Detecting Class 1 Integrons and Their Variable Regions in Escherichia coli Whole-Genome Sequences Reported from Andean Community Countries.

Various genetic elements, including integrons, are known to contribute to the development of antimicrobial resistance. Class 1 integrons have been identified in E. coli isolates and are associated with multidrug resistance in countries of the Andean Community. However, detailed information on the gene cassettes located on the variable regions of integrons is lacking. Here, we investigated the presence and diversity of class 1 integrons, using an in silico approach, in 2533 whole-genome sequences obtained from EnteroBase. IntFinder v1.0 revealed that almost one-third of isolates contained these platforms. Integron-bearing isolates were associated with environmental, food, human, and animal origins reported from all countries under scrutiny. Moreover, they were identified in clones known for their pathogenicity or multidrug resistance. Integrons carried cassettes associated with aminoglycoside (aadA), trimethoprim (dfrA), cephalosporin (blaOXA; blaDHA), and fluoroquinolone (aac(6')-Ib-cr; qnrB) resistance. These platforms showed higher diversity and larger numbers than previously reported. Moreover, integrons carrying more than three cassettes in their variable regions were determined. Monitoring the prevalence and diversity of genetic elements is necessary for recognizing emergent patterns of resistance in pathogenic bacteria, especially in countries where various factors are recognized to favor the selection of resistant microorganisms.

Dominance of ST131, B2, blaCTX-M-15, and papA-papC-kpsMII-uitA among ESBL Escherichia coli isolated from bloodstream infections in Quito, Ecuador: a 10-year surveillance study (2009-2019).

AIMS: This study aimed to examine antibiotic resistance and the epidemiology of extended-spectrum ß-lactamases (ESBL)-producing Escherichia coli associated with bloodstream infections over a period of 10 years. METHODS AND RESULTS: Isolates were collected from January 2009 to December 2019 and those testing for E. coli were included. Antibiotic susceptibility was tested using the VITEK® system. Selected isolates were further characterized by amplification of marker genes (virulence traits, phylogroups, and sequence types). A total of 166 ESBL-producing E. coli were recovered. The blaCTX-M-15 allele was the most abundant. Most of the isolates were resistant to ceftriaxone, cefepime, ceftazidime, ampicillin/sulbactam, piperacillin/tazobactam, and ciprofloxacin. No resistance to carbapenems was registered. More than 80% of bacteria were classified as extraintestinal pathogenic E. coli (ExPEC), and the combination of virulence traits:papA-papC-kpsMII-uitA was the most common. Phylogroup B2 was the most prevalent, and bacteria predominantly belonged to ST131. CONCLUSIONS: There was an increase in the ExPEC ESBL-E coli in bloodstream infections and the relationship between the isolates found in these infections during these 10 years.

An Overview of the Use and Applications of Limosilactobacillus fermentum in Broiler Chickens.

The implementation of government regulations on antibiotic use, along with the public's concern for drug resistance, has strengthened interest in developing alternatives not only aimed at preserving animal production but also at reducing the effects of pathogenic infections. Probiotics, in particular, are considered microorganisms that induce health benefits in the host after consumption of adequate amounts; they have been established as a potential strategy for improving growth, especially by stimulating intestinal homeostasis. Probiotics are commonly associated with lactic acid bacteria, and Limosilactobacillus fermentum is a well-studied species recognized for its favorable characteristics, including adhesion to epithelial cells, production of antimicrobial compounds, and activation of receptors that prompt the transcription of immune-associated genes. Recently, this species has been used in animal production. Different studies have shown that the application of L. fermentum strains not only improves the intestinal ecosystem but also reduces the effects caused by potentially pathogenic microorganisms. These studies have also revealed key insights into the mechanisms behind the actions exerted by this probiotic. In this manuscript, we aim to provide a concise overview of the effects of L. fermentum administration on broiler chicken health and performance.

Effects of Lactobacillus fermentum Administration on Intestinal Morphometry and Antibody Serum Levels in Salmonella-Infantis-Challenged Chickens.

