Intestinal Isolates Research Articles

Occurrence of virulence factors among human intestinal enterococcal isolates

The aim of this study was to investigate the frequency of enterococcal virulence factors among human intestinal Enterococcus faecalis strains and to find out whether the pattern differs from that seen in published reports on food and clinical isolates. The E. faecalis isolates were cultured from human faecal samples obtained from five ulcerative colitis patients in remission phase. The species identification was based on API120 strips and species-specific PCR primers. The isolates were further characterized using the pulsed-field gel electrophoresis. The presence of seven different known enterococcal virulence factors among the confirmed E. faecalis isolates were screened using PCR techniques and published primers. Among the 35 isolates representing nine different pulsotypes the most frequent virulence factors were cpd (33 isolates), agg (25 isolates), gelE (22 isolates) and esp (15 isolates). No complete sets of genes associated for the production of functional cytolysin were encountered indicating that intestinal enterococci may differ in this respect from clinical strains. According to the results, the commensal enterococcal strains appear to differ from clinical isolates in their complement of presumed virulence factors.

Overcoming the technological hurdles in the development of probiotic foods

While it is undoubted that clinical evidence supporting the health-promoting activity of probiotic cultures is of paramount importance, it is probably less well appreciated that the technological suitability of these strains is also critical to their exploitation. In this respect, it is not surprising that many human intestinal isolates, many of which are obligatively anaerobic grow very poorly outside their natural habitat, the human gut. Indeed, much of the human intestinal flora are at present unculturable and can only be studied using culture-independent approaches. Consequently, the large-scale cultivation and subsequent storage of probiotic lactobacilli and bifidobacteria in high numbers often presents a major bottleneck to the realization of their commercial potential. For this reason, intensive research efforts have recently focussed on protecting the viability of probiotic cultures both during product manufacture and storage and during gastric transit. These studies have demonstrated that cultures can be significantly protected via encapsulation in a variety of carriers, which include milk proteins and complex (prebiotic) carbohydrates. In many cases, the resultant products not only have better probiotic viability but can also be regarded as 'synbiotics' given the presence of probiotics and prebiotics (Roberfroid 1998). The physiological state of the probiotic cultures being added to a product can also be a major factor affecting overall culture viability. In this respect, the induction of stress responses in probiotic strains can have a dramatic effect on the ability of cultures to survive processing, such as freeze drying and spray drying and during gastric transit. Indeed, we have recently generated probiotic cultures that overexpress the heat shock proteins GroESL and have demonstrated improved performance of the culture under a variety of conditions including heat, spray drying and exposure to gastric acid (Desmond et al. 2004). The addition of various protective compounds to probiotic cultures can also improve their viability during manufacture - examples include glucose to energize cells on exposure to acid (Corcoran et al. 2005) and cryoprotectents such as inulin to improve survivability during freeze drying (Carvalho et al. 2004). In conclusion, a number of novel technologies are now emerging which can improve the viability of human intestinal strains for probiotic applications, which means that it may be possible to exploit many 'sensitive' cultures which hitherto have been difficult to propagate and maintain at high cell numbers.

The use of multilocus enzyme electrophoresis to characterise intestinal spirochaetes (Brachyspira spp.) colonising hens in commercial flocks

Multilocus enzyme electrophoresis (MLEE) was used to identify, examine genetic relationships and look at disease associations of a collection of 53 intestinal spirochaete isolates previously recovered from the faeces of adult hens on 14 farms in Qld, Australia. The MLEE results were compared with those previously obtained using species-specific PCR amplifications. The isolates were divided into five Brachyspira species groups by MLEE: Brachyspira murdochii (n=17), B. intermedia (n=15), B. pilosicoli (n=14), B. innocens (n=2) and "B. pulli" (n=1). Three new MLEE groups each containing single isolates also were identified. The results of the PCR assay for B. pilosicoli were concordant with the MLEE results, but the 23S rDNA-based PCR for B. intermedia had failed to detect 8 of the 15 isolates. The B. innocens/B. murdochii nox-based PCR had correctly identified all the isolates of B. murdochii, but did not identify either of the two B. innocens isolates. Using MLEE, isolates from two farms (14%) were identified as B. murdochii, whilst the pathogenic species B. intermedia and B. pilosicoli were present in hens from eight (57%) and five (36%) farms, respectively, and were identified together in four (29%) farms. All seven of the farms with production problems or wet litter were colonised with B. intermedia and/or B. pilosicoli. Six farms had multiple spirochaete isolates available for examination. Two broiler breeder farms both had five isolates of B. pilosicoli that shared the same MLEE electrophoretic type (ET), whilst one laying hen farm had three isolates of B. intermedia that all belonged to the same ET. Hence on each of these farms a predominant strain of a pathogenic species was present. On the other farms isolates of the same species were more diverse and belonged to different ETs. These results show that the epidemiology of intestinal spirochaetal infections in broiler breeder and laying hen flocks can vary considerably between farms, although the reasons for these differences were not established.

