A combination of the probiotic and prebiotic product can prevent the germination of Clostridium difficile spores and infection.

Clostridium difficile infection (CDI) is one of the most prevalent healthcare associated infections in hospitals and nursing homes. Different approaches are used for prevention of CDI. Absence of intestinal lactobacilli and bifidobacteria has been associated with C. difficile colonization in hospitalized patients. Our aim was to test a) the susceptibility of C. difficile strains of different origin and the intestinal probiotic Lactobacillus plantarum Inducia (DSM 21379) to various antimicrobial preparations incl. metronidazole, vancomycin; b) the susceptibility of C. difficile strains to antagonistic effects of the probiotic L. plantarum Inducia, prebiotic xylitol (Xyl) and their combination as a synbiotic (Syn) product; c) the suppression of germination of C. difficile spores in vitro and in vivo in animal model of C. difficile infection with Inducia, Xyl and Syn treatment. The VPI strain 10463 (ATCC 43255), epidemic strain (M 13042) and clinical isolates (n = 12) of C. difficile from Norway and Estonia were susceptible and contrarily L. plantarum Inducia resistant to vancomycin, metronidazole and ciprofloxacin. The intact cells of Inducia, natural and neutralized cell free supernatant inhibited in vitro the growth of tested C. difficile reference strain VPI and Estonian and Norwegian clinical isolates of C. difficile after co-cultivation. This effect against C. difficile sustained in liquid media under ampicillin (0.75 µg/ml) and Xyl (5%) application. Further, incubation of Inducia in the media with 5% Xyl fully stopped germination of spores of C. difficile VPI strain after 48 h. In infection model the 48 hamsters were administered ampicillin (30 mg/kg) and 10-30 spores of C. difficile VPI strain. They also received five days before and after the challenge a pretreatment with a synbiotic (single daily dose of L. plantarum Inducia 1 ml of 1010 CFU/ml and 20% xylitol in 1 ml by orogastric gavage). The survival rate of hamsters was increased to 78% compared to 13% (p = 0.003) survival rate of hamsters who received no treatment. When administered Xyl the survival rate of hamsters reached 56% vs.13% (p = 0.06). In both Syn (6/9, p = 0.003) and Xyl (3/9, p = 0.042) groups the number of animals not colonized with C. difficile significantly increased. In conclusion, the combination of xylitol with L. plantarum Inducia suppresses the germination of spores and outgrowth into vegetative toxin producing cells of C. difficile and reduces the colonization of gut with the pathogen. Putative therapeutical approach includes usage of the synbiotic during antimicrobial therapy for prevention of CDI and its potential to reduce recurrences of CDI.

Five-year prospective surveillance of nosocomial bloodstream infections in an Estonian paediatric intensive care unit.

BACKGROUND: Few studies provide rates of nosocomial bloodstream infections (BSIs) in mixed neonatal and paediatric intensive care units (PICUs). AIM: To determine the rate, pathogens and outcome of BSIs in an Estonian PICU. METHODS: Data were collected prospectively from 1st January 2004 to 31st December 2008 in the PICU of Tartu University Hospital. The definition criteria of the US Centers for Disease Control and Prevention were applied for the diagnosis of laboratory-confirmed BSI. FINDINGS: A total of 126 episodes of BSI were identified in 89 patients (74 neonates, eight infants, seven patients aged >1 year). Among neonates 42 (57%) had birth weight <1000 g. The overall incidence of BSI was 9.2 per 100 admissions, incidence density 12.8 per 1000 patient-days. Primary BSI was diagnosed in 92 episodes. Central line (CL)-associated BSI incidence density for neonates was 8.6 per 1000 CL-days with the highest incidence (27.4) among neonates with extremely low birth weight. The most common pathogens were coagulase-negative staphylococci (43%) and Serratia marcescens (14%). Resistance to meticillin was detected in four out of seven S. aureus isolates (all were part of an outbreak) and 23% of Enterobacteriaceae were extended spectrum beta-lactamase (ESBL)-producing strains. Overall case-fatality rate was 10%. CONCLUSION: We observed higher rates of BSIs in our mixed PICU than reported previously. High levels of antimicrobial resistance were detected. Future research should focus on the effects of infection control measures to prevent outbreaks and to decrease incidence of CL-associated BSI.

