Lenka Micenková

Bacteriocin synthesis in uropathogenic and commensal Escherichia coli: colicin E1 is a potential virulence factor

BackgroundBacteriocin production is an important characteristic of E. coli strains of human origin. To date, 26 colicin and 9 microcin types have been analyzed on a molecular level allowing molecular detection of the corresponding genes. The production incidence of 29 bacteriocin types and E. coli phylogroups were tested in a set of 361 E. coli strains isolated from human urinary tract infections (UTI) and in 411 control strains isolated from feces of patients without bacterial gut infection.ResultsProduction of 17 and 20 individual bacteriocin types was found in the UTI and control strains, respectively. Microcin H47 encoding determinants were found more often among UTI strains compared to controls (37.9% and 27.0% respectively, p = 0.02) and strains producing microcin H47 belonged predominantly to phylogroup B2 when compared to other bacteriocin producers (67.4% and 36.7%, respectively; p < 0.0001). Producers of 3 or more identified bacteriocin types were more common in the UTI group (20.0% compared to 12.4% in controls, p = 0.03). In the UTI strains, there was a markedly higher number of those producing colicin E1 compared to controls (22.1% to 10.2%, respectively, p = 0.0008). Moreover, colicin E1 production was more common in the UTI bacteriocinogenic strains with multi-producer capabilities. As shown by Southern blotting, pColE1 DNA was not recognized by the ColIa probe and vice versa suggesting that pColE1 was independently associated with pColIa in UTI strains.ConclusionE. coli strains isolated from human urinary tract infections showed increased incidence of microcin H47 and colicin E1 production, respectively. Moreover, colicin E1 itself appears to be a potentially important virulence factor of certain uropathogenic E. coli strains.

Bacteriocin-encoding genes and ExPEC virulence determinants are associated in human fecal Escherichia coli strains

BackgroundA set of 1181 E. coli strains of human fecal origin isolated in the South Moravia region of the Czech Republic was collected during the years 2007–2010. Altogether, 17 virulence determinants and 31 bacteriocin-encoding genes were tested in each of them.ResultsThe occurrence of bacteriocin-encoding genes was found to be positively correlated with the occurrence of E. coli virulence factors. Based on the presence of virulence factors and their combinations, E. coli strains were classified as non-pathogenic E. coli (n = 399), diarrhea-associated E. coli (n = 179) and ExPEC strains (n = 603). Non-pathogenic and diarrhea-associated E. coli strains had a low frequency of bacteriocinogeny (32.6% and 36.9%, respectively). ExPEC strains encoding S-fimbriae (sfa), P-fimbriae (pap) and having genes for aerobactin biosynthesis (aer, iucC), α-hemolysis (α-hly) and cytotoxic necrosis factor (cnf1) were often bacteriocinogenic (73.8%), had a high prevalence of bacteriocin multi-producers and showed a higher frequency of genes encoding microcins H47, M, V, B17 and colicins E1, Ia and S4.ConclusionsThe occurrence of bacteriocin-encoding genes and ExPEC virulence determinants correlate positively in E. coli strains of human fecal origin. Bacteriocin synthesis appears to modulate the ability of E. coli strains to reside in the human intestine and/or the virulence of the corresponding strains.

Characterization of Human Uropathogenic ESBL-Producing Escherichia coli in the Czech Republic: Spread of CTX-M-27-Producing Strains in a University Hospital

AIMS The purpose of this study was to characterize the extended-spectrum β-lactamase (ESBL)-producing uropathogenic Escherichia coli (UPEC) strains isolated in the South Moravia region of the Czech Republic. RESULTS Out of 109 ESBL-producing UPEC isolates, the CTX-M-15-producing E. coli O25b-ST131 was detected in 55 (50.5%) and the CTX-M-27-producing E. coli O25b-ST131 in 40 isolates (36.7%). Most isolates were distributed among three pulsed-field gel electrophoresis clusters and were characterized by low variability relative to antibiotic resistance patterns, in E. coli phylogroups and by the prevalence of virulence and bacteriocin determinants. Despite this, 14 groups of identical isolates (comprising a total of 41 isolates) were identified when all tested parameters of E. coli were combined. CONCLUSIONS Since the occurrence of E. coli B2-O25b-ST131 CTX-M-27 was only recently described in Asia, the frequent isolation of this lineage among patients in South Moravia suggests an efficient transfer of this clone from Asian countries. The limited variability of detected parameters of ESBL-producing UPEC strains is consistent with a common origin of the analyzed isolates, in which there is an ongoing process of genetic diversification.