Isolates from Normal Human Intestinal Flora but not Lactic Acid Bacteria Exhibit 7?- and 7?- Hydroxysteroid Dehydrogenase Activities

Ursodeoxycholic acid (UDCA)-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. UDCA is the 7β-OH epimer of the primary (i.e. synthesized by the liver) bile acid chenodeoxycholic acid (CDCA). Epimerization proceeds in two subsequent and reversible steps, catalysed by a 7α- and a 7β-hydroxysteroid dehydrogenase (7α- and 7β- HSDH), with 7oxo-lithocholic acid (7oxo-LCA) as the intermediate product. The aim of this study was to test the 7α- and 7β-HSDH activities of anaerobic whole cell cultures of a number of lactic acid bacteria and human intestinal isolates, using CDCA, UDCA and 7oxo- LCA as the substrates. Among 140 strains tested, 21 exhibited at least one of both 7-HSDH activities. 7α-HSDH activity was detected in six strains, 7β-HSDH in nine strains, and both activities in six other strains. All active strains were isolated from normal human and infant faeces. They belonged to the genera Clostridium , Eubacterium and Ruminococcus , whereas no strain of Lactobacillus , Bifidobacterium or Streptococcus was found to be active under our study conditions. The present study therefore revealed, for the first time, a number of normal human intestinal isolates supporting the epimerization of CDCA to UDCA, and further extended our knowledge of those intestinal bacteria which are responsible for 7α- or 7β-HSDH activity. Key words: screening, bile acids, epimerization, intestinal microflora, lactic acid bacteria, probiotics.

Isolates from normal human intestinal flora but not lactic acid bacteria exhibit 7α- and 7β-hydroxysteroid dehydrogenase activities

Ursodeoxycholic acid (UDCA)-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. UDCA is the 7β-OH epimer of the primary (i.e. synthesized by the liver) bile acid chenodeoxycholic acid (CDCA). Epimerization proceeds in two subsequent and reversible steps, catalysed by a 7α- and a 7β-hydroxysteroid dehydrogenase (7α- and 7β-HSDH), with 7oxo-lithocholic acid (7oxo-LCA) as the intermediate product. The aim of this study was to test the 7α- and 7β-HSDH activities of anaerobic whole cell cultures of a number of lactic acid bacteria and human intestinal isolates, using CDCA, UDCA and 7oxo-LCA as the substrates. Among 140 strains tested, 21 exhibited at least one of both 7-HSDH activities. 7α-HSDH activity was detected in six strains, 7β-HSDH in nine strains, and both activities in six other strains. All active strains were isolated from normal human and infant faeces. They belonged to the genera Clostridium, Eubacterium and Ruminococcus, whereas no strain of Lactobacillus, Bifidobacterium or Streptococcus was found to be active under our study conditions. The present study therefore revealed, for the first time, a number of normal human intestinal isolates supporting the epimerization of CDCA to UDCA, and further extended our knowledge of those intestinal bacteria which are responsible for 7α- or 7β-HSDH activity. Key words: screening, bile acids, epimerization, intestinal microflora, lactic acid bacteria, probiotics.

Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract.

To identify lactic acid bacteria (LAB) of porcine intestinal origin with anti-Salmonella activity. Samples were obtained from pig faeces and caeca and screened for the presence of anti-Salmonella LAB. The 11 most promising isolates were identified as belonging to the genera Lactobacillus and Pediococcus. The LAB exhibited large variation in their ability to survive in simulated gastric juice at pH 1.85. While Lactobacillus johnsonii species survived at levels of 80% for up to 30 min, Lactobacillus pentosus species declined to <0.001% in that time. All isolates tolerated porcine bile at a concentration of 0.3% (w/v), with some isolates capable of growth in the presence of up to 5% (w/v) bile. The ability of the LAB isolates to prevent Salmonella invasion of intestinal epithelial HT-29 cells varied, with reductions of between 30% (Lact. pentosus) and 80% (Lactobacillus murinus spp.) observed. LAB of porcine origin were observed to survive simulated passage through the GIT and inhibit growth of Salmonella and its invasion of the intestinal epithelium. The data demonstrate that some porcine intestinal LAB isolates may offer potential as probiotics for the reduction of Salmonella carriage in pigs.

