Bianca Castiglioni

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

ABSTRACT The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

Aim:  To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production.

High taxonomic level fingerprint of the human intestinal microbiota by Ligase Detection Reaction - Universal Array approach

BackgroundAffecting the core functional microbiome, peculiar high level taxonomic unbalances of the human intestinal microbiota have been recently associated with specific diseases, such as obesity, inflammatory bowel diseases, and intestinal inflammation.ResultsIn order to specifically monitor microbiota unbalances that impact human physiology, here we develop and validate an original DNA-microarray (HTF-Microbi.Array) for the high taxonomic level fingerprint of the human intestinal microbiota. Based on the Ligase Detection Reaction-Universal Array (LDR-UA) approach, the HTF-Microbi.Array enables specific detection and approximate relative quantification of 16S rRNAs from 30 phylogenetically related groups of the human intestinal microbiota. The HTF-Microbi.Array was used in a pilot study of the faecal microbiota of eight young adults. Cluster analysis revealed the good reproducibility of the high level taxonomic microbiota fingerprint obtained for each of the subject.ConclusionThe HTF-Microbi.Array is a fast and sensitive tool for the high taxonomic level fingerprint of the human intestinal microbiota in terms of presence/absence of the principal groups. Moreover, analysis of the relative fluorescence intensity for each probe pair of our LDR-UA platform can provide estimation of the relative abundance of the microbial target groups within each samples. Focusing the phylogenetic resolution at division, order and cluster levels, the HTF-Microbi.Array is blind with respect to the inter-individual variability at the species level.

Diacylglycerol acyltransferase 1, stearoyl-CoA desaturase 1, and sterol regulatory element binding protein 1 gene polymorphisms and milk fatty acid composition in Italian Brown cattle.

Several lipogenic genes have been shown to have effects on lipid metabolism: stearoyl CoA desaturase 1 (SCD1) catalyzes the desaturation of several fatty acids (FA) in the cis-Delta(9) position in mammary glands of ruminant animals, diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol synthesis in the mammary gland, and sterol regulatory element binding protein (SREBP-1) is a transcription factor that regulates expression levels of the SCD1 gene and other genes relevant to lipid and FA metabolism in adipose tissue and mammary gland. In this work, 351 Italian Brown cows were genotyped for polymorphisms in the SCD1, SREBP-1, and DGAT1 genes to reveal the allelic distribution in the population. Subsequently, effects on individual milk FA composition and on cis-9 unsaturated/saturated FA ratios, a proxy of mammary stearoyl CoA desaturase activity, were investigated. The genotypes of SCD1 (A293V) and DGAT1 (K232A) were determined by an approach based on the ligation detection reaction and a universal array, whereas the genotype of SREBP-1 (84-bp insertion-deletion) was revealed by PCR amplification of intron 5. The genotype analysis showed an unbalanced distribution of alleles within all genes, being the allele with higher gene frequency at 82, 84, and 98% for SCD1, SREBP-1, and DGAT1, respectively. Significant associations between SCD1 and DGAT1 polymorphisms and milk FA composition were found, whereas SREBP-1 polymorphism was not associated with milk FA composition. In particular, SCD1 showed significant association with C14:1 cis-9 and C14:1 cis-9/C14:0, which is considered the best proxy of the desaturation activity in mammary gland. The DGAT1 polymorphism had the strongest association with milk FA composition, which confirmed the key role of DGAT1 in lipid metabolism of mammary gland. However, the unbalanced distribution of alleles in all polymorphisms investigated suggested that the size of population should be increased to confirm the results of the present study.

Identification of hepatotoxin‐producing cyanobacteria by DNA‐chip

We developed a new tool to detect and identify hepatotoxin-producing cyanobacteria of the genera Anabaena, Microcystis, Planktothrix, Nostoc and Nodularia. Genus-specific probe pairs were designed for the detection of the microcystin (mcyE) and nodularin synthetase genes (ndaF) of these five genera to be used with a DNA-chip. The method couples a ligation detection reaction, in which the polymerase chain reaction (PCR)-amplified mcyE/ndaF genes are recognized by the probe pairs, with a hybridization on a universal microarray. All the probe pairs specifically detected the corresponding mcyE/ndaF gene sequences when DNA from the microcystin- or nodularin-producing cyanobacterial strains were used as template in the PCR. Furthermore, the strict specificity of detection enabled identification of the potential hepatotoxin producers. Detection of the genes was very sensitive; only 1-5 fmol of the PCR product were needed to produce signal intensities that exceeded the set background threshold level. The genus-specific probe pairs also reliably detected potential microcystin producers in DNA extracted from six lake and four brackish water samples. In lake samples, the same microcystin producers were identified with quantitative real-time PCR analysis. The specificity, sensitivity and ability of the DNA-chip in simultaneously detecting all the main hepatotoxin producers make this method suitable for high-throughput analysis and monitoring of environmental samples.

