  for 40–60 years group and 0.395 (95% CI: 0.235–0.666) for > 60 years group, respectively. We also found that the prevalence of infection caused by RGM and SGM differed significantly in resident and migrant population, and pulmonary RGM diseases were more common in migrant population than resident population . Notably, patients with pulmonary RGM diseases were significantly more likely to have bronchiectasis underlying noted than those with SGM diseases . In contrast, there were no other differences regarding TB history or comorbidities noted between SGM and RGM, respectively (P > 0.05).Comparison in demographic and clinical characteristics of NTM patients between SGM and RGM is summarized in Table M. avium complex and M. abscessus complex. As shown in Table M. avium complex, and the percentages of resistant strains were 4.2% (4/95) for M. avium and 3.8% (6/159) for M. intracellulare, respectively. Moxifloxacin and linezolid also showed potent activity against M. avium complex. There were 5 (5.3%) M. avium isolates and 8 (5.0%) M. intracellulare isolates resistant to moxifloxacin. For linezolid, the proportions of resistant isolates were 11.6% (11/95) for M. avium and 8.2% (13/159) for M. intracellulare, respectively. Of the antimicrobial agents tested, amikacin, clarithromycin, linezolid and tobramycin showed highly active against M. abscessus complex, and less than 5% of M. avium and M. intracellulare were resistant to each drug, respectively. In addition, cefoxitin had moderate activity against M. abscessus complex, and the percentages of cefoxitin -resistance were observed in 33.8% (46/136) of M. abscessus subspecies abscessus and 25.3% (24/95) of M. abscessus subspecies massiliense isolates. Statistical analysis revealed that there were no significant differences in the drug resistant rate between M. abscessus subspecies abscessus and M. abscessus subspecies massiliense (P > 0.05)  [M. abscessus complex. Despite occurring less frequently in Northern America and Europe [M. abscessus are in Asia; we thus speculate that Asian persons may also be more susceptible to M. abscessus infection. In line with our hypothesis, Adjemian and colleagues found that Asian persons have an increased risk for infection with M. abscessus than other ethnics [Pulmonary NTM disease is of increasing public health concern worldwide . This stes (85%) , Denmarkes (85%)  and Soutes (85%) . The secd Europe , 15, thid Europe , Taiwan d Europe  and Soutd Europe . The mard Europe . Interes ethnics . Hence, M. avium complex exceeds M. abscessus complex as the predominant causative agent of pulmonary NTM disease, which reflects the inherent difference in NTM prevalence between pulmonary diseases and colonization. The species difference of pulmonary NTM colonization may partly determine the frequency and manifestations of pulmonary NTM disease [M. avium complex was clinically more relevant than M. abscessus complex among patients meeting the ATS diagnostic criteria [M. avium complex compared with M. abscessus complex would increase the risk from NTM colonization to active disease in respiratory tract.Another interesting finding of this study is that the frequency of NTM species from pulmonary patients differs significantly from the observations from a recent laboratory-based study in Guangzhou . First,  disease . However disease . A popul disease . Similarcriteria . AlthougM. gordonae was the third frequently isolates species in South China on the basis of previous data, accounting for 22.5% of NTM isolates [M. gordonae in the present study. This finding confirms that this species is a rarely isolated weak pathogen, majorly contributing to patient colonization and culture contamination rather than patient disease [Second, isolates . In cont disease .Third, several rare geographically restricted NTM species identified by previous study was not associated with NTM diseases . AlthougM. avium complex lung disease was more common among males than females [Pulmonary NTM disease is not uncommon, particularly among elderly females . Our res females . The gen females . General females , which iNumerous studies have documented that pulmonary NTM infection more frequently affect elderly patients , 25. In M. avium complex is better equipped to grow in low-nutrient conditions than RGM by the development of more culturable biofilm [The association between bronchiectasis and NTM disease has been described by several reports –29. We f biofilm . AlthougM. avium infections are frequently encountered in AIDS patients [M. avium isolates in Southern China. Second, the clinical outcomes of pulmonary NTM patients were not collected in this study, because patients are not under follow-up for NTM diseases in China. As a consequence, we only analysed in vitro antibiotic susceptibility of NTM isolates rather than its correlation with treatment results. Therefore, further study is urgently needed to investigate the correlation between in vitro drug susceptibility and clinical outcomes among NTM patients. Third, another important explanation for poor response to macrolide-based chemotherapy for M. abscessus infections is the inducible macrolide resistance phenotype. Unfortunately, the routine detection of drug susceptibility for RGM only incubates 96-well microtiter plates for 3 days, whereas the detection of inducible resistance requires an extended incubation of plates with reading after 14 days of incubation. As a consequence, the resistance to clarithromycin for M. abscessus would be underestimated. Fourth, although cavitary is another major category regarding NTM pulmonary diseases, the