Alma L. Guerrero-Barrera

Waterborne pathogens: detection methods and challenges.

Waterborne pathogens and related diseases are a major public health concern worldwide, not only by the morbidity and mortality that they cause, but by the high cost that represents their prevention and treatment. These diseases are directly related to environmental deterioration and pollution. Despite the continued efforts to maintain water safety, waterborne outbreaks are still reported globally. Proper assessment of pathogens on water and water quality monitoring are key factors for decision-making regarding water distribution systems’ infrastructure, the choice of best water treatment and prevention waterborne outbreaks. Powerful, sensitive and reproducible diagnostic tools are developed to monitor pathogen contamination in water and be able to detect not only cultivable pathogens but also to detect the occurrence of viable but non-culturable microorganisms as well as the presence of pathogens on biofilms. Quantitative microbial risk assessment (QMRA) is a helpful tool to evaluate the scenarios for pathogen contamination that involve surveillance, detection methods, analysis and decision-making. This review aims to present a research outlook on waterborne outbreaks that have occurred in recent years. This review also focuses in the main molecular techniques for detection of waterborne pathogens and the use of QMRA approach to protect public health.

Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

BackgroundActinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae.ResultsFor the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure.ConclusionsIn conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.

Porcine Respiratory Disease Complex and Biofilms

The Porcine respiratory disease complex (PRDC) is a term used to describe polymicrobial respiratory infections in pigs. Respiratory diseases in pigs are common in modern pork production worldwide and are the responsible for major economic losses in the swine industry. Pathogens involved in respiratory disease in pigs vary significantly among farms, production sites, regions and countries, making generalizations about the PRDC treatment and difficult to control it. The interactions that occur on the cellular and molecular levels during concurrent infection of pigs with two or more respiratory pathogens are multi-faceted and convoluted. There are a variety of bacterial and viral pathogens commonly associated with the PRDC. The main associated bacteria include: Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus pasaruis, and Mycoplasma hyopneumoniae. Currently, it is known among microbiologists that biofilm formation is an universal attribute of microorganisms and the main way of life in nature that are causing problems such as developing diseases in animals and humans. Here, is reviewed the current knowledge of the major bacteria involved in this disease, their ability to form biofilms, as well as their importance on the infection process.

Amelogenin and enamelysin localization in human dental germs

Odontogenesis is extensively studied in animal models but less understood in human. In early amelogenesis, amelogenin constitutes 90% of enamel organic matrix, which is degraded by enamelysin and replaced by hydroxyapatite crystals. Here, amelogenin and enamelysin distribution changes during amelogenesis were shown by co-localization experiments by confocal microscopy. Early bell stage showed more amelogenin labeling than enamelysin, as free immune-reactive granular patches towards basal membrane between ameloblast and odontoblast. Increased amelogenin expression and secretion towards extracellular matrix formation region was found. Enamelysin distribution was perinuclear in early bell stage. During late bell stage, a decreasing amelogenin labeling in contrast with enamelysin increasing along the cells was found, suggesting specific temporal amelogenin degradation. Enamelysin was located initially around nuclei and later was found in all the ameloblast and stellate reticulum cytoplasm. Amelogenin was observed inside ameloblast, stellate reticulum, and intermediate stratum cells in the enamel as well as in the newly formed dentin extracellular matrix. In contrast, in dentin more amelogenin than enamelysin was found located close to the periphery.

Establishment and characterization of porcine aortic endothelial cell cultures with prolonged replicative lifespan by a non-enzymatic method

The aim of this work was the establishment and characterization of porcine aortic endothelial cell cultures with prolonged lifespan. Endothelial cells where isolated from porcine thoracic aorta and established in primary culture; after sub cultivation, the cells showed typical morphology of endothelial phenotype with cobblestone appearance and growth in monolayer; they were positive against anti-CD31 and anti-CD54 immunostaining and Ac-LDL-Dil uptake. The cells were able to migrate in culture and showed a normal growth curve. The phenotype of our in vitro model of endothelial cells was stable through subcultivation; so, it should be a valuable tool for diverse studies of the endothelial response against physiological and pathological stimulus, particularly it could be useful to study interactions of endothelium with pathogenic bacteria causing diseases in pigs, as they are difficult to study in vivo.

