Krista Lõivukene

Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens

Aim:  To develop in vitro assays for comparing the antagonistic properties and anti‐oxidative activity of probiotic Lactobacillus and Bifidobacterium strains against various entero‐ and urinary pathogens.

The limit of leucocytospermia from the microbiological viewpoint

Summary.  The aim of the study was to find out the correlation between white blood cell (WBC) counts in semen and quantitative composition of seminal microflora, and to establish the minimum WBC count associated with significant bacteriospermia. The research included 159 men with different WBC counts in their semen, 84 of them with chronic prostatitis/chronic pelvic pain syndrome. Semen samples were cultivated quantitatively for detecting anaerobic, microaerophilic and aerobic bacteria. Bryan‐Leishman stained slides were used for detecting WBC in semen. Seminal fluid was colonized by eight different microorganisms, and the total count of microorganisms in semen ranged from 102 to 107 CFU ml−1. A high frequency of anaerobic microorganisms was found. A positive correlation was observed between the WBC count and the number of different microorganisms, and also between the WBC count and the total count of microorganisms in semen sample. The receiver operating characteristic curve analysis demonstrated that the WHO‐defined WBC cut‐off point (1 × 106 WBC ml−1) has very low sensitivity for discriminating between patients with and without significant bacteriospermia, as a more optimal sensitivity/specificity ratio appears at 0.2 × 106 WBC ml−1 of semen. The quantitative microbiological finding of semen in the patients of National Institute of Health (NIH) categories IIIa and IV was very similar, i.e. a high number of different microorganisms and a high total count of microorganisms. In the control group (without leucocytospermia and prostatitis symptoms) both parameters were significantly lower.

Association of cagA and vacA Genotypes of Helicobacter pylori with Gastric Diseases in Estonia

ABSTRACT Gastric biopsy specimens from 156 adult patients from southern Estonia suffering from chronic gastritis, peptic ulcer disease, and perforated peptic ulcer were analyzed by PCR. The cagA gene was evenly distributed throughout 87% of the specimens from the patients with the different gastric diseases. The presence of the cagA gene correlated with that of vacA signal sequence type s1a (99%). However, no clear differences were found in the distribution of cagA and vacA genotypes among patients in Estonia with severe perforated peptic ulcer, uncomplicated peptic ulcer, or chronic gastritis.

Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people.

The higher counts or particular groups (Firmicutes/Bacteroidetes) of intestinal microbiota are related to host metabolic reactions, supporting a balance of human ecosystem. We further explored whether intestinal lactobacilli were associated with some principal cellular and metabolic markers of blood in 38 healthy >65-year-old persons. The questionnaire, routine clinical and laboratory data of blood indices as much as the oxidized low-density lipoprotein (ox-LDL) and baseline diene conjugates in low-density lipoprotein (BDC-LDL) of blood sera were explored. The PCR-based intestinal Lactobacillus sp. composition and counts of cultivable lactobacilli (LAB) were tested. The facultative heterofermentative lactobacilli (Lactobacillus casei and Lactobacillus paracasei) were the most frequent (89 and 97%, respectively) species found, while Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri were present in almost half of the elderly persons. The number of species simultaneously colonizing the individuals ranged from 1 to 7 (median 4). In elderly consuming probiotics the LAB counts were significantly higher than in these not consuming (median 7.8, range 4.2-10.8 vs. median 6.3, range 3.3-9.7 log cfu/g; p=0.005), adjusted (OR=1.71, CI95 1.04-2.82; p=0.035) for age and body mass index (BMI). The colonization by L. acidophilus was negatively related (r=-0.367, p=0.0275) to L. reuteri, staying significant after adjusting for age, sex and BMI (OR=0.16, CI95 0.04-0.73; p=0.018). However, the blood glucose concentration showed a tendency for a negative correlation for colonization with Lactobacillus fermentum (r=-0.309, p=0.062) adjusted for BMI (Adj. R(2)=0.181; p=0.013) but not for age and sex. The higher white blood cells (WBC) count was positively related (r=0.434, p=0.007) to presence of Lactobacillus reuteri adjusted for age, sex and BMI (Adj. R(2)=0.193, p=0.027). The lower values of ox-LDL were predicted by higher counts of cultivable lactobacilli adjusted by sex, age and BMI (r = -0.389, p = 0.016; Adj. R(2)=0.184 p=0.029). In conclusion, the pilot study of elderly persons shows that the intestinal lactobacilli are tightly associated with WBC count, blood glucose and content of ox-LDL which all serve as risk markers in pathogenesis of inflammation, metabolic syndrome and cardiovascular disease (CVD).

