Klasična i molekularna identifikacija termotolerantnih kampilobaktera iz piletine

Poultry meat samples from Slovenian retail market were examined for the presence of thermotolerant campylobacters. The isolates were identified by phenotypic and genotypic methods. ISO 10272 recommendations were followed for phenotypic identification. Different PCR assays, targeting species specific DNA regions in C. jejuni and C. coli, were checked for their applicability in identification. High degree of tested samples was positive (27/33), with significant proportion of C. coli (32 %) among identified strains. High percentage of C. jejuni strains (54 %) were hippurate negative. Phenotypic identification was therefore found to be inconvenient because of the presence of the strains with atypical phenotype and possible misinterpretation of test results. Multiplex PCR, targeting hippuricase gene in C. jejuni and species specific region in C. coli, was found to be an efficient method that allowed fast, simple and accurate identification of C. jejuni and C. coli. FlaA PCR is a reliable method to identify the group C. jejuni/C. coli, but it does not differentiate between the two species. CdtB PCR is inconvenient because of many false negative and some false positive results.Ispitana je prisutnost termotolerantnih bakterija roda Campylobacter u uzorcima piletine sa slovenskog tržišta. Izolirani su sojevi identificirani fenotipskim i genotipskim postupcima. Fenotipska je identifikacija provedena klasičnim postupkom ISO 10272. Da bi se identificirali određeni sojevi, ispitana je primjenjivost različitih PCR postupaka, korištenjem specifičnih DNA regija za vrste C. jejuni i C. coli. Veliki broj ispitivanih uzoraka bio je pozitivan (27/33), sa značajnim udjelom C. coli u tim dvama identificiranim sojevima (32 %). Veliki udjel sojeva C. jejuni (54 %) bio je hipurat negativan. Fenotipska je identifikacija stoga neprikladan postupak zbog prisutnosti sojeva s atipičnim fenotipom, a i zbog mogućnosti pogrešne interpretacije rezultata testova. Multipleks PCR-postupak, koristeći hipurikazni gen u C. jejuni i u specifičnoj regiji za vrstu C. coli, pokazao se uspješnim jer omogućava brzu, jednostavnu i točnu identifikaciju C. jejuni i C. coli. Postupak PCR s lokusom flagelarnoga gena (flaA PCR) pouzdan je postupak za identifikaciju C. jejuni/C. coli, ali se njime ne mogu razlikovati te dvije vrste. Postupak PCR s lokusom gena citoletalnog toksina (cdtB PCR) nije prikladan jer daje puno lažno negativnih, a i nešto lažno pozitivnih rezultata

Klasična i molekularna identifikacija termotolerantnih kampilobaktera iz piletine

Poultry meat samples from Slovenian retail market were examined for the presence of thermotolerant campylobacters. The isolates were identified by phenotypic and genotypic methods. ISO 10272 recommendations were followed for phenotypic identification. Different PCR assays, targeting species specific DNA regions in C. jejuni and C. coli, were checked for their applicability in identification. High degree of tested samples was positive (27/33), with significant proportion of C. coli (32 %) among identified strains. High percentage of C. jejuni strains (54 %) were hippurate negative. Phenotypic identification was therefore found to be inconvenient because of the presence of the strains with atypical phenotype and possible misinterpretation of test results. Multiplex PCR, targeting hippuricase gene in C. jejuni and species specific region in C. coli, was found to be an efficient method that allowed fast, simple and accurate identification of C. jejuni and C. coli. FlaA PCR is a reliable method to identify the group C. jejuni/C. coli, but it does not differentiate between the two species. CdtB PCR is inconvenient because of many false negative and some false positive results.Ispitana je prisutnost termotolerantnih bakterija roda Campylobacter u uzorcima piletine sa slovenskog tržišta. Izolirani su sojevi identificirani fenotipskim i genotipskim postupcima. Fenotipska je identifikacija provedena klasičnim postupkom ISO 10272. Da bi se identificirali određeni sojevi, ispitana je primjenjivost različitih PCR postupaka, korištenjem specifičnih DNA regija za vrste C. jejuni i C. coli. Veliki broj ispitivanih uzoraka bio je pozitivan (27/33), sa značajnim udjelom C. coli u tim dvama identificiranim sojevima (32 %). Veliki udjel sojeva C. jejuni (54 %) bio je hipurat negativan. Fenotipska je identifikacija stoga neprikladan postupak zbog prisutnosti sojeva s atipičnim fenotipom, a i zbog mogućnosti pogrešne interpretacije rezultata testova. Multipleks PCR-postupak, koristeći hipurikazni gen u C. jejuni i u specifičnoj regiji za vrstu C. coli, pokazao se uspješnim jer omogućava brzu, jednostavnu i točnu identifikaciju C. jejuni i C. coli. Postupak PCR s lokusom flagelarnoga gena (flaA PCR) pouzdan je postupak za identifikaciju C. jejuni/C. coli, ali se njime ne mogu razlikovati te dvije vrste. Postupak PCR s lokusom gena citoletalnog toksina (cdtB PCR) nije prikladan jer daje puno lažno negativnih, a i nešto lažno pozitivnih rezultata