radiological characteristics were not collected in this study due to the limited information in the medical records of patients. Nevertheless, this study provides important hints to help clinicians interpret laboratory results and recognize the risk factors associated with various NTM species.This study is subject to several limitations. First, HIV infection has been regarded as an independent risk factor for NTM infections , while tpatients , we may M. avium complex is the most predominate causative agent of pulmonary NTM disease in Southern China. Female, migrant population, the presence of bronchiectasis are independent risk factors for pulmonary diseases due to RGM. In addition, the prevalence of SGM increases significantly with advancing age. In view of the growing public health concern, further studies will be carried out to determine the association between in vitro susceptibility and treatment outcome among these NTM patients, which is essential to help clinicians select effective regimens for the treatment of NTM infections.In conclusion, our data illustrate that the


Aspergillus terreus extract against three mosquito species, namely Anopheles stephensi, Culex quinquefasciatus, and Aedes aegypti (Diptera: Culicidae). The isolated fungal strain was identified as A. terreus (GenBank accession no: KX694148.1) through morphological and molecular (phylogenetic) analysis. The morphological changes in the treated fourth instar larvae shown the demelanization of cuticle and shrinkage of the internal cuticle of anal papillae. The time duration of extract exposure against the larvae determines the level of toxicity. The extract treated larvae were displayed excitation, violent vertical and horizontal movements with aggressive anal biting behavior as the toxic effect on the neuromuscular system. The results of the biochemical analysis indicated that a decrease in the level of acetylcholinesterase, α-carboxylesterase, and β-carboxylesterase in extract treated fourth instar larvae of all tested mosquito species. The findings of histopathological investigation shown the disorganization of the abdominal region, mainly in mid, hindgut, and gastric caeca, loss of antenna, lateral hair, caudal hair, upper and lower head hairs in the mycelium extract treated An. stephensi, Cx. quinquefasciatus, and Ae. aegypti. The ovicidal bioassay test results showed the mosquito hatchability percentage was directly related to the concentrations of mycelium extract. Nil hatchability of mosquito eggs was noticed at 500 μg/ml concentration. The adulticidal activity of fungal mycelia ethyl acetate extract resulted in a dose-dependent activity (15 and 30 min recovery periods). The higher concentration of extract (1000 mg/L) acted as a repellent, the adult mosquitoes showed restless movement, uncontrolled/anesthetic flight at last died. The better adulticidal activity was observed in the ethyl acetate extract against An. stephensi, Cx. quinquefasciatus followed by Ae. aegypti with the best score of LD50 and LD90 values and nil mortality was found in the control. The results of smoke toxicity assay of the mycelia extract exhibited significant mortality rate against Ae. aegypti (91%), Cx. quinquefasciatus (89%), and An. stephensi (84%). In addition, the present investigation reported the stability and toxic effects of A. terreus mycelium ethyl acetate extract on Artemia nauplii. The swimming speed (0.88 mm s-1) of A. terreus was reduced with ethyl extract 24 h treatment whereas, the control A. nauplii showed the normal speed of 2.96 mm s-1. Altered behavior and swimming movement were observed in the 8 h A. terreus mycelium extract treated A. nauplii. A pale yellow color substance (metabolites) was found in the mid-gut region of the mycelial extract exposed A. nauplii. The outcome of the present study, suggest that the A. terreus metabolites might serve as an alternative, cost-effective, eco-friendly, and target specific mosquitocidal agent in the future.Vector-borne diseases like malaria, filariasis, and dengue are transmitted by mosquitoes and they cause global mortality and morbidity due to an increased resistance against commercial insecticides. The present study was aimed to evaluate the neurobehavioral toxicity, knock-down effect, histopathology, ovicidal, adulticidal, and smoke toxicity effect of  Culex quinquefasciatus is a most irritating vectors and important man-biting mosquitoes leads to cause allergic responses, including local skin and systemic diseases, including angioedema and urticaria -geodin, asterric acid, and butyrolactone I   , Beauver tenella , B. bassbassiana , Lagenidiganteum , and Chr lobatum  are wideAcetylcholinesterase (AChE) enzyme is sometimes called as true or specific cholinesterase and mainly found in nerve cells, skeletal, smooth muscle, various glands, and red blood cells in animals/insects . This maCarboxylesterases are ubiquitous non-specific enzymes it hydrolyse a variety of esters of carboxylic acids and play a major role in insect metabolic activity, i.e., regulation of juvenile hormone (JH), fat metabolism and mobilization, energy-related fat catabolism in muscles, cuticular wax synthesis and transport  mechanisArtemia sp. used as a nutritious live food source for a variety of marine organisms , acetylthiocholine iodide (AChEI), fast blue B, sodium dodecyl sulfate (SDS), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), α- and β- naphthyl acetate and fungal growth medium, potato dextrose agar (PDA), potato dextrose broth (PDB), and sabouraud dextrose broth (SDB) were procured from Hi-Media, Mumbai. The chemicals