key: cord-0267733-486ypxc1
authors: Kwasi, David A.; Babalola, Chinedum P.; Olubiyi, Olujide O.; Hoffmann, Jennifer; Uzochukwu, Ikemefuna C.; Okeke, Iruka N.
title: Antibiofilm agents with therapeutic potential against enteroaggregative Escherichia coli
date: 2021-11-05
journal: bioRxiv
DOI: 10.1101/2021.11.05.467448
sha: 041d1d90eb4677e03ed9ecf312e0ae5048bc471a
doc_id: 267733
cord_uid: 486ypxc1

Background Enteroaggregative Escherichia coli (EAEC) is a predominant but neglected enteric pathogen implicated in infantile diarrhoea and nutrient malabsorption. There are no non-antibiotic approaches to dealing with persistent infection by these exceptional colonizers, which form copious biofilms. We screened the Medicines for Malaria Venture Pathogen Box for chemical entities that inhibit EAEC biofilm formation. Methodology We used two EAEC strains, 042 and MND005E, in a medium-throughput crystal violet-based antibiofilm screen. Hits were confirmed in concentration-dependence, growth kinetic and time course assays and activity spectra were determined against a panel of genome-sequenced EAEC. Antibiofilm activity against isogenic EAEC mutants, molecular docking simulations and comparative genomic analysis were used to identify the mechanism of action of one hit. Principal findings In all, five compounds (1.25%) reproducibly inhibited biofilm accumulation by at least one strain by 30-85% while inhibiting growth by under 10%. Hits exhibited at least 10-fold greater antibiofilm activity than nitazoxanide, the only known EAEC biofilm inhibitor. Reflective of known EAEC heterogeneity, only one hit was active against both screen isolates, but three hits showed broad antibiofilm activity against a larger panel of strains. Mechanism of action studies point to the EAEC anti-aggregation protein (Aap), dispersin, as the target of compound MMV687800. Conclusions This study identified five compounds not previously described as anti-adhesins or Gram-negative antibacterials with significant and specific EAEC antibiofilm activity. One molecule, MMV687800, targets the EAEC Aap. In vitro small-molecule inhibition of EAEC colonization opens a way to new therapeutic approaches to preventing and treating EAEC infection. Author summary Diarrhoea accounts for over half a million deaths in children under five annually. It additionally contributes to childhood malnutrition as well as growth and development deficiencies, particularly in low-income countries. Enteroaggregative Escherichia coli (EAEC) causes diarrhoea that is often persistent and can also contribute to growth deficiencies in young children. EAEC is a neglected pathogen that is often resistant to antimicrobial drugs. Small molecules that block EAEC colonization may hold the key to interfering with EAEC disease without promoting antimicrobial resistance. We screened the Medicines for Malaria Ventures Pathogen Box for chemicals that can interfere with EAEC biofilm formation, a key colonization indicator. Our screen identified five biofilm-inhibiting molecules that did not interfere with bacterial viability and therefore are unlikely to exert strong pressure for resistance. Molecular biology and computational investigations point to the EAEC anti-aggregative protein, also known as dispersin, as a possible target for one of these hit molecules. Optimizing EAEC antibiofilm hits will create templates that can be employed for resolving EAEC diarrhoea and related infections.

µg/mL (81.45 µM) earlier reported to exhibit significant biofilm inhibition in EAEC [28] Nigeria [18, 33] were used and isogenic EAEC 042 mutants (Table 1) compounds were added, assay was set in triplicate for each hit concentration and optical 220 density was determined at 595 nm using a microplate spectrophotometer at 0 mins, 30mins, 221 then hourly up until the 8 th hour, incubating at 37°C between readings. 222 223

In order to obtain atomic level insight into and to validate the mechanism of action of the 225 identified hits, we employed a molecular docking protocol that screened the identified hit 226 molecules against the anti-aggregation protein (Aap) [36] , the likely antibiofilm target from 227 our mutant and compliment testing assays. The employed Aap structure (accession code 228 2JVU.pdb [36] ), solved using solution NMR, comprises 20 structurally distinct conformers 229 all of which were individually employed as receptor molecules, a strategy that allowed for the 230 incorporation of macromolecular flexibility in the docking protocol. For the docking 231 screening, three dimensional models were first generated for the five identified MMV 232 molecules using the ChemBioOffice Suite. Energy minimization with the steepest descent 233 algorithm was then performed on each model to resolve steric clashes and identify each 234 molecule's potential energy minimum from the sampled conformational landscape. Each 235 model was then saved in the P rotein D ata B ank (PDB) format. 236

