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.

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.

Presence of foodborne pathogens, extended-spectrum ß-lactamase -producing Enterobacteriaceae, and methicillin-resistant Staphylococcus aureus in slaughtered reindeer in northern Finland and Norway.

BACKGROUND: Various food-producing animals were recognized in recent years as healthy carriers of bacterial pathogens causing human illness. In northern Fennoscandia, the husbandry of semi-domesticated reindeer (Rangifer tarandus tarandus) is a traditional livelihood and meat is the main product. This study determined the presence of selected foodborne pathogens, methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in healthy semi-domesticated reindeer at slaughter in northern Finland and Norway. RESULTS: All 470 reindeer fecal samples tested negative for Salmonella spp., whereas L. monocytogenes was detected in 3%, Yersinia spp. in 10%, and Shiga toxins genes (stx1 and/or stx2) in 33% of the samples. Listeria monocytogenes isolates belonged to the serotype 1/2a (14/15) and 4b, Yersinia spp. were identified mainly as Y. kristensenii (30/46) and Y. enterocolitica (8/46), and stx2 predominated among the Shiga toxin genes (stx2 alone or in combination with stx1 was found in 25% of the samples). With regard to the frequency and distribution of stx1/stx2, striking differences were evident among the 10 different areas of origin. Hence, reindeer could constitute a reservoir for Shiga toxin-producing E. coli (STEC), but strain isolation and characterization is required for verification purposes and to assess the potential human pathogenicity of strains. On the other hand, the favorable antibiotic resistance profiles (only 5% of 95 E. coli isolates were resistant to one or more of the tested antibiotics) and the absence of MRSA and ESBL-producing Enterobacteriaceae (when applying selective methods) suggest only a limited risk of transmission to humans. CONCLUSIONS: Healthy semi-domesticated reindeer in northern Finland and Norway can be carriers of certain bacterial foodborne pathogens. Strict compliance with good hygiene practices during any step of slaughter (in particular during dehiding and evisceration) is therefore of central importance to avoid carcass contamination and to prevent foodborne pathogens from entering the food chain.

New dominant spa type t2741 in livestock-associated MRSA (CC398-MRSA-V) in Finnish fattening pigs at slaughter.

BACKGROUND: The emergence of livestock-associated MRSA has become a growing public health concern worldwide. Studies elucidating the population structure, as well as resistance phenotypes and virulence gene profiles of livestock-associated MRSA strains are needed to improve risk assessment and to develop effective control measures. The objective of this study therefore was to determine i) clonal complexes and spa types, as well as ii) resistance phenotypes and iii) virulence and resistance gene profiles of livestock-associated MRSA isolated from Finnish fattening pigs at slaughter. METHODS: Fifty MRSA isolates collected from Finnish fattening pigs at slaughter were characterized by spa typing and DNA microarray profiling. In addition, antimicrobial susceptibility testing was performed using the Kirby Bauer disk diffusion method. RESULTS: MRSA isolates were assigned to clonal complexes CC1 (n = 4) and CC398 (n = 46). One dominant spa type (t2741) was present in 33 out of 50 investigated isolates, originating from 15 out of 18 farms. The remaining isolates were assigned to spa types t034 (n = 7), t108 (n = 5), and t011 (n = 1). Although each herd exhibited isolates assigned to one clonal complex only, five herds harbored MRSA isolates of either two or three different spa types. All tested MRSA isolates were phenotypically resistant to penicillin, oxacillin, cefoxitin, and tetracycline. With the exception of the isolates assigned to t108, all isolates exhibited resistance to clindamycin. On the genomic level, all isolates exhibited mecA, blaZ/I/R, and tetK, and were assigned to SCCmec type V. Many isolates also harbored tetM (46/50 isolates), lnuB (41/50 isolates), ermB (26/50 isolates), and one isolate was positive for aadD. DNA microarray profiling showed that all isolates of the dominant CC398/t2741 MRSA-V type belonged to agr type I, capsule type 5, and were negative for fnbB. Interestingly, one isolate of CC398/t2741 MRSA-V was agr negative and also lacked hld. CONCLUSIONS: A new dominant LA-MRSA clone (CC398/t2741, SCCmec type V) was identified among fattening pigs in Finland. This is the first study identifying t2741 as a common spa type in LA-MRSA in pigs.