like ethyl acetate, dimethyl sulfoxide (10% DMSO) and others were obtained from Merck (Germany) and Sigma-Aldrich, United States.A. terreus from soils has been reported by 2O. Polymerase chain reaction  was performed in 50 μl reactions with the following concentrations: 5 U μl-1 of Invitrogen (Brazil) Taq DNA polymerase, 5X Invitrogen Buffer, 2 mM of dNTPs, 1.5 mM Mg2+, and 3 pmol μl-1 of each primer. The 5.8S rDNA (5′–GRAAGNAHADGTVGKAAYAWSG–3′) ITS primers (5′–TCCTNCGYTKATKGVTADGH–3′) (The isolation and identification (morphology and microscopy) steps of ADGH–3′)  were manA. terreus.Agarose gel electrophoresis (DNA) were done based on the modified method of The purification of PCR products was performed using DNA Wizard Clean-Up Kit . The purified PCR products were compared with Invitrogen’s Low DNA Mass Ladder for estimate the concentration of DNA required for sequencing. Using an Applied Biosystem ABI 3730 sequencer (Applied Biosystem) for sequencing the isolated DNA sample. The PCR product was sequenced in the forward and reverse directions, and consensus sequences were created by using BioEdit program version 7.0.9.0 .A. terreus were compared with the available data in NCBI. Multiple sequence alignment was performed with Clustal X software  sequences of the software , with gag method  and the g method . The nodAn. stephensi, Cx. quinquefasciatus, and Ae. aegypti were collected from Centre for Research in Medical Entomology (CRME-ICMR), Madurai and they were maintained in the Natural Drug Research Laboratory, Periyar University, Salem. The mosquitoes were reared in an appropriate equipment, i.e., dechlorinated tap water and maintained at 27 ± 2°C, 70–75% relative humidity (RH), under conditions of 14 h light:10 h dark (LD 14:10) photoperiod cycles without exposure of any pathogens or insecticides. The larvae fed on 3:1 ratio of dog biscuits and yeast powder as a food. Water has been changed every day to avoid scum formation, which may produce toxicity.Larvae of A. terreus were previously reported by Extraction of secondary metabolites, larvicidal, and pupicidal activity, FTIR, GCMS, and HPLC analysis of metabolites from A total of 30 fourth instar larvae were separately released in three replicates for each concentration. The cumulative larval mortality was recorded. Larvae was failed to move after probing with a blunt metallic probe in the thoracic segments is considered as dead and the dead larvae were collected for further study. The abnormal shape, size, discoloration, or failures to pupate  growth deformations of larvae were characterized. Pupal deformations , stage , and adult malformation  of mosquitoes were observed. All survival larvae in treated and control were maintained in the different beakers for further notifications (upto 6 days). The larvae were also monitored carefully for any changes in pigmentation pattern. During an experimental time, brewer’s yeast was not provided to adult mosquitoes .A. terreus against mosquito larvae were studied by the modified method of The neurobehavioral toxicity effect of ethyl acetate extract of The fourth instar larvae (both controls and treated) were taken separately and washed with double distilled water and the surface water was removed by blotting with tissue paper. Larvae (40 Nos) were homogenized with 4 ml phosphate buffered saline (PBS) contains a fresh Eppendorf tube. Then, it was homogenate using centrifugation at 8000 rpm for 15 min. After, the clear supernatant was collected and the pellet was discarded. The supernatant was stored on ice (4°C) until it will be used for performing biochemical assays .The acetylcholinesterase assay of the fourth instar (control and untreated) larval homogenate of selected mosquito was assayed by the method of The α- and β-carboxylesterase activity of the fourth instar larval homogenates was performed as per the modified method of A. terreus ethyl acetate extract treated and control larvae (fourth instar) were fixed in paraformaldehyde solution (10%) for 1 week followed by embedded in paraffin. Larval tissue blocks were sectioned (7 μm) using the microtome , mounted on the glass slide and stained with haematoxylin and eosin for visualization purpose  , 1.159 (0.002–7.977); 0.003 (0.000–0.823), 0.011 (0.000–1.370); and LD90 values of 0.336 (0.000–4.101), 0.785 (0.000–7.115), 0.232 (0.000–3.508), 0.563 (0.000–6.168) mg/ml, respectively. The control showed no mortality (Tables Ae. aegypti (91%) , Cx. quinquefasciatus (89%)  and An. stephensi (84%) . The cent percent of mortality (100%) was obtained in the commercial mosquito control.Nil knocked down mosquitoes were observed during the exposure of the extract at 40 min (test and control groups) Figure , due to A. terreus extract enhance in the smoke toxicity and it can be acted as effective alternative insecticide for An. stephensi, Cx. quinquefasciatus, and Ae. aegypti control. The smoke emitted from the extract exhibit a good knock down effect. However, when compared with the previous study  was carried out for the measurement of toxicity level. In control, A. nauplii showed an average speed of 2.96 mm s-1, after cultured in wells (8 and 48 h). After treated (at 24 h) with A. terreus extract, the swimming speed values were found to be 0.88 mm s-1. We observed behavioral changes in the swimming movement motionless phase and the A. nauplii position, after 8 h incubation treated with A. terreus mycelium extract. After 18 h exposure to the extract reflected slightly toxic cells,  and A. nauplii changes in swimming speed and their position on the water. The LC50 and LC90 values from the