Tetracycline resistance in lactobacilli isolated from Serbian traditional raw milk cheeses

The aim of this study was to investigate the presence of tetracycline resistance in lactobacilli isolated from traditional Serbian white brined raw milk cheeses (Homolje, Sjenica, Zlatar). Isolation of presumptive lactobacilli was initially performed using MRS-S agar without tetracycline, or supplemented with 16 and 64 A mu g/mL of tetracycline. Rep-PCR (GTG)(5) genotyping showed a high diversity of the isolates obtained, as examination of 233 isolates resulted in 156 different Rep-PCR fingerprints. Ninety out of 156 (57.69%) of the strains, representatives with different (GTG)(5) fingerprints, were identified by MALDI-TOF MS as lactobacilli, while 66 out of 156 (42.31%) strains were identified as members of other LAB genera. All except one out of 90 Lactobacillus isolates further tested by microdilution method, demonstrated unimodal distribution of tetracycline MIC values which were equal to or lower from the breakpoint MIC values (EFSA in EFSA J 10: 1-10, 2012. Only one Lb. paracasei isolate showed the presence of tet(M) gene, while the other analyzed tet genes [tet(A), tet(B), tet(C) tet(K), tet(L), tet(O) and tet(W)] were not detected in any of the isolates. The results of this study indicates that lactobacilli from traditional Serbian raw milk cheeses do not present considerable tetracycline resistance reservoirs. For final conclusions about the safety of these autochthonous cheeses regarding the possible tetracycline resistance transferability, the assessment of the entire cheese microbiota is needed

Influence of two feed supplements on technological properties of goat’s milk

Hranidba koza na dnevnoj bazi sastoji se od ispaše, sijena i/ili suplemenata. Suplementi su neophodni u hranidbi visokoproduktivnih pasmina koza u svrhu postizanja genetskog potencijala i uzgajivači moraju odabrati režim hranidbe koji osigurava proizvodnju velikih količina mlijeka bez neželjenih promjena u tehnološkoj kvaliteti mlijeka. U ovom istraživanju određivan je utjecaj dva komercijalno dostupna suplementa na sposobnost koagulacije kozjeg mlijeka te na reološka svojstva jogurta od kozjeg mlijeka. Ukupno 61 koza pasmine slovenska alpska koza hranjena je s dva suplementa tijekom tri godine. Prvi suplement (FS1) imao je viši udjel ječma i lucerne, dok je drugi suplement (FS2) imao dodane mineralne tvari i vitamine te više udjele pšenice i suncokretove pogače. Posljedično, FS1 je imao više sirovih vlakana, što je vrlo vjerojatno rezultiralo 15 % većom čvrstoćom, konzistencijom i kohezivnosti (P<0,05) jogurta iz skupine FS1 u odnosu na jogurte iz skupine FS2. K tomu je i vrijeme koagulacije (r) bilo kraće (P<0,05) kod jogurta iz skupine FS1 u usporedbi s jogurtima iz skupine FS2. Čvrstoća gruša 30 min nakon dodatka enzima (a30) također je bila veća u skupini FS1, iako te razlike nisu bile statistički značajne. Uzimajući sve navedeno u obzir, dobiveni rezultati ukazuju kako su koze hranjenje suplementom FS1 proizvodile mlijeko boljih tehnoloških svojstava nego koze hranjene suplementom FS2, unatoč činjenici da nisu utvrđene statistički značajne razlike u kemijskom sastavu mlijeka iz obiju navedenih skupina. Ova studija pokazala je da se pažljivim odabirom suplementa u hranidbi mogu poboljšati tehnološka svojstva kozjeg mlijeka. Međutim, potrebno je provođenje daljnjih istraživanja u svrhu određivanja specifičnih mehanizama koji su doveli do prethodno navedenih razlika.Goat’s daily diet is usually based on grazing, hay and/or feed supplements. Feed supplements are crucial in the diet of high productive goats to achieve their genetic potential and breeders must choose balanced feeding regime to produce large quantities of milk without affecting the technological quality of milk. In the present study, we evaluated the influence of two commercially available feed supplements on goat milk coagulation properties and rheological properties of yoghurts. Goats of the Slovenian Alpine breed (61) were fed with two feed supplements during the 3-year experiment. Feed supplement 1 (FS1) had higher proportions of barley and alfalfa, while feed supplement 2 (FS2) had added premix of minerals and vitamins and had higher proportions of wheat and sunflower meal. Con¬sequently, FS1 had more crude fibres, which is the most probable reason for approximately 15 % higher firmness, consistency and cohesiveness (P<0.05) of yoghurts in FS1 group, compared to the FS2 group. Moreover, the rennet coagulation time (r) was shorter (P<0.05) in the FS1 group, compared to the FS2 group. Curd firmness 30 min after enzyme addition (a30) was also higher in FS1 group although the results were not statistically significant. Taking together, our results indicate that goats fed with FS1 produced milk with better technological properties compared to those fed with FS2, despite the fact that there were no significant differences in chemical composition of milk from each group. We showed that careful selection of feed supplement’s constituents could improve technological properties of goat milk. However, further studies are needed to evaluate the mechanisms of the observed differences

