A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity.

The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections.

Phenotypic and genotypic antimicrobial resistance in Escherichia coli strains isolated from household dogs in Chile.

Introduction: Antimicrobial resistance (AMR) is a major threat to animal and public health worldwide; consequently, several AMR surveillances programs have been implemented internationally in both human and veterinary medicine, including indicator bacteria such as Escherichia coli. However, companion animals are not typically included in these surveillance programs. Nevertheless, there have been reports of increasing levels of antimicrobial resistance in E. coli strains isolated from dogs worldwide. In Chile, there is limited information available on AMR in E. coli isolated from companion animals, which prevents the establishment of objective prevention and control measures. Methods: For this reason, the aim of this study was to characterize the phenotypic and genotypic AMR of E. coli strains isolated from healthy household dogs in Chile. For this purpose, a multi-stage sampling was carried out in the Metropolitan Region of Chile, obtaining samples from 600 healthy dogs. These samples were processed using traditional bacteriology and molecular techniques to isolate E. coli strains. We assessed the minimal inhibitory concentration of 17 antimicrobials and conducted a search of six antimicrobial resistance genes, as well as class 1 and 2 integrons, in the isolated strains. Results: Two-hundred and twenty-four strains of E. coli were recovered, and 96.9% (n = 217) showed resistance to at least one drug and only 3.1% (n = 7) were susceptible to all analyzed antimicrobials. Most strains were resistant to cefalexin (91.5%, n = 205, 1st-generation cephalosporin), followed by ampicillin (68.3%, n = 153) and cefpodoxime (31.3%, n = 70, 3rd-generation cephalosporin). Moreover, 24.1% (n = 54) tested positive for extended-spectrum-ß-lactamases and 34.4% (n = 77) were multidrug resistant. As for the AMR genes, the most detected was qnrB (28.1%, n = 63), followed by blaCTX-M (22.3%, n = 50), and blaTEM-1 (19.6%, n = 44). Additionally, 16.1% (n = 36) harbored class 1 integrons. Our study shows that E. coli strains isolated from healthy household dogs exhibit resistance to several relevant drugs and also antimicrobial resistance genes considered critical for human health. These results can be used as a starting point for the prevention and control of antimicrobial resistance from companion animals. This background should be considered when formulating future resistance surveillance programs or control plans in which companion animals must be included.

Phytates as a natural source for health promotion: A critical evaluation of clinical trials.

Phytates are a type of organophosphorus compound produced in terrestrial ecosystems by plants. In plant feeds, phytic acid and its salt form, phytate, account for 60%-80% of total phosphorus. Because phytate is a polyanionic molecule, it can chelate positively charged cations such as calcium, iron, and zinc. Due to its prevalence in vegetal tissues and the fact that people consume plants, phytate was first considered a potential health benefit. This updated review aims to summarize the current data on the results of clinical trials of phytates on human health, highlighting both beneficial and undesirable effects. To obtain these updated data, published papers in electronic databases such as PubMed/MedLine, TRIP database, Wiley, Google Scholar, Baidu, and Scopus were searched. Study results have shown that phytate can have beneficial health effects such as antioxidant, anticancer potential and reduction of pathological calcifications in blood vessels and organs; but also, negative effects by reducing the absorption of minerals important for maintaining the homeostasis of the human body. According to these recent results derived from recent clinical studies, phytates may be a potential natural source for health benefits. To improve clinical efficacy and human health benefits, further dose-response studies are needed to determine effective therapeutic doses and potential interactions with conventional drugs.

Plastic residues produced with confirmatory testing for COVID-19: Classification, quantification, fate, and impacts on human health.