Afterword

This special issue of Food Technology and Biotechnology contains selected papers presented at the 11th Central European Congress on Food and Nutrition (CEFood 2022) entitled ’Food, technology and nutrition for healthy people in a healthy environment’, which was held from 27 to 30 September 2022 in Čatež ob Savi (Slovenia). Since 2002, CEFood congresses have been held biannually in different CEI countries, the 11th was held in Slovenia again after 20 years. The scientific and professional framework of the 1st CEFood congress in 2002 already provided a solid basis and guarantee for the CEFood congresses. In 2006, the European Federation of Food Science and Technology (EFFoST) expressed a clear wish to join this network, due to the good and efficient organisation based on the commitment of the main organisers of CEFood congresses and their teams. Above all, respect and gratitude towards all previous organisers are passed on from congress to congress by each previous main organiser becoming a member of the next programme and organising committee, as proposed at the very beginning. Throughout the centuries, Central Europe has always been a very influential melting pot of ideas for transnational and regional cooperation. At the end of the last century and the beginning of this century, there was a great spirit of cooperation in Central Europe, as first evidenced by the Alps-Adriatic Working Community and later by the Danube Initiatives – these two initiatives had a wide variety of cross-border motives for cooperation, not only political alliances. Obviously, various platforms had asserted a necessity for cooperation. However, the Central European initiative (CEI), established in Budapest in 1989, remains the one that provides a good framework for scientific research and educational cooperation in the area of food. The expansion of the European Union to Central Europe later stimulated a new visionary cooperation, not only in the North–South orientation, but also in the West–East orientation. This was the milestone for the establishment of the Central European Congress on Food and Nutrition (CEFood congress) in 2002 in Ljubljana. CEFood 2022 offered an excellent scientific programme with the best speakers from the Central European initiative countries. In addition, the congress offered other experiences that highlighted Slovenian cuisine and its gastronomic heritage. Therefore, the participants were able to identify and appreciate the latest advances along the food supply chain, including research and education with open discussions on current issues in this field. Environmental concerns were considered, guidance to the ongoing projects in food science, technology and nutrition was offered, and hands-on professionals, scholars, researchers and policy makers were involved. The outcome of the intensive work of the organisers are two publications: CEFood Congress Book with 336 pages and Food, nutrition and environment: Positions in Central European space with 176 pages. In 100 hours of scientific programme, participants enjoyed 8 plenary lectures, 11 keynote lectures and 34 oral presentations in 11 sessions, 5 flash presentations, 144 posters and 13 project presentations at the Central European Project Day, 11 contributions at the Central European Inspiration Food Day. The event was attended by 249 participants from 27 countries, including 233 from the CEI countries. The support by 12 sponsors provided 17 grants for students from economically disadvantaged countries. The scientific programme of the pre-congress event, the Central European Food Project Day, focused on the presentation of food-related R&D projects in the fields of food security, food technology and nutrition, and food safety. The main programme sessions covered the following topics: Food, nature and health; From technology to food; Before and after the harvest; Plants ‐ a staple food or an alternative?; Potentials and limitations of bioactive compounds; New food products, technologies and techniques; Can we improve food properties and prevent food fraud at the same time?; Food related health hazards and risks; Nutrition: is the point in the food or in the gut?; Biotechnology: evolution or revolution?; Food microbes: friends or foes?; Consumer’s food choices and risk management; and Food analytics. All scientific sessions were dedicated to Slovenian food professionals or scientists who have contributed in the past to the development and breakthroughs in the areas of science. The latest achievements in science and good practices were also shared in the World Cafe on food supplements, at the round table ’Edible insects ‐ food of the future?’ and in three workshops: ’Challenges for beer and wine in today’s food world’, ’Food and its safety in a fast changing world’ and ’Modern food: local vs. global, traditional vs. innovative in the “healthy” perspective’. In addition, the Central European Inspiration Food Day (CE-I-FooDay) offered younger professionals from each CEI country the opportunity to write a position paper, which was published in a special publication, and presented orally at CE-I-FooDay as an event after the CEFood 2022 Congress. Considering the current world and food situation, the CEFood congress provided an important contribution to new concepts and views of individual scientists/specialists/teachers in the field of food, technology and nutrition as a scientific advocate for healthy people in a healthy environment, in the future society from a food perspective. Concerning this special issue, a very rigorous selection from among sixteen submitted papers has resulted in the content in front of you - it brings a comprehensive review paper, two original scientific papers and a preliminary communication. The review article focuses on the application of minimal processing technologies for the production and preservation of tailor-made foods – with a range of approaches, including traditional and emerging technologies, and novel ingredients such as biomolecules from diverse sources, including microorganisms. Combined approaches based on the principles of hurdle technology produce effective synergistic effects that improve food safety and shelf-life extension of tailor-made foods while preserving their functional properties. The included scientific papers all target new products with improved functional characteristics – from the bakery raw materials, such as whole wheat flour with improved digestibility and antioxidant properties due to the effect of ultrasound and hydrothermal treatment, to the final goods with new functional ingredients, such as muffins with chia and lyophilised peach powder, and almond-based dairy-free milk alternative formulation fortified with plant extracts – all with the optimised quality attributes to meet the expectations of the contemporary consumer. We, the Guest Editors of this special issue, would like to thank the Editor-in-Chief of the journal, Professor Vladimir Mrša, for providing the valuable space in the journal to accommodate the selected contributions, to Zrinka Pongrac Habdija and Iva Grabarić Andonovski and the production team of FTB for their great support in completing this special issue. Our thanks also go to the reviewers for their insightful comments and for improving the quality of the contributions included in this issue. We would also like to thank all the authors of this special issue for their cooperation in its publication

Anti-Campylobacter Activities and Resistance Mechanisms of Natural Phenolic Compounds in Campylobacter

Background Campylobacter is a major foodborne pathogen and alternative antimicrobials are needed to prevent or decrease Campylobacter contamination in foods or food producing animals. The objectives of this study are to define the anti-Campylobacter activities of natural phenolic compounds of plant origin and to determine the roles of bacterial drug efflux systems in the resistance to these natural phenolics in Campylobacter jejuni. Methodology/Principal Findings Anti-Campylobacter activities were evaluated by an MIC assay using microdilution coupled with ATP measurement. Mutants of the cmeB and cmeF efflux genes and the cmeR transcriptional repressor gene were compared with the wild-type strain for their susceptibilities to phenolics in the absence and presence of efflux-pump inhibitors (EPIs). The phenolic compounds produced significant, but variable activities against both antibiotic-susceptible and antibiotic resistant Campylobacter. The highest anti-Campylobacter activity was seen with carnosic and rosmarinic acids in their pure forms or in enriched plant extracts. Inactivation of cmeB rendered C. jejuni significantly more susceptible to the phenolic compounds, while mutation of cmeF or cmeR only produced a moderate effect on the MICs. Consistent with the results from the efflux pump mutants, EPIs, especially phenylalanine-arginine β-naphthylamide and NMP, significantly reduced the MICs of the tested phenolic compounds. Further reduction of MICs by the EPIs was also observed in the cmeB and cmeF mutants, suggesting that other efflux systems are also involved in Campylobacter resistance to phenolic compounds. Conclusion/Significance Natural phenolic compounds of plant origin have good anti-Campylobacter activities and can be further developed for potential use in controlling Campylobacter. The drug efflux systems in Campylobacter contribute significantly to its resistance to the phenolics and EPIs potentiate the anti-Campylobacter activities of plant phenolic compounds

