Isolation, identification, and molecular characterization of probiotic bacteria from locally selected Ethiopian free range chickens gastrointestinal tract.

The poultry industry is facing continuous challenges with regard to increased feed costs and loss due to infectious disease. To overcome this challenge, several antibiotics have been used along with chicken feeds to promote growth. Nevertheless, the use of antibiotics as growth promoter has been banned in many countries, due to the concerns associated with potential risks of emerging and horizontal transfer of multidrug resistant genes to bacteria in animal tissues. The objective of this study was to identify and characterize potential probiotic bacteria strains from the gastrointestinal tract of free-range locally selected chickens. The bacterial isolates were screened, purified and characterized based on morphological, biochemical and molecular characteristics from 12 well-adopted free-range healthy young chickens. Low pH and bile salt tolerance, antagonistic activity, antibiotic activity, hemolysis activity, adhesion to the chicken intestine and carbohydrate fermentation tests was conducted to identify potential probiotic bacteria. Twelve bacterial isolates were screened based on their ability for their tolerance to low pH and bile salt. The isolates were identified by using 16S rRNA gene partial sequencing method. All screened isolates showed great survival percentage at low pH, that is (89.2 ± 0.75 to 97.1 ± 0.64) survived at 3 h and (83.6 ± 0.75 to 95.2 ± 0.63) at 6 h challenge at pH2. Isolate GCM112 was the least tolerant strain in 6.0% salt concentration at 12 and 24 h exposure time (82.1 ± 1.28 and 79.9 ± 1.96%) respectively. The result revealed no strain tests in this study exhibited α- and ß-hemolytic activity when cultured in sheep blood agar. Most isolated strains showed best growth at 37°C temperature and up to 4% NaCl concentration. Based on the reported result from in vitro data, GCH212 and GCM412 isolates were recognized as best potential probiotic bacteria for chicken against pathogens but further studies are needed on in vivo assessment on the health benefits in the real life situation.

Lactobacillus gasseri APC 678 Reduces Shedding of the Pathogen Clostridium difficile in a Murine Model.

Clostridium difficile is a common cause of health-care acquired diarrhea, resulting in a spectrum of disease from mild diarrhea to life-threatening illness. Sixty Lactobacillus strains were screened for anti-C. difficile activity using a co-culture method. Based on their ability to inhibit C. difficile, L. gasseri APC 678 and L. rhamnosus DPC 6111 were selected for study in a murine model of C. difficile infection. L. gasseri ATCC 33323, was included as a control. It was established that, relative to control mice not fed Lactobacillus, feeding with L. gasseri APC 678 resulted in a significant reduction by day 7 (8-fold, p = 0.017) of viable C. difficile VPI 10463 in the feces of mice. In contrast, neither L. rhamnosus DPC 6111 nor L. gasseri ATCC 33323 significantly reduced fecal C. difficile shedding. Sequencing of the cecal microbiota showed that in mice fed L. gasseri APC 678 there was a significant increase in bacterial diversity across a number of indices when compared to the control or other Lactobacillus-fed groups. There was no significant change in the relative abundance of Firmicutes or Bacteroidetes in the group fed L. gasseri APC 678 relative to the control, while the groups fed L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 showed a significant decrease in the relative abundance of Firmicutes (p = 0.002 and p = 0.019, respectively) and a significant increase in Bacteroidetes (p = 0.002 and p = 0.023, respectively). These results highlight the potential of L. gasseri APC 678 as a live therapeutic agent to target C. difficile infection.

Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract.

Thuricin CD is a two component narrow spectrum bacteriocin comprising two peptides with targeted activity against Clostridium difficile. This study examined the bioavailability of thuricin with a view to developing it as an effective antimicrobial against intestinal infection. One of the peptides, Trn-ß, was found to be degraded by the gastric enzymes pepsin and α-chymotrypsin both in vitro and in vivo, whereas Trn-α was resistant to digestion by these enzymes and hence was detected in the intestinal porcine digesta following oral ingestion by pigs. In order to determine if spores of the producing organism Bacillus thuringiensis DPC 6431 could be used to deliver the bacteriocin to the gut, spores were fed to 30 mice (approx. 10(8)-2×10(8) per animal) and their germination, growth and production of thuricin in the gastrointestinal tract (GIT) of the animals was monitored. Almost 99 % of the spores delivered to the GIT were excreted in the first 24 h and neither Trn-α nor Trn-ß was detected in the gut or faecal samples of the test mice, indicating that ingestion of B. thuringiensis spores may not be a suitable vehicle for the delivery of thuricin CD. When thuricin CD was delivered rectally to mice (n = 40) and C. difficile shedding monitored at 1, 6, 12 and 24 h post-treatment, there was a >95 % (>1.5 log units) reduction of C. difficile 027 in the colon contents of infected mice (n = 10) 1 h post-treatment compared with the control group (n = 10; P<0.001). Furthermore, 6 h post-treatment there was a further 1.5 log reduction in C. difficile numbers (n = 10) relative to the control group (n = 10; P<0.05). These results would suggest that rectal administration of thuricin may be a promising mode of delivery of thuricin CD to the colon.

