Lactobacillus in cecal microbiota of chickens. Compared to the control group (CT group), the relative abundance of genus of Alistipes, unclassified Rikenellaceae, Roseburia, and Anaeroplasma was enriched in the PEO group; that of genus Bacteroides, Lachnospiraceae, and unclassified Enterobacteriaceae was enriched in the cecal microbiota of the VIRG group. Untargeted metabolomics analyses revealed that the PEO treatment modified 102 metabolites and 3 KEGG pathways  in the cecal microbiota, and 81 metabolites and relevant KEGG pathways  in the serum of the chicken. Compared to the CT group, VIRG treatment group differed 217 metabolites and 10 KEGG pathways in cecal contents and 142 metabolites and 7 KEGG pathways in serum of chickens. Pearson’s correlation analysis showed that phyla Bacteroidetes and genus of Bacteroides, Alistipes, and unclassified Rikenellaceae (in the VIRG and PE group) were positively correlated with many lipid metabolites. However, phyla Firmicutes and genera Lactobacillus (higher in the CT group) were negatively correlated with the lipid and thymine metabolism, and positively correlated with hydroxyisocaproic acid, cytosine, and taurine. This study shows that dietary supplementation with PEO and VIRG altered the composition and metabolism profile of the cecal microbiota, modified the serum metabolism profile.This study investigated the cecal microbiota and serum metabolite profile of chickens fed with plant essential oils (PEO) or virginiamycin (VIRG) using high-throughput 16S rRNA gene sequencing and untargeted metabolomics approach. The main aim of this work was to explore the biochemical mechanisms involved in the improved growth performance of antibiotics and their alternatives in animal production. The results showed that both PEO and VIRG treatment significantly increased the relative abundance of phyla Bacteroidetes and decreased the abundance of phyla Firmicutes and genus of  Many studies have demonstrated that intestinal microbiota participates in many metabolic pathways, such as lipid metabolism and amino acid synthesis5. The mechanism by which PEO promote growth of may be alter gut microflora and hence improved absorption of nutrients6, increase absorption of micronutrients in the small intestine7 and reducing the deleterious effects of the microbial metabolites10. There are many alternatives to antibiotics, such as acidifiers, probiotics, oligosaccharides, and plant extracts, which play a growth promoting role by regulating gut microbes in pig and poultry11.The intestinal microbiota, the population of microorganisms that inhabit the intestine, plays an important role in the intestinal morphology, immunity, nutrient digestion and absorption, and host health13, serve as alternatives for antibiotics used in feed for their safety and limited residual effects15. Many studies have shown that PEO can decrease the number of E. coli and increase the amount of Lactobacilli in the intestine of broilers18. This has beneficial effects on the intestinal morphology and barrier, and the antioxidant in animals19. Also, Altop et al. (2018) reported the interactions of host gut–microbiota and co-metabolism on chickens that were fed with essential oils20. However, only a few studies have investigated the effects of PEO or antibiotics on the cecum microbiota and serum metabolites profile of the chicken.Plant essential oils (PEO), which can be extracted from plants by steam distillation, extrusion, or solvent extractionIn this study, the cecal microbial composition and metabolites in the cecum and serum of chickens that were fed with virginiaymicin (VIRG) or PEO were investigated through 16S rRNA gene sequencing and untargeted mass spectrometry (UPLC-Q-TOF/MS) metabolomic analysis. Comparing the differences in the cecal microbial composition and the potential chemical metabolites might help us understand how the dietary antibiotics or PEO alter the intestinal metabolism and health of the broiler chickens.High-throughput sequencing obtained 176,636 quality-controlled reads from 18 cecum samples from three treatments . After denoising, removing chimeras, and filtering low quality sequences, the sample had an average of 44,120 sequences, the average length of each sequence being 443 nucleotides. Based on the 97% identity level, these sequences were decomposed into 1,971 operational taxonomic units (OTUs). The 16S rRNA gene amplicon sequencing results were deposited in the Sequence Read Archive of the NCBI (accession number PRJNA553851).The diversity of the cecal microbiota in the three groups are shown in Fig. Lactobacillus, Faecalibacterium, Bacteroidaceae, and unclassified Rikenellaceae were the predominant genus in the cecal microbiota of the chicken. The relative abundance of Lactobacillus was lower, and that of unclassified Rikenellaceae was higher in the cecal microtia of the PEO group. The genus Bacteroides was higher in the VIRG group than in the CT group.Dietary treatment with PEO or VIRG Fig.  resultedAlistipes, unclassified Rikenellaceae, Anaeroplasma, unclassified Bacillaceae, and Roseburia were higher in the cecal microbiota of the PE group  value > 1, in 95% jack-knifed confidence intervals and P < 0.05, detailed information about the different biomarker metabolites has been shown in Supplementary Table Next, these different metabolites were conclusively identified using the in-house databases. Figure Compared to the CT group, dietary supplement with virginiamycin increased the levels of (S)-Equol, LysoPE (16:0/0:0), Caproic acid, D-Maltose, and ribitol in the cecum of the chicken. However, 9 other metabolites including nucleotides , Myristoleic acid, and Pyridoxal (Vitamin B6) decreased Fig. . About 1Rikenellaceae positively correlated with 1-Oleoyl-sn-glycero-3-phosphocholine, adenine, cytidine, and undecanoic acid in cecum  at the genus and phylum level in the cecal microbiota were found to be closely associated to the concentration of specific microbial metabolites in the cecum and serum of chickens Fig. . The higcum Fig.  and negacum Fig. . The genBacteroides positively correlated with LysoPE(16:0/0:0), salicylic acid, and tamsulosin in the cecum and betaine, taurochenodeoxycholate, and dopamine in the serum while negatively correlating with hydroxyisocaproic acid, cytosine, taurine, and Pyridoxal (Vitamin B6) in the cecum and taurine in the serum. The relative abundance of phylum Firmicutes positively correlated with hydroxyisocaproic acid, cytosine, taurine, and Pyridoxal (Vitamin B6) in the cecum and taurine in the serum, while negatively correlating with LysoPE(16:0/0:0) and (S)-Equol in the cecum and SOPC, betaine, and dopamine in the serum. The relative abundance of genera Lactobacillus positively correlated with hydroxyisocaproic acid and cytosine in the cecum and taurine, dodecanoic acid, citrate, and nicotinamide in the serum, while negatively correlating with LysoPE(16:0/0:0), (S)-Equol, and caprylic acid in the cecum and alpha-linolenic acid, cis--linolenic acid, linoleic acid, taurochenodeoxycholate, 13(S)-HODE, and dopamine in the serum.The high proportion of bacteria in the cecal microbiota of the VIRG group positively correlated with ribitol and (S)-Equol in the cecum and oleic acid, SOPC, and adynerin in the serum, and negatively correlated with cytidine, hydroxyisocaproic acid, and Pyridoxal (Vitamin B6) in the cecum. Additionally, the relative abundance of phylum Bacteroidetes and genera Based on these metabolites , the software Metaboanalyst 3.0 was used to enrich the relevant KEGG pathways. The relevant KEGG pathways enriched by metabolites between PEO within the CT group are shown in Table The relevant KEGG pathways enriched by metabolites between VIRG within the CT group are shown in Table 21. Our previous study has shown that dietary supplementation with PEO improved the growth and feed efficiency in broiler chickens22, which was consistent with other studies 24. PEO might promote growth by altering gut microflora and hence improving absorption of nutrients6, increasing the absorption of micronutrients in the small intestine7, stimulating the production and activity of digestive enzymes (such as trypsin and amylase)26, and stimulating digestive and physiology metabolism27.Since the ban on antibiotic growth-promotion (AGP) in many countries, several effective alternatives to AGPs have been developed in recent years. One of them, PEO or phytogenic additives are considered as appropriate candidates due to their safety benefitsLactobacillus and streptococcus in the cecal microbiota of chickens. Meanwhile, the relative abundance of phyla Bacteroidetes and genera Alistipes, Roseburia, and unclassified Rikenellaceae increased in the cecal microbiota of the PEO group. These finding were consistent with previous studies that reported that PEO decreased the number of Lactobacilli29. Next, some researchers reported that there was a significant increase in the Lactobacillus in the intestines of chickens fed with PEO or Macleaya cordata extracts18. These differences may be due to the difference in the source and breed of PEOs, feeding patterns, and sampling parts of the chicken14.This study shows that both PEO and VIRG treatments increased the relative abundance of phyla Bacteroidetes and decreased the relative abundance of phyla Firmicutes and genera of 2. Phyla Bacteroidetes are gram-negative bacteria that ferment polysaccharides and other indigestible carbohydrates and produce short-chain fatty acids (SCFAs) that are gut-friendly. Bacteroidetes are related to fat accumulation in chickens30. Alistipes and unclassified Rikenellaceae, which belong to phylum Bacteroidales, are generally considered beneficial to the host gut31. Alistipes can produce succinic acid and other long-chain fatty acids such as C1532. Rikenellaceae can produce propionic and succinic acids by fermentation of glucose, lactose, mannose, and melibiose, and formed the iso-methyl branched-chain fatty acid or long chain saturated acids33. Genus Roseburia belongs to the Lachnospiraceae family, a butyrate-producing organism, with a high capacity to form conjugated linoleic acid from linoleic acid34. Our Pearson’s correlation analyses revealed that phylum Bacteroidetes were positively correlated with LysoPE (16:0/0:0) and (s)-equol in the cecum and betaine, taurochenodeoxycholate, and dopamine in the serum, while negatively correlating with hydroxyisocaproic acid, cytosine, and taurine in the cecum and taurine in the serum. Moreover, unclassified Rikenellaceae and Alistipes were positively correlated with 1-Palmitoylglycerol, myristoleic acid, and Pyridoxal (Vitamin B6) in the cecum and D-Mannose in the serum. These metabolites participated in many biological functions. e.g. LysoPE (16:0/0:0), a lysophospholipid, that serves important signaling functions. Equol may enhance the actions of soy isoflavones, due to its greater affinity for estrogen receptors and have unique antiandrogenic and antioxidant activities5. Dopamine is a major transmitter in the extrapyramidal system of the brain and is important in regulating movement. A family of receptors (dopamine receptors) mediates action, which plays a major role in the reward-motivated behaviour35. The enrichment of the relative abundance of Phyla Bacteroidetes and genera Alistipes, unclassified Rikenellaceae might be related with biosynthesis of fatty acid and lipid metabolism. Recently, some researchers have reported that the increase in the proportion of Phyla Bacteroidetes is related to promoting animal growth performance. On the contrary, Although Lactobacillus is considered to be a beneficial probiotic to the host intestinal health