Pyrethroid genetic resistance in the dengue vector (Aedes aegypti) in Posadas, Argentina.

Pyrethroids are extensively used to control adult populations of the arboviral vector Aedes aegypti, raising concerns regarding the increasing frequency and distribution of insecticide resistance mutations (kdr: knock-down resistance) in the voltage-gated sodium channel gene (Nav). The widespread use of pyrethroids imposes a threat to the success of mosquito control and the environment. In this study, we investigated the presence of two kdr mutations (V1016I and F1534C) in the Nav gene and their distribution across four neighborhoods in Posadas, Argentina, with different Ae. aegypti abundance and contrasting socioeconomic status (SES). Alleles at each locus were interrogated using TaqMan SNP genotyping assays in DNA extracted from adult females collected in a longitudinal study. We report the presence of both pyrethroid resistance alleles (kdr 1016I = 29.08%; kdr 1534C = 70.70%) among adult females. The frequency of combined kdr genotypes reveals that approximately 70% of local adult females have enhanced resistance to pyrethroids. Both, the proportion of resistant adult females (with at least one kdr allele in each locus) and Ae. aegypti abundance showed an uneven distribution between neighborhoods with different SES (p < 0.001). In high-SES neighborhoods, we found more mosquitoes and a higher frequency of pyrethroid resistance, possibly as a consequence of different public health interventions, social habits, and insecticide use. This is the first report of kdr mutations in Ae. Aegypti in the northeast region of Argentina. Our results focus on the need for within-population (city) distribution analyses of kdr mutations and highlight the relevance of incorporating insecticide resistance monitoring within the Integrated Vector Management initiative.

Burkholderia cepacia complex: 11 years of surveillance in patients with Cystic Fibrosis in Posadas, Argentina

Abstract Cystic fibrosis patients with Burkholderia cepacia complex pulmonary infections have high morbidity and mortality. Worldwide, this disease is undergoing substantial epidemiological changes. Advances in the diagnosis and treatment have conditioned an increase in child sur-vival as well as in the proportion of affected adults. In order to know our reality, we refer to an epidemiological study in 64 CF patients during 11 years of surveillance, focusing on infections caused by Burkholderia species. Conventional and automated phenotypic tests, restriction fragment length polymorphism-recA, recA gene sequencing, and matrix-assisted laser desorp-tion ionization-time of flight (MALDI-TOF) mass spectrometry were applied. Bacterial isolates were also tested for antimicrobial susceptibility patterns. The prevalence of Burkholderia cepacia complex was 9.4%. Based on recA gene sequencing, the most common species identified were Burkholderia cenocepacia (67.3%) and Burkholderia vietnamiensis (20.3%). Ceftazidime and meropenem were the most active, inhibiting 53% and 46% of isolates, respectively. This report represents the first systematic study of Burkholderia infections in our CF population since beginning of monitoring and treatment and highlights the importance of continued longitudinal studies.

Resumen Los pacientes con fibrosis quística (FQ) con infecciones pulmonares causadas por especies del complejo Burkholderia cepacia tienen una alta morbimortalidad. En todo el mundo, esta enfermedad está experimentando cambios epidemiológicos sustanciales. Los avances en el diagnóstico y el tratamiento han condicionado un aumento en la supervivencia infantil, así como en la proporción de adultos afectados. Para conocer nuestra realidad, nos referimos a un estudio epidemiológico en 64 pacientes con FQ durante 11 años de vigilancia, focalizando las infecciones causadas por especies del género Burkholderia. Se aplicaron pruebas fenotípicas convencionales y automatizadas, polimorfismo de longitud de fragmentos de restricción-recA, secuenciación del gen recA y espectrometría de masa MALDI-TOF. Los aislados bacterianos también se analizaron para determinar los patrones de susceptibilidad antimicrobiana. La prevalencia de complejo B. cepacia fue del 9,4%. Con base en la secuenciación del gen recA, las especies más comunes identificadas fueron Burkholderia cenocepacia (67,3%) y Burkholderia vietnamiensis (20,3%). Ceftazidima y meropenem fueron los antibióticos más activos e inhibieron el 53 y el 46% de los aislamientos, respectivamente. Este informe representa el primer estudio sistemático de las infecciones por Burkholderia en nuestra población desde el comienzo de la monitorización y el tratamiento, y resalta la importancia de continuar los estudios de vigilancia longitudinales.

