[Well-known and new variants of pathogenic Escherichia coli as a consequence of the plastic genome]. / Znane i nowe warianty patogennych Escherichia coli jako konsekwencja plastycznego genomu.

E. coli is a diverse bacterial species encompassing commensal as well as intestinal and extraintestinal pathogenic strains. The ability to adapt to so many different niches in the host organism is determined by the extreme genomic plasticity of E. coli. The genetic diversity is due to a complex phylogenetic structure in which besides the well-known main groups A, B1, B2 and D, four new groups, C, E, F and Clad I, have been characterized recently. The mobile gene pool exchanged by horizontal gene transfer (HGT) is another important driving force in the evolution of E. coli. Pathogenicity of strains is conditioned by a specific repertoire of virulence factors located on the mobile genetic elements and transmitted by HGT. The environment changing constantly stimulates the formation of new virulence gene combinations that generate the formation, not observed so far, of new pathogenic clones of higher capacity for virulence and greater expansiveness. The presence of very similar virulence plasmids carrying conserved combinations of the virulence genes (CVP) among extraintestinal pathogenic strains in humans and birds has been observed. It indicates a real possibility of occurrence of common virulence factors. The increase in drug resistance among pathogenic E. coli is also reflected in the prevalence of highly expansive clones exhibiting both high virulence and resistance. The presented data indicate that further studies are required to determine the interdependencies of resistance and virulence at the genetic level to help improve our management of the infectious diseases caused by these bacteria.

Age as a factor influencing diversity of commensal E. coli microflora in pigs.

Commensal, intestinal E. coli microflora plays a role in maintenance of intestinal balance of the host, is responsible for defending against pathogenic E. coli. This study encompasses the analysis of BOX-PCR fingerprinting patterns, phylogenetic grouping and virulence genes prevalence among commensal E. coli isolates derived from healthy pigs. Altogether, 274 unique E. coli isolates were identified, 110 from weaned piglets (Piglets I and Piglets II) and 164 from adult sows (Sows I and Sows II). BOX-PCR analysis distinguished isolates from pigs in different age and indicated that during maturation the changes in E. coli microflora occurred. Phylogenetic grouping revealed significant differences between distribution of four phylogenetic groups among isolates derived from piglets and sows. In phylogenetic structure of isolates from the piglets group B1 prevailed significantly, while among isolates derived from the sows the majority of them were classified into phylogenetic group A. The identification of 17 virulence factors in E. coli isolates derived from healthy pigs was performed. Three of 13 intestinal (escV, ehxA, estII) and four extra-intestinal virulence genes (VGs) (hlyA, fimH, papA, sfaS) were detected in the porcine isolates. The percentage of VGs positive isolates among piglets is higher than among sows, moreover, the VGs occurring in E. coli isolates from piglets revealed greater diversity than that detected among isolates from sows.

The phenotypic and genotypic characteristics of antibiotic resistance in Escherichia coli populations isolated from farm animals with different exposure to antimicrobial agents.

The aim of the study was to determine the influence of the presence or the absence of antibiotic input on the emergence and maintenance of resistance in commensal bacteria from food producing animals. The research material constituted E. coli isolates from two animal species: swine at different age from one conventional pig farm with antibiotic input in young pigs and from beef and dairy cattle originated from organic breeding farm. The sensitivity to 16 antimicrobial agents was tested, and the presence of 15 resistance genes was examined. In E. coli from swine, the most prevalent resistance was resistance to streptomycin (88.3%), co-trimoxazole (78.8%), tetracycline (57.3%) ampicillin (49.3%) and doxycycline (44.9%) with multiple resistance in the majority. The most commonly observed resistance genes were: bla(TEM) (45.2%), tetA (35.8%), aadA1 (35.0%), sul3 (29.5%), dfrA1 (20.4%). Differences in phenotypes and genotypes of E. coli between young swine undergoing prevention program and the older ones without the antibiotic pressure occurred. A disparate resistance was found in E. coli from cattle: cephalothin (36.9%), cefuroxime (18.9%), doxycycline (8.2%), nitrofurantoin (7.7%), and concerned mainly dairy cows. Among isolates from cattle, multidrug resistance was outnumbered by resistance to one or two antibiotics and the only found gene markers were: bla(SHV), (3.4%), tetA (1.29%), bla(TEM) (0.43%) and tetC (0.43%). The presented outcomes provide evidence that antimicrobial pressure contributes to resistance development, and enteric microflora constitutes an essential reservoir of resistance genes.

Antibacterial Activity of Bacteriocinogenic Commensal Escherichia coli against Zoonotic Strains Resistant and Sensitive to Antibiotics.

