Antimicrobial Resistance of Common Zoonotic Bacteria in the Food Chain: An Emerging Threat

Antimicrobial resistance in the food chain is currently a subject of a major interest. The excessive use or rather misuse of antimicrobials coupled with a poor hygiene in the food production chain has led to a rise of resistant zoonotic bacteria, commonly transmitted by food. They pose a serious threat to human health. Campylobacteriosis is the leading bacterial food-borne illness and most commonly reported zoonosis in humans in the European Union for more than a decade. Salmonellosis is most frequently diagnosed in food-borne outbreaks. Fluoroquinolones are considered as critically important for treatment of severe cases of both zoonoses in humans. Due to an extremely prevalent resistant isolates, especially from broilers and meat, also the treatment of human Campylobacter infections with fluoroquinolones has become compromised. Salmonella isolates from poultry and poultry meat tend to be highly resistant to fluoroquinolones as well. Beside the resistance to this group of antibiotics, the threat of multiple drug resistant (MDR) Campylobacter and Salmonella strains is discussed in the light of most recent reports of animal, food and human clinical surveillance systems

Prevalence of antimicrobial resistance in bacteria intentionally added to agro-food chain

Protimikrobna zdravila so temelj moderne medicine, obenem pa njihovo učinkovitost ogroža naglo širjenje odpornosti. Tudi mlečnokislinske bakterije (MKB) in bifidobakterije so bile prepoznane kot rezervoar genov za odpornost (ARG), vendar dejansko tveganje, ki ga predstavljajo sevi, ki jih namensko dodajamo v agroživilsko verigo (SZPK), še ni raziskano. Na to vprašanje smo skušali odgovoriti v naši raziskavi. V analize smo vključili štiri skupine MKB in bifidobakterij ̶ poleg sevov iz starterskih kultur ali prehranskih dopolnil z živimi bakterijami (probiotikov) tudi izolate iz humanega mleka ali kolostruma, iz črevesne sluznice ali blata ter iz fermentiranih živil. Fenotipsko občutljivost smo ugotavljali za 474 sevov z mikrodilucijo in pokazali, da je odpornost pogostejša pri črevesnih izolatih. ARG in mobilne elemente (MGE) smo proučevali v celih genomih 1114 sevov s primerjalno genomiko. Ugotovili smo, da je 13,8 % sevov skupine SZPK vsebovalo pridobljene ARG, pri čemer so prevladovali tisti za tetraciklin, pogosto pa smo našli tudi mutacije. 75,5 % pridobljenih ARG smo povezali z MGE, katerih potencial za širjenje in vivo smo ovrednotili s pomočjo preiskovanja metagenomskih sekvenc. Ocenjujemo, da skupina sevov SZPK ne predstavlja pomembnega tveganja za širjenje odpornosti, saj so pridobljeni ARG in MGE pri teh sevih manj pogosti kot pri skupini humane črevesne sluznice/blata, vendar je pozornost potrebno nameniti predvsem posameznim probiotičnim sevom, ki vsebujejo elemente, za katere smo pokazali, da imajo potencial za širjenje med črevesno mikrobioto. Ti elementi so redkejši pri izolatih naravne mikrobiote iz fermentiranih živil in neznačilni za starterske kulture.Antimicrobials have become one of the foundations of modern medicine, yet the rapid spread of resistance threatens their effectiveness. Lactic acid bacteria (MKB) and bifidobacteria were recognised as a reservoir of resistance, but the actual risk posed by strains intentionally introduced into the agro-food chain (SZPK) has not yet been assessed. The aim of our study was to clarify whether these strains present a risk in terms of resistance. Four groups of MKB and bifidobacteria were included in the analyses ̶ in addition to strains from starter cultures or dietary supplements with live bacteria (probiotics), isolates from human milk or colostrum, intestinal mucosa or faeces, and fermented products. Phenotypic susceptibility was determined for 474 strains by microdilution and was most common in intestinal isolates. Antimicrobial resistance genes (ARG) and mobile genetic elements (MGE) were characterised in whole genomes of 1114 strains using comparative genomics. The results revealed that 13.8 % of the strains in the SZPK group contained acquired ARG, most frequently for tetracycline, however, mutations were also common. 75.5 % of acquired ARG were associated with MGE, whose potential for in vivo transmissibility was evaluated by examining metagenomic sequences. We confirmed that ARG and MGE were not as common in the SZPK group as in strains from human intestinal mucosa/faeces, suggesting that this group does not pose a significant threat. However, attention should be paid especially to individual probiotic strains that contain elements that have been shown to have high potential for transferability in the gut microbiota. These elements were uncommon in isolates of natural microbiota from fermented products and were atypical of starter cultures

