An effective antimicrobial strategy of colistin combined with the Chinese herbal medicine shikonin against colistin-resistant Escherichia coli.

IMPORTANCE: Infections caused by multidrug-resistant Escherichia coli (MDR E. coli) have become a major global healthcare problem due to the lack of effective antibiotics today. The emergence of colistin-resistant E. coli strains makes the situation even worse. Therefore, new antimicrobial strategies are urgently needed to combat colistin-resistant E. coli. Combining traditional antibiotics with non-antibacterial drugs has proved to be an effective approach of combating MDR bacteria. This study investigated the combination of colistin and shikonin, a Chinese herbal medicine, against colistin-resistant E. coli. This combination showed good synergistic antibacterial both in vivo and in vitro experiments. Under the background of daily increasing colistin resistance in E. coli, this research points to an effective antimicrobial strategy of using colistin and shikonin in combination against colistin-resistant E. coli.

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin.

BACKGROUND: The emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. METHODS: The synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. RESULTS: Gigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 µg/ml to 0.25 µg/ml), Salmonella 15E343 (MIC from 8 µg/ml to 1 µg/ml), K. pneumoniae 19-2-1 (MIC from 32 µg/ml to 2 µg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. CONCLUSION: Our results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

Cajanin stilbene acid: A direct inhibitor of colistin resistance protein MCR-1 that restores the efficacy of polymyxin B against resistant Gram-negative bacteria.

BACKGROUND: The resistance of Gram-negative bacteria to polymyxin B, caused by the plasmid-mediated colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase (MCR-1), is a serious threat to global public health. Therefore, it is urgent to find new drugs that can effectively alleviate polymyxin B resistance. Through the screening of 78 natural compounds, we found that cajanin stilbene acid (CSA) can significantly restore the susceptibility of polymyxin B to mcr-1 positive Escherichia coli (E. coli). PURPOSE: In this study, we tried to evaluate the ability of CSA to restore the susceptibility of polymyxin B towards the E. coli, and explore the mechanism of sensitivity recovery. STUDY DESIGN AND METHODS: Checkerboard MICs, time-killing curves, scanning electron microscope, lethal and semi-lethal models of infection in mice were used to assess the ability of CSA to restore the susceptibility of polymyxyn to E. coli. The interaction between CSA and MCR-1 was evaluated using surface plasmon resonance (SPR), and molecular docking experiments. RESULTS: Here, we find that CSA, a potential direct inhibitor of MCR-1, effectively restores the sensitivity of E. coli to polymyxin B. CSA can restore the sensitivity of polymyxin B to drug-resistant E. coli, and the MIC value can be reduced to 1 µg/ml. The time killing curve and scanning electron microscopy results also showed that CSA can effectively restore polymyxin B sensitivity. In vivo experiments showed that the simultaneous use of CSA and polymyxin B can effectively reduce the infection of drug-resistant E. coli in mice. SPR and molecular docking experiments confirmed that CSA strongly bound to MCR-1. The 17-carbonyl oxygen and 12- and 18­hydroxyl oxygens of CSA were the key sites binding to MCR-1. CONCLUSION: CSA is able to significantly restore the sensitivity of polymyxin B to E. coli in vivo and in vitro. CSA inhibits the enzymatic activity of the MCR-1 protein by binding to key amino acids at the active center of the MCR-1 protein.

Performance of an Escherichia coli phytase expressed in Lactococcus lactis on nutrient retention, bone traits and intestinal morphology in broiler chickens.