Thinning Partially Mitigates the Impact of Atlantic Forest Replacement by Pine Monocultures on the Soil Microbiome.

Forest replacement by exotic plantations drive important changes at the level of the overstory, understory and forest floor. In the Atlantic Forest of northern Argentina, large areas have been replaced by loblolly pine (Pinus taeda L.) monocultures. Plant and litter transformation, together with harvesting operations, change microclimatic conditions and edaphic properties. Management practices such as thinning promote the development of native understory vegetation and could counterbalance negative effects of forest replacement on soil. Here, the effects of pine plantations and thinning on physical, chemical and microbiological soil properties were assessed. Bacterial, archaeal, and fungal community structure were analyzed using a metabarcoding approach targeting ribosomal markers. Forest replacement and, to a lesser extent, thinning practices in the pine plantations induced significant changes in soil physico-chemical properties and associated shifts in bacterial and fungal communities. Most measured physical and chemical properties were altered due to forest replacement, but a few of these properties reached values similar to natural forests under the thinning operation. Fungal alpha diversity decreased in pine plantations, whereas bacterial alpha diversity tended to increase but with little statistical support. Shifts in community composition were observed for both fungal and bacterial domains, and were mostly related to changes in plant understory composition, soil carbon, organic matter, water content, pH and bulk density. Among several other changes, highly abundant phyla such as Proteobacteria (driven by many genera) and Mortierellomycota (mainly driven by Mortierella) decreased in relative abundance in the plantations, whereas Acidobacteria (mainly driven by Acidothermus and Candidatus Koribacter) and Basidiomycota (mainly driven by the ectomycorrhiza Russula) showed the opposite response. Taken together, these results provide insights into the effects of forest replacement on belowground properties and elucidate the potentially beneficial effect of thinning practices in intensive plantation systems through promoting the understory development. Although thinning did not entirely counterbalance the effects of forest replacement on physical, chemical and biological soil properties, the strategy helped mitigating the effects and might promote resilience of these properties by the end of the rotation cycle, if subsequent management practices compatible with the development of a native understory vegetation are applied.

Burkholderia cepacia complex: 11 years of surveillance in patients with Cystic Fibrosis in Posadas, Argentina.

Cystic fibrosis patients with Burkholderia cepacia complex pulmonary infections have high morbidity and mortality. Worldwide, this disease is undergoing substantial epidemiological changes. Advances in the diagnosis and treatment have conditioned an increase in child survival as well as in the proportion of affected adults. In order to know our reality, we refer to an epidemiological study in 64 CF patients during 11 years of surveillance, focusing on infections caused by Burkholderia species. Conventional and automated phenotypic tests, restriction fragment length polymorphism-recA, recA gene sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry were applied. Bacterial isolates were also tested for antimicrobial susceptibility patterns. The prevalence of Burkholderia cepacia complex was 9.4%. Based on recA gene sequencing, the most common species identified were Burkholderia cenocepacia (67.3%) and Burkholderia vietnamiensis (20.3%). Ceftazidime and meropenem were the most active, inhibiting 53% and 46% of isolates, respectively. This report represents the first systematic study of Burkholderia infections in our CF population since beginning of monitoring and treatment and highlights the importance of continued longitudinal studies.

Yerba mate (Ilex paraguariensis, A. St.-Hil.) de novo transcriptome assembly based on tissue specific genomic expression profiles.