Characterization of substrate specificity and type IV pili of gut bacteria

Pili tipa IV (PT4) so proteinski filamenti na površini nekaterih bakterij in arhej, ki sodelujejo v različnih procesih, med drugim so pri nekaterih bakterijah domnevno pomemben faktor pritrjanja na netopne substrate. V pričujoči študiji smo analizirali sekvence (osnutkov) genomov 118 taksonomsko raznolikih bakterijskih sevov, pretežno izoliranih iz prebavil (kot kontrolo smo vključili tudi nekaj sevov iz zunanjih ekosistemov), z namenom: (1) ugotoviti, ali lahko z »in silico« analizo nabora genov, ki kodirajo encime, aktivne na ogljikovih hidratih (CAZy), pravilno napovemo substratno specifičnost izbranih sevov, (2) ugotoviti, ali obstajajo povezave med nabori CAZy in prisotnostjo ter zgradbo (gruč) genov, ki domnevno kodirajo PT4a. Z algoritmom HMMer smo v osnutkih genomov izbranih sevov identificirali zapise za encime, ki so aktivni na ogljikovih hidratih (CAZy). Na osnovi števila domnevnih monospecifičnih CAZy smo napovedali sposobnost sevov za razgradnjo posameznega substrata. Da se nabor zapisov za CAZy dejansko odraža v substratni specifičnosti sevov, smo nato preverili (in potrdili) še eksperimentalno, z gojenjem 5 izbranih sevov. V drugem delu smo z algoritmoma BLAST in HMMer identificirali gene, ki kodirajo komponente PT4a (gene pil), ter jih glede na arhitekturno ohranjenost in aminokislinska zaporedja ohranjenih genov razvrstili v podskupine. Analiza je pokazala dobro ohranjene genske gruče pil pri mnogih celulolitičnih bakterijah iz različnih habitatov, vendar pa je slednje najti tudi pri nekaterih sevih z drugačnimi napovedanimi specifičnostmi.Type IV pili (TFP) are surface filamentous proteins of some bacteria and archea. Among numerous different functions they presumably have an important role in binding to insoluble fiber. In the present study we analyzed (draft) genome sequences of 118 taxonomically diverse bacterial strains, mostly isolated from gut (and some other ecosystems to control for the effect of the environment). The aim of our study was to (1): determine whether »in silico« analysis of genes encoding Carbohydrate Active enZymes (CAZy) reflects in substrate specificity of the selected strains, and (2) determine possible connections among CAZy genes and presence (structure) of putative pil gene clusters. CAZy genes were identified by HMMer algorithm. The number of putative monospecific CAZy was used as an indicator for substrate specificity of the strains. As a “proof of concept”, we verified (and confirmed) that a set of CAZy genes does predict the substrate specificity of 5 selected strains. In the second part of the study pil genes were identified by HMMer and BLAST algorithms. Conserved pil gene clusters were divided into subgroups according to the architecture conservation and amino acid similarities between sequences of pil genes. The results have shown that most of the (confirmed) cellulolytic strains encoded conserved pil gene clusters. However, these were also found in some strains with different predicted specificities