Protective effect by Bacillus smithii TBMI12 spores of Salmonella serotype enteritidis in mice.

Avoiding food-borne diseases by competitive exclusion agents is a proactive strategy. In the current paper, we report the use of Bacillus smithii TBMI12 spores as potential competitive exclusion agents. One group of mice was predosed for three successive days with 10(8) colony forming units of B. smithii TBMI12 spores followed by inoculation with 10(6) colony forming units of wild-type Salmonella enterica serotype Enteritidis cells. Microbial plate counts of the animals' livers and spleens showed that only 40% of the mice were infected with S. enterica serotype Enteritidis, while the control group was 100% infected. These results suggest that B. smithii TBMI12 spores may protect against infection by S. enterica serotype Enteritidis.

Epidemiology of nosocomial bloodstream infections in Estonia.

A prospective multicentre hospital-wide surveillance study was performed to investigate nosocomial bloodstream infections (BSIs) and to promote BSI surveillance in Estonia in 2004-2005. All patients from the acute care departments of two referral centres and one central hospital were included. A total of 549 episodes of BSI occurred in 507 patients (0.6 cases per 1000 patient-days). Of those, 55% occurred in intensive care units and 47% were catheter-associated infections. Of BSI cases, 24% occurred in patients with haematological malignancy. The in-hospital case-fatality rate was 31%. Of causative micro-organisms, 315 (53%) were Gram-positive aerobes, 232 (39%) were Gram-negative aerobes and 35 (6%) were fungi. Anaerobic bacteria accounted for 2%. The most common pathogens were coagulase-negative staphylococci (26%), Enterobacteriaceae (24%), enterococci (13%) and pseudomonas (10%). Eight percent of BSI were polymicrobial. Seven percent of Staphylococcus aureus isolates were meticillin resistant. Of pseudomonas isolates, 19%, 25%, 30% and 44% were resistant to ceftazidime, meropenem, piperacillin/tazobactam and imipenem, respectively. The incidence of BSI did not differ significantly from other reported studies. With the exception of relatively high antimicrobial resistance among pseudomonas, the overall resistance patterns of Estonian nosocomial bloodstream pathogens were similar to those seen in Nordic countries and lower than in Central and Southern Europe. This study contributes to the development and implementation of surveillance in Estonian hospitals.

Antimicrobial susceptibility and serogroup/serotype distribution of nasopharyngeal isolates of Streptococcus pneumoniae in healthy Estonian children in 1999-2003.

This study determined nasopharyngeal (NP) carriage rates of Streptococcus pneumoniae among healthy Estonian children, aged 1-7 years, and characterised the serotype/serogroup distribution and antibiotic susceptibility rates. NP swabs were collected from 685 previously healthy children attending 29 day care centres during the winters of 1999-2000 and 2003. The NP carriage rate of S. pneumoniae was 44%. Rates of penicillin and erythromycin non-susceptibility were low (both 6%), but high (67%) rates of co-trimoxazole resistance were found. Among the pneumococcal serotypes identified, 64% were included in or cross-reacted with the licensed heptavalent pneumococcal vaccine.

Surveillance of antimicrobial resistance of invasive pathogens: the Estonian experience.

The aim of the present study was to evaluate the needs for surveillance of invasive Gram-negative pathogens in Estonia. The antimicrobial susceptibility data of invasive isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella spp, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae and enterococci were collected in accordance with EARSS (European Antimicrobial Resistance Surveillance System) protocols. Despite the higher rate of Gram positive pathogens, their resistance to antimicrobials was low in contrast to the elevated resistance established for Gram negative pathogens. The higher resistance to antimicrobials was particularly associated with A. baumannii and P. aeruginosa. Also, the proportion of extended spectrum betalactamase (ESBL)-producing strains was 23% among Klebsiella spp. and 3.6% among E. coli. The inclusion of invasive Gram negative pathogens in antimicrobial resistance surveillance provides useful information concerning local pathogen susceptibility, as well as for the empirical treatment of suspected infections.

