Influence of Dietary Zinc Oxide and Copper Sulfate on the Gastrointestinal Ecosystem in Newly Weaned Piglets

Dietary doses of 2,500 ppm ZnO-Zn reduced bacterial activity (ATP accumulation) in digesta from the gastrointestinal tracts of newly weaned piglets compared to that in animals receiving 100 ppm ZnO-Zn. The amounts of lactic acid bacteria (MRS counts) and lactobacilli (Rogosa counts) were reduced, whereas coliforms (MacConkey counts) and enterococci (Slanetz counts, red colonies) were more numerous in animals receiving the high ZnO dose. Based on 16S rRNA gene sequencing, the colonies on MRS were dominated by three phylotypes, tentatively identified as Lactobacillus amylovorus (OTU171), Lactobacillus reuteri (OTU173), and Streptococcus alactolyticus (OTU180). The colonies on Rogosa plates were dominated by the two Lactobacillus phylotypes only. Terminal restriction fragment length polymorphism analysis supported the observations of three phylotypes of lactic acid bacteria dominating in piglets receiving the low ZnO dose and of coliforms and enterococci dominating in piglets receiving the high ZnO dose. Dietary doses of 175 ppm CuSO₄-Cu also reduced MRS and Rogosa counts of stomach contents, but for these animals, the numbers of coliforms were reduced in the cecum and the colon. The influence of ZnO on the gastrointestinal microbiota resembles the working mechanism suggested for some growth-promoting antibiotics, namely, the suppression of gram-positive commensals rather than potentially pathogenic gram-negative organisms. Reduced fermentation of digestible nutrients in the proximal part of the gastrointestinal tract may render more energy available for the host animal and contribute to the growth-promoting effect of high dietary ZnO doses. Dietary CuSO₄ inhibited the coliforms and thus potential pathogens as well, but overall the observed effect of CuSO₄ was limited compared to that of ZnO.     

Survival and retention of the probiotic Lactobacillus casei LAFTI L26 in the gastrointestinal tract of the mouse

AIMS: This study aimed to develop methods for the detection of the probiotic Lactobacillus casei LAFTI L26 (L26) from mouse faeces, and to determine the survival and retention time of L26 in the mouse gastrointestinal tract. METHODS AND RESULTS: A selective medium, de Man Rogosa Sharpe (MRS) + bromocresol green + vancomycin (MGV), was designed for the isolation and enumeration of L26 from faecal samples of mice. PCR primers were designed to confirm the identity of L26-like colonies on MGV. These primers did not produce PCR products from related organisms that grew on MGV. Following the administration of L26 to BALB/c mice, faecal samples were collected and analysed using the designed methods. Survival studies showed viable L26 cells to be present in the faeces of mice for >48 h. CONCLUSIONS: Our results suggest that L26 is able to survive and be retained within the digestive tract of mice for at least 48 h following oral administration. SIGNIFICANCE AND IMPACT OF THE STUDY: MGV allows effective recovery of L26 from the background microbiota, including lactobacilli of mice. PCR was used to confirm that L26-like colonies were correctly identified as L26. Given the long retention time of L26 in the gastrointestinal tract of mice, it would appear that this probiotic strain may survive in the human gastrointestinal tract.     

Risks Associated with High-Dose Lactobacillus rhamnosus in an Escherichia coli Model of Piglet Diarrhoea: Intestinal Microbiota and Immune Imbalances

Probiotic could be a promising alternative to antibiotics for the prevention of enteric infections; however, further information on the dose effects is required. In this study, weanling piglets were orally administered low- or high-dose Lactobacillus rhamnosus ACTT 7469 (10(10) CFU/d or 10(12) CFU/d) for 1 week before F4 (K88)-positive Escherichia coli challenge. The compositions of faecal and gastrointestinal microbiota were recorded; gene expression in the intestines was assessed by real-time PCR; serum tumour necrosis factor-α (TNF-α) concentrations and intestinal Toll-like receptor 4 (TLR4) were detected by ELISA and immunohistochemistry, respectively. Unexpectedly, high-dose administration increased the incidence of diarrhoea before F4(+)ETEC challenge, despite the fact that both doses ameliorated F4(+)ETEC-induced diarrhoea with increased Lactobacillus and Bifidobacterium counts accompanied by reduced coliform shedding in faeces. Interestingly, L. rhamnosus administration reduced Lactobacillus and Bifidobacterium counts in the colonic contents, and the high-dose piglets also had lower Lactobacillius and Bacteroides counts in the ileal contents. An increase in the concentration of serum TNF-α induced by F4(+)ETEC was observed, but the increase was delayed by L. rhamnosus. In piglets exposed to F4(+)ETEC, jejunal TLR4 expression increased at the mRNA and protein levels, while jejunal interleukin (IL)-8 and ileal porcine β-defensins 2 (pBD2) mRNA expression increased; however, these increases were attenuated by administration of L. rhamnosus. Notably, expression of jejunal TLR2, ileal TLR9, Nod-like receptor NOD1 and TNF-α mRNA was upregulated in the low-dose piglets after F4(+)ETEC challenge, but not in the high-dose piglets. These findings indicate that pretreatment with a low dose of L. rhamnosus might be more effective than a high dose at ameliorating diarrhoea. There is a risk that high-dose L. rhamnosus pretreatment may negate the preventative effects, thus decreasing the prophylactic benefits against potential enteric pathogens. Our data suggest a safe threshold for preventative use of probiotics in clinical practice.     