In March 2020, several international organizations started making recommendations regarding the need for prompt coronavirus-based diagnosis in order to prevent its spread among the world's population. The most widely used test for confirmation of COVID-19 is real-time PCR (RT-PCR). This technique uses plastic supplies in its procedures, which are 100% disposable to avoid cross-contamination and biological risks. The scientific community has become increasingly worried because of the environmental impacts associated with the current COVID-19 pandemic, such as medical plastic residues. We classified and estimated the amount of plastic residues generated as a consequence of COVID-19 diagnostic tests and analyzed the relationships of the plastics generated with number of confirmed cases, population size, and gross domestic product in each country. We evidenced that the RT-PCR generates 37.27 g of plastic residues per sample. All patients COVID-19 tested with RT-PCR have generated 15,439.59 tons of plastic residue worldwide, until August 2020. The plastic residues generated by the COVID-19 tests have no relation with size population or gross domestic product of the countries. There is also no relationship between the plastic residues generated by the COVID-19 tests and the confirmed cases. About 97% of the plastic residues from diagnostic tests for coronavirus are incinerated due to their hazardous nature to humans, but toxic chemicals are released into the environment during the process. In the short term, there is a need to reduce plastic waste and improve controls of gas emissions from incineration plants in countries where there is a deficit. In the long term, biodegradable biomedical manufacturing that are free of releasing toxic chemicals when they are incinerated, must be considered.

Phenotypic and Genotypic Antimicrobial Resistance in Non-O157 Shiga Toxin-Producing Escherichia coli Isolated From Cattle and Swine in Chile.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes bloody diarrhea and hemolytic-uremic syndrome in humans, and a major cause of foodborne disease. Despite antibiotic treatment of STEC infections in humans is not recommended, the presence of antimicrobial-resistant bacteria in animals and food constitutes a risk to public health, as the pool of genes from which pathogenic bacteria can acquire antibiotic resistance has increased. Additionally, in Chile there is no information on the antimicrobial resistance of this pathogen in livestock. Thus, the aim of this study was to characterize the phenotypic and genotypic antimicrobial resistance of STEC strains isolated from cattle and swine in the Metropolitan region, Chile, to contribute relevant data to antimicrobial resistance surveillance programs at national and international level. We assessed the minimal inhibitory concentration of 18 antimicrobials, and the distribution of 12 antimicrobial resistance genes and class 1 and 2 integrons in 54 STEC strains. All strains were phenotypically resistant to at least one antimicrobial drug, with a 100% of resistance to cefalexin, followed by colistin (81.5%), chloramphenicol (14.8%), ampicillin and enrofloxacin (5.6% each), doxycycline (3.7%), and cefovecin (1.9%). Most detected antibiotic resistance genes were dfrA1 and tetA (100%), followed by tetB (94.4%), bla TEM-1 (90.7%), aac(6)-Ib (88.9%), bla AmpC (81.5%), cat1 (61.1%), and aac(3)-IIa (11.1%). Integrons were detected only in strains of swine origin. Therefore, this study provides further evidence that non-O157 STEC strains present in livestock in the Metropolitan region of Chile exhibit phenotypic and genotypic resistance against antimicrobials that are critical for human and veterinary medicine, representing a major threat for public health. Additionally, these strains could have a competitive advantage in the presence of antimicrobial selective pressure, leading to an increase in food contamination. This study highlights the need for coordinated local and global actions regarding the use of antimicrobials in animal food production.

Virulence Genes, Shiga Toxin Subtypes, Serogroups, and Clonal Relationship of Shiga Toxin-Producing Escherichia Coli Strains Isolated from Livestock and Companion Animals.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes severe illness in humans and is an important cause of foodborne disease. In Chile, there is limited information on the virulence characteristics of this pathogen in livestock, and none in companion animals. The aim of this study was to characterize STEC strains isolated from cattle, swine, dogs, and cats, in Chile, in terms of the presence of Shiga toxin types and subtypes, virulence genes, serogroups, and clonality. One-thousand two-hundred samples were collected, isolating 54 strains (4.5%), where stx1a (68.5%) and ehxA (74.1%) were the most frequently detected virulence genes. Only one strain belonging to the most clinically relevant serogroups was identified. Pulsed field gel electrophoresis analysis showed high clonal diversity among strains isolated from cattle, while those from swine showed the same pattern. This study provides further evidence regarding cattle and swine in Chile as a potential source of a wide variety of STEC strains that could potentially cause severe illness in humans, and that companion animals do not seem to represent a relevant reservoir. It also argues that preventive and control strategies should not be focused on detecting serogroups, but instead, on detecting their determinants of virulence.

Association between cagA, vacAi, and dupA genes of Helicobacter pylori and gastroduodenal pathologies in Chilean patients.

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups-non-severe and severe gastric pathologies-and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54-21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59-4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26-7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09-0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.