There are no studies reporting the effects of Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) on intestinal architecture and immunoglobulin serum levels in chickens. Here, we measured these parameters and hypothesized whether probiotic administration could modulate the observed outcomes. Two-hundred 1-day-old COBB 500 male chicks were allocated into four groups: (I) the control, (II) the group treated with L. fermentum, (III) the group exposed to S. Infantis, and (IV) the group inoculated with both bacteria. At 11 days post infection, blood was gathered from animals which were then euthanized, and samples from the small intestine were collected. Intestinal conditions, as well as IgA and IgM serum levels, were assessed. S. Infantis reduced villus-height-to-crypt-depth (VH:CD) ratios in duodenal, jejunal, and ileal sections compared to control conditions, although no differences were found regarding the number of goblet cells, muc-2 expression, and immunoglobulin concentration. L. fermentum improved intestinal measurements compared to the control; this effect was also evidenced in birds infected with S. Infantis. IgM serum levels augmented in response to the probiotic in infected animals. Certainly, the application of L. fermentum elicited positive outcomes in S. Infantis-challenged chickens and thus must be considered for developing novel treatments designed to reduce unwanted infections.

A PMAxxTM qPCR Assay Reveals That Dietary Administration of the Microalgae Tetraselmis chuii Does Not Affect Salmonella Infantis Caecal Content in Early-Treated Broiler Chickens.

Salmonella enterica serovars cause infections in humans. S. enterica subsp. enterica serovar Infantis is considered relevant and is commonly reported in poultry products. Evaluating innovative approaches for resisting colonization in animals could contribute to the goal of reducing potential human infections. Microalgae represent a source of molecules associated with performance and health improvement in chickens. Tetraselmis chuii synthesizes fermentable polysaccharides as part of their cell wall content; these sugars are known for influencing caecal bacterial diversity. We hypothesized if its dietary administration could exert a positive effect on caecal microbiota in favor of a reduced S. Infantis load. A total of 72 one-day-old broiler chickens (COBB 500) were randomly allocated into three groups: a control, a group infected with bacteria (day 4), and a group challenged with S. Infantis but fed a microalgae-based diet. Caecal samples (n = 8) were collected two days post-infection. A PMAxxTM-based qPCR approach was developed to assess differences regarding bacterial viable load between groups. The inclusion of the microalga did not modify S. Infantis content, although the assay proved to be efficient, sensitive, and repeatable. The utilized scheme could serve as a foundation for developing novel PCR-based methodologies for estimating Salmonella colonization.

Antibiotic Resistance Awareness among Undergraduate Students in Quito, Ecuador.

The inappropriate use of antibiotics leads to antibiotic resistance, which reduces their efficacy. The education of undergraduates is likely to influence their practices. Assessing awareness is critical in the general effort to confront the spread of antibiotic resistance. This cross-sectional investigation was carried out using the questionnaire "Antibiotic resistance: Multi-country public awareness" developed by the World Health Organization. Students from different backgrounds at the Central University participated in the study (n = 733). The survey comprised five sections: demographics, knowledge, usage, sources of information, and attitudes. The rate of correct answers was 64.88%; differences were detected between programs of study (p < 0.001); effect size analysis showed that these differences cannot be considered large. Individuals from applied sciences scored higher than their counterparts from social studies. Mostly, interviewees were knowledgeable about usage, but mistakenly associated antibiotics with conditions such as cold/flu or viral illnesses; also, they associated antibiotic resistance with the patient and not with bacteria. Despite these misconceptions, positive attitudes were registered overall, and students generally adhered to common practices. They cited doctors/nurses and teachers as sources of information. As a consequence, it is recommended to develop courses that address deficient knowledge regarding antibiotic resistance, especially for individuals affiliated to social disciplines.

Administration of Dietary Microalgae Ameliorates Intestinal Parameters, Improves Body Weight, and Reduces Thawing Loss of Fillets in Broiler Chickens: A Pilot Study.

This pilot investigation aimed at studying the feasibility of using a low dose (0.2%) of dietary microalgae as a means of improving intestinal morphometry, body weight, and selected meat quality parameters in broilers. A total of 72 one-day-old ROSS 308 male chicks were randomly separated into four groups; three experimental pens in which the birds were fed with biomass from Tysochrysis lutea, Tetraselmis chuii, and Porphyridium cruentum over 30 days and a control group. T. chuii and P. cruentum had a positive effect with regard to body weight. In treated animals, duodenal and ileal sections showed characteristic tall and thin villi, with serrated surfaces and goblet cell differentiation. In both sections, values of the villus-height-to-crypt-depth ratio were increased by microalgae ingestion. The thawing weight loss of fillets was reduced in T. chuii-fed animals. The positive effects exerted by T. chuii and P. cruentum on intestinal architecture were associated with the improved body weight. Arguably, these outcomes exhibit the potential of using these species to enhance growth performance in broiler chickens by promoting gut homeostasis and thus nutrient absorption.

