Background and Aim: Understanding dental care in dogs has made remarkable progress in veterinary medicine. Therefore, this study aimed to analyze the academic literature published in veterinary dentistry from 1990 to 2023. Materials and Methods: A descriptive study was conducted using a scientometric approach and metadata from the Web of Science database. A search strategy adapted for this database was developed using MeSH and Emtree terms and the Boolean operators AND and OR. Using Bibliometrix, different metrics were evaluated to assess the scientific production of researchers and institutions and the impact of authors based on their publications. CiteSpace was also used for co-citation analysis and visualization of citation networks, trends, and patterns in this field of study over time. Results: The bibliometric study analyzed 211 documents from 50 different sources from 1990 to 2023, with an annual growth rate of 6.5%, covering the period 1990-2023. A total of 474 authors were identified, with an average of 2.82 coauthors per paper and 11.85% international coauthorships. The average age of the papers was 12.4 years and 4.55 citations per paper. The most common types of documents were articles (154 documents). Conclusion: Research in veterinary dentistry has shown steady growth from 1990 to 2023. Although there have been fluctuations in article production over the years, there has been a steady growth in article production in veterinary dentistry in general. The annual average number of citations per article has varied over the years, reaching 45 in 2015. However, the average number of citations per article has decreased significantly from 2021 to 2023.
The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a ß-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of ß-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of ß-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from ß-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that ß-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of ß-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.
AIM: To identify patterns and trends in the field of immunization, vaccination, and immunomodulation therapies for periodontitis. MATERIALS AND METHODS: Metadata were collected from the Scopus database on publications related to these topics from January 1986 to February 2024. Several types of papers were included in this study, a total of 22 publications. Data were extracted from relevant publications and loaded into SciVal for analysis that were used to identify trends and patterns in the data, including cross-country collaboration, thematic evolution, and keyword distribution. RESULTS: Mohsen Amin of Tehran University of Medical Sciences in Iran and S. Aadil Ahamed and Annie Kitty George of Saveetha Institute of Medical and Technical Sciences in India were found to be notable contributors in this field. India leads in terms of academic paper production, followed by Iran and China. The journals Expert Review of Vaccines and International Immunopharmacology have published significant papers in this field. CONCLUSIONS: According to Lotka's Law, most authors have written only one paper, reflecting the distribution of productivity in many academic and scientific fields. Collaborations were observed between Iran and Canada, Korea and New Zealand, and the United States and Belgium. This study provides useful insight into the predominant trends and patterns in the scientific literature in the field of immunization, vaccination, and immunomodulation therapies for periodontitis. CLINICAL SIGNIFICANCE: The findings of this study may help to understand the dynamics of the production on immunization, vaccination, and immunomodulation therapies could reduce the inflammation and progression of periodontitis, thus improving the patient's oral and overall health. How to cite this article: Mauricio F, Mendoza R, Silva H, et al. Overview, Trends, and Collaboration on Immunization, Vaccination, and Immunomodulation Therapies for Periodontitis: A Scientometric Study. J Contemp Dent Pract 2024;25(2):128-133.
Periodontitis , Vaccination , Humans , Iran , Immunization , Periodontitis/therapy , Immunomodulation
Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.
During the last century, the critically endangered cotton-top tamarin (Saguinus oedipus) has been threatened by multiple anthropogenic factors that drastically affected their habitat and population size. As the genetic impact of these pressures is largely unknown, this study aimed to establish a genetic baseline with the use of temporal sampling to determine the genetic makeup before detrimental anthropogenic impact. Genomes were resequenced from a combination of historical museum samples and modern wild samples at low-medium coverage, to unravel how the cotton-top tamarin population structure and genomic diversity may have changed during this period. Our data suggest two populations can be differentiated, probably separated historically by the mountain ranges of the Paramillo Massif in Colombia. Although this population structure persists in the current populations, modern samples exhibit genomic signals consistent with recent inbreeding, such as long runs of homozygosity and a reduction in genome-wide heterozygosity especially in the greater northeast population. This loss is likely the consequence of the population reduction following the mass exportation of cotton-top tamarins for biomedical research in the 1960s, coupled with the habitat loss this species continues to experience. However, current populations have not experienced an increase in genetic load. We propose that the historical genetic baseline established in this study can be used to provide insight into alteration in the modern population influenced by a drastic reduction in population size as well as providing background information to be used for future conservation decision-making for the species.
