M. Orsello

Probiotics: from research to consumer

Intestinal microflora has metabolic, trophic and protective functions, and can be modified in pathological conditions and by the exogenous administration of probiotics. Probiotics are defined as living microorganisms which resist gastric, bile, and pancreatic secretions, attach to epithelial cells and colonize the human intestine. In the last twenty years research has been focused on the identification of the role of planktonic flora and adhesive bacteria in health and disease, and on the requisite of bacterial strains to become probiotic product which can be marketed. Probiotics can be commercialized either as nutritional supplements, pharmaceuticals or foods, but the marketing as a pharmaceutical product requires significant time, complex and costly research, and the demonstration of a well-defined therapeutic target. This review examines the sequential steps of research which, from the identification of a possible probiotic strain, lead to its production and marketing, summarizing the whole process existing behind its development, through its growth in laboratory, the studies performed to test its resistance to human secretions and stability, microencapsulation technologies, and safety tests.

Endoscopy or surgery for malignant GI outlet obstruction

BACKGROUND The treatment of gastroduodenal outflow obstruction (GOO) caused by malignant diseases represents a significant challenge. Open surgical gastrojejunostomy (GJ) has been the treatment of choice, but it has high morbidity and mortality rates. More recently, endoscopic placement of self-expanding metallic stents (SEMS) has been proposed and the results of small, preliminary studies are encouraging. This study compared technical and clinical success, morbidity, mortality, and hospital stay in patients undergoing endoscopic and surgical treatment of GOO. METHODS Medical records of 60 consecutive patients with GOO seen between April 1997 and November 2002 were retrospectively reviewed. Because of extremely short life expectancy, 13 patients were treated by insertion of a double-lumen nasogastric-jejunal tube. The remaining 47 patients (28 men, 19 women; mean age 73.5 years, range 48-92 years) with unresectable pancreatic (33), gastric (7), metastatic lymph nodal (4), papillary (2), and biliary (1) tumors were treated by placement of a SEMS (24) or open surgical GJ (23). RESULTS The technical success rates were similar, but clinical success was lower in the GJ group (92% vs. 56%, p = 0.0067). The SEMS group had a shorter length of hospital stay (3.0 [1.4] days vs. 24.1 [10.3], p < 0.001). Thirty-day mortality was 30% in the GJ group, and 0% in the SEMS group ( p = 0.004). Morbidity was higher in the GJ compared with the SEMS group (61% vs. 17%, p = 0.0021). Mean survival was longer in the SEMS group (96.1 [9.6] days vs. 70.2 [36.2] days, p = 0.0165 for a single test of hypothesis; Bonferroni correction for a multiple testing removes this significance), consequently, out-of-hospital survival was longer for the SEMS group (93.2 [9.3] days vs. 46.0 [31.5] days, p < 0.001). None of the endoscopic procedures required the assistance of an anesthesiologist or the use of an operating room. CONCLUSIONS The results of this retrospective study suggest that SEMS insertion is better than surgical GJ for palliation of patients with GOO in terms of clinical success, morbidity, and mortality. Technical success rates were similar. SEMS placement should be proposed as the first-line treatment for relief of GOO. However, a randomized, comparative, prospective study of SEMS vs. laparoscopic GJ is needed.

The use of probiotics in healthy volunteers with evacuation disorders and hard stools: A double-blind, randomized, placebo-controlled study

Background Evacuation disorders and hard stools are common in industrialized countries, affecting on average 12% to 17% of the adult healthy population at any age. Dietary supplementation with probiotic microorganisms may be useful in reducing the disorder. Methods We performed a double-blind, randomized, placebo-controlled study to evaluate the effectiveness of 2 different probiotic blends, either mixed Lactobacillus plantarum LP01 (LMG P-21021) and Bifidobacterium breve BR03 (DSM 16604) or Bifidobacterium animalis subspecies lactis BS01 (LMG P-21384), in the management of evacuation disorders and intestinal discomfort. In a period of 5 years (2003 to 2008), the study involved 300 healthy volunteers (151 males and 149 females; age 24 to 71 y) with evacuation disorders and hard stools. In particular, subjects were divided into 3 groups: 80 subjects in the group A received placebo, 110 subjects in the group B received mixed L. plantarum LP01 and B. breve BR03 (2.5×109 colony-forming units/d of each strain), and 110 subjects in the group C received B. animalis subsp. lactis BS01 (5×109 colony-forming units/d) for 30 days. At the beginning of the observational study, the healthy status of volunteers was evaluated by a complete, laboratory and ultrasound study of the abdomen. The physical examination was repeated after 15 and 30 days. In particular, the main troubles typically associated with evacuation disorders and hard stools as well as abdominal bloating were considered as parameters of interest. Exclusion criteria were items of gastrointestinal diseases and antibiotics intake. Results Subjects treated with the mixed probiotic strains L. plantarum LP01 and B. breve BR03 or B. animalis subsp. lactis BS01 reported a significant improvement in the number of weekly bowel movements and in the main troubles associated with evacuations, particularly consistency of feces and ease of expulsion. Discomfort items such as abdominal bloating and anal itching, burning, or pain also registered a relevant improvement in the active groups receiving probiotics. Conclusions The intake of an effective amount of mixed L. plantarum LP01 and B. breve BR03 or B. animalis subsp. lactis BS01 for 30 days is able to significantly relieve the evacuation disorders and hard stools, thus providing a useful tool for the management of such condition, which is particularly widespread in industrialized countries at any age.