In Silico Detection of Antimicrobial Resistance Integrons in Salmonella enterica Isolates from Countries of the Andean Community.

Antimicrobial resistance genes are often associated with integrons, which promote their movement between and within DNA molecules. IntFinder 1.0 and I-VIP v1.2 were used for the detection of integrons and their associated resistance genes in assembled sequences and raw reads. A dataset comprising 1688 sequenced Salmonella enterica isolates from countries of the Andean Community was developed. A total of 749 and 680 integrons were identified by IntFinder 1.0 and I-VIP v1.2, respectively; class 2 integrons were the most abundant followed by class 1, whereas no class 3 integrons were detected. These elements were mainly associated with isolates from animal sources. S. Infantis ST32 contained the majority of integrons. Trimethoprim resistance genes (dfrA) were found in greater numbers than others, including aadA and bla genes. The presence of these resistance integrons may come as a response to antibiotic misuse, especially of co-trimoxazole. This represents a public health risk as novel resistant strains might appear due to gene dissemination. The information gathered from in silico studies not only contributes to our understanding of integron dynamics in pathogenic Salmonella, but also helps identify potential emergent patterns of resistance in the region, which is fundamental for developing pertinent antibiotic surveillance programs.

A Cross-Sectional Study to Assess Knowledge of COVID-19 among Undergraduate Students in North-Central Ecuador.

Adherence to preventive measures is influenced by people's knowledge, attitudes and practices towards a disease; therefore, assessing knowledge of COVID-19 is critical in the overall effort to contain the outbreak. This cross-sectional study was conducted among undergraduates (n = 3621) of different programs and different levels of education associated with universities in north-central Ecuador. The form consisted of 32 questions covering demographics, symptoms, detection, treatment, transmission, prevention and knowledge of the virus. The rate of correct answers was 75.5% (21.1 ± 5 out of 28), with differences observed regarding program of study, educational level and location of institution (α = 0.05), although effect size analyses showed that these differences could not be considered large. Multiple linear regression analyses showed that lower scores were associated with initial stages of education, careers related to social sciences and location of institution. Participants possessed sufficient knowledge about detection, transmission and prevention, although they overestimated fatality rate and were less confident about the characteristics of the virus and the effectiveness of traditional medicine. Consequently, future educational programs must place emphasis on addressing deficient knowledge. Certainly, improving COVID-19 literacy will promote the appropriate application of protective measures aimed at preventing the virus' spread.

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants.

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme's sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle's genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an 'Fe-only' nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.

A Simple Technology for Generating Marker-Free Chloroplast Transformants of the Green Alga Chlamydomonas reinhardtii.

The availability of routine methods for the genetic engineering of the chloroplast genome of Chlamydomonas reinhardtii is allowing researchers to explore the use of this microalga as a phototrophic cell platform for synthesis of high value recombinant proteins and metabolites. However, the established method for delivering transforming DNA into the algal chloroplast involves microparticle bombardment using an expensive "gene gun". Furthermore, selection of transformant lines most commonly involves the use of a bacterial antibiotic resistance gene. In this chapter, we describe a simple and cheap delivery method in which cell-DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. We also describe the use of plasmid expression vectors that target transgenes to a neutral site within the chloroplast genome between psbH and trnE2, and employ psbH as the selectable marker-thereby avoiding issues of unwanted antibiotic resistance genes in the resulting transgenic lines. Finally, we highlight a feature in our latest vectors in which the presence of a novel tRNA gene on the plasmid results in recognition within the chloroplast of UGA stop codons in transgenes as tryptophan codons. This feature simplifies the cloning of transgenes that are normally toxic to E. coli, serves as a biocontainment strategy restricting the functional escape of transgenes from the algal chloroplast to environmental microorganisms, and offers a simple system of temperature-regulated translation of transgenes.

An On-Line Cross-Sectional Questionnaire to Assess Knowledge of COVID-19 Pandemic among Citizens Tested for the SARS-CoV-2 Virus in Quito and Ibarra, Ecuador.

