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The marRAB operon is conserved in seven genera of enteric bacteria ( Escherichia, Shigella, Klebsiella, Enterobacter, Salmonella, Cronobacter, and Citrobacter). MarA is a transcriptional regulator affecting many genes involved in resistance to stresses, and MarR is an autorepressor of the operon, but a role for the marB gene has been unclear. A recent work reported that deletion of marB causes resistance to certain stresses and increases the amount of marA transcript. We show here that the small (216 bp) marB gene encodes a protein, not an sRNA, because two different stop codons within the predicted open reading frame of marB prevented plasmid-borne marB from complementing Δ marB::Kan. The Δ marB::Kan mutation did not increase the stability of the marA transcript, suggesting that MarB does not destabilize the marA transcript but rather reduces its rate of transcription. Placing the putative signal sequence of MarB upstream of signal-sequence-less alkaline phosphatase guided the phosphatase to its normal periplasmic location. We conclude that MarB is a small periplasmic protein that represses the marRAB promoter by an indirect mechanism, possibly involving a signal to one of the cytoplasmic regulators of that promoter. [ABSTRACT FROM AUTHOR]

Background: The prevalence and type of plasmids, resistance genes and integrons carried by two collections of multiresistant E. coli producing or not extended-spectrum β-lactamases have been compared. Rep-PCR was used to determine the clonal relationship of the organisms. Plasmids were classified according to their incompatibility. Class 1 and Class 2 integrons and antibiotic resistance genes were analysed by PCR and sequencing. Results: Both collections of organisms contained a large diversity of unrelated strains with some clones distributed in both groups of isolates. Large plasmids were identified in the two groups of organisms. Plasmids with replicons repK and repColE were more frequent among ESBL-producing isolates, while repFIA, repFII and repA/C replicons were more frequent in isolates lacking ESBL. Conjugative plasmids with repK and repA/C replicons coded for CTX-M-14 and CMY-2 β-lactamases, respectively. No significant differences were observed in the distribution of class 1 and class 2 integrons among multiresistant E. coli producing or not ESBL, and dfrA17-ant(3")-Ie was the cassette arrangement most commonly found. Conclusions: In the concrete temporal and geographical context of this study, multiresistant E. coli producing ESBL or other mechanisms of resistance were largely clonally diverse and present some differences in the types of harboured plasmids. Still, some clones were found in both ESBL-producing and -lacking isolates [ABSTRACT FROM AUTHOR]

Nineteen extended-spectrum β-lactamase (ESBL)-positive Escherichia coli strains recovered from food samples in Tunisia were characterized by multilocus sequence typing and phylogenetic typing, and the virulence gene and plasmid content were also determined. These strains presented unrelated pulsed-field gel electrophoresis patterns and contained genes coding for the following ESBLs (the number of strains is in parentheses): CTX-M-1 (15), CTX-M-14 (2), CTX-M-8 (1), and SHV-5 (1). Twelve different sequence types (STs) were identified among the 19 ESBL-positive strains, which included two new STs (ST2022 in 2 blaCTX-M-14-containing strains and ST1970 in 2 blaCTX-M-1containing strains). ST155 and ST602 were detected in four and three blaCTX-M-1-containing strains, respectively, and ST405 was detected in one blaCTX-M-8-Producing strain. All ESBL-positive strains were ascribed to the phylogenetic groups A and Bl. Most of the blaCTX-M-1-containing strains harbored an IncI1 plasmid, except for the four blaCTX-M-1-positive strains of beef origin and ST155, which harbored an IncN plasmid. The two blaCTX-M-14-containing strains contained an IncI1 plasmid. The virulence gene fimA was detected in all strains. Most strains also carried the aer gene, and six strains carried the eae gene. All strains were negative for the virulence genes sxt, papG-III, papC, hly, cnfl, and bfp. We conclude that ESBL-producing E. coli strains of food origin in Tunisia show high diversity and that plasmids harboring ESBL genes could be implicated in the dissemination of this resistance phenotype. [ABSTRACT FROM AUTHOR]

