Metagenomic analysis of MWWTP effluent treated via solar photo-Fenton at neutral pH: Effects upon microbial community, priority pathogens, and antibiotic resistance genes.

The effectiveness of advanced technologies on eliminating antibiotic resistant bacteria (ARB) and resistance genes (ARGs) from wastewaters have been recently investigated. Solar photo-Fenton has been proven effective in combating ARB and ARGs from Municipal Wastewater Treatment Plant effluent (MWWTPE). However, most of these studies have relied solely on cultivable methods to assess ARB removal. This is the first study to investigate the effect of solar photo-Fenton upon ARB and ARGs in MWWTPE by high throughput metagenomic analysis (16S rDNA sequencing and Whole Genome Sequencing). Treatment efficiency upon priority pathogens and resistome profile were also investigated. Solar photo-Fenton (30 mg L-1 of Fe2+ intermittent additions and 50 mg L-1 of H2O2) reached 76-86% removal of main phyla present in MWWTPE. An increase in Proteobacteria abundance was observed after solar photo-Fenton and controls in which H2O2 was present as an oxidant (Fenton, H2O2 only, solar/H2O2). Hence, tolerance mechanisms presented by this group should be further assessed. Solar photo-Fenton achieved complete removal of high priority Staphylococcus and Enterococcus, as well as Klebsiella pneumoniae and Pseudomonas aeruginosa. Substantial reduction of intrinsically multi-drug resistant bacteria was detected. Solar photo-Fenton removed nearly 60% of ARGs associated with sulfonamides, macrolides, and tetracyclines, and complete removal of ARGs related to ß-lactams and fluoroquinolones. These results indicate the potential of using solar-enhanced photo-Fenton to limit the spread of antimicrobial resistance, especially in developing tropical countries.

Combat of antimicrobial resistance in municipal wastewater treatment plant effluent via solar advanced oxidation processes: Achievements and perspectives.

This review aims to gather main achievements and limitations associated to the application of solar photocatalytic processes with regard to the removal of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from municipal wastewater treatment plant effluent (MWWTPE). Solar photocatalytic processes were chosen considering the context of developing tropical countries. Among these processes, solar photo-Fenton has been proved effective for the elimination of ARB from MWWTPE at neutral pH in bench and pilot scale and also under continuous flow. Yet, ARG removal varies as according to the gene. Irradiation intensity and matrix composition play a key role on treatment efficiency for this purpose. The use of sulfate radical in modified solar photo-Fenton is still incipient for ARB and ARG removal. Also, investigations related to ARB resistance profile and horizontal gene transfer rates after solar photo-Fenton treatment must be further analyzed. Regarding solar heterogeneous photocatalysis, TiO2 and TiO2-composites applied in suspension are the most commonly investigated for the removal of ARB and ARGs. Irradiation intensity, temperature and catalyst dosage affect treatment efficiency. However, most studies were performed in synthetic solutions using reduced sample volumes. Extended exposition times and addition of H2O2 to the system (solar/TiO2/H2O2) are required to prevent bacteria regrowth and ensure ARG abatement. In addition, enhancement of TiO2 with graphene or (semi)metals improved ARB elimination. Differences concerning irradiation intensity, matrix composition, catalyst dosage, and model ARB and ARGs used in studies analyzed in this review hinder the comparison of photocatalysts synthesized by various research groups. Finally, future research should aim at evaluating the efficiency of solar photocatalytic processes in real matrices originated from sewage treatment systems applied in developing countries; determining indicators of antimicrobial resistance in MWWTPE; and investigating ARB mutation rate as well as the removal of cell-free ARGs present in suspension in MWWTPE.

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

Genome of the avirulent human-infective trypanosome--Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts. METHODOLOGY/PRINCIPAL FINDINGS: The T. rangeli haploid genome is ∼ 24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins. CONCLUSIONS/SIGNIFICANCE: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.

Innate resistance against Toxoplasma gondii: an evolutionary tale of mice, cats, and men.

Recent studies have revealed remarkable species specificity of the Toll-like receptors (TLRs) TLR11 and TLR12 and the immunity-related GTPase (IRG) proteins that are essential elements for detection and immune control of Toxoplasma gondii in mice, but not in humans. The biological and evolutionary implications of these findings for the T. gondii host-pathogen relationship and for human disease are discussed.

Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Distinct genomic organization, mRNA expression and cellular localization of members of two amastin sub-families present in Trypanosoma cruzi.

BACKGROUND: Amastins are surface glycoproteins (approximately 180 residues long) initially described in Trypanosoma cruzi as particularly abundant during the amastigote stage of this protozoan parasite. Subsequently, they have been found to be encoded by large gene families also present in the genomes of several species of Leishmania and in other Trypanosomatids. Although most amastin genes are organized in clusters associated with tuzin genes and are up-regulated in the intracellular stage of T. cruzi and Leishmania spp, distinct genomic organizations and mRNA expression patterns have also been reported. RESULTS: Based on the analysis of the complete genome sequences of two T. cruzi strains, we identified a total of 14 copies of amastin genes in T. cruzi and showed that they belong to two of the four previously described amastin subfamilies. Whereas δ-amastin genes are organized in two or more clusters with alternating copies of tuzin genes, the two copies of ß-amastins are linked together in a distinct chromosome. Most T. cruzi amastins have similar surface localization as determined by confocal microscopy and western blot analyses. Transcript levels for δ-amastins were found to be up-regulated in amastigotes from several T. cruzi strains, except in the G strain, which is known to have low infection capacity. In contrast, in all strains analysed, ß-amastin transcripts are more abundant in epimastigotes, the stage found in the insect vector. CONCLUSIONS: Here we showed that not only the number and diversity of T. cruzi amastin genes is larger than what has been predicted, but also their mode of expression during the parasite life cycle is more complex. Although most T. cruzi amastins have a similar surface localization, only δ-amastin genes have their expression up-regulated in amastigotes. The results showing that a sub-group of this family is up-regulated in epimastigotes, suggest that, in addition of their role in intracellular amastigotes, T. cruzi amastins may also serve important functions during the insect stage of the parasite life cycle. Most importantly, evidence for their role as virulence factors was also unveiled from the data showing that δ-amastin expression is down regulated in a strain presenting low infection capacity.