72 research outputs found
Optimization of Production Parameters for Probiotic Lactobacillus Strains as Feed Additive
In animal nutrition, probiotics are considered as desirable alternatives to antibiotic growth promoters. The beneficial effects of probiotics primarily depend on their viability in feed, which demands technical optimization of biomass production, since processing and storage capacities are often strain-specific. In this study, we optimized the production parameters for two broiler-derived probiotic lactobacilli (L. salivarius and L. agilis). Carbohydrate utilization of both strains was determined and preferred substrates that boosted biomass production in lab-scale fermentations were selected. The strains showed good aerobic tolerance, which resulted in easier scale-up production. For the freeze-drying process, the response surface methodology was applied to optimize the composition of cryoprotective media. A quadratic polynomial model was built to study three protective factors (skim milk, sucrose, and trehalose) and to predict the optimal working conditions for maximum viability. The optimal combination of protectants was 0.14g/mL skim milk/ 0.08 g/mL sucrose/ 0.09 g/mL trehalose (L. salivarius) and 0.15g/mL skim milk/ 0.08 g/mL sucrose/ 0.07 g/mL (L. agilis), respectively. Furthermore, the in-feed stabilities of the probiotic strains were evaluated under different conditions. Our results indicate that the chosen protectants exerted an extensive protection on strains during the storage. Although only storage of the strains at 4 °C retained the maximum stability of both Lactobacillus strains, the employed protectant matrix showed promising results at room temperature
Dietary inulin affects the intestinal microbiota in sows and their suckling piglets
Background: Several studies have focused on the effects of dietary inulin on
the intestinal microbiota of weaned piglets. In the present study, inulin was
added to a diet for gestating and lactating sows, expecting not only effects
on the faecal microbiota of sows, but also on the bacterial cell numbers in
the gastrointestinal tract of their piglets during the suckling period. Sows
were fed a diet without (n = 11) or with (n = 10) 3% inulin, and selected
bacterial groups were determined in their faeces ante and post partum.
Suckling piglets, 8 per group, were euthanised on day 10 after birth to
analyse digesta samples of the gastrointestinal tract. Results: Dietary inulin
increased the cell numbers of enterococci, both, in the faeces of the sows
during gestation and lactation, and in the caecum of the piglets (P ≤ 0.05).
Moreover, higher cell numbers of eubacteria (stomach) and C. leptum (caecum),
but lower cell numbers of enterobacteria and L. amylovorus (stomach) were
detected in the digesta of the piglets in the inulin group (P ≤ 0.05).
Conclusions: In conclusion, inulin seems to have the potential to influence
the gastrointestinal microbiota of suckling piglets through the diet of their
mother, showing the importance of the mother-piglet couple for the microbial
development. Early modulation of the intestinal microbiota could be especially
interesting with regard to the critical weaning time
Influence of high- and low-fermentable dietary fibres in sows' diet on the colostrum potential against Clostridioides difficile toxin-induced effects in IPEC-J2 cells
Sow colostrum has been reported to protect the IPEC-J2 cells and piglet colon tissues from detrimental effect of Clostridioides difficile toxins. Since dietary fibre can influence the colostrum composition in sows, we hypothesised that it can also differentially affect the colostrum potential against C. difficile toxin-induced effects in IPEC-J2. IPEC-J2 were incubated with colostrum from sows fed either high-fermentable sugar beet pulp (SBP) or low-fermentable lignocellulose (LNC) fibres and in combination with the toxins and analysed by trans-epithelial electrical resistance (TEER) and cell viability using propidium iodide in flow cytometry. Toxins drastically decreased the integrity of IPEC-J2. Colostrum from the sows fed either SBP or LNC exerted protective effect against toxins on IPEC-J2 integrity and this effect was numerically superior in the SBP group. Differences in the percentages of TEER between different treatments were noted after 2 h (p = 0.043), 3 h (p = 0.017) and 4 h (p = 0.017) of incubation and a tendency for differences was noted after 5 h of incubation (p = 0.071). Colostrum from either SBP- or LNC-fed sows did not protect the IPEC-J2 from toxin-induced death. Colostrum of the sows fed either high-fermentable or low-fermentable fibres has a potential to protect IPEC-J2 from the loss of integrity, which may be important in protection from C. difficile-infection development in neonatal piglets
In vitro conjugation kinetics of AmpC, broad spectrum and extended-spectrum beta-lactamase-producing Escherichia coli donors and various Enterobacteriaceae recipients
Background: Extended spectrum beta-lactamase (ESBL)-producing enterobacteria pose a major hazard to public health. Due to the possibility of genetic transfer, ESBL genes might spread to pathogenic enterobacterial strains. Thus, information on possible genetic transfer between enterobacteria is of high interest. It was therefore the aim of this in vitro study to screen the capacity of a wide range of Enterobacteriaceae for differences in conjugation at different time points with five ESBL-producing Escherichia coli strains.
