Kai Truusalu

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.

Safety of a probiotic cheese containing Lactobacillus plantarum Tensia according to a variety of health indices in different age groups

Safety of the probiotic Lactobacillus plantarum strain Tensia (DSM 21380) was tested in vitro, in semihard Edam-type cheese, in an animal model and after consumption of the probiotic cheese in double-blind randomized placebo-controlled human intervention studies with different age groups. The susceptibility of L. plantarum Tensia to 8 antibiotics, and the presence of tetracycline (tet M, S, O, K, L) genes and class 1 integron was assessed by applying epsilometer-test and PCR-based methods. Production of biogenic amines by the probiotic strain in decarboxylation medium containing 1% of l-histidine, l-glutamine, l-ornithine, l-arginine, or l-lysine and in cheese was tested by gas chromatography. The biosafety of L. plantarum Tensia was evaluated on National Institutes of Health-line mice fed cheese containing Tensia at a concentration of 9.6 log cfu/g for 30 consecutive days. In human intervention trials in adults and the elderly, the effects of different doses of Edam-type cheese and the probiotic bacterium on BW, gut functionality indices, and host metabolism were evaluated. The strain L. plantarum Tensia was susceptible to all tested antibiotics and did not possess the tetracycline resistance-determining genes tet(L), tet(S) and tet(O), nor did it contain the integron (Int1) gene. However, the strain was tet(K) and tet(M) positive. Lactobacillus plantarum Tensia did not produce potentially harmful biogenic amines, such as histamine or cadaverine. The amount of tyramine produced in the cheese environment during ripening and after 15 wk of storage was below the clinically significant content. In the animal model, no translocation of the administered strain or other microbes into the blood or organs of mice was detected. No harmful effect was observed on body mass index, inflammatory markers, or serum lipidograms during human intervention trials with different age groups at a daily dose of 10.3 or 8.17 log cfu/serving for 3 wk. No negative effect on gastrointestinal welfare was observed, but the consumption of 100g/d for 3 wk caused hard stools from the second week of the trial. The content of total lactobacilli increased in feces, and the presence of the ingested probiotic strain was confirmed after the consumption of cheese. Thus, L. plantarum strain Tensia is suitable for generally recognized as safe (GRAS) and qualified presumption of safety (QPS) criteria because it did not have any undesirable characteristics. The regular semihard Edam-type cheese (fat content of 26%) with the probiotic additive at a daily dose of 50 g or in excess (100g) and with a probiotic daily dose of 10 log cfu for 3 wk was safe.

Persistence of Escherichia coli Clones and Phenotypic and Genotypic Antibiotic Resistance in Recurrent Urinary Tract Infections in Childhood

ABSTRACT We assessed the clonality of consecutive Escherichia coli isolates during the course of recurrent urinary tract infections (RUTI) in childhood in order to compare clonality with phenotypic antibiotic resistance patterns, the presence of integrons, and the presence of the sul1, sul2, and sul3 genes. Altogether, 78 urinary E. coli isolates from 27 children, who experienced recurrences during a 1-year follow-up after the first attack of acute pyelonephritis, were investigated. The MICs of sulfamethoxazole, trimethoprim-sulfamethoxazole (SXT), ampicillin, cefuroxime, cefotaxime, and gentamicin and the presence or absence of the intI gene for class 1 integrons and the sulfamethoxazole resistance-encoding genes sul1, sul2, and sul3 were determined. All E. coli strains were genotyped by pulsed-field gel electrophoresis. There were no significant differences in the prevalences of resistance to beta-lactams and SXT between initial and consecutive E. coli isolates (41 versus 45% and 41 versus 29%, respectively). However, the E. coli strains obtained after SXT administration more frequently carried two or more sul genes than the nonexposed strains (9/21 [43%] versus 11/57 [19%], respectively; P = 0.044). In 78% of the patients, the recurrence of unique clonal E. coli strains alone or combined with individual strains was detected. Phenotypic resistance and the occurrence of sul genes were more stable in clonal strains than in individual strains (odds ratios, 8.7 [95% confidence interval {95% CI}, 1.8 to 40.8] and 4.4 [95% CI, 1.1 to 17.7], respectively). Thus, in children with RUTIs, the majority of E. coli strains from consecutive episodes are unique persisting clones, with rare increases in the initially high antimicrobial resistance, the presence of sul genes, and the presence of integrons.