The effect of zinc oxide supplementation on the stability of the intestinal flora with special reference to composition of coliforms in weaned pigs

The effect of a dietary supplementation of zinc oxide (ZnO) on the stability of the intestinal flora and on the composition of coliforms in weaned pigs was investigated. Faecal floras were characterized by their metabolic activities and fermentative capacity (FC) using the Phene Plate generalized microplate. Coliforms were characterized by conventional enumeration and by the Phene Plate-RS plates. The latter measured FC, phenotypic diversity, persistence of each coliform strain in piglets, and similarity among the coliform populations within groups. From weaning onwards, the control pigs (n = 5) were fed a basal diet ad libitum, while experimental pigs (n = 5) were given the same food supplemented with 2500 ppm ZnO. Metabolic fingerprinting of faecal floras indicated marked differences between the composition of floras of treated and control pigs during the first 2 weeks post-weaning. The FC of faecal flora in both groups decreased as pigs aged, but it was significantly (P 相似文献   

Comparison of two selective media for the detection and enumeration of Lactobacilli in human faeces

The enumeration of faecal bacteria is an important requirement for many studies of bowel health. One approach is the use of selective culture media for the culture and identification of genera or species from faeces. This study compares the culture of Lactobacilli from dilution series of faecal samples from six healthy human volunteers on two commonly used media, LAMVAB and Rogosa agar. Colonies were counted after a 72-h anaerobic incubation at 37 degrees C, and colony morphology recorded by a single observer. DNA was isolated from a representative number of colonies and genus-specific PCR, single-stranded conformation polymorphism (SSCP) and DNA sequencing performed. Total colony counts ranged from <3.00 to 7.48 log(10) cfu/g of faeces for LAMVAB and 5.09 to 7.66 log(10) cfu/g for Rogosa. For each subject, the total colony count was higher on Rogosa than that obtained with LAMVAB agar. SSCP analysis and DNA sequencing indicated that colony morphology was not an accurate predictor of genus identity. Growth of two species, Lactobacillus acidophilus and Lactobacillus gasseri, was not supported on LAMVAB medium. Rogosa agar was more likely to support growth of non-Lactobacillus species. Therefore, neither medium gave a fully accurate representation of the Lactobacilli species present in human faecal samples.     

Effects of the antibiotic growth promoters flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂)

The 16S rDNA PCR-DGGE and rpoB quantitative PCR (RQ-PCR) techniques were used to evaluate the effects of dietary flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia. The fish were fed four diets: control, dietary flavomycin, florfenicol and their combination. After 8 weeks of feeding, 6 fish from each cage were randomly chosen for the analysis. The total number of intestinal bacteria was determined by RQ-PCR. The results showed that dietary antibiotics significantly influenced the intestinal microbiota and dramatically reduced the intensity of total intestinal bacterial counts. The intensity of some phylotypes (EU563257, EU563262 and EU563255) were reduced to non-detectable levels by both dietary antibiotics, while supplementation of florfenicol to the diet also reduced the intensity of the phylotypes EU563242 and EU563262, uncultured Mycobacterium sp.-like, uncultured Cyanobacterium-like and uncultured Cyanobacterium (EU563246). Dietary flavomycin only reduced the OTU intensity of one phylotype, identified as a member of the phylum Fusobacteria. The antibiotic combination only reduced the phylotypes EU563242 and EU563262. Based on our results, we conclude that the reduced effect of florfenicol on intestinal microbiota was stronger than that of flavomycin, and when flavomycin and florfenicol were added in combination, the effect of florfenicol overshadowed that of flavomycin.     

