Phenotypic zinc resistance does not correlate with antimicrobial multi-resistance in fecal E. coli isolates of piglets.

BACKGROUND: Following the ban on antimicrobial usage for growth promotion in animal husbandry in the EU, non-antimicrobial agents including heavy metal ions (e.g. zinc and copper), prebiotics or probiotics have been suggested as alternatives. Zinc has extensively been used in pig farming, particularly during weaning of piglets to improve animal health and growth rates. Recent studies, however, have suggested that high dietary zinc feeding during weaning of piglets increases the proportion of multi-drug resistant E. coli in the gut, contraindicating the appropriateness of zinc as an alternative. The underlying mechanisms of zinc effects on resistant bacteria remains unclear, but co-selection processes could be involved. In this study, we determined whether E. coli isolates from intestinal contents of piglets that had been supplemented with high concentrations of zinc acquired a higher tolerance towards zinc, and whether multi-drug resistant isolates tolerated higher zinc concentrations. In addition, we compared phenotypic zinc and copper resistance of E. coli isolates for possible correlation between phenotypic resistance/tolerance to different bivalent ionic metals. RESULTS: We screened phenotypic zinc/copper tolerance of 210 isolates (including antimicrobial resistant, multi-drug resistant, and non-resistant E. coli) selected from two, independent zinc-feeding animal trials by determining a zinc/copper minimal inhibitory concentration (Merlin, Bornheim-Hersel, Germany). In both trials, groups of piglets were supplemented either with high dietary zinc (> 2000 ppm) or control (50-70 ppm, background) concentrations. Our observations showed that high concentration zinc exposure did not have an effect on either zinc or copper phenotypic tolerance of E. coli isolates from the animals. No significant association was found between antimicrobial resistance and phenotypic zinc/copper tolerance of the same isolates. CONCLUSION: Our findings argue against a co-selection mechanism of antimicrobial drug-resistance and zinc tolerance after dietary zinc supplementation in weaning piglets. An explanation for an increase in multi-drug resistant isolates from piglets with high zinc dietary feeding could be that resistant bacteria to antimicrobial agents are more persistent to stresses such as zinc or copper exposure.

Cross-talk Between Host, Microbiome and Probiotics: A Systems Biology Approach for Analyzing the Effects of Probiotic Enterococcus faecium NCIMB 10415 in Piglets.

A comprehensive data-set from a multidisciplinary feeding experiment with the probiotic Enterococcus faecium was analyzed to elucidate effects of the probiotic on growing piglets. Sixty-two piglets were randomly assigned to a control (no probiotic treatment) and a treatment group (E. faecium supplementation). Piglets were weaned at 26 d. Age-matched piglets were sacrificed for the collection of tissue samples at 12, 26, 34 and 54 d. In addition to zootechnical data, the composition and activity of intestinal microbiota, immune cell types, and intestinal responses were determined. Our systems analysis revealed clear effects on several measured variables in 26 and 34 days old animals, while response patterns varied between piglets from different age groups. Correlation analyses identified reduced associations between intestinal microbial communities and immune system reactions in the probiotic group. In conclusion, the developed model is useful for comparative analyses to unravel systems effects of dietary components and their time resolution. The model identified that effects of E. faecium supplementation most prominently affected the interplay between intestinal microbiota and the intestinal immune system. These effects, as well as effects in other subsystems, clustered around weaning, which is the age where piglets are most prone to diarrhea.

Bacillus cereus var. Toyoi modulates the immune reaction and reduces the occurrence of diarrhea in piglets challenged with Salmonella Typhimurium DT104.