Antimicrobial susceptibilities of Bacteroides fragilis and Bacteroides thetaiotaomicron strains isolated from clinical specimens and human intestinal microbiota

Species of Bacteroides fragilis group bacteria are the most prevalent pathogens and have the highest resistance rates to antimicrobial agents among anaerobic bacteria. Infections due to these micro-organisms often originate from patient's own intestinal microbiota. The objective of the study was to determine and compare the susceptibility profiles of clinical and intestinal B. fragilis and B. thetaiotaomicron strains against certain antimicrobials. Isolates were identified by conventional methods and API-20 A. Susceptibility tests were performed according to recommendations of NCCLS (M 11-A4) agar dilution methods. Beta-lactamase production was determined with nitrocefin discs. Forty-five clinical isolates (33 B. fragilis and 12 B. thetaiotaomicron) were from following sites: blood (n:8), intra-abdominal abscess (n:7), soft tissue (n:26), and miscellaneous foci of infection (n:4). Fifty B. fragilis and 60 B. thetaiotaomicron isolates from intestinal microbiota of individuals with no history of antimicrobial treatment within last 30 days were also examined. Beta-lactamase production was detected in 93% of clinical and 99% of intestinal isolates. The organisms including intestinal isolates were uniformly susceptible to metronidazole. The MIC90s of other antibiotics and resistance rates of all clinical isolates to those antibiotics were as follows: 256μg/mL (93%) for ampicillin, 128μg/mL (13%) for piperacillin, 64μg/mL (11%) for cefoxitin, 1μg/mL (2%) for amoxicillin-clavulanate, 0.5μg/mL (2%) for imipenem, >256μg/mL (36%) for clindamycin, 8μg/mL (2%) for chloramphenicol. Intestinal isolates demonstrated similar resistance rates and MIC90s. Metronidazole, imipenem, amoxicillin-clavulanate seem to be effective drugs against these bacteria in Turkey.

Emerging nalidixic acid and ciprofloxacin resistance in non-typhoidal Salmonella isolated from patients having acute diarrhoeal disease.

BackgroundNon-typhoidal Salmonella are one of the key etiological agents of diarrhoeal disease. The appearance of multiple drug resistance along with resistance to quinolones in this bacterium poses a serious therapeutic problem, We determined the prevalence of nalidixic acid and ciprofloxacin resistance in non-typhoidal Salmonella isolated from faecal samples of patients with acute diarrhoeal disease attending the outpatient and inpatient departments of a hospital in Saudi Arabia during the years 1999 to 2002.MethodsNon-typhoidal Salmonella were isolated from faecal samples. Antimicrobial susceptibility was tested by the disc diffusion test MICs to nalidixic acid and ciprofloxacin were determined by the agar dilution method.ResultsDuring the study period, 524 strains of non-typhoidal Salmonella were isolated Strains belonging to serogroup C1 were the commonest (41.4%) followed by serogroups B and D (15.6% and 14.5%, respectively), Resistance to ampicillin was observed in 22.9% and to trimethoprim/sulfamethoxazole in 18.5% of the strains. Nalidixic acid resistance was encountered in 9.9% and ciprofloxacin resistance in 23% of the strains. Resistance to nalidixic acid significantly increased from 0.1% in 1999 to 5.5% in 2002 (P=0.0007) and ciprofloxacin resistance increased significantly from 0.1% in 1999 to 0.9% in 2002 (P=0.0001). MICs to nalidixic acid and ciprofloxacin were determined among 29 nalidixic acid-resistant strains of non-typhoidal Salmonella isolated during 2002. The MIC was >256 μg/mL to nalidixic acid and 8 to 16 μg/mL to ciprofloxacin.ConclusionThe increasing rates of antimicrobial resistance encountered among non-typhoidal Salmonella necessitate the judicious use of these drugs in humans. Moreover, these findings support the concern that the use of quinolones in animal feed may lead to an increase in resistance and should be restricted.

