Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein.

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.

PCR DGGE and RT-PCR DGGE show diversity and short-term temporal stability in the Clostridium coccoides-Eubacterium rectale group in the human intestinal microbiota.

As the Clostridium coccoides-Eubacterium rectale (Erec; clostridial phylogenetic cluster XIVa) group is one of the major groups of the human intestinal microbiota, DNA- and RNA-based population analysis techniques (denaturing gradient gel electrophoresis; DGGE) were developed and applied to assess the diversity and temporal stability (6 months-2 years) of this faecal clostridial microbiota in 12 healthy adults. The stability of the Erec group was compared with the stability of the predominant bacterial microbiota, which was also assessed with PCR-DGGE. In addition, the Erec group was quantified with a hybridization-based method. According to our results, the Erec group was diverse in each subject, but interindividual uniqueness was not as clear as that of the predominant bacteria. The Erec group was found to be temporally as stable as the predominant bacteria. Over 200 clones obtained from two samples proved the developed method to be specific. However, the amount of bacteria belonging to the Erec group was not related to the diversity of that same bacterial group. In conclusion, the newly developed DGGE method proved to be a valuable and specific tool for the direct assessment of the stability of the Erec group, demonstrating diversity in addition to short-term stability in most of the subjects studied.

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria.

The differences in faecal bacterial population between irritable bowel syndrome (IBS) and control subjects have been reported in several studies. The aim of the present study was to compare the predominant and clostridial faecal microbiota of IBS subjects and healthy controls by applying denaturing gradient gel electrophoresis (DGGE) and a recently developed multiplexed and quantitative hybridization-based technique, transcript analysis with the aid of affinity capture (TRAC). According to the results, the studied clostridial groups (Clostridium histolyticum, Clostridium coccoides-Eubacterium rectale, Clostridium lituseburense and Clostridium leptum) represented the dominant faecal microbiota of most of the studied subjects, comprising altogether 29-87% of the total bacteria as determined by the hybridized 16S rRNA. The C. coccoides-E. rectale group was the dominant subgroup of clostridia, contributing a mean of 43% of the total bacteria in control subjects and 30% (constipation type) to 50% (diarrhoea type) in different IBS symptom category subjects. The proportion of the C. coccoides-E. rectale group was found to be significantly lower in the constipation-type IBS subjects than in the control subjects. DNA-based PCR-DGGE and RNA-based RT-PCR-DGGE analyses targeted to the predominant bacterial population showed considerable biodiversity as well as uniqueness of the microbiota in each subject, in both control and IBS subject groups. The RT-PCR-DGGE profiles of the IBS subjects further indicated higher instability of the bacterial population compared to the control subjects. The observations suggest that clostridial microbiota, in addition to the instability of the active predominant faecal bacterial population, may be involved in IBS.

Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills.

The aim of this work was to characterize the cultivable obligate anaerobic bacterial population in paper mill environments. A total of 177 anaerobically grown bacterial isolates were screened for aerotolerance, from which 67 obligate anaerobes were characterized by automated ribotyping and 41 were further identified by partial 16S rDNA sequencing. The mesophilic isolates indicated 11 different taxa (species) within the genus Clostridium and the thermophilic isolates four taxa within the genus Thermoanaerobacterium and one within Thermoanaerobacter (both formerly Clostridium). The most widespread mesophilic bacterium was closely related to C. magnum and occurred in three of four mills. One mill was contaminated with a novel mesophilic bacterium most closely related to C. thiosulfatireducens. The most common thermophile was T. thermosaccharolyticum, occurring in all four mills. The genetic relationships of the mill isolates to described species indicated that most of them are potential members of new species. On the basis of identical ribotypes clay could be identified to be the contamination source of thermophilic bacteria. Automated ribotyping can be a useful tool for the identification of clostridia as soon as comprehensive identification libraries are available.

Probiotic and other functional microbes: from markets to mechanisms.

