Polyvalent Bacterial Lysate with Potential Use to Treatment and Control of Recurrent Urinary Tract Infections.

Overuse of antimicrobials has greatly contributed to the increase in the emergence of multidrug-resistant bacteria, a situation that hinders the control and treatment of infectious diseases. This is the case with urinary tract infections (UTIs), which represent a substantial percentage of worldwide public health problems, thus the need to look for alternatives for their control and treatment. Previous studies have shown the usefulness of autologous bacterial lysates as an alternative for the treatment and control of UTIs. However, a limitation is the high cost of producing individual immunogens. At the same time, an important aspect of vaccines is their immunogenic amplitude, which is the reason why they must be constituted of diverse antigenic components. In the case of UTIs, the etiology of the disease is associated with different bacteria, and even Escherichia coli, the main causal agent of the disease, is made up of several antigenic variants. In this work, we present results on the study of a bacterial lysate composed of 10 serotypes of Escherichia coli and by Klebsiella pneumoniae, Klebsiella aerogenes, Enterococcus faecalis, Proteus mirabilis, Citrobacter freundii, and Staphylococcus haemolyticus. The safety of the compound was tested on cells in culture and in an animal model, and its immunogenic capacity by analysing in vitro human and murine macrophages (cell line J774 A1). The results show that the polyvalent lysate did not cause damage to the cells in culture or alterations in the animal model used. The immunostimulatory activity assay showed that it activates the secretion of TNF-α and IL-6 in human macrophages and TNF-α in murine cells. The obtained results suggest that the polyvalent lysate evaluated can be an alternative for the treatment and control of chronic urinary tract infections, which will reduce the use of antimicrobials.

Characterization of Salmonella spp. and E. coli Strains Isolated from Wild Carnivores in Janos Biosphere Reserve, Mexico.

Enterobacteriaceae are considered one the most important zoonotic pathogens. In this study, we analyzed the characteristics of E. coli and Salmonella spp. strains present in carnivores from Janos Biosphere Reserve, Mexico. These microorganisms had been isolated from a wide range of domestic and free-range animals, including wild carnivores. Fifty-five individuals were sampled, and the presence of Salmonella and E. coli was determined by bacteriological standard methods. Strains isolated were characterized by molecular methods and in vitro infection assays. Eight different species of carnivores were captured, including coyotes (Canis latrans), gray fox (Urocyon cinereoargenteus), desert foxes (Vulpes macrotis), striped skunks (Mephitis mephitis), hooded skunks (Mephitis macroura), lynxes (Lynx rufus), raccoons (Procyon lotor), and badgers (Taxidea taxus). Salmonella spp. and E. coli were isolated from four species of carnivores. Five Salmonella spp. strains were isolated, and their molecular characterization revealed in three of them the presence of fimbrial and virulence genes associated with cell invasion. In vitro evaluation of these strains showed their capability to invade human Hep2 cells. Sixty-one E. coli strains were isolated; different serotypes and phylogroups were observed from these strains. Additionally, the presence of virulence genes showed differently.

Productive Performance and Cecum Microbiota Analysis of Broiler Chickens Supplemented with ß-Mannanases and Bacteriophages-A Pilot Study.

This study was conducted to evaluate the productive performance, intestinal health, and description of the cecum microbiota in broilers supplemented with ß-mannanases (MNs) and bacteriophages (BPs). Six hundred one-day-old broilers were divided into four groups and fed one of the following diets: CON-corn-soybean meal + 10 ppm enramycin (ENR); MN: CON + 500 ppm MN; BP: CON + 500 ppm BP; MN + BP: BP + 500 ppm MN. The BP and MN factors showed similar performances to ENR. MN improved the concentration of IgA in the jejunum at 35 days of age. The morphometric index (IM) of the thymus increased by adding MN, while BP increased the liver and thymus IM. The histological analysis showed that BP and MN improved the intestinal morphology. MN + BP showed a tendency to decrease the abundance of Proteobacteria and increase the abundance of Bacteroidetes, indicating better microbiota function. In conclusion, our results demonstrate that the combination of MN + BP has potential in poultry nutrition; however, we highly recommend further experiments to confirm this hypothesis.

Campylobacter fetus Induced Proinflammatory Response in Bovine Endometrial Epithelial Cells.

Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion in bovines and infertility that produces economic losses in livestock. In many infectious diseases, the immune response has an important role in limiting the invasion and proliferation of bacterial pathogens. Innate immune sensing of microorganisms is mediated by pattern-recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) and induces the secretion of several proinflammatory cytokines, like IL-1ß, TNF-α, and IL-8. In this study, the expression of IL-1ß, TNF-α, IL-8, and IFN-γ in bovine endometrial epithelial cells infected with C. fetus and Salmonella Typhimurium (a bacterial invasion control) was analyzed. The results showed that expression levels of IL-1ß and IL-8 were high at the beginning of the infection and decreased throughout the intracellular period. Unlike in this same assay, the expression levels of IFN-γ increased through time and reached the highest peak at 4 hours post infection. In cells infected with S. Typhimurium, the results showed that IL8 expression levels were highly induced by infection but not IFN-γ. In cells infected with S. Typhimurium or C. fetus subsp. fetus, the results showed that TNF-α expression did not show any change during infection. A cytoskeleton inhibition assay was performed to determine if cytokine expression was modified by C. fetus subsp. fetus intracellular invasion. IL-1ß and IL-8 expression were downregulated when an intracellular invasion was avoided. The results obtained in this study suggest that bovine endometrial epithelial cells could recognize C. fetus subsp. fetus resulting in early proinflammatory response.

Effect and Analysis of Bacterial Lysates for the Treatment of Recurrent Urinary Tract Infections in Adults.

Urinary tract infection (UTI) is a relevant public health problem, economically and socially affecting the lives of patients. The increase of antimicrobial bacterial resistance significantly hinders the treatment of UTIs, raising the need to search for alternative therapies. Bacterial lysates (BL) obtained from Escherichia coli and other pathogens have been used to treat different infectious diseases with promising results. This work aims to evaluate the effect and composition of an autologous BL for the treatment and control of recurrent UTIs in adults. The results show remission in 70% of the patients within the first three months after the administration of BL, while the infection is maintained under control for 6-12 months. The analysis by liquid chromatography-mass spectrometry (LC-MS) of the BL fractions recognized by the sera of patients shows the presence of cytosolic proteins, fimbriae, OMPs, and LPS. Our study demonstrates that the autologous BL contributed to the treatment and control of recurrent UTIs in adults, and its composition shows that different surface components of E. coli are potential immunogens that could be used to create a polyvalent protective vaccine.

Influence of a Polyherbal Mixture in Dairy Calves: Growth Performance and Gene Expression.

A polyherbal feed mixture containing (Achyrantes aspera, Trachyspermum ammi, Citrullus colocynthis, Andrographis paniculata, and Azadirachta indica) was evaluated in growing calves through blood chemistry, blood biometry, and gene expression during the pre-ruminant to weaning period. Forty Holstein calves (initial BW 45.6 ± 3.2 kg; 22.8 ± 0.9 days post birth) from a dairy farm were randomly assigned to the following treatments: 0, 3, 4, and 5 g/d of a polyherbal mixture, dosed in colloid gels with gelatin. Calves were housed in individual outdoor boxes with ad libitum access to a 21.5% CP calf starter and water and fed individually with a mixture of milk and a non-medicated milk replacer (22% CP). Blood samples were collected on day 59 for blood chemistry, blood biometry, and gene expression analysis in leukocyte through microarray assays. Immunoglobulins were quantified by enzyme-linked immunosorbent assay. The animals treated with the polyherbal mixture showed a quadratic effect on final body weight, daily weight gain, final hip height, and final thoracic girth. The best performance results were obtained with a treatment dose of 4 g/d. The serum IgG increased linearly with the treatment doses. Gene set enrichment analysis of upregulated genes revealed that the three biological processes with higher fold change were tight junction, mucin type O-Glycan biosynthesis, and intestinal immune network for IgA production. Also, these upregulated genes influenced arachidonic acid metabolism, and pantothenate and CoA biosynthesis. Gene ontology enrichment analysis indicated that the pathways enriched were PELP1 estrogen receptor interacting protein pathways, nuclear receptors in lipid metabolism and toxicity, tight junction, ECM-receptor interaction, thyroid hormone signaling pathways, vascular smooth muscle contraction, ribosome function, glutamatergic synapse pathway, focal adhesion, Hippo, calcium, and MAPK signaling pathways.