Several public health measures have been implemented to contain the SARS-CoV-2 outbreak. The adherence to control measures is known to be influenced by people's knowledge, attitudes and practices with regard to the disease. This study aimed at assessing COVID-19 knowledge in individuals who were tested for the virus. An online cross-sectional survey of 32 items, adapted to the national context, was conducted among 1656 Ecuadorians. The mean knowledge score was 22.5 ± 3 out of 28, with significant differences being observed with regard to educational attainment. People with postgraduate training scored higher than those with college, secondary and elementary instruction. Indeed, multiple linear regression revealed that lower scores were associated significantly with the latter three levels of education. Interviewees were knowledgeable about the symptoms, detection, transmission and prevention of the disease. However, they were less assertive regarding the characteristics of the virus as well as the usefulness of traditional and unproven treatments. These outcomes indicated a lack of knowledge in fundamental aspects of virus biology, which may limit the effectiveness of further prevention campaigns. Conclusively, educational and communicational programs must place emphasis on explaining the basic molecular characteristics of SARS-CoV-2; such information will certainly contribute to improve the public's adherence to control measures.

The Probiotic Lactobacillus fermentum Biocenol CCM 7514 Moderates Campylobacter jejuni-Induced Body Weight Impairment by Improving Gut Morphometry and Regulating Cecal Cytokine Abundance in Broiler Chickens.

This research was conducted to investigate if the administration of the probiotic Lactobacillus fermentum could influence body weight, intestinal morphometry and the cecal cytokine response in Campylobacter jejuni-infected chickens. Seventy-two 1-day old COBB 500 male chicks were allocated randomly into four experimental groups. (I) Control group (C), in which chicks were left untreated. (II) LB group, treated with L. fermentum. (III) Cj group, infected with C. jejuni and (IV) coexposure group in which both bacteria were administered. Body weight was registered and then all birds were slaughtered; samples from the small intestine and caecum were collected at 4- and 7-days post infection. The experiment lasted eleven days. Villi height and crypt depth ratios of the duodenum, jejunum and ileum were evaluated using appropriate software, while reverse transcription quantitative PCR (RT-qPCR) was utilized for assessing transcript levels of key cecal inflammatory cytokines (IL-1ß, IL-18, IL-17, IL-15, IL13 and IL-4). Campylobacter-infected birds showed lower body weight values than those supplemented with the probiotic; these birds, in turn, proved to be heavier than those reared under control conditions. L. fermentum administration improved morphometrical parameters of the duodenum, jejunum and ileum; in general, villi were larger and crypts deeper than those identified in control conditions. Moreover, the negative effects elicited by C. jejuni were not observed in chickens exposed to the probiotic. Significant differences were also determined with regards to transcript abundance of all evaluated cytokines in the caecum. C. jejuni induced a downregulation of the studied interleukins; however, such a response was heightened by administration of L. fermentum, with an increase rate of transcription that promoted a more effective response to a C. jejuni infection. The effects of experimental treatments proved to vary between sampling points. Conclusively, these results demonstrate that L. fermentum lessens the negative effects elicited by C. jejuni on body weight by alleviating the impact on intestinal morphometry and cecal cytokine response, which ultimately improve chicken growth performance.

Effects of Lactobacillus Fermentum Supplementation on Body Weight and Pro-Inflammatory Cytokine Expression in Campylobacter Jejuni-Challenged Chickens.

Due to the interest in using probiotic bacteria in poultry production, this research was focused on evaluating the effects of Lactobacillus fermentum Biocenol CCM 7514 administration on body weight gain and cytokine gene expression in chickens challenged with Campylobacter jejuni. One-hundred and eight 1-day old COBB 500 broiler chickens were equally assigned to four experimental groups at random. In the control group (C) chicks were left untreated, whereas in groups LB and LBCj a suspension of L. fermentum was administered. A suspension of C. jejuni was subsequently applied to groups Cj and LBCj. Body weight was registered, and the individuals were later slaughtered; cecum samples were collected at 12, 36 and 48 h post-infection (hpi). The entire experiment lasted seven days. Reverse transcription quantitative PCR (RT-qPCR) was used to determine expression levels of IL-1ß, IL-15, IL-17, and IL-18 at each time point. Pathogen-infected individuals were observed to weigh significantly less than those fed with the probiotic. Significant differences were also found in transcript abundance; expression of IL-15 was downregulated by the probiotic and upregulated by C. jejuni. The effects of bacterial treatments were time-dependent, as the expression profiles differed at later stages. The present outcomes demonstrate that L. fermentum both reduces the impact of C. jejuni infection on chicken body weight and regulates positively pro-inflammatory cytokine expression, which ultimately increase bird well-being and improves production.

Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii.

The chloroplast of microalgae such as Chlamydomonas reinhardtii represents an attractive chassis for light-driven production of novel recombinant proteins and metabolites. Methods for the introduction and expression of transgenes in the chloroplast genome (=plastome) of C. reinhardtii are well-established and over 100 different proteins have been successfully produced. However, in almost all reported cases the complexity of the genetic engineering is low, and typically involves introduction into the plastome of just a single transgene together with a selectable marker. In order to exploit fully the potential of the algal chassis it is necessary to establish methods for multigenic engineering in which many transgenes can be stably incorporated into the plastome. This would allow the synthesis of multi-subunit proteins and the introduction into the chloroplast of whole new metabolic pathways. In this short communication we report a proof-of-concept study involving both a combinatorial and serial approach, with the goal of synthesizing five different test proteins in the C. reinhardtii chloroplast. Analysis of the various transgenic lines confirmed the successful integration of the transgenes and accumulation of the gene products. However, the work also highlights an issue of genetic instability when using the same untranslated region for each of the transgenes. Our findings therefore help to define appropriate strategies for robust multigenic engineering of the algal chloroplast.

Lactobacillus fermentum Administration Modulates Cytokine Expression and Lymphocyte Subpopulation Levels in Broiler Chickens Challenged with Campylobacter coli.

This investigation was performed to assess the supplementation of probiotics on cytokine expression and lymphocyte subpopulation in Campylobacter coli challenged chickens. Thirty-six individuals were equally separated into four experimental treatments: C = untreated chickens, LB = probiotic control (Lactobacillus fermentum), Cc = Campylobacter-challenged control, LBCc = probiotic + Cc. All chicks were slaughtered and cecum samples were collected on day 4 postinfection. Gene expression analysis, using reverse transcription quantitative PCR (RT-qPCR), revealed significant differences in cytokine transcript expression between untreated and probiotic-treated chickens. In addition, flow cytometry was used to quantitate the levels of lymphocyte subpopulations. Principal component analysis showed that probiotic administration induced an overall downregulation of cytokine expression. C. coli exposure provoked a similar response to that of L. fermentum but to a lesser extent. Colonization of C. coli in the presence of the probiotic evoked a complex response with an upregulation of some type II cytokines, including interleukin IL-4 and IL-13, which could explain the increased presence of antibodies in both lamina propria and epithelium. Moreover, despite that the percentage of CD8 intraepithelial lymphocytes (IELs) was found to be higher, downregulation of proinflammatory cytokines IL-15, IL-16, and interferon γ was observed. This suggests that the detected CD8 are not effector cells but induced IELs, which release antimicrobial peptides, and are ready to be primed upon encountering antigen. These outcomes demonstrate that probiotic administration promotes a humoral response to a C. coli infection while dampening any potential inflammation mediated by effector T cells in 1-week-old chicks.

Assessing knowledge of genetics in undergraduate students in Quito, Ecuador.

Knowledge of genetics is crucial for understanding genetic and genomic tests and for interpreting personal genomic information. Despite this relevance, no data are available about the level of knowledge of genetics in an Ecuadorian population. This investigation sought to survey such knowledge in undergraduate students affiliated with private and public institutions in Quito, the capital city of Ecuador. A total of 350 individuals responded to a validated questionnaire measuring knowledge of genetics. Scores ranged from 45% to 87% (mean: 66.8%), and students achieved slightly better results when asked about genetics and diseases (mean score: 68.3%) than when asked about genetic facts (mean score: 64.9%). Additionally, no significant differences in performance were found among students from private and public institutions. Surprisingly, the lower score obtained (45%) was from a question about how chromosomes are passed to the next generation. The highly educated status of the surveyed population could explain the overall results; nonetheless, the possibility that the correct responses were given by chance cannot be ignored. Therefore, the actual knowledge of genetics among the participants might be different than that revealed by the percentages of correct answers. Consequently, to achieve the goal of ensuring informed decision-making concerning genetic and genomic tests, it seems evident that the national education programs of Ecuador require improvement in the teaching of genetic concepts.

Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.