Genetic heterogeneity and functional properties of intestinal bifidobacteria.

The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.

Relative ability of orally administered Lactobacillus murinus to predominate and persist in the porcine gastrointestinal tract.

Five porcine-derived Lactobacillus or Pediococcus isolates administered to pigs (n = 4), either singly or as a combination at approximately 10(10) CFU per day varied with respect to intestinal survival and persistence. Two Lactobacillus murinus strains survived best and were excreted at approximately 10(7) to 10(8) CFU/g of feces. In contrast, Pediococcus pentosaceus DPC6006 had the lowest fecal count at approximately 10(5) CFU/g and was excreted at a significantly lower level than both L. murinus strains. Fecal L. murinus DPC6003 counts were also significantly higher than both Lactobacillus salivarius DPC6005 and Lactobacillus pentosus DPC6004 ( approximately 10(6) CFU/g). The L. murinus strains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted approximately 10(7) CFU of the administered strains/g, with L. murinus predominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general, L. murinus DPC6002 and DPC6003 and L. pentosus DPC6004 predominated in the feces and the cecum while P. pentosaceus DPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecal Enterobacteriaceae counts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration, Enterobacteriaceae counts were reduced by approximately 87 to 98% in pigs fed L. salivarius DPC6005, P. pentosaceus DPC6006, L. pentosus DPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.

Genotypic and phenotypic studies of murine intestinal lactobacilli: species differences in mice with and without colitis.

Lactobacilli represent components of the commensal mammalian gastrointestinal microbiota and are useful as probiotics, functional foods, and dairy products. This study includes systematic polyphasic analyses of murine intestinal Lactobacillus isolates and correlation of taxonomic findings with data from cytokine production assays. Lactobacilli were recovered from mice with microbiota-dependent colitis (interleukin-10 [IL-10]-deficient C57BL/6 mice) and from mice without colitis (Swiss Webster and inducible nitric oxide synthetase-deficient C57BL/6 mice). Polyphasic analyses were performed to elucidate taxonomic relationships among 88 reference and murine gastrointestinal lactobacilli. Genotypic tests included single-locus analyses (16S ribosomal DNA sequencing and 16S-23S rRNA intergenic spacer region PCR) and genomic DNA profiling (repetitive DNA element-based PCR), and phenotypic analyses encompassed more than 50 tests for carbohydrate utilization, enzyme production, and antimicrobial resistance. From 20 mice without colitis, six Lactobacillus species were recovered; the majority of the mice were colonized with L. reuteri or L. murinus (72% of isolates). In contrast, only, L. johnsonii was isolated from 14 IL-10-deficient mice. Using an in vitro assay, we screened murine isolates for their ability to inhibit tumor necrosis factor alpha (TNF-alpha) secretion by lipopolysaccharide-activated macrophages. Interestingly, a subpopulation of lactobacilli recovered from mice without colitis displayed TNF-alpha inhibitory properties, whereas none of the L. johnsonii isolates from IL-10-deficient mice exhibited this effect. We propose that differences among intestinal Lactobacillus populations in mammals, combined with host genetic susceptibilities, may account partly for variations in host mucosal responses.

Prevalence of secreted autotransporter toxin gene among diffusely adhering Escherichia coli isolated from stools of children

In this report, we analyzed the prevalence of the sat gene in 336 Escherichia coli samples collected from stools of children with and without diarrhea in Brazil and in 100 uropathogenic E. coli strains. The results show a high correlation between diffusely adhering E. coli (DAEC) and the presence of sat (44%) in intestinal isolates. DAEC strain FBC114 expresses a 107-kDa protein, which showed 98% homology with Sat.

Prevalence, risk factors and molecular epidemiology of Brachyspira pilosicoli in humans on the island of Bali, Indonesia.