Objectives: To characterize the mechanisms implicated in the in vivo selection of quinolone and aminoglycoside resistance in a faecal Salmonella enterica serovar Typhimurium DT104B strain recovered after ciprofloxacin treatment of a hospitalized elderly patient with acute gastroenteritis. Methods: Two Salmonella Typhimurium isolates were obtained before (Se6) and after (Se20) treatment and they were typed by PFGE and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined by disc diffusion and agar dilution methods. Class 1, 2 and 3 integrons and resistance mechanisms were studied by PCR and sequencing. Plasmids were typed. Results: Both Salmonella Typhimurium strains were resistant to tetracycline, streptomycin and sulphonamides, while Se20 was also resistant to nalidixic acid, ciprofloxacin, norfloxacin, levofloxacin, ofloxacin, amikacin, tobramycin, kanamycin and trimethoprim. PFGE and MLST showed a clonal relationship between the strains, which belonged to the sequence type ST36. Both strains contained the repC-sul2-strA-strB structure and tet(A) and qnrS1 genes, and strain Se20 also contained the aac(6')-Ib-cr gene, the Ser83→Tyr substitution in GyrA and one class 1 integron with the dfrA17+aadA5 gene cassette arrangement lacking qacEΔ1+sul1. Two different transconjugants from Salmonella Se20 (TCSe20B and TCSe20L) harboured qnrS1 and sul2 genes and the class 1 integron. The TCSe20B strain also acquired the aac(6')-Ib-cr gene located on a non-typeable plasmid. qnrS1 was identified on a ColE-type plasmid and the class 1 integron on an IncI1-type plasmid. Conclusions: This is the first report of in vivo selection of the aac(6')-Ib-cr gene and the Ser83→Tyr change in GyrA in a qnrS1-positive Salmonella Typhimurium strain after ciprofloxacin treatment; the in vitro transfer of both plasmid-mediated quinolone resistance genes was also demonstrated. [ABSTRACT FROM AUTHOR]

To study the viability of a gyrA S83 stop mutation found in an Escherichia coli J53 ciprofloxacin-resistant strain (J53 CipR27), a pBR322 derivative was constructed with a TAG mutation in the bla gene knocking out ampicillin resistance. Ampicillin resistance was restored, suggesting that the strain contains tRNA suppressor activity able to suppress the UAG codon gyrA and allow viability. The method was applied to 22 unique clinical E. coli isolates, and all were found to have low-level suppressor activity. [ABSTRACT FROM AUTHOR]

Phenotypic and genotypic characterization of antimicrobial resistance was conducted for 98 Escherichia coli isolates recovered from 40 food samples of animal origin (poultry, sheep, beef, fish, and others) obtained in supermarkets and local butcheries in Tunis during 2004 and 2005. Susceptibility to 15 antimicrobial agents was tested by disk diffusion and agar dilution methods, the mechanisms of resistance were evaluated using PCR and sequencing methods, and the clonal relationship among isolates was evaluated using pulsed-field gel electrophoresis. High resistance was detected to tetracycline, sulphonamides, nalidixic acid, ampicillin, streptomycin, and trimethoprim-sulfamethoxazole (29 to 43% of isolates), but all isolates were susceptible to cefotaxime, ceftazidime, cefoxitin, azthreonam, and amikacin. One-third of the isolates had multiresistant phenotypes (resistance to at least five different families of antimicrobial agents). Different variants of blaTEM, tet, sul, dfrA, aadA, and aac(3) genes were detected in most of the strains resistant to ampicillin, tetracycline, sulphonamide, trimethoprim, streptomycin, and gentamicin, respectively. The presence of class 1 and class 2 integrons was studied in 15 sulphonamideresistant unrelated E. coli strains, and 14 of these strains harbored class 1 integrons with five different arrangements of gene cassettes, and a class 2 integron with the dfrAl + sat + aadA1 arrangement was found in one strain. This study revealed the high diversity of antimicrobial resistance genes, some of them included in integrons, in E. coli isolates of food origin. [ABSTRACT FROM AUTHOR]

Background: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. Results: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. Conclusions: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur. [ABSTRACT FROM AUTHOR]