Las intoxicaciones por herbicidas propanil son frecuentes en las diferentes áreas agrícolas, sobre todo en los campos de cultivos de arroz. Se presenta el caso de un paciente agricultor, sin factores de riesgo, quien sufrió una intoxicación moderada por propanil. Pese a las limitaciones diagnósticas en cada centro hospitalario, la sospecha y los datos clínicos al ingreso pueden ser suficientes para la elaboración diagnóstica y su respectivo manejo.
Poisoning by propanil herbicides are common in different agricultural areas, especially in rice fields. We present the case of a farmer patient, without risk factors, who suffered a moderate intoxication by propanil. Despite the diagnostic limitations in each hospital, the suspicion and clinical data on admission may be sufficient for the diagnosis and its respective management.
OBJECTIVES: Clostridioides difficile is a nosocomial pathogen that is associated with the use of antibiotics. One of the most worrying aspects of C. difficile infection is its ability to resist antimicrobial therapies, owing to spore formation. In several bacterial pathogens, proteases of the Clp family participate in phenotypes associated with persistence and virulence. This suggests that these proteins could be involved in virulence-related traits. In this study, we analysed the role of ClpC chaperone-protease of C. difficile in virulence-related traits by comparing the phenotypes of wild-type and mutant strains lacking the clpC gene (ΔclpC). METHODS: We performed biofilm, motility, spore formation, and cytotoxicity assays. RESULTS: Our results show significant differences between the wild-type and ΔclpC strains in all analysed parameters. CONCLUSIONS: Based on these findings, we conclude that clpC plays a role in the virulence properties of C. difficile.
Clostridioides difficile , Clostridioides difficile/genetics , Clostridioides/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms , Anti-Bacterial Agents/metabolism
The tropical dry forest is one of the world's most threatened ecosystems and is the habitat of the Robinson's Mouse Opossum (Marmosa robinsoni), a small marsupial within the Didelphidae family. This study aimed to describe cases of cuterebriases in free-ranging M. robinsoni by examining individuals caught in live animal traps. Sherman traps were deployed in four different sites over three different periods in five days. All animals passed through biometry, weighing, sampling parasites, and sampling feces. Only animals captured in the study site located close to the city were anesthetized and examined. The evaluation included blood samples and a clinical examination. Animals received anesthesia under physical restraint by intramuscular injections of ketamine and xylazine. For anesthetic reversion, the protocol was Yohimbine administered before release. In total, 8% (5/60) of all captured animals had fly larvae extracted from wounds. The molecular Barcode of the mitochondrial Cytochrome Oxidase I gene showed no match with any recognized species of Cuterebra. The animals weighed from 35 to 80 g and had lesions in the scapular region with parasites under their skin in sizes ranging from 1.3 to 2.2 cm. The animals with parasites were in good physical condition without evidence of disturbances in health conditions. This is compatible with literature, reporting little effect on population dynamics of other host species infected with Cuterebra larvae. The study included 24 animals captured in three areas far from any city, which showed no evidence of cuterebrid infection, suggesting that proximity to the city could increase exposure to cuterebriasis. There are reports of cuterebrids in M. robinsoni in Brazil; however, this is the first report of cuterebriasis in M. robinsoni in Colombia.