EFFICIENCY ANALYSIS AND PERCEIVED SERVICE QUALITY OF ALTERNATIVE AND COMPLEMENTARY MEDICINE FROM CONSUMERS PERSPECTIVE

Komplementarna in alternativna medicina (KAM) se v svetu vedno pogosteje uporablja kot dopolnilna oblika zdravljenja tradicionalne medicine. Namen našega magistrskega dela je bil oceniti stanje KAM v Sloveniji. Na osnovi primerjave zdravstvene zakonodaje v ZDA, Nemčiji in Sloveniji smo ugotovili, da je slovenska zakonodaja najmanj dodelana. Primerjava treh izbranih podjetij, ki izvajajo storitve s področja KAM je pokazala, da dejavnost izvajajo poleg drugih osnovnih dejavnosti in so ekonomsko gledano precej učinkovita. Z empirično raziskavo izvedeno s pomočjo anketnega vprašalnika smo ugotovili, da je več kot tretjina od 220 vprašanih poznala posamezne metode KAM, a zdravilca še niso obiskali. Med štirimi skupinami zdravilskih metod je 160 anketiranih uporabnikov KAM najbolje poznalo in menilo, da se najbolj pogosto uporabljajo biološko osnovane metode. Preučevanje obstoja vpliva posameznih dejavnikov na poznavanje in mnenje o pogostosti uporabe zdravilskih metod je pokazalo, da v večini primerov ne obstajajo statistično značilne razlike glede na posamezne dejavnike. Tam, kjer so se razlike pokazale, je šlo največkrat za razlike glede na spol in izobrazbo, v korist žensk in bolj izobraženih. Tako je lik slovenskega uporabnika KAM enovit za moške in ženske. Največ uporabnikov KAM je starih med 20 in 40 let, ima visokošolsko izobrazbo, letno zasluži do 10.000 evrov, največkrat si zdravi prehlad in je za storitve KAM pripravljena letno potrošiti do 200 evrov. Zadovoljstvo uporabnikov storitev KAM smo preverili s pomočjo modela ZPU (Zdravilec-Prostor-Učinek), ki smo ga izpeljali iz modela SERVQUAL. S pomočjo faktorske analize in multiple regresije smo ugotovili, da učinek terapije lahko ocenimo s karakteristikami zdravilca in prostora, v katerem se je terapija izvajala. Glede na velik interes uporabnikov za zdravljenje s KAM in visoke ocene zadovoljstva s tovrstnimi storitvami s strani uporabnikov, je pričakovati, da bo KAM še naprej, vsaj v takem obsegu kot do sedaj, prisotna na slovenskem tudi v prihodnje.Complementary and alternative medicine (CAM) has become a popular additional form of treatment throughout the world and within the field of traditional medicine. The aim of the master thesis is to estimate the state of CAM in Slovenia. When comparing health legislation in Germany, the USA and Slovenia, we found out that the Slovene is the least advanced. The analysis of three companies dealing with CAM has shown that the services are offered along with other basic activities and are – from an economic point of view – quite effective. A questionnaire has proven that more than a third of the 220 people questioned are familiar with certain CAM methods, nevertheless, they have not visited a practitioner yet. Among four different healing methods, 160 people claimed to be most familiar with CAM and thought that biologically based therapies are used most commonly. Studying the influence of certain factors on the knowledge and opinion concerning the usage of healing methods has shown that in most cases there are no statistically significant differences regarding those factors. However, some appeared when comparing gender and education – the results were in favour of women and people with a higher education. Consequently, the model of the Slovene CAM user applies to both, men and women. Most CAM users are 20 to 40 years old, well educated, earn approximately 10.000 Euros per year, most commonly treat a cold and are willing to spend up to 200 Euros per year for CAM services. Whether the users are satisfied with CAM services has been determined with the PRE model (practitioner-room-efficiency), which is based on the SERVQUAL model. The efficiency of the therapy is possible to express by the personal practitioner’s characteristics and the room properties. This findings have been leading out using factor analysis and multiple regression. As the users are very interested in CAM and satisfied with the offered services, we can expect that CAM will continue to be a possible form of treatment in Slovenia (perhaps even to a higher extent)

When the customer and the wine shelf meet

This study examines the importance of wine characteristics when choosing wine, such as the shape and colour of the bottle, type of wine cork, information on the wine label, price and ethnocentrism. Three research questions were proposed in this paper. A quantitative online survey in the form of 458 participants was carried out in Slovenia. Participants were asked questions relating to their wine consumption behaviour. The results of this study show the Spearman\u27s correlation coefficient between ethnocentrism and country-of-origin importance. These findings benefit winemakers who want to improve their wine sales and learn what characteristics of the wine label are significant

Harnessing omics big data in nine vertebrate species by genome-wide prioritization of sequence variants with the highest predicted deleterious effect on protein function