A feeding trial with sows and their piglets was performed with the probiotic feed additive Bacillus cereus var. Toyoi in two consecutive experimental periods. Sows (n = 8) were allocated into treatment (Bc) and control (CO) groups. Sows of Bc group (n = 4) were fed 3.14 × 10(5) cfu/g Bacillus cereus var. Toyoi with the diet from d 87 of pregnancy on. Their piglets received Bacillus cereus var. Toyoi supplemented feed (8.7 × 10(5) cfu/g) starting on d 14 of life and further on after weaning (6.5 × 10(5) cfu/g), whereas sows and piglets of the CO group remained untreated. One day after weaning, piglets from both groups (n = 24 each) were challenged orally with Salmonella Typhimurium DT104 (3 × 10(9) viable bacteria). Health status, shedding of B. cereus in the feces, and performance of the piglets were monitored. At 24 h, 72 h, 6 d, and 28 d postinfection (PI), six piglets from each group were euthanized and cell counts of Salmonellae were determined in the colon contents, mesenteric lymph nodes, and tonsils. Peripheral blood mononuclear cells and jejunal intraepithelial lymphocytes (IEL) were analyzed by flow cytometry. The incidence of scours was lower in the Bc group than in the CO group (P = 0.004). In addition, the fecal shedding of Salmonella was significantly lower in the Bc group at 25 d PI (P = 0.004). Shortly after infection, the γδ T cells were significantly less frequent in the blood of Bc piglets. For both CD8-positive γδ T cells (P = 0.033) and CD8-negative γδ T cells (P = 0.028), significant differences were observed. Furthermore, 28 d PI piglets from the treated group showed lower numbers of γδ T cells in the jejunal epithelium (P = 0.036). To investigate the role of intestinal γδ T cells during the infection with S. Typhimurium, IEL were gained from six healthy 40-d-old piglets and infected in vitro with S. Typhimurium. CD8ß cells and γδ T cells were detected by flow cytometry and the infection rates of both populations in the cell suspensions were compared. The infection rate (IR) of γδ T cells was higher in all six cell suspensions than the IR of CD8ß expressing T cells (P = 0.002). In conclusion, B. cereus var. Toyoi supplementation of sows and their piglets had a positive impact on the health status of the piglets after a challenge with Salmonella, likely due to an altered immune response marked by reduced frequencies of CD8+ γδ T cells in the peripheral blood and the jejunal epithelium.

Effects of a probiotic strain of Enterococcus faecium on the rate of natural chlamydia infection in swine.

Chlamydiae are obligately intracellular pathogens which cause infections associated with a broad range of diseases in both livestock and humans. In addition, a large proportion of animals may become persistently infected asymptomatic carriers and serve as reservoirs for other animals which also shed these potential zoonotic pathogens. Reducing the chlamydial load of animals is therefore of major importance, and since large-scale antibiotic treatment is neither desired nor feasible, alternative means of prevention are needed. Here we performed a study comparing the efficacy of a probiotic strain of Enterococcus faecium on the reduction of both the rate of natural infection and the shedding of chlamydiae in swine. The presence of Chlamydiaceae was detected by species-specific PCR of fecal samples of sows taken at three times prior to the birth of piglets. Piglets delivered from chlamydia-positive sows in either the control or the probiotic group were also examined for the frequency of chlamydiae at various ages. Eighty-five percent of the piglets from the control group were found to be chlamydia positive, whereas chlamydiae were found in only 60% of piglets from the probiotic group, results confirmed by fluorescence in situ hybridization and immunohistology, which showed higher rates of infection in the control group. In addition to the reduced frequency of chlamydia-positive piglets in the probiotic group, the time of appearance of positive samples was delayed. To our knowledge, these data show for the first time that a probiotic strain of E. faecium can reduce the rate of carryover infections of piglets by obligate intracellular pathogens.

Influence of a probiotic Enterococcus faecium strain on development of the immune system of sows and piglets.