The availability of plant phosphorus in the gut chicken can be improved by increasing phosphorus retention using phytase enzyme or a probiotic with phytase activity as an alternative. In this study, the efficacy of a recombinant probiotic, Lactococcus lactis (L. lactis), with a potential of phytase production was evaluated in broiler chickens. To this aim, 360 one-day-old male broiler Cobb 500 were divided into six treatments with six replicates and reared to 42 days of age. The experimental treatments included positive control diet containing adequate phosphorus (PC), negative control diet containing reduced available phosphorus (NC), negative control diet involving recombinant L. lactis (RLL), negative control diet containing both recombinant L. lactis and Lactobacillus salivarius (RLL + LBS), negative control diet including non-recombinant L. lactis (LL) and negative control diet containing Hostazym® . Growth performance, total tract apparent disappearance of phytate-P and nutrient retention, mineral content of the tibia and histomorphology of jejunum were evaluated at the age of 35 days. Based on the results, the phosphorus (P) deficiency in the diet reduced body weight (BW), average daily gain (ADG), length and strength of tibia and increased feed conversion ratio (FCR) compared to PC group. However, the supplementation of Hostazym® or RLL probiotic into the feed improved BW, ADG, FCR, disappearance of Phytate-P and retention of P, length and strength of the tibia in a level similar to PC treatment. Phosphorus content of tibia in the chickens fed P-deficient diets containing RLL was similar to that of the tibia in the control group. Excreta phytate and total P excretion of the chickens decreased when diets contained Hostazym® , RLL and RLL + LBS. In addition, the diet containing RLL + LBS probiotic increased villi height compared with other treatments (p < .05). Further, recombinant L. lactis could represent phytase activity in the gut environment of the chickens and could be an alternative to the commercial phytase.

Effects of a novel corn-expressed E. coli phytase on digestibility of calcium and phosphorous, growth performance, and bone ash in young growing pigs1.

Two experiments were conducted to test the hypothesis that a corn-expressed phytase increases growth performance, bone measurements, and nutrient digestibility by young growing pigs, if added to diets that are deficient in Ca and P. In Exp. 1, 60 pigs (initial BW: 10.78 ± 0.67 kg) were randomly allotted to 6 dietary treatments that included a positive control diet (PC; 0.70% total Ca and 0.60% total P) and a negative control diet (NC; 0.50% total Ca and 0.42% total P). Four additional diets were formulated by supplementing the NC diet with 250, 500, 1,000, or 1,500 phytase units (FTU)/kg. Diets were fed for 28 d and the individual BW of pigs on days 1 and 28 were recorded. Fecal samples were collected from days 25 to 27 to calculate apparent total tract digestibility (ATTD) of Ca and P. On the last day of the experiment, all pigs were euthanized, and the left femur was removed and analyzed for ash, Ca, and P. Results indicated that growth performance, ATTD of Ca and P, and bone ash measurements were reduced (P < 0.05) in NC fed pigs compared with PC fed pigs. However, growth performance, ATTD of Ca and P, and bone ash measurements were improved (linear and quadratic, P < 0.05) by including increasing concentrations of phytase to the NC diet. In Exp. 2, experimental procedures were similar to those used in Exp. 1. Forty-eight pigs (initial BW: 11.15 ± 0.85 kg) were randomly allotted to 6 dietary treatments in a 28-d experiment. Treatments included a PC diet, an NC diet, and 4 diets in which 500 or 1,000 FTU/kg of either the corn-expressed phytase or a commercial microbial phytase were added to the NC diet. Pigs fed the NC diet had reduced (P < 0.01) final BW, ADG, G:F, and bone ash concentrations compared with pigs fed the PC diet. When 500 FTU/kg phytase was fed, no differences were observed in growth performance or bone ash measurements between phytase sources, and there were no differences in growth performance among pigs fed 1,000 FTU/kg of either phytase source or the PC diet. However, regardless of concentration or source of phytase, pigs fed the PC diet had greater (P < 0.001) amount of bone ash, bone Ca, and bone P compared with pigs fed phytase diets. In conclusion, the corn-expressed phytase is effective in improving growth performance, Ca and P digestibility, and bone measurements in pigs fed diets that are deficient in Ca and P.

Effects of increasing concentrations of an Escherichia coli phytase on the apparent ileal digestibility of amino acids and the apparent total tract digestibility of energy and nutrients in corn-soybean meal diets fed to growing pigs.