BACKGROUND: The most common infusion in southern Latin-American countries is prepared with dried leaves of Ilex paraguariensis A. St.-Hil., an aboriginal ancestral beverage known for its high polyphenols concentration currently consumed in > 90% of homes in Argentina, in Paraguay and Uruguay. The economy of entire provinces heavily relies on the production, collection and manufacture of Ilex paraguariensis, the fifth plant species with highest antioxidant activity. Polyphenols are associated to relevant health benefits including strong antioxidant properties. Despite its regional relevance and potential biotechnological applications, little is known about functional genomics and genetics underlying phenotypic variation of relevant traits. By generating tissue specific transcriptomic profiles, we aimed to comprehensively annotate genes in the Ilex paraguariensis phenylpropanoid pathway and to evaluate differential expression profiles. RESULTS: In this study we generated a reliable transcriptome assembly based on a collection of 15 RNA-Seq libraries from different tissues of Ilex paraguariensis. A total of 554 million RNA-Seq reads were assembled into 193,897 transcripts, where 24,612 annotated full-length transcripts had complete ORF. We assessed the transcriptome assembly quality, completeness and accuracy using BUSCO and TransRate; consistency was also evaluated by experimentally validating 11 predicted genes by PCR and sequencing. Functional annotation against KEGG Pathway database identified 1395 unigenes involved in biosynthesis of secondary metabolites, 531 annotated transcripts corresponded to the phenylpropanoid pathway. The top 30 differentially expressed genes among tissue revealed genes involved in photosynthesis and stress response. These significant differences were then validated by qRT-PCR. CONCLUSIONS: Our study is the first to provide data from whole genome gene expression profiles in different Ilex paraguariensis tissues, experimentally validating in-silico predicted genes key to the phenylpropanoid (antioxidant) pathway. Our results provide essential genomic data of potential use in breeding programs for polyphenol content. Further studies are necessary to assess if the observed expression variation in the phenylpropanoid pathway annotated genes is related to variations in leaves' polyphenol content at the population scale. These results set the current reference for Ilex paraguariensis genomic studies and provide a substantial contribution to research and biotechnological applications of phenylpropanoid secondary metabolites.

Burkholderia puraquae sp. nov., a novel species of the Burkholderia cepacia complex isolated from hospital settings and agricultural soils.

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and ß-galactosidase activities.

First time identification of Pandoraea sputorum from a patient with cystic fibrosis in Argentina: a case report.

BACKGROUND: Pandoraea species are considered emerging pathogens in the context of cystic fibrosis (CF) and are difficult to identify by conventional biochemical methods. These multidrug resistant bacteria remain poorly understood particularly in terms of natural resistance, mechanisms of acquired resistance and impact on the prognosis of the disease and the lung function. Among them, Pandoraea sputorum has been previously described in few cases of CF patients from Spain, Australia, France and United States, underlining the need of more clinical data for a better knowledge of its pathogenicity. This is the first report relating to P. sputorum in a CF patient in Argentina. CASE PRESENTATION: Pandoraea sputorum was identified in a nine-year-old cystic fibrosis patient from Argentina, after treatment failure during an exacerbation. The isolates were successfully identified by combining molecular techniques based on 16S rRNA sequencing and mass spectrometry (MS) methods, after reassessing previous misidentified isolates by conventional methods. After first isolation of P. sputorum, patient's clinical condition worsened but later improved after a change in the treatment. Although isolates showed susceptibility to trimethoprim-sulfamethoxazole and imipenem, in our case, the antibiotic treatment failed in the eradication of P. sputorum. CONCLUSIONS: All combined data showed a chronic colonization with P. sputorum associated to a deterioration of lung function. We noted that the presence of P. sputorum can be underestimated in CF patients and MALDI-TOF MS appears to be a promising means of accurate identification of Pandoraea species.

Publish (in English) or perish: The effect on citation rate of using languages other than English in scientific publications.

There is a tendency for non-native English scientists to publish exclusively in English, assuming that this will make their articles more visible and cited. We tested this hypothesis by comparing the effect of language on the number of citations of articles published in six natural sciences journals from five countries that publish papers in either English or other languages. We analyzed the effect of language (English vs non-English), paper length, and year of publication on the number of citations. The articles published in English have a higher number of citations than those published in other languages, when the effect of journal, year of publication, and paper length are statistically controlled. This may result because English articles are accessible to a larger audience, but other factors need to be explored. Universities and scientific institutions should be aware of this situation and improve the teaching of English, especially in the natural sciences.

Chemical scaffolds with structural similarities to siderophores of nonribosomal peptide-polyketide origin as novel antimicrobials against Mycobacterium tuberculosis and Yersinia pestis.