Campylobacter fetus is Internalized by Bovine Endometrial Epithelial Cells.

Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Tilmicosin modulates the innate immune response and preserves casein production in bovine mammary alveolar cells during Staphylococcus aureus infection.

Tilmicosin is an antimicrobial agent used to treat intramammary infections against Staphylococcus aureus and has clinical anti-inflammatory effects. However, the mechanism by which it modulates the inflammatory process in the mammary gland is unknown. We evaluated the effect of tilmicosin treatment on the modulation of the mammary innate immune response after S. aureus infection and its effect on casein production in mammary epithelial cells. To achieve this goal, we used immortalized mammary epithelial cells (MAC-T), pretreated for 12 h or treated with tilmicosin after infection with S. aureus (ATCC 27543). Our data showed that tilmicosin decreases intracellular infection (P < 0.01) and had a protective effect on MAC-T reducing apoptosis after infection by 80% (P < 0.01). Furthermore, tilmicosin reduced reactive oxygen species (ROS) (P < 0.01), IL-1ß (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.05) production. In an attempt to investigate the signaling pathways involved in the immunomodulatory effect of tilmicosin, mitogen-activated protein kinase (MAPK) phosphorylation was measured by fluorescent-activated cell sorting. Pretreatment with tilmicosin increased ERK1/2 (P < 0.05) but decreased P38 phosphorylation (P < 0.01). In addition, the anti-inflammatory effect of tilmicosin helped to preserve casein synthesis in mammary epithelial cells (P < 0.01). This result indicates that tilmicosin could be an effective modulator inflammation in the mammary gland. Through regulation of MAPK phosphorylation, ROS production and pro-inflammatory cytokine secretion tilmicosin can provide protection from cellular damage due to S. aureus infection and help to maintain normal physiological functions of the bovine mammary epithelial cell.

Nivel de riesgo e incidencia de caries en niños atendidos en clínica estomatológica de pregrado / Risk level and caries incidence in children attended at a predoctoral stomatology clinic

Antecedentes: La caries tiene etiología multifactorial, se presenta por desbalance entre factores patológicos y protectores, es un problema de salud pública que en México afecta a 95% de los niños. Objetivo: Determinar la prevalencia de caries, establecer el nivel de riesgo y evaluar su relación con la incidencia de caries a los tres y seis meses. Material y métodos: Estudio observacional, descriptivo y longitudinal en clínica estomatológica de una universidad ubicada al sur de la Ciudad de México. Previo consentimiento informado, se realizó revisión bucal por dos cirujanos dentistas calibrados para el diagnóstico de caries/ICDAS (kappa 95.37%), llevada a cabo en sillón dental con luz artificial bajo principios para el control de infecciones. Se estableció nivel de riesgo mediante el Sistema Internacional para la Gestión de Caries/ICCMS empleando como indicadores: consumo de alimentos cariogénicos (Lipari/Andrade), pH (tiras Hidryon), prueba de saliva estimulada, experiencia de caries (CPOD/ceod), higiene bucal (O'Leary) y cultivo de Streptococcus mutans. Se realizó análisis estadístico para determinar medidas de tendencia central, dispersión y asociación entre variables mediante exacta de Fisher, STATA.SE/10. Resultados: La población en estudio fue de 42 niños con rango de edad de tres a 15 años, mediana de 7.04 (RIC = 5-9). La prevalencia de caries fue 95.23%, con estadios severos en 83.3%, el nivel de riesgo fue alto en 73.7% y se asoció con severidad de caries (p = 0.000), consumo de carbohidratos (p = 0.000), experiencia de caries (ceod) (p = 0.000) y S. mutans (p = 0.000). La incidencia de caries a los tres meses fue de 71.0% y a los seis meses de 11.1%. Conclusiones: La prevalencia de caries fue alta (95.23%) así como el riesgo de caries (73.7%), con elevada incidencia a tres meses (71%), aun cuando los pacientes estaban en tratamiento odontológico. La atención restaurativa prevalece a pesar de hacer diagnóstico de lesiones reversibles y determinación del nivel de riesgo (AU)

