Human cerebral organoids: cellular composition and subcellular morphological features.

Introduction: Human cerebral organoids (hCOs) derived from pluripotent stem cells are very promising for the study of neurodevelopment and the investigation of the healthy or diseased brain. To help establish hCOs as a powerful research model, it is essential to perform the morphological characterization of their cellular components in depth. Methods: In this study, we analyzed the cell types consisting of hCOs after culturing for 45 days using immunofluorescence and reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) assays. We also analyzed their subcellular morphological characteristics by transmission electron microscopy (TEM). Results: Our results show the development of proliferative zones to be remarkably similar to those found in human brain development with cells having a polarized structure surrounding a central cavity with tight junctions and cilia. In addition, we describe the presence of immature and mature migrating neurons, astrocytes, oligodendrocyte precursor cells, and microglia-like cells. Discussion: The ultrastructural characterization presented in this study provides valuable information on the structural development and morphology of the hCO, and this information is of general interest for future research on the mechanisms that alter the cell structure or function of hCOs.

Efficient generation of human cerebral organoids directly from adherent cultures of pluripotent stem cells.

Human cerebral organoids (hCOs) offer the possibility of deepening the knowledge of human brain development, as well as the pathologies that affect it. The method developed here describes the efficient generation of hCOs by going directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures to three-dimensional (3D) neuroepithelial tissue, avoiding dissociation and aggregation steps. This has been achieved by subjecting 2D cultures, from the beginning of the neural induction step, to dual-SMAD inhibition in combination with CHIR99021. This is a simple and reproducible protocol in which the hCOs generated develop properly presenting proliferative ventricular zones (VZs) formed by neural precursor and radial glia (RG) that differentiate to give rise to mature neurons and glial cells. The hCOs present additional cell types such as oligodendrocyte precursors, astrocytes, microglia-like cells, and endothelial-like cells. This new approach could help to overcome some of the existing limitations in the field of organoid biotechnology, facilitating its execution in any laboratory setting.

Low Levels of Amyloid Precursor Protein (APP) Promote Neurogenesis and Decrease Gliogenesis in Human Neural Stem Cells.

Amyloid precursor protein (APP) has been widely studied due to its association with Alzheimer's disease (AD). However, the physiological functions of APP are still largely unexplored. APP is a transmembrane glycoprotein whose expression in humans is abundant in the central nervous system. Specifically, several studies have revealed the high expression of APP during brain development. Previous studies in our laboratory revealed that a transient increase in APP expression induces early cell cycle exit of human neural stem cells (hNSCs) and directs their differentiation towards glial cells (gliogenesis) while decreasing their differentiation towards neurons (neurogenesis). In the present study, we have evaluated the intrinsic cellular effects of APP down-expression (using siRNA) on cell death, cell proliferation, and cell fate specification of hNSCs. Our data indicate that APP silencing causes cellular effects opposite to those obtained in previous APP overexpression assays, inducing cell proliferation in hNS1 cells (a model line of hNSCs) and favoring neurogenesis instead of gliogenesis in these cells. In addition, we have analyzed the gene and protein expression levels of ß-Catenin as a possible molecule involved in these cellular effects. These data could help to understand the biological role of APP, which is necessary to deepen the knowledge of AD.

Amyloid Precursor Protein (APP) Regulates Gliogenesis and Neurogenesis of Human Neural Stem Cells by Several Signaling Pathways.

Numerous studies have focused on the pathophysiological role of amyloid precursor protein (APP) because the proteolytic processing of APP to ß-amyloid (Aß) peptide is a central event in Alzheimer's disease (AD). However, many authors consider that alterations in the physiological functions of APP are likely to play a key role in AD. Previous studies in our laboratory revealed that APP plays an important role in the differentiation of human neural stem cells (hNSCs), favoring glial differentiation (gliogenesis) and preventing their differentiation toward a neuronal phenotype (neurogenesis). In the present study, we have evaluated the effects of APP overexpression in hNSCs at a global gene level by a transcriptomic analysis using the massive RNA sequencing (RNA-seq) technology. Specifically, we have focused on differentially expressed genes that are related to neuronal and glial differentiation processes, as well as on groups of differentially expressed genes associated with different signaling pathways, in order to find a possible interaction between them and APP. Our data indicate a differential expression in genes related to Notch, Wnt, PI3K-AKT, and JAK-STAT signaling, among others. Knowledge of APP biological functions, as well as the possible signaling pathways that could be related to this protein, are essential to advance our understanding of AD.