Is microencapsulation the future of probiotic preparations? The increased efficacy of gastro-protected probiotics

In a recent publication we assessed the kinetics of intestinal colonization by microencapsulated probiotic bacteria in comparison with the same strains given in an uncoated form. It’s well known, in fact, that microencapsulation of probiotics with specific materials is able to confer a significant resistance to gastric juice, thus protecting the cells during the gastric and duodenal transit and enhancing the probiotic efficacy of any supplementation. In any case, this was the first study reporting the fecal amounts of probiotics administered in a coated, protected form compared with traditional, uncoated ones. Here we discuss additional in vitro data of resistance of the same bacteria to gastric juice, human bile and pancreatic secretion and correlate them with the results of in vivo gut colonization. .

Fluorescence in situ hybridisation in the cytological diagnosis of pancreatobiliary tumours

Aims: To assess the sensitivity, specificity, positive and negative predictive values of fluorescence in situ hybridisation (FISH) and conventional cytology in identifying bile duct stricture malignancies. Methods: Brushing samples were collected from 64 patients by means of endoscopic retrograde cholangiopancreatography, and assessed cytologically and by means of a multi-probe FISH set. The cytological diagnoses were: positive, negative and suspicious, whereas criteria for FISH positivity were: more than five polysomic cells or more than 10 trisomic cells for chromosomes 3 or 7. Results: Forty-eight of the 64 patients showed histological or clinical signs of malignancy. The sensitivity of cytology was high (77%) if suspicious cases were considered positive, but was significantly lower than that of FISH if suspicious cases were considered negative (58% versus 90%; p < 0.05). The specificity of cytology was 81% (positive and suspicious) or 100% (negative and suspicious), and the specificity of FISH was 94% (p = 1). FISH yielded one false negative result (isolated chromosome 7 trisomy). FISH allowed a definite diagnosis of 9/12 cytologically inconclusive cases. Conclusions: Our findings suggest using FISH in the case of bile duct strictures cytologically negative or inconclusive; a FISH diagnosis of malignancy should only be made in the presence of polysomic pattern.

In vitro sensitivity of probiotics to human pancreatic juice.

Background The resistance of gut flora with probiotic activity to pancreatic juice is usually tested with artificial pancreatic fluid. Previous studies evaluated the sensitivity of diverse probiotics to human gastric and biliary secretion; none tested the resistance of probiotics to human pancreatic juice. As most bacteria sensitive to artificial pancreatic fluid in vitro have a high rate of isolation from feces, the resistance to human pancreatic juice could be higher. Aim The aim of this study was to compare the sensitivity of different strains of probiotics to artificial and human pancreatic juice. Materials and Methods The viability of 8 strains of Lactobacillus and 4 strains of Bifidobacterium was tested with standard artificial and human pancreatic juice withdrawn from 16 patients during endoscopic retrograde cholangiopancreatography procedure. The mortality rate (%) of various bacteria was measured after 5, 30, and 60 minutes contact time. The results were normalized for mortality rate induced by hypotonic condition and time exposure to 37°C. Results When incubated with artificial and human pancreatic juice, the mortality rate of all strains of Lactobacillus at 5, 30, and 60 minutes was 10.1 versus 7.6, 20.5 versus 19.7, and 28.6 versus 29.8, respectively; whereas the mortality rate of all strains of Bifidobacterium was 8.0 versus 9.2, 33.3 versus 28.9, and 42.2 versus 44.4, respectively. Conclusions All the tested strains were sensitive to artificial and human pancreatic juice depending on time contact. Bifidobacterium strains seem to be more sensitive than Lactobacillus strains in particular at higher time contact. There is no significant difference between sensitivity to simulated and human pancreatic juice. For this reason, probiotics activity may be tested with artificial pancreatic fluid using a standardized, easier, and less costly procedure.