Quantification of Clostridium difficile in Antibiotic-Associated-Diarrhea Patients

ABSTRACT Comparing culture- and non-culture-based methods for quantifying Clostridium difficile in antibiotic-associated-diarrhea patients, we found that the real-time PCR method correlated well with quantitative culture and was more sensitive. A positive association between the population levels of C. difficile and the presence of its toxins was found.

The occurrence of antimicrobial resistance and class 1 integrons among commensal Escherichia coli isolates from infants and elderly persons

BackgroundThe aim of our study was to compare the presence of the intI1 gene and its associations with the antibiotic resistance of commensal Escherichia coli strains in children with/without previous antibiotic treatments and elderly hospitalized/healthy individuals.MethodsOne-hundred-and-fifteen intestinal E. coli strains were analyzed: 30 strains from 10 antibiotic-naive infants; 27 from 9 antibiotic-treated outpatient infants; 30 from 9 healthy elderly volunteers; and 28 from 9 hospitalized elderly patients. The MIC values of ampicillin, cefuroxime, cefotaxime, gentamicin, ciprofloxacin, and sulfamethoxazole were measured by E-test and IntI1 was detected by PCR.ResultsOut of the 115 strains, 56 (49%) carried class 1 integron genes. Comparing persons without medical interventions, we found in antibiotic-naive children a significantly higher frequency of integron-bearing strains and MIC values than in healthy elderly persons (53% versus 17%; p < 0.01). Evaluating medical interventions, we found a higher resistance and frequency of integrons in strains from hospitalized elderly persons compared with non-hospitalized ones. Children treated with antibiotics had strains with higher MIC values (when compared with antibiotic-naive ones), but the integron-bearing in strains was similar. In most cases, the differences in resistance between the groups (integron-positive and negative strains separately) were higher than the differences between integron-positive and negative strains within the groups.ConclusionThe prevalence of integrons in commensal E. coli strains in persons without previous medical intervention depended on age. The resistance of integron-carrying and non-carrying strains is more dependent on influencing factors (hospitalization and antibiotic administration) in particular groups than merely the presence or absence of integrons.

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.

Antibiotic Susceptibility Patterns of Community- and Hospital-acquired Staphylococcus aureus and Escherichia coli in Estonia

