TY - JOUR A1 - Khan, Muhammad Moman A1 - Sidorczuk, Katarzyna A1 - Becker, Juliane A1 - Aleksandrowicz, Adrianna A1 - Baraniewicz, Karolina A1 - Ludwig, Christina A1 - Ali, Aamir A1 - Kingsley, Robert A. A1 - Schierack, Peter A1 - Kolenda, Rafał T1 - Characterization of clumpy adhesion of Escherichia coli to human cells and associated factors influencing antibiotic sensitivity JF - Microbiology Spectrum N2 - Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and non-pathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. KW - Escherichia coli KW - Clumpy adhesion KW - Motility KW - Antibiotic tolerance KW - Stress KW - Escherichia coli KW - Zelladhäsion KW - Phänotyp KW - Genanalyse Y1 - 2024 U6 - https://doi.org/10.1128/spectrum.02606-23 SN - 2165-0497 VL - 12 IS - 5 PB - American Society for Microbiology (ASM) CY - Washington, DC ER - TY - JOUR A1 - Ali, Aamir A1 - Khan, Muhammad Moman A1 - Kolenda, Rafal A1 - Olowe, Olugbenga Adekunie A1 - Weinreich, Jörg A1 - Li, Ganwu A1 - Schierack, Peter T1 - The role of AJB35136 and fdtA genes in biofilm formation by avian pathogenic Escherichia coli T1 - Die Rolle von AJB35136- und fdtA-Genen bei der Biofilmbildung von aviären pathogenen Escherichia coli N2 - Background Infections caused by avian pathogenic Escherichia coli (APEC) result in significant economic losses in poultry industry. APEC strains are known to form biofilms in various conditions allowing them to thrive even under harsh and nutrient-deficient conditions on different surfaces, and this ability enables them to evade chemical and biological eradication methods. Despite knowing the whole genome sequences of various APEC isolates, little has been reported regarding their biofilm-associated genes. A random transposon mutant library of the wild-type APEC IMT 5155 comprising 1,300 mutants was analyzed for biofilm formation under nutrient deprived conditions using Videoscan technology coupled with fluorescence microscopy. Seven transposon mutants were found to have reproducibly and significantly altered biofilm formation and their mutated genes were identified by arbitrary PCR and DNA sequencing. The intact genes were acquired from the wild-type strain, cloned in pACYC177 plasmid and transformed into the respective altered biofilm forming transposon mutants, and the biofilm formation was checked in comparison to the wild type and mutant strains under the same conditions. Results In this study, we report seven genes i.e., nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 and fdtA of APEC with significant contribution to biofilm formation. Reintroduction of AJB35136 and fdtA, reversed the altered phenotype proving that a significant role being played by these two O-antigen related genes in APEC biofilm formation. Presence of these seven genes across nonpathogenic E. coli and APEC genomes was also analyzed showing that they are more prevalent in the latter. N2 - Hintergrund Infektionen, die durch aviäre pathogene Escherichia coli (APEC) verursacht werden, führen zu erheblichen wirtschaftlichen Verlusten in der Geflügelindustrie. Es ist bekannt, dass APEC-Stämme unter verschiedenen Bedingungen Biofilme bilden, die es ihnen ermöglichen, selbst unter rauen und nährstoffarmen Bedingungen auf verschiedenen Oberflächen zu gedeihen. Diese Fähigkeit ermöglicht es ihnen, sich chemischen und biologischen Eradikationsmethoden zu entziehen. Obwohl die gesamten Genomsequenzen verschiedener APEC-Isolate bekannt sind, wurde bisher nur wenig über ihre Biofilm-assoziierten Gene berichtet. Eine zufällige Transposon-Mutantenbibliothek des Wildtyps APEC IMT 5155, die 1.300 Mutanten umfasst, wurde mit Hilfe der Videoscan-Technologie in Verbindung mit Fluoreszenzmikroskopie auf Biofilmbildung unter nährstoffarmen Bedingungen untersucht. Bei sieben Transposon-Mutanten wurde eine reproduzierbare und signifikant veränderte Biofilmbildung festgestellt, und ihre mutierten Gene wurden durch willkürliche PCR und DNA-Sequenzierung identifiziert. Die intakten Gene wurden dem Wildtyp-Stamm entnommen, in das pACYC177-Plasmid kloniert und in die jeweiligen veränderten biofilmbildenden Transposon-Mutanten transformiert, und die Biofilmbildung wurde im Vergleich zum Wildtyp und den mutierten Stämmen unter denselben Bedingungen überprüft. Ergebnisse In dieser Studie konnten wir sieben Gene, d.h. nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 und fdtA von APEC mit einem signifikanten Beitrag zur Biofilmbildung nachweisen. Die Wiedereinführung von AJB35136 und fdtA kehrte den veränderten Phänotyp um, was beweist, dass diese beiden mit dem O-Antigen verbundenen Gene eine wichtige Rolle spielen. KW - APEC KW - Biofilm KW - Gene KW - Complementation KW - Transposon mutant KW - VideoScan KW - Genkomplementierung KW - Transposon-Mutanten KW - Geflügelkrankheit KW - Escherichia coli KW - Biofilm KW - Komplementation KW - Transponson KW - Mutant Y1 - 2023 U6 - https://doi.org/10.1186/s12917-023-03672-7 ER -