Background: Caries has multifactorial etiology, presented by the imbalance between pathological/protective factors, is a public health problem, affecting 95% of children in México. Objective: To determine the prevalence of caries, to establish the level of risk and to evaluate its relation with incidence of caries at three and six months. Material and methods: Observational, descriptive and longitudinal study in the stomatologic clinic of the UAM-Xochimilco. Before informed consent, oral revision was performed by two dental surgeons calibrated for the diagnosis of caries/ICDAS (kappa 95.37%), carried out in dental chair with artificial light under principles for the control of infections. Risk level was established through the International System for Caries Management/ICCMS; using as indicators: consumption of cariogenic foods (Lipari/Andrade), pH (Hidryon strips), stimulated saliva test, caries experience (CPOD/ceod), oral hygiene (O'Leary) and culture of Streptococcus mutans. Statistical analysis was performed to determine measures of central tendency, dispersion and association between variables with Fisher's exact, STATA.SE/10. Results: The study population consisted of 42 children with ages ranging from 3 to 15 years, median of 7.04 (Q1 = 5-Q3 = 9). The prevalence of caries was 95.23%, with severe stages in 83.3%, the risk level was high in 73.7% and was associated with caries severity (p = 0.000), carbohydrate consumption (p = 0.000), caries experience (ceod) (p = 0.000) and S. mutans (p = 0.000). The incidence of caries at three months was 71.0% and at six months 11.1%. Conclusions: The prevalence of caries was high (95.23%), as well as the risk for caries (73.7%), and high incidence at three months (71%), despite the fact that the patients were in dental treatment. Restorative care prevails despite the diagnosis of reversible lesions and determination of the level of risk (AU)

Genetic Analysis Method for Staphylococcus chromogenes Associated with Goat Mastitis.

Mastitis in goats is mainly caused by coagulase-negative Staphylococcus (CNS). The identification methods for this group are based on evaluating the expression of phenotypic characteristics such as the ability to metabolize various substrates; however, this is disadvantageous as these methods are dependent on gene expression. In recent years, genotyping methods such as the Multiple Locus Variable-Number Tandem Repeat Analysis (MLVA) and gene identification have been useful for epidemiological study of several bacterial species. To develop a genotyping method, the genome sequence of Staphylococcus chromogenes MU970 was analysed. The analysis showed nine virulence genes described in Staphylococcus aureus. The MLVA was developed using four loci identified in the genome of S. chromogenes MU970. This genotyping method was examined in 23 strains of CNS isolated from goat mastitis. The rate of discrimination for MLVA was 0.8893, and the highest rates of discrimination per the index of Simpson and Hunter-Gaston were 0.926 and 0.968 for the locus 346_06, respectively. The virulence genes were present in all strains of S. chromogenes but not in other CNS. The genotyping method presented in this paper is a viable and easy method for typifying CNS isolates from mastitis cases in different regions and is an ideal mean of tracking this disease.

Genetic variability and structure of an isolated population of Ambystoma altamirani, a mole salamander that lives in the mountains of one of the largest urban areas in the world.

Amphibians are globally threatened by habitat loss and fragmentation; species within the order Ambystoma are not the exception, as there are 18 species of mole salamanders in México, of which 16 are endemic and all species are under some national or international status of protection. The mole salamander, Ambystoma altamirani is a microendemic species, which is distributed in central México, within the trans-Mexican volcanic belt, and is one of the most threatened species due to habitat destruction and the introduction of exotic species. Nine microsatellite markers were used to determine the genetic structure, genetic variability, effective population size, presence of bottlenecks and inbreeding coefficient of one population of A. altamirani to generate information which might help to protect and conserve this threatened species. We found two genetic subpopulations with significant level of genetic structure (FST = 0.005) and high levels of genetic variability (Ho = 0.883; He = 0.621); we also found a small population size (Ne = 8.8), the presence of historical (M = 0.486) and recent bottlenecks under IAM and TPM models, with a low, but significant coefficient of inbreeding (FIS = -0.451). This information will help us to raise conservation strategies of this microendemic mole salamander species.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity.

BACKGROUND: Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. OBJECTIVES: The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. METHODS: Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). RESULTS: The results obtained showed that the eight strains of C. jejuni, including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. CONCLUSIONS: This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA, cdtB and cdtC genes.

Genetic structure and diversity in an isolated population of an endemic mole salamander (Ambystoma rivulare Taylor, 1940) of central Mexico.