Dietary supplementation with zinc oxide decreases expression of the stem cell factor in the small intestine of weanling pigs

Dietary supplementation with a high level of zinc oxide (ZnO) has been shown to reduce the incidence of diarrhea in weanling pigs, but the underlying mechanisms remain largely unknown. Intestinal–mucosal mast cells, whose maturation and proliferation is under the control of the stem cell factor (SCF), play an important role in the etiology of diarrhea by releasing histamine. The present study was conducted to test the novel hypothesis that supplementing ZnO to the diet for weanling piglets may inhibit SCF expression in the small intestine, thereby reducing the number of mast cells, histamine release, and diarrhea. In Experiment 1, 32 piglets (28 days of age) were weaned and fed diets containing 100 or 3000 mg zinc/kg (as ZnO) for 10 days (16 piglets per group). In Experiment 2, two groups of 28-day-old piglets (8 piglets per group) were fed the 100- or 3000-mg zinc/kg diet as in Experiment 1, except that they were pair-fed the same amounts of feed. Supplementation with a high level of ZnO reduced the incidence of diarrhea in weanling piglets. Dietary Zn supplementation reduced expression of the SCF gene at both mRNA and protein levels, the number of mast cells in the mucosa and submucosa of the small intestine and histamine release from mucosal mast cells. Collectively, our results indicate that dietary supplementation with ZnO inhibits SCF expression in the small intestine, leading to reductions in the number of mast cells and histamine release. These findings may have important implications for the prevention of weaning-associated diarrhea in piglets.     

Microbiological quality assessment of Moroccan camel's milk and identification of predominating lactic acid bacteria

Samples of camel's milk collected from different zones of Morocco were analysed to evaluate their microbiological quality and to identify predominating lactic acid bacteria (LAB). The following average colony-forming units (c.f.u.s) of aerobic total count, enterococci, faecal and total coliforms, LAB, yeasts,Staphylococcus aureus and spores of sulphite-reducing clostridia were recorded: 6.2 × 10⁷, 2.9 × 10⁴, 1.6 × 10⁴, 7.0 × 10⁶, 1.0 × 10⁷, 3.8 × 10⁴, 1.3 × 10⁵ and 6.0 c.f.u./ml, respectively. The enumeration results were markedly variable and coliforms were not detected in 1 ml of some samples. Bacteriological identification revealed a definite dominance of enterococci with Enterococcus faecalis as the main representative species. Besides Enterococcus, other genera including Pediococcus (28.2%), Streptococcus (4%), Lactococcus (8%) and Leuconostoc(1%) were isolated on de Man, Rogosa and Sharp (MRS) agar.     

Selected culturable enteric bacterial populations are modified by diet acidification and the growth promotant Tylosin

AIMS: To determine the effect of diet acidification and an in-feed antibiotic growth promotant (Tylosin, Ty) on selected culturable bacterial populations in the gastrointestinal tract (GIT) of mice. METHODS AND RESULTS: Female C57Bl mice were given a standard diet supplemented with Acid Pak (AP) or Ty in the drinking water. After 21 days, lumen and adherent populations of Enterobacteriaceae, enterococci/streptococci, and lactic acid bacteria (LAB) from the ileum, caecum, colon and faeces were enumerated. General intestinal health was assessed by the frequency of haemolytic bacteria in the different intestinal compartments. Contrary to expectations, AP and Ty significantly increased haemolytic bacteria in the lumen of the caecum and colon (P<0.05). The small but significant growth-enhancing effect of Ty (P<0.05) was associated with decreases in enterococci/streptococci and surprisingly, LAB, as well as increases in coliforms. AP, which failed to improve growth rates, reduced coliforms, had limited effects on enterococci/streptococci, and specifically failed to promote the growth of LAB populations in all intestinal compartments. Ty supplementation was also associated with a significant increase in macrolide-resistant enterococci throughout the GIT. CONCLUSIONS: Dietary acidification is less effective than Ty in modulating the population dynamics of selected culturable populations of enteric bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The mouse can provide a useful experimental model to examine the effects of new dietary supplements, formulations or regimes on changes in microbial population dynamics, including monitoring for antibiotic resistance.     

