SK289178B6 - Microorganism strain Weissella cibaria 4/8 D37 CCM 9015, bacterial culture, cell-free supernatant of the strain and pharmaceutical composition containing this strain - Google Patents
Microorganism strain Weissella cibaria 4/8 D37 CCM 9015, bacterial culture, cell-free supernatant of the strain and pharmaceutical composition containing this strain Download PDFInfo
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Oblasť technikyThe field of technology
Predložený vynález sa týka izolovaného kmeňa mikroorganizmov Weissella cibaria Biocenol™ 4/8 D37 CCM 9015, ktorý je zároveň prostriedkom na prípravu topických preparátov na účely harmonizácie kožnej mikrobiocenózy.The present invention relates to the isolated strain of microorganisms Weissella cibaria Biocenol™ 4/8 D37 CCM 9015, which is also a means for the preparation of topical preparations for the purpose of harmonizing skin microbiocenosis.
Predložený vynález sa tiež týka kultúry mikroorganizmu získanej kultiváciou uvedeného mikroorganizmu alebo mikroorganizmov, ktoré ju obsahujú. Kmeň Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 bol izolovaný zo zdravej kože koňa.The present invention also relates to a microorganism culture obtained by cultivating said microorganism or microorganisms containing it. Weissella cibaria strain Biocenol™ 4/8 D37 CCM 9015 was isolated from healthy horse skin.
Doterajší stav technikyCurrent state of the art
Antimikrobiálna rezistencia predstavuje vážnu globálnu hrozbu pre ľudí a zvieratá (Cechini a kol. 2015). Výskyt a prenos mikroorganizmov rezistentných na antibiotiká je častejší pri lokálnej aplikácii antibiotík ako pri systémovej a je potrebné použiť alternatívy, akými sú napr. prospešné mikroorganizmy a ich bioaktívne látky (Nakamura a kol. 2007; Mihai a kol. 2018). Ak dôjde k dysbióze kože v dôsledku lokálnej antibiotickej liečby, môžu prospešné mikroorganizmy pôsobiť ako modulátory obnovujúce mikrobiálnu rovnováhu. Hlavným mechanizmom ich pozitívneho účinku je takzvaná kolonizačná rezistencia, ktorá zahŕňa napríklad kompetitívnu exklúziu, vytvorenie nevhodného prostredia na rast kožných patogénov a modifikáciu kožnej mikrobiocenózy zvýšením hustoty a diverzity prospešných zložiek kožnej mikrobioty (Walker 2008; Cinque a kol. 2011). Rod Weissella patrí do kmeňa Firmicutes, triedy Bacilli, radu Lactobacillales a čeľade Leuconostocaceae (Collins a kol. 1993) a je zahrnutý v skupine baktérií mliečneho kvasenia (Dillon a kol. 2014). Weissella spp. sa používajú hlavne v potravinárskom priemysle pri výrobe fermentovaných potravín a ako probiotiká (Zhang a kol. 2014; Kang a kol. 2019) hlavne kvôli ich antimikrobiálnej aktivite, ako je to v prípade určitých bakteriocinogénnych kmeňov Weissella paramesenteroides, Weissella hellenica a Weissella cibaria (Fusco a kol. 2015). W. cibaria CMU, ktorá inhibovala tvorbu biofilmu pri Streptococcus mutans, sa využíva ako prevencia proti tvorbe biofilmu v ústnej dutine (Kang a kol. 2016) a W. cibaria JW15 sa používa ako probiotikum pre psy (Sun a kol. 2019). Patentový spis KR101667496 (B1) opisuje využite kmeňa Weissella cibaria WIKIM28 izolovaného z kimchi na orálnu aplikáciu a ovplyvnenie imunitného systému pri liečbe atopickej dermatitídy. Patent KR20180124302 opisuje využite kmeňa Weissella cibaria AB160066 izolovaného z kimchi ako funkčnej potraviny.Antimicrobial resistance represents a serious global threat to humans and animals (Cechini et al. 2015). The occurrence and transmission of antibiotic-resistant microorganisms is more frequent with local application of antibiotics than with systemic, and it is necessary to use alternatives, such as e.g. beneficial microorganisms and their bioactive substances (Nakamura et al. 2007; Mihai et al. 2018). If skin dysbiosis occurs as a result of topical antibiotic treatment, beneficial microorganisms can act as modulators to restore the microbial balance. The main mechanism of their positive effect is the so-called colonization resistance, which includes, for example, competitive exclusion, the creation of an unsuitable environment for the growth of skin pathogens and the modification of the skin microbiocenosis by increasing the density and diversity of the beneficial components of the skin microbiota (Walker 2008; Cinque et al. 2011). The genus Weissella belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales, and family Leuconostocaceae (Collins et al. 1993) and is included in the group of lactic acid bacteria (Dillon et al. 2014). Weissella spp. are mainly used in the food industry in the production of fermented foods and as probiotics (Zhang et al. 2014; Kang et al. 2019) mainly due to their antimicrobial activity, as in the case of certain bacteriocinogenic strains of Weissella paramesenteroides, Weissella hellenica and Weissella cibaria (Fusco et al. 2015). W. cibaria CMU, which inhibited biofilm formation in Streptococcus mutans, is used as a prevention against biofilm formation in the oral cavity (Kang et al. 2016), and W. cibaria JW15 is used as a probiotic for dogs (Sun et al. 2019). Patent file KR101667496 (B1) describes the use of Weissella cibaria strain WIKIM28 isolated from kimchi for oral application and influencing the immune system in the treatment of atopic dermatitis. Patent KR20180124302 describes the use of Weissella cibaria strain AB160066 isolated from kimchi as a functional food.
Podstata vynálezuThe essence of the invention
Podstatou tohto vynálezu je kmeň mikroorganizmu Weissella cibaria Biocenol™ 4/8 D37 deponovaného v Českej zbierke mikroorganizmov Masarykovej univerzity Prírodovedeckej fakulty, Kamenice 5, budova A25, 625 00, Brno, Česká republika, pod číslom CCM 9015 podľa podmienok Budapeštianskej zmluvy.The essence of this invention is the strain of the microorganism Weissella cibaria Biocenol™ 4/8 D37 deposited in the Czech collection of microorganisms of the Masaryk University Faculty of Natural Sciences, Kamenice 5, building A25, 625 00, Brno, Czech Republic, under number CCM 9015 under the terms of the Budapest Treaty.
Mikroorganizmus bol izolovaný zo zdravej kože koňa. Podstatou tohto vynálezu je aj kultúra mikroorganizmu získaná kultiváciou tohto mikroorganizmu alebo mikroorganizmov.The microorganism was isolated from the healthy skin of a horse. The essence of this invention is also the culture of a microorganism obtained by cultivating this microorganism or microorganisms.
Vyznačuje sa produkciou prospešných substancií, antimikrobiálnou, antibiofilmovou a disperznou aktivitou proti patogénnym mikroorganizmom a je schopný prežívať na koži aj po ukončení topickej aplikácie; morfológia kolónie pri fakultatívne aeróbnom raste na selektívnom agare pre baktérie mliečneho kvasenia: guľatá, hladká, lesklá, mierne vypuklá, okraj súvislý, biela farba, priemer 1 až 3 mm. Kmeň netvorí hemolýzu. Pri stanovení minimálnych inhibičných koncentrácií (MIC) mikrodilučnou metódou s použitím mikrotitračných panelov VetMIC Lact-1 a Lact-2 na testovanie citlivosti baktérií (Statens Veterinarmedicinska Anstalt, Uppsala, Švédsko) proti antibiotikám stanovené EFSA (2012) pre fylogeneticky príbuzný rod Leuconostoc (Flórez a kol. 2016) je kmeň citlivý na tetracyklín, gentamycín, erytromycín, klindamycín, streptomycín, chloramfenikol, neomycín, kanamycín, ampicilín, penicilín, virginiamycín, linezolid, trimetoprim, ciprofloxacín a rifampicín. Kmeň produkuje najmä kyselinu mliečnu, v menších množstvách kyselinu octovú, acetoctovú, jantárovú a mravčiu. Neneutralizovaný bezbunkový supernatant (nnCFS) kmeňa úplne inhibuje rast a tvorbu biofilmu referenčného kmeňa Staphylococcus aureusIt is characterized by the production of beneficial substances, antimicrobial, antibiofilm and dispersion activity against pathogenic microorganisms and is able to survive on the skin even after the end of topical application; colony morphology during facultative aerobic growth on selective agar for lactic acid bacteria: round, smooth, shiny, slightly convex, continuous edge, white color, diameter 1 to 3 mm. The strain does not form hemolysis. When determining the minimum inhibitory concentrations (MIC) by the microdilution method using the VetMIC Lact-1 and Lact-2 microtiter panels for bacterial susceptibility testing (Statens Veterinarmedicinska Anstalt, Uppsala, Sweden) against antibiotics established by EFSA (2012) for the phylogenetically related genus Leuconostoc (Florez and col. 2016) is a strain sensitive to tetracycline, gentamicin, erythromycin, clindamycin, streptomycin, chloramphenicol, neomycin, kanamycin, ampicillin, penicillin, virginiamycin, linezolid, trimethoprim, ciprofloxacin, and rifampicin. The strain mainly produces lactic acid, with smaller amounts of acetic, acetoacetic, succinic and formic acids. Non-neutralized cell-free supernatant (nnCFS) of the strain completely inhibits the growth and biofilm formation of the reference strain Staphylococcus aureus
CCM 4223 (Česká zbierka mikroorganizmov Masarykovej univerzity, Brno) tvoriaceho biofilm a klinického izolátu meticilín rezistentného kmeňa S. aureus (MRSA). nnCFS spôsobuje disperziu predvytvoreného 24-hodinového biofilmu S. aureus CCM 4223 a MRSA v rozpätí 70 až 77 %. Neutralizovaný bezbunkový supernatant (nCFS) kmeňa inhibuje rast S. aureus CCM 4223 a MRSA v rozpätí 41 až 44 % a ich tvorbu biofilmu v rozpätí 95 až 97 %. Kmeň inhibuje rast grampozitívnych aj gramnegatívnych baktérií, konkrétne Escherichia coli O149 F4, Proteus mirabilis CCM 7188, Staphylococcus aureus CCM 4223 a Bacillus cereus CCM 869. Inhibičná aktivita nCFS kmeňa je negovaná po ošetrení katalázou aj trypsínom, čo naznačuje úlohu peroxidu vodíka a bakteriocínu v inhibičnej aktivite kmeňa. Kmeň je topicky aplikovaný samostatne alebo kultivovaný na nosiči, na ktorom je následne aplikovaný na kožu v čerstvom stave alebo v lyofilizovanej forme. Slúži na prevenciu dermatitíd alebo na harmonizáciu kožnej mikrobiocenózy po aplikácii antibiotík a/alebo kortikosteroidov. Kmeň prežíva na koži 7 a viac dní od ukončenia jeho topickej aplikácie.CCM 4223 (Czech Collection of Microorganisms of the Masaryk University, Brno) forming a biofilm and a clinical isolate of methicillin-resistant strain S. aureus (MRSA). nnCFS causes dispersion of preformed 24-hour biofilm S. aureus CCM 4223 and MRSA in the range of 70-77%. Neutralized cell-free supernatant (nCFS) of the strain inhibits the growth of S. aureus CCM 4223 and MRSA in the range of 41 to 44% and their biofilm formation in the range of 95 to 97%. The strain inhibits the growth of both Gram-positive and Gram-negative bacteria, namely Escherichia coli O149 F4, Proteus mirabilis CCM 7188, Staphylococcus aureus CCM 4223 and Bacillus cereus CCM 869. The inhibitory activity of the nCFS strain is negated after both catalase and trypsin treatment, suggesting the role of hydrogen peroxide and bacteriocin in the inhibition activity of the tribe. The strain is topically applied alone or cultured on a carrier, on which it is subsequently applied to the skin in fresh or lyophilized form. It serves to prevent dermatitis or to harmonize the skin microbiocenosis after the application of antibiotics and/or corticosteroids. The strain survives on the skin for 7 or more days from the end of its topical application.
Podstatu tohto vynálezu tvorí aj bakteriálna kultúra, ktorá obsahuje kmeň mikroorganizmu Weissella cibaria Biocenol™ 4/8 D37 deponovaného v Českej zbierke mikroorganizmov Masarykovej univerzity Prírodovedeckej fakulty, Kamenice 5, budova A25, 625 00, Brno, Česká republika, pod číslom CCM 9015.The essence of this invention is also a bacterial culture, which contains a strain of the microorganism Weissella cibaria Biocenol™ 4/8 D37 deposited in the Czech collection of microorganisms of the Masaryk University Faculty of Natural Sciences, Kamenice 5, building A25, 625 00, Brno, Czech Republic, under the number CCM 9015.
Ďalšou podstatou tohto vynálezu je aj farmaceutická kompozícia obsahujúca terapeuticky účinné množstvo uvedeného kmeňa mikroorganizmu Weissella cibaria Biocenol™ 4/8 D37 alebo bakteriálnu kultúru, a/alebo bezbunkový supernatant a farmaceuticky prijateľný nosič. Farmaceutická kompozícia môže obsahovať biologicky čistú čerstvú kultúru alebo vo forme na fosílnom nosiči, ktorým môže byť alginit, alebo môže obsahovať lyofilizovanú kultúru na nosiči alebo bezbunkový supernatant tejto kultúry. Takéto farmaceutické kompozície sú vhodné na topickú aplikáciu na kožu.Another essence of this invention is also a pharmaceutical composition containing a therapeutically effective amount of said strain of the microorganism Weissella cibaria Biocenol™ 4/8 D37 or a bacterial culture, and/or a cell-free supernatant and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain a biologically pure fresh culture or in a form on a fossil carrier, which may be alginite, or it may contain a lyophilized culture on a carrier or a cell-free supernatant of this culture. Such pharmaceutical compositions are suitable for topical application to the skin.
Fosílnym nosičom môže byť napríklad aj leonardit, smektit, bentonit alebo lignit.The fossil carrier can be, for example, leonardite, smectite, bentonite or lignite.
Príklady uskutočnenia vynálezuExamples of implementation of the invention
Príklad 1Example 1
Postup prípravy kmeňaProcedure for preparing the stem
Pomocou sterilnej odberovej súpravy je odobratý ster z kože koňa. Odberový tampón je ponorený do skúmavky s 2 ml fyziologického roztoku. Vzorky sú nariedené na -1 a -2. K 4,5 ml fyz. roztoku je pridaných 0,5 ml vzorky a pomocou pipety prenesených 0,5 ml do ďalšej skúmavky. Jednotlivé riedenia sú naočkované po 100 μl na Petriho misky so selektívnou pôdou pre rast baktérií mliečneho kvasenia. Naočkované platne sú umiestnené do anaerostatov s vyvíjačom anaeróbneho prostredia, vložené do termostatu a kultivované počas 48 hodín pri 27 °C. Po inkubácii sú kolónie morfologicky posúdené (guľaté, hladké, lesklé, mierne vypuklé, okraj súvislý, biela farba, priemer 1 - 3 mm) a odpichnuté na nanesenie do čiarky na Petriho misky so selektívnou pôdou pre rast baktérií mliečneho kvasenia. Po kultivácii za uvedených podmienok je z porastených kultúr zhotovený preparát farbením podľa Grama. Na posúdenie morfológie je použitý svetelný mikroskop s 1 000-násobným zväčšením. Weissella spp. sú pozorované ako grampozitívne sfarbené, nepohyblivé, nesporulujúce, krátke tyčinky. Katalázová skúška je negatívna.A swab is taken from the horse's skin using a sterile collection kit. The sampling swab is immersed in a test tube with 2 ml of physiological solution. Samples are diluted to -1 and -2. To 4.5 ml of physical 0.5 ml of the sample is added to the solution and 0.5 ml is transferred to another test tube using a pipette. Individual dilutions are inoculated at 100 μl on Petri dishes with selective medium for the growth of lactic acid bacteria. The inoculated plates are placed in anaerostats with an anaerobic environment developer, placed in a thermostat and cultured for 48 hours at 27°C. After incubation, the colonies are morphologically assessed (round, smooth, shiny, slightly convex, continuous edge, white color, diameter 1-3 mm) and pricked to apply to a line on Petri dishes with selective soil for the growth of lactic acid bacteria. After cultivation under the mentioned conditions, a preparation is made from the grown cultures by staining according to Gram. A light microscope with 1,000 times magnification is used to assess the morphology. Weissella spp. are observed as Gram-positive stained, non-motile, non-sporulating, short rods. Catalase test is negative.
Príklad 2Example 2
Stanovenie hemolytickej aktivityDetermination of hemolytic activity
Kolónie sú naočkované do čiarok na krvný agar a po uvedenej kultivácii je pozitivita izolátov indikovaná lýzou krvných buniek a bezfarebnou priehľadnou zónou okolo kolónií. Vybrané sú izoláty, ktoré netvoria hemolýzu.Colonies are inoculated into streaks on blood agar, and after said cultivation, the positivity of the isolates is indicated by blood cell lysis and a colorless transparent zone around the colonies. Isolates that do not cause hemolysis are selected.
Príklad 3Example 3
Genotypická identifikácia izolátovGenotypic identification of isolates
DNA je izolovaná zo solitárnych kolónií a 16S rRNA gény sú amplifikované pomocou PCR s použitím univerzálnych primérov Bac27F (5-AGAGTTTGATCMTGGCTCAG-3) a 1492R (5-CGGYTACCTTGTTACGACTT-3).DNA is isolated from solitary colonies and 16S rRNA genes are amplified by PCR using universal primers Bac27F (5-AGAGTTTGATCMTGGCTCAG-3) and 1492R (5-CGGYTACCTTGTTACGACTT-3).
Produkty amplifikácie sú následne sekvenované Sangerovou metódou v komerčnom laboratóriu. Bakteriálny druh je stanovený na základe konsenzuálnej sekvencie génu 16S rRNA a genotypizáciou pomocou online analýzy BLASTn.The amplification products are subsequently sequenced by the Sanger method in a commercial laboratory. The bacterial species is determined based on the consensus sequence of the 16S rRNA gene and genotyping using online BLASTn analysis.
Príklad 4Example 4
Detekcia inhibičných zón proti indikátorovým kmeňomDetection of inhibition zones against indicator strains
Inhibičné zóny sú testované diskovodifúznou metódou proti gramnegatívnym a grampozitívnym kmeňom. Veľkosť inhibičných zón kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 proti referenčným kmeňom Escherichia coli O149 F4 (Výskumný ústav veterinárneho lekárstva v Brne, Česká republika) - 18,33 ±2,89 mm, Proteus mirabilis CCM 7188 (Česká zbierka mikroorganizmov Masarykovej univerzity v Brne (CCM MUNI Brno, Česká republika) - 16,67 ±2,89 mm, Staphylococcus aureus CCM 4223 (CCM MUNI Brno, Česká republika) - 16,67 ±1,15 mm a Bacillus cereus CCM 869 (CCM MUNI Brno, Česká republika) - 26,33 ±1,15 mm.Inhibition zones are tested by the disc diffusion method against gram-negative and gram-positive strains. The size of the inhibition zones of the strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 against the reference strains Escherichia coli O149 F4 (Research Institute of Veterinary Medicine in Brno, Czech Republic) - 18.33 ±2.89 mm, Proteus mirabilis CCM 7188 (Czech Collection of Microorganisms Masaryk University in Brno (CCM MUNI Brno, Czech Republic) - 16.67 ±2.89 mm, Staphylococcus aureus CCM 4223 (CCM MUNI Brno, Czech Republic) - 16.67 ±1.15 mm and Bacillus cereus CCM 869 (CCM MUNI Brno, Czech Republic) - 26.33 ±1.15 mm.
Príklad 5Example 5
Stanovenie produkcie organických kyselínDetermination of organic acid production
Koncentrácie kyseliny mravčej, mliečnej, octovej, propiónovej, maslovej, valérovej, jantárovej, pyrohroznovej, α-ketoglutárovej, acetoctovej a β-hydroxymaslovej sú stanovené metódou kapilárnej izotachoforézy. Izolovaný kmeň Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 6 vykazoval najvyššiu produkciu kyseliny mliečnej - 119,77 ±2,47 mmol/l. Hodnoty produkcie ďalších organických kyselín: kyselina octová - 96,61 ±3,59 mmol/l, kyselina acetoctová - 35,07 ±1,66 mmol/l, kyselina jantárová - 8,69 ±0,52 mmol/l, kyselina mravčia - 2,08 ±1,88 mmol/l.Concentrations of formic, lactic, acetic, propionic, butyric, valeric, succinic, pyruvic, α-ketoglutaric, acetoacetic and β-hydroxybutyric acids are determined by the capillary isotachophoresis method. The isolated strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 6 showed the highest production of lactic acid - 119.77 ± 2.47 mmol/l. Production values of other organic acids: acetic acid - 96.61 ±3.59 mmol/l, acetoacetic acid - 35.07 ±1.66 mmol/l, succinic acid - 8.69 ±0.52 mmol/l, formic acid - 2.08 ±1.88 mmol/l.
Príklad 6Example 6
Testovanie antibiotickej rezistencieAntibiotic resistance testing
Pri stanovení minimálnych inhibičných koncentrácií (MIC) mikrodilučnou metódou s použitím mikrotitračných panelov VetMIC Lact-1 a Lact-2 proti antibiotikám stanovené EFSA (2012) pre fylogeneticky príbuzný rod Leuconostoc (Flórez a kol. 2016) je kmeň Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 citlivý na tetracyklín, gentamycín, erytromycín, klindamycín, streptomycín, chloramfenikol, neomycín, kanamycín, ampicilín, penicilín, virginiamycín, linezolid, trimetoprim, ciprofloxacín a rifampicín.When determining the minimum inhibitory concentrations (MIC) by the microdilution method using the VetMIC Lact-1 and Lact-2 microtiter panels against antibiotics established by EFSA (2012) for the phylogenetically related genus Leuconostoc (Flórez et al. 2016), the strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 susceptible to tetracycline, gentamicin, erythromycin, clindamycin, streptomycin, chloramphenicol, neomycin, kanamycin, ampicillin, penicillin, virginiamycin, linezolid, trimethoprim, ciprofloxacin, and rifampicin.
Príklad 7Example 7
Testovanie produkcie biosurfaktantovTesting the production of biosurfactants
Pomocou oil spreading testu podľa Morikawa a kol. (2000) bola otestovaná produkcia biosurfaktantov produkovaných do prostredia. Veľkosť zóny vyčírenia po odčítaní kontroly (modifikovaného bujónu, ktorý je selektívny pre baktérie mliečneho kvasenia, neobsahuje tween a má neutrálne pH) je 13 mm.Using the oil spreading test according to Morikawa et al. (2000) the production of biosurfactants produced in the environment was tested. The size of the clearing zone after subtraction of the control (modified broth that is selective for lactic acid bacteria, does not contain tween and has a neutral pH) is 13 mm.
Príklad 8Example 8
Príprava pH neneutralizovaného bezbunkového supernatantu (nnCFS) a neutralizovaného bezbunkového supernatantu (nCFS)Preparation of the pH of non-neutralized cell-free supernatant (nnCFS) and neutralized cell-free supernatant (nCFS)
Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 je naočkovaná na selektívnu pôdu pre baktérie mliečneho kvasenia a kultivovaná v anaerostate s vyvíjačom anaeróbneho prostredia počas 48 hodín pri 27 °C. Na prípravu štandardizovanej suspenzie sú použité 3 - 4 solitárne kolónie resuspendované v 5 ml fyz. roztoku a upravené na optickú denzitu McFarland 1. 0,5 ml každej suspenzie sa naočkuje do 50 ml modifikovaného bujónu (selektívneho pre baktérie mliečneho kvasenia, bez obsahu tweenu, s neutrálnym pH) a inkubuje v trepacom vodnom kúpeli pri 119 otáčkach za minútu, pri 37 °C počas 48 hodín. Po kultivácii sa kultúra centrifuguje pri 4 °C, 4 500 otáčkach za minútu počas 40 minút. Účinok organickej kyseliny je neutralizovaný úpravou pH nCFS na hodnotu 7 pomocou 10 M NaOH. pH nnCFS je v rozmedzí 3,4 až 4,0. nnCFS a nCFS sa prefiltrujú cez injekčný striekačkový filter s veľkosťou pórov 0,22 μm.Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 is inoculated onto a selective medium for lactic acid bacteria and cultured in an anaerostat with an anaerobic medium developer for 48 hours at 27°C. To prepare a standardized suspension, 3-4 solitary colonies resuspended in 5 ml of phys. solution and adjusted to a McFarland optical density of 1. 0.5 ml of each suspension is inoculated into 50 ml of modified broth (selective for lactic acid bacteria, tween-free, neutral pH) and incubated in a shaking water bath at 119 rpm, at 37 °C for 48 hours. After cultivation, the culture is centrifuged at 4°C, 4500 rpm for 40 minutes. The effect of the organic acid is neutralized by adjusting the pH of the nCFS to 7 using 10 M NaOH. The pH of nnCFS is in the range of 3.4 to 4.0. nnCFS and nCFS are filtered through a syringe filter with a pore size of 0.22 μm.
Príklad 9Example 9
Dynamika antimikrobiálnej aktivity kmeňaDynamics of antimicrobial activity of the strain
Dynamika antimikrobiálnej aktivity nCFS kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 proti biofilm tvoriacemu referenčnému kmeňu Staphylococcus aureus CCM 4223 (Česká zbierka mikroorganizmov Masarykovej univerzity, Brno) a klinickému izolátu meticilín rezistentného kmeňa S. aureus (MRSA) je testovaná turbidimetricky na prístroji Synergi 4 MultiMode microplate reader (BioTek Instruments Inc., USA) a meraná každé 4 hodiny počas 24 hodín pri 37 °C. Percento inhibície S. aureus CCM 4223 je po 8. hod. rastu 45 %, po 12. hod. 50 %, po 16. hod. 62 %, po 20. hod. 67 % a po 24. hod. 68 %. Percento inhibície S. aureus (MRSA) je po 8. hod. rastu 42 %, po 12. hod. 77 %, po 16. hod. a 20. hod. 80 % a po 24. hod. 84 %. Po 24 hod. rastu je signifikantne významný rozdiel (P < 0,0001) v raste S. aureus CCM 4223 a MRSA v prítomnosti nCFS oproti kontrole.The dynamics of the antimicrobial activity of the nCFS strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 against the biofilm-forming reference strain Staphylococcus aureus CCM 4223 (Czech Collection of Microorganisms of the Masaryk University, Brno) and the clinical isolate of the methicillin-resistant strain S. aureus (MRSA) is tested turbidimetrically on the device Synergi 4 MultiMode microplate reader (BioTek Instruments Inc., USA) and measured every 4 hours for 24 hours at 37 °C. The percentage of inhibition of S. aureus CCM 4223 is after 8 hours. growth 45%, after 12 o'clock 50%, after 4 p.m. 62%, after 8 p.m. 67% and after 24 hours 68%. The percentage of inhibition of S. aureus (MRSA) is after 8 hours. growth 42%, after 12 o'clock 77%, after 4 p.m. and 8 p.m. 80% and after 24 hours 84%. After 24 hours growth, there is a significant difference (P < 0.0001) in the growth of S. aureus CCM 4223 and MRSA in the presence of nCFS compared to the control.
Príklad 10Example 10
Antimikrobiálna, antibiofilmová a disperzná aktivita nnCFS a nCFS kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM 9015Antimicrobial, antibiofilm and dispersion activity of nnCFS and nCFS strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015
Antibiofilmová a antimikrobiálna aktivita nnCFS (pH 3,4 až 3,8) a nCFS (pH 6,99 až 7,1) proti kmeňu S. aureus CCM 4223 a klinickému izolátu MRSA je testovaná v mikrotitračných platničkách. Antibiofilmová aktivita je stanovená modifikovanou crystal violet assay podľa O'Toole a kol. (1999). Optická denzita je meraná turbidimetricky (OD550 nm). Disperzná aktivita nnCFS je stanovená na 24 hod. predformovanom biofilme S. aureus CCM 4223 a MRSA podľa Kaur a kol. (2018). nnCFS kmeňa úplne inhibuje rast a tvorbu biofilmu S. aureus CCM 4223 a MRSA, spôsobuje disperziu predvytvoreného 24-hodinového biofilmu S.Antibiofilm and antimicrobial activity of nnCFS (pH 3.4 to 3.8) and nCFS (pH 6.99 to 7.1) against S. aureus strain CCM 4223 and clinical isolate MRSA is tested in microtitre plates. Antibiofilm activity is determined by a modified crystal violet assay according to O'Toole et al. (1999). Optical density is measured turbidimetrically (OD550 nm). Dispersive activity of nnCFS is determined for 24 hours. preformed biofilm of S. aureus CCM 4223 and MRSA according to Kaur et al. (2018). nnCFS strain completely inhibits growth and biofilm formation of S. aureus CCM 4223 and MRSA, causes dispersion of preformed 24-hour biofilm of S.
aureus CCM 4223 a MRSA v rozpätí 70 až 77 %. nCFS kmeňa inhibuje rast S. aureus CCM 4223 a MRSA v rozpätí 41 až 44 % a tvorbu ich biofilmu v rozpätí 95 až 97 %.aureus CCM 4223 and MRSA in the range of 70 to 77%. The nCFS strain inhibits the growth of S. aureus CCM 4223 and MRSA in the range of 41 to 44% and their biofilm formation in the range of 95 to 97%.
Príklad 11Example 11
Antimikrobiálna aktivita nCFS kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 ošetreného trypsínom alebo katalázouAntimicrobial activity of nCFS strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 treated with trypsin or catalase
V prípade nCFS kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 je inhibičný účinok nCFS na rast S. aureus CCM 4223 100 % eliminovaný pridaním 0,5 mg/ml katalázy (Sigma-Aldrich, USA) a 1 mg/ml trypsínu (Sigma-Aldrich, USA). Po inkubácii pri 37 °C počas 24 hodín je antimikrobiálna aktivita ošetreného nCFS kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 meraná v Synergy 4 Multi-Mode microplate readeri (BioTek Instruments Inc., USA) pri optickej denzite 550 nm. Pokus sa uskutočňuje trikrát a výsledky sa uvádzajú ako priemer ± smerodajná odchýlka.In the case of nCFS strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015, the inhibitory effect of nCFS on the growth of S. aureus CCM 4223 is 100% eliminated by the addition of 0.5 mg/ml catalase (Sigma-Aldrich, USA) and 1 mg/ml trypsin ( Sigma-Aldrich, USA). After incubation at 37 °C for 24 h, the antimicrobial activity of the nCFS-treated strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 is measured in a Synergy 4 Multi-Mode microplate reader (BioTek Instruments Inc., USA) at an optical density of 550 nm. The experiment is performed in triplicate and the results are reported as mean ± standard deviation.
Príklad 12Example 12
Experiment v podmienkach in vivoExperiment in in vivo conditions
Kmeň Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 je kultivovaný na selektívnej pôde pre baktérie mliečneho kvasenia počas 48 hod. pri 27 °C s vyvíjačom anaeróbneho prostredia. Po kultivácii je kmeň prenesený do selektívneho bujónu pre baktérie mliečneho kvasenia a kultivovaný v termostate pri 27 °C počas 18 hodín, za stáleho trepania (119 rpm) na trepačke. Bujón s pomnoženou kultúrou je centrifugovaný počas 55 minút, pri 4 500 otáčkach a 4 °C. Supernatant je vyliaty, sediment je rozpustený vo fyz. roztoku a napipetovaný po 5 ml k 95 ml selektívneho bujónu pre baktérie mliečneho kvasenia. Kultivuje sa pri 27 °C za stáleho trepania na trepačke (119 rpm) v termostate počas 18 hodín. Po kultivácii je radiačne vysterilizovaný alginit zaliaty bujónom s pomnoženou kultúrou a kultivácia prebieha počas 48 hodín pri izbovej teplote. Na overenie bezpečnosti aplikácie prospešného kmeňa a alginitu (nosič prospešného kmeňa) na zdravú kožu je použitý kmeň Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 zmiešaný s radiačne vysterilizovaným alginitom v koncentrácii 9 x 109 KTJ (kolónie tvoriacich jednotiek)/g kultivačného média, ktorý je zmiešaný s radiačne vysterilizovaným alginitom. Na končatiny 6 koní je nanášaný pomocou jednorazových nesterilných vyšetrovacích rukavíc 1-krát denne ráno počas 14 dní a prekrytý cellonou, ovínadlom a copolou tak, aby boli nosičom pokryté palmárne/plantárne strany končatiny v ohybe sponkového kĺbu. Je sledované začervenanie kože, zvýšenie teploty kože, výskyt pruritu, bolesti, lézií v mieste aplikácie. Počas a po aplikácii uvedeného kmeňa na alginite na zdravú kožu distálnej časti končatín neboli pri 6 koňoch pozorované žiadne sledované kožné reakcie. Kmeň prežíva na koži koňa 7 a viac dní od ukončenia jeho topickej aplikácie.Weissella cibaria strain Biocenol™ 4/8 D37 CCM 9015 is cultured on a selective medium for lactic acid bacteria for 48 hours. at 27 °C with an anaerobic environment developer. After cultivation, the strain is transferred to selective broth for lactic acid bacteria and cultured in a thermostat at 27°C for 18 hours, with constant shaking (119 rpm) on a shaker. The broth with the multiplied culture is centrifuged for 55 minutes, at 4,500 revolutions and 4 °C. The supernatant is poured out, the sediment is dissolved in phys. solution and pipetted 5 ml to 95 ml of selective broth for lactic acid bacteria. It is cultured at 27 °C with constant shaking on a shaker (119 rpm) in a thermostat for 18 hours. After cultivation, the radiation-sterilized alginite is covered with broth with the multiplied culture and the cultivation takes place for 48 hours at room temperature. To verify the safety of the application of the beneficial strain and alginite (carrier of the beneficial strain) to healthy skin, the strain Weissella cibaria Biocenol™ 4/8 D37 CCM 9015 mixed with radiation-sterilized alginite at a concentration of 9 x 109 KTJ (colony forming units)/g culture medium is used. which is mixed with radiation sterilized alginite. It is applied to the limbs of 6 horses using disposable non-sterile examination gloves 1 time a day in the morning for 14 days and covered with a cellona, a dressing and a copola so that the carrier covers the palmar/plantar sides of the limb in the bend of the staple joint. Redness of the skin, increase in skin temperature, appearance of pruritus, pain, lesions at the application site are monitored. During and after the application of the said strain on alginite to the healthy skin of the distal part of the limbs, no observed skin reactions were observed in 6 horses. The strain survives on the horse's skin for 7 or more days from the end of its topical application.
Rovnaký experiment je vykonaný na 6 koňoch s dermatitídou na distálnej časti končatiny. Pri koňoch s akútnymi mokvavými eróziami v oblasti sponky došlo po 1 dni aplikácie kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM stabilizovaného na alginite v koncentrácii 9 x 109 KTJ/g k vysušeniu lézií, po týždni aplikácie k ich zmenšeniu približne o polovicu. Po dvoch týždňoch aplikácie kmeňa stabilizovaného na alginite boli lézie vyhojené a nepalpovateľné. Po 7 dňoch od ukončenia aplikácie kmeňa stabilizovaného na alginite zostali vyliečené akútne lézie nepalpovateľné. Kmeň prežíval na postihnutých miestach 7 a viac dní od ukončenia jeho topickej aplikácie. V sledovanom období 6 mesiacov po ukončení aplikácie kmeňa Weissella cibaria Biocenol™ 4/8 D37 CCM stabilizovaného na alginite nedošlo k recidíve ochorenia. Kmeň Weissella cibaria Biocenol™ 4/8 D37 CCM bol na 7. deň od ukončenia jeho topickej aplikácie prítomný vo vzorkách kožných sterov v počtoch 3,39 x 103 ±0,20 KTJ/ml riediaceho roztoku.The same experiment is performed on 6 horses with dermatitis on the distal part of the limb. In horses with acute wet erosions in the area of the clip, after 1 day of application of Weissella cibaria strain Biocenol™ 4/8 D37 CCM stabilized on alginite in a concentration of 9 x 109 KTJ/gk, the lesions dried up, after a week of application they were reduced by approximately half. After two weeks of application of the strain stabilized on alginite, the lesions were healed and not palpable. After 7 days from the end of the application of the strain stabilized on alginite, the healed acute lesions remained unpalpable. The strain survived on the affected areas for 7 or more days from the end of its topical application. In the monitored period of 6 months after the end of the application of Weissella cibaria strain Biocenol™ 4/8 D37 CCM stabilized on alginite, there was no recurrence of the disease. Weissella cibaria strain Biocenol™ 4/8 D37 CCM was present in skin swab samples at 3.39 x 10 3 ±0.20 KTJ/ml dilution solution on the 7th day after the end of its topical application.
Výsledky amplikonového sekvenovania preukázali, že kmeň Weissella cibaria Biocenol™ 4/8 D37 CCM stabilizovaný na alginite negatívne neovplyvňuje mikrobiotu kože a zvyšuje druhovú rôznorodosť kožných baktérií.The results of amplicon sequencing demonstrated that Weissella cibaria Biocenol™ 4/8 D37 CCM strain stabilized on alginite does not negatively affect the skin microbiota and increases the species diversity of skin bacteria.
Priemyselná využiteľnosťIndustrial applicability
Podľa tohto vynálezu je možné produkovať Weissella cibaria Biocenol™ 4/8 D37 CCM 9015. Uvedené účinky ho predurčujú na využitie pri prevencii recidív dermatitíd a harmonizáciu kožnej mikrobiocenózy po topickej terapii. Mikroorganizmus je využiteľný na harmonizáciu mikrobiocenóz a ako súčasť farmaceutických prípravkov na rekolonizáciu kože. Kultúra mikroorganizmu je využiteľná na topickú aplikáciu v čerstvom stave, kultiváciu na fosílnom nosiči a topickú aplikáciu, kultiváciu na fosílnom nosiči s následnou lyofilizáciou a topickou aplikáciou.According to this invention, it is possible to produce Weissella cibaria Biocenol™ 4/8 D37 CCM 9015. The mentioned effects predetermine it for use in the prevention of recurrences of dermatitis and harmonization of skin microbiocenosis after topical therapy. The microorganism can be used to harmonize microbiocenoses and as part of pharmaceutical preparations for skin recolonization. The culture of the microorganism can be used for topical application in the fresh state, cultivation on a fossil carrier and topical application, cultivation on a fossil carrier with subsequent lyophilization and topical application.
Claims (18)
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