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The CRISPR-Cas technology can be used to disable drug resistance genes. Since carbapenem resistance can be mediated by multiple resistance determinants, we here investigated the extent of re-sensizitation when targeting the bla KPC carbapenemase gene and assessed possible effects on porins and efflux pumps. While full re-sensitization was achieved in a laboratory strain of Escherichia coli solely equipped with bla KPC , resistance reduction in a clinical isolate of Klebsiella michiganensis was achieved in 63% of analyzed transformants, which was a consequence of plasmid copy number reduction and decreased bla KPC gene expression. Damages in the Cas9, as well as alterations in carbapenem-resistance promoting genes including ompK36 downregulation and mutations in the acrB gene were found, likely preventing more efficient re-sensitization. Hence, interference with a single resistance gene promoted the emergence of clonal variants that exhibit alterations in outer membrane proteins. Those bacterial countermeasures, however, were not sufficient to restore the original carbapenem-resistant phenotype.

Background While the macrophage polarization is well characterized in helminth infections, the natural heterogeneity of monocytes with multiple cell phenotypes might influence the outcome of neglected diseases, such hookworm infection. Here, we report the profile of monocytes in human hookworm infections as a model to study the regulatory subpopulation of monocytes in helminth infections. Methods Blood samples were collected from 19 Necator americanus -infected individuals and 13 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated, and immunophenotyping was conducted by flow cytometry. The expressions of genes encoding human nitric oxide synthase (iNOS), interleukin 4 (IL-4), arginase-1 (Arg-1) and glyceraldehyde 3-phosphate dehydrogenase were quantified by qPCR. Plasma levels of IL-4 were determined by sandwich ELISA. Unpaired t-tests or Mann-Whitney tests were used depending on the data distribution. Results Hookworm infected individuals (HWI) showed a significant increase in the number of monocytes/mm 3 (555.2 ± 191.0) compared to that of the non-infected (NI) individuals (120.4 ± 44.7) ( p  < 0.0001). While the frequencies of CD14 + IL-10 + and CD14 + IL-12 + cells were significantly reduced in the HWI compared to NI group ( p  = 0.0289 and p  < 0.0001, respectively), the ratio between IL-10/IL-12 producing monocytes was significantly elevated in HWI ( p  = 0.0004), indicating the potential regulatory activity of these cells. Measurement of IL-4 levels and gene expression of IL-4 and Arg-1 (highly expressed in alternatively activated macrophages) revealed no significant differences between the NI and HWI groups. Interestingly, individuals from the HWI group had higher expression of the iNOS gene (associated with a regulatory profile) (20.27 ± 2.97) compared to the NI group (11.28 ± 1.18, p  = 0.0409). Finally, individuals from the HWI group had a significantly higher frequency of CD206 + CD23 + IL-10 + (7.57 ± 1.96) cells compared to individuals from the NI group (0.35 ± 0.09) ( p  < 0.001), suggesting that activated monocytes are a potential source of regulatory cytokines during hookworm infection. Conclusions Natural hookworm infection induces a high frequency of circulating monocytes that present a regulatory profile and promote the downmodulation of the proinflammatory response, which may contribute to prolonged survival of the parasite in the host.

To date, the investigation of genes involved in Al resistance has focused mainly on microarrays and short periods of Al exposure. We investigated genes involved in the global response under Al stress by tracking the expression profile of two inbred popcorn lines with different Al sensitivity during 72 h of Al stress. A total of 1003 differentially expressed genes were identified in the Al-sensitive line, and 1751 were identified in the Al-resistant line, of which 273 were shared in both lines. Genes in the category of “response to abiotic stress” were present in both lines, but there was a higher number in the Al-resistant line. Transcription factors, genes involved in fatty acid biosynthesis, and genes involved in cell wall modifications were also detected. In the Al-resistant line, GST6 was identified as one of the key hub genes by co-expression network analysis, and ABC6 may play a role in the downstream regulation of CASP-like 5. In addition, we suggest a class of SWEET transporters that might be involved in the regulation of vacuolar sugar storage and may serve as mechanisms for Al resistance. The results and conclusions expand our understanding of the complex mechanisms involved in Al toxicity and provide a platform for future functional analyses and genomic studies of Al stress in popcorn.