and growth36, our results showed that the proportion of Lactobacillus in the cecum negatively correlated with lipid metabolites, such as LysoPE (16:0/0:0) and (S)-Equol in the cecum and alpha-linolenic acid, all cis--linolenic acid, linoleic acid, taurochenodeoxycholate, and dopamine in the serum; and positively correlated with some metabolites which accelerated lipid peroxidation (such as hydroxyisocaproic acid and cytosine), down-regulated KEGG pathway related to primary bile acid biosynthesis and citrate cycle (TCA cycle). These results were consistent with De Boever et al.37, who reported that Lactobacillus impaired lipid absorption and consequently resulted in the dietary energy losses37. Recently, some reports showed that some Lactobacillus strains had a negative influence on the growth performance38, some strains of Lactobacillus are retailed as weight loss probiotics while others are reported with the ability to reduce obesity for increasing appetite and feed consumption40. Therefore, alteration of intestinal flora may result to improve of animal performance.The intestinal microbiota plays multiple roles in the intestinal morphology, immunity, nutrient absorption and metabolism, and host health41. In the blood of chickens of PEO group, six lipid metabolites (such as LysoPC (18:1(9Z)) and LysoPE (16:0/0:0)) enriched fatty acid biosynthesus, Lyso PCs perform essential functions in the lipid metabolism of organisms42; 1-Palmitoylglycerol is the biosynthetic precursor of phosphatidic acid: the major component of lysophosphatidic acid, which is a pluripotent lipid mediator which controls growth, motility, and differentiation43. In addition, D-Mannose up-regulated KEGG pathway of fructose and mannose metabolism and ABC transporters in serum of the PEO group. ABC transporters utilize the energy of ATP binding and hydrolysis to transport various substrates across cellular membranes44. Above all, dietary supplementation with PEO alters the cecal and blood metabolic pathways, The biosynthesis pathways of fatty acids and unsaturated fatty acids that are closely related to host lipid metabolism might be an important mechanism for growth promoters of PEO13.Metabolomic analysis revealed that chenodeoxycholate and glycocholic acid up-regulated the KEGG pathway of primary bile acid biosynthesis in the cecal contents of PEO group, Primary bile acids are also critical to the digestion and absorption of fat46. Recently, extensive research on the effects of antibiotics on intestinal biochemicals has been carried out using culture-independent methods and metabolomic analysis47. Many reports showed that the relative abundance of phyla Bacteroidetes increased and that of genus Lactobacillus decreased in the intestines of chickens after the administration of antibiotic50, Higher numbers of lactobacilli were previously implicated in broiler growth depression due to competition in nutrient uptake or impaired fat absorption48. Some other studies showed that proportion of phyla Firmicutes and genus of Lactobacillus increased in the gut of the chicken after administration of antibiotics53. Metabolomic analysis showed that 4-EOTC treatment improved the synthesis of lysophosphatidylcholine (LysoPC), as indicated by the lipid biomarkers LysoPC (16:0), LysoPC (18:3), LysoPC (20:3), and LysoPC (20:4) in the blood of Wistar rats. VIRG treatment altered 218 biochemicals  in the ileum of chickens, including many long chain saturated and polyunsaturated fatty acids; several lysophospholipids also increased in the process10. This observation was corroborated by our data that VIRG treatment increased the relative content of phyla Bacteroidetes and decreased that of genus Lactobacillus in the cecum, enriched lipid metabolites and biosynthesis of fatty acid and linoleic acid, and reduced some metabolites (such as urea) that may be toxic or harmful to growth of host.The mechanism of improving performance of AGPs is related to reduced incidences of subclinical infections, stability of the microbial ecology, thinning of the intestinal wall, suppression of inflammation, and the reduction of bioamines and toxins produced by the bacteria54. Arachidonic acid is catalyzed by cyclooxygenase (COX) to generate prostaglandins (PGs), which regulate fever, inflammation, smooth muscle contraction, and translocation of water and salt in the kidney56. In addition, PEO treatment increases the biosystem of Pyridoxal (VB6) and Pantothenate in the cecal contents. Pyridoxal participates protein and sugar metabolism57, and the active form of VB6 is linked with adipogenesis58. These results indicate that PEO might alter different lipid metabolites and vitamins from AGP.Compared to the VIRG group, PEO group also increased the content of D-Mannose and arachidonic acid, docosapentaenoic acid, linoleic acid in the cecum of chickens. These metabolites are essential fatty acids with several biological functions: as the precursor that are metabolized by various enzymes to a wide range of bioactive components for growth and proper healthIn summary, this study investigated the microbial profile and metabolites in the cecum and serum of chickens fed with PEO or VIRG by combined microbiome and metabolomic analysis. Our results demonstrated that PEO treatment increased the relative abundance of phyla Bacteroidetes and genera Alistipes, unclassified Rikenellaceae and decreased that of phyla Firmicutes and genus of Lactobacillus in the cecum. Many Lipid metabolites and KEGG pathway of fatty acid biosynthesis were enriched in cecum and serum of chickens in the PEO and VIRG group. These finding provide better understanding of the mechanism of promoting performance of PEO or VIRG, which could further provide useful information for developing an effective and safe alternative to AGP in poultry industry.PEO product purchased from Guangdong Ruisheng Technology Co., Ltd , which contained 65% silicon dioxide and 30% plant essential oils. The composition of plant essential oil was analysis by GC/MS , the relative content of the active components were 78.3% Cinnamic dehyde (RT = 33.6), 4% Isophorone (RT = 22.9), and eugenol (2.7%)  were randomly divided into three groups, and each group was replicated six times with 12 chickens in each replicate. Each group was fed with: (1) a basal diet (CT), without any additives; (2) a basal diet with 400 mg/kg (PEO); (3) a basal diet with 30 mg/kg virginiamycin (VIRG). Virginiamycin purchased from Phibro Animal Health Corporation (USA). The corn-soybean-based basal diet was used, and the nutritional level of the diet was prepared following the NRC (1994) feeding standards. All experiments were performed in accordance with the approved guidelines and regulations. The diet composition and nutrient levels are shown in Table 4 were raChickens were reared in a coop (100 cm × 80 cm) with two water nipples per pen. Chickens were raised routinely and immunized according to the normal immunization procedures. The diet was available ad libitum throughout the trial period. The temperature of the house was maintained at the optimum temperature of broiler chickens at 33–34 °C for the first week, and decreased from the second week by 3 °C per week until it reached 22 °C. All experimental protocols were approved by the China Agricultural University Animal Care Committee (permit number SYXK20171208).At 28 days of age, six chickens per treatment group (one chicken per repeat) were randomly selected to collected blood from the left brachial vein. After blood was collected, the chicken was injected intravenously pentobarbital sodium (30 mg/kg body weight) and cervical dislocation was executed about 2 g of cecal digestive fluid was collected and placed in two sterilizing tubes, immediately frozen in liquid nitrogen and stored at −80 °C. One sample was used for DNA extraction and Pyrosequencing, and second one for global metabolomic analysis. The blood samples were centrifuged for 15 min . serum sample was stored at −80 °C for UPLC-Q-TOF/MS analysis.Total bacterial genomic DNA was extracted from each digested sample using the Fast DNA SPIN extraction kits . DNA extracts were stored at −20 °C prior to further analysis. Extracted DNAs were measured using a NanoDrop ND-1000 spectrophotometer  and agarose gel electrophoresis.59. The V3-V4 region of 16S rRNA genes was amplified using the 338F-806R primer set (338F: 55′-ACTCCTACGGGAGGCAGCA-3′. 806R:5′-GGACTACHVGGGTWTCTAAT-3′). The sample specificity 7-bp barcode was added to the primer for multiple sequencing. Thermal cycle included initial denaturation of 98 °C for 2 min, followed by 25 cycles consisting of denaturation of 98 °C for 15 s, annealing at 55 °C for 30 s, extension of 72 °C for 30 s, and final extension of 72 °C for 5 min. PCR amplifiers were purified using Agencourt AMPure Beads  and quantified using PicoGreen dsDNA Assay Kit 59. After the individual sample was quantified, the amplicon were collected in equal amount, and pair-end 2×300 bp sequencing was performed using the Illlumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd 5.PCR amplification was performed according to the method of Jinfeng Songet al. (2010)60. Briefly, raw sequencing reads with complete barcode matches were assigned to the appropriate sample and identified as valid. Sequences < 150 bp long, with average Phred scores < 20, containing ambiguous bases, or with mononucleotide repeats longer than 8 bp were considered low-quality and were excluded from further analysis61. Paired-end reads were assembled using FLASH62. After chimera detection, UCLUST63 was used to group the remaining high-quality sequences into OTUs based on a minimum sequence identity of 97%. A representative sequence was identified for each OTU using default parameters. The OTUs were taxonomically classified using BLAST; each representative sequence was searched against the Greengenes Database64 and the best hit was selected65. An OTU table was used to record the abundance and taxonomic affiliation membership of every OTU in each sample. OTUs representing less than 0.001% of all of the sequences across all of the samples were ignored. To maintain a constant sequencing depth across all of the samples, an averaged, rounded, rarefied OTU table was generated by re-sampling an average of 100 OTU subsets at a minimum sequencing depth of 90%. This averaged OTU table was used for all of the subsequent analyses. Bioinformatics analyses of the sequence data were primarily performed in QIIME and R. v3.2.0. Raw reads were denoised and then cleaned to remove chimeras and low-quality sequences. OTU-level alpha diversity indices, such as Shannon diversity index, Chao1 richness estimate, and observed species richness were calculated based on the OTU table in QIIME, and OTU-level rank abundance curves were generated to compare the evenness and richness of OTUs among samples. Principal component analysis (PCA) was performed at the genus level13. LEfSe  was performed to detect differential abundant taxa across groups using the default parameters66. Operational taxonomic units were clustered with a 97% similarity threshold. Alpha diversity analysis included Shannon diversity index, Chao1 richness estimate, and observed species richness. Principal component analysis (PCA) was performed at the genus level13. LEfSe  was performed to detect differential abundant taxa across groups using the default parameters66.The sequencing data were processed using the Quantitative Insights Into Microbial Ecology  pipeline, following Caporaso 67, the samples were re-dissolved in 100 μL of acetonitrile/water . The same procedure was conducted using 100 mg sample from frozen cecal contents. One