Pathogenic Microorganisms Linked to Fresh Fruits and Juices Purchased at Low-Cost Markets in Ecuador, Potential Carriers of Antibiotic Resistance.

The pathogenic microorganisms linked to fresh fruits and juices sold out in retail low-cost markets raise safety concerns as they may carry multidrug-resistant (MDR) genes. To evaluate the microbiological quality and safety of highly consumed fruits and derivatives in Imbabura Province, Ecuador, ready-to-eat strawberries (5 independent batches; n = 300 samples), and gooseberries (5 separate batches; n = 500 samples), purchased from a local fruit farm grower and low-cost retail market, along with 20 different natural fruit- and vegetables-based juices (3 independent batches; n = 60 samples) purchased from food courts located within the low-cost markets were analyzed. Bacteriological analysis showed that the microbial quality was lower as several indicators (n = 984) consisting of total coliforms (TCOL), total aerobes (AEROB), Enterobacter spp. (ENT), Shigella spp., (SHIGA), yeasts (YE), and molds (M) were detected. Staphylococcus spp. (STAPHY) was found in both fruits regardless of origin, while Escherichia coli (EC) isolates were found in strawberries but not gooseberries. Salmonella spp. (SALM) were detected in juices only. Antibiotic susceptibility testing showed multidrug resistance of several isolates. The hemolytic pattern revealed that 88.89% of EC and 61.11% of ENT isolates were beta-hemolytic. All STAPHY isolates were beta-hemolytic while SALM and SHIGA were alpha-hemolytic. Plasmid curing assay of MDR isolates (ENT, EC, SALM, and STAPHY) showed that the antibiotic resistance (AR) was highly indicative of being plasmid-borne. These results raise concerns about the consumption of MDR bacteria. However, good agricultural and industrial practices, behavioral change communication, and awareness-raising programs are necessary for all stakeholders along the food production and consumption supply chain.

Genome Characterization of Lactiplantibacillus plantarum Strain UTNGt2 Originated from Theobroma grandiflorum (White Cacao) of Ecuadorian Amazon: Antimicrobial Peptides from Safety to Potential Applications.

The genome characterization of the Lactiplantibacillus plantarum strain UTNGt2, isolated from wild copoazu or white cacao (Theobroma grandiflorum), is described. A total of 31 contigs is assembled with a total length of 3,264,448 bases, with all contigs matching the core genome of different groups in the database. The genome size is 3,540,752 bases with GC content of 44.53% and the genome repeat sequences constitute around 457,386 bases of the assembly. The UTNGt2 matches the Lactiplantibacillus plantarum genome with 99% identity. The genome contains 3115 genes, 3052 protein-coding genes, assigned with the EggNOG database. On the basis of the results, 745 proteins are classified with an unknown function, from which 128 proteins have no match in the BLASTN database. It also contains 57 tRNAs, 5 copies of 5S rRNA, and 1 copy of tmRNA. Based on gene prediction and annotation results, 9.4% of proteins are involved in carbohydrate transport and metabolism and 8.46% in transcription, 2.36% are responsible for defense mechanisms, 0.5% are responsible for the biosynthesis of secondary metabolites, transport, and catabolism, while 25.11% have an unknown function. The genome revealed the presence of genes involved in riboflavin and folate production, the presence of CRISPR/Cas genes, phage sequences, the absence of acquired antibiotics resistance genes, virulence, and pathogenic factors, suggesting that UTNGt2 is a safe strain. Its highly antimicrobial capacity is related to the presence of two bacteriocin clusters (class IIc) of the sactipeptide class (contig 4) and plantaricin E class (contig 22), as detected by the BAGEL 4 webserver. Several RiPP-like peptides (non-bactericidal ribosomally produced and post-translationally modified peptides), polyketides (PKs), and terpenes were predicted. Whole-genome sequencing analysis revealed that the UTNGt2 strain has diverse bacteriocins with a high inhibitory capacity, thus it is a bacteriocinogenic strain. Considering the safety profile, UTNGt2 is a nonpathogenic, nonvirulent strain with valuable biotechnological traits and can be further exploited for its probiotic and antimicrobial potential in the food industry or as a potential producer strain of antimicrobial peptides as an alternative to conventional antibiotics.

A Novel Weissella cibaria Strain UTNGt21O Isolated from Wild Solanum quitoense Fruit: Genome Sequence and Characterization of a Peptide with Highly Inhibitory Potential toward Gram-Negative Bacteria.