Antimicrobial susceptibility of common pathogens from community-acquired lower respiratory tract infections in Estonia.

Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae (n = 49), Haemophilus influenzae (n = 66) and Moraxella catarrhalis (n = 25) from adults with community-acquired lower respiratory tract infections (CALRTI) was assessed in a national surveillance study in Southern Estonia during 2000-2003. In S. pneumoniae, no penicillin resistance and only 2.0% resistance to clarithromycin was present, although intermediate resistance to trimethoprim-sulphamethoxazole (TMP-SMX) reached 24.5%. Beta-lactamase production rate was 96.0% in M. catarrhalis, but only 3.0% in H. influenzae. Both Gram-negative strains lacked resistance to ciprofloxacin. Clarithromycin resistance was 12% in M. catarrhalis but lacking among H. influenzae (except for 18.2% intermediate resistance). Resistance to TMP-SMX was 12.0% and 24.2% in M. catarrhalis and H. influenzae, respectively. In summary, a favorable resistance pattern of the main CALRTI pathogens is preserved in Estonia to core antibacterials. Nevertheless, use of TMP-SMX in CALRTI is discouraged and clinical response should be carefully monitored, when clarithromycin is chosen against M. catarrhalis or H. influenzae.

Surveillance of antimicrobial resistance of invasive pathogens: the Estonian experience.

The aim of the present study was to evaluate the needs for surveillance of invasive Gram-negative pathogens in Estonia. The antimicrobial susceptibility data of invasive isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella spp, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae and enterococci were collected in accordance with EARSS (European Antimicrobial Resistance Surveillance System) protocols. Despite the higher rate of Gram positive pathogens, their resistance to antimicrobials was low in contrast to the elevated resistance established for Gram negative pathogens. The higher resistance to antimicrobials was particularly associated with A. baumannii and P. aeruginosa. Also, the proportion of extended spectrum betalactamase (ESBL)-producing strains was 23% among Klebsiella spp. and 3.6% among E. coli. The inclusion of invasive Gram negative pathogens in antimicrobial resistance surveillance provides useful information concerning local pathogen susceptibility, as well as for the empirical treatment of suspected infections.

Toenail onychomycosis in Estonia.

The aim of our study was to evaluate the clinical features, predisposing factors and pathogens of toenail onychomycosis in Estonia. During study period we interviewed and examined 436 dermatological patients with clinical signs of toenail onychomycosis in all counties of Estonia. In 69% of cases, the clinical diagnosis of onychomycosis was confirmed by the mycological analysis. The most common clinical symptoms of onychomycosis both in mycologically proven and non-proven cases were discolorization of nail plate, hyperkeratosis and brittle nails. The number of infected toenails positively correlated with patients' age. On average, patient had 5.4 infected nails. In 78% of culture-positive cases, a dermatophyte was isolated as the causative agent, in 10% yeasts and in 7% moulds. In 6% of culture-positive cases we reported mixed infections. Trichophyton rubrum was the most common pathogen. The high occurrence of mixed infections, clinical symptoms characteristic to long lasting or chronic infection and high number of infected toenails indicate that Estonian patients have more advanced stage of toenail onychomycosis compared with other western and central European countries.

Isolation of Neosartorya pseudofischeri from blood: first hint of pulmonary Aspergillosis.

We report a case of invasive pulmonary aspergillosis caused by Neosartorya pseudofischeri S. W. Peterson [anamorph Aspergillus thermomutatus (Paden) S. W. Peterson]. The diagnosis was initially based on a positive blood culture for a strain isolated from a neutropenic patient by means of a BACTEC 9050 blood culture system. The final diagnosis was established based on X-ray and computer tomography scan results as well as the detection of Aspergillus antigen in the patient's serum.

Dissemination of bacteria in multiple organs associated with apoptosis and macrophage activity in different stages of experimental sepsis.