milliliter of cold methanol/acetonitrile  was added to each sample and mixed. The mixture was separated by centrifugation for 15 min . The supernatant was dried under vacuum centrifuge. Through the vortex dissolved in 100 μL acetonitrile and water , and under the 49 °C to 12,000 g centrifugal for 15 minutes. Monitor the stability and repeatability of the instrument analysis, quality control (QC) samples from the pool by 10 μL per sample and analysis along with other samples. Every 6 samples were periodically inserted into QC samples for analysis68.The serum (100 μL) was added to 400 μL of ice-chilled methanol/acetonitrile  and centrifuged for 20 min  to remove the protein. The supernatant was dried in a vacuum centrifuge. For LC-MS analysis69. In the ESI positive and negative mode, the mobile phase contained A = 25 mM ammonium acetate and 25 mM aqueous ammonium hydroxide, B = acetonitrile. The gradient was 85% B for 1 minute, followed by a linear drop to 65% in 11 minutes, then a drop to 40% in 0.1 minutes for 4 minutes, and ultimately increased to 85% in 0.1 minutes for 5 minutes with a rebalancing period.Determination of cecal contents and serum by using a UHPLC  coupled to a quadrupole time-of-flight (AB Sciex TripleTOF 6600) in Shanghai Applied Protein Technology Co., Ltd. According to Wenqiang Fan’s method, the HILIC separation was carried out samples were analyzed by using a 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 µm column 70. The production scan is acquired using information dependent acquisition (IDA) with the selection of high sensitivity mode. The parameters were set as follows71: the collision energy (CE) was fixed at 35 V with ±15 eV; declustering potential (DP), 60 V(+) and −60V(−); exclude isotopes within 4 Da; and 10 candidate ions to monitor per cycle.ESI conditions were set as follows: the ion source Gas1 (Gas1) was 60, the ion source Gas2 (Gas2) was 60, the curtain gas (CUR) was 30, the source temperature was 600 °C, and the ion spray voltage fluctuation (ISVF) was ±5500 V. In the MS only acquisition, the instrument was set to collect data in the range of 60–1000 Da, and the TOF MS scanning accumulation time was set to 0.20 s/spectrum. In the automatic MS/MS acquisition, the instrument was set at the range of m/z and above 25–1000 Da, and the production scanning accumulation time was set at 0.05 s/spectrum67. For peak selection, the following parameters were used: centWave m/z= 25 ppm, peak width = c , prefilter = c . For peak grouping, bw = 5, mzwid = 0.025, minfrac = 0.5 were used71. CAMERA (Collection of Algorithms of MEtabolite pRofile Annotation) was used for annotation of isotopes and admixtures. In the extracted ion features, only more than 50% of the variables of at least one set of non-zero measurements were retained. The variation coefficient (CV) of metabolites in the QC samples was set at a threshold value of 30%, which was used as the standard for evaluating the repeatability of the metabolomics data set. Compound identification of metabolites was performed by comparing the accuracy of m/z value (<25 ppm), and MS/MS spectra with an in-house database established using reliable standards72.The raw MS data (wiff. scan files) were converted to MzXML files using ProteoWizard MSConvert before importing it into freely available XCMS software71. The importance of each variable in the OPLS-DA model in the projection (VIP) value was calculated to indicate its contribution to the classification. Metabolites with the VIP score > 1 were further applied to Student’s t-test at univariate level to measure the significance of each metabolite, the P values less than 0.05 were considered as statistically significant. The correlative analysis between different cecal microbial species and microbial or serum metabolites was used for calculating by Spearman’s correlations coefficient13.After normalizing the processed data to the total peak intensity, it was uploaded before importing into SIMCA-P , for multivariate data analysis, including pareto scale principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA)Supplementary Table 1.Supplementary Table 2.


In this study, we developed a perfusable microfluidic platform suitable for robust and reproducible 3D cell spheroid formation and tissue maturation. The geometry of the device was optimized through computational fluid dynamic (CFD) simulations to improve cell trapping. Experimental data were used in turn to generate a model able to predict the number of trapped cells as a function of cell concentration, flow rate, and seeding time. We demonstrated that tuning non-geometrical parameters it is possible to control the size and shape of 3D cell spheroids generated using articular chondrocytes (ACs) as cellular model. After seeding, cells were cultured under perfusion at different flow rates , which induced the formation of conical and spherical spheroids. Wall shear stress values on cell spheroids, computed by CFD simulations, increased accordingly to the flow rate while remaining under the chondroprotective threshold in all configurations. The effect of flow rate on cell number, metabolic activity, and tissue-specific matrix deposition was evaluated and correlated with fluid velocity and shear stress distribution. The obtained results demonstrated that our device represents a helpful tool to generate stable 3D cell spheroids which can find application both to develop advanced in vitro models for the study of physio-pathological tissue maturation mechanisms and to obtain building blocks for the biofabrication of macrotissues.Three-dimensional (3D) cell spheroids are being increasingly applied in many research fields due to their enhanced biological functions as compared to conventional two-dimensional (2D) cultures. 3D cell spheroids can replicate tissue functions, which enables their use both as  In part studies . Moreoveprinting . In thisTraditional methods for 3D cell spheroid formation include the culture on non-adhesive substrates, the use of rotating vessel bioreactors, the hanging-drop method, and the centrifugation in conical tubes. However, all these approaches are characterized by a limited control over the size and geometry of 3D cell spheroids. In the last years, several microwell platforms have been developed by microfabrication technologies to overcome this limitation , findingCompared to static microwell systems, microfluidics offers the advantage to modulate additional parameters, such as flow rate and shear stress. The effect of these parameters is strictly dependent on the chip design. For example, it has been shown that the presence of microgrooves within microchannel strongly influences the fluid dynamic environment. Moreover, the modulation of microgrooves geometry (width and height) determines microcirculation areas and microscale shear stresses, in turn affecting cell trapping . Howeverin vivo  modeling is a powerful tool that is being applied to assist microfluidic platforms design, allowing to unravel the factors determining specific hydrodynamic patterns, and study the influence of fluid dynamics on cell behavior . Interesin vivo . Based oThe aim of this study was to control cell trapping and 3D cell spheroid formation by tuning non-geometrical parameters within a perfused microfluidic environment through a computational–experimental approach. Here, articular chondrocytes (ACs) were used since chondrocytes are known as 3D spheroid-forming cells and as responsive to biophysical cues. CFD modeling was exploited to optimize chip geometry, while cell concentration, flow rate, and seeding time were modulated to control and generate a predictive model of cell trapping. CFD modeling was subsequently used to study the influence of fluid flow and shear stress on 3D cell spheroid formation and tissue maturation.In this study, we evaluated the influence of multiple non-geometrical parameters, such as cell concentration, flow rate, and seeding time on cell trapping in a concave microfluidic chamber. Computational modeling was used to predict cell trapping and correlate the formation and biological response of 3D spheroids of human ACs with the fluid dynamic features associated to different flow rates.A microfluidic concave chamber was conceived, resembling the conical end-tip of a standard 1.5 ml tube routinely used to generate 3D cell spheroids. Computational modeling was used to optimize its geometry. At first, a preliminary chamber design was realized with a desktop CNC milling machine  through a carbide round mill (Ø 400 μm). The chamber surface was characterized by the presence of micro-burrs. Images of the chamber were acquired using an IX71 inverted microscope (Olympus) and analyzed to reproduce the actual geometry in the numerical model. CFD modeling was exploited to verify whether a simplified (smooth) geometry of the chamber could be adopted in the simulations to replace the actual design. Chamber geometries were discretized by GAMBIT (Ansys Inc.) adopting a tetrahedral meshing scheme with about 2,000,000 elements. The steady-state Navier–Stokes equations for an incompressible fluid were then solved using the finite volume code ANSYS FLUENT (Ansys Inc.). In all the simulations, the following conditions were applied: no-slip boundary at walls, zero pressure at the outlet, and flat average velocity profiles at the inlet port. The velocity fields calculated by CFD simulations in both configurations were compared at different depths. The microfluidic chip geometry was then analyzed by CFD modeling to verify whether the inclusion of an additional groove between the microchannels and the seeding chamber could be a suitable option to reduce the flow velocity in proximity of the chamber, hence favoring cell trapping by sedimentation.Based on the results of the CFD simulations, the microfluidic device was designed to include a channel allowing for cell loading and medium perfusion, a groove to reduce the velocity of flowing cells, and a concave chamber for cell trapping and 3D spheroid culture.The microfabrication consisted in the manufacturing of two layers obtained by replica molding of poly(dimethylsiloxane) (PDMS) and assembled together to obtain a continuous fluidic domain . The top4 cells/cm2 in complete medium (CM) added with 5 ng/ml FGF-2 and 1 ng/ml TGF-β1  undergoing routine arthroplasty surgical procedures, with patients’ informed consent. ACs were isolated by macroscopically non-fibrillated regions of cartilage by enzymatic digestion of minced tissue. After digestion, cells were plated at 10l TGF-β1 . After 2Before use, microfluidic devices were cleaned by flowing 70% ethanol at 10 μl/min for 3 min and rinsing with phosphate buffer solution (PBS), and finally filled with CM. Cells were seeded using a syringe pump . Different combinations of cell concentrations , flow rates , and seeding times  were tested as non-geometrical parameters able to modulate cell docking.A statistical software  was used to analyze the interactions between the analyzed non-geometrical parameters and to predict their effect on cell trapping. The number of trapped cells represented the output, whereas cell concentration, flow rate, and seeding time represented the input variables. All the experimental conditions and the relative number of trapped cells were included in the model. Experiments in an additional condition  were performed to test the model using the simulation tool provided by JMP with the following specifications: random error, five repetitions. The interaction plot and the correlation plot were drawn using the software to evaluate the interaction between cell concentration and flow rate or seeding