A novel Weissella cibaria strain UTNGt21O from the fruit of the Solanum quitoense (naranjilla) shrub produces a peptide that inhibits the growth of both Salmonella enterica subsp. enterica ATCC51741 and Escherichia coli ATCC25922 at different stages. A total of 31 contigs were assembled, with a total length of 1,924,087 bases, 20 contig hits match the core genome of different groups within Weissella, while for 11 contigs no match was found in the database. The GT content was 39.53% and the genome repeats sequences constitute around 186,760 bases of the assembly. The UTNGt21O matches the W. cibaria genome with 83% identity and no gaps (0). The sequencing data were deposited in the NCBI Database (BioProject accessions: PRJNA639289). The antibacterial activity and interaction mechanism of the peptide UTNGt21O on target bacteria were investigated by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with different concentrations (1×, 1.5× and 2× MIC) of the peptide applied alone or in combination with chelating agent ethylenediaminetetraacetic acid (EDTA) at 20 mM. The results indicated a bacteriolytic effect at both early and late target growth at 3 h of incubation and total cell death at 6 h when EDTA was co-inoculated with the peptide. Based on BAGEL 4 (Bacteriocin Genome Mining Tool) a putative bacteriocin having 33.4% sequence similarity to enterolysin A was detected within the contig 12. The interaction between the peptide UTNGt21O and the target strains caused permeability in a dose-, time- response manner, with Salmonella (3200 AU/mL) more susceptible than E. coli (6400 AU/mL). The results indicated that UTNGt21O may damage the integrity of the cell target, leading to release of cytoplasmic components followed by cell death. Differences in membrane shape changes in target cells treated with different doses of peptide were observed by transmission electronic microscopy (TEM). Spheroplasts with spherical shapes were detected in Salmonella while larger shaped spheroplasts with thicker and deformed membranes along with filamentous cells were observed in E. coli upon the treatment with the UTNGt21O peptide. These results indicate the promising potential of the putative bacteriocin released by the novel W. cibaria strain UTNGt21O to be further tested as a new antimicrobial substance.

Inhibitory Effect of Substances Produced by Native Lactococcus lactis Strains of Tropical Fruits towards Food Pathogens.

The use of peptides produced by lactic acid bacteria (LAB) as antimicrobial agents in food emerged from the increasing need of replacing chemicals with natural substances to ensure their safety and quality. A total of 30 LAB belonging to the genus Lactococcus sp. (10) and Enterococcus sp. (20) were isolated from native fruits of Ecuador subtropical rainforest. Among Lactococcus species, the isolates assigned Gt28, Gt29, and Ella8, identified as Lactococcus lactis subsp. lactis with 99% identity, showing highly inhibitory potential against four food pathogens were further characterized. The treatment of cell-free supernatant with proteolytic enzymes indicated the protein nature of released components, which displayed a broad antimicrobial activity against Gram-positive and -negative bacteria. Polymerase chain reaction analysis indicated the presence of lacticin 3147 gene in all isolates, lactococcin M gene in Gt28 and Gt29 but not in Ella8 and lactococcin A gene in Gt28 only. The antimicrobial activity was not linked to the presence of structural nisin gene as no amplification product was obtained. Treatment of Salmonella enterica ATCC 51741 and Escherichia coli ATCC 25922 at both vegetative and exponential phase of growth with the cell-free supernatant of Gt28 resulted in complete inactivation upon 3 h suggesting its bactericidal mode of action. An increment on inhibitory activity occurred when partial purified bacteriocin Gt28 was combined with ethylenediaminetetraacetic acid rather than bacteriocin only, indicating that the cells were sensitized in vitro by the chelator agent acting synergistically to induce the killing of pathogenic cells.

Characterization of Bacteriocin-Producing Lactic Acid Bacteria Isolated from Native Fruits of Ecuadorian Amazon.

Tropical, wild-type fruits are considered biodiverse "hotspots" of microorganisms with possible functional characteristics to be investigated. In this study, several native lactic acid bacteria (LAB) of Ecuadorian Amazon showing highly inhibitory potential were identified and characterized. Based on carbohydrate fermentation profile and 16S rRNA gene sequencing, seven strains were assigned as Lactobacillus plantarum and one strain as Weissella confusa. Using agar-well diffusion method the active synthetized components released in the neutralized and hydroxide peroxide eliminated cell-free supernatant were inhibited by proteolytic enzymes, while the activity was maintained stable after the treatment with catalase, lysozyme, α-amylase and lipase suggesting their proteinaceous nature. The inhibitory activity was stimulated by acidic conditions, upon exposure to high heat and maintained stable at different ranges of sodium chloride (4-10%). The DNA sequencing analysis confirmed the presence of plw structural gene encoding for plantacirin W in the selected L. plantarum strains. Moreover, we showed that the active peptides of Cys5-4 strains contrast effectively, in a bactericidal manner, the growth of food borne E. coli UTNEc1 and Salmonella UTNSm2, with about tree fold reduction of viable counts at the early stage of the target cell growth. The results indicated that the bacteriocin produced by selected native lactic acid bacteria strains has elevated capacity to suppress several pathogenic microorganisms implying their potential as antimicrobial agents or food preservatives.

Proteina C reactiva en recién nacidos sanos Hospital Simón Bolívar / Reactive C protein in newborn

La concentración sérica de proteína fué medida por aglutinación en 71 recién nacidos sanos a las 24 horas de edad, atendidos en el Hospital Regional Simón Bolívar, durante los meses de Mayo a Julio de 1992. Se utilizó una muestra que confiere un 95 por ciento de confiabilidad estadística; encontrando en 67 pacientes niveles de proteína C reactiva menores de 6 miligramos por litro, en un paciente entre 6 y 12 miligramos por litro, en dos pacientes entre 13 y 18 y en un paciente entre 42 y 48 miligramos por litro. Ninguno de los pacientes tenía riesgo de infección y tampoco la adquirió durante el período nenonatal temprano, Con los resultados obtenidos concluímos que el 98 por ciento de los pacientes recién nacidos sanos de dicho hospital presentan niveles entre 0 y 18 miligramos por litro; concentraciones que se pueden tomar como referencia para estudios de sensibilidad y especificidad frente al diagnóstico y exclusión de sepsis neonatal temprana y para el seguimiento de pacientes infectados durante el tratamiento