bioassay results of ethyl acetate extract against A. terreus strain was presented in Table 50 and LC90 values of A. terreus mycelia extract was noted against A. nauplii larvae treatments, i.e., 38.483, 27.595 (24 h) and 90.017, 53.710 μg/ml (48 h). The occurrence of dark yellow color substance in the gut region of the Artemia sp. treated with A. terreus mycelium ethyl acetate extract clearly indicates the accumulation of extract  did not show any significant changes in the gut region , similar to caused by chemical insecticides (organophosphate or carbamate). Interestingly, Ageratum conyzoides extract remarkably accelerated the growth of Anopheles sp. larvae into pupae resulted into deficient melanization and uneven dead larval-pupal intermediates. Similarly, Achyranthes aspera extract treated with Ae. aegypti larvae showed modified behavioral symptoms viz: excitation and restlessness, observe violent anal self-biting behavior. Likewise, Ae. aegypti larvae treated with the Apium graveolens extract.Mycoinsecticides are better as well as alternative insecticidal agents due to their least contamination level, low toxicity to humans and various other advantages . AbamectAe. aegypti larvae while treating with metabolite than untreated larvae  induced by Ageratum conyzoides extract.We observed the larvae having symptoms of tremors and muscle paralysis are the major sign related to neurobehavioral toxicity. The excessive wriggling movements made by d larvae . A. terrd larvae . In our An. stephensi, Cx. quinquefasciatus, and Ae. aegypti) treated with mycelium extract obtained from A. terreus (500 μg/ml) showed morphological and behavioral changes. After 30 min of exposure, the larvae were found be restless and exhibited sluggish movements, while increasing exposure time. The treated larvae showed morphological changes in the anal papillae region and cuticle layer. Larvae treated with A. terreus mycelium ethyl acetate extract developed dramatic lesions affected mainly in the epithelial layer of the midgut than the control. The cross section of the larvae midgut illustrates disruption in the appearance of columnar cells, swelling, and extruding masses of cellular molecules in the mid gut position. Recently, Bacillus licheniformis exopolysaccharide treated An. stephensi and Ae. aegypti larval showed histological damages of midgut, muscles, and shrinkage in the abdominal regions. Likewise, Penicillium oxalicum showed the damage of microvilli, midgut lumen, peritrophic membrane, and epithelial cells of tested Culex sp. mosquito larvae. Interestingly, Aspergillus clavatus metabolites against Cx. quinquefasciatus larvae. Similarly, Ae. aegypti, An. Stephensi, and Cx. quinquefasciatus. Recently, Ae. aegypti (fourth-instar larvae) had swelling in the gut lumen, reducing intercellular contacts and degeneration of nuclei, after treated with P. daleae mycelium extract.The fourth instar larvae of selected mosquitoes (Ae. aegypti larvae treated with the Bt-based product (Vectobar). The present study results clearly stated that a significant reduction of acetylcholinesterase level activity in the larvae, treated with A. terreus mycelium ethyl acetate extract than control larvae. Similarly, Dendrolimus tabulaeformis larvae treated with Beauveria bassiana spore suspension.Different types of enzymes namely oxidases, reductases, and esterases used by the mosquito for detoxifying the pesticide compounds . Among tAn. stephensi, Cx. quinquefasciatus, and Ae. aegypti) larval homogenates were exposed to A. terreus mycelia extract found to be the disturbed physiological constituents are evident for reduction of detoxification enzymes. This observation suggests that direct toxic effect of A. terreus mycelia ethyl acetate metabolites in the protein synthetic machinery of the mosquito larvae. The α- and β-carboxylesterase activity level was gradually decreased while the formation of fourth instar larvae of three mosquitoes. The exposure of larvae to the A. terreus mycelia metabolite had a dose dependent manner at the level of both carboxylesterase activities. Likewise, Ae. aegypti larvae by The mosquito (An. stephensi (500 μg/ml), Cx. quinquefasciatus (300 μg/ml), and Ae. aegypti (300 μg/ml). The average percent of hatchability of eggs were observed, after 48 h treatment. Overall, the ovicidal activity was decreased with an increase in concentration of A. terreus mycelium ethyl acetate extract. Also, Ae. aegypti. Similarly, Cx. gelidus and Cx. tritaeniorhynchus revealed 100% ovicidal (no egg hatchability) observed in 1,000 ppm.The zero percentage of ovicidal and egg hatchability results were observed in all tested mosquitoes, i.e., A. terreus mycelial extract was showed good adulticidal activity against Ae. aegypti the maximum LD50 and LD90 values found than the Cx. quinquefasciatus and An. stephensi adults. F. pallidoroseum spores against adult mosquitoes. Whereas, the present investigation studied A. terreus mycelia ethyl acetate metabolite having remarkable adulticidal effects against three targeted mosquitoes. The maximum smoke toxicity was recorded (within 40 min) of different mosquitoes, i.e., Ae. aegypti (91%), Cx. quinquefasciatus (89%), and An. stephensi (84%), while commercial mosquito coil showed 100% adult mortality. Recently, Salmonella bongori metabolites showed 62% mortality against Cx. quinquefasciatus compared with positive control (pyrethrin). Also, In contrast, A. nauplii. A. nauplii show a normal swimming speed in the wells. After 24 h treated with mycelia ethyl extract of A. terreus, the swimming speed of organisms has changed as motionless. Likewise, Artemia