Effect of pH in the survival of Lactobacillus salivarius strain UCO_979C wild type and the pH acid acclimated variant

Background Bacterial acclimation involves cellular changes permitting the survival of a microorganism to prolonged acid pH exposure. The general aim of this work is to support this idea by determining the effect of pH in the survival of the human gastric derived probiotic strain Lactobacillus salivarius UCO_979C-1 (wild type) and L. salivarius UCO_979C-2 (acclimation to pH 2.6), which possesses anti-Helicobacter pylori properties. Results To assess this aim, the exopolysaccharide production through the phenol-sulfuric acid method was evaluated. Moreover, morphological and structural changes by transmission and scanning electron microscopy were observed. The bacterial survival was measured by viable count. The results showed that the acclimated variant strain synthesized higher levels of exopolysaccharide (690 ± 0.03 mg/L) more than the wild type (450 ± 0.12 mg/L). In addition, the acclimated variant preserved the viable count at pH 2.6 for 48 h, whereas the wild type strain decreases after 6 h and was non-viable at 24 h. Conclusion The results suggest that the acid stress acclimation of the strain L. salivarius UCO_979C-1 modified some cellular properties making this strain potentially useful as a gastric probiotic.

Prevalencia de Helicobacter pylori y de genes asociados a virulencia en biopsias gástricas / Prevalence of Helicobacter pylori virulence genes in gastric biopsies in Chilean patients over a 10-year period

Introducción: La detección de genes asociados a virulencia en Helicobacter pylori constituye un buen marcadorgenético para predecir riesgo de enfermedades asociadas a la persistencia del patógeno. Objetivos: Determinar la prevalencia de cagA, vacA, babA2, iceA y dupA en pacientes chilenos, durante 10 años de seguimiento. Métodos: Se analizaron las biopsias gástricas de 1 577 pacientes (183 niños) obtenidas entre enero de 2003 y diciembre de 2007, mediante PCR convencional y cultivo bacteriano. En 374 individuos positivos se investigó la prevalencia de cagA, vacA (s1a, s1b, s2, m1, m2, i1 e i2), babA2, iceA (1 y 2) y dupA. Resultados: La prevalenciade H. pylori en adultos fue 48.7% y un 23.7% de los pacientes presentó más de un cepa bacteriana. La prevalenciapor genes fue: cagA 29.4%, vacAm1 52.7%, vacAm2 61.8%, vacAs1a 46.5%, vacAs1b 28.3%, vacAs2 41.7%, vacAi1 30.9%, vacAi2 12.0%, babA23.5%, iceA1 30.5%, iceA2 61.2% y dupA 28.9%. Se observó un 90% de concordancia en la prevalencia delos genes hpy, cagA, babA2, iceA e iceA2, y un 67.6% para vacAs1a, cuando se comparó biopsia y cultivo como fuente de ADN. Conclusiones: La mayoría de los genes de virulencia han mantenido su prevalencia en el tiempo,excepto vacAm1 y babA2 que la han aumentado. Sin embargo, la prevalencia de babA2 continúa siendo muy baja.

Background: Genes associated with virulence in Helicobacter pylori are good markers for prediction of risk of developing diseases due to persistent infection. Aim: To establish the prevalence of cagA, vacA, babA2,iceA and dupA among Chilean patients over a 10-year period. Methods: 1 577 gastric biopsies (183 children), collected from January 2003 to December 2007, were analyzed by conventional PCR and bacteriological culture. The prevalence of the genes cagA, vacA (s1a, s1b, s2, m1, m2, i1 and i2), babA2, iceA (1 and 2) and dupA were investigated in 374 positive individuals. Results: Prevalence of H. pylori in adults was 48.7%, with 23.7% of them presenting more than one infecting strain. Prevalence of genes was as follows: cagA 29.4%, vacAm152.7%, vacAm2 61.8%, vacAs1a 46.5%, vacAs1b 28.3%, vacAs2 41.7%, vacAi1 30.9%, vacAi2 12.0%, babA2 3.5%, iceA1 30.5%, iceA2 61.2% and dupA 28.9%. Ninety percent of agreement was observed in the prevalence of genes hpy, cagA, babA2, iceA and iceA2, by using DNA from both sources, but was only 67.6% for vacAs1a gene. Conclusion: The results suggest that all the genes conserved their prevalence in this period with the exception of vacAm1 and babA2, in which there was increased prevalence. Nonetheless, the prevalence of the gene babA2 continues to be very low.