Results: Conjugation frequencies for five potential E. coli donor strains producing the enzymes CTX-M-1, CTX-M-15, SHV-12, TEM-1, TEM-52 and CMY-2, and six potential recipient strains commonly detected in the gastrointestinal tract of poultry (E. coli, Serratia marcescens subsp. marcescens, Enterobacter cloacae, Salmonella (S.) enterica serovar Typhimurium and Proteus mirabilis) were obtained. Different combinations of donor and recipient strains were co-incubated for between 0 and 22 h and spread on selective agar. Conjugation frequencies were calculated as transconjugants per donor. Some donor and recipient strain combinations did not perform plasmid transfer within 22 h. Hence, the recipient Proteus mirabilis did not accept plasmids from any of the given donors and the E. coli ESBL10716 donor was unable to transfer its plasmid to any recipient. Enterobacter cloacae only accepted the plasmids from the donors E. coli ESBL10708 and E. coli ESBL10716 while E. coli ESBL10708 did not transfer its plasmid to Serratia marcescens subsp. marcescens. E. coli IMT11716 on the other hand did not perform conjugation with the donor E. coli ESBL10689. The remaining mating pairs differed in conjugation frequency, ranging from 10− 5 to 10− 9 transconjugants/donor. The earliest conjugation events were detected after 4 h. However, some mating pairs turned positive only after 22 h of coincubation.
Conclusion: A suitable mating pair for future in vivo studies to combat transfer of antibiotic resistance to pathogenic bacteria in broiler chicken was determined. The results of this study also suggest that the kinetic of conjugation differs between mating pairs and is independent of species origin. This should be considered when performing conjugation experiments
The Impact of Dietary Zinc Oxide on the Bacterial Diversity of the Small Intestinal Microbiota of Weaned Piglets
Dietary zinc oxide is often used in pharmacological concentrations to promote
health as well as performance of weaned piglets due to its bacteriostatic
effects. This study was conducted to provide an in depth analysis of the
bacterial composition in weaned piglets fed different amounts of dietary zinc
oxide. Piglets were fed diets containing 57 (low), 164 (medium) or 2425 (high)
mg/kg dietary zinc. Zinc above the basal dietary level was supplied from
analytical grade zinc oxide (ZnO). DNA was extracted from stomach and ileum
digesta samples of 32 and 53d old animals (n=4 per group) and used to generate
bar-coded 16S ribosomal DNA amplicons for deep sequencing analysis. A total of
9 phyla, 40 orders, 75 families and 328 genera were detected in 8.76 x 105
sequencing reads. Firmicutes, Bacteroidetes and Proteobacteria were the
dominant phyla, but no significant differences between treatment groups were
observed. Lactobacillales (16.3-59.9%), Bacteroidales (2.2-59.1%),
Clostridiales (0.05-70.2%) and Selenomonadales (2.6-17.5%) were found as the
dominating order. Noteworthy changes on the order level were found for
numerically or significantly increased ratios of Clostridiales, but
significantly decreased Lactobacillales in the high dietary zinc group. The
bacterial diversity for the high dietary zinc diet was significantly higher
for the total microbiota than the medium or low zinc diet. However,
Lactobacillales diversity decreased, while Clostridiales and Enterobacteriales
diversity increased significantly. Principal component analysis confirmed
changes in the microbiota, most notably for the high dietary zinc treatment.