Antibacterial Susceptibility of Intestinal Lactobacilli of Healthy Children

We investigated the antibacterial susceptibility of intestinal lactobacilli of Estonian and Swedish children aged 1-2 y. Sixty isolates (10 species) of lactobacilli (29 Estonian and 31 Swedish strains) were tested against ampicillin, cefuroxime, cefoxitin, gentamicin, ciprofloxacin, tetracycline, vancomycin, metronidazole and erythromycin. We observed that intestinal lactobacilli do not display uniform susceptibility to antibiotics. None of the tested lactobacilli was resistant to ampicillin, gentamicin and erythromycin. Single strains were resistant to cefuroxime and tetracycline, about half of the strains to cefoxitin and ciprofloxacin and 73% of the strains to vancomycin. All studied strains were resistant to metronidazole. Most of the strains investigated were resistant to two or three antibiotics out of nine. Some differences in susceptibility were noted between strains belonging to different fermentation types. No differences in susceptibility were found between Estonian and Swedish isolates. Metronidazole, cefoxitin, vancomycin and ciprofloxacin seem to be safer for gastrointestinal lactoflora than other tested antibiotics in both countries.We investigated the antibacterial susceptibility of intestinal lactobacilli of Estonian and Swedish children aged 1?2 y. Sixty isolates (10 species) of lactobacilli (29 Estonian and 31 Swedish strains) were tested against ampicillin, cefuroxime, cefoxitin, gentamicin, ciprofloxacin, tetracycline, vancomycin, metronidazole and erythromycin. We observed that intestinal lactobacilli do not display uniform susceptibility to antibiotics. None of the tested lactobacilli was resistant to ampicillin, gentamicin and erythromycin. Single strains were resistant to cefuroxime and tetracycline, about half of the strains to cefoxitin and ciprofloxacin and 73% of the strains to vancomycin. All studied strains were resistant to metronidazole. Most of the strains investigated were resistant to two or three antibiotics out of nine. Some differences in susceptibility were noted between strains belonging to different fermentation types. No differences in susceptibility were found between Estonian and Swedish isolates. Metronidazole, cefoxitin, vancomycin and ciprofloxacin seem to be safer for gastrointestinal lactoflora than other tested antibiotics in both countries.

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.

Anaerobic seminal fluid micro-flora in chronic prostatitis/chronic pelvic pain syndrome patients.

We investigated the seminal micro-flora of 116 men. Eighty-four men had chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), and 34 of them were also leukocytospermic. Thirty-two asymptomatic men formed the control group. Micro-organisms were found in all of the 116 seminal fluid specimens. More than 20 different micro-organisms were found in both groups. Neisseria gonorrhoeae and Chlamydia trachomatis were not found. A high frequency of anaerobic bacteria was found in all groups (68-79%), and in most of the specimens, anaerobic micro-organisms were equal to or outnumbered the aerobic strains. We found 1-8 different micro-organisms in each semen sample, the total count of micro-organisms ranged from 10(2) to 10(7)/mL of semen. Both parameters were significantly higher in leukocytospermic CP/CPPS (NIH IIIA category) patients (median=5 different micro-organisms; total median count 5 x 10(4)) than in the control group (median=3 different micro-organisms; total median count 10(3)). In the CP/CPPS patients, the prevalence and/or count of some opportunistic bacteria was higher than in the control group. To show that the micro-organisms do not originate from the urethra, first voided urine was also investigated in 17 prostatitis patients and 15 controls. One patient had significantly fewer micro-organisms (median 1 vs. 4) and a lower total count of micro-organisms (median 10(2) vs. 10(4)/mL) in the first-catch urine than in the seminal fluid. We found only one third of the micro-organisms to be similar in urine and semen while anaerobic bacteria and some aerobic opportunists were infrequent in urine. Semen is a suitable specimen for the diagnosis of prostatitis.