BACKGROUND: Gram negative sepsis is reported to induce massive translocation of bacteria into tissues, which associates with decreased macrophage function and increased macrophage apoptosis. AIMS: The objective of this study was to detect the translocation of bacteria into different organs and to evaluate macrophage activity and the apoptosis of macrophages in the liver during different stages of sepsis and to correlate these parameters. MATERIAL: Wistar rats (n = 43) were inoculated intraperitoneally with an E. coli and divided into 5 groups, which were killed at different times. METHODS: Counts of translocated bacteria in tissues were evaluated by using morphological and bacteriological methods. Macrophage activity and apoptotic cells in the liver were studied by applying immunohistochemical methods. RESULTS: The counts of E. coli were the highest in the organs and blood 6 h after the onset of sepsis, being in correlation with the highest counts of apoptotic cells in the liver and the falling counts of activated macrophages. The counts of microbes show a new wave of elevation in tissues by 120th h. CONCLUSIONS: The massive penetration of bacteria, the depressed macrophage response in early sepsis following the increased rate of apoptotic macrophages, the different rate of bacterial multiplication in tissues and blood and the second wave of the multiplication of bacteria in tissues in late sepsis all refer to the significance of developing immune dysfunction.

Antibiotic resistance as an indicator of bacterial chlorhexidine susceptibility.

The antibiotic and chlorhexidine (CHX) susceptibility of 70 distinct clinical isolates: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus (not MRSA), Streptococcus pyogenes and Enterococcus faecalis (10 of each) were tested using minimal bactericidal (MBC) and/or minimal inhibitory (MIC) concentrations. Non-fermentative bacteria tolerated CHX at high concentrations; Gram-positive cocci, especially S. pyogenes, were the most susceptible. We found a good correlation between CHX and antibiotic susceptibility in both MIC and MBC among Gram-negative bacteria, and mainly in MBC among Gram-positive bacteria. Resistance to ciprofloxacin, imipenem, cefotaxime, ceftazidime, gentamicin and aztreonam appeared to indicate increased CHX resistance among Gram-negative bacteria. This finding gives clinicians the ability to predict CHX susceptibility according to routine antibiotic resistance testing.

Antibiotic usage and resistance - trends in Estonian University Hospitals.

The use of antibiotics, type of infections and resistance of prevalent bacteria was surveyed in Tartu University hospitals. The data from two ICUs (1995 and 1998), surgical and internal medicine departments (1998) were compared. Overall antibiotic usage in the ICUs and in the hospital as a whole had increased. There was a significant increase in Gram-positive bacterial infections and a decrease in Gram-negative infections in the ICUs. At the same time, susceptibility to several antibiotics decreased in most of the prevalent Gram-negative aerobes in the ICUs (Acinetobacter spp., Pseudomonas spp., Klebsiella spp.). Exceptions to this were the greater susceptibility of Pseudomonas spp. to gentamicin and Acinetobacter spp. to imipenem. Some changes in the predominant bacterial populations did not correlate to changes in antibiotic use.

Translocation of indigenous microflora in an experimental model of sepsis.

Translocation of viable bacteria from gut to bloodstream and other sterile body sites during shock has been demonstrated in several experimental and clinical studies. The factors causing translocation and its incidence at different stages of shock are not known. The aim of the study was to evaluate the importance of several factors causing translocation of indigenous microflora in an experimental model of septic shock based on intraperitoneal Escherichia coli sepsis in rats. Counts of inoculated E. coli and translocated bacteria in different locations, gut morphology and haematological values were evaluated at different stages of sepsis. Sepsis developed in all animals and E. coli achieved the highest counts in blood 6 h after inoculation. Translocation was commonest at 6 and 12 h after inoculation. Frequently translocating bacteria were lactobacilli, bifidobacteria, bacteroides and peptostreptococci. In early sepsis, translocation was associated with high E. coli counts in blood, yet in late sepsis the opposite correlation was present. Low infiltration by neutrophils in the ileum and decreased mitotic activity in the colon were associated with a high translocation rate. In early sepsis, translocation was associated with low lymphocyte counts, but in late sepsis, with low neutrophil counts. Translocation of bacteria (including anaerobes) that colonise the gut in high counts takes place during sepsis. Putative influencing factors such as activity of the primary disease (bacterial counts in blood), gut morphology or haematological values seem to have different impacts on translocation, depending on the stage of the disease.