time and to compare experimental and predicted results.2 and perfused with serum-free chondrogenic medium  or a solid sphere (Ø 1 mm).Computational models were gleaned on the different aggregate geometries generated upon the tested flow conditions. Specifically, three models were developed: a filled chamber, a disk-shaped aggregate, and a spherical aggregate. The filled chamber model was generated creating a 3D surface above the concave chamber. The other models were developed by designing solid bodies inside the chamber: a disk (Øex 485 nm–λem 535 nm) using a Victor X3 Plate Reader (Perkin Elmer).Immediately after seeding, the top layer of the chip was punched (Ø 2 mm) and cells trapped in the concave chamber were retrieved using a micropipette. Similarly, cell aggregates were retrieved from the microfluidic devices after 14 days of culture and digested with papain . DNA quantification was performed by CyQUANT kit, according to the manufacturer’s instructions. Fluorescence was measured (λTM cell viability assay (Life Technologies). Briefly, cell spheroids were transferred to a 96-well plate and incubated with 10% Alamar BlueTM in HG-DMEM . After incubation, the supernatant was harvested and fluorescence was measured (λex 540 nm–λem 580 nm) using a Victor X3 Plate Reader. Fluorescence values were then normalized on the DNA content to obtain the metabolic activity per cell, defined as normalized metabolic activity.Cell metabolic activity was assessed through the Alamar BlueAfter papain digestion, GAG content of cell aggregates was measured by dimethylmethylene blue assay  with chondroitin sulfate as standard. Absorbance was read (λ 525 nm) using a Victor X3 Plate Reader.Cell aggregates were fixed in 4% neutral buffered formalin, dehydrated in a graded series of alcohols, individually embedded in paraffin, and sectioned at 4 μm. Sections were stained with hematoxylin and eosin to visualize cell and extracellular matrix distribution and with alcian blue to detect GAG deposition. Images were acquired using an Olympus IX71 microscope.n ≥ 3). Data were analyzed using GraphPad Prism. Data distribution was evaluated by means of D’Agostino and Pearson normality test. Comparison between two groups was performed by t-test for parametric or non-parametric data, depending on data normality. Comparison of multiple groups was performed by univariate analysis of variance (ANOVA) followed by Bonferroni’s or Dunnet’s post hoc tests for parametric and non-parametric data, respectively. Differences were considered significant when p < 0.05.Data are presented as mean + standard deviation (SD), except where otherwise noted. Each experimental condition was repeated at least three times  changed from 46.34 μm/s without groove to 38.17 μm/s with groove (−18%). The average velocity decreased also at z = 200 μm from 35.37 μm/s without groove to 33.87 μm/s with groove (−4.2%). Based on the results of CFD optimization, the microfluidic chip was fabricated including the upstream and downstream groove  were applied for 14 days . A repreWe compared cell number, normalized metabolic activity, and GAG production in cell aggregates cultured at 100 and 500 μl/min. In both conditions, cell number increased after 14 days compared to day 0  and the spherical (Ø 1 mm) cell aggregate generated at 100 and 500 μl/min, respectively  were able to promote spheroid formation. Noticeably, even when cultured with the highest flow rate, the maximum shear stress (148.83 mPa) imposed on cells was well below 1 Pa, the threshold above which shear stress loses the chondroprotective potential and begins to be detrimental for cell survival (in vitro is controlled largely by diffusion. For this reason, mass transfer of nutrients within static culture systems is limited, while it is increased by fluid motion in dynamic culture systems (The conical chamber configuration proved to be suitable to support cell spheroid formation within 24 h after cell seeding, due to the synergic action of fluid flow and gravity sedimentation. Additionally, our device allowed the biophysical stimulation of the developing constructs, which resulted in the formation of stable cell spheroids, when adequate levels of shear stress were applied. Indeed, very low shear stress levels did not yield the formation of a stable spheroid. This result indicates that in our system cell trapping survival . Interessurvival . Our hypsurvival . Alcian survival . It mustsurvival . Based osurvival , we can  systems . In acco systems , it is p systems . This hyin vitro models related to different research fields and to produce mature building blocks for tissue biofabrication.The described device meets the requirements for an easily accessible and consistent process to generate and culture 3D cell spheroids in a microfluidic set-up that could be upscaled to increase the throughput of the system. Our system allowed predicting the dimension and shape of the generated spheroid, proving to be highly reproducible and flexible. The particular advantage of the current system is the possibility to achieve different outcomes by simply tuning non-geometrical parameters without the need to modify the chip design. Considering the wide application of 3D cell spheroids, this platform holds a great potential both to generate The datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.FP, MP, MR, and MM contributed to the conception and design of the study. SL, FP, GT, and MP performed the experiments and analyzed the data. SB and VM performed the computational analysis and analyzed the relative data. LZ provided the cartilage biopsies for articular chondrocyte isolation. SL, FP, GT, and VM wrote the manuscript. All authors contributed to manuscript revision and read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lineages of Firmicutes, including Clostridiales, have been frequently detected in oil reservoirs, but are typically not considered impactful or relevant due to their spore-forming nature. Here we show, using metagenomics, a high temperature oil reservoir of marine salinity contains a microbial population that is predominantly from within the Order Clostridiales. These organisms form an oil-reservoir specific clade based on the phylogenies of both 16S rRNA genes and ribosomal proteins, which we propose to name UPetromonas tenebris, meaning they are single-celled organisms from dark rocks. Metagenome-assembled genomes (MAGs) of these Petromonas sp. were obtained and used to determine that these populations, while capable of spore-formation, were also likely replicating in situ in the reservoir. We compared these MAGs to closely related genomes and show that these subsurface Clostridiales differ, from the surface derived genomes, showing signatures of the ability to degrade plant-related compounds, whereas subsurface genomes only show the ability to process simple sugars. The estimation of in-situ replication from genomic data suggest that UPetromonas tenebris lineages are functional in-situ and may be specifically adapted to inhabit oil reservoirs.Oil reservoirs have been shown to house numerous microbial lineages that differ based on the  It is thought that temperatures in source rocks or reservoirs greater than 80 °C act to deter hydrocarbon degradation and sterilize microbial life3. As sterile crude oil and gases migrate from the hot source rocks and cool they accumulate and may be populated by existing subsurface communities in rock or flowing subterranean waters. Determining the indigenous microbial community structures in these reservoirs can be difficult because of the presence of pipeline and infrastructure contaminants, and the indigenous communities themselves can be altered in terms of member representation and activity by the injection of seawater or gases and chemical additives used for secondary recovery4.Oil reservoirs are unique subsurface environments that can be hostile to life due to high temperatures, pressures, and salinity  as carbon and energy sources15. Hydrocarbon enrichment cultures from oil reservoirs have stimulated clostridial lineages16, and clostridial hydrocarbon degradation genes have been observed in surficial oil-contaminated environments17. Previous metagenomic studies of oil reservoirs show clostridial lineages containing genes initially classified as hydrocarbon degradation genes, but well-characterized anaerobic hydrocarbon-degrading genes, bssA and assA, were not detected. Instead, robust signatures of polysaccharide, peptide, and fatty acid degradation were seen as well as robust pathways of sugar fermentation4. To our knowledge, few studies have assessed the portion of sporulated and vegetative cells of Clostridiales in oil reservoirs.Microbes can cause souring or consume significant quantities of lower molecular weight hydrocarbons, degrading the quality of the petroleum products. Given the potential for degradation of resources, there is interest in understanding the structure and source of these communities, and the metabolisms they are employingUPetromonas tenebris was the dominant microbial signature in this subsurface environment, and in-situ replication of this organism can be detected via metagenomics.In this study, we sampled production wells from Galveston 209, a mature oil field ~32 km south of Galveston Island, Texas. Well temperatures of this oil field range from 80 to 160° Celsius. Discovered in 1983, the field was extensively developed off two platforms (A & B) with a cumulative production over 23 MBO (million barrels of oil) and 300 GCF (gas cubic feet) out of stacked pay of Lower to Middle Miocene shallow marine sandy reservoirs (~2.1 to 4.6 km depth) with outer shelf mudstone seals, all deposited in a wave-dominated deltaic setting. Production data indicates that there is a strong water drive through well-connected, continuous reservoirs. Traps are in low-relief fault-dependent closes on basinward-dipping listric normal faults. In this study, microbial communities in the produced fluids (mixture of oil and water) of these hot wells were examined via metagenomics. Functional annotation and the generation of metagenome assembled genomes (MAGs) were used to examine potential community metabolisms. A novel clostridial lineage which we propose to name Produced fluids were collected using sterile technique at the drilling rig offshore Texas. Fluids came from 4 distinct wells that had temperatures ranging from 88 to 102° C , with the following parameters: removal of low quality sequence ; removal of ambiguous nucleotides: No ambiguous nucleotides allowed; removal of terminal nucleotides: 2-12 nucleotides from either end to minimize sequencing errors and enriched 5mers; removal of sequences on length: minimum length 60 nucleotides. Whenever one read of a read pair was excluded due to the quality trim, the entire pair was excluded. Trimmed, paired reads were assembled using IDBA-UD version 1.1.1. with the following settings:–mink 40–maxk 120–step 20–min_contig 30020. A maximum likelihood phylogenetic tree of 16S rRNA gene was inferred from these sequences using Mega version 5 using default parameters for alignment and tree construction with 500 bootstrap replicates21.The phylogeny of metagenome community members was determined using EMIRGE (Expectation-Maximization Iterative Reconstruction of Genes from the Environment) based on the reconstructed 16S rRNA gene sequences from unassembled data22. The taxonomical uniqueness of each resulting MAG was initially determined using Phylosift version 1.0.123 with the default parameters. The level of potential contamination and strain heterogeneity in each MAG was evaluated using CheckM 1.0.6 with