Pathogen detection in milk samples by ligation detection reaction-mediated universal array method

This paper describes a new DNA chip, based on the use of a ligation detection reaction coupled to a universal array, developed to detect and analyze, directly from milk samples, microbial pathogens known to cause bovine, ovine, and caprine mastitis or to be responsible for foodborne intoxication or infection, or both. Probes were designed for the identification of 15 different bacterial groups: Staphylococcus aureus, Streptococcus agalactiae, nonaureus staphylococci, Streptococcus bovis, Streptococcus equi, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus uberis, Streptococcus pyogenes, Mycoplasma spp., Salmonella spp., Bacillus spp., Campylobacter spp., and Escherichia coli and related species. These groups were identified based on the 16S rRNA gene. For microarray validation, 22 strains from the American Type Culture Collection or other culture collections and 50 milk samples were tested. The results demonstrated high specificity, with sensitivity as low as 6 fmol. Moreover, the ligation detection reaction-universal array assay allowed for the identification of Mycoplasma spp. in a few hours, avoiding the long incubation times of traditional microbiological identification methods. The universal array described here is a versatile tool able to identify milk pathogens efficiently and rapidly.

Characterization of Staphylococcus aureus strains isolated from Italian dairy products by MALDI-TOF mass fingerprinting

Staphylococcus aureus is a known pathogen, causing serious food‐borne intoxications due to the production of enterotoxins, being otherwise a major cause of mastitis. In this sense, the detection of S. aureus is an important issue for the food industry to avoid health hazards and economic losses. The present work applied MALDI‐TOF MS for the classification of 40 S. aureus strains, 36 isolated from Italian dairy products and four from human samples. All isolated strains were clearly identified as S. aureus by their spectral fingerprints. The peak masses m/z 3444, 5031, and 6887 were determined to be specific biomarkers for S. aureus. Furthermore, clustering of the peak mass lists was successfully applied as a typing method, resulting in eight groups of strains. This is the first time that a detailed spectral comparison was carried out and characteristic peak masses were determined for every spectral group. Three strains exhibited a peak at m/z 6917 instead of m/z 6887, which was related to four polymorphisms in their 16S rRNA sequences. However, the grouping obtained by MALDI‐TOF MS fingerprinting could not be related to toxin production or to the origin of the strains.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources

BackgroundGene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific.ResultsIngenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response.ConclusionsThis meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with low within-herd prevalence of intra-mammary infections in dairy cows: Genotyping of isolates

Staphylococcus aureus is one of the most common mastitis-causing pathogens worldwide. In the last decade, livestock-associated methicillin-resistant S. aureus (LA-MRSA) infections have been described in several species, included the bovines. Hence, this paper investigates the diffusion of MRSA within Italian dairy herds; the strains were further characterized using a DNA microarray, which detects 330 different sequences, including the methicillin-resistance genes mecA and mecC and SCCmec typing. The analysis of overall patterns allows the assignment to Clonal Complexes (CC). Overall 163 S. aureus isolates, collected from quarter milk samples in 61 herds, were tested. MRSA strains were further processed using spa typing. Fifteen strains (9.2%), isolated in 9 herds (14.75%), carried mecA, but none harboured mecC. MRSA detection was significantly associated (P<0.011) with a within-herd prevalence of S. aureus intra-mammary infections (IMI) ≤5%. Ten MRSA strains were assigned to CC398, the remaining ones to CC97 (n=2), CC1 (n=2) or CC8 (n=1). In 3 herds, MRSA and MSSA co-existed: CC97-MRSA with CC398-MSSA, CC1-MRSA with CC8-MSSA and CC398-MRSA with CC126-MSSA. The results of spa typing showed an overall similar profile of the strains belonging to the same CC: t127-CC1, t1730-CC97, t899 in 8 out of 10 CC398. In the remaining 2 isolates a new spa type, t14644, was identified. The single CC8 was a t3092. The SCCmec cassettes were classified as type IV, type V or type IV/V composite. All or most strains harboured the genes encoding the β-lactamase operon and the tetracycline resistance. Streptogramin resistance gene was related to CC398. Enterotoxin and leukocidin genes were carried only by CC1, CC8 and CC97-MRSA. The persistence of MRSA clones characterized by broader host range, in epidemiologically unrelated areas and in dairy herds with low prevalence of S. aureus IMI, might enhance the risk for adaptation to human species.

Two efficient polymeric chemical platforms for oligonucleotide microarray preparation.

ABSTRACT In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step with appropriate silanes (γ-aminopropyltriethoxysilane-APTES or 3-glycid-oxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.