Microcin determinants are associated with B2 phylogroup of human fecal Escherichia coli isolates

Escherichia coli strains are classified into four main phylogenetic groups (A, B1, B2, and D) and strains of these phylogroups differ in a number of characteristics. This study tested whether human fecal E. coli isolates belonging to different phylogroups differ in prevalence of bacteriocinogenic isolates and prevalence of individual bacteriocinogenic determinants. A set of 1283 fecal E. coli isolates from patients with different diseases was tested for the presence of DNA regions allowing classification into E. coli phylogroups and for the ability to produce bacteriocins (23 colicins and 7 microcins). Of the isolates tested, the most common was phylogroup B2 (38.3%) followed by phylogroups A (28.3%), D (26.3%) and B1 (7.2%). Altogether, 695 bacteriocin producers were identified representing 54.2% of all tested isolates. The highest prevalence of bacteriocin producers was found in group B2 (60.3%) and the lowest in group B1 (44.6%). Determinants encoding colicins E1, Ia, and microcin mV were most common in phylogroup A, determinants encoding microcins mM and mH47 were most common in phylogroup B2, and determinant encoding mB17 was most common in phylogroup D. The highest prevalence of bacteriocinogeny was found in phylogroup B2, suggesting that bacteriocinogeny and especially the synthesis of microcins was associated with virulent and resident E. coli strains.

Human Escherichia coli isolates from hemocultures: Septicemia linked to urogenital tract infections is caused by isolates harboring more virulence genes than bacteraemia linked to other conditions

Escherichia coli is the most common cause of bloodstream infections and community-acquired sepsis. The main aim of this study was to determine virulence characteristics of E. coli isolates from hemocultures of patients with a primary disease of urogenital tract, digestive system, a neoplastic blood disease, or other conditions. Results from a set of 314 E. coli isolates from hemocultures were compared to data from a previously published analysis of 1283 fecal commensal E. coli isolates. Genetic profiling of the 314 E. coli isolates involved determination of phylogenetic group (A, B1, B2, D, C, E, and F), identification of 21 virulence factors, as well as 30 bacteriocin-encoding determinants. Pulsed-field gel electrophoresis was used to analyze clonal character of the hemoculture-derived isolates. The E. coli isolates from hemocultures belonged mainly to phylogenetic groups B2 (59.9%) and D (21.0%), and less frequently to phylogroups A (10.2%) and B1 (5.7%). Commonly detected virulence factors included adhesins (fimA 92.0%, pap 47.1%, and sfa 26.8%), and iron-uptake encoding genes (fyuA 87.9%, fepC 79.6%, aer 70.7%, iucC 68.2%, and ireA 13.7%), followed by colibactin (pks island 31.5%), and cytotoxic necrotizing factor (cnf1 11.1%). A higher frequency of microcin producers (and microcin M determinant) and a lower frequency of colicin Ib and microcin B17 was found in hemoculture-derived isolates compared to commensal fecal isolates. E. coli isolates from hemocultures harbored more virulence genes compared to fecal E. coli isolates. In addition, hemoculture E. coli isolates from patients with primary diagnosis related to urogenital tract were clearly different and more virulence genes were detected in these isolates compared to both fecal isolates and hemoculture-derived isolates from patients with blood and gastrointestinal diseases.

Identification of AHK2- and AHK3-like cytokinin receptors in Brassica napus reveals two subfamilies of AHK2 orthologues

Cytokinin (CK) signalling is known to play key roles in the regulation of plant growth and development, crop yields, and tolerance to both abiotic stress and pathogen defences, but the mechanisms involved are poorly characterized in dicotyledonous crops. Here the identification and functional characterization of sensor histidine kinases homologous to Arabidopsis CK receptors AHK2 and AHK3 in winter oilseed rape are presented. Five CHASE-containing His kinases were identified in Brassica napus var. Tapidor (BnCHK1-BnCHK5) by heterologous hybridization of its genomic library with gene-specific probes from Arabidopsis. The identified bacterial artificial chromosome (BAC) clones were fingerprinted and representative clones in five distinct groups were sequenced. Using a bioinformatic approach and cDNA cloning, the precise gene and putative protein domain structures were determined. Based on phylogenetic analysis, four AHK2 (BnCHK1-BnCHK4) homologues and one AHK3 (BnCHK5) homologue were defined. It is further suggested that BnCHK1 and BnCHK3, and BnCHK5 are orthologues of AHK2 and AHK3, originally from the B. rapa A genome, respectively. BnCHK1, BnCHK3, and BnCHK5 displayed high affinity for trans-zeatin (1-3nM) in a live-cell competitive receptor assay, but not with other plant hormones (indole acetic acid, GA3, and abscisic acid), confirming the prediction that they are genuine CK receptors. It is shown that BnCHK1 and BnCHK3, and BnCHK5 display distinct preferences for various CK bases and metabolites, characteristic of their AHK counterparts, AHK2 and AHK3, respectively. Interestingly, the AHK2 homologues could be divided into two subfamilies (BnCHK1/BnCK2 and BnCHK3/BnCHK4) that differ in putative transmembrane domain topology and CK binding specificity, thus implying potential functional divergence.