AIM: To perform a bibliometric analysis of the scientific production related to the use of probiotics in oral health. MATERIALS AND METHODS: A retrospective, descriptive, observational study with a scientometric approach was conducted. Metadata from scientific articles were retrieved from the Web of Science (WoS) Core Collection and analyzed using Medical Subject Headings (MeSH) and Emtree terms. The search strategy included the terms related to probiotics and oral health, and articles published between 2013 and 2022 were analyzed. Different scientometric indicators were used to analyze the production, impact, and network collaboration. RESULTS: A total of 485 articles were retrieved from 241 sources, with an average annual growth rate of 10.72%. The manuscripts received an average citation per doc of 15.2. Authors collaborated on an average of 5.76 coauthors per paper. The number of articles published increased progressively from 2013 to 2022. The year 2022 had the highest number of articles published (90), while in 2013 only 36 articles were published. There was a progressive decrease in the average number of citations per article, from 32.44 in 2013 to 2.41 in 2022. CONCLUSION: The production of articles on probiotics in dentistry has grown steadily, with an annual growth rate of 10.72%. The most productive countries were the United States, Italy, and China. The importance of international scientific collaborations and the need for more research in this field is emphasized. CLINICAL SIGNIFICANCE: The findings of this study may help researchers identify areas that require further study and lead to more rapid and efficient advances in the use of probiotics to improve oral health. Its clinical importance lies in its value and ability to inform and guide future research to advance this field of science. How to cite this article: Mayta-Tovalino F, Espinoza-Carhuancho F, Alvitez-Temoche D, et al. A 10-year Scientometric Study of the Emerging Patterns and Spatial-Temporal Trends of Probiotics in Dentistry. J Contemp Dent Pract 2023;24(12):981-986.
Bibliometrics , Probiotics , United States , Retrospective Studies , Oral Health , Dentistry , Probiotics/therapeutic use
In mold making, the mold surface roughness directly affects the surface roughness of the produced part. To achieve surface roughness below 0.8 µm, the cost of surface finish is high and time-consuming. One alternative to the different grinding and polishing steps is laser polishing (LP). This study investigates and models the LP of tool steel (X38CrMoV5-1-DIN 1.2343), typical for the mold industry, having an initial rough surface obtained by electrical discharge machining. The microstructures of the re-melted layer and heat-affected zone due to the LP process were also studied. Four parameters: the laser spot size, velocity, maximum melt pool temperature and overlapping were investigated via a design of experiments (DoE) approach, specifically a factorial design. The responses were line roughness (Ra), surface roughness (Sa), and waviness (Wa). The surface topography was measured before and after the LP process by white light profilometer or confocal microscopy. DoE results showed that the selected factors interact in a complex manner, including the interactions, and depend on the responses. The DoE analysis of the results revealed that the roughness is mainly affected by the velocity, temperature and overlap. Based on a first DoE model, an optimization of the parameters was performed and allowed to find optimum parameters for the LP of the rough samples. The optimum conditions to minimize the roughness are a spot size of 0.9 mm, a velocity of 50 mm/s, a temperature of 2080 °C and an overlap of 90%. By using these parameters, the roughness could be reduced by a factor of almost 8 from 3.8 µm to approximately 0.5 µm. Observations of the microstructure reveal that the re-melted layer consists of columnar grains of residual austenite. This can be explained by the carbon intake of the electro-machined surface that helps stabilize the austenitic phase.
The study of the stress responses in bacteria has given us a wealth of information regarding the mechanisms employed by these bacteria in aggressive or even non-optimal living conditions. This information has been applied by several researchers to identify molecular targets related to pathogeny, virulence, and survival, among others, and to design new prophylactic or therapeutic strategies against them. In this study, our knowledge of these mechanisms has been summarized with emphasis on some aquatic pathogenic bacteria of relevance to the health and productive aspects of Chilean salmon farming (Piscirickettsia salmonis, Tenacibaculum spp., Renibacterium salmoninarum, and Yersinia ruckeri). This study will aid further investigations aimed at shedding more light on possible lines of action for these pathogens in the coming years.
Micrococcaceae , Virulence Factors , Aquaculture , Chile
BACKGROUND: The Barcode of Life initiative was originally motivated by the large number of species, taxonomic difficulties and the limited number of expert taxonomists. Colombia has 1,610 freshwater fish species and comprises the second largest diversity of this group in the world. As genetic information continues to be limited, we constructed a reference collection of DNA sequences of Colombian freshwater fishes deposited in the Ichthyology Collection of the University of Antioquia (CIUA), thus joining the multiple efforts that have been made in the country to contribute to the knowledge of genetic diversity in order to strengthen the inventories of biological collections and facilitate the solution of taxonomic issues in the future. NEW INFORMATION: This study contributes to the knowledge on the DNA barcodes and occurrence records of 96 species of Colombian freshwater fishes. Fifty-seven of the species represented in this dataset were already available in the Barcode Of Life Data System (BOLD System), while 39 correspond to new species to the BOLD System. Forty-nine specimens were collected in the Atrato River Basin and 708 in the Magdalena-Cauca asin during the period 2010-2020. Two species (Loricariichthysbrunneus (Hancock, 1828) and Poeciliasphenops Valenciennes, 1846) are considered exotic to the Atrato, Cauca and Magdalena Basins and four species (Oncorhynchusmykiss (Walbaum, 1792), Oreochromisniloticus (Linnaeus, 1758), Parachromisfriedrichsthalii (Heckel, 1840) and Xiphophorushelleri Heckel, 1848) are exotic to the Colombian hydrogeographic regions. All specimens are deposited in CIUA and have their DNA barcodes made publicly available in the BOLD online database. The geographical distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).