the “lineage_wf” option24. The VizBin program25 was then used to visually refine the MAGs to minimize outlier scaffolds. Close relative genomes of the clostridial MAGs were downloaded from NCBI . Average nucleotide identity (ANI) between the MAGs and the two reference genomes were calculated using PyANI26 implemented in Anvio v5.527, by following the procedure described here (http://merenlab.org/2016/11/08/pangenomics-v2/). Pair-wise average amino acid identity (AAI) was calculated as one-way AAI and two-way AAI using the online tool AAI calculator (http://enve-omics.ce.gatech.edu/aai/).Metagenome assembly of individual samples were subjected to binning using MaxBin version 1.4.2 with the max iteration of 20028 from each MAG were extracted using the Geneious software  from the PROKKA annotation. Also included for phylogenetic comparison are genomes from closely related microbial groups downloaded from National Center for Biotechnology Information (NCBI): E. coli, H. congolese, T. gondwanense, D. alkaliphilum, C. cellulovorans, C. hydrogeniformans, C. acetium, C. forminaceticum, C. sporogenes, P. caminithermalis, M. halophilus. Ribosomal proteins were concatenated and aligned with MAFFT v7.39229 for each of the recovered genomes. Only those genomes that had over 50% of the ribosomal proteins were used in the analysis. The alignment output was used to generate maximum likelihood phylogenetic trees with 100 iterations using FastTree v2.1.1130.A collection of 16 ribosomal proteins31. COG (Clusters of Orthologous Groups) data was obtained via a local copy of the RAMMCAP  using the updated version of the 2014 COG database from NCBI32.PROKKA (prokaryotic annotation) version 1.11 was used to annotate the metagenomes and MAGshttp://www.kegg.jp/blastkoala/), between July and October, 201733.The presence or absence of functional genes in metabolic pathways of both cultured metagenome and MAGs were predicted using the BlastKOALA web service provided by the KEGG: Kyoto Encyclopedia of Genes and Genomes website (http://cys.bios.niu.edu/dbCAN/), and based on the CAZyDB released on March 17, 201536.The Carbohydrate Active Enzymes (CAZY) annotation was performed via HMMER searches against the dbCAN release 4.0 HMM (hidden Markov model) database , both of which are moderately thermophilic and halophilic and were initially isolated from deep-sea hydrothermal vent systems  and P. carminithalis (4.1 Mb). The number of annotated genes of the MAGs ranged from 2,815–2,959 (Table Metagenome assembled genomes (MAGs) of kM Table . The est59 Table .Table 3UCaminicella sporogenes and Paramaledivibacter caminithermalis, along with Maledivibacter halophilus  are 99-100% among the 4 MAGs, but the MAGs share only 74-75% ANI and 51-61% average amino acid identity (AAI) with the cultured genomes (Table UPetromonas tenebris as the MAGs fit the suggested metrics for establishment of a new genus and species40. The name comes from “petra” (rock), “monas” (single celled organism) and “tenebris” (dark) since the phylogenetic trees show relatives of these organisms are all found in oil reservoirs, the U indicates its uncultivated status.As such, we provisionally name the organism Ca. sporogenes and P. carminithermalis show that individual MAGs of UPetromonas tenebris have different levels of overlap with cultured relatives, with an average of 323 and 197 unique genes, respectively  shared with either ly Table . These uCaminicella, Maledivibacter, and Paramaledivibacter genera, with the remainder coming from other Clostridiales or Firmicutes groups  between each of the cultured genomes and between the cultured genomes and the reservoir MAGs Table . The res2 fixation pathway found in three of the four reservoir MAGs, but not in the other genomes  found in Archaeoglobus species, which may be used to anaerobically metabolize some hydrocarbons41. However, the annotated pyruvate formate lyase genes found in the UPetromonas tenebris MAGs contain a different active site that was found in more typical carbohydrate fermenters, leading to the conclusion that these are indeed typical pyruvate formate lyase genes used in the majority of anaerobic bacteria41. We examined the metabolic profile of the MAGs via standard annotation by PROKKA and also via individual BLAST analysis for known anaerobic hydrocarbon degradation genes43. We found no evidence for other anaerobic hydrocarbon metabolism pathways, except that the gamma subunit of acetophenone carboxylase, an enzyme in the ethylbenzene degradation pathway, was present in each of the MAGs. However, the gene encoding ethylbenzene dehydrogenase, the initial enzyme in this pathway44, could not be found. As such, based on current genomes and annotations, UPetromonas tenebris seems not to be capable of utilizing hydrocarbons. We do caveat that as seen in the Archaeoglobus case41, genes may exist for hydrocarbon degradation that are either unannotated or misannotated and full proof cannot be given until a culture is tested.Both UPetromonas tenebris MAGs came from spores or active, vegetative cells we estimated the index of replication (iRep) of the MAGs in the four oil reservoirs. The resulting iRep indexes ranged from 1.35-1.40 for the reservoir MAGs. For reference, this iRep value is comparable to the median values seen in other environments, including soil (1.34) and human gut systems (1.37–1.42)37, and should be interpreted as a measurement indicative of growth. The value indicates 35–40% of these cells were replicating in situ at time of sampling.To determine if the Caminicella/Paramaledivibacter clades of thermophilic and halophilic Clostridiales, and form a distinct clade with other uncultured organisms found in high-temperature oil reservoirs (Fig. UPetromonas tenebris (Figs. Ca. sporogenes in Oman at temperatures near 60 °C45 and formation water from the high temperature Ekofisk oil field in the North Sea46. While these relatives do not grow at temperatures as high as observed in this environment, hyperthermophilic clostridial species have been documented from oil wells previously47. However, no genomic information is available for comparison to these MAGs.The hot and salty oil reservoirs described here represent a challenging environment for microbial growth. Using metagenomic analysis, we found the dominant species in these reservoirs is related to the thermophilic irs Fig. . Due to is Figs.  and 3. Rin-situ environment. We cannot refute the hypothesis that spores may be germinating en route to oil processing inside pipelines, however, we note that communities which show infrastructure influence are typically much more complex, reflecting the increase in electron acceptors and metals available within pipelines, as well as industrially introduced materials4. As such, the entirety of the data presented suggests these are active in-situ.It is unlikely that these MAGs represent infrastructure contaminants or sporulated, inactive cells. First, 35–40% of these cells were in the process of replication. Additionally, the DNA was readily extractable and this species dominated the community, also suggesting it was not heavily sporulated. Considering the apparent lack of hydrocarbon consumption by these cells, it is plausible that clostridial spores present in the subsurface may have germinated as the oil seeped upward from hotter source rocks to a slightly cooler reservoir formation, where water was present. However, we interpret the low diversity of the community to be reflective of the challenging Methanothermococcus methanogens which use hydrogen and formate as electron donors48, and Desulfallas species such as D. gibsonae and D. geothermicum, which utilize simple organic compounds, including some carbohydrates and/or fatty acids, and alcohols such as ethanol, propanol, and butanol, as electron donors and sulfur compounds as electron acceptors, producing carbon dioxide or acetate as end products49. Also present is Petrotoga mobilis, which is a fermenter of a variety of carbohydrates including xylan50. This system may be a syntrophic methanogenic system in which the UPetromonas and Petrotoga ferment complex organic compounds, with Desulfallas and Methanothermococcus scavenging the fermentation products. Based on the analysis of genomes, no alkane metabolizing partner has been detected, compared to other syntrophic methanogenesis systems that were explored via enrichment cultures51. Therefore, UPetromonas tenebris represent one of the keystone members of the microbial community inhabiting this harsh environment.Other organisms in this system in lower abundance Fig.  include UPetromonas tenebris MAGs show some distinctions between wells. In particular, they contained between 8-13 unique genes per genome (Fig. Despite being retrieved from a similar geologic formation, the ome Fig. . They weome Fig.  and 3 anome Fig. . The varome Fig. . Overallome Fig.  and 3 anome Fig. , despiteUPetromonas tenebris MAGs are the most abundant organisms in this reservoir system, they show signatures of replication, and potential end products of their fermentative metabolism have built up in this system. Yet they lack potential hydrocarbon processing pyruvate formate lyase genes seen in the cultured relatives, and none of the other less abundant organisms have signatures of directly processing hydrocarbons either. Nevertheless, the UPetromonas tenebris in this reservoir have become a dominant species in the environment, likely through fermentation, are growing in-situ and may be responsible for the increase of organic acids in the reservoirs. Due to their potential influence in reservoirs, Firmicutes and clostridial relatives should be regarded as key players in reservoir microbiomes, not only as sporulated, inactive cells. Furthermore, presence of these organisms in produced fluids, drilling fluids, or naturally seeped fluids could be an indicator of the temperature of a connected subsurface reservoir, with applications to oil and gas exploration and development52.The


