RESUMO
Antibiotic resistance has emerged as a global threat to public health, generating a growing interest in investigating the presence of antibiotic-resistant bacteria in environments influenced by anthropogenic activities. Wastewater treatment plants in hospital serve as significant reservoirs of antimicrobial-resistant bacteria, where a favorable environment is established, promoting the proliferation and transfer of resistance genes among different bacterial species. In our study, we isolated a total of 243 strains from 5 hospital wastewater sites in Mexico, belonging to 21 distinct Gram-negative bacterial species. The presence of ß-lactamase was detected in 46.9% (114/243) of the isolates, which belonging to the Enterobacteriaceae family. We identified a total of 169 ß-lactamase genes; blaTEM in 33.1%, blaCTX-M in 25.4%, blaKPC in 25.4%, blaNDM 8.8%, blaSHV in 5.3%, and blaOXA-48 in 1.1% distributed in 12 different bacteria species. Among the 114 of the isolates, 50.8% were found to harbor at least one carbapenemase and were discharged into the environment. The carbapenemase blaKPC was found in six Citrobacter spp. and E. coli, while blaNDM was detected in two distinct Enterobacter spp. and E. coli. Notably, blaNDM-1 was identified in a 110 Kb IncFII conjugative plasmid in E. cloacae, E. xiangfangensis, and E. coli within the same hospital wastewater. In conclusion, hospital wastewater showed the presence of Enterobacteriaceae carrying a high frequency of carbapenemase blaKPC and blaNDM. We propose that hospital wastewater serves as reservoirs for resistance mechanism within bacterial communities and creates an optimal environment for the exchange of this resistance mechanism among different bacterial strains. IMPORTANCE: The significance of this study lies in its findings regarding the prevalence and diversity of antibiotic-resistant bacteria and genes identified in hospital wastewater in Mexico. The research underscores the urgent need for enhanced surveillance and prevention strategies to tackle the escalating challenge of antibiotic resistance, particularly evident through the elevated frequencies of carbapenemase genes such as blaKPC and blaNDM within the Enterobacteriaceae family. Moreover, the identification of these resistance genes on conjugative plasmids highlights the potential for widespread transmission via horizontal gene transfer. Understanding the mechanisms of antibiotic resistance in hospital wastewater is crucial for developing targeted interventions aimed at reducing transmission, thereby safeguarding public health and preserving the efficacy of antimicrobial therapies.
Assuntos
Proteínas de Bactérias , Citrobacter , Enterobacter , Hospitais , Águas Residuárias , beta-Lactamases , Águas Residuárias/microbiologia , beta-Lactamases/genética , Proteínas de Bactérias/genética , Citrobacter/genética , Citrobacter/enzimologia , Citrobacter/efeitos dos fármacos , Citrobacter/isolamento & purificação , Enterobacter/genética , Enterobacter/efeitos dos fármacos , Enterobacter/isolamento & purificação , Enterobacter/enzimologia , Antibacterianos/farmacologia , MéxicoRESUMO
Background: The axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model vertebrates. Methods: The most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other vertebrates. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human, Xenopus tropicalis, Danio rerio (zebrafish), and eight tetrapod reference genomes were used for comparison. Results: Canonical A. mexicanum heavy chain (IGH), lambda (IGL), sigma (IGS), and the putative surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes and there is no clan I IGHV genes. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality. Conclusion: The A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of Ig gene loss which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.
Assuntos
Ambystoma mexicanum , Imunoglobulinas , Animais , Ambystoma mexicanum/genética , Genoma , Genômica , Imunoglobulinas/genéticaRESUMO
The use of colistin in food-producing animals favors the emergence and spread of colistin-resistant strains. Here, we investigated the occurrence and molecular mechanisms of colistin resistance among E. coli isolates from a Mexican piglet farm. A collection of 175 cephalosporin-resistant colonies from swine fecal samples were recovered. The colistin resistance phenotype was identified by rapid polymyxin test and the mcr-type genes were screened by PCR. We assessed the colistin-resistant strains by antimicrobial susceptibility test, pulse-field gel electrophoresis, plasmid profile, and mating experiments. Whole-Genome Sequencing data was used to explore the resistome, virulome, and mobilome of colistin-resistant strains. A total of four colistin-resistant E. coli were identified from the cefotaxime-resistant colonies. All harbored the plasmid-borne mcr-1 gene, which was located on conjugative 170-kb IncHI-2 plasmid co-carrying ESBLs genes. Thus, high antimicrobial resistance rates were observed for several antibiotic families. In the RC2-007 strain, the mcr-1 gene was located as part of a prophage carried on non-conjugative 100-kb-plasmid, which upon being transformed into K. variicola strain increased the polymyxin resistance 2-fold. The genomic analysis showed a broad resistome and virulome. Our findings suggest that colistin resistance followed independent acquisition pathways as clonal and non-genetically related mcr-1-harboring strains were identified. These E. coli isolates represent a reservoir of antibiotic resistance and virulence genes in animals for human consumption which could be potentially propagated into other interfaces.