sp. Fusarium isolates (No: 24) exhibit various levels of toxicity on the brine shrimp, based on the role of fusaproliferin and beauvericin compounds  of Penicillium sp. extract against the brine shrimp mortality, which is quite lower toxicity. Likewise, 50 value of 6-O-methylaverufin (produced by the endophytic fungus A. versicolor) was 0.5 μg/ml. Another study, A. nidulans exhibited strong toxicity against A. salina, with LC50 values ranged from 4.5 to 7.1 μM.Biotoxicity assay of fungal metabolites was conducted with brine shrimp ompounds . The preA. terreus (KX694148.1) through ITS rDNA sequencing and phylogenetic tree analysis. The present study revealed that significant to moderate toxic effect of A. terreus mycelia extract on different stages of mosquitoes such as egg, larvae, pupa, and adults of Ae. aegypti, Cx. quinquefasciatus, and An. stephensi. A. terreus extract exhibited strong neurobehavioral toxicity, knock-down effect, the ovicidal and adulticidal effect on An. stephensi, Cx. quinquefasciatus, and Ae. aegypti in a dose and time-dependent manner. The exposure of A. terreus extract caused significant behavioral changes, growth disturbance and morphological abnormalities on tested mosquito species. The activities of vital mosquito enzymes such as α- and β-carboxylesterase and AchE were decreased upon A. terreus extract treatment. The histopathological study shows A. terreus extract affected the larval development and completely damaged the digestive system of mosquito. The smoke of A. terreus extract induced the time-dependent adult death/toxicity and behavioral changes in mosquitoes. A. terreus extract showed considerable toxic effect, behavioral changes and anomalies on A. nauplii larvae. The outcome of the present investigation concludes that ethyl acetate extract of A. terreus contains promising mosquito control principles which may be used in the control of mosquito in the future. Further studies on isolation and identification of bioactive constituents from A. terreus extract is under process.A fungal strain isolated from soil was identified as All applicable national, international, and/or institutional standard protocols for the concern and utilized the insects/crustaceans were maintained and experiments conducted.CR and DN planned the research, performed the experiments, shared chemicals and analysis tools, and wrote the manuscript. CR analyzed the statistics. RS and MK contributed significant work pertaining to the analysis of data and finally, all the authors has read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


OBJECTIVES/SPECIFIC AIMS: Urinary tract infections (UTIs) serve as one of the most common infections affecting women. With rising reports of antibiotic resistance (ABR), which can prolong illness and limit treatment options, the Infectious Disease Society of America recommends using local resistance patterns to shape empirical treatment selection. Although no studies have evaluated ABR in Ureaplasma spp. urinary isolates in college-aged women, regional studies in the Southeast United States have found levels of tetracycline resistance in over 30% of Ureaplasma spp. clinical isolates. Thus, this study aims to determine the antibiogram for 73 Ureaplasma spp. and 10 Mycoplasma hominis isolates collected from women with first-time UTI against a panel of 9 antibiotics, and assess resistant isolates for genetic mechanisms associated with resistance. METHODS/STUDY POPULATION: This study used archival samples and data collected from college-aged women with first-time UTI recruited to participate in a prospective cohort study conducted at a student healthcare facility from 2001 to 2006 in Florida. Ureaplasma spp. and M. hominis isolates cultured from urine samples collected at the initial clinical presentation and for any recurrent UTI were evaluated for susceptibility to a panel of 9 antibiotics (8 for M. hominis) using validated microbroth and agar dilution methods, respectively. Ureaplasma spp. isolates were tested against azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, doxycycline, gentamicin, levofloxacin, and tetracycline. M. hominis isolates underwent the same testing, with the addition of linezolid and exclusion of azithromycin and erythromycin, as M. hominis is intrinsically resistance to 14 and 15-membered macrolides and azilides. PCR and Sanger sequencing were employed to identify molecular mechanisms associated with resistance. RESULTS/ANTICIPATED RESULTS: Of the 73 Ureaplasma spp. isolates, 1 isolate was resistant to levofloxacin (MIC: 4 µg/mL) and 1 to tetracycline (MIC: 8 µg/mL). All M. hominis isolates were sensitive. For the Ureaplasma spp. isolates, MIC90s were highest against gentamicin (32 µg/mL) and lowest against doxycycline (0.25 µg/mL). PCR amplification identified tetM present in the tetracycline resistant isolate, an established gene associated with tetracycline resistance in Ureaplasma spp. A S83W mutation within the quinolone-resistance-determining region (QRDR) of parC was detected in the levofloxacin resistant isolate. DISCUSSION/SIGNIFICANCE OF IMPACT: Overall, antibiotic resistance in this population of college-aged women with first-time UTI was low. A previous study detected a novel S83W substitution in a perinatal Ureaplasma spp. isolate from Japan, and provided in silico evidence that a S83W change would prevent levofloxacin from binding to its target. However, that study was unable to cultivate the isolate. Our study has provided the corresponding phenotypic evidence that a S83W substitution results in quinolone resistance in Ureaplasma spp.