The study of the stress responses in bacteria has given us a wealth of information regarding the mechanisms employed by these bacteria in aggressive or even non-optimal living conditions. This information has been applied by several researchers to identify molecular targets related to pathogeny, virulence, and survival, among others, and to design new prophylactic or therapeutic strategies against them. In this study, our knowledge of these mechanisms has been summarized with emphasis on some aquatic pathogenic bacteria of relevance to the health and productive aspects of Chilean salmon farming (Piscirickettsia salmonis, Tenacibaculum spp., Renibacterium salmoninarum, and Yersinia ruckeri). This study will aid further investigations aimed at shedding more light on possible lines of action for these pathogens in the coming years.
Virulence Factors , Micrococcaceae , Chile , Aquaculture
Abstract Background: Two biotypes of Aberdeen Angus cattle breed, known as Old Type and New Type, that differ in their origin and beef production are formally recognized. In Colombia, this breed has been commercialized for approximately 80 years. Studies on the origin, kinship and levels of genetic diversity of this breed in Colombian herds are scarce, yet important for planning crossing and management strategies. Objective: To measure the genetic diversity and structure of two Colombian herds of Old Type and New Type biotypes of Aberdeen Angus from Huila and Cundinamarca provinces and assess mitochondrial introgression with other breeds. Methods: A set of ten microsatellites and sequences of the Mitochondrial Control Region were characterized. Estimators of genetic diversity and population differentiation along with tests of population assignment were applied. Results: Nuclear loci were highly polymorphic as shown by the Polymorphic Information Content (0.599) and the Probability of Identity (1.896 10-08). Both populations were highly diverse and clearly differentiated into two groups corresponding to the Old Type and New Type phenotypes. In contrast, mitochondrial data failed to distinguish these two groups and showed extensive admixture. Conclusions: This study optimized a set of ten highly polymorphic nuclear markers that may be used for parentage and population genetic studies of Aberdeen Angus. Genetic differentiation in these loci agreed with phenotypic differences of the Old and New Types. However, mitochondrial data indicated ancestry of multiple European breeds in the origin of Colombian Aberdeen Angus.
Resumen Antecedentes: Dentro de la raza Aberdeen Angus existen dos biotipos conocidos como Old Type y New Type, las cuales difieren en su origen y producción de carne. En Colombia, esta raza se ha venido comercializando desde hace aproximadamente 80 años. No obstante, aún no se han realizado estudios sobre su origen, parentesco y niveles de diversidad genética de esta raza en hatos colombianos, lo cual es importante para planear estrategias de cruce y manejo. Objetivo: Medir la diversidad y estructura genética de dos hatos colombianos de Aberdeen Angus Old Type y New Type de Huila y Cundinamarca y evaluar la introgresión mitocondrial con otras razas. Métodos: Se caracterizó un grupo de diez loci microsatélite y se secuenció la Región Control Mitocondrial. Se aplicaron estimadores de diversidad genética y diferenciación poblacional, junto con pruebas de asignación poblacional. Resultados: Los loci microsatélite fueron altamente polimórficos, tal como lo indicaron el Contenido de Información Polimórfica (0,599) y la Probabilidad de Identidad (1,896 10-08). Las poblaciones evaluadas de Aberdeen Angus en Colombia fueron altamente diversas y se diferenciaron claramente en dos grupos correspondientes a los fenotipos Old Type y New Type. En contraste, los datos mitocondriales no recobraron estos dos grupos y mostraron una amplia mezcla genética. Conclusiones: Este estudio optimizó un grupo de diez marcadores altamente polimórficos que pueden ser usados para estudios de parentesco y genética poblacional de Aberdeen Angus. La diferenciación genética en loci nucleares concordó con las diferencias fenotípicas entre Old y New Types, pero los datos mitocondriales indicaron ancestría de múltiples razas europeas en el origen del Aberdeen Angus colombiano.