RESUMO
Insect neuropeptides, play a central role in the control of many physiological processes. Based on an analysis of Nyssorhynchus albimanus brain transcriptome a neuropeptide precursor database of the mosquito was described. Also, we observed that adipokinetic hormone/corazonin-related peptide (ACP), hugin and corazonin encoding genes were differentially expressed during Plasmodium infection. Transcriptomic data from Ny. albimanus brain identified 29 pre-propeptides deduced from the sequences that allowed the prediction of at least 60 neuropeptides. The predicted peptides include isoforms of allatostatin C, orcokinin, corazonin, adipokinetic hormone (AKH), SIFamide, capa, hugin, pigment-dispersing factor, adipokinetic hormone/corazonin-related peptide (ACP), tachykinin-related peptide, trissin, neuropeptide F, diuretic hormone 31, bursicon, crustacean cardioactive peptide (CCAP), allatotropin, allatostatin A, ecdysis triggering hormone (ETH), diuretic hormone 44 (Dh44), insulin-like peptides (ILPs) and eclosion hormone (EH). The analysis of the genome of An. albimanus and the generated transcriptome, provided evidence for the identification of myosuppressin neuropeptide precursor. A quantitative analysis documented increased expression of precursors encoding ACP peptide, hugin and corazonin in the mosquito brain after Plasmodium berghei infection. This work represents an initial effort to characterize the neuropeptide precursors repertoire of Ny. albimanus and provides information for understanding neuroregulation of the mosquito response during Plasmodium infection.
RESUMO
Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes mosquitoes, which causes Chikungunya fever. Three CHIKV genotypes have been identified: West African, East-Central-South African and Asian. In 2014, CHIKV was detected for the first time in Mexico, accumulating 13,569 confirmed cases in the following three years. Studies on the molecular diversification of CHIKV in Mexico focused on limited geographic regions or investigated only one structural gene of the virus. To describe the dynamics of this outbreak, we analyzed 309 serum samples from CHIKV acute clinical cases from 15 Mexican states. Partial NSP3, E1, and E2 genes were sequenced, mutations were identified, and their genetic variability was estimated. The evolutionary relationship with CHIKV sequences sampled globally were analyzed. Our sequences grouped with the Asian genotype within the Caribbean lineage, suggesting that the Asian was the only circulating genotype during the outbreak. Three non-synonymous mutations (E2 S248F and NSP3 A437T and L451F) were present in our sequences, which were also identified in sequences of the Caribbean lineage and in one Philippine sequence. Based on the phylogeographic analysis, the viral spread was reconstructed, suggesting that after the introduction through the Mexican southern border (Chiapas), CHIKV dispersed to neighboring states before reaching the center and north of the country through the Pacific Ocean states and Quintana Roo. This is the first viral phylogeographic reconstruction in Mexico characterizing the CHIKV outbreak across the country.
Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Variação Genética , Epidemiologia Molecular , Aedes/virologia , Animais , Região do Caribe , Febre de Chikungunya/epidemiologia , Surtos de Doenças , Genótipo , México/epidemiologia , Mutação , Oceano Pacífico , Filogenia , FilogeografiaRESUMO
The heat shock protein family 70 (Hsp70) comprises chaperone proteins that play major multiple roles in Plasmodium asexual and sexual development. In this study, we analyzed the expression of Hsp70-1 in gametocytes, gametes, zygotes, and its participation in ookinete formation and their transition into oocysts. A monoclonal antibody against recombinant Hsp70-1 revealed its presence in zygotes and micronemes of ookinetes. Compared to wild type parasites, Hsp70-1 knockout ookinetes produced fewer oocysts in Plasmodium-susceptible Anopheles albimanus mosquitoes. This may indicate a defective transformation of ookinetes into oocysts in the absence of Hsp70-1. The presence of this protein in micronemes suggests its participation in mosquito infection, probably aiding to the adequate structural conformation of proteins in charge of motility, recognition and invasion of the insect midgut epithelium.
Assuntos
Anopheles/parasitologia , Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Animais , Trato Gastrointestinal/parasitologia , Vetores Genéticos/genética , Estágios do Ciclo de Vida , Masculino , Fenótipo , Plasmodium berghei/crescimento & desenvolvimento , Ratos , Zigoto/metabolismoRESUMO
Using molecular and whole-genome sequencing tools, we investigated colistin-resistant Escherichia coli isolates from wild sea lions. Two unrelated E. coli colistin-resistant isolates, ST8259 and ST4218, were identified, both belonging to the B2 phylogroup and different serotypes. Polymorphisms in PmrA, PmrB, and PhoQ proteins were identified, and the role of PmrB and PhoQ in contributing to colistin resistance was determined by complementation assays. However, the mutations characterized in the present study are not involved in colistin resistance, which have been described in E. coli isolates from clinical settings. Therefore, the acquired mutations in pmrB and phoQ genes in resistance to colistin in bacteria related to marine environment animals are different. This work contributes to the surveillance and characterization of colistin resistance in Escherichia coli obtained from animals from aquatic environments.
Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Genoma Bacteriano , Leões-Marinhos/microbiologia , Animais , Animais Selvagens/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/genética , Testes de Sensibilidade Microbiana , Sorogrupo , Sequenciamento Completo do GenomaRESUMO
The origin and function of blood IgM+IgD+CD27+ B cells is controversial, and they are considered a heterogeneous population. Previous staining of circulating B cells of healthy donors with rotavirus fluorescent virus-like particles allowed us to differentiate two subsets of IgM+IgD+CD27+: IgMhi and IgMlo B cells. Here, we confirmed this finding and compared the phenotype, transcriptome, in vitro function, and Ig gene repertoire of these two subsets. Eleven markers phenotypically discriminated both subsets (CD1c, CD69, IL21R, CD27, MTG, CD45RB, CD5, CD184, CD23, BAFFR, and CD38) with the IgMhi phenotypically resembling previously reported marginal zone B cells and the IgMlo resembling both naïve and memory B cells. Transcriptomic analysis showed that both subpopulations clustered close to germinal center-experienced IgM only B cells with a Principal Component Analysis, but differed in expression of 78 genes. Moreover, IgMhi B cells expressed genes characteristic of previously reported marginal zone B cells. After stimulation with CpG and cytokines, significantly (p < 0.05) higher frequencies (62.5%) of IgMhi B cells proliferated, compared with IgMlo B cells (35.37%), and differentiated to antibody secreting cells (14.22% for IgMhi and 7.19% for IgMlo). IgMhi B cells had significantly (p < 0.0007) higher frequencies of mutations in IGHV and IGKV regions, IgMlo B cells had higher usage of IGHJ6 genes (p < 0.0001), and both subsets differed in their HCDR3 properties. IgMhi B cells shared most of their shared IGH clonotypes with IgM only memory B cells, and IgMlo B cells with IgMhi B cells. These results support the notion that differential expression of IgM and IgD discriminates two subpopulations of human circulating IgM+IgD+CD27+ B cells, with the IgMhi B cells having similarities with previously described marginal zone B cells that passed through germinal centers, and the IgMlo B cells being the least differentiated amongst the IgM+CD27+ subsets.
Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Adulto , Perfilação da Expressão Gênica , Humanos , Imunoglobulina D/genética , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Fenótipo , Análise de Componente Principal , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Carbapenem-resistant Gram-negative bacteria isolated in Venezuela have been poorly characterized. The present study characterized a total of 34 isolates obtained from 27 patients; five of these patients were multi-infected. The bacterial species identified were Klebsiella pneumoniae (17), Pseudomonas aeruginosa (9), and Acinetobacter baumannii (8). From these isolates, 85% were identified as carbapenemase-producing bacteria, and the identified carbapenemase genes were blaKPC-2 (10/29 [34.4%]), blaVIM-type (7/29 [24.1%]), blaOXA-23 (7/29 [24.1%]), blaNDM-1 (8/29 [27.5%]), and the coexistence of blaOXA-23/blaNDM-1 (2/29 [6.8%]). Patient 1 was multi-infected by K. pneumoniae ST11 and ST2413 isolates harbouring the blaNDM-1 and blaKPC-2 genes, respectively. The other patients were multi-infected by two or three different bacterial species such as ESBL-producing K. pneumoniae isolates, P. aeruginosa harbouring the blaVIM-type gene, K. pneumoniae ST147 harbouring the blaKPC-2 gene and by A. baumannii harbouring the blaOXA-23 gene. The blaNDM-1 gene in A. baumannii is flanked by an uncommon genetic structure, whereas blaNDM-1 gene in K. pneumoniae revealed a common structure described in different plasmids from Enterobacteriaceae isolates. This study provides new information about the epidemiology of carbapenemase-producing bacteria in clinical setting in Venezuela.
Assuntos
Proteínas de Bactérias/biossíntese , Bactérias Gram-Negativas/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/biossíntese , Acinetobacter baumannii , Adulto , Feminino , Genes Bacterianos/genética , Bactérias Gram-Negativas/enzimologia , Humanos , Klebsiella pneumoniae , Masculino , Pessoa de Meia-Idade , Pseudomonas aeruginosa , VenezuelaRESUMO
Antibody class switch recombination (CSR) to IgG, IgA, or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iµ and acceptor IH exons, located 5' upstream of each CH coding gene. Although, our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce. We analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA, and the Sequence Read Archive using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as, coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iµ, Iγ3, and Iγ1 in humans in response to vaccination. We mapped IH transcription for the human IGH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called "immune privileged" tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased Iδ transcription in ileal mucosa in Crohn's disease with ulceration was found. In conclusion, CSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e., cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.