The purpose of this study was to investigate the prevalence and epidemiology of the anaerobic intestinal spirochaete Brachyspira pilosicoli amongst Indonesians living in rural and urban settings on the island of Bali. Faecal samples (n = 992) were collected on two occasions, 4 months apart, from people living in four traditional farming villages, one peri-urban location and one urban area. Samples were cultured anaerobically on selective agar and intestinal spirochaete isolates were confirmed to be B. pilosicoli by using a species-specific PCR. Forty-eight of the 121 isolates obtained were typed by using PFGE. A questionnaire was administered to participants and analysed in order to identify potential risk factors for colonization. Overall prevalence of carriage on the two visits was 11.8 and 12.6 %, respectively. Prevalence at different locations varied from 3.3 to 23.4 %, with the highest prevalence occurring in the peri-urban location. Considerable strain diversity was found, with the 48 isolates being divided into 44 PFGE types. There was no significant association between colonization and ownership of animals, contact with animals, farming, age or gender. On the first visit, colonization was significantly more common in people who used well water compared to those who used tap water. On the second visit, colonization was significantly more common in people with wet faeces than in those with normal faeces.

Helicobacter sp. flexispira 16S rDNA taxa 1, 4 and 5 and Finnish porcine Helicobacter isolates are members of the species Helicobacter trogontum (taxon 6).

The term 'flexispira' refers to micro-organisms with a particular morphology: fusiform-shaped with helical periplasmic fibrils and bipolar tufts of sheathed flagella. Two flexispira taxa have been formally named, Helicobacter bilis and Helicobacter trogontum, a third named species is Helicobacter aurati and eight additional 16S rRNA sequence-based flexispira taxa have been described by Dewhirst et al. (Int J Syst Evol Microbiol 50, 1781-1787, 2000) and given the provisional designation Helicobacter sp. flexispira taxa 1-5, 7, 8 and 10. In the present study, seven gastric or intestinal flexispira isolates from seven Finnish pigs originating from different farms were characterized. Morphologically, all these porcine isolates had typical flexispira morphology. Analysis of the 16S rDNA sequences of five isolates showed that they were most closely related to the sequences of flexispira taxa 4 and 5 and H. trogontum (taxon 6), but less closely related to taxa 1-3 and 8, H. bilis and H. aurati. Phenotypic characterization, analysis of RFLPs of 16S and 23S rDNAs and SDS-PAGE profiles revealed that all of the porcine isolates, reference strains of flexispira taxa 1, 4 and 5 and the type strain of H. trogontum (ATCC 700114T) had highly related characteristics that differed from those of the reference strains of taxa 2, 3 and 8 and H. bilis. Furthermore, a high DNA-DNA binding rate was found, in dot-blot hybridization studies, between the Finnish porcine strains, taxa 1, 4 and 5 reference strains and H. trogontum ATCC 700114T. In conclusion, polyphasic characterization of novel porcine flexispira isolates and previously described taxa 1, 4 and 5 reference strains showed that they all belong to a validly described species, H. trogontum, and that the taxonomy of known flexispiras is less complicated than proposed on the basis of 16S rDNA sequence analysis.

In situ production of exopolysaccharides during Sourdough fermentation by cereal and intestinal isolates of lactic acid bacteria.

EPS formed by lactobacilli in situ during sourdough fermentation may replace hydrocolloids currently used as texturizing, antistaling, or prebiotic additives in bread production. In this study, a screening of >100 strains of cereal-associated and intestinal lactic acid bacteria was performed for the production of exopolysaccharides (EPS) from sucrose. Fifteen strains produced fructan, and four strains produced glucan. It was remarkable that formation of glucan and fructan was most frequently found in intestinal isolates and strains of the species Lactobacillus reuteri, Lactobacillus pontis, and Lactobacillus frumenti from type II sourdoughs. By the use of PCR primers derived from conserved amino acid sequences of bacterial levansucrase genes, it was shown that 6 of the 15 fructan-producing lactobacilli and none of 20 glucan producers or EPS-negative strains carried a levansucrase gene. In sourdough fermentations, it was determined whether those strains producing EPS in MRS medium modified as described by Stolz et al. (37) and containing 100 g of sucrose liter(-1) as the sole source of carbon also produce the same EPS from sucrose during sourdough fermentation in the presence of 12% sucrose. For all six EPS-producing strains evaluated in sourdough fermentations, in situ production of EPS at levels ranging from 0.5 to 2 g/kg of flour was demonstrated. Production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria. Thus, the use of these organisms in bread production may allow the replacement of additives.

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.

