Molecular Characterization of Escherichia coli Strains Isolated from Different Food Sources

Hrana predstavlja mogući izvor patogenih i na antibiotik otpornih sojeva bakterije Escherichia coli, pa je u radu analizirano 84 izolata iz uzoraka hrane identificiranih 2007. i 2008. godine na Nacionalnom institutu za javno zdravstvo u Sloveniji. Pomoću metode PCR (lančana reakcija polimeraze) prema Clermontu izolati su razvrstani u filogenetske skupine i podskupine. Četrdeset i dva (50 %) su izolata svrstana u filogenetsku skupinu A, a njih trideset (35,7 %) u skupinu B1. Te dvije skupine uglavnom obuhvaćaju komenzale crijevne mikroflore. Deset je izolata (11,9 %) svrstano u skupinu D, a samo njih dva (2,4 %) u skupinu B2. U tim se skupinama uglavnom nalaze ekstraintestinalni patogeni sojevi E. coli. Sojevi su zatim analizirani ne bi li se utvrdila prisutnost različitih virulentnih gena i plazmidnih gena otpornosti na kinolone (qnr). U jednom je izolatu (1,2 %) otkriven virulentni gen stx1, kod njih pet (6 %) gen stx2, a u sljedećih pet izolata (6 %) oba gena, stx1 i stx2. U osam je izolata (9,5 %) pronađen gen ehxA, a u njih tri (3,7 %) gen eae. Svi su ti geni karakteristični za patotip STEC, koji proizvodi Shiga toksin. Gen fimH pronađen je u sedamdeset i četiri (88,1 %) izolata. Od virulentnih gena tipičnih za ekstraintestinalne patogene sojeve E. coli, gen hra je pronađen u devet (11 %), gen ompTAPEC u osam (9,5 %), a gen iha u šest (7 %) izolata. U jednom su izolatu otkriveni geni kpsMTII, sfa, usp i vat, dok geni hlyA, bmaE, cnf, hpb, sat i plazmidni geni otpornosti na kinolone qnr nisu pronađeni u ispitanim izolatima. Rezultati pokazuju da razni prehrambeni proizvodi zaista predstavljaju izvor intestinalnih, te u manjoj mjeri ekstraintestinalnih patogenih sojeva E. coli.Since food represents a possible source of pathogenic and antibiotic-resistant Escherichia coli strains, we analyzed 84 isolates from food samples identified in 2007 and 2008 at the National Institute of Public Health in Slovenia. Using polymerase chain reaction (PCR), the isolates were classified into phylogenetic groups and subgroups following the Clermont method. Forty-two (50 %) and thirty (35.7 %) isolates were classified into commensal gut phylogenetic groups A and B1, respectively. Only ten (11.9 %) and two (2.4 %) isolates were assigned to the phylogenetic groups D and B2, which include mainly extraintestinal pathogenic E. coli strains. The strains were further analyzed for the presence of various virulence genes and plasmid-mediated quinolone resistance qnr genes. Virulence genes stx1, stx2, both stx1 and stx2, ehxA and eae associated with Shiga-toxin producing E. coli were detected in one (1.2 %), five (6 %), five (6 %), eight (9.5 %) and three (3.7 %) isolates, respectively. Seventy-four (88.1 %) isolates carried the gene fimH, whereas virulence genes characteristic of extraintestinal pathogenic E. coli, hra, ompTAPEC and iha, were detected in nine (11 %), eight (9.5 %) and six (7 %) isolates, respectively. Genes kpsMTII, sfa, usp and vat were discovered in single isolates, whereas hlyA, bmaE, cnf, hbp and sat, as well as plasmid- mediated quinolone resistance genes qnr, were not detected in the analyzed strains. Our results show that various food items are indeed a source of intestinal and, albeit to a lesser extent, of extraintestinal pathogenic E. coli strains

Two Tales of Prokaryotic Genomic Diversity: Escherichia coli and Halophiles

Jedna je od općenitih značajki prokariota velika raznolikost genoma, na što utječu mutacije, horizontalni prijenos gena, prisutnost bakteriocina i bakterijskih virusa. Iznimna se genomska raznolikost razvila kao posljedica izlaganja mikroorganizama stresu i njihove prilagodbe različitim uvjetima okoliša. U radu su predstavljena dva primjera raznolikosti prokariota: genetska varijabilnost jedinki vrste Escherichia coli i raznolikost mikroorganizama prisutnih u slanim staništima, praćena raspravom o zdravstvenim problemima uzrokovanim unosom kontaminirane hrane i mogućnosti primjene mikroorganizama u biotehnologiji.Prokaryotes are generally characterized by vast genomic diversity that has been shaped by mutations, horizontal gene transfer, bacteriocins and phage predation. Enormous genetic diversity has developed as a result of stresses imposed in harsh environments and the ability of microorganisms to adapt. Two examples of prokaryotic diversity are presented: on intraspecies level, exemplified by Escherichia coli, and the diversity of the hypersaline environment, with the discussion of food-related health issues and biotechnological potential

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations

Escherichia coli Bacteriocins: Antimicrobial Efficacy and Prevalence among Isolates from Patients with Bacteraemia

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin

The bacterial SOS response.