Intracardiac tumors in children are relatively rare, but their clinical consequences may include severe outflow tract obstruction, embolism, cardiac insufficiency, or rhythm disturbances. In some cases, the tumor may constitute part of a genetic condition and prompt additional investigations, as well as a modification of therapeutic management. Herein, we present a molecularly confirmed familial case of Gorlin syndrome with an early cardiac tumor as a presenting sign. We provide detailed clinical characteristics of the affected individuals and a useful review of syndromic causes of pediatric cardiac tumors in clinical practice. Cardiac tumors are benign or malignant neoplasms that may originate in all the three layers of heart tissue  gene, which is part of the Hedgehog (Hh) molecular pathway  (OMIM:109400) Gorlin . NBCCS iHerein, we show a molecularly confirmed familial case of Gorlin syndrome consisting of an infant proband presenting with intracardiac tumor, polydactyly, and macrocephaly and the father with features of macrocephaly, mitten-type syndactyly of fingers, polydactyly, and maxillary cysts. We describe current and future targeted treatment options in our familial case and provide a review of syndromic causes of pediatric intracardiac tumors.We present a case of an infant girl with multiple dysmorphic features and a cardiac tumor of unknown origin diagnosed postnatally. The girl was born preterm at 36 weeks’ gestational age with the birth weight of 4000 g by caesarean section due to maternal hypertension. The Apgar score was 3 and 5 at 1 and 5 min after birth, respectively. After initial resuscitation, the transthoracic echocardiography was performed revealing a tumor in the left ventricle. The neonate was referred to the department of pediatric cardiology for subsequent diagnostics.On admission, the child was in a serious but stable condition with normal resting heart rate of 150 beats per minute (bpm) and arterial blood pressure of 81/68 mmHg. Blood oxygen saturation was 92–94%, and capillary refill time was normal. Macrocephaly with head circumference of 38.5 cm (> 97th centile), bilateral polydactyly of fingers with unilateral polydactyly of toes, and a natal tooth on lower gingiva were observed Fig. . DyspneaLaboratory testing revealed anaemias (hemoglobin concentration of 10.8 g/dL) and significantly increased NT-proBNP level   attached to the posterolateral wall of left ventricle with the areas of hyper- and hypodensity Fig. . ModeratCardiac magnetic resonance confirmed echocardiographical findings and revealed possible diaphragm and parietal pericardium invasion. Contractility of left ventricle wall was impaired by presence of the tumor mass. Peripheral contrast enhancement suggested the presence of tumor capsule, and the lack of central enhancement suggested necrosis. Additionally, oval mass  localized between the anterior and lateral wall of left ventricle with peripheral contrast enhancement similar to the main tumor was described; however, the connection between them could not be excluded. Imaging studies suggested rhabdomyoma, fibroma, or sarcoma.Subsequent treatment included administration of spironolactone, furosemide, and captopril. Hemodynamically significant PDA was closed with PDA occluder. Signs of dyspnea decreased. Because of the high risk of thromboembolic events, cardiac tamponade, arrhythmia, and possible presence of metastases, the biopsy during the procedure was not performed.Magnetic resonance imaging of central nervous system showed no tumors. Rhabdomyoma associated with tuberous sclerosis could still not be excluded; thus, therapy with sirolimus was started. After 4 weeks, no significant changes in tumor size were observed, and sirolimus therapy was discontinued. Pericardial drainage due to fluid accumulation was performed. Subsequent specimen cytology was negative for tumor cells.Additionally, wide QRS extra beats were observed during hospitalization in the standard ECG monitoring. Twenty-four-hour Holter monitoring revealed a few episodes of ventricular tachycardia up to 200 bpm. Metoprolol was administered, and during subsequent tests, only single premature ventricular beats were observed. Pending genetic test results the infant was discharged in a good general condition.The 29-year-old father of the proband girl presented with macrocephaly  and frontal bossing, mitten-hand–type bilateral syndactyly, and unilateral postaxial hand polydactyly Fig. . He had Nextera Flex for Enrichment sample preparation kit combined with TruSeq DNA Exome (45 Mb) probes  according to the manufacturer’s instruction. Library was pair-end sequenced (2× 100 bp) on HiSeq 1500 (Illumina) to the mean depth 83×, 98.7% of target was covered ≥ 10×, and 94.4% was covered ≥ 20×. Bioinformatics analysis of raw ES data and variant prioritization were performed as previously described  was performed using ASH1L, CDKN2A, SIX4, and PTCH1 genes for verification and family study. Variants in ASH1L, CDKN2A, and SIX4 were inherited from the healthy mother, while the variant in the PTCH1 gene ) was inherited from the affected father. The p.(Leu923Pro) variant in PTCH1 was absent from the proband’s grandparents’ blood. Therefore, we concluded that this variant occurred de novo in the proband’s father and was transmitted to the proband in an autosomal dominant manner of inheritance  is the most frequent syndromic presentation of cardiac tumors, and mTOR inhibitors are effective in treating CNS and kidney neoplasms of TS (Curatolo and Moavero PTCH1 gene creating a substitution p.(Leu923Pro). According to Varsome, most missense changes in PTCH1 are of uncertain significance. However, this particular variant is absent from GnomAD or our in-house databases of variants, and in silico analyses are predictive of its pathogenicity. Moreover, the variant was excluded in healthy paternal grandparents of our proband. Thus far, somatic mutation p.Leu923Pro has only been found in childhood medulloblastomas as the Sonic Hedgehog (SHH) pathway is likely involved in progression of these tumors (Parsons et al. In the described proband and her affected father, we have identified through ES a missense variant NM_000264.4:c.2768T>C in the In summary, we present a familial case of Gorlin syndrome characterized by the presence of a rare intracardiac tumor in the proband. Clearly, the syndromic diagnosis enabled optimization of therapeutic management, and it may support introduction of specific prophylactic measures. Similar personalized management should be introduced once the diagnosis of any of the genetic syndromes presenting with primary intracardiac tumors and discussed here is reached.


The valorization of minor accessions of olive is potentially a good way to improve the qualitative production of a specific territory. Olive oils of four minor accessions  produced in the same area of the Calabria region were characterized for the principal qualitative analyses at two drupe harvesting periods (October and November). Good quality in terms of free acidity, peroxides, spectrophotometric indexes, and fatty acid composition was observed in olive oils produced at both drupe harvesting times, with the exception of those of Tonda di Filogaso, which showed a free acidity level over the legal limit for extra virgin olive oil in the second harvesting time. All of the olive oils possessed at both production periods averagely abundant total polyphenols (460–778 mg/kg) and tocopherols (224–595 mg/kg), and the amounts changed in the experimental years for expected different environmental variations. Ottobratica Cannavà and Ottobratica Calipa clones showed some peculiar qualitative characteristics , distancing themselves from the principal variety of reference, Ottobratica. In view of the recognized importance of the right lifestyle, primarily resulting in healthy eating, the daily consumption of olive oil is highly recommended for its dotation in monounsaturated fatty acids, in particular oleic acid, and antioxidant compounds, proven to reduce the incidence of cardiovascular and age-associated diseases . Olive vThe Italian olive heritage contains over 500 varieties; many of these are in Calabria , a regioThis paper aims to investigate for the first time the chemical characteristics of olive oils from four minor olive accessions, Ciciarello, Tonda di Filogaso, Ottobratica Calipa, and Ottobratica Cannavà, present in the Tyrrenian Southern area of Calabria. Ottobratica Calipa and Ottobratica Cannavà are in particular two genotypes selected within the Ottobratica population variety in the last decades by the olive growers of this specific territory of the Calabria region . The stuThe studied olive oils were obtained from four olive accessions  in a fifteen-year-old olive grove located in Gioia Tauro Plain, an important olive growing area located on the Tyrrhenian side of the Calabria region . Ottobratica cultivar was also submitted to the research as a reference for its related clones. Previous morphological and molecular characterization studies conducted on the two clones of Ottobratica  averted The experiment was carried out considering three blocks of the four accessions, each composed of three olive trees. About 10–12 kg of drupes were sampled from each block in two harvesting times: October (O) and November (N) of 2017, 2018, and 2019.−1) through a mobile phase of methanol/acetonitrile (50:50). The detector was set at a 290 nm excitation wavelength and 330 nm emission wavelength. The identification and quantification were performed by calibration curve, using pure α-tocopherol as the standard and concentrations ranging from 10 to 500 mg kg−1. Results were expressed as mg kg−1. Determination of total polyphenols was performed following Baiano et al. [v/v) and 2 mL of hexane were added to 5 g of oil samples and mixed with a vortex (10 min). The hydro-alcoholic phase containing phenols was separated from the oil phase by several centrifugations; 100 μL of phenolic extract were mixed with 100 μL of Folin–Ciocal teau reagent (2N) and, after 4 min, with 800 μL of an aqueous solution of Na2CO3 (5%). The mixture was heated in a 40 °C water bath for 20 min, and the total phenol content was determined calorimetrically at 750 nm. The total phenolic content was expressed as milligrams of gallic acid equivalents per kilogram of oil. The total antioxidant activity of the olive oils was detected by 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/Trolox equivalent antioxidant capacity (TEAC), according to Re et al. [The oil yield (% oil dry weight) was determined in drupes after stone removing by extraction with petroleum ether in a Soxhlet apparatus . For the olive oil extraction, about 15 kg of drupes were milled with a hammer mill. The obtained paste was mixed at a temperature below 20–25 °C for 30 min and pressed using a hydraulic press (pressure up to 200 bar) in a small olive oil press mill Mini 30 system . After centrifugation and filtration through paper, olive oils were then stored in dark glass bottles at room temperature and analyzed for the total free acidity value, peroxide index, and UV light absorption coefficients according to EC regulations ,22. Pigme et al. , and 2,2e et al. . Fatty ap < 0.05) were obtained by one-way analysis of variance (ANOVA) and multivariate analysis. Pearson’s coefficient was used to study the correlation among qualitative parameters of olive oils. SPSS Software  was used for statistical elaboration.The results of the analyses were elaborated as mean ± standard deviations of three sampling years for two harvesting times. Significant differences . The range observed in oils produced in October was 0.31–0.57%; at November, it tended to increase in all of the samples, exceeding the 0.8% in some years, except for Ottobratica Cannavà oils. The other productions were affected probably by a different varietal response to some negative environmental factors linked to a specific year : 2018 fo2/kg, with significant differences between the two harvesting times only in Ottobratica Cannavà oils. Higher peroxide values were noted in the oils of clones compared to those from Ottobratica cv in October, whereas an opposite result was detected for the productions of November (6.63 mEq O2/kg in oils from Ottobratica). Spectrophotometric indices denoted olive oil productions of good quality at both harvesting times without significant differences, with the only exception of Tonda di Filogaso olive oils.Peroxide values of oils were in the range of 2.50–5.58 mEq Op < 0.05) elaboration (data not shown). Olive oils from Ciciarello showed the highest oleic/linoleic (11–13) and monounsaturated/polyunsaturated acid (MUFA/PUFA) (10–12) ratios, confirming the previously discussed results for fatty acid quantification. The antioxidant compositions of olive oil from minor accessions is reported in The major fatty acid in olive oil is oleic acid; in our study, its content varied with significance from 68.15% (Tonda di Filogaso oils in November) to 75.79% (Ciciarello oils in October). The followed principal detected fatty acids were palmitic acid (C16:0), quantified from 13.23% to 15.74%, and then linoleic acid (C18:2) that varied from 5.96 to 10.06%; both components were similar among minor varieties. The stearic acid (C18:0) was significantly higher in the