Incorporation of Actinobacillus pleuropneumoniae in Preformed Biofilms by Escherichia coli Isolated From Drinking Water of Swine Farms

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, represents one of the most important health problems in the swine industry worldwide and it is included in the porcine respiratory disease complex. One of the bacterial survival strategies is biofilm formation, which are bacterial communities embedded in an extracellular matrix that could be attached to a living or an inert surface. Until recently, A. pleuropneumoniae was considered to be an obligate pathogen. However, recent studies have shown that A. pleuropneumoniae is present in farm drinking water. In this study, the drinking water microbial communities of Aguascalientes (Mexico) swine farms were analyzed, where the most frequent isolated bacterium was Escherichia coli. Biofilm formation was tested in vitro; producing E. coli biofilms under optimal growth conditions; subsequently, A. pleuropneumoniae serotype 1 (strains 4074 and 719) was incorporated to these biofilms. Interaction between both bacteria was evidenced, producing an increase in biofilm formation. Extracellular matrix composition of two-species biofilms was also characterized using fluorescent markers and enzyme treatments. In conclusion, results confirm that A. pleuropneumoniae is capable of integrates into biofilms formed by environmental bacteria, indicative of a possible survival strategy in the environment and a mechanism for disease dispersion.

An evaluation of multidrug-resistant Escherichia coli isolates in urinary tract infections from Aguascalientes, Mexico: cross-sectional study

BackgroundUropathogenic Escherichia coli (UPEC) are one of the main bacteria causing urinary tract infections (UTIs). The rates of UPEC with high resistance towards antibiotics and multidrug-resistant bacteria have increased dramatically in recent years and could difficult the treatment.MethodsThe aim of the study was to determine multidrug-resistant bacteria, antibiotic resistance profile, virulence traits, and genetic background of 110 E. coli isolated from community (79 isolates) and hospital-acquired (31 isolates) urinary tract infections. The plasmid-mediated quinolone resistance genes presence was also investigated. A subset of 18 isolates with a quinolone-resistance phenotype was examined for common virulence genes encoded in diarrheagenic and extra-intestinal pathogenic E. coli by a specific E. coli microarray.ResultsFemale children were the group most affected by UTIs, which were mainly community-acquired. Resistance to trimethoprim–sulfamethoxazole, ampicillin, and ampicillin–sulbactam was most prevalent. A frequent occurrence of resistance toward ciprofloxacin (47.3%), levofloxacin (43.6%) and cephalosporins (27.6%) was observed. In addition, 63% of the strains were multidrug-resistant (MDR). Almost all the fluoroquinolone (FQ)-resistant strains showed MDR-phenotype. Isolates from male patients were associated to FQ-resistant and MDR-phenotype. Moreover, hospital-acquired infections were correlated to third generation cephalosporin and nitrofurantoin resistance and the presence of kpsMTII gene. Overall, fimH (71.8%) and fyuA (68.2%), had the highest prevalence as virulence genes among isolates. However, the profile of virulence genes displayed a great diversity, which included the presence of genes related to diarrheagenic E. coli. Out of 110 isolates, 25 isolates (22.7%) were positive to qnrA, 23 (20.9%) to qnrB, 7 (6.4%) to qnrS1, 7 (6.4%) to aac(6′)lb-cr, 5 (4.5%) to qnrD, and 1 (0.9%) to qnrC genes. A total of 12.7% of the isolates harbored blaCTX-M genes, with blaCTX-M-15 being the most prevalent.ConclusionsUrinary tract infection due to E. coli may be difficult to treat empirically due to high resistance to commonly used antibiotics. Continuous surveillance of multidrug resistant organisms and patterns of drug resistance are needed in order to prevent treatment failure and reduce selective pressure. These findings may help choosing more suitable treatments of UTI patients in this region of Mexico.

Expresión de Tuftelina en Gérmenes Dentales Humanos

La tuftelina es una proteina secretada en la matriz adamantina en desarrollo durante la formacion del esmalte. Su funcion continua sin esclarecerse, aunque se presume que juega un papel importante en la biomineralizacion de esmalte y dentina, asi como en el desarrollo del organo dental. Con el presente estudio se identifico su localizacion en las diferentes estructuras de germenes dentales de fetos humanos, conforme a los resultados se observo su expresion en el estadio pre-secretor observandose en el citoplasma de los ameloblastos, reticulo estrellado, papila dental, asi como en el estrato intermedio; en el secretor se identifico principalmente en la union amelodentinaria, y en la superficie externa del esmalte, observando una marcada expresion de la proteina en la porcion basal del proceso odontoblastico, pero no en la matriz extracelular de la dentina. De acuerdo a los resultados obtenidos se puede considerar que su expresion se presenta tanto en la amelogenesis, como en la odontogenesis en tejidos sin mineralizar.