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi.

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial resolution of a cell or tissue for microscopic imaging. This is performed by physically expanding a sample with off-the-shelf chemicals and common lab equipment. Chagas disease is a widespread and pressing public health concern caused by T. cruzi. The disease is prevalent in Latin America and has become a significant problem in non-endemic regions due to increased migration. The transmission of T. cruzi occurs through hematophagous insect vectors belonging to the Reduviidae and Hemiptera families. Following infection, T. cruzi amastigotes multiply within the mammalian host and differentiate into trypomastigotes, the non-replicative bloodstream form. In the insect vector, trypomastigotes transform into epimastigotes and proliferate through binary fission.The differentiation between the life cycle stages requires an extensive rearrangement of the cytoskeleton and can be recreated in the lab completely using different cell culture techniques. We describe here a detailed protocol for the application of U-ExM in three in vitro life cycle stages of Trypanosoma cruzi, focusing on optimization of the immunolocalization of cytoskeletal proteins. We also optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that has enabled us to mark different parasite structures.

Prevalence and Population Diversity of Listeria monocytogenes Isolated from Dairy Cattle Farms in the Cantabria Region of Spain.

Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. Here we show the prevalence and transmission of L. monocytogenes in dairy farms in the Cantabria region, on the northern coast of Spain. A total of 424 samples was collected from 14 dairy farms (5 organic and 9 conventional) and 211 L. monocytogenes isolates were recovered following conventional microbiological methods. There were no statistically significant differences in antimicrobial resistance ratios between organic and conventional farms. A clonal relationship among the isolates was assessed by pulsed field gel electrophoresis (PFGE) analysis and 64 different pulsotypes were obtained. Most isolates (89%, n = 187) were classified as PCR serogroup IVb by using a multiplex PCR assay. In this case, 45 isolates of PCR serogroup IVb were whole genome-sequenced to perform a further analysis at genomic level. In silico MLST analysis showed the presence of 12 sequence types (ST), of which ST1, ST54 and ST666 were the most common. Our data indicate that the environment of cattle farms retains a high incidence of L. monocytogenes, including subtypes involved in human listeriosis reports and outbreaks. This pathogen is shed in the feces and could easily colonize dairy products, as a result of fecal contamination. Effective herd and manure management are needed in order to prevent possible outbreaks.

The transcription factor LEF1 interacts with NFIX and switches isoforms during adult hippocampal neural stem cell quiescence.

Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical ß-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active ß-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence.

Metabolic Reprogramming Helps to Define Different Metastatic Tropisms in Colorectal Cancer.

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.

Ultrastructure Expansion Microscopy (U-ExM) in Trypanosoma cruzi: localization of tubulin isoforms and isotypes.

Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. For the first time, I report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as α-tubulin and ß-tubulin. Also, I immunolocalized acetylated and tyrosinated α-tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.

Neurogenesis Is Increased in Human Neural Stem Cells by Aß40 Peptide.

Amyloid-ß 40 peptides [Aß1-40 (Aß40)] are present within amyloid plaques in the brains of patients with Alzheimer's disease (AD). Even though Aß peptides are considered neurotoxic, they can mediate many biological processes, both in adult brains and throughout brain development. However, the physiological function of these Aß peptides remains poorly understood, and the existing data are sometimes controversial. Here, we analyze and compare the effects of monomeric Aß40 on the biology of differentiating human neural stem cells (human NSCs). For that purpose, we used a model of human NSCs called hNS1. Our data demonstrated that Aß40 at high concentrations provokes apoptotic cellular death and the damage of DNA in human NSCs while also increasing the proliferation and favors neurogenesis by raising the percentage of proliferating neuronal precursors. These effects can be mediated, at least in part, by ß-catenin. These results provide evidence of how Aß modulate/regulate human NSC proliferation and differentiation, suggesting Aß40 may be a pro-neurogenic factor. Our data could contribute to a better understanding of the molecular mechanisms involved in AD pathology and to the development of human NSC-based therapies for AD treatment, since these results could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.

Essential Bromodomain TcBDF2 as a Drug Target against Chagas Disease.

Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of TcBDF2 in T. cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results point to TcBDF2 as a druggable target against T. cruzi.

Evaluation of peri-implant soft and hard tissues behavior in screw-retained crowns by the biologically oriented preparation technique (BOPT): Ambispective longitudinal analytical study.

