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The objective of the study: to evaluate the effectiveness of diagnosis of tuberculosis and mycobacteriosis in bronchobiopsy specimens obtained during navigation by radial endobronchial ultrasonography (rEBUS) in patients with peripheral lung lesions without bacterial excretion.Subjects and methods. A retrospective analysis of the diagnostic effectiveness of bronchoscopic examination with biopsies was carried out in 179 patients (75 men and 104 women) suffering from pulmonary tuberculosis or mycobacteriosis without bacterial excretion; peripheral lung lesions had been visualized by computed tomography (CT). The patients were divided into two groups: 93 underwent bronchoscopy with biopsies with rEBUS navigation, 86 underwent bronchoscopy with classical biopsies and preliminary CT navigation. Each patient underwent multiple biopsies, at least one fluid biopsy (bronchoalveolar lavage or bronchial lavage), and one tissue biopsy (transbronchial lung biopsy or brush biopsy). Specimens collected by all types of bronchobiopsy were sent for microbiological and cytological tests, specimens of pulmonary transbronchial biopsy were additionally sent for histological examination.Results. The diagnosis of tuberculosis was verified by bronchobiopsy in 106 (67.5%) of 158 patients with tuberculosis, but statistically significantly more often in the group with rEBUS navigation versus the group without it – 81.9% (68/83) versus 50.7% (38/75), respectively (pχ2 < 0.01). The diagnosis of non-tuberculous mycobacteriosis was verified by bronchobiopsy in 13 (61.9%) of 21 patients, in the group with rEBUS navigation – in 80.0% (8/10) patients, in the group without it – in 45.5% (5/11) (pφ > 0.05). The use of rEBUS navigation while collecting bronchobiopsy specimens made it possible to increase the etiological verification of tuberculosis using the following microbiological methods: microscopy – from 14.7 to 49.4% (pχ2 < 0.01), molecular genetic – from 41.3 to 72.3% ( pχ2 < 0.01), culture (Bactec MGIT960) – from 44.0 to 67.5% (pχ2 < 0.01) The greatest enhancement of diagnostic effectiveness was achieved in the specimens of bronchoalveolar lavage and bronchial lavage – from 33.3 to 71.1% (pχ2 < 0.01) and in brush biopsy specimens – from 25.6 to 57.6% (pχ2 < 0.01).

Background The PhageDx™Cronobacter Assay is based on the infection of Cronobacter spp. by specific bacteriophages and expression of a luciferase reporter gene. Results are generated in as little as 18.5 h for powdered infant formula (PIF). Objective An AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Cronobacter Assay for the detection of Cronobacter in 10, 100, and 300 g milk- and soy-based PIF test portions. Method The performance of the PhageDx method was compared to the ISO 22964:2006/2017 Microbiology of the Food Chain—Horizontal Method for the Detection of Cronobacter spp. and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 29 Cronobacter: 2012. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted. Results There was no significant difference between the 10, 100, or 300 g test portions for the milk and soy PIF matrixes between the PhageDx Cronobacter Assay, the ISO 22964:2006/2017, and the FDA BAM Chapter 29 Cronobacter: 2012 methods. The reporter bacteriophages were specific for Cronobacter and infected 75 strains in inclusivity testing. They did not infect 35 non-Cronobacter bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least 3 months. Conclusions Work in the submitting and independent laboratories demonstrated that the PhageDx Cronobacter Assay meets the qualifications for PTM status. Highlights The PhageDx Cronobacter Assay is a rapid, simple, and specific test that has shown equivalence to both the FDA BAM and ISO reference methods for detecting Cronobacter spp. in PIF.

Background The PhageDx™ Listeria Assay is a simple, specific, and sensitive assay based on the infection of Listeria spp. by selected bacteriophages and the resultant expression of a luciferase reporter gene. Results are generated in as little as 24.5 h for stainless steel and ceramic environmental surfaces. Objective An AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Listeria Assay for the detection of Listeria on stainless steel and ceramic surfaces. Method The performance of the PhageDx method was compared to that of the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM) Ch. 10. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted. Results Inclusivity testing demonstrated that the reporter bacteriophages were specific for Listeria ssp. and detected 58/61 Listeria strains tested, including all 34 L. monocytogenes strains. The reporter bacteriophage also was shown to not detect 46/47 non-Listeria bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least 6 months. Matrix studies on stainless steel and ceramic surfaces showed that there was no significant difference between the PhageDx Listeria Assay and the FDA/BAM Chapter 10 reference method. Conclusions and Highlights The validation study demonstrates that the PhageDx Listeria Assay is an effective method for the detection of Listeria spp. on stainless steel and ceramic environmental surfaces and meets the qualifications for AOAC PTM status.

Legionnaires’ disease has been recognized since 1976 and Legionella pneumophila still accounts for more than 95% of cases. Approaches in countries, including France, suggest that focusing risk reduction specifically on L. pneumophila is an effective strategy, as detecting L. pneumophila has advantages over targeting multiple species of Legionella. In terms of assays, the historically accepted plate culture method takes 10 days for confirmed Legionella spp. results, has variabilities which affect trending and comparisons, requires highly trained personnel to identify colonies on a plate in specialist laboratories, and does not recover viable-but-non-culturable bacteria. PCR is sensitive, specific, provides results in less than 24 h, and determines the presence/absence of Legionella spp. and/or L. pneumophila DNA. Whilst specialist personnel and laboratories are generally required, there are now on-site PCR options, but there is no agreement on comparing genome units to colony forming units and action limits. Immunomagnetic separation assays are culture-independent, detect multiple Legionella species, and results are available in 24 h, with automated processing options. Field-use lateral flow devices provide presence/absence determination of L. pneumophila serogroup 1 where sufficient cells are present, but testing potable waters is problematic. Liquid culture most probable number (MPN) assays provide confirmed L. pneumophila results in 7 days that are equivalent to or exceed plate culture, are robust and reproducible, and can be performed in a variety of laboratory settings. MPN isolates can be obtained for epidemiological investigations. This accessible, non-technical review will be of particular interest to building owners, operators, risk managers, and water safety groups and will enable them to make informed decisions to reduce the risk of L. pneumophila.

Background Reveal® 3-D for Peanut is an immunochromatographic, lateral flow test for qualitative detection of peanut residue in food manufacturing and food preparation settings. The test can detect low ppm levels of peanut in clean-in-place (CIP) rinses and in swabs from environmental surfaces and can serve as a tool in managing allergen risk. Objective The objective of the study was to validate the lateral flow method for detection of peanut in CIP rinses, specifically water, peroxyacetic acid/hydrogen peroxide, and quaternary ammonium compound rinses, and in swabs taken from stainless steel and plastic surfaces. Methods CIP rinses spiked with low levels of peanut were tested, as were surfaces inoculated with peanut. Specificity and assay interference were assessed in testing of food commodities with and without added peanut. Assay robustness and test kit stability and consistency testing were also performed. Results Results demonstrated that the lateral flow test can detect peanut in CIP rinses in the range of 2–4 ppm and in environmental surface swabs in the range of 3–4 µg/100 cm2. Results of specificity testing with 29 common food items showed lack of cross-reactivity, and potential assay interference only from walnut. Data from stability trials supports expiration dating for the kit of up to 23 months post-manufacture. Conclusions and Highlights The lateral flow test is a sensitive, specific, and rapid method for detection of low levels of peanut residue in CIP rinses and environmental samples and can be an important component in a comprehensive allergen risk management program.

A qualitative 3 min one-step assay for detecting beta-lactam, sulfonamide, and tetracycline antibiotics was validated following milk screening test guidelines developed by FDA-CVM, AOAC-RI, and IDF. The validated 90% detection levels with 95% confidence were: penicillin G 2 part per billion (ppb); amoxicillin 4 ppb; ampicillin 9 ppb; ceftiofur plus metabolites 50 ppb; cloxacillin 9 ppb; cephapirin 15 ppb; sulfadimethoxine 8 ppb; sulfamethazine 9 ppb; chlortetracycline 34 ppb; oxytetracycline 53 ppb; and tetracycline 42 ppb. Detection levels were lower than U.S. and Canadian allowable limits for milk and were consistent with most European Maximum Residue Limits. Tests of raw commingled cows’ milk indicated a low positive error rate of

Background Soleris®Enterobacteriaceae is a growth-based, automated method for detection of Enterobacteriaceae in food. Objective A study was conducted to validate the Soleris method for detection of Enterobacteriaceae in select foods (pasteurized milk, yogurt, mozzarella cheese, ice cream, dried milk, pasteurized liquid egg, frozen cooked chicken, deli ham, lettuce, and dry dog food) at a threshold of ≥ 10 CFU/g of product. Methods Inclusivity and exclusivity of the Soleris method were assessed by testing 55 and 38 target and non-target bacterial strains, respectively. Matrix testing was performed with one naturally contaminated and nine inoculated foods. Efficacy of the Soleris method was compared to that of the ISO 21528-2:2017 direct plating reference method using probability of detection analysis. Independent laboratory testing was conducted to verify method performance in two matrixes (yogurt and deli ham). Method robustness, stability, and lot-to-lot consistency of the Soleris reagents were also assessed. Results Inclusivity of the Soleris test was 91% and exclusivity was 100%. In matrix testing, there were no significant differences in the number of positive results obtained with the Soleris and reference methods for any of the matrixes examined. Overall, of 370 test portions, there were 176 positive results by the Soleris method and 177 positive results by the reference procedure. Conclusions Soleris Enterobacteriaceae is an effective method for detection of Enterobacteriaceae in the foods evaluated, with performance equivalent to that of the ISO 21528-2:2017 reference method. Highlights The Soleris method offers the advantages of labor savings and results within 18 h.

Kisla sirotka je tekoč stranski produkt, ki nastane pri fermentaciji ali kisli koagulaciji mleka z dodatkom organske ali anorganske kisline. V primerjavi z obdelavo sladke sirotke je predelava kisle sirotke veliko zahtevnejša, predvsem zaradi večje vsebnosti mlečne kisline, ki otežuje proces sušenja. Kisla sirotka je zato pogosteje obravnavana kot odpadek in predstavlja ekološko obremenitev oziroma strošek za proizvajalca. Namen magistrskega dela je bil raziskati uporabnost kisle sirotke, ki nastane pri industrijski izdelavi skute, za gojenje bakterij s probiotičnim potencialom. V manjšem merilu smo najprej pokazali, da so bili vsi izbrani sevi sposobni rasti v gojišču iz kisle sirotke. Obogatitev kisle sirotke s kvasnim ekstraktom in/ali mešanico mineralov je izboljšala rast sevov Lactiplantibacillus plantarum in seva Lacticaseibacillus rhamnosus IM239, ni pa vplivala na rast seva Lactobacillus paragasseri IM105. V večjem merilu smo nato z gojenjem v bioreaktorju pokazali, da je bil izplen živih bakterij boljši, če tekom fermentacije vrednosti pH nismo uravnavali. Ugotovili smo tudi, da so testirane bakterije, odvzete na začetku stacionarne faze rasti, bolje preživele liofiliziranje, če smo jim tekom gojenja uravnavali vrednost pH na 6,5, medtem ko pri kulturah, odvzetih po 24-ih urah, vpliv vrednosti pH ni bil tako izrazit. Z raziskavo smo tako potrdili potencial kisle sirotke za industrijsko gojenje mlečnokislinskih bakterij. Acid whey is a liquid fraction that remains after the fermentation of milk or the addition of organic or mineral acids to milk. The processing of acid whey is much more challenging than the processing of sweet whey, mainly because of the higher lactic acid content, which obstructs the drying process. Acid whey is, therefore, more often considered as waste, and as such as an ecological burden for the environment and an unwanted cost for the manufacturer. The purpose of this study was to investigate the possibility of using acid whey as a culture medium for probiotic bacteria. The acid whey used was the by-product of an industrial-sized production of cottage cheese. We first showed on a smaller scale that the chosen bacterial strains were able to grow in an acid whey medium. The addition of yeast extract and/or a mixture of minerals improved the growth of the Lactiplantibacillus plantarum strains and the Lacticaseibacillus rhamnosus IM239 strain, but it did not affect the growth of the Lactobacillus paragasseri IM105 strain. Then we showed on a larger scale that the yield of live bacteria was higher if the pH value was not regulated during the fermentation in a bioreactor. We also showed that the bacteria samples collected at the beginning of the stationary growth phase had a higher survival rate during freeze-drying if their pH value was adjusted to 6,5 during the fermentation. However, in the samples collected after 24 hours the pH value had a smaller effect on the survival rate during freeze-drying. With the study we thus confirmed that acid whey can be used as a growth medium for the cultivation of lactic acid bacteria.

Background ThePhageDx™ E. coli O157:H7 Assay originally required a 5 h enrichment period and a 10 mL test sample for 25 g ground beef. The proposed method modifications seek to include beef trim (375 g) matrix and a new method procedure for 25 g ground beef comprised of 6–7 h enrichment and 1 mL test sample. Objective To validate the method modifications for PhageDx E. coli O157:H7 Assay to include beef trim (375 g) matrix and modify enrichment time and test sample size for 25 g ground beef. Method For both matrixes, pre-warmed tryptic soya broth (TSB; 42 ± 1°C) was added in a 3:1 (media:sample size) ratio, blended, and enriched for 9–10 h (beef trim, 375 g) or 6 h (ground beef, 25 g) at 42 ± 1°C. One milliliter samples were transferred to a microfuge tube, centrifuged, and the supernatant removed. The pellet was resuspended in 0.2 mL of TSB and phage reagent was added. Samples were incubated for 2 h at 37 ± 1°C. After infection, the sample was centrifuged, and 150 µl of the supernatant was transferred to a 96-well plate. Then, lysis buffer and luciferase substrate were added and the sample read on a luminometer to determine the presence or absence of E. coli O157:H7. Results No significant differences were found between the PhageDx E. coli O157:H7 Assay method modifications and the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) reference method. Conclusions The independent study demonstrated that the PhageDx E. coli O157:H7 Assay method modifications meet the qualifications for Performance Tested Method SM status. Highlights The PhageDx E. coli O157:H7 Assay is a rapid detection method capable of detecting E. coli O157:H7 in 25 g ground beef and 375 g beef trim samples in as little as 8.5 h and 11.5 h total turn around time, respectively.

Okratoksin A (OTA) se smatra jednim od najopasnijih mikotoksina koji se nalaze u prehrambenim proizvodima, a kao glavni izvori OTA u ljudskoj prehrani navode se žitarice i vino. Istraživanja su pokazala da ima nefrotoksično, karcinogeno, imunotoksično, mutageno i neurotoksično djelovanje te je klasificiran kao mogući ljudski karcinogen. Osim preventivnih postupaka koji se primjenjuju kako bi se smanjio rizik od kontaminacije, potrebno je stalno kontrolirati razinu mikotoskina u hrani. Metode koje se koriste za smanjenje OTA dijele se na fizikalne, kemijske i mikrobiološke metode. Zbog ograničene primjene fizikalnih i kemijskih metoda, koriste se mikrobiološke metode koje su ekološki prihvatljive i učinkovite. Ochratoxin A (OTA) is considered as one of the most dangerous mycotoxins found in food products, and cereal and wine are reported as a main source of OTA in the human diet. Previous studies have shown that it has nephrotoxic, carcinogenic, immunotoxic, mutagenic and neurotoxic effects and it has been classified as a possible human carcinogenic. Besides preventive procedures that are applied to reduce the risk of contamination, what is necessary is a constant control of the levels of mycotoxins in food. The methods used to reduce OTA are physical, chemical or microbiological. Due to the limited application of physical and chemical methods, more environmentally friendly and efficient methods are more used.

Background Peel PlateTM Enterobacteriaceae Bacteria (EB) is dried selective media on a 47 mm plastic plate that produces enzyme substrate colored colonies on rehydration and incubation for 24 h and up to 48 h at 37 ± 1°C. Purpose The method validation compared quantification of EB to reference methods ISO 21528:2017 Parts 1 and 2. Methods Matrixes compared were whole milk, skim powdered milk, vanilla ice cream, butter, infant formulas (soy- and dairy-based), infant cereals ± probiotic, environmental sponge swab of stainless steel surface, and poultry carcass rinse with two different peptone buffers. Results In inclusivity and exclusivity studies, the method detected 54 of 54 EB strains and did not detect 30 of 30 non-EB strains. In matrix studies, the claimed foods were tested at three contamination levels using paired analysis between the reference and Peel Plate EB methods. Colony-forming units per gram or mL [CFU/g (mL)] were log10 transformed for statistical analysis. The candidate method and reference method were shown to be equivalent by the performance requirement of all 95% confidence intervals on mean difference falling between −0.5 and +0.5 log10 CFU/g (mL). An international collaborative study with dried infant formula spiked with Cronobacter sakazakii at log10 CFU/g (mL) 1.05, 2.31, and 3.21 levels, produced method differences −0.16, 0.15, and 0.18 log10 CFU/g (mL) with repeatabilities (r) = 0.33, 0.20, and 0.12 log10 CFU/g (mL) and reproducibilities (R) = 0.45, 0.26, and 0.18 log10 CFU/g (mL). Conclusions Based on these evaluations, the candidate method is considered equivalent to the reference methods at both the 24 h and 48 h incubation periods at 37 ± 1°C. Highlights Ready to use Enterobacteriaceae method equivalent to ISO-21528:2017 Parts 1 and 2; EB test colored colonies at 37°C for 24 h are equivalent at 48 h incubation; Singlet determined CFU/mL are statistically the same as duplicate average results; EB test validated for infant formula and dairy products including with probiotics; EB test for environmental surfaces and poultry carcass rinses using peptone buffers.

Sensitive methods for enumeration of Listeria monocytogenes (L. monocytogenes) are needed to verify compliance with microbiological criteria in ready-to-eat foods. Here, we assessed the reference EN ISO 11290-2 method and three modifications of it with lower threshold levels for enumeration in terms of specificity, false results and practical limitations for use. Two of the methods, called the EURL and the Cyprus protocols, use membrane filtration to obtain a more concentrated test suspension, and the third, called the Norway protocol, uses less diluent. This study included 18 samples of foods naturally contaminated with L. monocytogenes at concentrations of 0.2-80 CFU/g. All four tested methods yielded valid results with good repeatability (Fisher’s test, p, FR; PDF; corresponding author: lena.barre@anses.fr, {"references":["AFNOR. 1998. Analyse des produits agricoles et alimentaires – Procédure de validation intra-laboratoire d'une méthode alternative par rapport à une méthode de référence – Cas de méthodes d'analyse quantitatives. AFNOR NF V03-110; Association française de normalisation, La Plaine Saint-Denis, France","AFNOR. 2009. Poissons transformés - Méthode pour le dénombrement de Listeria monocytogenes aux faibles niveaux de contamination dans le saumon fumé et la truite fumée.","NF V45-008. Association française de normalisation, La Plaine Saint-Denis, France","Anonymous. 2000. Rapport de la commission d'étude des risques liés à Listeria monocytogenes, juillet 2000, AFSSA. Maisons Alfort : Agence française de sécurité sanitaire des aliments, 143 p.","Barre L, Brasseur E, Doux C, Lombard B, Gnanou Besse N. 2015. Sensitive enumeration of Listeria monocytogenes and other Listeria species in various naturally contaminated matrices using a membrane filtration method. Food microbiology 48:171-177.","Baudouin N, Lombard B, Audinet N, Gnanou Besse N. 2010. Enumeration of Listeria monocytogenes at low contamination levels in several food matrices using a membrane filtration method. Food Analytical Methods 3:54-64.","Buchanan RL, Gorris LGM, Hayman MM, Jackson TC, Whiting RC. 2017. A review of Listeria monocytogenes: An update on outbreaks, virulence, dose-response, ecology, and risk assessments. Food Control, 75:1-13, 10.1016/j.foodcont.2016.12.016","EFSA. 2007. Scientific opinion of the panel on biological hazards on a request from the European Commission on Request for updating the former SCVPH opinion on Listeria monocytogenes risk related to ready-to-eat foods and scientific advice on different levels of Listeria monocytogenes in ready-to-eat foods and the related risk for human illness. EFSA Journal 59:1-42.","European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC). 2012. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2010. EFSA Journal 10(3):2597.","EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards), Ricci A, Allende A, Bolton D, Chemaly M, Davies R, Fernández Escámez PS, Girones R, Herman L, Koutsoumanis K, Nørrung B, Robertson L, Ru G, Sanaa M, Simmons M, Skandamis P, Snary E, Speybroeck N, Ter Kuile B, Threlfall J, Wahlström H, Takkinen J, Wagner M, Arcella D, Da Silva Felicio MT, Georgiadis M, Messens W, Lindqvist R. 2018. Scientific Opinion on the Listeria monocytogenes contamination of ready-to-eat foods and the risk for human health in the EU. EFSA Journal 16(1):5134, 173 pp.","Gnanou Besse N, Beaufort A, Rudelle S, Denis C, Lombard B. 2008. Evaluation of an enumeration method for Listeria monocytogenes at low contamination levels in cold-smoked salmon. 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Microbiology of food and animal feeding stuffs - Horizontal method for detection and enumeration of Listeria monocytogenes, Part 2. Enumeration method, Amendment 1: 2004. Modification of the enumeration medium. International Standard ISO 11290-2. Geneva: International Organisation for Standardisation.","Jadhav S, Bhave M, Palombo EA. 2012. Methods used for the detection and subtyping of Listeria monocytogenes. Journal of microbiological methods 88(3):327-341.","Skjerdal T, Reitehaug E, Eckner K. 2014. Development of performance objectives for Listeria monocytogenes contaminated salmon (Salmo salar) intended used as sushi and sashimi based on analysis of naturally contaminated samples. International Journal of Food Microbiology 184:8-13","Välimaa AL, Tilsala-Timisjärvi A, Virtanen E. 2015. Rapid detection and identification methods for Listeria monocytogenes in the food chain–A review. Food Control 55:103-114."]}

* This record is given in both English and Spanish Microbiological methods for the analytical control of antibiotic residues are based on detecting the only common characteristic among all antibiotics, their ability to inhibit bacterial growth. Therefore, over the past 30 years in Spain, they have played an important role in the control of these residues within the framework of the National Residue Monitoring Plan in food. Its simplicity, flexibility, low cost and multi-residue detection and sample processing capabilities made them the methods of choice for undertaking the analysis of antibiotic residue screening. The validation of some of the antibiotic residue screening methods is complex given that intact tissue is used as a test sample. Therefore, the five-plate screening method validation was completed through collaborative trials in which the National Reference Laboratory and the official control laboratories participated, so that their validation was shared and the accreditation of the method was facilitated according to the ISO/IEC 17025 standard. As a National Reference Laboratory for antibiotic residues in food, the National Centre for Food (CNA) of the Spanish Agency for Food Safety and Nutrition (AESAN) has carried out a whole series of activities to serve the official control laboratories, including: laboratory communications, conferences, courses, intercomparison and collaborative tests and supplying materials for analysis. The current development of multi-residue physical-chemical instrumental methods and with detection limits better adjusted to the maximum antibiotic residue limits established in the European Union has resulted in a turning point and has limited the applicability of traditional microbiological methods for detecting antibiotic residues in the official control of the same, ES; PDF; pfefsa@aesan.gob.es

Število probiotičnih izdelkov tako na slovenskem kot tudi svetovnem trgu vsako leto strmo narašča. Raziskave kakovosti in ustreznosti probiotičnih prehranskih dopolnil pogosto pokažejo neskladnost med deklariranimi in ugotovljenimi podatki o številu in vrsti probiotičnih bakterij v izdelkih. Razen tega neredko poročajo o zdravstvenih trditvah, ki jih na probiotičnih prehranskih dopolnilih ne bi smelo biti. Namen magistrskega dela je bil analizirati nekatere probiotične izdelke, ki so naprodaj v lekarnah in specializiranih trgovinah na slovenskem trgu. Pod drobnogled smo vzeli 19 izdelkov ter s pomočjo konvencionalne metode štetja na selektivnih gojiščih in molekularno-bioloških metod preverili skladnost glede števila in prisotnosti deklariranih bakterij. Prav tako smo preverili, ali imajo izdelki ustrezne deklaracije. Na vseh izdelkih so bile navedene bakterijske vrste, ne pa tudi posamezni sevi. Na večini deklaracij so bili le podatki za skupno število probiotičnih bakterij, ne pa za posamezno bakterijsko vrsto. Le devet od devetnajstih izdelkov je vsebovalo zadostno število bakterij in le osem izdelkov je vsebovalo vse deklarirane bakterijske vrste. Prisotnost deklariranih bakterijskih vrst smo ugotavljali z metodo PCR. Standardne gojitvene metode zaradi omejenega izbora selektivnih gojišč večinoma ne omogočajo razlikovanja med posameznimi bakterijskimi vrstami, so pa uporabne za ugotavljanje skupnega števila probiotičnih bakterij. Domneva, da so na slovenskem trgu probiotični izdelki, ki ne ustrezajo kriterijem kakovosti in ustreznosti, se je izkazala za upravičeno. The number of probiotic products on Slovenian and global market is increasing every year. Research on the quality and adequacy of probiotic food supplements often shows many discrepancies between the number and identity of probiotic bacteria present in the product and those claimed on the label. Often, there have also been health claims observed, which are not allowed on probiotic food supplements. The purpose of this study was to analyze some of the probiotic products avaliable in pharmacies and specialised stores on the Slovenian market. We examined 19 products and used conventional plate counting and molecular methods in order to verify if the number of bacteria corresponds to the number on the label and if declared species are present in the product. We have also checked whether the declarations on the products were appropriate. Names of bacterial species were labeled on all products, however many of them lacked the designation of the bacterial strain. Most of the products had only the total number of bacteria listed but not also the number of each species. Only nine out of nineteen products contained sufficient total number of probiotic bacteria and only eight products contained all of the claimed bacterial species. We used PCR method to detect individual bacterial species. Conventional methods due to the lack of selective growth media often do not allow differentiation between related bacterial speices but are more usefull for the enumeration of total probiotic bacteria. Our hypothesis that there are probiotic products on Slovenian market which doesn´t meet criteria of quality and adequacy, was confirmed.

V magistrskem delu smo spremljali odziv bakterije Bacillus subtilis PS-216 na signalni peptid ComX, ki sproži odziv na kvorum (zadostno število) celic. Optimizirali smo protokol za pripravo rekombinatnega peptida ComX v bakteriji Escherichia coli ED367 in ga uspeli izboljšati tako, da smo kulturo najprej namnožili do visoke celične gostote in nato v naslednji stopnji inducirali sintezo ComX z IPTG. Tako pripravljen ComX shranjen v pufru SS in BSA je bil relativno stabilen pri temperaturi 4 °C in razpolovna doba ComX je bila 100 dni. Odziv surfaktinskega promotorja (PsrfAA) na ComX smo spremljali s pomočjo reporterskega gena yfp ali gena, ki kodira -galaktozidazo. Primerjali smo odziv na ComX dveh biosenzorjev (BM1456 in BM1455), ki ne proizvajata signala in ki smo jima dodali ComX: biosenzor 1 je imel gen comQ prekinjen z antibiotičnim označevalcem biosenzor 2 je imel gen comQ prekinjen s tehnologijo brez označevalca. Naši rezultati so potrdili, da je biosenzor 1 bolj občutljiv na ComX (k= 2,7 ± 0,2) kot biosenzor 2 (k=3,5 ± 0,2). Izboljšali smo tudi metodo za kvantitativno analizo odziva biosenzorja na ComX s konfokalno mikroskopijo na nivoju posamezne celice. Rezultati so pokazali, da se B. subtilis odziva šele po treh urah izpostavljenosti ComX, ko je populacija v eksponentni fazi rasti, odziv se poveča na prehodu v stacionarno fazo (po 4 urah) do maksimalne jakosti in je primerljiv odzivu v stacionari fazi rasti (po 6h urah inkubacije). We studied a quorum sensing (QS) response to the ComX signaling peptide in Bacillus subtilis PS-216. First of all, we optimized the protocol for synthesis of signal peptide ComX from Escherichia coli ED367 to reach bigger yield. Bacteria were firstly incubated in rich medium to reach high cell density and then followed IPTG induction of cell culture in minimal medium. Furthermore, we also checked stability of pure ComX signal peptide over time stored in SS buffer with BSA at 4 °C. ComX signal peptide in buffer was relatively stable (DT50~100 days). To study the response of B. subtilis PS-216 to ComX, we used biosensors that do not produce the peptide and carry a fluorescence reporter (yfp) fused to the surfactin promoter (PsrfAA), known to be under the QS control. We used two different mutant strains B. subtilis PS-216 to compare quorum sensing response. One strain had inactivation of comQ gene with antibiotic marker (BM1456), the other strain had deletion of comQ gene with markerless technology (BM1455). Our results show oversensitivity of biosensor 1, BM1456 (k= 2,7 ± 0,2) to biosensor 2, BM1455 (k=3,5 ± 0,2). Cell response to ComX was measured at 3 different incubation times – 3 h, 4 h, and 6 h. We observed cell response to ComX after 3 h incubation (exponential growth phase), with stronger response to ComX at late growth phase (4 h) and at stationary phase (6 h).

The presence of Clostridium botulinum spores in 59 honey samples originating from different regions of the Republic of Serbia was studied. In addition to microbiological methods, after enrichment, centrifugation and membrane filtration, molecular methods (PCR methods) were utilized. The number of spores in PCR positive samples was estimated by the most probable number (MPN) method. PCR confirmed C. botulinum spores in 5 (8.47%) honey samples. MPN of spores varied from 20/kg to 204/kg honey. PCR was more sensitive than cultural methods. Natural honey contamination with C. botulinum spores is low-level and not homogeneous, and therefore, PCR methods require multiple sub-sampling.

Bakterijski biofilmi predstavljajo velik problem v medicini in živilski industriji. Bakterijski biofilm je vir kontaminacije v prehranski verigi, problem predstavlja tudi kot mehanska ovira, hkrati pa je zelo težko odstranljiv. Agregacijska sposobnost in površinska hidrofobnost bakterij sta ključna dejavnika za nastanek biofilma. Agregacija mikroorganizmov je ključna za nastanek mikrokolonij, površinska hidrofobnost bakterij pa je v povezana z bakterijsko adhezivnostjo. To je virulentna lastnost bakterij. Pri nalogi smo uporabili različne seve Campylobacter jejuni. Ta bakterija je pogosta povzročiteljica zoonoze, bolezni, ki se prenaša posredno ali neposredno iz živali na ljudi. Pri poskusih smo primerjali različne seve in njihove kombinacije s testi agregacije s pomočjo merjenja optične gostote in izračuna agregacije in ko-agregacije. Stopnjo adhezivnosti na polistirenski površini pa smo ocenili s pomočjo primerjave dveh postopkov - s kristalvijoličnim barvilom ter s štetjem kolonij na trdnih gojiščih. Rezultati so pokazali, da je bakterijska agregacija pri bakteriji C. jejuni sevnospecifična lastnost. Ko-agregacija sevov se razlikuje od sposobnosti agregacije posameznih sevov. Naši rezultati pa niso potrdili, da je sposobnost agregacije in ko-agregacije v korelaciji s sposobnostjo adhezivnosti in ko-adhezivnosti. Bacterial biofilms are a big problem in medicine and food industry. A bacterial urce of contamination in a food chain, it presents a problem as a mechanical obstacle and it is very difficult to remove. Aggregational capability of bacteria and bacterial surface hydrophobicity are key factors for the formation of biofilm. Microbiological aggregation is necessary for microcolony formation, while bacterial surface hydrophobicity is related to bacterial adhesiveness. This is a virulent trait of bacteria. We used different strains of Campylobacter jejuni. This bacteria is a common cause of zoonoses, a disease which spreads directly or indirectly from animals to people. In the experiments, we compared different strains and their combinations in a test of aggregation with measuring optical density and a later calculation of aggregation and co-aggregation. The level of bacterial adhesiveness on a polystyrene surface was evaluated with using Kristal violet staining and plate counting. Results showed that bacterial aggregation of C. jejuni is a strain-specific trait. Bacterial co-aggregation is different from the capability of bacterial aggregation of individual strains. Our results did not confirm that the capability of bacterial aggregation and co-aggregation is in correlation with the capability of bacterial adhesiveness and co-adhesiveness.

Als onderdeel van een Europees project werd de methode voor de telling van Clostridium perfringens in voedingsmiddelen en diervoeders, zoals beschreven in ISO 7937 (1997) gevalideerd. Een ringonderzoek werd georganiseerd om de precisie, zijnde de herhaalbaarheid (r) en de reproduceerbaarheid (R), van deze methode te bepalen. Zeventien laboratoria in 13 Europese landen analyseerden kaas, vlees, gedroogd diervoeder en een referentiemateriaal volgens een standaard protocol. Alle monsters waren kunstmatig besmet om het gewenste besmettingsniveau (blanco, laag, midden en hoog) en homogeniteit te verkrijgen. De studie bevatte twee technieken voor de bevestiging van vermoedelijk Clostridium perfringens, zodat mogelijke verschillen in prestatie gedetecteerd konden worden: 1) lactose-sulfiet medium (ISO 7937) en 2) lactose-gelatine medium en beweeglijkheid-nitraat medium (facultatief in EN 13401). De prestatiekenmerken werden berekend volgens zowel ISO 5725 (1994) als volgens prEN ISO 16140 (2000). De herhaarbaarheid (r) en reproduceerbaarheid (R) werden voor elk monster type, concentratieniveau en bevestigingsmethode afzonderlijk berekend. De precisie varieerde per monstertype. De analyse van de RMs leverde de beste prestatiekenmerken op

The European Drinking Water Directive defines reference methods for the enumeration of microbiological parameters in drinking water. The method to be used for Escherichia coli and coliforms is the membrane filtration technique on Lactose TTC agar with Tergitol 7. Many technical drawbacks of the procedure, as well as its limitations regarding the recent taxonomy of coliforms, make it necessary to evaluate alternative methods. Two alternative assays, a chromogenic media (m-ColiBlu24®) and a defined substrate technology-DST test (Colilert 18/Quanty Tray™) were compared with the ISO standard with attention to the phenotypic characteristic of the isolates. Results showed that the ISO method failed to detect an important percentage of coliforms and E. coli while m-ColiBlu24® and Colilert 18 provided results in a shorter time allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps.

U radu su opisane mikrobiološke te enzimatske metode uklanjanja bentazona, pendimetalina i terbutilazina. Odabrani herbicidi nalaze se u tri različite kemijske skupine jer se razlikuju po mehanizmu djelovanja. Bentazon se ubraja u benzotiadiazinone, pendimetalin u dinitroaniline, a terbutilazin u triazine. Benzotiadiazinoni i triazini inhibitori su fotosinteze, dok su dinitroanilini inhibitori diobe biljnih stanica. Razgradnja terbutilazina u tlu ovisi i o temperaturi te se brže razgrađuje na višim temperaturama. Uklanjanje pendimetalina iz vode moguće je korištenjem bakterija koje imaju sposobnost razgradnje tog herbicida. Kao postupak razgradnje u literaturi spominje se bioremedijacija koja djeluje kao prirodni proces mikrobiološke razgradnje odnosno kao interakcija mikroorganizama i sastojaka otpada, primjenom bioreaktora te tzv. „zelena tehnologija“ koja podrazumijeva biljke kao faktore razgradnje različitih onečišćujućih tvari. Postupci uklanjanja provode se kako bi se smanjio utjecaj na zdravlje ljudi te sačuvao prirodni okoliš. This paper describes the microbiological and enzymatic methods for removing bentazone, pendimethalin and terbuthylazine. Selected herbicides are divided in three different chemical groups differing in the mechanism of action. Bentazon is included in the benzothiadiazinones, pendimethalin in the dinitroaniline and terbuthylazine in the benzothiadiazinones. Benzothiadizinones and triazines are inhibitors of photosynthesis while dinitroanilines are inhibitors of plant cell division. Terbuthylazine degradation in the soil also depends on the temperature and is quickly degraded at higher temperatures. Pendimethalin can be removed from water using bacteria that have the ability of herbicide degradation. As a process of degradation bioremediation is mentioned, this acts as a natural process of microbial degradation or by the interaction of microorganisms and components of waste, by using a bioreactor. Removal processes are carried out in order to reduce the impact on human health and preserve the natural environment.

Background: Mycoplasma synoviae is an important avian pathogen which can cause both respiratory disease and synovial joint inflammation (synovitis) in poultry. Mycoplasmas spp. may cause the respiratory system infection in ostriches with symptoms such as inflammation of the nose, trachea and also damages of lungs. Objectives: The current study aimed to use the M. synoviae specific Polymerase Chain Reaction (PCR) and microbiological methods in order to isolate and identify M. synoviae from suspected ostriches in Kerman Province, Iran, and compare the two methods (microbiological and PCR) employed to confirm Mycoplasmal contamination of ostrich lungs. Materials and Methods: Fifty three samples of different parts of lung and trachea were immediately collected after slaughtering the ostriches in Kerman Province six months. Samples were cultured in the same conditions in pleuropneumonia-like organism (PPLO) broth and to isolate and identify M. synoviae, PCR and microbiological methods were conducted. The identified isolates were confirmed by specific amplification of 16S rRNA gene (163 and 207 base pair). Results: In the current study, 25 and 17 out of 53 ostrich samples were identified as Mycoplasma-positive in the PCR and microbiological methods, respectively; and 13 out of 25 the mentioned Mycoplasma-positive samples were also confirmed by PCR method. Conclusions: The current study showed that PCR method is time consuming, effective, and efficient method to detect M. synoviae infection in ostriches. PCR method could be recommended as an alternative for culturing; M. synoviae was isolated from ostriches for first time in Kerman Province, Iran.

R3-Nordic, the Nordic Society of Cleanroom Technology, is a non-profit, independent association for the promotion of new technologies in cleanroom technology and contamination control in the Nordic countries. The aim of the annual R3Nordic Symposium is to provide knowledge within the pharmaceutical, food and electronic industries as well as hospitals and hospital pharmacies. This year the sessions at the 45th R3Nordic Symposium are Pharma, Hospital and General Sessions and the presentations deal with construction and design, planning, auditing, contamination control, cleanroom technology and management, sterilization techniques, cleaning of clean rooms, protective clothing, monitoring techniques, rapid test methods and regulations in clean and controlled rooms. The venue of the annual symposium 2014 is Naantali Spa in Naantali. The persons involved in the Programme Committee are Satu Salo, Kari Leonsaari, Leila Kakko, Sirkka Malmioja, Antti Mikkola, Raimo Pärssinen and Gun Wirtanen. The editors of the proceedings would like to express their gratitude to the speakers for preparing the abstracts published in the journal Renhetsteknik 1/14 as well as the extended abstracts or full papers published in these electronic proceedings.