Insight into the diversity and function of the human intestinal microbiota has been stimulated by clinical studies with bacteria that exhibit specific functions and which are marketed as probiotics to positively affect our health. Initial efforts concentrated on establishing sound scientific support for the efficacy of these probiotic bacteria, which mainly include Lactobacillus and Bifidobacterium species. Following these evidence-based functional approaches, considerable research is now focused on the mechanisms of action of probiotic bacteria. The mechanisms identified to date mainly relate to the stimulation of host defence systems, immune modulation and the competitive exclusion of pathogens. Recent efficacy, molecular and genomics-based studies have also been reported for some probiotic strains that have found their position in the market place.

Methodologies for the characterization of microbes in industrial environments: a review.

There is growing interest in research and development to develop novel tools to study, detect, and characterize microbes and their communities in industrial environments. However, knowledge about their validity in practical industrial use is still scarce. This review describes the advantages and limitations of traditional and molecular methods used for biofilm and/or planktonic cell studies, especially those performed with Listeria monocytogenes, Bacillus cereus, and/or Clostridium perfringens. In addition, the review addresses the importance of isolating the microorganisms from the industrial environment and the possibilities and future prospects for exploiting the described methods in the industrial environment.

Bioprotectives and probiotics for dry sausages.

The microbial stability of dry sausages is determined by the combination and timing of different factors referred to as the hurdle-concept. However, the hurdles present in dry sausage are not sufficient to prevent the survival of Listeria monocytogenes or enterohemorrhagic Escherichia coli O157:H7. Recently bioprotective lactic acid bacteria, which in addition to the production of antimicrobial lactic acid, have been found to contribute to the safety of the dry sausage by producing antimicrobial peptide, i.e. bacteriocins and other low-molecular-mass compounds. Furthermore, the possibilities to use probotics in dry sausage manufacturing process has been addressed recently. As one possible mode of action for probiotics is the production of antimicrobial compounds, lactic acid bacteria may act as both probiotic and bioprotective culture as well as fermenting agent in meat product, such as dry sausage.

Microbial Evaluation of the Biotransfer Potential from Surfaces with Bacillus Biofilms after Rinsing and Cleaning Procedures in Closed Food-Processing Systems.

The biotransfer potential in food processing is defined as the ability of the microorganisms present on equipment surfaces both before and after cleaning procedures to contaminate products during processing. Growth of Bacillus biofilms was detectable on both stainless-steel and Teflon© surfaces after all the cleaning procedures. B. cereus and B. subtilis cells adhered more firmly to unsoiled than to soiled surfaces, whereas B. thuringiensis adhered most firmly to soiled surfaces. The results showed that the removal of biofilms from surfaces was more difficult from stainless steel because the microorganisms were attached more tenaciously to rough surfaces. Biofilms were cleaned most effectively from unsoiled surfaces with a simple rinsing procedure; thus the mechanical forces of the flow are very important in the cleaning. The chemical and heat treatments used for cleaning had the greatest impact on the abundance of living microorganisms. All the procedures tested did not remove biofilm material effectively from the surfaces. Significantly more cells were removed when the alkaline phase in the alkaline-acid treatment of the cleaning-in-place (CIP) procedure was prolonged, and ethylene diaminetetraacetic acid (EDTA) was used.

Epifluorescence Image Analysis and Cultivation of Foodborne Biofilm Bacteria Grown on Stainless Steel Surfaces.

In this study, biofilm was grown on stainless steel surfaces (AISI 304) for 2, 5, and 10 d at 25°C in slime broth inoculated with the food spoilage microbes Bacillus subtilis , Listeria monocytogenes , Pediococcus pentosaceus , and Pseudomonas fragi . The biofilm developing on steel surfaces were investigated using conventional plating, quantitative glycocalyx determination, and epifluorescence microscopy with image analysis. The results showed that B. subtilis and P. fragi could easily be cultivated after 2 d growth. After a growth period of 10 d, the cells were difficult to cultivate from the surface, and the growth was detected better by microscopy. L. monocytogenes , on the other hand, could easily be detected by cultivation after 2, 5, and 10 d. The greatest amount of slime was produced by P. pentosaceus , as was also shown by epifluorescence microscopy.