This study has shown that pharmacological doses of high dietary zinc can
drastically alter the bacterial composition and development of the microbiota
in weaned piglets. The quantitative shift of bacterial groups due to high
dietary zinc was most pronounced one week after weaning, while the more
developed microbiota in older animals seemed to be able to adapt to high
concentrations of dietary zinc
Synergistic Effects of Probiotics and Phytobiotics on the Intestinal Microbiota in Young Broiler Chicken
Probiotics and phytobiotics have been studied as in-feed antibiotic alternatives for decades, yet there are no studies on their possible symbiotic effects. In the present study, newly hatched chickens were fed with feeds supplemented either with host-specific Lactobacillus strains (L. agilis and L. salivarius), commercial phytobiotics, or combinations of both. After 13 days of life, crops and caecums were analyzed for bacterial composition (16S rDNA sequencing, qPCR) and activity (bacterial metabolites). Crop and caecum samples were also used to study the ex vivo survival of a broiler-derived extended-spectrum beta-lactamase (ESBL) producing Escherichia coli strain. In the crop, combinations of probiotics and phytobiotics, but not their single application, increased the dominance of lactobacilli. The single application of phytobiotics reduced the metabolite concentrations in the crop, but certain combinations synergistically upregulated the metabolites. Changes in the qualitative and quantitative composition of the caecal microbiota were less pronounced than in the crop. Acetate concentrations were significantly lower for phytobiotics or the L. agilis probiotic strain compared to the control group, but the L. salivarius probiotic showed significantly higher acetate concentrations alone or in combination with one phytobiotic. The synergistic effects on the reduction of the ex vivo survival of an ESBL producing E. coli strain in crop or caecum contents were also observed for most combinations. This study shows the beneficial synergistic effects of probiotics and phytobiotics on the intestinal bacterial composition and their metabolic activity in young broilers. The reduced survival of potentially problematic bacteria, such as ESBL-producing E. coli further indicates that combinations of probiotics and phytobiotics may lead to a more enhanced functionality than their individual supplementation
Nutrition Related Stress Factors Reduce the Transfer of Extended-Spectrum Beta-Lactamase Resistance Genes between an Escherichia coli Donor and a Salmonella Typhimurium Recipient In Vitro
The transfer of extended spectrum β-lactamase (ESBL)-genes occurs frequently between different bacteria species. The aim of this study was to investigate the impact of nutrition related stress factors on this transfer. Thus, an Escherichia coli donor and a Salmonella Typhimurium recipient were co-incubated for 4 h in media containing different levels of the stress factors’ pH, osmolality, copper, zinc and acetic, propionic, lactic, and n-butyric acid, as well as subtherapeutic levels of cefotaxime, sulfamethoxazole/trimethoprim, and nitrofurantoin. Conjugation frequencies were calculated as transconjugants per donor, recipient, and total bacterial count. A correction factor for the stress impact on bacterial growth was used. Acetic, lactic, and n-butyric, acid, as well as pH, showed no significant impact. In contrast, increasing concentrations of propionate, zinc, copper, and nitrofurantoin, as well as increased osmolality reduced conjugation frequencies. Sulfamethoxazole/trimethoprim and cefotaxime showed increased transconjugants per donor, which decreased after correction for stress. This study showed, for the model mating pair, that conjugation frequencies decreased under different physiological stress conditions, and, thus, the hypothesis that stress factors may enhance conjugation should be viewed with caution. Furthermore, for studies on in vitro gene transfer, it is vital to consider the impact of studied stressors on bacterial growth
Timely approaches to identify probiotic species of the genus Lactobacillus
Over the past decades the use of probiotics in food has increased largely due
to the manufacturer's interest in placing "healthy" food on the market based
on the consumer's ambitions to live healthy. Due to this trend, health
benefits of products containing probiotic strains such as lactobacilli are
