High-Throughput, Sequence-Based Analysis of the Microbiota of Greek Kefir Grains from Two Geographic Regions

Pozadina istraživanja. Kefir je prirodni probiotički napitak koji se tradicionalno dobiva fermentacijom mlijeka s kefirnim zrncima. Kefirna zrnca sadržavaju združenu populaciju bakterija i kvasaca uklopljenih u proteinsko-polisaharidni matriks. Geografsko porijeklo kefirnih zrnaca može bitno utjecati na njihov mikrobni sastav, a time i na svojstva dobivenog napitka. Iako u literaturi nalazimo detaljne podatke o sastavu kefirnih zrnaca iz različitih geografskih područja, nema dostupnih informacija o mikrobiomu grčkog kefira. Stoga je svrha ovoga rada bila ispitati strukturu i raznolikost bakterijske zajednice grčkih kefirnih zrnaca. Eksperimentalni pristup. Metodom sekvenciranja visoke propusnosti ispitani su struktura i raznolikost sastava kefirnih zrnaca iz dvaju geografski udaljenih područja u Grčkoj, radi boljeg razumijevanja raznolikosti bakterijskih populacija u zrncima. Ovaj pristup ispitivanju metagenoma, koji ne ovisi o kulturi, za sekvenciranje koristi gen 16S rRNA sa segmentima oko V4 varijabilne regije. Rezultati i zaključci. Koljeno Firmicutes (kojem pripadaju i bakterije mliječno-kiselog vrenja), s 99 % sekvenci u oba uzorka kefirnih zrnaca, izrazito je dominiralo među identificiranim bakterijama. Na razini porodice, sekvence Lactobacillaceae bile su zastupljene s više od 98 % jedinica (engl. operational taxonomic units, OTU), zatim slijede: Ruminococcaceae, Lahnospiraceae, Bacteroidaceae, te ostale, manje zastupljene porodice. Relativno je mali broj vrsta bakterija dominirao, od kojih je nazastupljenija bila bakterija Lac¬tobacillus kefiranofaciens, i to s 95,0 % jedinica u kefiru A i 96,3 % jedinica u kefiru B. Međutim, pronađena je vrlo raznolika subdominantna populacija u oba uzorka zrnaca, uključujući i vrste bakterija koje su dosad bile povezivane s probavnim traktom ljudi i životinja. a za neke od njih se smatra da imaju probiotička svojstva (Faecalibacterium spp., Bacteroides spp. i Blautia spp.). Razlike u profilima bakterijskih vrsta između dvaju uzoraka kefirnih zrnaca su vrlo male, što potvrđuje izrazitu homogenost uzoraka usprkos njihovoj velikoj geografskoj udaljenosti. Novina i znanstveni doprinos. Ovaj rad po prvi put detaljno istražuje raznolikost bakterijskih vrsta i njihovo bogatstvo u grčkom kefiru.Research background. Kefir is a natural probiotic drink traditionally produced by milk fermentation using kefir grains. Kefir grains are composed of a complex population of bacteria and yeasts embedded in a polysaccharide-protein matrix. The geographic origin of kefir grains may largely influence their microbial composition and the associated kefir drink properties. Although the detailed bacterial composition of kefir grains from several geographic regions has been reported, to date, analogous data about the microbiome of Greek kefir are lacking. Hence, the aim of this study is to investigate the structure and the diversity of the bacterial community of Greek kefir grains. Experimental approach. The bacterial community structure and diversity of two different kefir grains from distant geographic regions in Greece were examined via high-throughput sequencing analysis, a culture-independent metagenomic approach, targeting the 16S rRNA V4 variable region, in order to gain a deeper understanding of their bacterial population diversities. Results and conclusions. Firmicutes (a phylum that includes lactic acid bacteria) was strikingly dominant amongst the identified bacterial phyla, with over 99 % of the sequences from both kefir grains classified to this phylum. At the family level, Lactobacillaceae sequences accounted for more than 98 % of the operational taxonomic units (OTUs), followed by Ruminococcaceae, Lahnospiraceae, Bacteroidaceae and other bacterial families of lesser abundance. Α relatively small number of bacterial genera dominated, with Lactobacillus kefiranofaciens being the most abundant in both kefir grains (95.0 % of OTUs in kefir A and 96.3 % of OTUs in kefir B). However, a quite variable subdominant population was also present in both grains, including bacterial genera that have been previously associated with the gastrointestinal tract of humans and animals, some of which are believed to possess probiotic properties (Faecalibacterium spp., Bacteroides spp., Blautia spp.). Differences among the bacterial profiles of the two grains were very small indicating a high homogeneity despite the distant geographic origin. Novelty and scientific contribution. This is the first study to deeply explore and report on the bacterial diversity and species richness of Greek kefir