Mycobacterium tuberculosis (Mtb) and Yersinia pestis (Yp) produce siderophores with scaffolds of nonribosomal peptide-polyketide origin. Compounds with structural similarities to these siderophores were synthesized and evaluated as antimicrobials against Mtb and Yp under iron-limiting conditions mimicking the iron scarcity these pathogens encounter in the host and under standard iron-rich conditions. Several new antimicrobials were identified, including some with increased potency in the iron-limiting condition. Our study illustrates the possibility of screening compound libraries in both iron-rich and iron-limiting conditions to identify antimicrobials that may selectively target iron scarcity-adapted bacteria and highlights the usefulness of building combinatorial libraries of compounds having scaffolds with structural similarities to siderophores to feed into antimicrobial screening programs.

A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis.

BACKGROUND: Yersinia pestis is the causative agent of plague and a potential agent of bioterrorism and biowarfare. The plague biothreat and the emergence of multidrug-resistant plague underscore the need to increase our understanding of the intrinsic potential of Y. pestis for developing antimicrobial resistance and to anticipate the mechanisms of resistance that may emerge in Y. pestis. Identification of Y. pestis genes that, when overexpressed, are capable of reducing antibiotic susceptibility is a useful strategy to expose genes that this pathogen may rely upon to evolve antibiotic resistance via a vertical modality. In this study, we explored the use of a multicopy suppressor, Escherichia coli host-based screening approach as a means to expose antibiotic resistance determinant candidates in Y. pestis. RESULTS: We constructed a multicopy plasmid-based, Y. pestis genome-wide expression library of nearly 16,000 clones in E. coli and screened the library for suppressors of the antimicrobial activity of ofloxacin, a fluoroquinolone antibiotic. The screen permitted the identification of a transcriptional regulator-encoding gene (robAYp) that increased the MIC99 of ofloxacin by 23-fold when overexpressed from a multicopy plasmid in Y. pestis. Additionally, we found that robAYp overexpression in Y. pestis conferred low-level resistance to many other antibiotics and increased organic solvent tolerance. Overexpression of robAYp also upregulated the expression of several efflux pumps in Y. pestis. CONCLUSION: Our study provides proof of principle for the use of multicopy suppressor screening based on the tractable and easy-to-manipulate E. coli host as a means to identify antibiotic resistance determinant candidates of Y. pestis.

Small molecules with structural similarities to siderophores as novel antimicrobials against Mycobacterium tuberculosis and Yersinia pestis.

Drugs inhibiting the iron scarcity-induced, siderophore-mediated iron-scavenging systems of Mycobacterium tuberculosis (Mtb) and Yersinia pestis (Yp) may provide new therapeutic lines of defense. Compounds with structural similarities to siderophores were synthesized and evaluated as antimicrobials against Mtb and Yp under iron-limiting conditions, which mimic the iron scarcity these pathogens encounter and must adapt to in the host, and under standard iron-rich conditions for comparison. New antimicrobials were identified, some of which warrant exploration as initial leads against potentially novel targets and small-molecule tools to assist in the elucidation of targets specific to iron-scarcity adapted Mtb and Yp.

Mycobacterial phenolic glycolipid virulence factor biosynthesis: mechanism and small-molecule inhibition of polyketide chain initiation.

Phenolic glycolipids (PGLs) are polyketide-derived virulence factors produced by Mycobacterium tuberculosis, M. leprae, and other mycobacterial pathogens. We have combined bioinformatic, genetic, biochemical, and chemical biology approaches to illuminate the mechanism of chain initiation required for assembly of the p-hydroxyphenyl-polyketide moiety of PGLs. Our studies have led to the identification of a stand-alone, didomain initiation module, FadD22, comprised of a p-hydroxybenzoic acid adenylation domain and an aroyl carrier protein domain. FadD22 forms an acyl-S-enzyme covalent intermediate in the p-hydroxyphenyl-polyketide chain assembly line. We also used this information to develop a small-molecule inhibitor of PGL biosynthesis. Overall, these studies provide insights into the biosynthesis of an important group of small-molecule mycobacterial virulence factors and support the feasibility of targeting PGL biosynthesis to develop new drugs to treat mycobacterial infections.

The dimycocerosate ester polyketide virulence factors of mycobacteria.