Resumo Antecedentes: Dentro da raça Aberdeen Angus há dois biótipos conhecidos como Old Type e New Type, que diferem em sua origem e produção de carne. Na Colômbia, esta raça é comercializada há aproximadamente 80 anos. Entretanto, estudos sobre a origem, o parentesco e os níveis de diversidade genética desta raça em rebanhos colombianos ainda não foram realizados, o que é importante para o planejamento de cruzamentos e estratégias de manejo. Objetivo: Medir a diversidade genética e a estrutura de dois rebanhos colombianos de biótipos de Old Type e New Type de Aberdeen Angus de Huila e Cundinamarca e avaliar a introgressão mitocondrial com outras raças. Métodos: Um grupo de dez loci de microssatélites foi caracterizado e a Região de Controle Mitocondrial foi sequenciada. As estimativas de diversidade genética e diferenciação populacional foram aplicadas, juntamente com testes de designação populacional. Resultados: Os locus microssatélites foram altamente polimórficos, conforme indicado pelo Conteúdo de Infomação Polimórfica (0,599) e Probabilidade de Identidade (1,896 10-08). As populações avaliadas de Aberden Angus na Colômbia eram altamente diversificadas e claramente diferenciadas em dois grupos correspondentes aos fenótipos do Old Type e New Type. Em contraste, os dados mitocondriais não recuperaram esses dois grupos e mostraram um amplo mix genético. Conclusões: Este estudo otimizou um grupo de dez marcadores altamente polimórficos que podem ser usados para estudos genéticos de parentesco e população de Aberdeen Angus. A diferenciação genética nos loci nucleares concordou com as diferenças fenotípicas entre os Old e New Types, mas os dados mitocondriais indicam ancestralidade de várias raças européias na origem do Aberdeen Angus colombiano.
Yersinia ruckeri causes outbreaks of enteric redmouth disease in salmon aquaculture all over the world. The transient antibiotic tolerance exhibited by bacterial persisters is commonly thought to be responsible for outbreaks; however, the molecular factors underlying this behavior have not been explored in Y. ruckeri. In this study, we investigated the participation of the RNA chaperone Hfq from Y. ruckeri in antibiotic persistence. Cultures of the hfq-knockout mutant (Δhfq) exhibited faster replication, increased ATP levels and a more reductive environment than the wild type. The growth curves of bacteria exposed to sublethal concentrations of ampicillin, oxolinic acid, ciprofloxacin and polymyxin B revealed a greater susceptibility for the Δhfq strain. The time-kill curves of bacteria treated with the antibiotics mentioned above and florfenicol, using inoculums from exponential, stationary and biofilm cultures, demonstrated that the Δhfq strain has significant defects in persister cells production. To shed more light on the role of Hfq in antibiotic persistence, we analyzed its dependence on the (p)ppGpp synthetase RelA by determining the persister cells production in the absence of the relA gene. The ΔrelA and ΔrelAΔhfq strains displayed similar defects in persister cells formation, but higher than Δhfq strain. Similarly, stationary cultures of the ΔrelA and ΔrelAΔhfq strains exhibited comparable levels of ATP but higher than that of the Δhfq strain, indicating that relA is epistatic over hfq. Taken together, our findings provide valuable information on antibiotic persistence in Y. ruckeri, shedding light on the participation of Hfq in the persistence phenomenon.