Antibiotic resistance concerns various areas with high consumption of antibiotics, including husbandry. Resistant strains are transmitted to humans from livestock and agricultural products via the food chain and may pose a health risk. The commensal microbiota protects against the invasion of environmental strains by secretion of bacteriocins, among other mechanisms. The present study aims to characterize the bactericidal potential of bacteriocinogenic Escherichia coli from healthy humans against multidrug-resistant and antibiotic-sensitive strains from pigs and cattle. Bacteriocin production was tested by the double-layer plate method, and bacteriocin genes were identified by the PCR method. At least one bacteriocinogenic E. coli was detected in the fecal samples of 55% of tested individuals, adults and children. Among all isolates (n = 210), 37.1% were bacteriocinogenic and contained genes of colicin (Col) Ib, ColE1, microcin (Mcc) H47, ColIa, ColM, MccV, ColK, ColB, and single ColE2 and ColE7. Twenty-five E. coli carrying various sets of bacteriocin genes were further characterized and tested for their activity against zoonotic strains (n = 60). Strains with ColE7 (88%), ColE1-ColIa-ColK-MccH47 (85%), MccH47-MccV (85%), ColE1-ColIa-ColM (82%), ColE1 (75%), ColM (67%), and ColK (65%) were most active against zoonotic strains. Statistically significant differences in activity toward antibiotic-resistant strains were shown by commensal E. coli carrying MccV, ColK-MccV, and ColIb-ColK. The study demonstrates that bacteriocinogenic commensal E. coli exerts antagonistic activity against zoonotic strains and may constitute a defense line against multidrug-resistant strains.

Extended Phylogeny and Extraintestinal Virulence Potential of Commensal Escherichia coli from Piglets and Sows.

Commensal Escherichia coli, naturally occurring in the intestinal tract, can be the origin of extraintestinal pathogenic E. coli (ExPEC) strains. ExPEC causes high mortality and significant economic losses in the swine industry in several countries and poses a serious threat to public health worldwide. The aim of this study was to analyze the extended phylogenetic structure and extraintestinal virulence potential in two groups of commensal E. coli isolates from post-weaning piglets and sows. The phylogenetic assignment to eight groups was determined using the revised Clermont phylogenetic typing method in quadruplex PCR. Identification of extraintestinal virulence genes (VGs) and adhesin operon genes was performed using multiplex or simplex PCR. The revised phylogenetic assignment allowed us to distinguish E. coli with significantly higher (groups C and F) or lower (group E) virulence potential in isolates from piglets. The majority of the tested VGs occurred more frequently in isolates from piglets than from sows, with statistically significant differences for seven genes: fimH, papAH, iutA, iroN, ompT, traT, and iss. Complete operons for type I and P fimbriae significantly prevailed among E. coli from piglets. This study provides insight into the extended phylogenetic structure of porcine commensal E. coli and showed that these strains, particularly from piglets, constitute a considerable reservoir of extraintestinal VGs and may increase the potential risk of extraintestinal infections.

[The interactions of actin cell and membrane skeleton proteins with lipids]. / Bialka aktynowego szkieletu komórki i blony partnerami lipidów.

The results of many studies indicate that many cytoskeletal proteins interact with lipids, or are regulated by phosphoinositides. Proteins may associate with membranes through specific domains, amphipathic helices and undefined motifs that interact through electrostatic or hydrophobic interactions. The interaction between specific proteins and certain lipids affect stabilization of lipid microdomains, which may provide an anchor for cytoskeletal proteins. In vitro, proteins that sever or depolymerize actin filaments, such as gelsolin, villin, cofilin and profilin, are inactivated by PI(4,5)P2. Conversely, proteins like e.g. vinculin, talin, alpha-actinin, ezrin, N-WASP, WAVE that promote actin polymerization and link actin filaments to each other, are activated by this lipid. The major components of red blood cell membrane skeleton: spectrin and protein 4.1, also their nonerythroid counterparts interact with lipids, and those interactions may be regulated by phosphoinositides.

Complexity of Antibiotic Resistance in Commensal Escherichia coli Derived from Pigs from an Intensive-Production Farm.