The influence of the probiotic bacterium Enterococcus faecium SF68 on the immune system and the intestinal colonization of pigs were determined in a feeding experiment with sows and piglets. Mucosal immunity of the developing piglets was monitored by isolation and detection of intestinal lymphocyte cell populations from the proximal jejunal epithelium and the continuous Peyers patches by the use of flow cytometry. The levels of intestinal IgA in both groups of piglets were compared, as well as total IgG in the serum of sows and piglets. Feces of the sows and intestinal contents of the piglets were taken for determination of total anaerobe and coliform bacterial counts in both probiotic and control groups. Villus length and depth of the crypts were measured in the jejunum of sacrificed piglets to monitor the development of the intestinal mucosal surface amplification. Total serum IgG of the sows appeared to be unaffected. Piglets of both groups showed similar IgG levels up to 5 weeks after birth with a slight tendency toward lower values in the probiotic group. At an age of 8 weeks the total IgG levels of the probiotic animals were significantly lower (p<0.01). No differences were observed in the populations of CD4+ and CD8+ T cells in the Peyers patches. However, the levels of cytotoxic T cells (CD8+) in the jejunal epithelium of piglets of the probiotic group were significantly reduced. The depth of the jejunal crypts and length of the villi were similar in both groups, suggesting the relative T-cell population differences were not due to alterations in the epithelial cell numbers. The total anaerobe and coliform bacterial populations were not significantly affected by the probiotic treatment, either in sows or in the piglets. However, a remarkable decline in the frequency of beta-haemolytic and O141 serovars of Escherichia coli was observed in the intestinal contents of probiotic piglets, suggesting an explanation for the reduction in cytotoxic T-cell populations.

Comparison of DeltarelA strains of Escherichia coli and Salmonella enterica serovar Typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis.

The growth recovery of Escherichia coli K-12 and Salmonella enterica serovar Typhimurium DeltarelA mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type E. coli K-12 and S. enterica serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (ilvGM) and III (ilvIH) isozymes, respectively, DeltarelA derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the DeltarelA strains. In the E. coli K-12 MG1655 DeltarelA background, correction of the preexisting rph-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery. S. enterica serovar Typhimurium LT2 DeltarelA strains were fully complemented by elevated basal ppGpp levels in an S. enterica serovar Typhimurium LT2 DeltarelA spoT1 mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by relA-dependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the E. coli K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp.

Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs.

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless lambda cl and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cl synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine-Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.

Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence.

It has previously been proposed that Escherichia coli ribosomal protein S1 is required for the translation of highly structured mRNAs. In this study, we have examined the influence of structural features at or near the start codon of different mRNAs. The requirement for ribosomal protein S1 for translation initiation was determined when (i) the ribosome-binding site (RBS) was either preceded by a 5' non-translated leader sequence; (ii) the RBS was located 5' proximal to a mRNA start codon; and (iii) the start codon was the 5' terminal codon as exemplified by leaderless mRNAs. In vitro translation studies revealed that the leaderless lambda cl mRNA is translated with Bacillus stearothermophilusribosomes, naturally lacking a ribosomal protein S1 homologue, whereas ompA mRNA containing a 5' leader is not. These studies have been verified by toeprinting with E. coli ribosomes depleted for S1. We have shown that S1 is required for ternary complex formation on ompA mRNA but not for leaderless mRNAs or for mRNAs in which the RBS is close to the 5' end.

Downstream box-anti-downstream box interactions are dispensable for translation initiation of leaderless mRNAs.

The downstream box (db) with complementarity to a segment in the penultimate stem of 16S rRNA has been suggested to serve as a recognition element for the ribosome. For some mRNAs, the db has been proposed to act synergistically with the Shine and Dalgarno sequence (SD), while for the leaderless lambda(cI) mRNA it has been suggested to substitute for the SD in translation initiation. To test whether the db-anti-db interaction is required for translation initiation, we have used three different leaderless transcripts, the lambda(cI), phage P2 gene V and Tn1721 tetR mRNA. Using primer extension inhibition analysis (toeprinting), we show that the db does not influence translation initiation complex formation in vitro. In an attempt to demonstrate the simultaneous interactions between the db and anti-db and between the anticodon of initiator tRNA and the start codon, respectively, chemical probing has been employed on cI translation initiation complexes. These studies did not reveal a protection of the bases comprising the putative db in cI. In addition, kinetic toeprinting experiments and in vivo expression studies with cI mRNA showed that the db is dispensable for the initial interaction between ribosome and cI mRNA in the pathway towards formation of the initiation complex.

The RNA chain elongation rate of the lambda late mRNA is unaffected by high levels of ppGpp in the absence of amino acid starvation.