promoted and probiotic strains have been established in many different
products with their numbers increasing steadily. Probiotics are used as
starter cultures in dairy products such as cheese or yoghurts and in addition
they are also utilized in non-dairy products such as fermented vegetables,
fermented meat and pharmaceuticals, thereby, covering a large variety of
products.To assure quality management, several pheno-, physico- and genotyping
methods have been established to unambiguously identify probiotic
lactobacilli. These methods are often specific enough to identify the
probiotic strains at genus and species levels. However, the probiotic ability
is often strain dependent and it is impossible to distinguish strains by basic
microbiological methods.Therefore, this review aims to critically summarize
and evaluate conventional identification methods for the genus Lactobacillus,
complemented by techniques that are currently being developed.1\. Auflag
Developing Gut Microbiota Exerts Colonisation Resistance to Clostridium (syn. Clostridioides) difficile in Piglets
Clostridium (syn. Clostridioides) difficile is considered a pioneer colonizer and may cause gut infection in neonatal piglets. The aim of this study was to explore the microbiota-C. difficile associations in pigs. We used the DNA from the faeces of four sows collected during the periparturient period and from two to three of their piglets (collected weekly until nine weeks of age) for the determination of bacterial community composition (sequencing) and C. difficile concentration (qPCR). Furthermore, C. difficile-negative faeces were enriched in a growth medium, followed by qPCR to verify the presence of this bacterium. Clostridium-sensu-stricto-1 and Lactobacillus spp. predominated the gut microbiota of the sows and their offspring. C. difficile was detected at least once in the faeces of all sows during the entire sampling period, albeit at low concentrations. Suckling piglets harboured C. difficile in high concentrations (up to log10 9.29 copy number/g faeces), which gradually decreased as the piglets aged. Enrichment revealed the presence of C. difficile in previously C. difficile-negative sow and offspring faeces. In suckling piglets, the C. difficile level was negatively correlated with carbohydrate-fermenting bacteria, and it was positively associated with potential pathogens. Shannon and richness diversity indices were negatively associated with the C. difficile counts in suckling piglets. This study showed that gut microbiota seems to set conditions for colonisation resistance against C. difficile in the offspring. However, this conclusion requires further research to include host-specific factors
High dietary zinc supplementation increases the occurrence of tetracycline and sulfonamide resistance genes in the intestine of weaned pigs
Background Dietary zinc oxide is used in pig nutrition to combat post weaning
diarrhoea. Recent data suggests that high doses (2.5 g/kg feed) increase the
bacterial antibiotic resistance development in weaned pigs. Therefore, the aim
of this study was to investigate the development of enterobacterial antibiotic
resistance genes in the intestinal tract of weaned pigs. Findings Weaned pigs
were fed diets for 4 weeks containing 57 (low), 164 (intermediate) or 2425
(high) mg kg−1 analytical grade ZnO. DNA extracts from stomach, mid-jejunum,
terminal ileum and colon ascendens were amplified by qPCR assays to quantify
copy numbers for the tetracycline (tetA) and sulfonamide (sul1) resistance
genes in Gram-negative bacteria. Overall, the combined data (n = 336) showed
that copy numbers for tetracycline and sulfonamide resistance genes were
significantly increased in the high zinc treatment compared to the low (tetA:
p value < 10−6; sul1: p value = 1 × 10−5) or intermediate (tetA: P < 1.6 ×
10−4; sul1: P = 3.2 × 10−4) zinc treatment. Regarding the time dependent
development, no treatment effects were seen 1 week after weaning, but
significant differences between high and low/intermediate zinc treatments
evolved 2 weeks after weaning. The increased number of tetA and sul1 copies
was not confined to the hind gut, but was already present in stomach contents.
Conclusions The results of this study suggest that the use of high doses of
dietary zinc beyond 2 weeks after weaning should be avoided in pigs due to the
possible increase of antibiotic resistance in Gram-negative bacteria
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