Genetic and Phenotypic Features to Screen for Putative Adherent-Invasive Escherichia coli

To date no molecular tools are available to identify the adherent-invasive Escherichia coli (AIEC) pathotype, which has been associated with Crohn’s disease and colonizes the intestine of different hosts. Current techniques based on phenotypic screening of isolates are extremely time-consuming. The aim of this work was to search for signature traits to assist in rapid AIEC identification. The occurrence of at least 54 virulence genes (VGs), the resistance to 30 antibiotics and the distribution of FimH and ChiA amino acid substitutions was studied in a collection of 48 AIEC and 56 non-AIEC isolated from the intestine of humans and animals. χ2 test was used to find frequency differences according to origin of isolation, AIEC phenotype and phylogroup. Mann–Whitney test was applied to test association with adhesion and invasion indices. Binary logistic regression was performed to search for variables of predictive value. Animal strains (N = 45) were enriched in 12 VGs while 7 VGs were more predominant in human strains (N = 59). The prevalence of 15 VGs was higher in AIEC (N = 49) than in non-AIEC (N = 56) strains, but only pic gene was still differentially distributed when analyzing human and animal strains separately. Among human strains, three additional VGs presented higher frequency in AIEC strains (papGII/III, iss and vat; N = 22) than in non-AIEC strains (N = 37). No differences between AIEC/non-AIEC were found in FimH variants. In contrast, the ChiA sequence of LF82 was shared with the 35.5% of AIEC studied (N = 31) and only with the 7.4% of non-AIEC strains (N = 27; p = 0.027). Binary logistic regression analysis, using as input variables all the VGs and antibiotic resistances tested, revealed that typifying E. coli isolates using pic gene and ampicillin resistance was useful to correctly classify strains according to the phenotype with a 75.5% of accuracy. Although there is not a molecular signature fully specific and sensitive to identify the AIEC pathotype, we propose two features easy to be tested that could assist in AIEC screening. Future work using additional strain collections would be required to assess the applicability of this method

Genetics and transcriptomics of adherent-invasive Escherichia coli (AIEC): new approaches to uncover molecular markers for its rapid identification

The adherent-invasive Escherichia coli (AIEC) pathotype could play a role in the course of Crohn’s disease. This is characterized by its capacity to adhere to and to invade intestinal epithelial cells as well as to replicate and survive within macrophages. At present, identification of the AIEC pathotype relies on time-consuming techniques based on the phenotypic screening of cultured bacteria, which are not standardized. In this thesis, we focused on the study of AIEC genetics in order to look for key characteristics that could assist the development of a molecular tool for the identification of the pathotype. To sum up, the results of this work provide meaningful information that contributes to our understanding of AIEC genomics. In this case, two putative molecular markers resulting from a combination of genetic and/or phenotypic features have been presented and these could assist in AIEC screening. Finally, gene expression results provide new insights to better describe genes putatively involved in AIEC virulenceEl patotip adherent-invasiu d’Escherichia coli (AIEC) podria jugar un paper en el transcurs de la malaltia de Crohn. Aquest es caracteritza per tenir capacitat d’adhesió i invasió a cèl·lules de l’epiteli intestinal a més de replicar-se i sobreviure en macròfags. Actualment la única manera d’identificar aquests bacteris és analitzant aquestes característiques fenotípiques, un mètode poc estandarditzat i que requereix molt temps i dedicació. En la present tesi ens hem centrat en estudiar genèticament el patotip AIEC per tal de buscar característiques clau que puguin ajudar en el desenvolupament d’una eina molecular per a la seva identificació. En resum, els resultats d'aquest treball proporcionen informació significativa que contribueix a la comprensió de la genètica del patotip AIEC. En aquest cas, s'han presentat dos possibles marcadors moleculars resultants d'una combinació de característiques genètiques i/o fenotípiques que podrien ajudar en la detecció d’AIEC. Finalment, els resultats d'expressió gènica proporcionen noves idees per descriure millor els gens implicats en la virulència del patotip AIECPrograma de Doctorat en Biologia Molecular, Biomedicina i Salu

Study of a classification algorithm for AIEC identification in geographically distinct E. coli strains

Adherent-invasive Escherichia coli (AIEC) have been extensively implicated in Crohn\u27s disease pathogenesis. Currently, AIEC is identified phenotypically, since no molecular marker specific for AIEC exists. An algorithm based on single nucleotide polymorphisms was previously presented as a potential molecular tool to classify AIEC/non-AIEC, with 84% accuracy on a collection of 50 strains isolated in Girona (Spain). Herein, our aim was to determine the accuracy of the tool using AIEC/non-AIEC isolates from different geographical origins and extraintestinal pathogenic E. coli (ExPEC) strains. The accuracy of the tool was significantly reduced (61%) when external AIEC/non-AIEC strains from France, Chile, Mallorca (Spain) and Australia (82 AIEC, 57 non-AIEC and 45 ExPEC strains in total) were included. However, the inclusion of only the ExPEC strains showed that the tool was fairly accurate at differentiating these two close pathotypes (84.6% sensitivity; 79% accuracy). Moreover, the accuracy was still high (81%) for those AIEC/non-AIEC strains isolated from Girona and Mallorca (N = 63); two collections obtained from independent studies but geographically close. Our findings indicate that the presented tool is not universal since it would be only applicable for strains from similar geographic origin and demonstrates the need to include strains from different origins to validate such tools

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells

IntroductionAdherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn’s disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers.MethodsComparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers.ResultsComparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection.DiscussionThis is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification

Amino Acid Substitutions and Differential Gene Expression of Outer Membrane Proteins in Adherent-Invasive Escherichia coli.