An experiment was conducted to test the hypothesis that inclusion of increasing concentrations of an Escherichia coli phytase to a corn-soybean meal (SBM) diet results in improved digestibility of DM, GE, CP, NDF, ADF, macrominerals, microminerals, and AA. Twenty-four growing barrows (initial BW: 37.0 ± 1.4 kg) were equipped with a T-cannula in the distal ileum and placed individually in metabolism crates, and allotted to a 2-period switch-back design with 6 diets and 4 replicate pigs per diet in each period. The positive control diet was a corn-SBM diet that contained limestone and dicalcium phosphate to meet the requirement for standardized total tract digestible (STTD) P and Ca (0.31% STTD P and 0.70% Ca). A negative control diet that was similar to the positive control diet, with the exception that no dicalcium phosphate was used, was also formulated, and this diet contained 0.16% STTD P and 0.43% Ca. Four additional diets were formulated by adding 500, 1,000, 2,000, or 4,000 units of microbial phytase (FTU) to the negative control diet. Each period lasted 14 d. Fecal and urine samples were collected from the feed provided from days 6 to 11 of each period following 5 d of adaptation to the diets. Ileal digesta were collected for 8 h on days 13 and 14. Results indicated that addition of the E. coli phytase to the negative control diet tended to quadratically improve the apparent ileal digestibility of Phe (P = 0.086) and Asp (P = 0.054), and linearly increased (P < 0.05) the apparent total tract digestibility (ATTD) of ADF, K, and Fe. Microbial phytase also quadratically increased (P < 0.05) the ATTD of NDF and Mg, and linearly and quadratically increased (P < 0.05) the ATTD and retention of Ca and P. However, no effects of the phytase on ATTD of GE or the concentration of DE were observed. In conclusion, the increased absorption of several minerals including Ca, P, K, Mg, and Fe that was observed as increasing concentrations of an E. coli phytase was added to a corn-SBM meal diet indicates that the dietary provision of these minerals may be reduced if phytase is fed.

EspA-loaded mesoporous silica nanoparticles can efficiently protect animal model against enterohaemorrhagic E. coli O157: H7.

In the present study, the application of mesoporous silica nanoparticles (MSNPs) loaded with recombinant EspA protein, an immunogen of enterohaemorrhagic E. coli, was investigated in the case of BALB/c mice immunization against the bacterium. MSNPs of 96.9 ± 15.9 nm in diameter were synthesized using template removing method. The immunization of mice was carried out orally and subcutaneously. Significant immune responses to the antigen were observed for the immunized mice when rEspA-loaded MSNPs were administered in both routes in comparison to that of the antigen formulated using a well-known adjuvant, i.e. Freund's. According to the titretitre of serum IL-4, the most potent humoral responses were observed when the mice were immunized subcutaneously with antigen-loaded MSNPs (244, 36 and 14 ng/dL of IL-4 in the serum of mice immunized subcutaneously or orally by antigen-loaded MSNPs, and subcutaneously by Freund's adjuvant formulated-antigen, respectively). However, the difference in serum IgG and serum IgA was not significant in mice subcutaneously immunized with antigen-loaded MSNPs and mice immunized with Freund's adjuvant formulated-antigen. Finally, the immunized mice were challenged orally by enterohaemorrhagic E. coli cells. The amount of bacterial shedding was significantly reduced in faecesfaeces of the animals immunized by antigen-loaded MSNPs in both subcutaneous and oral routes.

Factors derived from Escherichia coli Nissle 1917, grown in different growth media, enhance cell death in a model of 5-fluorouracil-induced Caco-2 intestinal epithelial cell damage.

We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LB+) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer.

Preserving catalytic activity and enhancing biochemical stability of the therapeutic enzyme asparaginase by biocompatible multilayered polyelectrolyte microcapsules.

The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the long-term storage stability of the encapsulated enzyme were investigated. We show that encapsulation of l-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37 °C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.

Vaccination of pregnant cows with EspA, EspB, γ-intimin, and Shiga toxin 2 proteins from Escherichia coli O157:H7 induces high levels of specific colostral antibodies that are transferred to newborn calves.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of intestinal disease and hemolytic uremic syndrome, a serious systemic complication that particularly affects children. Cattle are primary reservoirs for EHEC O157:H7 and the main source of infection for humans. Vaccination of cattle with different combinations of bacterial virulence factors has shown efficacy in decreasing EHEC O157:H7 shedding. It is, therefore, important to demonstrate whether vaccination of pregnant cows with EHEC O157:H7 induces high titers of transferable antibodies to avoid early colonization of calves by the bacteria. In this study we evaluated the ability of EspA, EspB, the C-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C280) and inactivated Shiga toxin (Stx) 2 proteins to induce specific antibodies in colostrum and their passive transference to colostrum-fed calves. Friesian pregnant cows immunized by the intramuscular route mounted significantly high serum and colostrum IgG responses against EspB and γ-intimin C280 that were efficiently transferred to their calves. Antibodies to EspB and γ-intimin C280 were detected in milk samples of vaccinated cows at d 40 postparturition. Significant Stx2-neutralizing titers were also observed in colostrum from Stx2-vaccinated cows and sera from colostrum-fed calves. The results presented showed that bovine colostrum with increased levels of antibodies against EHEC O157:H7 may be obtained by systemic immunization of pregnant cows, and that these specific antibodies are efficiently transferred to newborn calves by feeding colostrum. Hyperimmune colostrum and milk may be an alternative to protect calves from early colonization by EHEC O157:H7 and a possible key source of antibodies to block colonization and toxic activity of this bacterium.

Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis.

Nitrobenzodiazepine (NBDZ) is an addictive drug of the abused substances that causes severe neurological effects and even death. Bacterial type I nitroreductase NfsB (EC 1.5.1.34) has been reported to catalyze NBDZ into inactive metabolite 7-amino-benzodiazepine (7ABDZ) with promising activity, so as to become an attractive candidate for treatment of NBDZ overdose and addiction. Here, we investigate the nitroreduction of an NBDZ, flunitrazepam (FZ), by various mutants of NfsB designed from the solved crystal structure and characterize their in vitro and in vivo potency. Conformational changes occurred in the active site of N71S/F124W in contrast to the wild-type, including the flipping on the aromatic rings of W124 and F70 as well as the extension on the hydrogen bond network between flavin mononucleotide (FMN) and S71, which allow the significant enlargement in the active site pocket. In the complex structure of N71S/F124W and nicotinamide (NIA), stacking sandwich attractions of W124-FMN-NIA were also found, implying the importance of W124 in substrate accessibility. Consequently, N71S/F124W exhibited increased 7AFZ production in vitro with nearly no toxicity and reduced 50% sleeping time (hypnosis) in vivo. Taken together, we demonstrate for the first time that N71S/F124W can serve as an effective antidote for NBDZ-induced hypnosis and provide the molecular basis for designing NfsB and the like in the future.

The effects of supplementary bacterial phytase on dietary true metabolisable energy, nutrient digestibility and endogenous losses in precision fed turkeys.

1. A total of 40 female BUT9 turkeys were used in a precision-feeding assay to investigate the effect of dietary phytase on true metabolisable energy corrected for N retention (TME(N)), coefficients of true dry matter (TDMD), mineral, amino acid and nitrogen (TND) digestibilities and the excretion of endogenous mucin, measured as sialic acid (SA). 2. Five treatments were used in this study: control (C), C + 250 phytase units (FTU) per kg feed, C + 500 FTU, C + 2500 FTU, and glucose only for endogenous losses estimation. Diets were formulated to be nutritionally adequate with the exception that the P content was relatively low (3·6 g/kg non-phytate P). 3. Inclusion of phytase increased TND in a quadratic manner with the optimum being achieved at approximately 500 FTU, at which TND was 37 % greater than in the control. The concentration of SA in the excreta decreased linearly with increased phytase supplementation. Dietary TME(N), TDMD and true mineral digestibility coefficients were not significantly affected by phytase supplementation. 4. Phytase inclusion increased digestibility coefficients for indispensable, dispensable and total amino acids in a linear manner. The scale of the response to phytase was greatest with threonine and least with lysine digestibility, suggesting a specific mechanism of action that benefits gut health. 5. The strong negative relationship between secretion of SA and threonine digestibility suggests that a large part of the threonine benefit may be from reduced mucin synthesis. This supports the hypothesis that dietary phytase may play a role in improving the health status of the intestine and, as a result, reduces the maintenance energy requirements of turkeys.

The effects of supplementary bacterial phytase on dietary energy and total tract amino acid digestibility when fed to young chickens.