Clinical experience with probiotics in the elderly on total enteral nutrition.

Background: Recent data support that after 2 years of age, intestinal microflora remains relatively constant over time, except in elderly people, who harbor fewer bifidobacteria and a higher population of fungi and enterobacteria than young adults. Diet supplementation with probiotics may improve the nutritional status and reduce the impaired immunity associated with aging. The goal of this study was to establish the effect on bifidobacteria fecal counts, and some clinical parameters, of bifidobacteria supplementation to elderly patients in total parenteral nutrition. Methods: Thirteen patients (6 men and 7 women; mean age, 69 years; range, 65–76 years) affected by permanent vegetative status (PVS) and fed by total enteral nutrition (TEN) were studied. Bifidobacteria and clostridia were investigated by microbiologic and molecular biology methods in stool specimens collected twice at basal time (T–2 and T0) and after 12 and 15 days (T12 and T15, respectively). Seven patients with basal bifidobacteria counts less than 10−7 were supplemented with Bifidobacterium longum W 11 for 12 days. The remaining 6 patients were used as control subjects. For 1 month diarrhea and fever episodes, use of antibiotics, and nutritional status (BMI) were assessed. Results: In the 7 patients with bifidobacteria counts less than 107, the administration of B. longum W 11 resulted in a 1 log increase in 6 of 7 patients at T12. No statistically significant difference in episodes of fever or diarrhea, use of antibiotics, or BMI was observed between the treatment and control groups. Conclusion: The administration of B. longum W11 in PVS patients fed by TEN is effective in increasing the population of bifidobacteria. Larger studies with longer follow-up could demonstrate the influence of these microbiologic changes in a clinical setting.

The innovative potential of Lactobacillus rhamnosus LR06, Lactobacillus pentosus LPS01, Lactobacillus plantarum LP01, and Lactobacillus delbrueckii Subsp. delbrueckii LDD01 to restore the "gastric barrier effect" in patients chronically treated with PPI: A pilot study

Background: Gastroesophageal reflux disease is a very widespread condition. In Europe, it is estimated that about 175 million people suffer from this disease and have to chronically take drugs to increase gastric pH. The proton pump inhibitors (PPIs) such as omeprazole, lansoprazole, and esomeprazole are the most widely used drug typology in this regard. However, the inhibition of normal gastric acid secretion has important side effects, the most important being bacterial overgrowth in the stomach and duodenum with a concentration of >105 viable cells/mL. As a major consequence of this, many harmful or even pathogenic bacteria contained in some foods could survive the gastric transit and colonize either the stomach itself, the duodenum, or the gut, where they could establish acute and even chronic infections with unavoidable consequences for the host’s health. In other words, the “gastric barrier effect” is strongly reduced or even disrupted. To date, there are no real strategies to deal with this widespread, although still relatively little known, problem. The aim of this study was to confirm the gastric bacterial overgrowth in long-term PPI consumers and to assess the efficacy of some probiotic bacteria, belonging to both genera Lactobacillus and Bifidobacterium, in the reduction of gastric and duodenal bacterial overgrowth, therefore partially restoring the gastric barrier effect against foodborne pathogenic bacteria. Methods: For this purpose, probiotics with a strong demonstrated inhibitory activity on gram-negative bacteria, such as Escherichia coli, were tested in a human intervention trial involving a total of 30 subjects treated with PPIs for either 3 to 12 consecutive months (short-term) or >12 consecutive months (long-term). An additional 10 subjects not taking PPIs were enrolled and used as a control group representing the general population. Four selected probiotics Probiotical SpA (Novara, Italy), namely Lactobacillus rhamnosus LR06 (DSM 21981), Lactobacillus pentosus LPS01 (DSM 21980), Lactobacillus plantarum LP01 (LMG P-21021), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106) were administered for 10 days to 10 subjects treated with PPIs for >12 months (group B). In the 60 mg formulation, N-acetylcysteine was included as well in light of its well-known mechanical effects on bacterial biofilms. Gastroscopies were performed at the beginning of the study (d0) in all the groups (A, B, C, and D) and after 10 days (d10) in group B only; that is, at the end of probiotics intake. The total viable cells and total Lactobacillus were quantified in gastric juice and duodenal brushing material from all subjects. The results were compared among all the groups and with the control subjects (group D) to confirm the bacterial overgrowth. A comparison was made also between d0 and d10 in group B to quantify the efficacy of the 4 probiotics administered for 10 days. Fecal samples were collected from all groups at d0, including subjects not treated with PPIs, and in group B only at d10. Specific bacterial classes, namely enterococci, total coliforms, E. coli, molds, and yeasts were quantified in all fecal specimens. Results: The results collected confirmed the strong bacterial overgrowth in the stomach and duodenum of people treated with PPIs compared with subjects with a normal intragastric acidity. It is also worth noting that the bacterial cell counts in subjects who underwent a long-term treatment with a PPI were greater than the results from subjects taking these drugs for 3 to 12 months. The intake of 4 specific probiotic strains with a marked antagonistic activity towards 5 E. coli bacteria, including the enterohaemorrhagic O157:H7 strain, and an effective amount of N-acetylcysteine (NAC) was able to significantly reduce bacterial overgrowth in long-term PPI-treated subjects. Total lactobacilli represented the major percentage of bacterial counts, thus demonstrating the ability of such bacteria to colonize the stomach and the duodenum, at least temporarily, and to consequently restore the gastric barrier effect. A significant decrease in fecal enterococci, total coliforms, E. coli, molds, and yeasts in subjects treated with PPIs was recorded at the end of probiotics supplementation (d10) compared with baseline (d0) in group B. This is a further confirmation of the barrier effect also exerted at the stomach level. Conclusions: PPIs are the most widely sold and used drugs in the world. However, the chronic use of these pharmacological molecules exposes the subject to the risk of foodborne infections as most pathogens are able to survive the gastric transit in a condition of significantly decreased acidity.