The intestinal microflora in allergic Estonian and Swedish 2-year-old children.

BACKGROUND: The prevalence of allergic diseases seems to have increased particularly over the past 35-40 years. Furthermore, allergic disease is less common among children in the formerly socialist countries of central and Eastern Europe as compared with Western Europe. It has been suggested that a reduced microbial stimulation during infancy and early childhood would result in a slower postnatal maturation of the immune system and development of an optimal balance between TH1- and TH2-like immunity. AIMS: To test the hypothesis that allergic disease among children may be associated with differences in their intestinal microflora in two countries with a low (Estonia) and a high (Sweden) prevalence of allergy. METHODS: From a prospective study of the development of allergy in relation to environmental factors, 29 Estonian and 33 Swedish 2-year-old children were selected. They were either nonallergic (n = 36) or had a confirmed diagnosis of allergy (n = 27) as verified by typical history and at least one positive skin prick test to egg or cow's milk. Weighed samples of faeces were serially diluted (10-2-10-9) and grown under anaerobic conditions. The counts of the various genera and species were calculated for each child. In addition, the relative amounts of the particular microbes were expressed as a proportion of the total count. RESULTS: The allergic children in Estonia and Sweden were less often colonized with lactobacilli (P < 0.01), as compared with the nonallergic children in the two countries. In contrast, the allergic children harboured higher counts of aerobic micro-organisms (P < 0. 05), particularly coliforms (P < 0.01) and Staphylococcus aureus (P < 0.05). The proportions of aerobic bacteria of the intestinal flora were also higher in the allergic children (P < 0.05), while the opposite was true for anaerobes (P < 0.05). Similarly, in the allergic children the proportions of coliforms were higher (P < 0. 05) and bacteroides lower (P < 0.05) than in the nonallergic children. CONCLUSIONS: Differences in the indigenous intestinal flora might affect the development and priming of the immune system in early childhood, similar to what has been shown in rodents. The role of intestinal microflora in relation to the development of infant immunity and the possible consequences for allergic diseases later in life requires further study, particularly as it would be readily available for intervention as a means for primary prevention of allergy by the administration of probiotic bacteria.

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.

Intestinal microflora of Estonian and Swedish infants.

The intestinal microflora of 1-y-old healthy Estonian (n = 27) and Swedish infants (n = 29) was studied by quantitative culture of faecal samples. The major differences were high counts of lactobacilli and eubacteria in the former and increased numbers of clostridia in the latter babies. Bifidobacteria and anaerobic cocci prevailed equally in both groups, while eubacteria and enterococci were the major microorganisms in many Estonian infants and bacteroides and clostridia in many Swedish infants. The microflora of the Estonian infants was in many aspects similar to the flora prevailing in infants of western Europe in the 1960s. The results suggest a shift in the intestinal microflora among infants in western industrialized countries.

Inhibition of adhesion of Clostridium difficile to Caco-2 cells.

For many microorganisms, including Clostridium difficile, mucosal association is an important factor influencing intestinal colonisation and subsequent infection. Inhibition of adhesion of C. difficile to intestinal mucosa could be a new promising strategy for prevention and treatment of antibiotic-associated diarrhoea. We investigated the possibilities of influencing the adhesion of C. difficile by xylitol and bovine colostrum whey. Caco-2 cells and C. difficile cells were incubated with 1%, 5% and 10% solutions of xylitol and colostrum. Our study revealed that both xylitol and colostrum inhibited the adhesion of C. difficile to Caco-2 cells. Inhibition by xylitol was dose-dependent. When compared to the control, the count of adherent C. difficile decreased 3.4 times when treated with 1% xylitol, 12 times when 5% xylitol was applied, and 18.7 times when treated with 10% xylitol. The inhibition of adherence by colostrum was partially dose-dependent: 3.1 times in the case of 1%, and 5.5 times in the cases of 5% and 10% colostrum. Further experimental and clinical studies are needed for the application of xylitol and colostrum in the treatment and prophylaxis of pseudomembraneous colitis.