Pathotype and antibiotic resistance gene distributions of Escherichia coli isolates from broiler chickens raised on antimicrobial-supplemented diets.

The impact of feed supplementation with bambermycin, monensin, narasin, virginiamycin, chlortetracycline, penicillin, salinomycin, and bacitracin on the distribution of Escherichia coli pathotypes in broiler chickens was investigated using an E. coli virulence DNA microarray. Among 256 E. coli isolates examined, 59 (23%) were classified as potentially extraintestinal pathogenic E. coli (ExPEC), while 197 (77%) were considered commensal. Except for chlortetracycline treatment, the pathotype distribution was not significantly different among treatments (P > 0.05). Within the 59 ExPEC isolates, 44 (75%) were determined to be potentially avian pathogenic E. coli (APEC), with the remaining 15 (25%) considered potentially "other" ExPEC isolates. The distribution within phylogenetic groups showed that 52 (88%) of the ExPEC isolates belonged to groups B2 and D, with the majority of APEC isolates classified as group D and most commensal isolates (170, 86%) as group A or B1. Indirect assessment of the presence of the virulence plasmid pAPEC-O2-ColV showed a strong association of the plasmid with APEC isolates. Among the 256 isolates, 224 (88%) possessed at least one antimicrobial resistance gene, with nearly half (107, 42%) showing multiple resistance genes. The majority of resistance genes were distributed among commensal isolates. Considering that the simultaneous detection of antimicrobial resistance tet(A), sulI, and bla(TEM) genes and the integron class I indicated a potential presence of the resistance pAPEC-O2-R plasmid, the results revealed that 35 (14%) of the isolates, all commensals, possessed this multigene resistance plasmid. The virulence plasmid was never found in combination with the antimicrobial resistance plasmid. The presence of the ColV plasmid or the combination of iss and tsh genes in the majority of APEC isolates supports the notion that when found together, the plasmid, iss, and tsh serve as good markers for APEC. These data indicate that different resistant E. coli pathotypes can be found in broiler chickens and that the distribution of such pathotypes and certain virulence determinants could be modulated by antimicrobial agent feed supplementation.

Antibiotic resistance and virulence genes in commensal Escherichia coli and Salmonella isolates from commercial broiler chicken farms.

Antibiotic resistance patterns and the presence of antibiotic and virulence determinants in 74 sorbitol-negative Escherichia coli and 62 Salmonella isolates from nine different broiler chicken farms were investigated. Each farm was supplied by one of three companies that used different antimicrobial agents in feed for growth promotion. The isolates were identified by API 20E for E. coli and by serological tests for Salmonella. The susceptibility of the isolates to antibiotics was determined by Sensititre using the Clinical and Laboratory Standards Institute's breakpoints. Fifty-two E. coli isolates (70.3%) and nine Salmonella isolates (14.52%) were multiresistant to at least nine antibiotics. The multiresistant isolates were evaluated for the presence of tetracycline resistance, integron class 1, and blacMY 2 genes by PCR. Of the 74 E. coli isolates, 55 were resistant to amoxicillin and ceftiofur. Among these 55 resistant E. coli isolates, 45 (81.8%) and 22 (40.0%) were positive for blacMY-2 and qacEdeltal-Sull genes, respectively. Tetracycline resistance was found in 56 isolates (75.8%) among which 12 (21.4%) and 24 (42.9%) gave positive results for tetA and tetB, respectively. Virulence genes (iss, tsh, and traT), aerobactin operon (iucC), and the eaeA gene were detected in some E. coli strains. Among the 27 amoxicillin- and ceftiofur-resistant Salmonella isolates, the blacMY-2 gene was detected in 22 isolates. The class 1 integron gene (qacEdeltal-Sull) was not detected in any Salmonella isolates, whereas the invasin (inv) and virulence (spy) genes were found in 61 (98.4%) and 26 (42%) of the Salmonella isolates, respectively. This study indicated that multiple antibiotic-resistant commensal E. coli and Salmonella strains carrying virulence genes can be found on commercial broiler chicken farms and may provide a reservoir for these genes in chicken production facilities. Except for the presence of tetB, there was no significant effect of feed formulations on the phenotypic or genotypic characteristics of the isolates.

Improved bioavailability of vitamin D3 using a ß-lactoglobulin-based coagulum.

Vitamin D3 (D3) was encapsulated within a water-soluble matrix, formed by promoting the ßlg/D3 complex by acidification. The capacity of the ßlg-based coagulum to increase the long term stability of D3 in cold storage, upon exposure to intensive UV-light, and in the presence and absence of intestinal proteases, was evaluated. Additionally, the impact of the sequestration of D3 within the matrix of ßlg-based coagulum on its bioavailability was determined in vivo with force-fed rats. The water solubility, long-term storage and UV-light stability of D3 were significantly increased (p < 0.0001) due to the high encapsulation efficiency (94.5 ± 1.8%). The ßlg-based coagulum was not rapidly disrupted by the proteases in the intestines, leading to a slow release of D3, increased uptake of D3 and subsequent enhancement of the bioavailability of D3 in rats.

Self-assembly of ß-lactoglobulin and egg white lysozyme as a potential carrier for nutraceuticals.