A multistep cytological approach for patients with jaundice and biliary strictures of indeterminate origin

Aims Fluorescence in situ hybridisation (FISH) increases the sensitivity for detecting pancreatobiliary tract cancer over routine cytology. In this study, diagnostic accuracy and costs of cytology and FISH in detecting cancer in patients with jaundice with biliary strictures were assessed. Methods Brushing specimens from 109 patients with jaundice were obtained during endoscopic retrograde cholangiopancreatography and examined by cytology and FISH. The specimens were considered FISH-positive for malignancy if at least five polysomic cells or 10 cells with homozygous or heterozygous 9p21/p16 deletion were detected. Definitive diagnosis of the stricture as benign or malignant relied on surgical pathology (45 cases) or clinical-radiological follow-up >18 months (64 cases). We calculated costs of cytology and FISH based on the reimbursement from the Piedmont region, Italy (respectively, €33 and €750). Results Ninety of 109 patients had evidence of malignancy (44 pancreatic carcinomas, 36 cholangiocarcinomas, 5 gallbladder carcinomas, 5 other cancers), while 19 had benign strictures. Routine cytology showed 42% sensitivity, but 100% specificity for the diagnosis of malignancy, while FISH-polysomy showed 70% sensitivity with 100% specificity and FISH-polysomy plus homozygous or heterozygous 9p21/p16 deletion showed 76% sensitivity with 100% specificity. The cost per additional correct diagnosis of cancer obtained by FISH, in comparison with cytology, was €1775 using a sequential cytological approach (ie, performing FISH only in patients with negative or indeterminate cytology). Conclusions FISH should be recommended as the second step in detecting cancer in patients with jaundice with pancreatobiliary tract strictures and cytology negative or indeterminate for malignancy.

Prospective evaluation of early endoscopic ultrasonography for triage in suspected choledocholithiasis: Results from a large single centre series

BACKGROUND Endoscopic ultrasonography is accurate, safe, and cost-effective in diagnosing common bile duct stones, thus suggesting the possibility to avoid invasive endoscopic retrograde cholangiopancreatography. AIM To prospectively evaluate the diagnostic and therapeutic performance of early endoscopic ultrasonography in suspected choledocholithiasis. PATIENTS AND METHODS All consecutive patients presenting to the Emergency Department with suspicion of choledocholithiasis between January 2010 and January 2012 were evaluated and categorized as low, moderate, or high probability of choledocholithiasis, according to accepted criteria. Endoscopic endosonography was carried out within 48 h from the admission and endoscopic retrograde cholangiopancreatography was performed soon in case of confirmed choledocholithiasis. RESULTS Overall 179 patients were included: 48 (26.8%) were classified as low, 65 (36.3%) as moderate, and 66 (36.9%) as high probability of choledocholithiasis. Of the 86 patients with common bile duct stones at endoscopic endosonography, endoscopic retrograde cholangiopancreatography confirmed the finding in 79 (92%). By multivariate analysis only the common bile duct diameter proved an independent predictor of common bile duct stones. CONCLUSIONS Early endoscopic endosonography is accurate in identifying choledocholithiasis allowing immediate endoscopic treatment and significant spare of unnecessary endoscopic retrograde cholangiopancreatography. This approach can be useful as a triage test to select patients not needing endoscopic retrograde cholangiopancreatography, allowing, in selected cases, their early discharge.