The appearance of multi-resistant strains has contributed to reintroducing polymyxin as the last-line therapy. Although polymyxin resistance is based on bacterial envelope changes, other resistance mechanisms are being reported. Outer membrane vesicles (OMVs) are nanosized proteoliposomes secreted from the outer membrane of Gram-negative bacteria. In some bacteria, OMVs have shown to provide resistance to diverse antimicrobial agents either by sequestering and/or expelling the harmful agent from the bacterial envelope. Nevertheless, the participation of OMVs in polymyxin resistance has not yet been explored in S. Typhi, and neither OMVs derived from hypervesiculating mutants. In this work, we explored whether OMVs produced by the hypervesiculating strains Salmonella Typhi ΔrfaE (LPS synthesis), ΔtolR (bacterial envelope) and ΔdegS (misfolded proteins and σ E activation) exhibit protective properties against polymyxin B. We found that the OMVs extracted from S. Typhi ΔtolR and ΔdegS protect S. Typhi WT from polymyxin B in a concentration-depending manner. By contrast, the protective effect exerted by OMVs from S. Typhi WT and S. Typhi ΔrfaE is much lower. This effect is achieved by the sequestration of polymyxin B, as assessed by the more positive Zeta potential of OMVs with polymyxin B and the diminished antibiotic's availability when coincubated with OMVs. We also found that S. Typhi ΔtolR exhibited an increased MIC of polymyxin B. Finally, we determined that S. Typhi ΔtolR and S. Typhi ΔdegS, at a lesser level, can functionally and transiently transfer the OMV-mediated polymyxin B resistance to susceptible bacteria in cocultures. This work shows that mutants in genes related to OMVs biogenesis can release vesicles with improved abilities to protect bacteria against membrane-active agents. Since mutations affecting OMV biogenesis can involve the bacterial envelope, mutants with increased resistance to membrane-acting agents that, in turn, produce protective OMVs with a high vesiculation rate (e.g., S. Typhi ΔtolR) can arise. Such mutants can functionally transfer the resistance to surrounding bacteria via OMVs, diminishing the effective concentration of the antimicrobial agent and potentially favoring the selection of spontaneous resistant strains in the environment. This phenomenon might be considered the source for the emergence of polymyxin resistance in an entire bacterial community.
Growing evidence indicates that small noncoding RNAs (sRNAs) play important regulatory roles during bacterial infection. In Salmonella Typhimurium, several sRNAs are strongly up-regulated within macrophages, but little is known about their role during the infection process. Among these sRNAs, the well-characterized paralogs RyhB-1 and RyhB-2 are two regulators of gene expression mainly related with the response to iron availability. To investigate the role of the sRNAs RyhB-1 and RyhB-2 from S. Typhimurium in the infection of RAW264.7 macrophages, we analyzed several phenotypic traits from intracellular mutant strains lacking one and both sRNAs. Deletion of RyhB-1 and/or RyhB-2 resulted in increased intracellular survival and faster replication within macrophages. The bacterial metabolic status inside macrophages was also analyzed, revealing that all the mutant strains exhibited higher intracellular levels of ATP and lower NAD+/NADH ratios than the wild type. Expression analyses from bacteria infecting macrophages showed that RyhB-1 and RyhB-2 affect the intra-macrophage expression of bacterial genes associated with the Salmonella pathogenicity island 1 (SPI-1) and the type III secretion system (T3SS). With a two-plasmid system and compensatory mutations, we confirmed that RyhB-1 and RyhB-2 directly interact with the mRNAs of the invasion chaperone SicA and the regulatory protein RtsB. Altogether, these results indicate that the RyhB homologs contribute to the S. Typhimurium virulence modulation inside macrophages by reducing the intracellular growth and down-regulating the SPI-1 gene expression.
Small noncoding RNAs (sRNAs) are important regulators of gene expression and physiology in bacteria. RyhB is an iron-responsive sRNA well characterized in Escherichia coli and conserved in other Enterobacteriaceae. In this study, we identified and characterized two RyhB homologs (named RyhB-1 and RyhB-2) in the fish pathogen Yersinia ruckeri. We found that, as in other Enterobacteriaceae, both RyhB-1 and RyhB-2 are induced under iron starvation, repressed by the Fur regulator, and depend on Hfq for stability. Despite these similarities in expression, the mutant strains of Y. ruckeri lacking RyhB-1 (ΔryhB-1) or RyhB-2 (ΔryhB-2) exhibited differential phenotypes. In comparison with the wild type, the ΔryhB-1 strain showed a hypermotile phenotype, reduced biofilm formation, increased replication rate, faster growth, and increased ATP levels in bacterial cultures. By contrast, in salmon cell cultures, the ΔryhB-1 strain exhibited an increased survival. On the other hand, the ΔryhB-2 strain was non-motile and showed augmented biofilm formation as compared to the wild type. The expression of a subset of RyhB conserved targets, selected from different bacterial species, was analyzed by quantitative RT-PCR in wild type, ΔryhB-1, ΔryhB-2, and ΔryhB-1 ΔryhB-2 strains cultured in iron-depleted media. RyhB-1 negatively affected the expression of most analyzed genes (sodB, acnA, sdhC, bfr, fliF, among others), whose functions are related to metabolism and motility, involving iron-containing proteins. Among the genes analyzed, only sdhC and bfr appeared as targets for RyhB-2. Taken together, these results indicate that Y. ruckeri RyhB homologs participate in the modulation of the bacterial physiology with non-redundant roles.