oils of Tonda di Filogaso obtained in November (3.44%) than in the other samples. The other fatty acids that significantly varied among the samples were C16:1, among the unsaturated ones, from 0.99 to 1.64% and C20:0, among the saturated ones, from 0.34 to 0.46%, as evidenced by the Tukey’s post hoc test (p < 0.01) differences of pigment amounts were observed among the samples; the olive oils of Ciciarello and Tonda di Filogaso obtained in October were the richest in chlorophylls . Ciciarello olive oils were even the richest in total carotenes (7.16 ± 2.28 mg/kg). The oils extracted in November showed reduced pigment amounts and, in particular, a major reduction was observed in Ottobratica Calipa olive oils (TCL: 2.53 ± 1.23 mg/kg and TCA: 2.26 ± 0.90 mg/kg). ANOVA data elaboration showed variations for pigments between harvesting times, except in the oils of the Ottobratica Cannavà clone (Significant (và clone .p < 0.05) higher than those resulted in the cultivar population of reference (Ottobratica cv).Chlorophyll and carotenoid amounts were also significantly , followed by Ottobratica Calipa olive oils, whereas lower amounts were detected in those from Tonda di Filogaso (224–227 mg/kg) as confirmed by literature . A signiThe antioxidant activity of the oils was analyzed by the reaction against two radicals, DPPH and ABTS. The obtained results denoted a higher response with the second antioxidant assay (23.29–45.67%) than the DPPH radical (13.80–34.71%). The largest differences among the varieties were significantly observed in the oils produced in October for ABTS assays .p < 0.05). It is interesting to note that good oils from Ottobratica clones could be obtained at both harvesting times, without significant variations among each other (p > 0.05) and, different from Ottobratica, their reference variety. The oils of this last production in November denoted the previously discussed variability during the years and, on average, poor quality (free acidity of 1.18 ± 1.08%). Peroxides and extinction coefficients complied with the regulation limits for the extra virgin olive oil [The olive oil accumulation on fruits during ripening follows the triglyceride-forming biosynthesis pathway up to the achievement of full drupe maturation. The olive oil yield in fruits is influenced by the choice of the right harvesting time for each variety and by several growing conditions, such as water availability . In our live oil . Fatty alive oil . The fatThe molecules responsible for olive oil color are pigments belonging to chlorophyll and carotenoid compounds. Their quantification is important to determine not only the sensorial characters and consumer acceptability of olive oils, but also their antioxidant potentiality; olive oil chlorophylls react as radical scavengers in dark storage and as pro-oxidant (sensitizer pigments) in light. Carotenes instead protect cells against the light, with oxygen and sensitizer pigment effects having the ability to quench singlet oxygen and excited sensitizer molecules. Moreover, they can also react as antioxidants under conditions other than photosensitization . The totIn particular, the total content can range from 2 to 40 mg/kg for chlorophylls and from zero to a few mg/kg for carotenoids ,37. Olivp < 0.00) in the olive oils. From this data elaboration that considered overall data for all of the three years of experimentation, it was noted that, among the other qualitative parameters, in particular the total carotene content was affected exclusively by the drupe ripening, as the literature [Finally, a multivariate data analysis was performed to evidence the influence of varietal characteristics or drupe harvesting times to the olive oil quality . Resultsterature  confirmsThis study allowed the characterization of the oil productions from four minor olive accessions grown in the same area of Calabria, with the aim to compare the qualitative differences measured during three years of observations, excluding climatic variables due to different environmental conditions. For some of these (oils from Tonda di Filogaso and Ciciarello cv), harvesting times significantly affected the results for free acidity and total pigments. All of the oil productions obtained in October possessed the chemical parameters to be classified as extra virgin olive oils. Comparing all of the studied accessions, olive oils from Ciciarello cv and Calipa and Cannavà clones also showed good quality when extracted in November. This is interesting for growing practices in the same studied area and for new knowledge about the potentiality of the two new clones obtained from the Ottobratica cultivar. In particular, the oils of the Ottobratica Cannavà clone showed better quality for free fatty acidity at both harvesting times, with oleic acid content and total antioxidants (polyphenols and tocopherols) with respect to the cultivar of reference, Ottobratica, largely diffused in the considered territory.


Lactobacillus casei, Pediococcuspentosaceus, Lactobacillusplantarum, Weissellaparamesenteroides, Lactobacillusrhamnosus, Lactococcuslactis, and Lactococcusgarvieae. In addition, Enterococcus and Streptococcus species were also isolated. Season seemed to affect the genus/species of LAB since Lactobacillus were mainly isolated from winter samples, while Lactococcus and Enterococcus species were the main genera identified in summer samples. Regarding the geographical distribution, the majority of the Lactobacillus spp. were isolated from the South-eastern region in both seasons. In conclusion, through this study the diversity of indigenous LAB in the Maltese cow milk was monitored for the first time and highlighted that the microbial communities are affected by seasonality and geographical distribution of the farms.A geographical and seasonal assessment of indigenous lactic acid bacteria (LAB) in Maltese cow milk was conducted in this study. To investigate this, milk was collected from different regions of Malta during winter and summer seasons. Total viable counts (TVC) and LAB population were enumerated. Afterwards, LAB were isolated and identified by molecular methods. According to the results, similar TVC were enumerated on winter and summer samples, while highest LAB population was detected on summer samples. LAB isolates were grouped in seven different clusters which were assigned to  Milk is a highly nutritious product, and thus it can serve as an optimum growth medium for a large variety of different microbes . An incr CFU//mL . Most daThe aim of this study was to characterize the microbiota of raw Maltese cow milk for the first time and to assess the effect of season and region on microbial diversity. To our knowledge no information is available for the LAB diversity of raw milk from the country of Malta. The only available information about Maltese cow milk is its physicochemical characteristics ,7. To acA two factorial experiment was designed for the seasonal and geographical assessment of microbial communities of raw Maltese cow milk. Firstly, the Maltese Islands were divided into five regions, namely: Southern region, South-eastern region, Central region, Northern region, and Gozo. Raw milk samples were collected from all around Malta and Gozo and the region of the farms was noted. For the seasonal assessment, raw milk samples were collected from the local herdsmen in both winter  of 2015 and summer  of 2016. Samples were collected in a tightly sealed sterile plastic bottle using a sterile scoop directly from the herdsmen’s tanks as soon as the raw cow’s milk was delivered from the farm to the local dairy industry. The enumeration of bacteria from the raw milk samples was performed on the same day that the samples were collected. Raw milk aliquots of each herdsman were retrieved from the refrigerator (4 °C) and a serial dilution in ¼ strength Ringer‘s solution  was performed on each sample. The appropriate dilution was plated out on Tryptic Soya Agar (TSA)  and Lactobacillus selective agar (LSA)  for the detection of Total Viable Counts (TVC) and LAB, respectively. The TVC and LAB population expressed on log cfu/ mL after incubation of TSA and LSA at 30 °C and 37 °C respectively for 48 h. After microbial enumeration, colonies from LSA medium were isolated; in order for the study to be close to a national survey, 10% of colonies grown on LSA were recovered and purified by successive subcultures on LSA. Purity of the cultures was also checked on Brain Heart Infusion agar (BHI) , and pure isolates were stored in vials with TSB and glycerol (20% v/v) in a freezer at −80 °C, until further use. Prior use, each isolate was cultured in BHI broth at 37 °C for 24 h.5 (GTGGTGGTGGTGGTG). PCR amplifications were conducted into a thermal cycler  in a final volume of 25 µL containing DNA TOP polymerase  (1.25 U), 1× blend mastermix buffer, dNTPs (0.2 mM from each), primer (2 µΜ), magnesium chloride (2 mM), DNA (2 µL) and ddH2O. PCR reactions consisted οf an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of denaturation at 90 °C, 30 sec, primer annealing at 40 °C, 1 min and primer extension at 72 °C, 8 min, and concluded by a final extension step at 72 °C for 16 min. PCR products were separated in a 1.5% agarose gel 1× TAE buffer for 90 min at 100V. A 500 base pair ladder was chosen as marker. After visualization of bands under ultraviolet (UV) trans-illuminator, conversion, normalization, and further analysis were performed using the Sørensen–Dice coefficient and UPGMA (un-weighted pair group method with arithmetic mean) cluster analysis with Bionumerics software, version 6.1 . DNA was extracted according to the protocol described by the Accuprep Genomic DNA extraction kit  and was stored in the −20 °C freezer until further use. For strain differentiation, all isolates were subjected to rep-PCR using primer GTG2O. PCR reactions consisted οf an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C, 30 sec, primer annealing at 56 °C, 30 sec and primer extension at 72 °C, 1 min, and concluded by a final extension step at 72 °C for 5 min. After PCR product purification pureLink DNA purification kit  according to the manufacturer recommendations, cycle sequencing uses the BigDye Terminator v3.1 Sequencing Buffer . The cycle sequencing mix was prepared by mixing the following: 2 μL 2.5X ready reaction premix (ddNTPs), 1 μL 5× BigDye sequencing buffer (polymerase), 1 μL 10 pmol primer, template and 6 μL water. This freshly prepared cycle sequencing reaction mix (9 μL) was added to a reaction tube followed by the addition of 1 μL of the purified PCR products. This tube was placed in a thermal cycler , which was set to perform the following sequencing cycles: initial denaturation step at 96 °C for 1 min, followed by 25 cycles of denaturation at 96 °C, 10 sec, primer annealing at 50 °C, 5 sec and primer extension at 60 °C, 6 min. Afterwards, MagSi-DNA cleanFIX kit reagent (10 μL) , was placed for the removal of unused dNTPs, in the tube consisting of the cycle sequencing product, as described by the manufacturer . Representative isolates with genetically different rep-PCR patterns i.e., 22 from the winter samples and 13 isolates from the summer samples were chosen for further analysis. The representative isolates were subjected to species identification with 16S-PCR and Sanger sequencing. The PCR reaction mix was prepared in a final volume of 25 μL in a thermal cycler : 1× blend master mix buffer, dNTPs (1 mM of each), 0.2 μM forward and reverse primers (P1 and P4 respectively) , DNA TOP polymerase (1.25 U), DNA (2 μL) and ddHThe nucleotide base sequence of the 16S rRNA gene of the isolated LAB were determined