Platelets play a fundamental role in the increased atherothrombotic risk related to central obesity since they show hyperactivation and lower sensitivity to antiplatelet therapy in obese patients. The main goal of this study was to identify platelet biomarkers related to the risk of atherothrombosis in obese patients, confirm platelet activation levels in these patients, and identify altered activation pathways.Platelets were obtained from cohorts of obese patients and age- and sex-matched lean controls. Biochemical and proteome analyses were done by two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and immunoblotting. Functional and mechanistic studies were conducted with aggregation assays and flow cytometry.We confirmed an up-regulation of αIIb and fibrinogen isoforms in platelets from obese patients. A complementary platelet aggregation approach showed platelets from obese patients are hyper-reactive in response to collagen and collagen-related peptide (CRP), revealing the collagen receptor Glycoprotein VI (GPVI) signalling as one of the altered pathways. We also found the active form of Src (pTyr418) is up-regulated in platelets from obese individuals, which links proteomics to aggregation data. Moreover, we showed that CRP-activated platelets present higher levels of tyrosine phosphorylated PLCγ2 in obese patients, confirming alterations in GPVI signalling. In line with the above, flow cytometry studies show higher surface expression levels of total GPVI and GPVI-dimer in obese platelets, both correlating with BMI.Our results suggest a higher activation state of SFKs-mediated signalling pathways in platelets from obese patients, with a primary involvement of GPVI signalling.  •Higher expression of the active form of Src (pTyr418) in obese patients platelets.•Increased aggregation levels of obese platelets in response to GPVI activation.•GPVI-activated platelets show higher levels of tyr-phosphorylated PLCγ2 in obesity.•Higher surface expression levels of GPVI in obese platelets, correlating with BMI. Financial support from the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia , and the European Regional Development Fund (ERDF) is also gratefully acknowledged. SMJ and RWF were supported by British Heart Foundation, grants SP/10/011/28199 and RG/15/4/31268.This work was supported by the M.N. Barrachina performed research, analyzed data and wrote the paper. A.M. Sueiro supervised clinical work and analyzed data. F.F. Casanueva supervised clinical work. I. Izquierdo and L. Hermida-Nogueira performed experiments; E. Guitián performed mass spectrometry analyses; R. Farndale, M. Moroi and S.M. Jung contributed with key reagents and analytical tools. M. Pardo contributed to the study design and revised the manuscript. A. García designed the study, contributed with key reagents and analytical tools, and wrote the paper.


Caenorhabditis elegans is a classic system for functional genetics with poor ecology. In order to help bridge these disciplines, here we describe the natural history of a close relative of C. elegans, Caenorhabditis inopinata, that is associated with the fig Ficus septica and its pollinating Ceratosolen wasps.Biotic interactions are ubiquitous and require information from ecology, evolutionary biology, and functional genetics in order to be understood. However, study systems that are amenable to investigations across such disparate fields are rare. Figs and fig wasps are a classic system for ecology and evolutionary biology with poor functional genetics; Caenorhabditis, fresh F. septica figs from four Okinawan islands were sampled, dissected, and observed under microscopy. C. inopinata was found in all islands where F. septica figs were found. C.i nopinata was routinely found in the fig interior and almost never observed on the outside surface. C. inopinata was only found in pollinated figs, and C. inopinata was more likely to be observed in figs with more foundress pollinating wasps. Actively reproducing C. inopinata dominated early phase figs, whereas late phase figs with emerging wasp progeny harbored C. inopinata dauer larvae. Additionally, C. inopinata was observed dismounting from Ceratosolen pollinating wasps that were placed on agar plates. C. inopinata was not found on non-pollinating, parasitic Philotrypesis wasps. Finally, C. inopinata was only observed in F. septica figs among five Okinawan Ficus species sampled.To understand the natural context of fig-associated C. inopinata, and they suggest a natural history where this species proliferates in early phase F. septica figs and disperses from late phase figs on Ceratosolen pollinating fig wasps. While consistent with other examples of nematode diversification in the fig microcosm, the fig and wasp host specificity of C. inopinata is highly divergent from the life histories of its close relatives and frames hypotheses for future investigations. This natural co-occurrence of the fig/fig wasp and C. inopinata study systems sets the stage for an integrated research program that can help to explain the evolution of interspecific interactions.These are the first detailed field observations of The online version of this article (10.1186/s12898-018-0182-z) contains supplementary material, which is available to authorized users. Interactions at a broad range of scales structure the organization of biological systems. Within ecology, the biotic environment is a major determinant of the distribution and abundance of both species and communities, and so understanding the origins and maintenance of interspecific interactions is a key goal within the field. Yet, interspecific