This study compares the susceptibility patterns of Staphylococcus aureus and Escherichia coli isolated from patients with hospital-acquired and outpatient infections. A total of 902 isolates of S. aureus and 1,114 of E. coli were collected in five different Estonian medical centers between January 1997 and November 1997. Strains were grouped into two different categories, depending on whether they had been obtained from inpatients or outpatients. Compared to S. aureus strains isolated from inpatients, the strains from outpatients were significantly more resistant to erythromycin (25.3% vs. 17.9%), tetracycline (33.5% vs. 22.4%) and trimethoprim-sulfamethoxazole (13.9% vs. 7.9%). The overall prevalence of oxacillin-resistant S. aureus was 10.4%, with no significant differences noted between isolates recovered from inpatients and outpatients. In the case of E. coli, significantly more isolates from inpatients (42.8%) than from outpatients (34.4%) were ampicillin-resistant. Inpatient isolates of E. coli were also more resistant to cefotaxime (9.3%) and nitrofurantoin (11.2%) than outpatient strains (0% and 3.1%, respectively). Analysis showed remarkable co-resistance among both inpatient and outpatient strains of S. aureus and E. coli. Multiple resistant S. aureus and E. coli strains represented 15.1% and 17.3%, respectively of the organisms examined in this study. With respect to E. coli, significantly more multiresistant isolates were found in inpatient than outpatient isolates (20.4% vs. 8.9%). Our results indicate that the distinction between community-acquired and hospital infections due to S. aureus and E. coli may not be valid in Estonia.This study compares the susceptibility patterns of Staphylococcus aureus and Escherichia coli isolated from patients with hospital-acquired and outpatient infections. A total of 902 isolates of S. aureus and 1,114 of E. coli were collected in five different Estonian medical centers between January 1997 and November 1997. Strains were grouped into two different categories, depending on whether they had been obtained from inpatients or outpatients. Compared to S. aureus strains isolated from inpatients, the strains from outpatients were significantly more resistant to erythromycin (25.3% vs. 17.9%), tetracycline (33.5% vs. 22.4%) and trimethoprim?sulfamethoxazole (13.9% vs. 7.9%). The overall prevalence of oxacillinresistant S. aureus was 10.4%, with no significant differences noted between isolates recovered from inpatients and outpatients. In the case of E. coli, significantly more isolates from inpatients (42.8%) than from outpatients (34.4%) were ampicillin-resistant. Inpatient isolates of E. coli were also more resistant to cefotaxime (9.3%) and nitrofurantoin (11.2%) than outpatient strains (0% and 3.1%, respectively). Analysis showed remarkable co-resistance among both inpatient and outpatient strains of S. aureus and E. coli. Multiple resistant S. aureus and E. coli strains represented 15.1% and 17.3%, respectively of the organisms examined in this study. With respect to E. coli, significantly more multiresistant isolates were found in inpatient than outpatient isolates (20.4% vs. 8.9%). Our results indicate that the distinction between community-acquired and hospital infections due to S. aureus and E. coli may not be valid in Estonia.

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.

The Escherichia coli phylogenetic group B2 with integrons prevails in childhood recurrent urinary tract infections

The aim of our study was to characterize the phylogenetic groups of Escherichia coli, antibiotic resistance, and containment of class 1 integrons in the first attack of pyelonephritis and in subsequent recurrences in young children. Altogether, 89 urine E. coli isolates from 41 children with urinary tract infection (UTI) were studied for prevalence and persistence of phylogenetic groups by pulsed‐field gel electrophoresis (PFGE), antibacterial resistance by minimal inhibitory concentrations (MIC) and class 1 integrons by PCR. Phylogenetic group B2 was most common (57%), followed by D (20%), A (18%) and B1 (5%). Overall resistance to betalactams was 61%, trimethoprim‐sulfamethoxazole 28%, and was not associated with phylogenetic groups. According to PFGE, the same clonal strain persisted in 77% of patients. The persistence was detected most often in phylogenetic group B2 (70%). Phylogenetic group B2 more often contained class 1 integrons than group A. Integron positive strains had higher MIC values of cefuroxime, cefotaxime, and gentamicin. In conclusion, phylogenetic group B2 was the most common cause of the first episode of pyelonephritis, as well as in case of the persistence of the same strain and contained frequently class 1 integrons in childhood recurrent UTI. An overall frequent betalactam resistance was equally distributed among phylogenetic groups.

IL‐22 neutralizing autoantibodies impair fungal clearance in murine oropharyngeal candidiasis model

Protection against mucocutaneous candidiasis depends on the T helper (Th)17 pathway, as gene defects affecting its integrity result in inability to clear Candida albicans infection on body surfaces. Moreover, autoantibodies neutralizing Th17 cytokines have been related to chronic candidiasis in a rare inherited disorder called autoimmune polyendocriopathy candidiasis ectodermal dystrophy (APECED) caused by mutations in autoimmune regulator (AIRE) gene. However, the direct pathogenicity of these autoantibodies has not yet been addressed. Here we show that the level of anti‐IL17A autoantibodies that develop in aged Aire‐deficient mice is not sufficient for conferring susceptibility to oropharyngeal candidiasis. However, patient‐derived monoclonal antibodies that cross‐react with murine IL‐22 increase the fungal burden on C. albicans infected mucosa. Nevertheless, the lack of macroscopically evident infectious pathology on the oral mucosa of infected mice suggests that additional susceptibility factors are needed to precipitate a clinical disease.