Antibiotics in animal husbandry are used to maintain welfare, but lead to the generation of resistant strains. We analyzed commensal multidrug-resistant Escherichia coli from pigs at the beginning and end of the production cycle in a farm with a farrow-to-finish system in order to investigate whether clonal spread or horizontal gene transfer constitutes the main factor responsible for the prevalence of resistance in this environment. Among 380 isolates, 56 multidrug-resistant E. coli with a similar resistant phenotype were selected for more detailed investigations including a genomic similarity analysis and the detection of mobile elements. Isolates carried blaTEM-1, aadA1, strA/B, tetA, tetB, tetC, dfrA1, dfrA5, dfrA7, dfrA12, sul1, sul2, sul3, and qnrS resistance genes, with the common co-occurrence of genes encoding the same resistance phenotype. A pulse-field gel electrophoresis analysis of the genomic similarity of multidrug-resistant E. coli showed ≤65% similarity of most of the tested strains and did not reveal a dominant clone responsible for the prevalence of resistance. Class 1 and 2 integrons and transposons 7 and 21 were detected among mobile elements; however, some were truncated. Plasmids were represented by 11 different incompatibility groups (K, FIB, I1, FIIA, FIC, FIA, Y, P, HI1, B/O, and T). Genetic resistance traits were unevenly spread in the clonal groups and suggested the major rearrangement of genetic material by horizontal gene transfer. The present results revealed that in commensal E. coli from pigs in a homogeneous farm environment, there was no dominant clone responsible for the spread of resistance and persistence in the population.

Comparison of Commensal Escherichia coli Isolates from Adults and Young Children in Lubuskie Province, Poland: Virulence Potential, Phylogeny and Antimicrobial Resistance.

Commensal Escherichia coli population is a dynamic structure which may be important in the pathogenesis of extraintestinal infections. The aim of this study was the comparison of genetic diversity of commensal E. coli isolates from two age group-adults and young children. E. coli strains were isolated on MacConkey agar and identified by biochemical tests. Determination of four major phylogenetic groups, identification of virulence genes and antimicrobial resistance determinants were performed by using multiplex or simplex PCR. Phenotypic analysis of resistance was based on disc-diffusion method. The prevalence of virulence genes was significantly higher among isolates from adults than from young children. Phylogroup B2 predominated among E. coli from adults, whereas phylogroup A was the most common in isolates from young children. The analyses of antimicrobial resistance revealed that resistance to at least one antimicrobial agent and multidrug-resistance were detected significantly more frequent in the isolates from adults than from young children. This study documented that the commensal E. coli isolates from adults showed greater genetic diversity than from young children and constitutes a substantial reservoir of the virulence genes typical for extraintestinal pathogenic E. coli.

Spectrin-phospholipid interactions. Existence of multiple kinds of binding sites?

The object of this paper is to review briefly the studies on the interactions of erythroid and non-erythroid spectrins with lipids in model and natural membranes. An important progress on the identification of lipid-binding sites has recently been made although many questions remain still unanswered. In particular, our understanding of the physiological role of such interactions is still limited. Another important issue is the occurrence of spectrins in membrane rafts, how they are attached to the raft and what is their function in rafts.

Mapping of an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine mono- and bi-layer binding site in erythroid beta-spectrin.

It has been shown previously that binding of vesicles and monolayers containing PE (phosphatidylethanolamine) by either erythroid or non-erythroid spectrin proved sensitive to inhibition by purified erythrocyte ankyrin. We tested the lipid-binding affinities of the purified ankyrin-binding domain of beta-spectrin and of its truncated mutants in four ways, by analysing: (1) penetration of 'loose' PE/PC (phosphatidylcholine) monolayers; (2) binding to liposomes in suspension; (3) competition with spectrin for liposomes; and (4) binding of a PE/PC monolayer in a surface plasmon resonance system. The results obtained indicated that the full-length ankyrin-binding domain bound PE/PC mono- and bi-layers with moderate affinity, penetrated monolayers and competed with spectrin for liposomes. Moreover, its truncated mutants that retained the N-terminal part, in contrast with those lacking eight or 38 N-terminal residues (which bound lipid mono- and bi-layers with lower affinity), bound PE/PC mono- and bi-layers with an affinity and capacity comparable with those of the full-length ankyrin-binding domain, and this activity was inhibited by purified erythrocyte ankyrin. The full-length domain, in contrast with the mutant lacking 38 N-terminal residues, induced a small increase in the fluidity of PE/PC membranes when probed with 5'-doxyl stearate, similar to the effect of purified spectrin. Therefore we conclude that the binding site for PE-rich lipids, which is sensitive to ankyrin inhibition, is located in a 38-residue N-terminal fragment of the beta-spectrin ankyrin-binding domain, and that the first eight residues play a key role in this activity.

Antimicrobial resistance in commensal Escherichia coli from pigs during metaphylactic trimethoprim and sulfamethoxazole treatment and in the post-exposure period.