BACKGROUND: Clinical and radiographic evaluation of soft and hard tissues around convergent collar implants and shoulderless abutments. MATERIAL AND METHODS: Ambispective longitudinal analytical study with a sample size of 32 implants in 21 patients treated in a private dental clinic. Patients were divided into two groups: Prama Implants or group 1 (n=21) and Shelta implants combined with XA abutment or group 2 (n=11). Probing depth, horizontal mucosa thickness, peri-implant bone loss, plaque and bleeding after one-and two-year follow up are analyzed. RESULTS: In group 1, mean probing depth value was 1.67 mm (±0.58) and mean horizontal mucosa thickness value was 2.71 (±0.96). In group 2 mean probing depth was 2.18 (±0.40) and mean horizontal mucosa thickness value was 3.27 mm (±1.19). In group 1 an 85.7% of peri-implant bone level was maintained and a 14.3% increased. In group 2 a 100% of peri-implant bone level was maintained. In group 1 a 19% presented plaque when crowns were removed and in group 2 a 18.2% presented plaque. Neither of two groups presented spontaneous bleeding when crowns were removed. A 52.4% presented probing bleeding in group 1 and a 45.4% in group 2. CONCLUSIONS: Biologically guided crowns design seems to provide peri-implant hard and soft tissue stability. Key words:Biologic width, peri-implant soft tissue, marginal bone loss, transmucosal implant, convergent collar, BOPT (biological oriented preparation technique), BOPT abutment, soft tissue stability.

Update on the Biological Relevance of Lysine Acetylation as a Novel Drug Target in Trypanosomatids.

The number of acetylated proteins identified from bacteria to mammals has grown exponentially in the last ten years, and it is now accepted that acetylation is a key component in most eukaryotic signaling pathways and is as important as phosphorylation. The enzymes involved in this process are well described in mammals; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids, several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this review article, we present an update of the most important known acetylated proteins in trypanosomatids, analyzing the acetylomes available. Also, we summarize the information available regarding acetyltransferases and deacetylases in trypanosomes and their potential use as chemotherapeutic targets.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing ß-galactosidase.

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi. T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission. This paper describes an assay based on measuring the activity of the cytoplasmic ß-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red ß-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a ß-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

Oligomeric and Fibrillar Species of Aß42 Diversely Affect Human Neural Stem Cells.

Amyloid-ß 42 peptide (Aß1-42 (Aß42)) is well-known for its involvement in the development of Alzheimer's disease (AD). Aß42 accumulates and aggregates in fibers that precipitate in the form of plaques in the brain causing toxicity; however, like other forms of Aß peptide, the role of these peptides remains unclear. Here we analyze and compare the effects of oligomeric and fibrillary Aß42 peptide on the biology (cell death, proliferative rate, and cell fate specification) of differentiating human neural stem cells (hNS1 cell line). By using the hNS1 cells we found that, at high concentrations, oligomeric and fibrillary Aß42 peptides provoke apoptotic cellular death and damage of DNA in these cells, but Aß42 fibrils have the strongest effect. The data also show that both oligomeric and fibrillar Aß42 peptides decrease cellular proliferation but Aß42 oligomers have the greatest effect. Finally, both, oligomers and fibrils favor gliogenesis and neurogenesis in hNS1 cells, although, in this case, the effect is more prominent in oligomers. All together the findings of this study may contribute to a better understanding of the molecular mechanisms involved in the pathology of AD and to the development of human neural stem cell-based therapies for AD treatment.

Optimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi strain.

There is an urgent need to develop safer and more effective drugs for Chagas disease, as the current treatment relies on benznidazole (BZ) and nifurtimox (NFX). Using the Trypanosoma cruzi Dm28c strain genetically engineered to express the Escherichia coli ß-galactosidase gene, lacZ, we have adapted and validated an easy, quick and reliable in vitro assay suitable for high-throughput screening for candidate compounds with anti-T. cruzi activity. In vitro studies were conducted to determine trypomastigotes sensitivity to BZ and NFX from Dm28c/pLacZ strain by comparing the conventional labour-intensive microscopy counting method with the colourimetric assay. Drug concentrations producing the lysis of 50% of trypomastigotes (lytic concentration 50%) were 41.36 and 17.99 µM for BZ and NFX, respectively, when measured by microscopy and 44.74 and 38.94 µM, for the colourimetric method, respectively. The optimal conditions for the amastigote development inhibitory assay were established considering the parasite-host relationship (i.e. multiplicity of infection) and interaction time, the time for colourimetric readout and the incubation time with the ß-galactosidase substrate. The drug concentrations resulting in 50% amastigote development inhibition obtained with the colourimetric assay were 2.31 µM for BZ and 0.97 µM for NFX, similar to the reported values for the Dm28c wild strain (2.80 and 1.5 µM, respectively). In summary, a colourimetric assay using the Dm28c/pLacZ strain of T. cruzi has been set up, obtaining biologically meaningful sensibility values with the reference compounds on both trypomastigotes and amastigotes forms. This development could be applied to high-throughput screening programmes aiming to identify compounds with anti-T. cruzi in vitro activity.

Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression.

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

Soft tissue thickness evaluation in screw-retained crowns by the biologically oriented preparation technique (BOPT).

BACKGROUND: Intraoral scanner evaluation (3Shape, TRIOS®) of soft tissue thickness around convergent collar implants and shoulderless abutments. MATERIAL AND METHODS: Ambispective longitudinal analytical study with a sample size of 26 implants in 17 patients treated in a private dental clinic. Pacients were divided into two groups: Prama Implants or group 1 (n=19) and Shelta implants combined with XA abutment or group 2 (n=7). Thickness changes after one- and two-year follow-up were analyzed. RESULTS: In group 1 baseline mean thickness was 6.53 mm (±1.06) and follow-up mean thickness was 8.06 mm (±0.98), in group 2 initial mean thickness was 7.66 mm (±1.09) and follow-up mean thickness was of 8.42 mm (±1.03). CONCLUSIONS: Biologically guided crowns design seems to significantly increase the soft tissue volumen around convergent morphology implants. Key words:Biologic width, peri-implant soft tissue, marginal bone loss, transmucosal implant, convergent collar, BOPT (biological oriented preparation technique), BOPT abutment, soft tissue stability, intraoral scanner.

Effect directed synthesis of a new tyrosinase inhibitor with anti-browning activity.

The inhibition of enzymatic browning is an attractive target to elevate the quality of foods. The objective of this work is to describe a novel platform for the discovery of tyrosinase inhibitors, based on (a) one-pot preparation of a library of thiosemicarbazide compounds, (b) biological evaluation using tyrosinase TLC bioautography, (c) inhibitor identification via mass spectrometry coupled to bioautography. During these proof-of-concept experiments, the approach led to the straightforward identification of a new thiosemicarbazone with improved tyrosinase inhibition properties and fresh-cut apple slices antibrowning effect when compared to kojic acid. In conclusion, the platform represents an interesting strategy for the discovery of this type of inhibitors.

Analysis of Benzalkonium Chloride Resistance and Potential Virulence of Listeria monocytogenes Isolates Obtained from Different Stages of a Poultry Production Chain in Spain.

ABSTRACT: Listeria monocytogenes can survive in food production facilities and can be transmitted via contamination of food during the various stages of food production. This study was conducted to compile the results of three independent previous studies on the genetic diversity of L. monocytogenes in a poultry production company in Spain and to determine the potential virulence and sanitizer resistance of the strains by using both genotype and phenotype analyses. L. monocytogenes was detected at three production stages: a broiler abattoir, a processing plant, and retail stores marketing fresh poultry products from the same company. These three stages spanned three locations in three provinces of Spain. A set of 347 L. monocytogenes isolates representing 39 subtypes was obtained using pulsed-field gel electrophoresis (PFGE). A total of 28 subtypes (68%) had a full-length internalin A gene, and two subtypes had a phenotype with low potential for virulence because of a mutation in the prfA gene. A total of 32 subtypes (82%) were classified as benzalkonium chloride resistant (BAC-R) and contained the resistance determinant bcrABC (21 subtypes, 54%) or the resistance gene qacH (11 subtypes, 28%). A total of 13 persistent BAC-R subtypes (minimum of 3 months between the first and last sample from with the isolate was recovered) were identified at the abattoir and processing plant. The three production stages shared a unique subtype (PFGE type 1), which had the mutation in the prfA gene and the bcrABC resistance determinant. Whole genome sequencing revealed this subtype to be sequence type 31. Limited genetic diversity was noted in the isolates studied, including some subtypes that were persistent in the environment of the investigated facilities. Given the high prevalence of BAC-R subtypes, these results support the association between resistance to biocides and persistence of L. monocytogenes.