Microarray based genetic profiling of Staphylococcus aureus isolated from abattoir byproducts of pork origin

Many parts of pork meat processing are currently not used for human consumption in Switzerland, although they are of great nutritional value. Therefore, data on the occurrence of pathogenic organisms on byproducts is extremely scarce and the prevalence and population structure of Staphylococcus aureus on meat processing sidestreams is unknown. Hence, abattoir byproducts of pork origin including ear, forefoot, heart, intestine, liver, rib bone, sternum, bladder, stomach, hind foot and tongue originating from six abattoirs were screened for S. aureus. The obtained isolates were investigated by spa typing and DNA microarray analysis to reveal their genomic profile and population structure. The prevalence of S. aureus was generally low with a mean of 8%. In total, 40 S. aureus strains were detected and assigned to 12 spa types (t015, t1491, t1778, t091, t337, t899, t2922, t7439, t1333, t208, t4049, t034) and seven clonal complexes (CC1, CC7, CC9, CC30, CC45, CC49, CC398). Detected enterotoxin genes included sea, seb, sec, seh, sel and egc encoded toxin genes seg, sei, sem, sen, seo, and seu. None of the isolates harbored genes conferring methicillin resistance, but blaZ/I/R genes causing penicillin resistance were frequently found. In addition, strains from CC398 exhibited tetM and tetK, conferring tetracycline resistance. Similarity calculations based on microarray profiles revealed no association of clonal complexes with particular body parts, but revealed a certain correspondence of clonal complex and originating abattoir

Searching for new plastic-degrading enzymes from the plastisphere of alpine soils using a metagenomic mining approach

Plastic materials, including microplastics, accumulate in all types of ecosystems, even in remote and cold environments such as the European Alps. This pollution poses a risk for the environment and humans and needs to be addressed. Using shotgun DNA metagenomics of soils collected in the eastern Swiss Alps at about 3,000 m a.s.l., we identified genes and their proteins that potentially can degrade plastics. We screened the metagenomes of the plastisphere and the bulk soil with a differential abundance analysis, conducted similarity-based screening with specific databases dedicated to putative plastic-degrading genes, and selected those genes with a high probability of signal peptides for extracellular export and a high confidence for functional domains. This procedure resulted in a final list of nine candidate genes. The lengths of the predicted proteins were between 425 and 845 amino acids, and the predicted genera producing these proteins belonged mainly to Caballeronia and Bradyrhizobium. We applied functional validation, using heterologous expression followed by enzymatic assays of the supernatant. Five of the nine proteins tested showed significantly increased activities when we used an esterase assay, and one of these five proteins from candidate genes, a hydrolase-type esterase, clearly had the highest activity, by more than double. We performed the fluorescence assays for plastic degradation of the plastic types BI-OPL and ecovio® only with proteins from the five candidate genes that were positively active in the esterase assay, but like the negative controls, these did not show any significantly increased activity. In contrast, the activity of the positive control, which contained a PLA-degrading gene insert known from the literature, was more than 20 times higher than that of the negative controls. These findings suggest that in silico screening followed by functional validation is suitable for finding new plastic-degrading enzymes. Although we only found one new esterase enzyme, our approach has the potential to be applied to any type of soil and to plastics in various ecosystems to search rapidly and efficiently for new plastic-degrading enzymes.ISSN:1932-620

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Plastic materials, including microplastics, accumulate in all types of ecosystems, even in remote and cold environments such as the European Alps. This pollution poses a risk for the environment and humans and needs to be addressed. Using shotgun DNA metagenomics of soils collected in the eastern Swiss Alps at about 3,000 m a.s.l., we identified genes and their proteins that potentially can degrade plastics. We screened the metagenomes of the plastisphere and the bulk soil with a differential abundance analysis, conducted similarity-based screening with specific databases dedicated to putative plastic-degrading genes, and selected those genes with a high probability of signal peptides for extracellular export and a high confidence for functional domains. This procedure resulted in a final list of nine candidate genes. The lengths of the predicted proteins were between 425 and 845 amino acids, and the predicted genera producing these proteins belonged mainly to Caballeronia and Bradyrhizobium. We applied functional validation, using heterologous expression followed by enzymatic assays of the supernatant. Five of the nine proteins tested showed significantly increased activities when we used an esterase assay, and one of these five proteins from candidate genes, a hydrolase-type esterase, clearly had the highest activity, by more than double. We performed the fluorescence assays for plastic degradation of the plastic types BI-OPL and ecovio® only with proteins from the five candidate genes that were positively active in the esterase assay, but like the negative controls, these did not show any significantly increased activity. In contrast, the activity of the positive control, which contained a PLA-degrading gene insert known from the literature, was more than 20 times higher than that of the negative controls. These findings suggest that in silico screening followed by functional validation is suitable for finding new plastic-degrading enzymes. Although we only found one new esterase enzyme, our approach has the potential to be applied to any type of soil and to plastics in various ecosystems to search rapidly and efficiently for new plastic-degrading enzymes.</div

Amino acid sequences of the proteins from candidate genes A to I and from the positive control gene Z.

The signal peptides are indicated in red letters. (PDF)</p

<i>Escherichia coli</i> codon optimized nucleotide sequences of candidate genes A to I and the positive control gene Z used for cloning.

Escherichia coli codon optimized nucleotide sequences of candidate genes A to I and the positive control gene Z used for cloning.</p

P2Rank prediction for the positive control protein Z (PLA depolymerase PlaM4; Mayumi et al. 2008).

The main pocket on the structure is shown in red. Pocket score: 13.9, probability score: 0.72, amino acids count: 23, pocket conservation score: 1.66. (TIF)</p

Model predictions for the protein from candidate gene F (hydrolase-type esterase).

(A) AlphaFold2 prediction of the protein structure. Blue corresponds to very high (>90%), light blue to high (80%), green to moderate (70%), yellow to low (60%), and red to very low ((B) P2Rank prediction for the AlphaFold2 model. The main pocket on the structure is shown in red.</p

Workflow for functional validation of the proteins from candidate genes.

For abbreviations see the Materials and Methods section.</p

SDS-PAGE of proteins from transformed <i>Escherichia coli</i> strains containing candidate genes A, B, C, F and H before and after induction of expression with IPTG (isopropyl ß-D-1-thiogalactopyranoside).

Red arrows indicate the proteins of the candidate genes bound to the maltose-binding protein (43 kDa): A: 122 kDa, B: 92 kDa, C: 97 kDa, F: 88 kDa, H: 134 kDa. (TIF)</p