Characterization of plant-derived lactococci on the basis of their volatile compounds profile when grown in milk.

A total of twelve strains of lactococci were isolated from grass and vegetables (baby corn and fresh green peas). Ten of the isolates were classified as Lactococcus lactis subsp. lactis and two as Lactococcus lactis subsp. cremoris based on 16S rDNA sequencing. Most of the plant-derived strains were capable of metabolising a wide range of carbohydrates in that they fermented D-mannitol, amygdalin, potassium gluconate, l-arabinose, d-xylose, sucrose and gentibiose. None of the dairy control strains (i.e. L. lactis subsp. cremoris HP, L. lactis subsp. lactis IL1403 and Lactococcus lactis 303) were able to utilize any of these carbohydrates. The technological potential of the isolates as flavour-producing lactococci was evaluated by analysing their growth in milk and their ability to produce volatile compounds using solid phase micro-extraction of the headspace coupled to gas chromatography-mass spectrometry (SPME GC-MS). Principal component analysis (PCA) of the volatile compounds clearly separated the dairy strains from the plant derived strains, with higher levels of most flavour rich compounds. The flavour compounds produced by the plant isolates among others included; fatty acids such as 2- and 3-methylbutanoic acids, and hexanoic acid, several esters (e.g. butyl acetate and ethyl butanoate) and ketones (e.g. acetoin, diacetyl and 2-heptanone), all of which have been associated with desirable and more mature flavours in cheese. As such the production of a larger number of volatile compounds is a distinguishing feature of plant-derived lactococci and might be a desirable trait for the production of dairy products with enhanced flavour and/or aroma.

Gut solutions to a gut problem: bacteriocins, probiotics and bacteriophage for control of Clostridium difficile infection.

Clostridium difficile infection (CDI) is a major cause of morbidity and mortality among hospitalized patients and imposes a considerable financial burden on health service providers in both Europe and the USA. The incidence of CDI has dramatically increased in recent years, partly due to the emergence of a number of hypervirulent strains. The most commonly documented risk factors associated with CDIs are antibiotic usage leading to alterations of the gut microbiota, age >65 years and long-term hospital stay. Since standard therapies for antibiotic-associated diarrhoea and CDI have limited efficacy, there is now an urgent need for alternative therapeutics. In this review, we outline the current state of play with regard to the potential of gut-derived bacteriocins, probiotics and phage to act as antimicrobial agents against CDI in the human gut.

Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells.

UNLABELLED: Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (φNH-4) and a podovirus (φMR299-2). Both phages were active against clinical isolates of P. aeruginosa. Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 10(7) to 2 × 10(7) P. aeruginosa. Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients. IMPORTANCE: Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

Genome of a virulent bacteriophage Lb338-1 that lyses the probiotic Lactobacillus paracasei cheese strain.

There is a lack of fundamental knowledge about the influence of bacteriophage on probiotic bacteria and other commensals in the gut. Here, we present the isolation and morphological and genetic characterization of a virulent narrow-host-range bacteriophage, phiLb338-1. This phage was isolated from fresh sewage and was shown to infect the probiotic cheese strain Lactobacillus paracasei NFBC 338. Electron microscopy studies revealed that phiLb338-1 is a member of the Myoviridae family, with an isometric head, a medium-sized contractile tail, and a complex base plate. Genome sequencing revealed a 142-kb genome with 199 open reading frames. Putative functions could be assigned to 22% of the open reading frames; these had significant homology to genes found in the broad-host-range SPO1-like group of phages which includes the Enterococcus faecalis phage phiEF24C, Listeria phage A511, and Lactobacillus plantarum phage LP65. Interestingly, no significant genomic similarity was observed between the phage and the probiotic host strain. Future studies will determine if the presence of bacteriophage phiLb338-1 or others in the human or animal gut plays an antagonistic role against the probiotic effect of beneficial bacteria.