by Sanger sequencing. The Sanger sequencing was carried out by pipetting the final supernatant into a 96-well plate . The 3500 Genetic Analyzer , was used to perform Sanger sequencing by capillary electrophoresis and analysis involved the inputting of the sequences produced into an NCBI BLAST database.Data and statistical analysis were performed using GraphPad Prism v8.0.1 . TVC and total LAB counts of milk samples collected in summer and winter for all regions were tested for normality using the D′ Agostino and Pearson test and the Shapiro Wilk’s test. Pearson  and Spearman  tests were applied to assess correlation for the TVC and the viable counts of LAB between summer and winter samples. Finally, two-way ANOVA analysis using a mixed model and Sidak’s post-hoc test for multiple comparisons was performed to determine any significant difference between microbial counts of milk samples collected in summer and winter or different geographical locations. The microbiota of raw Maltese cow milk was investigated and the effect of season and region on microbial counts and microbial diversity was assessed for the first time through the current study. A total of 50 raw Maltese cow milk samples was collected, 25 samples in summer and 25 samples in winter . For thep < 0.0001 based on Two-way ANOVA analysis of pooled geographical samples) between the winter (0.75–3.20 log CFU/mL) and summer samples (2.59–4.33 log CFU/mL)  except the Central region in the summer samples. No correlation between summer and winter samples was found between the TVC (Pearson r = 0.2002) and the total LAB counts (Spearman r = 0.1423). Analysis for significant differences between different geographical regions results in a significant decrease in TVC when compared to the central region (p < 0.05). No significant difference in LAB counts was observed between geographical samples collected in different seasons  . The mul seasons . More spLactobacillus, Pediococcus, Weissella, Lactococcus, Enterococcus, Streptococcus, Staphylococcus, and Bacillus. In Lactobacillus genus while the majority of the isolates recovered from summer samples were identified as enterococci and Lactobacillus spp. The 262 isolates from raw Maltese cow milk were grouped in 32 clusters based on rep-PCR patterns . The repLactobacillus casei was the most common recovered species in samples collected from Gozo, Northern and South-eastern region, while Lactobacillus casei was also detected in the rest of the regions. Furthermore, Lactobacillus plantarum and Pediococcus pentosaceus were recovered in higher numbers from samples collected from Central and Southern region, respectively. Lactobacillus rhamnosus was fairly recovered from Gozo samples while it was sporadically detected in the south-eastern and southern regions. Weissella paramesenteroides was isolated from samples collected from Gozo, Northern and Southern region. It has to be noted that Bacillus cereus and Staphylococcus sp. were isolated from samples under the Northern region on the geographical distribution map. The geographical distribution of isolates recovered from LSA medium from raw Maltese milk during winter period at species level is shown in Enterococcus was the most common recovered genus in samples collected from Northern (Enterococcus sp.) and Central  region, while its was also detected in high numbers in the rest of the regions. In Gozo samples, Enterococcus casseliflavus and Weissella paramesenteroides were recovered in higher numbers, with the species Enterococcus casseliflavus to be detected only in Gozo samples. However, Weissella paramesenteroides was not recovered from Northern region. In the case of Lactococcus lactis, the species was recovered in higher numbers from samples collected from South-eastern and Southern region, while it was not detected in Central region. In samples from Southern region, Lactobacillus plantarum was not detected, while Lactobacillus rhamnosus was recovered only from this region. Pediococcus pentosaceus was sporadically detected in Gozo, Northern and South-eastern region. Finally, it has to be noted that Bacillus cereus and Streptococcus suis were isolated from the Southern region.The geographical distribution of isolates recovered from LSA medium from raw Maltese milk during summer period at species level is shown in Weissella paramesenteroides MM27.2 was the strain that was the most commonly isolated, where a total of 22 similar isolates were recovered from both the summer and winter samples, i.e., 15 from the summer and 7 from the winter samples. In winter, this isolate was found in samples from Gozo, Northern and Southern region, while it was detected in all regions but Central region in summer samples. The next most common isolates were Enterococcus faecalis MMS8.5 and Lactococcus lactis MMS2.9, where a total of 19 and 16 similar isolates were found respectively, in the summer samples only. Enterococcus faecalis was isolated from all regions but Northern region; while Lactococcus lactis was isolated from samples of Gozo, South-eastern and Southern region. Lactobacillus plantarum MM12.8 and MM8.2, Lactobacillus casei (except strain MM10.2) and Pediococcus pentosaceus MM19.5 and MM23.2 were also fairly common in the winter isolates Pediococcus pentosaceus MM19.5 was only detected in Gozo samples in both periods. Other isolates that were only detected in Gozo samples are Enterococcus casseliflavus (summer), Weissella paramesenteroides MM27.1 (winter) and Enterococcus malodoratus (summer). Similarly, Lactobacillus casei MM14.5 was only detected in winter samples of South-eastern region.Regarding the frequency of isolation, Raw milk was collected from different registered herdsmen from all around Malta and Gozo delivering milk to the local dairy industry. There was quite a different range of bacteria found in each sample indicating that each sample has its own unique bacterial level. The TVC in the milk can indicate the hygienic practices of the farm. In an earlier study, the effect of hygiene practices and disinfection procedures on microbial composition of raw milk was highlighted . This is of importance as, an extremely high bacterial count before pasteurization can overwhelm the thermal destruction capacity of the pasteurizer, which can result in pasteurized milk with high bacterial numbers, hence being of inferior quality and which may have a reduced shelf life. Microbial enumeration in this study indicated that some farms have adequate hygienic practice, while other farms require a drastic decrease of the TVC to improve the overall quality of the milk supplied to the local dairy industry. It seems that increased awareness, especially during the summer period, should aim to decrease or prevent contamination of raw milk with bacteria, especially pathogenic bacteria and will result in better quality raw milk supplied to the dairy industry. Statistical analysis performed in this study confirmed that although the summer samples had slightly higher counts of bacteria, there was still no significant difference between the TVC in summer and winter in most raw milk samples. In some cases, the TVC were higher in winter than in summer samples. On the other hand, since only a few differences were noted between summer and winter bacterial counts, these results also indicate that seasonality is not a critical factor for the levels of bacteria in raw milk. No significant difference between summer and winter population of total bacteria in raw milk samples have been also reported earlier ,9. HowevAlthough data collected in this study show that the average summer raw milk samples had higher counts of LAB than winter samples, it was also determined that the level at which the bacteria decrease (from summer to winter samples), was different for all the farms. Furthermore, for a small percentage of farms, the winter raw milk samples showed higher counts than summer raw milk samples. In an earlier study, variations in bacterial counts of raw cow’s milk were observed in 10 different farms in South Dakota, which were also related to the sampling season . SimilarLactobacillus, Lactococcus, Bacillus, Enterococcus, Streptococcus, and also Staphylococcus species. In a similar study, a large number of isolates were identified as Lactococcus lactis subsp. lactis, while Lactococcus lactis subsp. biovar diacetlyactis, Lactococcus lactis subsp. cremoris. Lactoccocus lactis subsp. lactis Lactobacillus and enterococci were found in raw milk in western Algeria [Lactococcus piscium, Lactococcus lactis, Staphylococcus pasteuri, Staphylococcus warneri, and Staphylococcus epidermidis were detected in raw cow’s milk collected from four farms in Campania [Enterococcus and L. lactis were detected [Enterococcus spp., Lactococcus spp., Lactobacillus spp., Streptococcus spp., and Leuconostoc spp. were also detected in raw camel milk [Lactobacillus paracasei, Lactococcus lactis, Carnobacterium maltaromaticum, Leuconostoc, Enterococcus and Streptococcus were detected in raw donkey milk [It was reported before that microbial enumeration is not enough to highlight the differences in microbial community . This st Algeria . LactocoCampania . Certaindetected . Enterocmel milk . In the key milk .Lactobacillus) and summer (mainly Enterococcus) samples. Furthermore, it was observed that some species were only found in the winter sample i.e., Staphylococcus sp. or in the summer samples i.e., Enterococcus casseliflavus, Enterococcus faecalis, Enterococcus malodoratus, Streptococcus suis. Similarly different strains of the same species i.e., Bacillus cereus detected only in the winter samples and in summer samples. Furthermore, season seemed to affect the presence of specific strains of the same species, for example in the case of Lactobacillus casei five strains were detected only in winter samples while the sixth strain was isolated from both seasons. In another study, where species of the genera Lactobacillus, Pediococcus and Lactococcus (previously grouped as Streptococcus) were isolated from 20 samples of raw Sudanese milk, Pediococcus was isolated widely from the winter samples [In this study, different genera were the most commonly detected in winter  and Enterococcus, respectively. These results allow us to come to the conclusion that there is a difference between the natural microbiota present in raw Maltese milk during the winter and summer seasons. In conclusion, population dynamics strengthen our knowledge that differences in microbial communities could be observed although similar microbial counts were enumerated. In addition, it was also highlighted that the effect of season and geographical distribution on microbial communities could not be underestimated in microbial studies.Through the present study, the diversity of indigenous lactic acid bacteria in the Maltese cow milk was monitored for the first time. Microbial enumeration of the LAB population resulted in the detection of higher LAB counts in summer when compared to the winter sample. It was also concluded that samples from different herdsmen had different initial TVC and LAB counts. These results enabled the conclusions that in the summer, higher temperatures provide a more adequate environment for LAB to thrive and also, seasonality is not a critical factor determining the total bacterial levels in raw milk. A wide range of bacteria were isolated and identified from the raw cow’s milk. Seasonal differences at total LAB and species/strain level between summer and winter raw milk samples were noted. In total, a variety of different LAB were isolated from the winter and summer samples, where the majority of them were identified as