Gasteiger atomic charges, needed for a more accurate computation of electrostatics of the 237 binary interactions, were calculated using AutoDock Tool [37, 38] verified pDAK24 clone was used to transform LV1 (042Δaap), LV2 (042ΔaapΔhra1) and 273 LTW1 (042ΔaapΔaafA) [40] and the resulting aap mutant complements were tested for 274 biofilm formation and inhibition. 275

Antibiofilm spectra of the five hits identified in this study were investigated using EAEC 277 reference strain 042 [33] , 60A from Mexico [41] and 25 other EAEC strains identified and 278 characterized in our laboratory in Nigeria from an ongoing diarrhoea case-control study [18] . 279

Virulence gene profiles of strains in each group were retrieved from whole genome sequence 280 data using VirulenceFinder database [42] then compared to identify genes which were unique 281 to each group for each hit. 282 283

Data from biofilm inhibition assays (average of three replicates) were analyzed by comparing 285 inhibition / percentage inhibitions for tests and controls and significant differences were 286 inferred from Fisher's exact test (to identify genes which are unique to strains whose biofilms 287 were inhibited by hits) and student's t-test analysis (for biofilm inhibition assay). 288 289

The 400-compound Pathogen Box chemical library was screened for EAEC biofilm 293 formation inhibitors in a medium throughput assay. Initial hit selection criteria were that 294 molecules must demonstrate at least 30% biofilm inhibition and under 10% growth inhibition 295 at a concentration of 5 µM [43, 44] . Applying these criteria, we identified five compounds 296 which reproducibly inhibited accumulation of biofilm biomass by EAEC 042 and/ or 297 MND005E by 30-85% while inhibiting growth by ≤ 10% (Figure 1a-d) . As shown in Table  298 1, the compounds possessed diverse molecular architectures and chemical properties. 299

Consistent with the known heterogeneity of EAEC strains, the two EAEC test strains, which 300 were selected because they were phylogenetically distant and had largely non-overlapping 301 suites of virulence factors, retrieved different hits from the library. Compounds 302 MMV687800, MMV688978 and MMV687696 inhibited biofilm formation of strain 042 303 only, MMV000023, was active against MND005E while MMV688990 showed activity 304 against both strains. Nitazoxanide (MMV688991), the only previously reported EACE 042 305 biofilm inhibitor [28] , is also contained within Pathogen Box but did not meet our hit criteria. 306

Significant antibiofilm activity was previously reported for NTZ at 15 µg/ml (48.8µM), 20 307 µg/ml (65.15µM) and 25 µg/ml (81.45µM) with an estimated growth inhibition of up 50% 308 [28] . Based on this information, we conducted comparative biofilm inhibition assays for our 309 hits (at 5 µM) and NTZ at 48.8 µM, 65.15 µM and 81.45 µM. We observed similar patterns 310 of antibiofilm activity and growth inhibition with NTZ at these concentrations as reported 311 earlier by Shamir et al. [28] . Additionally, the five validated hits in this screen were found 312 active at concentrations at least 10 times lower than those of nitazoxanide (NTZ) as shown in 313 ruling out Hra1 as a target for this compound. A slightly lower degree of inhibition was seen 355 in the 042ΔaafA mutant (3.4.14) but this was not statistically significantly different from the 356 inhibition produced on the wild type (Figure 5a ). For the aap mutant, 042Δaap (LV1), 357 inhibition occurred to a significantly lower degree. When we tested aap double mutants, we 358 found that inhibition was similarly impaired in the 042ΔaapΔhra1 (LV2) and 042ΔaapΔaaf 359 (LTW1), with the latter showing no significant difference (p > 0.05) in biofilm formation in 360 the presence or absence of MMV687800 (Figure 5a ). Thus, the data point to aap as a likely 361 target for MMV687800 and aaf may or may not be a minor target. Following the hypothesis 362 that the antibiofilm activity of MMV687800 could involve aap, we set up biofilm assays with 363 MMV687800 against EAEC 042, LV1, LV2, LTW1 alone and with the mutants 364 complemented with pDAK24. As is known for these strains, biofilm inhibition was low in 365 mutants 042ΔaapΔhra1 (LV2) and 042ΔaapΔaaf (LTW1) double mutants compared to the 366 wild type strain 042 ( Figure 6 ). Conversely, notable reductions in biofilm formation were 367 observed in the construct, LV1(pDAK24), LV2(pDAK24) and LTW1(pDAK24) (Figure 6 ), 368

where aap deletions were complemented in trans. 369

Inhibition was not completely abrogated in aap mutants, hence it is probable that there is 370 more than one MMV687800 target in 042 and collectively, the data suggest that aafA could 371 represent a second target. 372 373 Antibiofilm spectra of hit compounds 374

