Effects of freshwater sponge Ephydatia fluviatilis on conjugative transfer of antimicrobial resistance in Enterococcus faecalis strains in aquatic environments.

Filter feeding is a biotic process that brings waterborne bacteria in close contact with each other and may thus support the horizontal transfer of their antimicrobial resistance genes. This laboratory study investigated whether the freshwater sponge Ephydatia fluviatilis supported the transfer of vancomycin resistance between two Enterococcus faecalis strains that we previously demonstrated to exhibit pheromone responsive plasmid conjugation. Microcosm experiments exposed live and dead colonies of laboratory-grown sponges to a vancomycin-resistant donor strain and a rifampicin-resistant recipient strain of Ent. faecalis. Enterococci with both resistance phenotypes were detected on double selection plates. In comparison to controls, abundance of these presumed transconjugants increased significantly in water from sponge microcosms. Homogenized suspensions of sponge cells also yielded presumed transconjugants; however, there was no significant difference between samples from live or dead sponges. Fluorescent in situ hybridization analysis of the sponge cell matrix using species-specific probes revealed the presence of enterococci clusters with cells adjacent to each other. The results demonstrated that sponge colonies can support the horizontal transfer of antimicrobial resistance although the mechanism underlying this process, such as binding of the bacteria to the sponge collagen matrix, has yet to be fully elucidated.

Microbial biosurfactants: current trends and applications in agricultural and biomedical industries.

Synthetic surfactants are becoming increasingly unpopular in many applications due to previously disregarded effects on biological systems and this has led to a new focus on replacing such products with biosurfactants that are biodegradable and produced from renewal resources. Microbially derived biosurfactants have been investigated in numerous studies in areas including: increasing feed digestibility in an agricultural context, improving seed protection and fertility, plant pathogen control, antimicrobial activity, antibiofilm activity, wound healing and dermatological care, improved oral cavity care, drug delivery systems and anticancer treatments. The development of the potential of biosurfactants has been hindered somewhat by the myriad of approaches taken in their investigations, the focus on pathogens as source species and the costs associated with large-scale production. Here, we focus on various microbial sources of biosurfactants and the current trends in terms of agricultural and biomedical applications.

Interspecies transfer of vancomycin, erythromycin and tetracycline resistance among Enterococcus species recovered from agrarian sources.

BACKGROUND: Enterococci are now well recognised for their ability to transfer antibiotic resistance and for their association with nosocomial infections, but less is known regarding their relevance in the wider environment. Enterococcus faecalis and Enterococcus faecium were isolated from a range of agrarian associated sources (low-flow water, septic tank, poultry litter, high flow water, slurry/soil) and were assessed for latent ability to transfer antimicrobial resistance. RESULTS: The isolates were tested for phenotypic clumping in the presence of cell-free supernatant from other isolates. Some isolates were identified which demonstrated clumping, indicating that they possessed peptide sex pheromone conjugal machinery. All isolates were also tested for antibiotic resistance phenotypes using both disc diffusion and minimum inhibitory concentration (MIC) assays. These tests revealed that the enterococci demonstrated both phenotypic clumping and antibiotic resistance phenotypes. Based on these selection criteria, the isolates were identified as having the potential for horizontal gene transfer and were used to investigate the transfer of multiple antibiotic resistance phenotypes. Conjugal transfer of antibiotic resistance phenotypes was determined using a solid agar mating method followed by a standard antibiotic selection test resulting in different transfer patterns. An interspecies conjugal transfer of vancomycin resistance from E. faecalis to E. faecium was identified while the remaining reactions were within the same species. Transfer efficiencies ranging from 2 × 10-1 to 2.3 × 10-5 were determined based on the reactions of three donor isolates (MF06036, MF0410 and MF06035) and two recipient isolates (MW01105Rif and ST01109Rif), with the transfer of vancomycin, erythromycin and tetracycline resistance genes. CONCLUSIONS: The conjugation reactions and selection conditions used in this study resulted in a variety of co-transferred resistance phenotypes suggesting the presence of different mobile elements in the set of natural isolates. This study highlights the potential for extensive horizontal gene transfer in a previously neglected reservoir for enterococci.

Relationship between dietary-induced changes in intestinal commensal microflora and duodenojejunal myoelectric activity monitored by radiotelemetry in the rat in vivo.