Antibacterial abilities of intestinal bacteria from larval and juvenile Japanese flounder against fish pathogens

: The present study was undertaken to examine the antibacterial abilities of intestinal bacteria isolated from juveniles and larvae of Japanese flounder Paralichthys olivaceus. Newly hatched larvae of flounder were held in a 100 L plastic circular tank and fed rotifiers, Artemia nauplii and commercial feeds, depending on the developmental stage of the fish. Genera Aeromonas, Moraxella and Vibrio were predominantly isolated from the intestinal tracts of Japanese flounder at larval and juvenile stages, whereas Aeromonas, Bacillus, coryneforms, Moraxella, Pseudomonas and Vibrio were detected at high densities in live diets and artificial feeds. Antibacterial bacteria accounted for 1.7–24.3% of the intestinal isolates against Lactococcus garvieae, Pasteurella piscicida, Vibrio anguillarum and V. vulnificus. In particular, as much as 53.3% of Vibrio spp. other than Vibrio-swarmer isolated from 197-day-old juveniles inhibited the growth of P. piscicida. These results suggest that intestinal bacteria having antibacterial activity play a role in the prevention of infectious diseases.

Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production.

Interaction of macrophages with bacteria is a stimulus for production of cytokines such as IL-10 and IL-12. IL-12 stimulates T cell and natural killer (NK) cell cytotoxicity and interferon-gamma (IFN-gamma) production. IL-10 opposes the T cell-stimulating action of IL-12, decreases the release of proinflammatory cytokines from macrophages, and stimulates B cells. We have studied the capacity of human intestinal isolates from the three Lactobacillus species dominating on the human gastrointestinal mucosa, L. plantarum, L. rhamnosus and L. paracasei ssp. paracasei, to induce production of IL-10 and IL-12 from human blood mononuclear cells, or monocytes. Whole killed lactobacilli were potent stimulators of IL-12 over a wide range of bacterial concentrations. Lactobacillus paracasei gave the highest levels of IL-12 (1.5 ng/ml in response to 5 x 106 bacteria/ml), roughly 10 times more than obtained by stimulation with L. rhamnosus or L. plantarum. Escherichia coli induced on average < 50 pg/ml of IL-12 regardless of the bacterial concentration used. The secretion of free p40 subunit IL-12 followed the same pattern as the secretion of p70 (bioactive IL-12) with regard to the efficiency of different bacteria as stimulators. Escherichia coli was the most efficient trigger of IL-10 production, inducing 0.5 ng/ml IL-10 after stimulation with 5 x 106 bacteria/ml. Lactobacillus rhamnosus induced the highest levels of IL-10 among the lactobacilli (0.5 ng/ml) compared with 0.1 ng/ml evoked by L. plantarum or L. paracasei, but 10 times more bacteria were required for optimal stimulation than with E. coli. When neutralizing anti-IL-10 antibodies were added to the cultures, the IL-12-inducing capacity of L. rhamnosus was increased markedly, while that of E. coli remained low. The results show that mucosa-associated lactobacilli can be potent stimulators of IL-12, and thus potentially of cell-mediated immunity, if they pass over the gut epithelial barrier and interact with cells of the gut immune system.

Entamoeba histolytica: Genetic Diversity of Clinical Isolates from Bangladesh as Demonstrated by Polymorphisms in the Serine-Rich Gene

Ayeh-Kumi, P. F., Ali, I. M., Lockhart, L. A., Gilchrist, C. A., Petri, W. A., Jr., and Hague, R. 2001. Entamoeba histolytica: Genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene Experimental Parasitology99, 80–88. The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.

Comparative study on biochemical activity of the intestinal isolates Lactobacillus sp. V3 and Bifidobacterium sp. A71 in different substrates

The two intestinal isolates Lactobacillus sp. V3 and Bifidobacterium sp. A71 were selected for soymilk fermentation according to their acidification activity in soymilk. Beetroot juice and carrot juice were chosen for soymilk supplementation as additional sources of carbohydrates and brewer's yeast as an extra source of nitrogen. The fermentation was carried out for eight hours at 42?C. The fermentation was monitored by standard analytical and microbiological tests for changes of acidity (decreasing pH and increasing acid content), the contents of soluble dry substances, sugars and ?-amino acids as well as changes in the number of viable cells. The samples were collected at the beginning and subsequently every two hours until the end of the fermentation. The results showed that there were differences between the tested isolates in terms of their ability to ferment soymilk. The mix with brewer's yeast had a better stimulating effect on the growth of both strains compared to those with juices alone. In addition, the carrot juice stimulated the growth of Bifidobacterium sp. A71 better than beetroot juice, while the opposite effect was found for the growth of Lactobacillus sp. V3.