Along with the two EAEC strains used in our preliminary screen (EAEC strains 042 [33] and 375 MND005E [18] ), we investigated antibiofilm activity of hits against, 60A an EAEC isolate 376 from Mexico [39.41] and 24 EAECs strains from our laboratory [18] , which form moderate 377 or strong biofilms and have been whole genome sequenced ( Since data from the isogenic 042 mutant biofilm inhibition assay additionally suggests that 397 MMV687800 could have a second target (likely AAF/II), all five investigated hits were 398 docked against AAF/ II major and minor subunits. The five hits and NTZ demonstrated 399 different degrees of moderately thermodynamically favourable binding to the EAEC (AAF/II 400 major and minor subunits), the only EAEC adhesins whose solution structure have been 401 resolved [8] . Hits demonstrated stronger binding to non-Gd site cavities present on the 402 surfaces of the EAEC proteins (Table 4) . NTZ, initially shown to inhibit aggregative 403 adherence fimbria and type I pili assembled by the Chaperone Usher (CU) pathway in EAEC 404 [28] was subsequently proven to interfere with the folding of the usher beta-barrel domain in 405 the outer membrane [45] . It was recently reported for its selective activity in disrupting beta-406 barrel assembly machine (BAM)-mediated folding of the outer membrane usher protein in 407 uropathogenic Escherichia. coli (UPEC) [46] ). In our screening it demonstrated the highest 408 affinity (-6.6 kcal/mol) for AAF/II closely followed by MMV687800 with ∆ G of -5.7 409 kcal/mol. With focused binding interaction (that is, focusing on the Gd site), the strongest 410 binding interaction with AAF/II major subunit among the hits was obtained with 411 MMV687800 (-5.7 kcal/ mol), MMV000023 (-5.5 kcal/mol), then MMV688978 (-5.2 412 kcal/mol) ( Table 4 ). In all, Nitazoxanide, reported to interfere with AAF/ II assembly [28] , 413 outperformed all five test compounds with respect to the strength of interaction with the AAF 414 /II binding site even though the differences are at best marginal. 415 416 417

Biofilms contribute significantly to pathogenesis of several bacterial infections [47] [48] [49] [50] [51] . They 419 are major players thwarting host defense and the activity of antimicrobials against many 420 microorganisms [52] and will consequently require a novel approach to target them during 421

infections. EAEC are known to form copious biofilms, which enhances their persistence 422 during infection [53, 54] hence antibiofilm agents could be particularly effective [55] . As 423 antibiofilm agents attack a colonization process and not bacterial viability, it is hoped that, 424 unlike conventional antibiotics, they will exert less selection pressure for antimicrobial 425 resistance [56] . 426 427 Whilst antibiofilm activity of a few synthetic and natural small molecules has been 428 demonstrated in vitro [52, [57] [58] [59] [60] [61] [62] and in vivo [55, 63, 64] , for other pathogens, only one study 429 reported significant antibiofilm activity of a compound, nitazoxanide, against EAEC biofilms 430 [28] . Shamir et al (2010) discovered the antibiofilm activity of NTZ when they were 431 evaluating its growth inhibitory activity and subsequently showed that this antiparasitic 432 compound inhibits assembly of AAF/II. NTZ has subsequently been shown to interfere with 433 pilus usher function [45, 46] and Bolick et al (2013) [55] were able to show that it reduces 434 diarrhoea in vivo, and shedding of EAEC at concentrations not inhibiting growth. These data 435 suggest that antiadhesive agents have therapeutic potential against EAEC. By systematically 436 screening for EAEC antibiofilm activity, this study has uncovered many more inhibitors that 437 are significantly more potent than NTZ. At least one of them, MMV687800 inhibits EAEC-438 specific targets and so is unlikely to have deleterious effects on the normal flora. 439

The five hits we identified from the MMV's pathogen box reproducibly inhibited biofilm 441 formation by one or two EAEC strains by 30-85% while inhibiting growth by ≤ 10 at 5 µM. 442

Our hit rate for biofilm inhibitors that do not inhibit growth (1.25%), from a curated library, 443 provides support for this drug discovery approach. Targeting EAEC biofilms without 444 compromising their viability holds a high potential since hits from our screen are unlikely to 445 select for antibacterial resistance due to selection pressure for resistant strains. Giardia lamblia and Cryptosporidium parvum in a pathogen box screen [73] . prolificans [74] . MMV687696, has been reported by MMV to possess anti-tuberculosis 476 activity. 477

The antiparasitic compound nitazoxanide, also a component of Pathogen Box, which was 478 previously reported to inhibit biofilm by EAEC 042 [28] was not recovered as hit from our 479 screen and we verified that it is indeed inactive at 5 µM, our screen concentration. We re-480 tested nitazoxanide at concentrations for which antibiofilm activity was previously recorded 481 and observed significant concentration-dependent biofilm inhibition at 15, 20 and 25 µg/ml 482 (48.8, 65.15 and 81.4 µM) but with estimated growth inhibition of up to 50% [28] . 483