Quorum sensing is a cell-cell communication mechanism mediated by chemical signals that leads to differential gene expression in response to high population density. Salmonella is unable to synthesize the autoinducer-1 (AI-1), N-acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. This study aimed to evaluate the fatty acid and protein profiles of Salmonella enterica serovar Enteritidis PT4 578 throughout time of cultivation in the presence of AHL. The presence of N-dodecanoyl-homoserine lactone (C12-HSL) altered the fatty acid and protein profiles of Salmonella cultivated during 4, 6, 7, 12 and 36 h in anaerobic condition. The profiles of Salmonella Enteritidis at logarithmic phase of growth (4 h of cultivation), in the presence of C12-HSL, were similar to those of cells at late stationary phase (36 h). In addition, there was less variation in both protein and fatty acid profiles along growth, suggesting that this quorum sensing signal anticipated a stationary phase response. The presence of C12-HSL increased the abundance of thiol related proteins such as Tpx, Q7CR42, Q8ZP25, YfgD, AhpC, NfsB, YdhD and TrxA, as well as the levels of free cellular thiol after 6 h of cultivation, suggesting that these cells have greater potential to resist oxidative stress. Additionally, the LuxS protein which synthesizes the AI-2 signaling molecule was differentially abundant in the presence of C12-HSL. The NfsB protein had its abundance increased in the presence of C12-HSL at all evaluated times, which is a suggestion that the cells may be susceptible to the action of nitrofurans or that AHLs present some toxicity. Overall, the presence of C12-HSL altered important pathways related to oxidative stress and stationary phase response in Salmonella.

Background Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira spp. This zoonotic disease is distributed globally and affects domestic animals, including cattle. Leptospira interrogans serogroup Sejroe serovar Hardjo and Leptospira borgpetersenii serogroup Sejroe serovar Hardjo remain important species associated with this reproductive disease in livestock production. Previous studies on Brazilian livestock have reported that L. interrogans serovar Hardjo is the most prevalent leptospiral agent in this country and is related to clinical signs of leptospirosis, which lead to economic losses in production. Here, we described the isolation of three clinical strains (Norma, Lagoa and Bolivia) obtained from leptospirosis outbreaks that occurred in Minas Gerais state in 1994 and 2008. Results Serological and molecular typing using housekeeping ( secY and 16SrRNA) and rfb locus (ORF22 and ORF36) genes were applied for the identification and comparative analysis of Leptospira spp. Our results identified the three isolates as L. interrogans serogroup Sejroe serovar Hardjo and confirmed the occurrence of this bacterial strain in Brazilian livestock. Genetic analysis using ORF22 and ORF36 grouped the Leptospira into serogroup Sejroe and subtype Hardjoprajitno. Genetic approaches were also applied to compare distinct serovars of L. interrogans strains by verifying the copy numbers of the IS1500 and IS1533 insertion sequences (ISs). The IS1500 copy number varied among the analyzed L. interrogans strains. Conclusion This study provides evidence that L. interrogans serogroup Sejroe serovar Hardjo subtype Hardjoprajitno causes bovine leptospirosis in Brazilian production. The molecular results suggested that rfb locus (ORF22 and ORF36) could improve epidemiological studies by allowing the identification of Leptospira spp. at the serogroup level. Additionally, the IS1500 and IS1533 IS copy number analysis suggested distinct genomic features among closely related leptospiral strains.

Staphylococcus aureus is a leading cause of bovine mastitis worldwide. Despite some improved understanding of disease pathogenesis, progress towards new methods for the control of intramammary infections (IMI) has been limited, particularly in the field of vaccination. Although herd management programs have helped to reduce the number of clinical cases, S. aureus mastitis remains a major disease burden. This review summarizes the past 16 years of research on bovine S. aureus population genetics, and molecular pathogenesis that have been conducted worldwide. We describe the diversity of S. aureus associated with bovine mastitis and the geographical distribution of S. aureus clones in different continents. We also describe studies investigating the evolution of bovine S. aureus and the importance of host-adaptation in its emergence as a mastitis pathogen. The available information on the prevalence of virulence determinants and their functional relevance during the pathogenesis of bovine mastitis are also discussed. Although traits such as biofilm formation and innate immune evasion are critical for the persistence of bacteria, the current understanding of the key host-pathogen interactions that determine the outcome of S. aureus IMI is very limited. We suggest that greater investment in research into the genetic and molecular basis of bovine S. aureus pathogenesis is essential for the identification of novel therapeutic and vaccine targets.

The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).