Interdigestive intestinal motility, and especially phase III of the migrating myoelectric/motor complex (MMC), is responsible for intestinal clearance and plays an important role in prevention of bacterial overgrowth and translocation in the gut. Yet previous results from gnotobiotic rats have shown that intestinal microflora can themselves affect the characteristics of the myoelectric activity of the gut during the interdigestive state. Given that the composition of the intestinal microflora can be altered by dietary manipulations, we investigated the effect of supplementation of the diet with synbiotics on intestinal microflora structure and the duodenojejunal myoelectric activity in the rat. To reduce animal distress caused by restraint and handling, which can itself affect GI motility, we applied radiotelemetry for duodenojejunal EMG recordings in conscious, freely moving rats. Thirty 16-month-old Spraque-Dawley rats were used. The diet for 15 rats (E group) was supplemented with chicory inulin, Lactobacillus rhamnosus and Bifidobacterium lactis. The remaining 15 rats were fed control diet without supplements (C group). Three rats from each group were implanted with three bipolar electrodes positioned at 2, 14 and 28 cm distal to the pylorus. After recovery, two 6 h recordings of duodenojejunal EMG were carried out on each operated rat. Subsequently, group C rats received feed supplements and group E rats received only control diet for 1 week, and an additional two 6 h recordings were carried out on each of these rats. Non-operated C and E rats were killed and samples of GI tract were collected for microbiological analyses. Supplementation of the diet with the pro- and prebiotics mixture increased the number of bifidobacteria, whereas it decreased the number of enterobacteria in jejunum, ileum, caecum and colon. In both caecum and colon, the dietary supplementation increased the number of total anaerobes and lactobacilli. Treatment with synbiotics increased occurrence of phase III of the MMC at all three levels of the small intestine. The propagation velocity of phase III in the whole recording segment was also increased from 3.7 +/- 0.2 to 4.4 +/- 0.2 cm min(-1) by dietary treatment. Treatment with synbiotics increased the frequency of response potentials of the propagated phase III of the MMC at both levels of the jejunum, but not in the duodenum. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of phase I and phase III. Using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet.

Effect of feed particle size and feed processing on morphological characteristics in the small and large intestine of pigs and on adhesion of Salmonella enterica serovar Typhimurium DT12 in the ileum in vitro.

A 2 x 2 factorial experiment with pigs was undertaken to investigate the effect of particle size (fine and coarse) and feed processing (pelleted and nonpelleted) on morphological characteristics in the small intestine, cecum, and colon of pigs and on the adhesion of Salmonella enterica serovar Typhimurium DT12 to the ileum in vitro. Ninety-six pigs (average BW = 33 +/- 7 kg) were fed the experimental diets. After 4 wk, 24 pigs were selected (six pigs per diet) and euthanized, and tissue samples were taken from the mid and distal small intestine, cecum, and distal colon. The effects of particle size and feed processing on villus height and crypt depth in the small intestine were minor. Feeding coarse diets increased (P = 0.05) the crypt depth in the colon. The crypt depth was 420 +/- 12 and 449 +/- 12 microm in pigs fed finely and coarsely ground feed, respectively. Pigs fed pelleted diets had a larger (P = 0.01) staining area for neutral mucins, as well as for acidic and sulfomucins on the villi of the distal small intestine than pigs fed nonpelleted diets. The area was 41, 46, and 33% larger for neutral, acidic, and sulfomucins, respectively. The mucin-staining areas of the crypts in the cecum and the colon were not affected by the experimental diets. Examination of lectin binding characteristics of the distal small intestine and the cecum did not reveal any differences between the experimental diets. Using a pig intestine organ culture model, Salmonella adhered less (P < 0.05) to the ileal tissue of pigs fed the nonpelleted diets than to those fed pelleted diets; the adherence was 60% less in these pigs. Results of this study suggest that pigs fed pelleted diets secrete mucins that are capable of binding Salmonella enterica serovar Typhimurium DT12 and thereby allowing for colonization. Therefore, pigs fed a nonpelleted diet are better protected against Salmonella infections than pigs fed a pelleted diet.

A bioreactor system to study survival of Salmonella Typhimurium in pig gut content.

The batch culture system included six bioreactors. Three bioreactors containing stomach slurry were maintained at pH 4.5 and 6 respectively. Bioreactors containing small intestine slurry were maintained at pH 5.6 and 7 respectively. The bioreactors were inoculated with 10 ml of viable Salmonella. The bioreactors were maintained for 6 hours. Samples of 10 ml were taken at 0 time and at 1, 2, 4 and 6 hours. The samples were analysed for the presence of Salmonella and SCFA. In the stomach samples Salmonella numbers increased at pH 6 but fell at pH 4. In the small intestine sample Salmonella numbers increased at pH 6 and 7. In terms of SCFA production, in the stomach, with samples at pH 6 there was little change in the amounts of lactate, succinate and formate to that detected at 0 time, however levels of acetate did increase slightly. In the small intestine samples levels of succinate and formate increased slightly up to 4 hours, levels of acetate increased significantly from 0 to 6 hours. In terms of the specific growth rates of the individual strains, both strains grew at pH 6 in the stomach content and to a greater extent in the small intestinal content. A bactericidal effect was observed at pH 4 in the stomach content while neither killing nor growth occurred at pH 5 either in the stomach or the small intestine content. Both strains grew well in the small intestine content at pH 7, showing generation times of up to 24 min.