1. Four diets were offered to broiler chickens from 7 to 17 d of age; these included a phosphorus-adequate positive control (PC) (4·7 g/kg available P), a sub-optimal P negative control (NC, 2·5 g/kg available P) with (500 and 12500 FTU/kg) and without phytase. Dietary apparent metabolisable energy (AME), dietary net energy for production (NEp), the efficiency of AME retention (Kre), heat production and total tract amino acid digestibility coefficients were determined. The determination of NEp involved a comparative slaughter technique in which growing chickens were fed the experimental diets ad libitum. 2. Feed intake, weight gain and feed conversion efficiency increased significantly in a dose dependent manner in response to dietary phytase activity. Overall, the NEp of the phytase supplemented diets significantly improved by approximately 15·6% compared with the negative control, while dietary AME was unaffected. Although phytase did not affect AME, the large increase in the NEp demonstrated that dietary phytases improves energy utilisation, i.e. diverting more energy, not accounted for in the AME procedure, for production. This is largely a result of the stimulatory effect that phytase has on feed intake rather than on digestibility of the diet. 3. Overall, the diet supplemented with 12500 FTU had 6·4% significant improvement in total tract digestibility coefficients of the total amino acids compared with the negative control. With regard to individual amino acids, the impact of phytase was far more pronounced for threonine, an important component of the gastrointestinal mucin, than for other amino acids. 4. Dietary NEp was more highly correlated with performance criteria than dietary AME and seems to be a more sensitive way to evaluate broiler response to phytase supplementation.

Alkaline phosphatase-polyresorcinol complex: characterization and application to seed coating.

An alkaline phosphatase (EC 3.1.3.1) from Escherichia coli ATCC27257 was immobilized by copolymerization with resorcinol. The phosphatase-polyresorcinol complex synthesized retained about 74% of the original enzymatic activity. The pH and temperature profile of the immobilized and free enzyme revealed a similar behavior. Kinetic parameters were determined: K(m) and K(i) values were 2.44 and 0.423 mM, respectively, for the phosphatase-polyresorcinol complex and 1.07 and 0.069 mM, respectively, for free phosphatase. The thermal and storage stabilities of the immobilized phosphatase were higher than those of the native one. On addition to soil, free enzyme was completely inactivated in 4 days, whereas the phosphatase-polyresorcinol complex was comparatively stable. Barley seed coated with the immobilized enzyme exhibited higher rhizosphere phosphatase activity. Under pot culture conditions, an increase in the soil inorganic phosphorus was detected when the seed was encapsulated with the phosphatase-polyresorcinol complex, and a positive influence on biomass and inorganic phosphorus concentration of shoot was observed.

Distribution of supplemental Escherichia coli AppA2 phytase activity in digesta of various gastrointestinal segments of young pigs.

The objective of this study was to determine the functional location and disappearance of activity of a supplemental Escherichia coli AppA2 phytase and its impact on digesta P and Ca concentrations in the gastrointestinal tract of pigs. In Exp. 1, 18 pigs (8.3 +/- 0.2 kg of BW) were allotted to 3 groups (n = 6 each) and fed a low-P (0.4%) corn-soybean meal, basal diet (BD), BD + phytase [500 units (U)/kg of feed], or BD + inorganic P (iP, 0.1%) for 4 wk. In Exp. 2, 30 pigs (14.5 +/- 0.2 kg of BW) were allotted to 3 groups (n = 10 each) and fed BD, BD + 500 U of phytase/kg of feed, or BD + 2,000 U of phytase/kg of feed for 2 wk. Five or six pigs from each treatment group were killed at the end of both experiments to assay for digesta phytase activity and soluble P concentration in 6 segments of the digestive tract and digesta total P and Ca concentrations in stomach and colon. Compared with pigs fed BD, pigs fed BD + 500 U of phytase/kg of feed in Exp. 1 and BD + 2,000 U of phytase/kg of feed in Exp. 2 had greater (P < 0.05) phytase activities in the digesta of the stomach and upper jejunum (2 m aborally from the duodenum). No phytase activity was detected in the digesta of the lower jejunum (2.12 m cranial to the ileocecal junction) or ileum from any of the treatment groups in either trial. Concentrations of digesta-soluble P peaked in the upper jejunum of pigs fed BD in Exp. 1 and 2, but showed gradual decreases between the stomach and the upper jejunum of pigs fed BD + phytase or BD + iP. In both experiments, pigs fed only BD had greater (P < 0.05) colonic digesta phytase activity and soluble P concentrations than those fed phytase. In Exp. 2, total colonic digesta P or Ca concentrations, or both, of pigs displayed a phytase-dose-dependent reduction (P < 0.05). In conclusion, supplemental dietary AppA2 mainly functioned in the stomach and was associated with a reduced phytase activity in colonic digesta of weanling pigs.