Effects of alternative feed additives to medicinal zinc oxide on productivity,diarrhoea incidence and gut development in weaned piglets

The use of medicinal zinc oxide (ZnO) must be phased out by 2022, thus prompting an urgent need for alternative strategies to prevent diarrhoea in weaner piglets. The objectives of this study were to assess the impact on weaner piglet performance, diarrhoea incidence and gut development, when (1) dietary ZnO supplementation was substituted by alternative commercial products based on macroalgae, specific probiotics or synbiotics, or (2) dietary ZnO inclusion was reduced from 2500 to 1500 ppm. A total of 4680 DLY piglets (DanBred, Herlev, Denmark), weaned around 35 days of age, were randomly assigned according to sex and BW to six different dietary treatment groups. A basal diet was supplemented with no ZnO (NC= negative control), 2500 ppm ZnO (PC= positive control), 1500 ppm ZnO (RDZ= reduced dose of ZnO) or commercial macroalgae (OceanFeed™ Swine =OFS), probiotic Miya-Gold or synbiotic GærPlus products. The piglets entered and exited the weaner unit at ~7.0 and 30 kg BW, respectively. In-feed ZnO was provided the first 10 days post-weaning, while the alternative supplements were fed throughout the weaner period. As expected, the average daily feed intake, average daily weight gain (ADG), feed conversion ratio (FCR) and diarrhoea incidence were improved in the PC compared to NC group (P< 0.05) during phase 1 consistent with improved indices of villi development observed in subgroups of piglets sacrificed 11 days post-weaning. Reduction of ZnO to 1500 ppm lowered ADG (P< 0.05) and slightly increased incidence of diarrhoea during the first 10 days after weaning (but not later) without affecting FCR. None of the three alternative dietary additives, including a 10-fold increased dose of GærPlus than recommended, improved piglet performance, gut health and gut development above that of NC piglets. The OFS piglets sacrificed 11 days after weaning had significantly lower weights of hindgut tissue and contents compared to the PC group, consistent with antimicrobial activity of the product, which was detected from anaerobicin vitrofermentation. In conclusion, dietary ZnO supplementation during the first 10 days post-weaning may be reduced from 2500 to 1500 ppm without major negative implications for weaner piglet performance and health in herds under a high management level. However, none of the alternative dietary supplements were able to improve piglet performance or gut health, when ZnO was omitted from the diet.     

Fluoride Exposure Aggravates the Testicular Damage and Sperm Quality in Diabetic Mice: Protective Role of Ginseng and Banaba

The current study was conducted to investigate the effects of dietary zinc oxide (ZnO) on the antioxidant capacity, small intestine development, and jejunal gene expression in weaned piglets. Ninety-six 21-day-old piglets were randomly assigned to three dietary treatments. Each treatment had eight replicates with four piglets per replicate. The piglets were fed either control diet (control) or control diet supplemented with in-feed antibiotics (300 mg/kg chlortetracycline and 60 mg/kg colistin sulfate) or pharmacological doses of ZnO (3000 mg/kg). The experiment lasted 4 weeks. Blood samples were collected at days 14 and 28, while intestinal samples were harvested at day 28 of the experiment. Dietary high doses of ZnO supplementation significantly increased the body weight (BW) at day 14 and average daily gain (ADG) of days 1 to 14 in weaned piglets, when compared to control group (P < 0.05). The incidence of diarrhea of piglets fed ZnO-supplemented diets, at either days 1 to 14, days 14 to 28, or the overall experimental period, was significantly decreased in comparison with those in other groups (P < 0.05). Supplementation with ZnO increased the villus height of the duodenum and ileum in weaned piglets and decreased the crypt depth of the duodenum, when compared to the other groups (P < 0.05). Dietary ZnO supplementation decreased the malondialdehyde (MDA) concentration at either day 14 or day 28, but increased total superoxide dismutase (T-SOD) at day 14, when compared to that in the control (P < 0.05). ZnO supplementation upregulated the messenger RNA (mRNA) expression of zonula occludens-1 (ZO-1) and occludin in the jejunum mucosa of weaned piglets, compared to those in the control (P < 0.05). The pro-inflammatory cytokine interleukin-lβ (IL-1β) mRNA expression in the jejunum mucosa was downregulated in the ZnO-supplemented group, compared with the control (P < 0.05). Both in-feed antibiotics and ZnO supplementation decreased the mRNA expression of interferon-γ (IFN-γ), but increased the mRNA expression of transforming growth factor-β (TGF-β), in the jejunum mucosa of piglets, when compared to those in the control (P < 0.05). In summary, supplemental ZnO was effective on the prevention of post-weaning diarrhea (PWD) in weaned piglets and showed comparative growth-promoting effect on in-feed antibiotics, probably by the mechanism of improvement of the antioxidant capacity, restoration of intestinal barrier function and development, and modulation of immune functions.     