Serological tests are preferentially used for the diagnosis of Chagas’ disease (CD) during the chronic phase because of the low parasitemia and high anti-Trypanosoma cruzi antibody titers. However, the current methods showed several disadvantages, as contradictory or inconclusive results, mainly related to the characteristics of the antigens used, in general, crude or whole parasites, but also due to antigen production protocol and the experimental conditions used in serological tests. Thus, better-quality serological assays are urgently needed. Here, we performed a wide immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted proteome based on genome sequence from T. cruzi strains to will be applied as synthetic peptides in the serodiagnosis of the chronic CD. Three B-cell epitopes derived from mucin-associated surface protein (MASP) family, expressed in both infective parasite stages, trypomastigote and amastigotes, conserved in T. cruzi strains, and highly divergent as compared with Leishmania spp. proteome, were selected for this study. The results demonstrated that synthetic peptide 2 and a mixture of peptides (Mix II: peptides 2 and 3) were able to identify all chronic CD cases, indeterminate or Chagas cardiomyopathy clinical presentation, and simultaneously able to discriminate infections caused by Leishmania parasites, with high accuracy (98.37 and 100.00%, respectively) and agreement (kappa index = 0.967 and 1.000, respectively) with direct methods as compared to current diagnostic pipeline performed by reference laboratories in Brazil. This study represents an interesting strategy for the discovery of new antigens applied to serologic diagnosis of infectious diseases and for the technological development of platforms for large-scale production of diagnostic tests.

Most polymeric plastics used as food packaging are obtained from petroleum or made with non-biodegradable synthetic molecules, which slowly degrade and leach into the environment, resulting in the accumulation of microplastics along the trophic chains. To mitigate these impacts, biodegradable packaging derived from agro-industrial biomass residues has emerged as a promising alternative. In this study, bio-based methylcellulose films reinforced with cellulose nanocrystals (CNCs) extracted from low-quality coffee beans were developed and fully characterized. The extracted CNCs presented a needle-like morphology, with an average height of 7.27 nm and a length of 221.34 nm, with 65.75% crystallinity, were stable at pH 7–8, and presented thermogravimetric mass loss of 8.0%. Methylcellulose films containing 0.6% w/w of CNC were produced by casting and characterized in terms of thermal, mechanical, and optical properties. Notably, the incorporation of CNCs resulted in significantly more flexible and less rigid films, as evidenced by the higher elongation at break (57.90%) and lower Young’s modulus (0.0015 GPa) compared to neat methylcellulose film. The tensile strength was not affected (p > 0.05). Additionally, the MCNC 0.6% films effectively blocked UV light in the 200–300 nm range without compromising transparency. Altogether, these findings underscore the MCNC 0.6% film as a flexible, biodegradable packaging material suitable for food industry application.

Background The clinical outcome of malaria depends on the delicate balance between pro-inflammatory and immunomodulatory cytokine responses triggered during infection. Despite the numerous reports on characterization of plasma levels of cytokines/chemokines, there is no consensus on the profile of these mediators during blood stage malaria. The identification of acute phase biomarkers might contribute to a better understanding of the disease, allowing the use of more effective therapeutic approaches to prevent the progression towards severe disease. In the present study, the plasma levels of cytokines and chemokines and their association with parasitaemia and number of previous malaria episodes were evaluated in Plasmodium vivax -infected patients during acute and convalescence phase, as well as in healthy donors. Methods Samples of plasma were obtained from peripheral blood samples from four different groups: P. vivax -infected, P. vivax -treated, endemic control and malaria-naïve control. The cytokine (IL-6, IL-10, IL-17, IL-27, TGF-β, IFN-γ and TNF) and chemokine (MCP-1/CCL2, IP-10/CXCL10 and RANTES/CCL5) plasma levels were measured by CBA or ELISA. The network analysis was performed using Spearman correlation coefficient. Results Plasmodium vivax infection induced a pro-inflammatory response driven by IL-6 and IL-17 associated with an immunomodulatory profile mediated by IL-10 and TGF-β. In addition, a reduction was observed of IFN-γ plasma levels in P. vivax group. A lower level of IL-27 was observed in endemic control group in comparison to malaria-naïve control group. No significant results were found for IL-12p40 and TNF. It was also observed that P. vivax infection promoted higher levels of MCP-1/CCL2 and IP-10/CXCL10 and lower levels of RANTES/CCL5. The plasma level of IL-10 was elevated in patients with high parasitaemia and with more than five previous malaria episodes. Furthermore, association profile between cytokine and chemokine levels were observed by correlation network analysis indicating signature patterns associated with different parasitaemia levels. Conclusions The P. vivax infection triggers a balanced immune response mediated by IL-6 and MCP-1/CCL2, which is modulated by IL-10. In addition, the results indicated that IL-10 plasma levels are influenced by parasitaemia and number of previous malaria episodes.