The prevalence of trimethoprim (TMP) and sulfamethoxazole (SMX) resistance in commensal E. coli from pigs was tested in this study. E. coli was derived from three groups of piglets in successive stages of metaphylactic therapy and from two groups of sows 10 and 18 weeks after the treatment. MIC values of TMP and SMX were determined for a total of 352 strains. The presence of resistance genes (dfrA1, dfrA5, dfrA7, dfrA12, dfrA17, sul1, sul2, sul3) and class 1 and 2 integron-associated dfrA gene cassettes was tested. Resistance to TMP was very high during the administration of the antimicrobial (from 97 to 100%) and amounted to 86% and 69% in the post-exposure period; MIC > 32 mg/L. The isolates from all groups of pigs were resistant to sulfamethoxazole, with MIC > 1028 mg/L. The dfrA1 and sul1 genes (as part of integrons) dominated in E. coli from piglets, but the dfrA12 and sul1 genes were prevalent in E. coli from sows. Coexistence of the different dfrA genes was detected in 71 isolates from all groups of swine. Transcription analysis revealed that most of these genes were not transcribed, particularly gene cassettes of class 1 integrons. The research revealed a high level of resistance associated with the metaphylactic treatment, persistence and circulation of resistance in bacterial populations. Diverse genetic background with multiple and not transcribed resistance genes was observed.

Prevalence of virulence determinants and antimicrobial resistance among commensal Escherichia coli derived from dairy and beef cattle.

Cattle is a reservoir of potentially pathogenic E. coli, bacteria that can represent a significant threat to public health, hence it is crucial to monitor the prevalence of the genetic determinants of virulence and antimicrobial resistance among the E. coli population. The aim of this study was the analysis of the phylogenetic structure, distribution of virulence factors (VFs) and prevalence of antimicrobial resistance among E. coli isolated from two groups of healthy cattle: 50 cows housed in the conventional barn (147 isolates) and 42 cows living on the ecological pasture (118 isolates). The phylogenetic analysis, identification of VFs and antimicrobial resistance genes were based on either multiplex or simplex PCR. The antimicrobial susceptibilities of E. coli were examined using the broth microdilution method. Two statistical approaches were used to analyse the results obtained for two groups of cattle. The relations between the dependent (VFs profiles, antibiotics) and the independent variables were described using the two models. The mixed logit model was used to characterise the prevalence of the analysed factors in the sets of isolates. The univariate logistic regression model was used to characterise the prevalence of these factors in particular animals. Given each model, the odds ratio (OR) and the 95% confidence interval for the population were estimated. The phylogroup B1 was predominant among isolates from beef cattle, while the phylogroups A, B1 and D occurred with equal frequency among isolates from dairy cattle. The frequency of VFs-positive isolates was significantly higher among isolates from beef cattle. E. coli from dairy cattle revealed significantly higher resistance to antibiotics. Some of the tested resistance genes were present among isolates from dairy cattle. Our study showed that the habitat and diet may affect the genetic diversity of commensal E. coli in the cattle. The results suggest that the ecological pasture habitat is related to the increased spreading rate of the VFs, while the barn habitat is characterised by the higher levels of antimicrobial resistance among E. coli.

Ankyrins, multifunctional proteins involved in many cellular pathways.

Ankyrins, originally discovered as components of the erythrocyte membrane appeared to be a family of animal proteins encoded in mammalian cells by three related genes. Developmentally regulated, tissue specific posttranscriptional processing generates a great variety of isoforms which seem to play specific role in various cells and subcellular structures, being involved, for example, in membrane skeleton organisation, ionic transport, maintenance of cell polarity as well as cell-cell adhesion regulation. The interaction between the membrane skeleton and cytoplasmic domains of transmembrane proteins plays a fundamental role in membrane integrity and stability as well as in many cellular processes. Once the cDNA sequence of red blood cell ankyrin was determined it became clear that "ankyrin-repeat" motifs are present in many proteins whose function is rather unrelated to the membrane skeleton, e.g. transcription factors. Ankyrins are a multigene family of intracellular, structural proteins that link several integral membrane proteins and the spectrin-based membrane cytoskeleton. The anion exchanger, Na+-K+ ATPase, a voltage dependent Na+ channel, an Na+/Ca2+-exchanger, and adhesion molecules have been reported to interact with ankyrin in nonerythroid cells. Ankyrin was first found to link integral membrane proteins to the underlying spectrin-actin based membrane skeleton in the human erythrocyte. It was subsequently described in a variety of vertebrate cells and tissues, including brain, epithelia, and skeletal muscle. Variable cellular localisation of these membrane proteins may be possible due to different relative affinities of various isoforms of ankyrin for target proteins.