Bacterial Physiological Phenomena , Fish Diseases/microbiology , RNA, Bacterial/genetics , RNA, Small Untranslated/genetics , Yersinia ruckeri/genetics , Yersinia ruckeri/physiology , Animals , Bacterial Proteins/genetics , Biofilms/growth & development , Escherichia coli/genetics , Fishes , Gene Deletion , Gene Expression Regulation, Bacterial , Homeostasis , Iron/metabolism , Phenotype , Yersinia Infections
Salmonella Typhimurium is a facultative, intracellular pathogen whose products range from self-limited gastroenteritis to systemic diseases. Food ingestion increases biomolecules' concentration in the intestinal lumen, including amino acids such as cysteine, which is toxic in a concentration-dependent manner. When cysteine's intracellular concentration reaches toxic levels, S. Typhimurium expresses a cysteine-inducible enzyme (CdsH), which converts cysteine into pyruvate, sulfide, and ammonia. Despite this evidence, the biological context of cdsH's role is not completely clear, especially in the infective cycle. Since inside epithelial cells both cdsH and its positive regulator, ybaO, are overexpressed, we hypothesized a possible role of cdsH in the intestinal phase of the infection. To test this hypothesis, we used an in vitro model of HT-29 cell infection, adding extra cysteine to the culture medium during the infective process. We observed that, at 6 h post-invasion, the wild type S. Typhimurium proliferated 30% more than the ΔcdsH strain in the presence of extra cysteine. This result shows that cdsH contributes to the bacterial replication in the intracellular environment in increased concentrations of extracellular cysteine, strongly suggesting that cdsH participates by increasing the bacterial fitness in the intestinal phase of the S. Typhimurium infection.
Toxin-antitoxin systems are known to be involved in many bacterial functions that can lead to growth arrest and cell death in response to stress. Typically, toxin and antitoxin genes of type I systems are located in opposite strands, where the antitoxin is a small antisense RNA (sRNA). In the present work we show that the sRNA IsrA from Salmonella Typhimurium down-regulates the expression of its overlapping gene STM0294.1n. Multiple sequence alignment and comparative structure analysis indicated that STM0294.1n belongs to the SymE toxin superfamily, and the gene was renamed iasE (IsrA-overlapping gene with similarity to SymE). The iasE expression was induced in response to mitomycin C, an SOS-inducing agent; conversely, IsrA overexpression repressed the iasE expression even in the presence of mitomycin C. Accordingly, the inactivation of IsrA with an anti-IsrA RNA expressed in trans abrogated the repressive effect of IsrA on the iasE expression. On the other hand, iasE overexpression, as well as the blockage of the antisense IsrA function, negatively affected bacterial growth, arguing for a toxic effect of the iasE gene product. Besides, a bacterial lysate obtained from the iasE-overexpressing strain exhibited endoribonuclease activity, as determined by a fluorometric assay based on fluorescent reporter RNAs. Together, these results indicate that the IasE/IsrA pair of S. Typhimurium constitutes a functional type I toxin-antitoxin system.
Bacterial Proteins/genetics , RNA, Antisense/genetics , RNA, Bacterial/genetics , SOS Response, Genetics/genetics , Salmonella typhimurium/genetics , Amino Acid Sequence , Antitoxins/genetics , Bacterial Toxins/genetics , Endoribonucleases/metabolism , Gene Expression Regulation, Bacterial , Mitomycin/metabolism , Models, Molecular , Mutation , Open Reading Frames/genetics , Protein Conformation , Protein Folding