Metronidazole and Clarithromycin Susceptibility and the Subtypes of VacA of Helicobacter pylori Isolates in Estonia

The prevalence of metronidazole and clarithromycin resistance of Helicobacter pylori strains under different growth conditions (microaerophilic or anaerobic preincubation) was tested in 56 patients suffering from gastritis and peptic ulcer. vacA subtypes were detected in 46 H. pylori strains and were subsequently compared with the antibiotic resistance pattern. From 56 isolates, 26 proved resistant and 30 sensitive to metronidazole. The patients with peptic ulcer and gastritis were infected with both metronidazole-sensitive and metronidazole-resistant strains. In anaerobic preincubation all the strains were sensitive to metronidazole (MIC < 8 mg/l). All the strains were clarithromycin-sensitive (MIC < 2 mg/l). In the patients with gastritis and peptic ulcer s1 was the predominant vacA subtype. Comparison of vacA subtypes with the diagnoses revealed no correlation; different virulence factors such as vacA subtypes and antibiotic resistance to metronidazole in a microaerophilic milieu proved unrelated.The prevalence of metronidazole and clarithromycin resistance of Helicobacter pylori strains under different growth conditions (microaerophilic or anaerobic preincubation) was tested in 56 patients suffering from gastritis and peptic ulcer. vacA subtypes were detected in 46 H. pylori strains and were subsequently compared with the antibiotic resistance pattern. From 56 isolates, 26 proved resistant and 30 sensitive to metronidazole. The patients with peptic ulcer and gastritis were infected with both metronidazole-sensitive and metronidazole-resistant strains. In anaerobic preincubation all the strains were sensitive to metronidazole (MIC<8 mg/l). All the strains were clarithromycin-sensitive (MIC<2 mg/l). In the patients with gastritis and peptic ulcer s1 was the predominant vacA subtype. Comparison of vacA subtypes with the diagnoses revealed no correlation; different virulence factors such as vacA subtypes and antibiotic resistance to metronidazole in a microaerophilic milieu proved unrelated.

Higher blood glucose level associated with body mass index and gut microbiota in elderly people

Background Some dominant bacterial divisions of the intestines have been linked to metabolic diseases such as overweight and diabetes. Objective A pilot study aimed to evaluate the relations between the culturable intestinal bacteria with body mass index (BMI) and some principal cellular and metabolic markers of blood in people older than 65. Design Altogether 38 generally healthy elderly people were recruited: ambulatory (n=19) and orthopedic surgery (n=19). Questionnaires on general health, anthropometric measurements, routine clinical and laboratory data, and quantitative composition of cultivable gut microbiota were performed. Results Blood glucose level was positively correlated with BMI (r=0.402; p=0.014). Higher blood glucose level had negative correlation with relative share of intestinal anaerobic bacteria such as bacteroides (r=−0.434; p=0.0076) and gram-positive anaerobic cocci (r=−0.364; p=0.027). In contrast, the relative share of bifidobacteria (r=0.383; p=0.019) and staphylococci (r=0.433; p=0.008) was positively correlated to blood glucose level. In elderly people, a higher blood glucose concentration was predicted by the reduction of the anaerobes’ proportion (adj. sex, age, and BMI R2=0.192, p=0.028) and that of Bacteroides sp. (adj. R2=0.309, p=0.016). Conclusion A tight interplay between increased BMI, level of blood glucose, and the reduced proportion of cultivable bacteroides is taking place in the gut microbiota of elderly people.