<p>(A) During normal growth the LexA transcriptional repressor downregulates the SOS response genes. (B) Various endogenous and exogenous triggers induce the SOS response, resulting in drug resistance, tolerance, persistence in host, virulence-factor synthesis, and dissemination of both resistance and virulence factor genes.</p

The DNA damage inducible SOS response is a key player in the generation of bacterial persister cells and population wide tolerance

Population-wide tolerance and persisters enable susceptible bacterial cells to endure hostile environments, including antimicrobial exposure. The SOS response can play a significant role in the generation of persister cells, population-wide tolerance, and shielding. The SOS pathway is an inducible DNA damage repair system that is also pivotal for bacterial adaptation, pathogenesis, and diversification. In addition to the two key SOS regulators, LexA and RecA, some other stressors and stress responses can control SOS factors. Bacteria are exposed to DNA-damaging agents and other environmental and intracellular factors, including cigarette smoke, that trigger the SOS response at a number of sites within the host. The Escherichia coli TisB/IstR module is as yet the only known SOS-regulated toxin–antitoxin module involved in persister formation. Nevertheless, the SOS response plays a key role in the formation of biofilms that are highly recalcitrant to antimicrobials and can be abundant in persisters. Furthermore, the dynamic biofilm environment generates DNA-damaging factors that trigger the SOS response within the biofilm, fueling bacterial adaptation and diversification. This review highlights the SOS response in relation to antimicrobial recalcitrance to antimicrobials in four clinically significant species, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium tuberculosis

Global transcriptional responses to the bacteriocin colicin M in Escherichia coli

ABSTRACT: BACKGROUND: Bacteriocins are protein antimicrobial agents that are produced by all prokaryotic lineages. Escherichia coli strains frequently produce the bacteriocins known as colicins. One of the most prevalent colicins, colicin M, can kill susceptible cells by hydrolyzing the peptidoglycan lipid II intermediate, which arrests peptidoglycan polymerization steps and provokes cell lysis. Due to the alarming rise in antibiotic resistance and the lack of novel antimicrobial agents, colicin M has recently received renewed attention as a promising antimicrobial candidate. Here the effects of subinhibitory concentrations of colicin M on whole genome transcription in E. coli were investigated, to gain insight into its ecological role and for purposes related to antimicrobial therapy. RESULTS: Transcriptome analysis revealed that exposure to subinhibitory concentrations of colicin M altered expression of genes involved in envelope, osmotic and other stresses, including genes of the CreBC two-component system,exopolysaccharide production and cell motility. Nonetheless, there was no induction of biofilm formation or genes involved in mutagenesis. CONCLUSION: At subinhibitory concentrations colicin M induces an adaptive response primarily to protect the bacterial cells against envelope stress provoked by peptidoglycan damage. Among the first induced were genes of the CreBC two-component system known to promote increased resistance against colicins M and E2, providing novel insight into the ecology of colicin M production in natural environments. While an adaptive response was induced nevertheless, colicin M application did not increase biofilm formation, nor induce SOS genes, adverse effects that can be provoked by a number of traditional antibiotics, providing support for colicin M as a promising antimicrobial agent

The <em>Escherichia coli</em> SOS Response: Much More than DNA Damage Repair

The Escherichia coli SOS response is an inducible DNA damage repair pathway controlled by two key regulators, LexA, a repressor and RecA, an inducer. Upon DNA damage RecA is activated and stimulates self cleavage of LexA, leading to, in E. coli, derepresion of approximately 50 SOS genes. The response is triggered by exogenous and endogenous signals that bacteria encounter at a number of sites within the host. Nevertheless, besides regulating DNA damage repair the SOS response plays a much broader role. Thus, SOS error prone polymerases promote elevated mutation rates significant for genetic adaptation and diversity, including antibiotic resistance. Here we review the E. coli SOS response in relation to recalcitrance to antimicrobials, including persister and biofilm formation, horizontal gene tranfer, gene mobility, bacterial pathogenicity, as well SOS induced bacteriocins that drive diversification. Phenotypic heterogeneity in expression of the SOS regulator genes, recA and lexA as well as colicin activity genes is also discussed