relationships taken as an aggregate are composed of millions of interactions between individual organisms , 2, and Heliconius butterflies . To. ToC. inuni Fig. . F. septh Mapbox .Fig. 1CaFicus, wasp, and nematode species from natural collections (see below) were initially identified via morphological characteristics. Subsequently, DNA was isolated from some ethanol-preserved, F. septica-derived wasp and nematode specimens and sequenced to verify genus identity. For wasp samples, preserved animals were washed three times in PBS and subsequently crushed with a pestle in a 1.5 mL Eppendorf tube. DNA was then isolated from the suspension with a Qiagen Blood and Tissue DNeasy kit. For worm DNA samples, preserved single individuals were washed three times in PBS and digested with 5% Proteinase K in Tris–EDTA buffer for 1 h at 58 °C. This solution was immediately used for PCR after a 10 min, 95 °C incubation for enzyme deactivation. For wasp and nematode identification, the mitochondrial cytochrome oxidase I (COI) locus was amplified with primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [Parasitodiplogaster salicifoliae (GenBank accession KP015022) and Schistonchus guangzhouensis  were retrieved from GenBank. Sequences of Pristionchus pacificus, Caenorhabditis japonica, and C. elegans were retrieved from WormBase [C. inopinata COI sequence was retrieved from the genome assembly (https://www.ncbi.nlm.nih.gov/nuccore?term=382947%5BBioProject%5D) [ATCA-3′) . PCR reahouensis ; GenBankWormBase . The C. ject%5D) . Sequencject%5D) .C. inopinata presence and fig pollination status. Whereas in May 2016, figs were additionally scored for fig developmental stage, wasp foundress number, and surface nematodes. Unless otherwise noted, the data reported in this study are derived from the larger 2016 set. A fraction of F. septica figs (131/250 dissected figs) were initially washed with tap water before dissection in order to interrogate the presence of fig surface nematodes. Dissected figs were then assayed for fig developmental stage, foundress number (in only 169/250 of dissected F. septica figs), and the presence of C. inopinata under a dissection microscope. Caenorhabditis nematodes exhibit a stereotypical pharyngeal morphology that was used for species identification [C. inopinata animals were binned into reproductive phase . The diematodes , and theematodes –63, we aF. septica figs were caught in a plastic bag  fig of Ficus septica in Okinawa, Japan [F. septica figs were collected from additional Okinawan islands . Also, between-island differences in fig and plant Caenorhabditis occupancy could not be detected in 2015  compared to that of those subsequently dissected . Thus, C. inopinata is associated with the fig interior and not its surface.Ficus are renowned for their classic mutualism with pollinating fig wasps [Ficus-associated nematodes that require such wasps to complete their life cycle [Caenorhabditis, F. septica figs were also queried for their pollination status, which can be ascertained by the presence of developing seed or pollinating wasp progeny. In both field work seasons, C. inopinata animals were never observed in unpollinated F. septica figs (Tables C. inopinata). Thus, C. inopinata likely requires pollinating fig wasps in order to thrive.Plants of the genus ig wasps , and thefe cycle . To inteF. septica fig was noted. Typically, female pollinating wasps enter the fig, pollinate it, lay eggs in the fig ovules, and die [C. inopinata increases with foundress wasp number  than in those without . Thus, higher foundress number is associated with C. inopinata fig occupancy, suggestive that these nematodes disperse on pollinating fig wasps.In addition to pollination status, the number of foundress pollinating wasps per  and die . In a nu and die , 28, 65.Caenorhabditis nematodes can undergo alternative developmental trajectories depending on environmental conditions [F. septica figs were binned into five developmental stages based on wasp presence and development .Fig. 7AC. inopinata, a close relative of the model genetic organism C. elegans. What has been observed in this Caenorhabditis study, together with the known biology of the fig microcosm, can then be used to inform hypotheses regarding the evolution of interspecific relationships in both systems.The intricacy of the fig microcosm has facilitated decades of evolutionary and ecological field studies , 30. It Caenorhabditis species typically proliferate on rotting plants and disperse on invertebrate carriers. And although the features defining niche specialization in this group remain uncertain, it seems clear that there is variation in its extent. Some species appear limited in their geographic range , despite intensive sampling, have only been observed dispersing on one insect species in a highly host-specific manner [C. inopinata in the fresh figs of a single species of Ficus and observations of its dispersal via pollinating wasps reveals a dramatic shift in substrate from rotting plants to fresh figs. This intimate coupling further reveals an added instance of carrier host-specificity in this group. Further, this niche shift has coincided with extreme morphological and developmental divergence [ast Asia ), whereaast Asia . Interspast Asia . Furthermunities , consistmunities . There iyriapods . Other sc manner , 86. Thevergence , 47, sugBursaphelenchus sycophilus is a fig parasite that evolved from fungal-feeders [Parasitodiplogaster nematodes are fig wasp parasites that likely evolved from bacteria-feeders [Teratodiplogaster and Parasitodiplogaster clades rapidly diverged