Occurrence and antibiotic resistance of enterotoxigenic Staphylococcus aureus in raw ovine and caprine milk in Greece

International audienceAbstractOvine (n = 140) and caprine (n = 35) raw bulk tank milk samples from farms in central Greece were examined for the occurrence of enterotoxigenic Staphylococcus aureus. The S. aureus isolates were screened for staphylococcal enterotoxin (SE) production, the presence of enterotoxin genes, antibiotic resistance (AR), and methicillin resistance. S. aureus was isolated from 24.3% and 31.4% of ovine and caprine milk samples, respectively. Among the S. aureus isolates of ovine milk (n = 34) and caprine (n = 11) milk, the enterotoxigenic (SEA-SED) isolates were 21 (61.8%) and 7 (63.6%) for the ovine and caprine milk, respectively. Most toxigenic isolates harbored more than one toxin gene and a total of 11 distinct toxinotypes were detected. The most frequent toxin-gene combinations were “sec, tst” (8 isolates), “seb, seg, sei, tst” (4), “seb, seg, sei” (3), and “seb” (3). Six isolates displayed multiple AR towards up to five antimicrobials. Among ovine milk isolates, the highest resistance frequency was observed towards erythromycin (11.8% of the isolates) and tetracycline (8.8%). Among caprine milk isolates, the most frequent resistance was observed towards erythromycin (18.2%). One methicillin-resistant S. aureus (MRSA) isolate was detected in an ovine milk sample and belonged to spa type t4038. This spa type has been isolated for the first time in Greece and, to our knowledge, has not been previously reported among MRSA isolates from raw milk or dairy products worldwide

The role of high frequency intra-daily data, daily range and implied volatility in multi-period Value-at-Risk forecasting

In this paper, we assess the informational content of daily range, realized variance, realized bipower variation, two time scale realized variance, realized range and implied volatility in daily, weekly, biweekly and monthly out-of-sample Value-at-Risk (VaR) predictions. We use the recently proposed Realized GARCH model combined with the skewed student distribution for the innovations process and a Monte Carlo simulation approach in order to produce the multi-period VaR estimates. The VaR forecasts are evaluated in terms of statistical and regulatory accuracy as well as capital efficiency. Our empirical findings, based on the S&P 500 stock index, indicate that almost all realized and implied volatility measures can produce statistically and regulatory precise VaR forecasts across forecasting horizons, with the implied volatility being especially accurate in monthly VaR forecasts. The daily range produces inferior forecasting results in terms of regulatory accuracy and Basel II compliance. However, robust realized volatility measures such as the adjusted realized range and the realized bipower variation, which are immune against microstructure noise bias and price jumps respectively, generate superior VaR estimates in terms of capital efficiency, as they minimize the opportunity cost of capital and the Basel II regulatory capital. Our results highlight the importance of robust high frequency intra-daily data based volatility estimators in a multi-step VaR forecasting context as they balance between statistical or regulatory accuracy and capital efficiency

Role of the Glycine Betaine and Carnitine Transporters in Adaptation of Listeria monocytogenes to Chill Stress in Defined Medium

The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4°C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4°C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature

Three Transporters Mediate Uptake of Glycine Betaine and Carnitine by Listeria monocytogenes in Response to Hyperosmotic Stress

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress

Identification of OpuC as a Chill-Activated and Osmotically Activated Carnitine Transporter in Listeria monocytogenes

The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. (13)C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress

Genetic characterization of two methicillin-resistant Staphylococcus aureus spa type t127 strains isolated from workers in the dairy production chain in Greece

Methicillin-resistant Staphylococcus aureus (MRSA) constitutes a constant threat for the public health. Aim of the present study was to analyse the whole genome sequences of two MRSA strains belonging to Staphylococcus protein A (spa) type t127 isolated from humans working in two distantly located dairy production farms in Greece. MRSA strains were isolated from the nasal cavity of a food handler in a milk industry in Epirus, northwestern Greece (E-MRSA), and a person working in a cattle farm in Thrace, northeastern Greece (T-MRSA). Whole genome sequences taken using next generation sequencing were analysed for resistance and virulence genes applying various bioinformatic tools. Both isolates were assigned to ST1-IVa-t127 type, and they were transferring genes conferring resistance to tetracycline, beta-lactams, and aminoglycosides; T-MRSA was carrying additional genes leading to macrolide, lincosamide and streptogramin B (MLSB) resistance. Both isolates were carrying three plasmid replicon types, reps, rep7 and rep16, while T-MRSA harboured also rep10 and rep15. E-MRSA carried scn and sak genes which were absent from T-MRSA. In conclusion, the genetic characterization of two unrelated ST1-IVa-t127 MRSA strains isolated from humans in close contact with livestock in Greece can be used as basis for further epidemiological and evolutionary studies