Effects of nondigestible oligosaccharides on Salmonella enterica serovar Typhimurium and nonpathogenic Escherichia coli in the pig small intestine in vitro.

An in vitro intestinal tissue model was developed for the investigation of bacterial association in the pig small intestine under different dietary regimes. In preliminary experiments, jejunal and ileal tissue was taken from Danish Landrace pigs fed standard diet and inoculated with either Salmonella or nonpathogenic Escherichia coli strains. Higher numbers of salmonellae associated with the ileal tissues, but the numbers did not reach significance. Hence, jejunal sections were inoculated with nonpathogenic E. coli and ileal sections were inoculated with salmonellae in the presence of mannose or commercial nondigestible oligosaccharides (NDO) at 2.5%. There was a significant decrease in E. coli associated with the jejunum in the presence of mannose (P < 0.05). Furthermore, in pigs fed a diet supplemented with commercial NDO at 4% there was a significant reduction in the numbers of E. coli in jejunal organ cultures of pigs fed the FOS diet (P < 0.05). There was a reduction, though not a significant one, in the association of Salmonella sp. to the ileal sections of pigs fed the commercial FOS diet. The feeding of commercial GOS or its addition to organ cultures did not affect E. coli or Salmonella numbers.

Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo.

The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm. enteritidis 857. Con A significantly increased numbers of Salm. typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone. Con A had much less effect on rats infected with Salm. enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study. GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm. typhimurium S986 and significantly improved rat growth. GNA had little effect on infection by Salm. enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.

Salmonella enterica var Typhimurium and Salmonella enterica var Enteritidis express type 1 fimbriae in the rat in vivo.

In a series of experiments rats were dosed with purified type 1 fimbriae from Salmonella enterica var Enteritidis or with fimbriated cultures of either S. enterica var Typhimurium or S. enterica var Enteritidis. Paraffin-wax embedded histological sections of jejunal and ileal tissue were taken and stained by the streptavidin biotin complex (sABC) staining technique for the detection of salmonella and type 1 fimbriae. On oral infection with Enteritidis and Typhimurium both bacteria were shown to be closely associated with the rat ileal epithelium and expressed type 1 fimbriae, thus clearly demonstrating that type 1 fimbriae are expressed by salmonellae in vivo. Moreover, association with the ileum was also shown to occur when purified type 1 fimbriae were orally administered to rats. Our results suggest that type 1 fimbriae alone or in combination with other fimbriae may play an important role in the early stages of infection with these pathogenic bacteria.

A rat model of infection by Salmonella typhimurium or Salm. enteritidis.

Salmonellosis in the rat has many similarities with the disease in humans, with the ileum thought to be the main site of colonization/invasion in both species. Thus, the rat may be a useful way to study the mechanism of infection by these pathogenic bacteria. A series of infection trials carried out with Hooded Lister rats showed that a salmonella infection persisted for an extended period of time and that salmonellae bind to the small intestinal epithelium as early as 4 h after intragastric intubation. Reinfection from the large intestine may not therefore initially play a significant role in the salmonella infection process. The rat model may therefore provide a means to test in vivo interventionist strategies, designed to block binding of the pathogens in the gastrointestinal tract.

Salmonella typhimurium and Salmonella enteritidis induce gut growth and increase the polyamine content of the rat small intestine in vivo.

The effects of infection by Salmonella enteritidis and S. typhimurium on the small and large intestines, liver, spleen and mesenteric nodules of rats were studied in vivo. Both Salmonella serotypes persisted and proliferated in the gastrointestinal tract and invaded sub-epithelial tissues, mainly the ileum, leading to the systemic distribution of these pathogens. Coincidental with infection, the rate of crypt cell proliferation increased resulting in substantial growth of the small intestine. The extent of this and the accompanying accumulation of polyamines was particularly dramatic in the ileum where there was also some disruption of the villus epithelium. It is possible that these effects of the infection on the metabolism and morphology of the small bowel, which strongly resembled the changes induced by some plant lectins, may facilitate the colonisation and invasion of the gut by Salmonellae.