Antibiotic resistance in Salmonella enterica serovar Typhimurium exposed to microcin-producing Escherichia coli

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.     

Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium

AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity. CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.     

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G₁₆, -G₁₈, -G₂₀, -G₂₂, and -G₂₄). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G₂₀ clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.     

CorA Affects Tolerance of Escherichia coli and Salmonella enterica Serovar Typhimurium to the Lactoperoxidase Enzyme System but Not to Other Forms of Oxidative Stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg²⁺ transporter and at least three other inducible open reading frames belonged to the Mg²⁺ regulon, repression of the Mg stimulon by Mg²⁺ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni²⁺, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni²⁺-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.     

1-methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism

In Salmonella enterica serovar Typhimurium a mutation in the purF gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a purF mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from S. enterica specific for leucine, proline, and arginine contain 1-methylguanosine (m(1)G37) adjacent to and 3' of the anticodon (position 37). The formation of m(1)G37 is catalyzed by the enzyme tRNA(m(1)G37)methyltransferase, which is encoded by the trmD gene. Mutations in this gene, which result in an m(1)G37 deficiency in the tRNA, in a purF mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m(1)G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m(1)G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.     

The acyl homoserine lactone receptor, SdiA, of Escherichia coli and Salmonella enterica serovar Typhimurium does not respond to indole

In this study, we tested the hypothesis that the SdiA proteins of Escherichia coli and Salmonella enterica serovar Typhimurium respond to indole. While indole was found to have effects on gene expression and biofilm formation, these effects were not sdiA dependent. However, high concentrations of indole did inhibit N-acyl-l-homoserine lactone (AHL) sensing by SdiA. We conclude that SdiA does not respond to indole but indole can inhibit SdiA activity in E. coli and Salmonella.     

Growth rate toxicity phenotypes and homeostatic supercoil control differentiate Escherichia coli from Salmonella enterica serovar Typhimurium

Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella, showing disruption of supercoiling near the terminus and replicon failure at 42 degrees C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella. Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.     

Membrane topology of the ZntB efflux system of Salmonella enterica serovar Typhimurium

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.     

Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Exposed to Microcin-Producing Escherichia coli

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.     

Isolation of spectinomycin resistance mutations in the 16S rRNA of Salmonella enterica serovar Typhimurium and expression in Escherichia coli and Salmonella

Two single-base mutations in 16S rRNA conferring high-level resistance to spectinomycin were isolated on a plasmid-borne copy of the rrnD operon from Salmonella enterica serovar Typhimurium. Neither of the mutations (C1066U and C1192U) had appreciable effects on cell growth, but each had differential effects on resistance to spectinomycin and fusidic acid. Both mutations also conferred resistance to spectinomycin in Escherichia coli strains containing deletions of all seven chromosomal rrn operons and expressing plasmid-encoded Salmonella rRNA exclusively. In contrast, when expressed in E. coli strains containing intact chromosomal rrn operons, the strains were sensitive to spectinomycin. However, chromosomal mutations arose that allowed expression of the rRNA-dependent spectinomycin resistance phenotype. It is proposed that in heterogeneous rRNA populations, the native E. coli rRNA out-competes the heterologous Salmonella rRNA for binding to ribosomal proteins, translation factors, or ribosome assembly, thus limiting entry of the antibiotic-resistant 30S subunits into the functioning ribosome pool.     

Isolation of Spectinomycin Resistance Mutations in the 16S rRNA of Salmonella enterica serovar Typhimurium and Expression in Escherichia coli and Salmonella

Two single-base mutations in 16S rRNA conferring high-level resistance to spectinomycin were isolated on a plasmid-borne copy of the rrnD operon from Salmonella enterica serovar Typhimurium. Neither of the mutations (C1066U and C1192U) had appreciable effects on cell growth, but each had differential effects on resistance to spectinomycin and fusidic acid. Both mutations also conferred resistance to spectinomycin in Escherichia coli strains containing deletions of all seven chromosomal rrn operons and expressing plasmid-encoded Salmonella rRNA exclusively. In contrast, when expressed in E. coli strains containing intact chromosomal rrn operons, the strains were sensitive to spectinomycin. However, chromosomal mutations arose that allowed expression of the rRNA-dependent spectinomycin resistance phenotype. It is proposed that in heterogeneous rRNA populations, the native E. coli rRNA out-competes the heterologous Salmonella rRNA for binding to ribosomal proteins, translation factors, or ribosome assembly, thus limiting entry of the antibiotic-resistant 30S subunits into the functioning ribosome pool. Received: 28 September 2001 / Accepted: 26 March 2002     

Differences in enzymatic properties allow SodCI but not SodCII to contribute to virulence in Salmonella enterica serovar Typhimurium strain 14028

Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth. By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter. In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is "tethered" within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.     

Effects of nondigestible oligosaccharides on Salmonella enterica serovar Typhimurium and nonpathogenic Escherichia coli in the pig small intestine in vitro

An in vitro intestinal tissue model was developed for the investigation of bacterial association in the pig small intestine under different dietary regimes. In preliminary experiments, jejunal and ileal tissue was taken from Danish Landrace pigs fed standard diet and inoculated with either Salmonella or nonpathogenic Escherichia coli strains. Higher numbers of salmonellae associated with the ileal tissues, but the numbers did not reach significance. Hence, jejunal sections were inoculated with nonpathogenic E. coli and ileal sections were inoculated with salmonellae in the presence of mannose or commercial nondigestible oligosaccharides (NDO) at 2.5%. There was a significant decrease in E. coli associated with the jejunum in the presence of mannose (P < 0.05). Furthermore, in pigs fed a diet supplemented with commercial NDO at 4% there was a significant reduction in the numbers of E. coli in jejunal organ cultures of pigs fed the FOS diet (P < 0.05). There was a reduction, though not a significant one, in the association of Salmonella sp. to the ileal sections of pigs fed the commercial FOS diet. The feeding of commercial GOS or its addition to organ cultures did not affect E. coli or Salmonella numbers.