Development of a differential medium for bile salt hydrolase-active Lactobacillus spp.

An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments.     

应用rep-PCR分型技术筛选潜在治疗性乳杆菌

分离鉴定阴道弯曲乳酸杆菌并对其进行基因分型分析和产H2O2能力测定,初步筛选具有潜在防治女性生殖道感染的弯曲乳酸杆菌菌株。将健康妇女阴道分泌物接种到de Man,Rogosa and Sharpe (MRS) 培养基,分离培养乳酸杆菌。通过16S rRNA序列进行乳酸杆菌分类鉴定,重复序列片段PCR扩增方法进行弯曲乳酸杆菌的基因分型,并进一步采用pH直接测酸法和辣根过氧化物酶催化四甲基联苯胺与过氧化氢反应显色法检测了10株弯曲乳酸杆菌产酸和产H2O2能力。经过序列比对鉴定,共得到65株乳酸杆菌。其中,弯曲乳杆菌Lactobacillus crispatus 19株,詹氏乳杆菌Lactobacillus jensenii 17株,发酵乳杆菌Lactobacillus fermentum 12株;rep-PCR分型发现不同种类的乳酸杆菌和同一种弯曲乳酸杆菌均表现为不同的带型指纹图;10株弯曲乳酸杆菌均产酸,其中T22-3和T29-5两株弯曲乳酸杆菌产H2O2量最高。结果表明个体阴道内乳酸杆菌分布具有差异,弯曲乳酸杆菌具有种内多样性,产H2O2丰富的T22-3和T29-5两株弯曲乳酸杆菌有可能作为防治女性生殖道感染的有益菌株。     

Distribution of indicator bacteria and Vibrio parahaemolyticus in sewage-polluted intertidal sediments.

The impact of a sewage point source on the bacterial densities in an intertidal mud flat in Boston Harbor, Mass., was investigated. The area, Savin Hill Cove, acts as a receiving basin for a combined storm and sewage outlet (CSO). Preliminary examination of sediments and overlying water at high tide demonstrated that fecal coliforms were present in sediments at abundances 2 to 4 orders of magnitude higher than in the overlying water column. The following bacterial counts were determined from sediments along a sampling transect extending 460 m from the CSO: total bacteria by epifluorescent microscopy, heterotrophic bacteria by plate counts on nutrient-rich and nutrient-poor media, fecal coliforms and enterococci by membrane filtration, and Vibrio parahaemolyticus by a most-probable-number technique with a resuscitation step. Median sediment grain size, average tidal exposure, carbon/nitrogen ratio, and total organic carbon were also measured. All bacterial indices, except for V. parahaemolyticus, declined significantly with distance from the outfall. Multiple regression analysis indicated that tidal exposure (low tides) may affect densities of total bacteria. Fecal coliforms and enterococci were still present in appreciable numbers in sediments as far as 460 m away from the CSO. In contrast, V. parahaemolyticus densities did not correlate with the other bacterial counts nor with any of the environmental parameters examined. These results indicate that intertidal sediments which adjoin point sources of pollution are severely contaminated and should be considered as potentially hazardous reservoirs of sewage-borne diseases.     

Development of a differential medium for bile salt hydrolase-active Lactobacillus spp

An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments.     

Distribution of indicator bacteria and Vibrio parahaemolyticus in sewage-polluted intertidal sediments

The impact of a sewage point source on the bacterial densities in an intertidal mud flat in Boston Harbor, Mass., was investigated. The area, Savin Hill Cove, acts as a receiving basin for a combined storm and sewage outlet (CSO). Preliminary examination of sediments and overlying water at high tide demonstrated that fecal coliforms were present in sediments at abundances 2 to 4 orders of magnitude higher than in the overlying water column. The following bacterial counts were determined from sediments along a sampling transect extending 460 m from the CSO: total bacteria by epifluorescent microscopy, heterotrophic bacteria by plate counts on nutrient-rich and nutrient-poor media, fecal coliforms and enterococci by membrane filtration, and Vibrio parahaemolyticus by a most-probable-number technique with a resuscitation step. Median sediment grain size, average tidal exposure, carbon/nitrogen ratio, and total organic carbon were also measured. All bacterial indices, except for V. parahaemolyticus, declined significantly with distance from the outfall. Multiple regression analysis indicated that tidal exposure (low tides) may affect densities of total bacteria. Fecal coliforms and enterococci were still present in appreciable numbers in sediments as far as 460 m away from the CSO. In contrast, V. parahaemolyticus densities did not correlate with the other bacterial counts nor with any of the environmental parameters examined. These results indicate that intertidal sediments which adjoin point sources of pollution are severely contaminated and should be considered as potentially hazardous reservoirs of sewage-borne diseases.     