Effects of Low Temperature, Starvation and Oxidative Stress on the Physiology of Campylobacter jejuni Cells

Campylobacter jejuni is a food-borne pathogen that causes diarrheal diseases in humans and animals. Still unknown mechanisms allow the cells to overcome significant stresses despite the absence of several traditional stress response genes. C. jejuni is problematic because of its ability to adapt and survive in various conditions of extraintestinal environment, common to food production and supply chain. The authors demonstrated that preliminary exposure to 25 °C did not influence the heat resistance of C. jejuni cells at 55 °C. In addition, changes of morphology, culturability and viability were noticeable for fed and starved cultures exposed to low temperature and oxidative stresses. All physiology changes were influenced by starvation and, to a lesser extent, by other stresses. Starvation was the most significant factor in C. jejuni survival, coccoid cell formation and especially culturability. Temperature downshift, oxidative stress and starvation, usually in combination with other factors, can trigger transition of bacteria to a viable but nonculturable state. Results showed substantial variability in responses to stresses applied, indicating no correlation between low temperature and oxidative stress response of campylobacters. Understanding of microbial physiology after exposure to various stresses is essential for food safety

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

The Prevalence of Multiple Antibiotic Resistance in Campylobacter spp. From Retail Poultry Meat

Macrolides and fluoroquinolones are regarded as drugs of choice for the treatment of human Campylobacter infections. The use of antimicrobials for this purpose as well as in food animal production has resulted in the resistance of Campylobacter spp. to selected antibiotics. Since poultry is one of the most important sources of human Campylobacter infections the use of antibiotics in animal production can shorten the effective therapeutic life of antibiotics for human use. During 2001–2003, over 220 strains of C. jejuni and C. coli were isolated from 60 poultry meat samples from the retail market in Slovenia and further characterized by phenotypic and molecular methods. In this study, 55 sample-representative strains were tested for susceptibility to eight different antibiotics (ampicillin, amoxycillin/ clavulanic acid, ciprofloxacin, erythromycin, gentamicin, nalidixic acid, pefloxacin and tetracycline). Phenotypic procedures (disc diffusion test, E-test) as well as molecular detection of mutations (mismatch amplification mutation assay (MAMA) polymerase chain reaction (PCR)) in case of ciprofloxacin resistance were used. When assuming the results about antibiotic resistance, only 38.2 % of strains tested were susceptible to all antibiotics tested. Regarding ciprofloxacin, 58.2 % of tested strains were found to be resistant (minimal inhibitory concentration, MIC>4 mg/mL). The occurrence of resistance was much higher in C. coli (75.9 %) than in C. jejuni (38.5 %) isolates. The resistance rates to pefloxacin, nalidixic acid, erythromycin and tetracycline were 58.2, 49.1, 14.5 and 12.7 %, respectively. Eleven percent of strains were resistant to erythromycin and ciprofloxacin and 12.7 % of strains were resistant to tetracycline and quinolones. The results show the need for monitoring the prevalence and antibiotic resistance of zoonotic bacteria such as Campylobacter as well as the multiresistance phenomenon of Campylobacter isolates from food in our country