Self-assembly structures of ß-lactoglobulin (ßlg) and egg protein lysozyme (Lyso) were developed, using electrostatic interactions between the two oppositely charged proteins. Different ßlg/Lyso concentration ratios were essayed at pH 6.8 to select the optimal ratio for the proteins co-precipitation, which behaviour was then studied at varying pH values. Optimal ßlg/Lyso concentration ratio, prepared at pH 7.5, was selected for protein co-precipitation. As a result, a structure with a mean diameter of 7.1±2.5 µm was formed, as indicated by static light scattering. Furthermore, the SEM images showed that ßlg and Lyso self-assembled to form a microsphere. Vitamin D3, used as a model nutraceutical, was successfully entrapped in the ßlg/Lyso microspheres with an encapsulation efficiency of 90.8±4.8%. Therefore, the ßlg/Lyso microspheres can serve as a potential food-grade vehicle for bioactives in the formulation of food products and pharmaceuticals.

Food protein-based microspheres for increased uptake of vitamin D3.

To protect vitamin D3 during cold storage and exposure to UV-light, vitamin D3 has been entrapped in microspheres formed by bovine protein ß-lactoglobulin (ßlg) and lysozyme (Lyso) from egg white. The behaviour of the ßlg/Lyso microspheres in simulated intestinal fluid and their impact on the kinetic release of D3 were determined. The impact of the D3-loaded ßlg/Lyso microspheres on the bioavailability of D3 was evaluated in vivo by force-feeding rats. The data indicate that the ßlg/Lyso microspheres effectively improved the stability of D3, which was readily released in the intestines. The release kinetics were accelerated in the presence of proteolytic enzymes. The bioavailability of D3 was improved, as confirmed by the significant increase in the serum levels of 25-hydroxy-D3 in rats. The current work demonstrates that water soluble proteins were used to substantially increase the bioavailability of the lipophilic vitamin, and thus can serve in the oral delivery of D3.

Increased stability and protease resistance of the ß-lactoglobulin/vitamin D3 complex.

The stability of the ß-lactoglobulin (ßlg)/vitamin D3 (D3) complex at 4°C and upon exposure to UV-C light, and in simulated intestinal fluid, were studied in vitro. Caco-2 cells were used to demonstrate the passage of the ßlg/D3 complex across the monolayers. Furthermore, an in vivo experiment was conducted by force-feeding rats with the free D3 and ßlg/D3 complex, with subsequent determination of the plasma concentration of 25-hydroxy-D3. The ßlg/D3 complex significantly improved the stability of the vitamin at 4 °C and when exposed to UV-C light. The resistance of ßlg to proteases was increased, indicating a mutual protective effect. The ßlg/D3 complex crossed the monolayers, which was confirmed by the significant increase in the concentration of 25-hydroxy-D3 in rats fed the ßlg/D3 complex compared to the ones fed the free D3. Therefore, the current study suggests that the ßlg/D3 complex can effectively be used for the fortification of milk products and low-fat content foods to improve the intake and bioavailability of D3.

Impact of feed supplementation with antimicrobial agents on growth performance of broiler chickens, Clostridium perfringens and enterococcus counts, and antibiotic resistance phenotypes and distribution of antimicrobial resistance determinants in Escherichia coli isolates.

The effects of feed supplementation with the approved antimicrobial agents bambermycin, penicillin, salinomycin, and bacitracin or a combination of salinomycin plus bacitracin were evaluated for the incidence and distribution of antibiotic resistance in 197 commensal Escherichia coli isolates from broiler chickens over 35 days. All isolates showed some degree of multiple antibiotic resistance. Resistance to tetracycline (68.5%), amoxicillin (61.4%), ceftiofur (51.3%), spectinomycin (47.2%), and sulfonamides (42%) was most frequent. The levels of resistance to streptomycin, chloramphenicol, and gentamicin were 33.5, 35.5, and 25.3%, respectively. The overall resistance levels decreased from day 7 to day 35 (P < 0.001). Comparing treatments, the levels of resistance to ceftiofur, spectinomycin, and gentamicin (except for resistance to bacitracin treatment) were significantly higher in isolates from chickens receiving feed supplemented with salinomycin than from the other feeds (P < 0.001). Using a DNA microarray analysis capable of detecting commonly found antimicrobial resistance genes, we characterized 104 tetracycline-resistant E. coli isolates from 7- to 28-day-old chickens fed different growth promoters. Results showed a decrease in the incidence of isolates harboring tet(B), bla(TEM), sulI, and aadA and class 1 integron from days 7 to 35 (P < 0.01). Of the 84 tetracycline-ceftiofur-resistant E. coli isolates, 76 (90.5%) were positive for bla(CMY-2). The proportions of isolates positive for sulI, aadA, and integron class 1 were significantly higher in salinomycin-treated chickens than in the control or other treatment groups (P < 0.05). These data demonstrate that multiantibiotic-resistant E. coli isolates can be found in broiler chickens regardless of the antimicrobial growth promoters used. However, the phenotype and the distribution of resistance determinants in E. coli can be modulated by feed supplementation with some of the antimicrobial agents used in broiler chicken production.