Human activities are affecting the distribution of species worldwide by causing fragmentation and isolation of populations. Isolation and fragmentation lead to populations with lower genetic variability and an increased chance of inbreeding and genetic drift, which results in a loss of biological fitness over time. Studies of the genetic structure of small and isolated populations are critically important for management and conservation decisions. Ambystoma rivulare is a micro-endemic Mexican mole salamander from central Mexico. It is found in the most ecologically disturbed region in Mexico, the Trans-Mexican Volcanic Belt. The goal of this study of the population genetics of the micro-endemic mole salamander was to provide information to be used as a basis for future research and conservation planning of this species and other species of the Ambystoma genus in Mexico. The structural analysis found two subpopulations, one for each river sampled, with no signs of admixture and very high levels of genetic differentiation. Medium to high levels of heterozygosity and few alleles and genotypes were observed. Evidence of an ancestral genetic bottleneck, low values of effective population size, small inbreeding coefficients, and low gene flow were also found.

Genetic variability and structure of jaguar (Panthera onca) in Mexican zoos.

Genealogical records of animals (studbook) are created to avoid reproduction between closely related individuals, which could cause inbreeding, particularly for such endangered species as the Panthera onca (Linnaeus, 1758). Jaguar is the largest felid in the Americas and is considered an important ecological key species. In Mexico, wild jaguar populations have been significantly reduced in recent decades, and population decline typically accompany decreases in genetic variation. There is no current census of captive jaguars in Mexico, and zoos do not follow a standardized protocol in breeding programs based on genetic studies. Here, we emphasise the importance of maintaining an adequate level of genetic variation and propose the implementation of standardised studbooks for jaguars in Mexico, mainly to avoid inbreeding. In addition, achieving the aims of studbook registration would provide a population genetic characterisation that could serve as a basis for ex situ conservation programmes.

Genetic diversity and genetic structure of an endemic Mexican Dusky Rattlesnake (Crotalus triseriatus) in a highly modified agricultural landscape: implications for conservation.

It is necessary to determine genetic diversity of fragmented populations in highly modified landscapes to understand how populations respond to land-use change. This information will help guide future conservation and management strategies. We conducted a population genetic study on an endemic Mexican Dusky Rattlesnake (Crotalus triseriatus) in a highly modified landscape near the Toluca metropolitan area, in order to provide crucial information for the conservation of this species. There was medium levels of genetic diversity, with a few alleles and genotypes. We identified three genetically differentiated clusters, likely as a result of different habitat cover type. We also found evidence of an ancestral genetic bottleneck and medium values of effective population size. Inbreeding coefficients were low and there was a moderate gene flow. Our results can be used as a basis for future research and C. triseriatus conservation efforts, particularly considering that the Trans-Mexican Volcanic Belt is heavily impacted by destructive land-use practices.

Cytokine patterns in paediatric patients presenting serious gastrointestinal and respiratory bacterial infections.

In the adaptive immune response, the types of cytokines produced define whether there is a cellular (T1) or a humoral (T2) response. Specifically, in the T1 response, interleukin 2 (IL-2), interferon γ (IFN-γ) and tumor necrosis factor ß (TNF-ß) are produced, whereas in the T2 response, IL-4, IL-5, IL- 6, IL-10 and IL-13 are primarily produced. Cytokines are primarily involved in the regulation of immune system cells. The aim of the present study was to evaluate the cytokine patterns (Type 1/Type 2) and TNF-α expression levels in children with severe gastrointestinal and respiratory bacterial infections. The enzyme-linked immunosorbent assay (ELISA) technique was used to identify the cytokines and the infectious agents. The results obtained demonstrated that, in general, children with bacterial infections experienced an increase in IL-2, IFN-γ and IL-4 concentrations and a decrease in TNF-α, IL-5 and IL-6 concentrations when compared to healthy children. Specifically, type 1 cytokines and an increased TNF-α concentration were found in children with gastrointestinal infections. However, patients with respiratory infections showed increased concentrations of both T2 (IL-4, IL-6 and IL-10) and T1 (IL-2 and IFN-γ) components. Thus, it was concluded that children with gastrointestinal infections exclusively developed a T1 response, whereas children with respiratory infections developed a T1/T2 response to fight the infection.