Harnessing the genomics big data requires innovation in how we extract and interpret biologically relevant variants. Currently, there is no established catalog of prioritized missense variants associated with deleterious protein function phenotypes. We report in this study, to the best of our knowledge, the first genome-wide prioritization of sequence variants with the most deleterious effect on protein function (potentially deleterious variants [pDelVars]) in nine vertebrate species: human, cattle, horse, sheep, pig, dog, rat, mouse, and zebrafish.The analysis was conducted using the Ensembl/BioMart tool. Genes comprising pDelVars in the highest number of examined species were identified using a Python script. Multiple genomic alignments of the selected genes were built to identify interspecies orthologous potentially deleterious variants, which we defined as the ‘‘ortho-pDelVars.’’ Genome-wide prioritization revealed that in humans, 0.12% of the known variants are predicted to be deleterious. In seven out of nine examined vertebrate species, the genes encoding the multiple PDZ domain crumbs cell polarity complex component (MPDZ) and the transforming acidic coiled-coil containing protein 2 (TACC2) comprise pDelVars. Five interspecies ortho-pDelVars were identified in three genes.These findings offer new ways to harness genomics big data by facilitating the identification of functional polymorphisms in humans and animal models and thus provide a future basis for optimization of protocols for whole genome prioritization of pDelVars and screening of orthologous sequence variants. The approach presented here can inform various postgenomic applications such as personalized medicine and multiomics study of health interventions (iatromics)

Characterization of antimicrobial resistance in lactobacilli and bifidobacteria used as probiotics or starter cultures based on integration of phenotypic and in silico data

Lactic acid bacteria and bifidobacteria deliberately introduced into the food chain may act as a reservoir of antimicrobial resistance genes (ARGs), which is considered a safety concern. In the present study, resistance to antimicrobials of commercial probiotic strains, probiotic candidate strains, and starter cultures (n = 20) was characterised based on integration of phenotypic and in silico data. Minimum inhibitory concentrations (MICs) of 16 antimicrobials were determined for lactobacilli and bifidobacteria that were isolated from pharmaceutical products or obtained from the manufacturers or culture collections. Using different databases and bioinformatic tools, we predicted ARGs, mutations, genomic islands, and mobile genetic elements (MGEs) in their whole genome sequences. In addition, a comprehensive in silico analysis of the prevalence of the tetW gene and its genetic environment across lactobacilli and bifidobacteria (n = 1423) was conducted. Several strains exhibited phenotypic resistance to kanamycin, tetracycline, chloramphenicol, quinupristin-dalfopristin, ciprofloxacin, or neomycin. These resistances, however, did not always correspond to the presence of ARGs and vice versa. We detected an acquired tetW gene in four commercial strains of Bifidobacterium animalis subsp. lactis, whereas homologs of antimicrobial resistance (AR) proteins were predicted in all 20 proteomes. The prevalence of the tetW gene, which was often flanked by MGEs, was higher in analysed bifidobacteria (31.9%) than lactobacilli (6.3%). In addition, sequences flanking tetW were associated with putative genomic islands and were conserved in several strains, including potential pathogens. Our findings provide an insight into AR of probiotics, probiotic candidates, and starter cultures with an emphasis on tetracycline and into the safety of these strains in the context of AR

When the Customer and the Wine Shelf Meet: Factors of Ethnocentrism When Selecting a Bottle of Wine

This study examines the importance of wine characteristics when choosing wine, such as the shape and colour of the bottle, type of wine cork, information on the wine label, price and ethnocentrism. Three research questions were proposed in this paper. A quantitative online survey in the form of 458 participants was carried out in Slovenia. Participants were asked questions relating to their wine consumption behaviour. The results of this study show the Spearman’s correlation coefficient between ethnocentrism and country-of-origin importance. These findings benefit winemakers who want to improve their wine sales and learn what characteristics of the wine label are significant

Genetic Polymorphisms Modifying Oxidative Stress Are Associated with Disease Activity in Rheumatoid Arthritis Patients

Reactive oxygen and nitrogen species are involved in the pathology of rheumatoid arthritis (RA). Polymorphisms in genes coding for superoxide dismutases (SOD2 and SOD3), catalase (CAT), tumor necrosis factor-alpha (TNFA) and inducible NO synthase (NOS2A) may influence RA activity. We determined SOD2 Ala-9Val, SOD3 Arg213Gly, CAT C-262T, TNFA G-308A, TNFA C-857T and NOS2A (CCTTT)n polymorphisms in 327 RA patients. Carriers of CAT -262T and TNFA -308A allele had lower mean disease activity score of 28 joint count (DAS28) values than patients with CAT -262CC and TNFA -308GG genotypes (p = 0.014 and p = 0.046, respectively). Patients with the combination of CAT -262T and TNFA -308A allele had lower mean DAS28 values and a higher probability for low disease activity than non-carriers (p = 0.003, OR = 3.585, 95% CI = 1.538–8.357). Our results suggest that CAT and TNFA polymorphisms alone and in combination influence the activity of RA