Variations in the sequence and/or the expression of outer membrane proteins (OMPs) may modulate bacterial virulence. OmpA and OmpC have been involved in the interaction of adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells (IECs). Scarce data exist about OMPs sequence variants in a collection of AIEC strains, and no study of OMPs expression during infection exists. We aimed to determine whether particular mutations or differential expression of OMPs are associated with AIEC virulence. The ompA, ompC, and ompF genes in 14 AIEC and 30 non-AIEC strains were sequenced by Sanger method, and the protein expression profile was analyzed by urea-SDS-PAGE. Gene expression was determined during in vitro bacterial infection of intestine-407 cells by RT-qPCR. The distribution of amino acid substitutions in OmpA-A200V, OmpC-S89N, V220I, and W231D associated with pathotype and specific changes (OmpA-A200V, OmpC-V220I, D232A, OmpF-E51V, and M60K) correlated with adhesion and/or invasion indices but no particular variants were found specific of AIEC. OMPs protein levels did not differ according to pathotype when growing in Mueller-Hinton broth. Interestingly, higher OMPs gene expression levels were reported in non-AIEC growing in association with cells compared with those non-AIEC strains growing in the supernatants of infected cultures (p 0.146). Despite no particular OMPs sequence variants have been found as a common and distinctive trait in AIEC, some mutations could facilitate a better interaction with the host. Moreover, the different behavior between pathotypes regarding OMPs gene expression at different stages of infection could be related with the virulence of the strains

Prevalence, Abundance, and Virulence of Adherent-Invasive Escherichia coli in Ulcerative Colitis, Colorectal Cancer, and Coeliac Disease

Adherent-invasive E. coli (AIEC) has largely been implicated in the pathogenesis of Crohn's disease (CD). E. coli strains with similar genetic backgrounds and virulence genes profiles have been associated with other intestinal disorders, such as ulcerative colitis (UC), colorectal cancer (CRC), and coeliac disease (CeD), but the role of AIEC in these diseases remains unexplored. We aimed to assess the distribution, abundance, and pathogenic features of AIEC in UC, CRC, and CeD.This work was funded by the Spanish Ministry of Education and Science through projects SAF2010-15896, SAF2013-43284-P, and SAF2107-82261-P (MINECO/AEI/FEDER/UE) and the grant AGL2017-88801-P from the Spanish Ministry of Science and Innovation (MICINN, Spain). MLS is a Serra Húnter Fellow.Peer reviewedSAF2010-15896, SAF2013-43284-P, and SAF2107-82261-P (MINECO/AEI/FEDER/UE) and the grant AGL2017-88801-

Nous resultats de les sals de bor per lluitar contra la resistència antimicrobiana

Els antibiòtics són medicaments que s'utilitzen enfront de les infeccions produïdes per bacteris, tant en animals com en persones. Usar-los de manera inadequada pot generar resistència, cosa que significa que deixen de ser efectius. La resistència antimicrobiana és avui dia una de les majors amenaces per a la salut mundial. En aquest article, el grup de recerca de Clara Viñas de l'Institut de Materials de Barcelona (ICMAB-CSIC) en col·laboració amb altres universitats (UdG, Córdoba, Durham), presenta el seu treball en el qual s'ha avaluat les propietats antimicrobianes de les sals de bor. Els resultats semblen ser prometedors per a quatre soques de bacteris i tres de fongs.Los antibióticos son medicamentos que se utilizan frente a las infecciones producidas por bacterias, tanto en animales como en personas. Usarlos de manera inadecuada puede generar resistencia, lo que significa que dejan de ser efectivos. La resistencia antimicrobiana es hoy en día una de las mayores amenazas para la salud mundial. En este artículo, el grupo de investigación de Clara Viñas del Instituto de Materiales de Barcelona (ICMAB-CSIC) en colaboración con otras universidades (UdG, Córdoba, Durham), presenta su trabajo en el que se ha evaluado las propiedades antimicrobianas de las sales de boro. Los resultados parecen ser prometedores para cuatro cepas de bacterias y tres de hongos

Metallacarborane Assemblies as Effective Antimicrobial Agents, Including a Highly Potent Anti-MRSA Agent

The salts Na[ROC(O)Ph], Na[1-ROC(O)-1,12-C2B10H11], K2[1,4-(ROC(O))2-C6H4], and K2[1,12-(ROC(O))2-1,12-C2B10H10], where R is the cobaltabis(dicarbollide)-diethylene glycol group [3,3′-Co(8-(OCH2CH2OCH2CH2)-1,2-C2B9H10)(1′,2′-C2B9H11)]−, were synthesized from the corresponding carboxylate salt and the zwitterion [3,3′-Co(8-(CH2CH2O)2-1,2-C2B9H10)(1′,2′-C2B9H11)]. The dianion in K2[1,12-(ROC(O))2-1,12-C2B10H10] showed at least one K+ cation to be tightly bound to the dianion via K···O and K···H–B interactions with both p-carborane and cobaltabis(dicarbollide) clusters on the basis of NMR and MS data and support of hybrid-DFT computations. An evaluation of the antimicrobial properties of these compounds revealed all of the salts to be highly effective antibacterial agents for four Gram-positive bacteria strains (standard minimum inhibitory concentration, MIC, of 1 mg/L for Na[ROC(O)Ph]; Na[3]) and antifungal agents for three Candida albicans strains (MIC 4 mg/L for the salts K2[4] and K2[6]). One of the four Gram-positive bacteria strains tested was a life-threatening superbug methicillin-resistant Staphylococcus aureus (MRSA) isolate, which is resistant to many commercial antimicrobial drugs. The cobaltabis(dicarbollide) derivative Na[3] has a remarkable inhibitory effect on the MRSA strain with an MIC of only 1 mg/L and a minimum bactericidal concentration (MBC) of 2 mg/L, thus suggesting its potential as an antibacterial agent against MRSA