in mouth morphology, consistent with their divergent nutrient sources  [Pristionchus, the evolution of fig-association is connected with dramatic plasticity in mouth morphologies, with a single species having up to five different morphotypes [Caenorhabditis reveals rapid changes in morphology upon fig-association, with C. inopinata being nearly twice as long as its close relatives [C. inopinata, it may be unsurprising that its biology so divergent from its close relatives.Nematodes have evolved to live in association with the fig microcosm at least nine times independently  , 87. In photypes . And Caeelatives , 47. Furelatives  as well elatives , 43, 44.C. inopinata has only been observed dispersing on pollinating fig wasps  and wasps (fig ovules). Male/female Caenorhabditis species tend to be incredibly diverse with enormous population sizes, and C. brenneri is among the most diverse eukaryotes known [C. inopinata should reduce diversity, as has been seen in C. japonica, another Caenorhabditis male/female species with high host-specificity [C. elegans displays reduced diversity, low global population structure, yet high local structure [C. inopinata is dispersed by wasps that can migrate over long distances while exhibiting small founder populations  revealing no isolation by distance over 1000 km of southeast Asia [Schistonchus caprifici nematodes among Turkish [F. septica and its pollinating Ceratosolen wasps themselves have been undertaken across Taiwan and the islands of southeast Asia [Ceratosolen bisculatus as the only pollinator of F. septica in Okinawa [Ceratosolen pollinate F. septica across Taiwan, the Philippines, and Okinawa [F. septica, revealing a potential example of incipient local co-diversification [Ceratosolen species vary in pigmentation color (with some species black and others yellow), but only one black pollinator species was recovered in Okinawa . No yelC. inopinata also displays differences in developmental timing and developmental decision-making from their close relatives [Caenorhabditis observed on invertebrate carriers are in the dauer stage [C. inopinata disperses on pollinating wasps to travel to new figs, it is reasonable to suspect that their divergence in developmental timing and decision-making are related to these features of fig biology. Although it is unclear how many generations are produced within a single fig, C. inopinata may have faced selective pressure to slow its developmental rate in order to match progeny production with the timing of wasp emergence. Further, given that dispersal on pollinating wasps is likely critical for C. inopinata propagation, the decision to enter into dauer may be more dependent on fig and/or wasp chemical cues than those related to stress and population density, which would explain their rarity in laboratory rearing conditions. In addition, fig-associated nematodes often disperse on fig wasps at specific stages in their development: Schistonchus fig parasites disperse at the fourth larval stage, and Parasitodiplogaster fig wasp parasites disperse at the dauer larval (or infective juvenile) stage [elatives . Their delatives , and dauer stage , it is ler stage , and thae) stage . Thus thC. inopinata on fig and fig wasp fitness remains an open question. Unlike the fig parasite Schistonchus [Parasitodiplogaster [C. inopinata is unlikely to inflict direct harm on figs or wasps as a parasite. This is because C. inopinata maintains its typical Caenorhabditis pharyngeal morphology throughout the reproductive stages observed in fresh figs , and prdiameter , and Fic average . C. inopvae Fig. , the per in figs –38, 40. erbivory . As measerbivory , the intcreasing , 109, thC. inopinata was found dispersing on pollinating Ceratosolen wasps, and not Philotrypesis parasitoid wasps emerging from figs of the same tree . The mobug host , and sim beetles . In addiemolymph , and sinlogaster , 43. Theps Figs. , 6. In aematodes , althougematodes , 114. Thocarbons . SimilarC. elegans that is associated with figs and fig wasps. The fig–fig wasp system is a legendary study system in evolution and ecology, and C. elegans is a legendary one in model systems genetics. Here then is a serendipitous convergence of research organisms that can facilitate the conceptual connection of their respective disciplines. The functional genetics of C. inopinata has the potential to inform the molecular basis of how ecologically-relevant phenotypes are generated, whereas the evolution and ecology of the fig system can inform how population-level and environmental forces sort said variation. This all begins with a simple understanding of where and how this organism lives in nature.The elegance of contemporary molecular biology resides in the explanatory power generated by conceptual continuity across multiple hierarchical levels   among different islands Fisher’s exact test p-values. Table S3. Differences in C. inopinata fig occupancy (excluding pollinated figs) among different islands Fisher’s exact test p-values. Table S4.C. inopinata occupancy in Ficus septica figs in 2015. Table S5. Differences in C. inopinata plant and fig occupancy in different field seasons Fisher’s exact test p-values. Table S6. Differences in C. inopinata fig occupancy given different foundress number Fisher’s exact test p-values. Table S7. Differences in the presence of reproductive stage C. inopinata given different fig stages Fisher’s exact test p-values. Table S8. Differences in the presence of dauer stage C. inopinata given different fig stages Fisher’s exact test p-values. Table S9. Repeated convergence of fig-association in nematodes.