Isolation and characterization of novel Lactobacillus spp. with promising gastro-intestinal survival and adhesion properties

Klopper, Kyle Brent (2017-12)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH SUMMARY: The human gastrointestinal tract (GIT) is a complex organ system, and is closely associated with immunological and hormonal functions. A delicate balance needs to be maintained between the selective and beneficial colonization of allochthonous and autochthonous microorganisms, which contribute to the preservation of gut homeostasis and protect the host against infections. Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2, isolated from the feces of healthy humans, formed biofilms on a hydrophobic abiotic surface (polystyrene) under static conditions and were selected for further studies. Meaningful differences in cell surface properties were observed between the isolates, with strain HFI-K2 exhibiting a significantly greater basic surface property, in addition to a significantly higher surface hydrophobicity (37.71 %, p˂ 0.05) compared to that recorded for strain HFI-LD5 (8.82 %, p˂ 0.05). The hydrophobic nature of L. rhamnosus HFI-K2 in conjunction with better biofilm formation, may contribute to a greater GIT colonization ability. Neither of the two strains isolated degraded mucus, and their growth was not irreversibly inhibited when exposed to acidic conditions (pH 2.5) and bile salts (0.5 % and 1.0 % w/v), suggesting that they may survive conditions in the GIT. To confirm planktonic and sessile survival of L. rhamnosus HFI-K2 and L. reuteri HFI-LD5 in the human GIT, the effect of three simulated, fasting-state gastrointestinal fluids (gastric fluid, pH 2, 2 h exposure; intestinal fluid, pH 7.5, 6 h exposure and colonic fluid pH 7, 24h exposure) on both free-living and attached cell viability of the strains was assessed. Exposure to simulated gastric juice had the greatest effect on both planktonic cell viability and biofilm metabolic activity. The sequential introduction of the simulated gastrointestinal fluids initiated the detachment of biofilm biomass, accompanied by a decrease in the metabolic activity, as recorded by changes in CO2 production, by the use of the carbon dioxide measurement system (CEMS). However, as soon as the exposure was halted and sterile culture medium was reintroduced, the remaining biofilm biomass responded by producing CO2, followed by the recovery of biofilm biomass and re-establishment of pre-exposure activity within 24 h. In contrast to the complete loss of planktonic L. rhamnosus HF1-K2 viability after exposure to gastric juice, biofilms of this strain not only recovered within 24 h after exposure, but also exhibited increased metabolic activity after recovery. To our knowledge, this is the first study to assess the effect of simulated, fasting-state gastrointestinal fluids on lactobacilli biofilms. Monitoring of CO2 production as a real-time indicator of metabolic activity in a biofilm provided insight to the differential survival responses of lactic acid bacteria under fasting-state gastrointestinal conditions. The ability of L. reuteri HFI-LD5 and L. rhamnosus HFI-K2 to survive acid, bile and simulated gastrointestinal fluid induced stresses, coupled with biofilm formation under dynamic flow conditions, may contribute to improved survival and persistence of these strains within the human GIT. These characteristics, especially those exhibited by L. rhamnosus HFI-K2, are promising indicators for the application of these isolates as probiotic supplements.

AFRIKAANS OPSOMMING: Die mens se spysverteringskanaal (SVK) is ‘n komplekse orgaanstelsel en is nou verbind met immunologiese en hormonale funksies. ‘n Delikate balans moet gehandhaaf word tussen die selektiewe en voordelige kolonisasie deur inheemse en indringer mikroörganismes, wat bydra tot die instandhouding van derm homeostase en die beskerming teen mikrobiese infeksies. Lactobacillus reuteri HFI-LD5 en Lactobacillus rhamnosus HFI-K2, geïsoleer uit gesonde menslike feces, vorm biofilms onder statiese toestande op ʼn hidrofobiese, abiotiese oppervlak (polistireen), en gekies vir verdere studies. Betekenisvolle verskille in seloppervlak eienskappe is tussen die isolate waargeneem, waar L. rhamnosus HFI-K2 'n aansienlik hoër basiese, tesame met 'n aansienlik hoër hidrofobiese, seloppervlak (37.71 % vs 8.82 %, p˂ 0.05) getoon het. Die hidrofobiese aard van L. rhamnosus HFI-K2 asook die beter vermoë om biofilms onder statiese groei te vorm, kan bydra tot beter kolonisasie van die SVK. Nie een van die twee isolate was daartoe instaat om slym af te breek nie. Blootstelling aan suurtoestande (pH 2.5) en galsoute (0.5 % and 1.0 % w/v) het nie ‘n noemenswaardige effek op enige van die isolate gehad nie, wat verder dui op die potensiaal om oorlewing onder SVK toestande. Om oorlewing in die SVK te bevestig, is die effek van drie gesimuleerde, vastende staat SVK vloeistowwe (maagvloeistof, pH 2, 2 h blootstelling; dermvloeistof, pH 7.5, 6 h blootstelling en kolonvloeistof pH 7, blootstelling 24 h) op die lewensvatbaarheid van beide vrydrywende en oppervlak-geassosieerde selle van beide isolate bepaal. Blootstelling aan gesimuleerde maagvloeistof het die grootste invloed op die lewensvatbaarheid van beide vrydrywende en biofilm-geassosieerde metaboliese aktiwiteit getoon. Blootstelling van L. reuteri HFI-LD5 en L. rhamnosus HFI-K2 biofilms aan hierdie toestande het die verlies van biofilm biomassa geïnisieer, en was vergesel deur 'n afname in metaboliese aktiwiteit, soos bepaal deur veranderinge in CO2-produksie deur die gebruik van die CO2 meting stelsel (CEMS). Sodra blootstelling gestaak is deur die invloei van steriele groeimedium, het die oorblywende biofilm biomassa gereageer met produksie van CO2, gevolg deur die herstel van biofilm biomassa en hervestiging van voorblootstelling aktiwiteit binne 24 uur. In teenstelling met die volledige verlies van vrydrywende L. rhamnosus HF1-K2 lewensvatbaarheid na blootstelling aan maagsap, het oppervlak geassosieerde selle van hierdie stam nie net herstel binne 24 uur na blootstelling nie, maar verhoogde metaboliese aktiwiteit na herstel getoon. Sover ons kennis strek, is hierdie die eerste studie om die invloed van gesimuleerde, vastende staat SVK vloeistowwe op melksuurbakterie biofilms te evalueer. Die gebruik van CO2 produksie as 'n riële aanwyser van biofilm metaboliese aktiwiteit bied insig in die differensiële oorlewingsreaksie van melksuurbakterieë onder gesimuleerde vastende staat maag kondisies. Die vermoë van beide L. reuteri HFI-LD5 en L. rhamnosus HFI-K2 om suur, gal en gesimuleerde maagvloeistof-geïnduseerde spanning te oorleef, tesame met die vermoë om biofilms te vorm onder dinamiese vloeitoestande, kan bydra tot verbeterde oorlewing en voortbestaan van hierdie stamme in die menslike SVK. Hierdie eienskappe, veral dié van L. rhamnosus HFI-K2, is belowende aanwysers vir die inkorporering van hierdie isolate in probiotiese aanvullings.

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