Proteins with spectrin motifs which do not belong to the spectrin-alpha-actinin-dystrophin family.

Using several consensus sequences for the 106 amino acid residue alpha-spectrin repeat segment as probes we searched animal sequence databases using the BLAST program in order to find proteins revealing limited, but significant similarity to spectrin. Among many spectrins and proteins from the spectrin-alpha-actinin-dystrophin family as well as sequences showing a rather high degree of similarity in very short stretches, we found seven homologous animal sequences of low overall similarity to spectrin but showing the presence of one or more spectrin-repeat motifs. The homology relationship of these sequences to alpha-spectrin was further analysed using the SEMIHOM program. Depending on the probe, these segments showed the presence of 6 to 26 identical amino acid residues and a variable number of semihomologous residues. Moreover, we found six protein sequences, which contained a sequence fragment sharing the SH3 (sarc homology region 3) domain homology of 42-59% similarity. Our data indicate the occurrence of motifs of significant homology to alpha-spectrin repeat segments among animal proteins, which are not classical members of the spectrin-alpha-actinin-dystrophin family. This might indicate that these segments together with the SH3 domain motif are conserved in proteins which possibly at the early stage of evolution were close cognates of spectrin-alpha-actinin-dystrophin progenitors but then evolved separately.

Type 1 fimbriae in commensal Escherichia coli derived from healthy humans.

Type 1 fimbriae are one of the most important factors of Escherichia coli adaptation to different niches in the host. Our study indicated that the genetic marker--fimH gene occurred commonly in commensal E. coli derived from healthy humans but expression of the type 1 fimbriae was not observed. Identification of fim structural subunit genes (fimA-fimH) and recombinase fimE and fimB genes showed that many of the strains were carrying an incomplete set of genes and the genes expression study revealed that in strains with complete set of fim genes, the fimC gene, encoding the chaperone protein, was not expressed.

Key amino acid residues of ankyrin-sensitive phosphatidylethanolamine/phosphatidylcholine-lipid binding site of ßI-spectrin.

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of ß-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that "opening" of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.

Structural insight into an ankyrin-sensitive lipid-binding site of erythroid beta-spectrin.

It was recently shown that the region within beta-spectrin responsible for interactions with ankyrin includes a lipid-binding site which displayed sensitivity to inhibition by ankyrin. We studied its structure by constructing a series of single and double spin-labeled beta-spectrin-derived peptides and analyzing their spin-spin distances via electron paramagnetic resonance spectroscopy and the Fourier deconvolution method. The results indicate that the whole ankyrin-sensitive lipid-binding site of beta-spectrin exhibits a helical conformation revealing a distinct 3(10)-helix contribution at its N-terminus. The start of the helix was located five residues upstream along the sequence compared to the theoretical predictions. A model based on the obtained data provides direct evidence that the examined lipid-binding site is a highly amphipathic helix, which is correlated with the specific conformation of its N-terminal fragment.

Lipid-binding role of betaII-spectrin ankyrin-binding domain.

It is known that erythroid and non-erythroid spectrins binding of vesicles and monolayers containing PE proved sensitive to inhibition by red blood cell ankyrin. We now show that the bacterially-expressed recombinant peptides representing betaII(brain)-spectrin's ankyrin-binding domain and its truncated mutants showed lipid-binding activity, although only those containing a full-length amino terminal fragment showed high to moderate affinity towards phospholipid mono- and bilayers and a substantial sensitivity of this binding to inhibition by ankyrin. These results are in accordance with our published data on betaI-spectrin's ankyrin-binding domain [Hryniewicz-Jankowska A, et al. Mapping of ankyrin-sensitive, PE/PC mono- and bilayer binding site in erythroid beta-spectrin. Biochem J 2004;382:677-85]. Moreover, we tested also the effect of transient transfection of living cells of several cell-lines with vectors coding for GFP-conjugates including betaII and also betaI full-length ankyrin-binding domain and their truncated fragments on the membrane skeleton organization. The transfection with constructs encoding full-length ankyrin-binding domain of betaII and betaI spectrin resulted in increased aggregation of membrane skeleton and its punctate appearance in contrast to near normal appearance of membrane skeleton of cells transiently transfected with GFP control or construct encoding ankyrin-binding domain truncated at their N-terminal region. Our results therefore indicate the importance of N-terminal region for lipid-binding activity of the beta-spectrin ankyrin-binding domain and its substantial role in maintaining the spectrin-based skeleton distribution.