Raik-Hiio Mikelsaar

Bacterial translocation, intestinal microflora and morphological changes of intestinal mucosa in experimental models of Clostridium difficile infection.

Bacteraemia and subsequent sepsis is one possible complication of Clostridium difficile infection. The aim of this study was to examine a correlation between bacterial translocation with morphological changes of intestinal mucosa and shifts of intestinal microflora in experimental models of C. difficile infection. A mouse model was used to study post-antibiotic shifts and mild C. difficile infection, and hamsters were used to study fatal enterocolitis. The influence of pro- and pre-biotics (lactobacilli and xylitol) were also studied in the hamster model. The quantitative composition of luminal and mucosal microflora was evaluated in different intestinal loci, inflammatory changes of mucosa were estimated in histological sections and bacterial translocation was detected in samples from blood, liver, spleen and mesenteric lymph nodes. In cases of mild C. difficile infection, the extent of disturbance of intestinal microflora appeared to be a more important promoting factor in translocation than inflammatory activity in the mucosa. Translocation was frequent in fatal enterocolitis, with facultative species predominating in the intestinal mucosa and also C. difficile in some cases. The combination of lactobacilli and xylitol had some protective effect against C. difficile infection in these models.

Eradication of Salmonella Typhimurium infection in a murine model of typhoid fever with the combination of probiotic Lactobacillus fermentum ME-3 and ofloxacin.

BackgroundThe aim of the study was to detect whether in experimental Salmonella enterica Typhimurium infection the probiotic Lactobacillus fermentum ME-3 in combination with fluoroquinolone therapy would eradicate S. Typhimurium, prevent the development of liver and spleen granulomas and improve the indices of oxidative stress in the ileum mucosa.The selected bacteriological, histological and biochemical methods were applied.ResultsCombined treatment with L. fermentum ME-3 and ofloxacin eradicated Salmonella Typhimurium from blood, ileum and liver, decreased the number of animals with liver and spleen granulomas and reduced the value of lipid peroxides in the ileum mucosa. Higher total counts of intestinal lactobacilli in all experimental groups were associated with the absence of liver granulomas.ConclusionThe antimicrobial and antioxidative probiotic L. fermentum ME-3 combined with ofloxacin enhances the eradication of experimental S. Typhimurium infection. These observations on probiotic and antimicrobial co-action may serve as basis to develop new strategies for treatment of invasive bacterial infections of the gut.

Molecular modelling of secondary and tertiary structures of hyaluronan, compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution

Electron microscopy shows that hyaluronan (HA) forms sheets and tube-like structures in solution. Molecular modelling by Tartu plastic space-filling atomic models revealed that hydroxymethyl and carboxylate groups of HA anti-parallel chains can be joined by H-bonds. Using these bonds, HA molecules can be modelled as sheets and tubules. These tertiary structures have three kinds of lateral contact: (1) antiparallel chains stacked by hydrophobic patches; (2) parallel chains joined by both stacking interactions and H-bonds; and (3) crossing chains joined by H-bonds and stacking interactions. Sheet and tubular structures may explain some viscoelastic and biological properties of HA.

Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

ABSTRACT Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA− TcdB+ strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.

Studies of crystalline native celluloses using potential energy calculations

Energies for various trial packing arrangements of unit cells for the Iα and Iβ phases of native cellulose discovered by Sugiyamaet al. were evaluated. Both a rigid-ring method, PLMR, and the full-optimization, molecular mechanics program, MM3(90), were used. For both phases the models that had the lowest PLMR energy also had the lowest MM3 energy. Both calculated models have the chains packed ‘up’, O6s intg positions, and the same sheets of hydrogen-bonded chains. The Iβ structure model is essentially identical to that proposed previously for ramie cellulose by Woodcock and Sarko. It is also the same as the best parallel model previously proposed that was based on the X-ray data of Mann, Gonzalez and Wellard, once the various unit cell conventions are considered. Also, the energies from both methods for all three celluloses, Iα, Iβ and II, are in the order that rationalizes their relative stabilites.

Immunological, antioxidative, and morphological response in combined treatment of ofloxacin and Lactobacillus fermentum ME-3 probiotic in Salmonella Typhimurium murine model

Truusalu K, Kullisaar T, Hütt P, Mahlapuu R, Aunapuu M, Arend A, Zilmer M, Mikelsaar R‐H, Mikelsaar M. Immunological, antioxidative, and morphological response in combined treatment of ofloxacin and Lactobacillus fermentum ME‐3 probiotic in Salmonella Typhimurium murine model. APMIS 2010; 118: 867–72.

Macroscopic oropharyngeal signs indicating impaired defensive function of palatine tonsils in adults suffering from recurrent tonsillitis.

The present study explored in adults suffering from recurrent tonsillitis the association between macroscopic oropharyngeal signs of recurrent inflammation, immunomorphology of palatine tonsils (counts of neutrophils by CD4 and macrophages by CD68 monoclonal antibodies in tonsillar microcompartments) and the occurrence of post‐tonsillectomy bacteremia. The study involved 50 adults (31 females and 19 males) with recurrent tonsillitis. According to predominance of either inflammatory changes or evidence of sclerotic process in palatine tonsils and surrounding tissue macroscopic at oropharyngeal examination, the patients were divided into groups with ‘inflammatory‐type’ and ‘sclerotic‐type’ tonsils. Biochemically detected mean collagen content was significantly higher in ‘sclerotic‐type’ tonsils than in ‘inflammatory‐type’ tonsils (p=0.001). Post‐tonsillectomy bacteremia was found in 22 patients (44%). A noteworthy finding was the higher recovery of anaerobes from blood cultures than in previous studies. A logistic regression analysis revealed that the post‐tonsillectomy bacteremia was strongly associated with ‘sclerotic‐type’ tonsils (p=0.0015) and with low counts of neutrophils in tonsillar tissue (p=0.047). We conclude that macroscopic oropharyngeal signs of sclerotic process in palatine tonsils indicate impaired tonsillar defense, in terms of lowered counts of neutrophils, increasing the risk of post‐tonsillectomy bacteremia.