Recent advances in the study of mycobacterial lipids indicate that the class of outer membrane lipids known as dimycocerosate esters (DIMs) are major virulence factors of clinically relevant mycobacteria including Mycobacterium tuberculosis and Mycobacterium leprae. DIMs are a structurally intriguing class of polyketide synthase-derived wax esters discovered over seventy years ago, yet, little was known until recently about their biosynthesis. Availability of several mycobacterial genomes has accelerated progress toward clarifying steps in the DIM biosynthetic pathway and it is our belief that reviewing the bases of our current knowledge will clarify outstanding issues and help direct future endeavors.

Pseudomonas aeruginosa SoxR does not conform to the archetypal paradigm for SoxR-dependent regulation of the bacterial oxidative stress adaptive response.

SoxR is a transcriptional regulator that controls an oxidative stress response in Escherichia coli. The regulator is primarily activated by superoxide anion-dependent oxidation. Activated SoxR turns on transcription of a single gene, soxS, which encodes a transcriptional regulator that activates a regulon that includes dozens of oxidative stress response genes. SoxR homologues have been identified in many bacterial species, including the opportunistic pathogen Pseudomonas aeruginosa. However, the expected SoxR partner, SoxS, has not been found in P. aeruginosa. Thus, the primary gene target(s) of P. aeruginosa SoxR is unknown and the involvement of this regulator in the oxidative stress response of the bacterium remains unclear. We utilized transcriptome profiling to identify the P. aeruginosa SoxR regulon and constructed and characterized an unmarked P. aeruginosa DeltasoxR mutant. We provide evidence indicating that P. aeruginosa SoxR activates a six-gene regulon in response to O(2)(.-)-induced stress. The regulon includes three transcriptional units: (i) the recently identified mexGHI-ompD four-gene operon, which encodes a multidrug efflux pump system involved in quorum-sensing signal homeostasis; (ii) gene PA3718, encoding a probable efflux pump; and (iii) gene PA2274, encoding a probable monooxygenase. We also demonstrate that P. aeruginosa SoxR is not a key regulatory player in the oxidative stress response. Finally, we show that P. aeruginosa SoxR is required for virulence in a mouse model of intrapulmonary infection. These results demonstrate that the E. coli-based SoxRS paradigm does not hold in P. aeruginosa and foster new hypotheses for the possible physiological role of P. aeruginosa SoxR.

Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis.

Mycobacterium tuberculosis and Yersinia pestis, the causative agents of tuberculosis and plague, respectively, are pathogens with serious ongoing impact on global public health and potential use as agents of bioterrorism. Both pathogens have iron acquisition systems based on siderophores, secreted iron-chelating compounds with extremely high Fe3+ affinity. Several lines of evidence suggest that siderophores have a critical role in bacterial iron acquisition inside the human host, where the free iron concentration is well below that required for bacterial growth and virulence. Thus, siderophore biosynthesis is an attractive target in the development of new antibiotics to treat tuberculosis and plague. In particular, such drugs, alone or as part of combination therapies, could provide a valuable new line of defense against intractable multiple-drug-resistant infections. Here, we report the design, synthesis and biological evaluation of a mechanism-based inhibitor of domain salicylation enzymes required for siderophore biosynthesis in M. tuberculosis and Y. pestis. This new antibiotic inhibits siderophore biosynthesis and growth of M. tuberculosis and Y. pestis under iron-limiting conditions.

Crystal structure of PapA5, a phthiocerol dimycocerosyl transferase from Mycobacterium tuberculosis.

Polyketide-associated protein A5 (PapA5) is an acyltransferase that is involved in production of phthiocerol and phthiodiolone dimycocerosate esters, a class of virulence-enhancing lipids produced by Mycobacterium tuberculosis. Structural analysis of PapA5 at 2.75-A resolution reveals a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase, and VibH, a non-ribosomal peptide synthesis condensation enzyme. The PapA5 active site includes conserved histidine and aspartic acid residues that are critical to PapA5 acyltransferase activity. PapA5 catalyzes acyl transfer reactions on model substrates that contain long aliphatic carbon chains, and two hydrophobic channels were observed linking the PapA5 surface to the active site with properties consistent with these biochemical activities and substrate preferences. An additional alpha helix not observed in other acyltransferase structures blocks the putative entrance into the PapA5 active site, indicating that conformational changes may be associated with PapA5 activity. PapA5 represents the first structure solved for a protein involved in polyketide synthesis in Mycobacteria.

Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5.

Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acyltransferase [corrected]. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin.