Karakterizacija bakteriocinogenih sojeva bakterija mliječne kiseline iz tradicionalnog slovenskog sira ‘Tolminc’

The purpose of this research was to examine traditional Slovenian ‘Tolminc’ cheese for the presence of lactic acid bacteria that produce several bacteriocins. The presence of gene determinants for different bacteriocins in this type of cheese and in the cultivable population of ‘Tolminc’ microbiota, have already been demonstrated, as well as its antimicrobial activity. Due to the difficulties in connecting the presence of gene determinants for bacteriocins with the observed antimicrobial activity it was decided to examine in this study the same features on the level of individual bacteriocinogenic strains. Like in previous results, enterococci and their bacteriocins prevailed in cheese microbial consortia. None of isolated strains inhibited growth of Staphylococcus aureus, while the other indicator strains were inhibited in a strain specific manner. Most of isolated strains carried gene determinants for cytolysin. On the basis of gene determinants for bacteriocins, antimicrobial activity, phenotyping by PhP (PhenePlateTM) system and PCR identification, some similarities found were among Enterococcus isolates.Cilj ovog rada bio je provjeriti prisutnost sojeva bakterija mliječne kiseline koji proizvode različite bakteriocine u tradicionalnom slovenskom siru ‘Tolminc’. Prisutnost genskih determinanti za pojedine bakteriocine u ovoj vrsti sira i izoliranoj populaciji mikrobiote sira ‘Tolminc’ već je bila prikazana, a također i njihova antimikrobna aktivnost. Zbog poteškoća pri povezivanju detektiranih genskih determinanti za bakteriocine i antimikrobne aktivnosti, odlučeno je u ovom radu analizirati ista svojstva i kod pojedinačnih bakteriocinogenih sojeva. Slično prethodnim istraživanjima, enterokoki i njihovi bakteriocini najbolje su bili zastupljeni. Nijedan od izoliranih sojeva nije inhibirao bakteriju Staphylococcus aureus, dok su ostale indikatorske mikroorganizme inhibirali različito. Većina sojeva nosila je genske determinante za bakteriocin citolizin. Na temelju genskih determinanti za bakteriocine, antimikrobne aktivnosti, fenotipizacije s PhP (PhenePlateTM) sistemom i identifikacije roda i vrste sojeva, mogu se naći neke sličnosti između Enterococcus sojeva

Clinical characteristics of patients with tick-borne encephalitis (Tbe) : A European multicentre study from 2010 to 2017

Funding Information: Conflicts of Interest: W.Z. received financial support from GSK, Pfizer, Merck, and Sanofi for organizing the “Graz Vaccination Day”. Funding Information: Funding: This study was financially supported by Land Steiermark (Office of the Regional Government of Styria, Department of Health Care and Science, Unit of Science and Research, Austria). D.R., L.K. and M.P. were supported by the Czech Ministry of Health (grant No. NV19-05-00457). P.B. and F.S. were supported by the Slovenian Research Agency (grant. No P3-0296). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Tick-borne encephalitis (TBE) virus is a major cause of central nervous system infections in endemic countries. Here, we present clinical and laboratory characteristics of a large international cohort of patients with confirmed TBE using a uniform clinical protocol. Patients were recruited in eight centers from six European countries between 2010 and 2017. A detailed description of clinical signs and symptoms was recorded. The obtained information enabled a reliable classification in 553 of 555 patients: 207 (37.3%) had meningitis, 273 (49.2%) meningoencephalitis, 15 (2.7%) meningomyelitis, and 58 (10.5%) meningoencephalomyelitis; 41 (7.4%) patients had a peripheral paresis of extremities, 13 (2.3%) a central paresis of extremities, and 25 (4.5%) had single or multiple cranial nerve palsies. Five (0.9%) patients died during acute illness. Outcome at discharge was recorded in 298 patients. Of 176 (59.1%) patients with incomplete recovery, 80 (27%) displayed persisting symptoms or signs without recovery expectation. This study provides further evidence that TBE is a severe disease with a large proportion of patients with incomplete recovery. We suggest monitoring TBE in endemic European countries using a uniform protocol to record the full clinical spectrum of the disease.publishersversionPeer reviewe

How Can We Advance Integrative Biology Research in Animal Science in 21st Century?:Experience at University of Ljubljana from 2002 to 2022

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health

Clinical characteristics and immune responses in post-encephalitic syndrome after tick-borne encephalitis

Izhodišča. O dejavnikih, ki določajo, kakšna bosta potek in izid klopnega meningoencefalitisa (KME), o (dolgo)trajnih posledicah prebolele bolezni (postencefalitisni sindrom) in o mehanizmih, ki so povezani z neugodnim potekom in izidom bolezni, je malo znanega. Namen. Namen raziskave je opredeliti klinične značilnosti akutne bolezni, ugotoviti, kateri klinični in/ali laboratorijski parametri so povezani s težjim potekom bolezni in/ali neugodnim izidom, oceniti (dolgo)trajne posledice, ki jih pušča prebolela bolezen, in osvetliti osnovne imunske značilnosti KME. Metode dela. V raziskavo smo vključili 717 odraslih bolnikov, ki so bili v obdobju od 1. 1. 2007 do 31. 12. 2012 na Kliniki za infekcijske bolezni in vročinska stanja zdravljeni zaradi KME. Pri podskupini 420 bolnikov, ki smo jih povabili na kontrolni pregled leta 2014, 2–7 let po prebolelem KME, smo ocenili dolgoročni izid bolezni. V raziskavo smo vključili tudi kontrolno skupino oseb, ki so se z bolniki ujemale glede na starost in v preteklosti niso prebolele KME. Imunske odzive smo v obdobju akutne bolezni ocenili v serumu in možganski tekočini, 2 meseca in 2–7 let po preboleli bolezni pa le v serumu. Določili smo koncentracije 24 citokinov/kemokinov, ki zastopajo prirojene ter pridobljene Th1-, Th17- ter B-celične odzive. Rezultati. Multipla regresija je pokazala, da so s težjim potekom KME značilno povezani višja starost bolnikov (razmerje obetov (RO) 1,26, 95 % interval zaupanja (IZ) 1,11–1,44P = 0,001), predhodno cepljenje proti KME (RO 14,23, 95 % IZ 1,72–117,87P = 0,014), nizka koncentracija protiteles IgG proti virusu KME v serumu (RO 0,85, 95 % IZ 0,75–0,96P = 0,009), večja koncentracija levkocitov v krvi (RO 1,45, 95 % IZ 1,13–1,85P = 0,004) ter večja koncentracija C-reaktivnega proteina v serumu (ocenjeni koeficient 1,20, 95 % IZ 0,48–1,91P = 0,001). Pogostost postencefalitisnega sindroma se zmanjšuje s časom in se stabilizira 12 mesecev po preboleli bolezni, ko ima postencefalitisni sindrom tretjina bolnikovkasneje se zmanjšuje le še njegova teža. Neugoden izid 6 mesecev po preboleli bolezni je povezan s koncentracijo levkocitov v možganski tekočini (RO 1,43, 95 % IZ 1,07–1,93, P = 0,017), po 12 mesecih z izidom bolezni po 6 mesecih (RO 497,89, 95 % IZ 39,42–6289,16, P < 0,001) in 2–7 let po preboleli bolezni z izidom bolezni po 12 mesecih (RO 38,56, 95 % IZ 8,40–177,14, P < 0,001). Bolniki so 2&#82107 let po prebolelem KME navedli večje število nespecifičnih simptomov v primerjavi s kontrolnimi preiskovanci, ki niso preboleli KME, prisotnost vsakega od ponujenih simptomov pa bolj pogosto in bolj stalno. Slabše so ocenili tudi kakovost telesnega in duševnega zdravja, vendar so bile razlike med skupinama majhne in niso bile statistično značilne. Koncentracije velike večine citokinov/kemokinov v serumu in možganski tekočini se v času akutne bolezni značilno razlikujejo: koncentracije citokinov/kemokinov prirojenega in Th1-pridobljenega imunskega odziva so večje v možganski tekočini, tiste, povezane s pridobljenim Th17- in B-celičnim imunskim odzivom, pa v serumu. Večina predstavnikov prirojenega in pridobljenega Th1-imunskega odziva je povezana s težo akutne bolezni, z neugodnim izidom bolezni pa nismo dokazali statistično značilnih povezav. Sklep. Raziskava, temelječa na velikem številu dobro definiranih odraslih bolnikov s KME, je omogočila določitev dejavnikov, ki so povezani s težkim potekom in neugodnim izidom KME, ter oceno pogostosti in kliničnih značilnosti postencefalitisnega sindroma. Omogočila je tudi vpogled v imunske značilnosti akutne bolezni ter v vnetne parametre, povezane s težko akutno boleznijo in neugodnim izidom.Background. Information on parameters associated with the severity of tick-borne encephalitis (TBE), on the long-term outcome (post-encephalitic syndrome), and on the mechanisms responsible for the unfavorable clinical course and outcome of TBE are limited. Aims. The aims of the study were to describe the clinical characteristics of acute TBE, to determine the clinical and/or laboratory parameters associated with the severity of acute illness and/or unfavorable outcome, to assess the frequency and severity of post-encephalitic syndrome at different time points after TBE, and to highlight the basic immune characteristics of TBE. Methods. Seven hundred seventeen adult patients, diagnosed with TBE and hospitalized at the Department of Infectious Diseases, University Medical Centre Ljubljana, in the period from 1. 1. 2007 to 31. 12. 2012, qualified for the study. In a subgroup of 420 patients who participated in a follow-up visit in 2014, i.e. 2–7 years after TBE, we assessed the long-term outcome of the disease. The study also included control group of subjects of the same age (± 5 years). Immune responses were evaluated in serum and cerebrospinal fluid in the period of acute illness, and in the serum 2 months and 2–7 years after the onset of illness. The concentrations of 24 cytokines /chmokines that represent innate and adaptive Th1, Th17 and B cell responses were determined. Results. Multivariate regression showed that patient age (odds ratio (OR) 1.26, 95% confidence interval (CI) 1.11–1.44P = 0.001), previous vaccination against TBE (OR 14.23, 95% CI 1.72–117.87P = 0.014), low level of specific TBE virus serum IgG antibodies (OR 0.85, 95% CI 0.75–0.96P = 0.009), blood leukocyte count (OR 1.45, 95% CI 1.13–1.85P = 0.004), and serum C-reactive protein level (estimated coefficient 1.20, 95% CI 0.48–1.91P = 0.001) were associated with severe acute illness. The frequency of post-encephalitic syndrome diminished over time and stabilized 12 months after the acute illness when it was present in one-third of patientswhereas the severity of post-encephalitis syndrome continued to decline. Unfavourable outcome at 6 months was associated with cerebrospinal fluid leukocyte count (OR 1.43, 95% CI 1.07–1.93, P = 0.017), at 12 months with the disease outcome at 6 months (OR 497.89, 95% CI 39.42–6289.16, P < 0.001), and at the final visit with disease outcome at 12 months (OR 38.56, 95% CI 8.40–177.14, P < 0.001). Unspecific symptoms 2–7 years after TBE were more frequent and more constant in patients than in the control group. Similarly, patients have poorly assessed their physical and mental health, but the differences between the groups were small and were not statistically significant. The concentrations of most cytokines/chemokines in serum and cerebrospinal fluid were significantly different during the time of acute illness: cytokines and chemokines associated with innate immune responses and Th1 adaptive immune responses were significantly higher in cerebrospinal fluid, whereas mediators associated with Th17 and B cell immune responses were generally higher in serum. The majority of studied cytokines/chemokines of innate and adaptive Th1 immune responses were associated with the severity of acute illness, while statistically significant correlations with the unfavorable outcome of the disease have not been found. Conclusion. A study based on a large number of well-defined adult patients with TBE enabled the identification of parametrs associated with the severity of acute illness and/or unfavourable (long-term) outcome of TBE, and the assessment of the frequency and severity of postencephalitic syndrome. It also provided an insight into the immune characteristics of acute illness and inflammatory parameters associated with severe acute illness and an unfavorable outcome of TBE

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

Evaluation of human milk microbiota by 16S rRNA gene next-generation sequencing (NGS) and cultivation/MALDI-TOF mass spectrometry identification

The aim of the present study was to characterize human milk microbiota (HMM) with 16S rRNA gene amplicon next-generation sequencing and cultivation/matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) identification approaches. We analyzed 31 human milk samples from healthy Slovenian mothers. To check the accuracy of MALDI-TOF MS identification, several colonies representing most abundant genera and those, which could not be reliably identified by MALDITOF, were subjected to Sanger sequencing of their 16S rRNA gene. We showed that cultivation/MALDI-TOF MS was a suitable tool for culture-dependent determination of HMM. With both approaches, Staphylococcus and Streptococcus were found as predominant genera in HMM and the abundance of Staphylococcus was associated with decreased microbial diversity. In addition, we characterized factors that might influence HMM. The use of a breast pump was significantly associated with composition of HMM, higher microbial load, and lower abundance of cultivable staphylococci. Moreover, our study suggests that administration of probiotics to the suckling infant might influence HMM by increased abundance of lactobacilli and the presence of viable probiotic bacteria in human milk. However, since our study was observational with relatively small sample size, more targeted studies are needed to study possible transfer of probiotics to the mammary gland via an external route and the physiological relevance of these events

Evaluation of dietary supplements containing viable bacteria by cultivation/MALDI-TOF mass spectrometry and PCR identification