Selection of fecal enterococci exhibiting tcrB-mediated copper resistance in pigs fed diets supplemented with copper

Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene, has been reported in Enterococcus faecium and E. faecalis. A longitudinal field study was undertaken to determine the relationship between copper supplementation and the prevalence of tcrB-positive enterococci in piglets. The study was done with weaned piglets, housed in 10 pens with 6 piglets per pen, fed diets supplemented with a normal (16.5 ppm; control) or an elevated (125 ppm) level of copper. Fecal samples were randomly collected from three piglets per pen on days 0, 14, 28, and 42 and plated on M-Enterococcus agar, and three enterococcal isolates were obtained from each sample. The overall prevalence of tcrB-positive enterococci was 21.1% (38/180) in piglets fed elevated copper and 2.8% (5/180) in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MICs of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. The restriction digestion of the genomic DNA of E. faecium or E. faecalis with S1 nuclease yielded a band of ～194-kbp size to which both tcrB and the erm(B) gene probes hybridized. A conjugation assay demonstrated cotransfer of tcrB and erm(B) genes between E. faecium and E. faecalis strains. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to that in piglets fed normal copper suggests that supplementation of copper in swine diets selected for resistance.     

Enumeration and identification of pediococci in powder-based products using selective media and rapid PFGE

In this study, pediococci selective medium (PSM) was evaluated for the enumeration of Pediococcus acidilactici and Pediococcus pentosaceus from probiotic animal feed and silage inoculants. PSM is based on the complex basal medium MRS supplemented with cysteine hydrochloride, novobiocin, vancomycin, and nystatin. No significant change in electivity was observed when pediococci where recovered from culture or powder-based products following incubation at 37 degrees C under anaerobic conditions for 24 h. The medium was suitable for the enumeration of pediococci in samples also containing bacilli, bifidobacteria, enterococci, lactobacilli, lactococci, propionibacteria, streptococci, and yeast components. However, to inhibit Lactobacillus plantarum and Lactobacillus casei, ampicillin was added and the revised medium, termed PSM+A, was also considered to be suitably elective for pediococci recovered from powder. In addition, a rapid PFGE protocol is presented, which allows Pediococcus species and strain verification from colonies in less than 3 days.     

Identification and characterization of potential performance-related gut microbiotas in broiler chickens across various feeding trials

Three broiler feeding trials were investigated in order to identify gut bacteria consistently linked with improvements in bird performance as measured by feed efficiency. Trials were done in various geographic locations and varied in diet composition, broiler breed, and bird age. Gut microbial communities were investigated using microbial profiling. Eight common performance-linked operational taxonomic units (OTUs) were identified within both the ilea (180, 492, and 564-566) and ceca (140-142, 218-220, 284-286, 312, and 482) across trials. OTU 564-566 was associated with lower performance, while OTUs 140-142, 482, and 492 were associated with improved performance. Targeted cloning and sequencing of these eight OTUs revealed that they represented 26 bacterial species or phylotypes which clustered phylogenetically into seven groups related to Lactobacillus spp., Ruminococcaceae, Clostridiales, Gammaproteobacteria, Bacteroidales, Clostridiales/Lachnospiraceae, and unclassified bacteria/clostridia. Where bacteria were identifiable to the phylum level, they belonged predominantly to the Firmicutes, with Bacteroidetes and Proteobacteria also identified. Some of the potential performance-related phylotypes showed high sequence identity with classified bacteria (Lactobacillus salivarius, Lactobacillus aviarius, Lactobacillus crispatus, Faecalibacterium prausnitzii, Escherichia coli, Gallibacterium anatis, Clostridium lactatifermentans, Ruminococcus torques, Bacteroides vulgatus, and Alistipes finegoldii). The 16S rRNA gene sequence information generated will allow quantitative assays to be developed which will enable elucidations of which of these phylotypes are truly performance related. This information could be used to monitor strategies to improve feed efficiency and feed formulation for optimal gut health.     

Diversity and dynamics of lactobacilli populations during ripening of RDO Camembert cheese

The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.