Human Escherichia coli strains of different geographical and time source: bacteriocin types and their gene sequences are population‐specific

Bacteriocin production was tested in two sets of Escherichia coli strains: one isolated in 1978 from healthy children of rubber tree tappers and Indians in Amazonia, Brazil (n = 35), and the second one isolated in 2004 from healthy adult volunteers in the Moravia region, Czech Republic (n = 53). Although the occurrence of bacteriocin production was rather similar in both groups (54.3% and 43.4% respectively), the spectra of bacteriocin-encoding determinants in both groups were different. Altogether, 12 different bacteriocin-encoding determinants were found among the tested strains. The occurrence of colicin E1- and Y-genes was higher (P = 0.02 and P = 0.009 respectively) while the occurrence of microcin V gene was lower (P = 0.02) among Amazonian strains compared with Moravian strains. The colicin-encoding determinants of colicins Ia, M, Y and E1 were amplified from Amazonian and Moravian producer strains and sequenced, as were chromosomal 16S rRNA, gyrB and tonB genes. While sequence alignments of chromosomal loci revealed no clusters with respect to geographical origin of strains, the colicin-encoding genes were very similar among the strains of each origin but different between the two geographic groups.

Unique Activity Spectrum of Colicin FY: All 110 Characterized Yersinia enterocolitica Isolates Were Colicin FY Susceptible

Colicin FY is a plasmid encoded toxin that recognizes a yersinia-specific outer membrane protein (YiuR) as a receptor molecule. We have previously shown that the activity spectrum of colicin FY comprises strains of the genus Yersinia. In this study, we analyzed the activity of colicin FY against 110 Yersinia enterocolitica isolates differing in geographical origin and source. All isolates were characterized through analysis of 16S rRNA genes, serotyping, biotyping, restriction profiling of genomic DNA, detection of virulence markers and susceptibility to antibiotics. This confirmed the broad variability of the collection, in which all 110 Y. enterocolitica isolates, representing 77 various strains, were inhibited by colicin FY. Although isolates showed variable levels of susceptibility to colicin FY, it was not associated with any strain characteristic. The universal susceptibility of Y. enterocolitica strains to colicin FY together with the absence of activity towards strains outside the Yersinia genus suggests potential therapeutic applications for colicin FY.

Colicins U and Y inhibit growth of Escherichia coli strains via recognition of conserved OmpA extracellular loop 1

Interactions of colicins U and Y with the OmpA (Outer membrane protein A) receptor molecule were studied using site-directed mutagenesis and colicin binding assay. A systematic mutagenesis of the colicin-susceptible OmpA sequence from Escherichia coli (OmpAEC) to the colicin-resistant OmpA sequence from Serratia marcescens (OmpASM) was performed in regions corresponding to extracellular OmpA loops 1-4. Susceptibility to colicins U and Y was significantly affected by the OmpA mutation in loop 1. As with functional analysis, a decrease in binding capacity of His-tagged colicin U was found for recombinant OmpA with a mutated segment in loop 1 compared to control OmpAEC. To verify the importance of the identified amino acid residues in OmpA loop 1, we introduced loop 1 from OmpAEC into OmpASM, which resulted in the substantial increase of susceptibility to colicins U and Y. In addition, colicins U and Y were tested against a panel of 118 bacteriocin non-producing strains of four Escherichia species, including E. coli (39 strains), E. fergusonii (10 strains), E. hermannii (42 strains), and E. vulneris (27 strains). A majority (82%) of E. coli strains was susceptible to colicins U and Y. Interestingly, colicins U and Y also inhibited all of the 30 tested multidrug-resistant E. coli O25b-ST131 isolates. These findings, together with the fact that OmpA loop 1 is important for bacterial virulence and is evolutionary conserved, offer the potential of using colicins U and Y as specific anti-OmpA loop 1 directed antibacterial proteins.

The effect of butyrate-supplemented parenteral nutrition on intestinal defence mechanisms and the parenteral nutrition-induced shift in the gut microbiota

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ) and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10 and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/ pathogenic genera, which might contribute to its overall beneficial effect.