In this study, the effects of high levels of guanosine tetraphosphate (ppGpp) on the decay and RNA chain elongation kinetics of the bacteriophage lambda late transcript in Escherichia coli were examined in the absence of amino acid starvation. The accumulation, mRNA decay kinetics, and RNA chain elongation rate of the lambda late mRNA were determined after heat induction of lambdacI857 lysogens in the presence of high levels of ppGpp induced from a RelAalpha fragment-overproducing plasmid. The accumulation kinetics and elongation rate determinations of the late mRNA were made at long times after induction to allow a new steady state of transcriptional activities under conditions of elevated intracellular levels of ppGpp. The results indicate no prolonged or significant effect on either mRNA decay or the RNA chain elongation rate of the late mRNA as a result of elevated ppGpp levels. Surprisingly, the RNA chain elongation rate determinations indicate an RNA polymerase processivity of approximately 90-100 nucleotides/s for the lambda late transcript despite the presence of high levels of ppGpp. The results are discussed in terms of various models for regulation of stable and messenger RNA synthesis in E. coli.

Ternary complex formation on leaderless phage mRNA.

The phage Lambda PRM promoter-derived cI mRNA and phage P2 gene V mRNA are transcribed beginning with the A residue of the AUG start codon. Using lacZ fusion analysis we have assessed the effects of alterations in the immediate downstream coding region on the translational efficiency of these mRNAs. Mutations, including deletions of the putative downstream box of either cI or gene V mRNAs, showed no significant reduction in expression of the different lacZ fusions. Primer extension inhibition analysis suggests a role of ribosomal protein S1 in cI mRNA recognition.

Evaluation of the E. coli ribosomal rrnB P1 promoter and phage-derived lysis genes for the use in a biological containment system: a concept study.

A concept study devised for the development of a biological containment system has been conducted. We show that the lysis genes of different phage origin function in a variety of bacteria. They may therefore be suited for conditional suicide cassettes. Moreover, we tested whether the Escherichia coli rrnB P1 promoter could function as an environmentally responsive element sensing poor growth conditions expected after an accidental release of E. coli production strains from a bioreactor. Mimicking poor nutrient conditions by production of the alarmone guanosine tetraphosphate (ppGpp) with a plasmid encoded ppGpp synthetase I, the rrnB P1 promoter activity was completely turned off. These experiments suggested that the rrnB P1 promoter may be used as an efficient biosensor for altered growth conditions. A concept for a conditional suicide system employing the rrnB P1 promoter and phage-derived lysis genes as key components is discussed.

Dual translational start motif evolutionarily conserved in the holin gene of Bacillus subtilis phage phi 29.

Holins represent phage encoded lysis functions required for transit of the phage murein hydrolases to the periplasm. The Lambda S, phage 21 S, and P22 13 holin genes contain a dual translational start motif, beginning with Met1-Lys2-X-Met3. In all cases both start codons at the 5' end of the respective holin gene are utilized. The resulting polypeptides have opposing functions, with the longer product acting as an inhibitor of the shorter one. The 131-codon gene 14 of Bacillus subtilis phage phi 29 encodes the holin function, whereas the downstream gene 15 codes for a lysozyme. phi 29 Gene 14 begins with Met1-Lys2-Met3. Here, we present in vitro and in vivo evidence for the expression of two protein 14 species consisting of 129 and 131 amino acids, respectively. These data suggest that the lysis control mechanism based on two holin species, which has been shown to be operational in the temperature Escherichia coli phages Lambda and 21, and in the Salmonella typhimurium phage P22, is evolutionarily conserved in the lytic B. subtilis phage phi 29.

Toxic effects of high levels of ppGpp in Escherichia coli are relieved by rpoB mutations.

A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action. To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter. The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp. In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases. At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates. After continued incubation, these colonies form blue sectors of faster growing mutant cells. Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase. One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis. The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid. The presence of this plasmid conferred increased resistance to overproduction of ppGpp. These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.

Age and social composition factors as explanations for cleavages in socio-political values.

While age group differences on social and political values have been frequently documented in survey data, it is often claimed that these differences are due to social composition factors rather than to age itself. In this analysis we test for the effects of age vs. social composition in explaining variation in four attitudinal dependent variables. Using a sample especially drawn to study generational differences, and employing a multivariate statistical model, it was found that age differences were only modestly reduced by social composition variables. It is concluded that nonartifactual age group differences do exist on the dependent variables.