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

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

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 different primers for PCR-DGGE analysis of cheese-associated enterococci

Enetrococci represent an imprtant part of bacterial microbiota in different types of artisanal cheeses, made from either raw or pasteurized milk. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of ribosomal DNA is currently one of the most frequently used fingerprinting method to study diversity and dynamics of microbial communities and also a tool for microbial identification. Among several primer pairs for DGGE analysis publushes so far, six primer pairs amplifying differnet variable regions of 16S rDNA were selected and applied in our DGGE analysis of 12 species belonging to genus Eneterococcus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactoccocus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactococcus lactis). When DGGE procedures were optimized, the same set of primers was used for DGGE analysis of five cheese samples. Our study demonstates that the use of different primer pairs generate significant differences in DGGE analysis of enterococcal population, consequently, appropriate primers regarding the purpose of analysis can be selected. For differentiation and identification of pure enterococcal isolates, primer pair P1V1/P2V1 showed the most promising results since all 12 enterococcal isolates gave distincive DGGE fingerprints, but with multiple bands, patternstherefore these primers do not seem to be appropriate for identification of enterococcal species in mixed cultures. Use of primer pairs HDA1/HDA2 and V3f/V3r amplifying V3 region showed better potential for detection and identification of enterococci in mixed communities, but since some bacterial species showed the same fingerprint, for clear identification combination of DGGE and some other method (e.g. species specific PCR) or combined DGGE analysis using two primer pairs generating distinctive results should be used

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

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

Genomic insights into antibiotic resistance and mobilome of lactic acid bacteria and bifidobacteria

Lactic acid bacteria (LAB) and Bifidobacterium sp. (bifidobacteria) can carry antimicrobial resistance genes (ARGs), yet data on resistance mechanisms in these bacteria are limited. The aim of our study was to identify the underlying genetic mechanisms of phenotypic resistance in 103 LAB and bifidobacteria using whole-genome sequencing. Sequencing data not only confirmed the presence of 36 acquired ARGs in genomes of 18 strains, but also revealed wide dissemination of intrinsic ARGs. The presence of acquired ARGs on known and novel mobile genetic elements raises the possibility of their horizontal spread. In addition, our data suggest that mutations may be a common mechanism of resistance. Several novel candidate resistance mechanisms were uncovered, providing a basis for further in vitro studies. Overall, 1,314 minimum inhibitory concentrations matched with genotypes in 92.4% of the caseshowever, prediction of phenotype based on genotypic data was only partially efficient, especially with respect to aminoglycosides and chloramphenicol. Our study sheds light on resistance mechanisms and their transferability potential in LAB and bifidobacteria, which will be useful for risk assessment analysis