The Prevalence of Multiple Antibiotic Resistance in Campylobacter spp. From Retail Poultry Meat

Macrolides and fluoroquinolones are regarded as drugs of choice for the treatment of human Campylobacter infections. The use of antimicrobials for this purpose as well as in food animal production has resulted in the resistance of Campylobacter spp. to selected antibiotics. Since poultry is one of the most important sources of human Campylobacter infections the use of antibiotics in animal production can shorten the effective therapeutic life of antibiotics for human use. During 2001–2003, over 220 strains of C. jejuni and C. coli were isolated from 60 poultry meat samples from the retail market in Slovenia and further characterized by phenotypic and molecular methods. In this study, 55 sample-representative strains were tested for susceptibility to eight different antibiotics (ampicillin, amoxycillin/ clavulanic acid, ciprofloxacin, erythromycin, gentamicin, nalidixic acid, pefloxacin and tetracycline). Phenotypic procedures (disc diffusion test, E-test) as well as molecular detection of mutations (mismatch amplification mutation assay (MAMA) polymerase chain reaction (PCR)) in case of ciprofloxacin resistance were used. When assuming the results about antibiotic resistance, only 38.2 % of strains tested were susceptible to all antibiotics tested. Regarding ciprofloxacin, 58.2 % of tested strains were found to be resistant (minimal inhibitory concentration, MIC>4 mg/mL). The occurrence of resistance was much higher in C. coli (75.9 %) than in C. jejuni (38.5 %) isolates. The resistance rates to pefloxacin, nalidixic acid, erythromycin and tetracycline were 58.2, 49.1, 14.5 and 12.7 %, respectively. Eleven percent of strains were resistant to erythromycin and ciprofloxacin and 12.7 % of strains were resistant to tetracycline and quinolones. The results show the need for monitoring the prevalence and antibiotic resistance of zoonotic bacteria such as Campylobacter as well as the multiresistance phenomenon of Campylobacter isolates from food in our country

Učestalost antibiotičke i biocidne rezistentnosti bakterija Campylobacter coli i Campylobacter jejuni iz različitih izvora

The increasing levels of antimicrobial resistance among foodborne bacteria are recognised as an important emerging public health problem. Reduced susceptibility to biocides also appears to be increasing. A potential concern is the possibility that the widespread use of biocides is responsible for the selection and maintenance of antibiotic-resistant bacteria. Here, we examine the prevalence of erythromycin, ciprofloxacin, triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride, trisodium phosphate and sodium dodecyl sulphate resistance among 27 isolates of Campylobacter coli and 15 isolates of Campylobacter jejuni from food, animal, human and environmental water sources. These antimicrobial susceptibilities were determined by the broth microdilution method. In the 42 Campylobacter strains studied, different antibiotic resistance levels were seen. The resistance to erythromycin and ciprofloxacin was observed in 14.3 % of Campylobacter strains. A higher rate of erythromycin resistance and multi-resistance was observed among isolated C. coli than among C. jejuni strains. Similar situations were seen for triclosan. Conversely, the level of benzalkonium chloride resistance was higher in C. jejuni than in C. coli. No correlation between biocide and antibiotic resistance was observed. This study does not provide evidence to confirm that tolerance to biocides is connected to antibiotic resistance in Campylobacter.U javnom zdravstvu važan problem predstavlja sve učestalija rezistencija bakterija tj. uzročnika trovanja hranom na antimikrobne agense. Isto tako se smanjuje njihova osjetljivost na biocidne pripravke. Postoji opravdana sumnja da zbog rasprostranjene primjene biocida dolazi do selekcije i preživljavanja bakterija koje su otporne na djelovanje antibiotika. U radu je ispitana učestalost pojave rezistencije na eritromicin, ciprofloksacin, triklosan, benzalkonijev klorid, klorheksidin diacetat, cetilpiridinijev klorid, trinatrijev fosfat i natrijev dodecil-sulfat u 27 izolata Campylobacter coli i 15 izolata Campylobacter jejuni iz različitih izvora, tj. iz hrane, vode, životinja i čovjeka. Antimikrobna osjetljivost određena je mikrodilucijskom metodom. Otpornost na antibiotike zamijećena je u 42 ispitana soja vrste Campylobacter. Od ispitanih je sojeva njih 14,3 % bilo otporno na eritromicin i ciprofloksacin. Više je izolata C. coli od onih C. jejuni imalo veću otpornost na eritromocin, te je uz to bilo otporno i na ciprofloksacin. Isti je rezultat dobiven primjenom triklosana. Za razliku od antibiotičke otpornosti, rezistencija na biocidne pripravke izraženija je u C. jejuni nego u C. coli. Nije zamijećena korelacija između biocidne i antibiotičke rezistencije. Ispitivanjem se nije uspjelo dokazati da je otpornost na biocide povezana s antibiotičkom rezistencijom bakterija vrste Campylobacter

Antimicrobial and Resistance Modulatory Activity of Alpinia katsumadai Seed Phenolic Extract, Essential Oil and Post-Distillation Extract

Antimikrobna otpornost patogenih mikroorganizama predstavlja ozbiljan problem u proizvodnji hrane, a naročito zbog rastućeg broja multirezistentnih sojeva. Sve je veća potreba za novim i efikasnim antimikrobnim agensima i modulatorima otpornosti radi prevladavanja ovog problema, pri čemu biljke predstavljaju bogat izvor takvih spojeva. U ovom je radu ispitan antimikrobni učinak fenolnog ekstrakta, eteričnog ulja i destilacijskog ekstrakta sjemenki biljke Alpinia katsumadai, te njihov utjecaj na modulaciju otpornosti sojeva Campylobacter jejuni i Staphylococcus aureus. Ekstrakt sjemenki i postdestilacijski ekstrakt imali su relativno snažan antimikrobni učinak na bakteriju C. jejuni, dok je bakterija S. aureus pokazala veću otpornost. Fenolni ekstrakt, eterično ulje i destilacijski ekstrakt A. katsumadai u kombinaciji sa svim ispitanim antimikrobnim agensima (ciprofloksacin, eritromicin, triklosan, soli žučne kiseline i etidijev bromid) uspješno su modulirali otpornost svih sojeva C. jejuni. Učinak je ekstrakata i eteričnog ulja na bakteriju S. aureus ovisio o bakterijskom soju i primjenjenom antimikrobnom agensu, ali je uspješno smanjena otpornost svih sojeva na soli žučne kiseline. Eterično je ulje sjemenki biljke A. katsumadai potaklo akumulaciju etidijevog bromida unutar bakterijskih stanica, čime je dokazano da se može upotrijebiti kao inhibitor efluksne pumpe bakterija C. jejuni i S. aureus.Antimicrobial resistance of food-related bacterial pathogens is becoming a serious problem, especially after the emergence of multidrug-resistant strains. To overcome this problem, new and effective antimicrobials or resistance modulators are highly needed and plant kingdom represents a valuable source of these compounds. We investigated antimicrobial and resistance modulatory activity of the phenolic extract, essential oil and post-distillation extract of Alpinia katsumadai seeds against Campylobacter jejuni and Staphylococcus aureus. Among the tested plant formulations, A. katsumadai seed phenolic extract and post-distillation extract showed moderate antimicrobial activity against C. jejuni, while S. aureus was more resistant. When evaluating resistance modulatory potential of A. katsumadai phenolic extract, essential oil and post-distillation extract in C. jejuni against ciprofloxacin, erythromycin, triclosan, bile salts and ethidium bromide, plant formulations exhibited modulatory activity in combination with all antimicrobials. Modulation of resistance was more strain- and antimicrobial-specific in S. aureus, but very efficient in the case of reduced resistance to bile salts. Essential oil from A. katsumadai seeds efficiently increased intracellular ethidium bromide accumulation and was thus confirmed as potential inhibitor of efflux pumps in C. jejuni and S. aureus