The insufficient quality of products containing beneficial live bacteria in terms of content and viability of labelled microorganisms is an often-reported problem. The aim of this work was to evaluate the quality of dietary supplements containing viable bacteria available in Slovenian pharmacies using plate counting, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and species- or subspecies-specific PCR with DNA isolated from consortia of viable bacteria, from individual isolates, or directly from the products. Twelve percent of the products (3 of 26) contained insufficient numbers of viable bacteria. Eighty-three of the labelled species (111 in total) were confirmed by PCR with DNA from the product; 74% of these were confirmed by PCR with DNA from viable consortium, and 65% of these were confirmed by MALDI-TOF MS analysis of colonies. Certain species in multi-strain products were confirmed by PCR with DNA from viable consortia but not by MALDI-TOF MS, suggesting that the number of isolates examined (three per labelled strain) was too low. With the exception of Lacticaseibacillus casei and closely related species (Lacticaseibacillus rhamnosus and Lacticaseibacillus zeae), PCR and MALDI-TOF identification results agreed for 99% of the isolates examined, although several MALDI-TOF results had lower score values (1.700–1.999), indicating that the species identification was not reliable. The species L. zeae, which appeared in 20 matches of the Biotyper analysis, was identified as L. rhamnosus by PCR. The MALDI-TOF MS analysis was also unsuccessful in detecting Lactobacillus acidophilus La-5 and Bacillus coagulans due to missing peaks and unreliable identification, respectively. Mislabelling was detected by both methods for two putative L. casei strains that turned out to belong to the species Lacticaseibacillus paracasei. PCR remains more successful in subspecies-level identification as long as the database of MALDI-TOF MS spectra is not expanded by building in-house databases. The lack of positive PCR results with viable consortia or colonies, but positive PCR results with DNA isolated directly from the products observed in 10% (11/112) of the labelled strains, suggests the presence of non-culturable bacteria in the products. MALDI-TOF MS is a faster and simpler alternative to PCR identification, provided that a sufficient number of colonies are examined. Generation of in-house library may further improve the identification accuracy at the species and sub-species level

Early Lyme borreliosis in patients treated with tumour necrosis factor-alfa inhibitors

The study evaluated the course and outcome of erythema migrans in patients receiving tumour necrosis factor-alpha (TNF-α) inhibitors. Among 4157 adults diagnosed with erythema migrans in the period 2009–2018, 16 (2.6%) patients were receiving TNF-α inhibitors (adalimumab, infliximab, etarnecept, golimumab), often in combination with other immunosuppressants, for rheumatic (13 patients) or inflammatory bowel (three patients) disease. Findings in this group were compared with those in 32 sex- and age-matched immunocompetent patients diagnosed with erythema migrans in the same years. In comparison with the control group, the immunocompromised patients had a shorter incubation period (7 vs. 14 daysp = 0.0153), smaller diameter of erythema migrans (10.5 vs. 15.5 cmp = 0.0014), and more frequent comorbidities other than immune-mediated diseases (62.5% vs. 25%, p = 0.0269), symptoms/signs of disseminated Lyme borreliosis (18.8% vs. 0%, p = 0.0324), and treatment failure (25% vs. 0%, p = 0.0094). After retreatment with an antibiotic, the clinical course of Lyme borreliosis resolved. Continuing TNF inhibitor treatment during concomitant borrelial infection while using identical approaches for antibiotic treatment as in immunocompetent patients resulted in more frequent failure of erythema migrans treatment in patients receiving TNF inhibitors. However, the majority of treatment failures were mild, and the course and outcome of Lyme borreliosis after retreatment with antibiotics was favourable

Early Lyme Borreliosis in Patients Treated with Tumour Necrosis Factor-Alfa Inhibitors

The study evaluated the course and outcome of erythema migrans in patients receiving tumour necrosis factor-alpha (TNF-&alpha;) inhibitors. Among 4157 adults diagnosed with erythema migrans in the period 2009&ndash;2018, 16 (2.6%) patients were receiving TNF-&alpha; inhibitors (adalimumab, infliximab, etarnecept, golimumab), often in combination with other immunosuppressants, for rheumatic (13 patients) or inflammatory bowel (three patients) disease. Findings in this group were compared with those in 32 sex- and age-matched immunocompetent patients diagnosed with erythema migrans in the same years. In comparison with the control group, the immunocompromised patients had a shorter incubation period (7 vs. 14 days; p = 0.0153), smaller diameter of erythema migrans (10.5 vs. 15.5 cm; p = 0.0014), and more frequent comorbidities other than immune-mediated diseases (62.5% vs. 25%, p = 0.0269), symptoms/signs of disseminated Lyme borreliosis (18.8% vs. 0%, p = 0.0324), and treatment failure (25% vs. 0%, p = 0.0094). After retreatment with an antibiotic, the clinical course of Lyme borreliosis resolved. Continuing TNF inhibitor treatment during concomitant borrelial infection while using identical approaches for antibiotic treatment as in immunocompetent patients resulted in more frequent failure of erythema migrans treatment in patients receiving TNF inhibitors. However, the majority of treatment failures were mild, and the course and outcome of Lyme borreliosis after retreatment with antibiotics was favourable