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.     

No genetic barriers between Salmonella enterica serovar typhimurium and Escherichia coli in SOS-induced mismatch repair-deficient cells

Conjugational crosses trigger SOS induction in Escherichia coli F(-) cells mated with Salmonella enterica serovar Typhimurium Hfr donors. Using an epigenetic indicator of SOS induction, we showed that a strong SOS response occurring in a subpopulation of mated mismatch repair-deficient cells totally abolishes genetic barriers between these two genera.     

Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104

Epidemic strain Salmonella typhimurium DT104 is characterized by various multiresistance patterns. At least some of the resistance genes are organized as integrons. Resistance genes of DT104 isolates can be efficiently transduced by P22-like phage ES18 and by phage PDT17 which is released by all DT104 isolates so far analyzed. Cotransduction tests demonstrate that the resistance genes, although not organized in a unique integron, are tightly clustered on the Salmonella chromosome. The spread of resistance genes in this strain by generalized transduction is discussed.     

Characterization of a T7-like lytic bacteriophage (phiSG-JL2) of Salmonella enterica serovar gallinarum biovar gallinarum

PhiSG-JL2 is a newly discovered lytic bacteriophage infecting Salmonella enterica serovar Gallinarum biovar Gallinarum but is nonlytic to a rough vaccine strain of serovar Gallinarum biovar Gallinarum (SG-9R), S. enterica serovar Enteritidis, S. enterica serovar Typhimurium, and S. enterica serovar Gallinarum biovar Pullorum. The phiSG-JL2 genome is 38,815 bp in length (GC content, 50.9%; 230-bp-long direct terminal repeats), and 55 putative genes may be transcribed from the same strand. Functions were assigned to 30 genes based on high amino acid similarity to known proteins. Most of the expected proteins except tail fiber (31.9%) and the overall organization of the genomes were similar to those of yersiniophage phiYeO3-12. phiSG-JL2 could be classified as a new T7-like virus and represents the first serovar Gallinarum biovar Gallinarum phage genome to be sequenced. On the basis of intraspecific ratios of nonsynonymous to synonymous nucleotide changes (Pi[a]/Pi[s]), gene 2 encoding the host RNA polymerase inhibitor displayed Darwinian positive selection. Pretreatment of chickens with phiSG-JL2 before intratracheal challenge with wild-type serovar Gallinarum biovar Gallinarum protected most birds from fowl typhoid. Therefore, phiSG-JL2 may be useful for the differentiation of serovar Gallinarum biovar Gallinarum from other Salmonella serotypes, prophylactic application in fowl typhoid control, and understanding of the vertical evolution of T7-like viruses.     

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.     

Detection of Salmonella enterica serovar typhimurium by using a rapid, array-based immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 x 10(4) CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 x 10(3) CFU/g.     

Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract

Salmonella enterica subsp. enterica serovar Newport resistant to the extended-spectrum cephalosporins (ESCs) and other antimicrobials causes septicemic salmonellosis in humans and animals and is increasingly isolated from humans, animals, foods, and environmental sources. Mechanisms whereby serovar Newport bacteria become resistant to ESCs and other classes of antimicrobials while inhabiting the intestinal tract are not well understood. The present study shows that 25.3% of serovar Newport strains isolated from the turkey poult intestinal tract after the animals were dosed with Escherichia coli harboring a large conjugative plasmid encoding the CMY-2 beta-lactamase and other drug resistance determinants acquired the plasmid and its associated drug resistance genes. The conjugative plasmid containing the cmy-2 gene was transferred not only from the donor E. coli to Salmonella serovar Newport but also to another E. coli serotype present in the intestinal tract. Laboratory studies showed that the plasmid could be readily transferred between serovar Newport and E. coli intestinal isolates. Administration of a single dose of ceftiofur, used to prevent septicemic colibacillosis, to 1-day-old turkeys did not result in the isolation of ceftiofur-resistant E. coli or Salmonella serovar Newport. There was a remarkable association between serotype, drug resistance, and plasmid profile among the E. coli strains isolated from the poults. This study shows that Salmonella serovar Newport can become resistant to ESCs and other antibiotics by acquiring a conjugative drug resistance plasmid from E. coli in the intestines.     

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.     

Multiple antibiotic resistance (mar) locus in Salmonella enterica serovar typhimurium DT104

In order to understand the role of the mar locus in Salmonella with regard to multiple antibiotic resistance, cyclohexane resistance, and outer membrane protein F (OmpF) regulation, a marA::gfp reporter mutant was constructed in an antibiotic-sensitive Salmonella enterica serovar Typhimurium DT104 background. Salicylate induced marA, whereas a number of antibiotics, disinfectants, and various growth conditions did not. Increased antibiotic resistance was observed upon salicylate induction, although this was shown to be by both mar-dependent and mar-independent pathways. Cyclohexane resistance, however, was induced by salicylate by a mar-dependent pathway. Complementation studies with a plasmid that constitutively expressed marA confirmed the involvement of mar in Salmonella with low-level antibiotic resistance and cyclohexane resistance, although the involvement of mar in down regulation of OmpF was unclear. However, marA overexpression did increase the expression of a ca. 50-kDa protein, but its identity remains to be elucidated. Passage of the marA::gfp reporter mutant with increasing levels of tetracycline, a method reported to select for mar mutants in Escherichia coli, led to both multiple-antibiotic and cyclohexane resistance. Collectively, these data indicate that low-level antibiotic resistance, cyclohexane resistance, and modulation of OMPs in Salmonella, as in E. coli, can occur in both a mar-dependent and mar-independent manner.     

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.     

Resuscitation of Salmonella enterica serovar typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the sigma(s) subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.     

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.     

Characterization of grvA, an antivirulence gene on the gifsy-2 phage in Salmonella enterica serovar typhimurium

The lambdoid phage Gifsy-2 contributes significantly to Salmonella enterica serovar Typhimurium virulence. The phage carries the periplasmic superoxide dismutase gene, sodCI, and other unidentified virulence factors. We have characterized the gene grvA, a single open reading frame inserted in the opposite orientation in the tail operon of the Gifsy-2 phage. Contrary to what is observed with classic virulence genes, grvA null mutants were more virulent than wild type as measured by intraperitoneal competition assays in mice. We have termed this effect antivirulence. Wild-type grvA in single copy complemented this phenotype. However, grvA(+) on a multicopy plasmid also conferred the antivirulence phenotype. Neither a grvA null mutation nor the grvA(+) plasmid conferred a growth advantage or disadvantage in laboratory media. The antivirulence phenotype conferred by the grvA null mutation and the grvA(+) plasmid required wild-type sodCI but was independent of other virulence factors encoded on Gifsy-2. These results suggest that in a wild-type situation, GrvA decreases the pathogenicity of serovar Typhimurium in the host, most likely by affecting resistance to toxic oxygen species. These virulence phenotypes were independent of functional Gifsy-2 phage production. Our data suggest that the contribution of Gifsy-2 is a complicated sum of both positive virulence factors such as sodCI and antivirulence factors such as grvA.