Consequently, the validated hits obtained from the screen in this study, which lack growth 484

inhibition activity at the concentration tested, were at least 10 times more active in biofilm 485 inhibition than nitazoxanide the only known EAEC biofilm inhibitor, which has subsequently 486 been shown to be effective against experimental infections in a weaned mouse model [55] . 487

Unlike NTZ, our five hits inhibited biofilm formation at concentrations that did not produce 488 significant growth inhibition, pointing to the possibility of biofilm-specific targets and 489 minimal, if any, cross resistance with clinical antibacterials [44, 56] . For three of the 490 compounds for which we could get sufficient chemical to test, inhibition was largely 491 concentration dependent, again suggesting that specific biofilm factors are targeted. When 492 those factors are EAEC-specific, use of antibiofilm agents is unlikely to disrupt the normal 493 flora, including other E. coli, which are protective against enteric infection. 494

Biofilm formation is a complex and stepwise process involving numerous bacterial factors, 495 which vary among and even within pathotypes. Early-stage contributors include adhesins, 496 flagella and secreted protein autotransporters. At late-exponential phase, the accumulation of 497 quorum sensing signals leads to the activation of other genes. Late-stage biofilm factors 498 include adhesins with greater permanence, components that comprise or requite a 499 macromolecular matrix as well as antiagregation proteins which can be co-opted to release 500 bacteria from the biofilm. 501

The temporal patterns of biofilm inhibition and EAEC inhibition spectra of compounds 503 MMV687800, MMV688978, MMV687696, MMV000023 and MMV688990 were different, 504 implying that they likely target different contributors to EAEC biofilm formation. EAEC 505 expressed many surface factors involved in host adherence and biofilm formation 506 [21,40,75.76] . Any of these, or their regulators, could be direct targets. Overlaying activity 507 spectra data with virulence factor profiles of genome sequenced EAEC strains provided 508 preliminary insights to mechanism of action. For MMV687800, subsequent testing of five 509 isogenic mutants of EAEC 042 provided further insight. Aap is an antiaggregation protein or 510 dispersin that allows bacteria to detach from old biofilms and seed new ones. Mutants in aap 511

show increased biofilm formation but impaired colonization [21, 40] . Biofilm formation by 512 aap mutant (LV1: 042Δaap) was significantly less inhibited by MMV687800 than wildtype. 513 Additionally (LV2: 042ΔaapΔhra1) double mutants [40] were inhibited in biofilm formation 514 to a proportionally lower degree and inhibition seen in (LTW1: 042ΔaapΔaaf) was 515 statistically insignificant compared to controls no compound at all (p = 0.11). This 516 phenotype could be complemented in trans and thus molecular Koch's postulates [77] are 517 fulfilled for aap as an MMV687800 target. 518

To preliminarily determine whether the interaction between MMV687800 and Aap could be 520 direct, we independently determined binding affinities of hits with twenty Aap conformers 521 using molecular docking techniques since the solution structure of Aap has been resolved by 522 NMR [35] . The strong binding interaction sustained for hit MMV687800 and in the outcome 523 of the molecular computational docking experiments against 20 conformational instances of 524 the dispersin (Aap) in Figure 7 indicates a high significance for the obtained affinities. 525 MMV687800 demonstrated moderately strong interaction (in some cases

with Aap. This additionally suggests that aap is one of the surface factors targeted by 527

The aafA gene encodes the structural subunit of AAF/II fimbriae and Hra1, the heat-resistant 529 agglutinin 1 is an outer-membrane protein involved in autoaggregation that serves as an 530 accessory colonization factor [40, 75, 78] . MMV687800 showed a small, insignificant 531 reduction of activity in the aafA mutant 3.4.14 [79] , but not the hra1 mutant, which we 532 initially discounted, given the robust phenotype with Aap. However, the reduction in 533 MMV687800 biofilm inhibition was visible with the aap aafA mutant LTW1, and 534 complementation of this mutant with aap alone LTW1(pDAK24) could not fully restore 535 biofilm inhibition. These data, and the comparative genomic data which shows that aafA-D 536 are unique to biofilm inhibition groups alone (data not shown), strengthen the likelihood that 537 AAF/II is implicated in MMV687800 biofilm inhibition, albeit to a lower degree than Aap. 538

The five hit compounds demonstrated stronger binding to non-Gd site cavities present on the 539 surfaces of AAF/II (Table 4 ). Docking outcome however revealed NTZ, known to inhibit 540 AAF/II assembly exhibited highest affinity (-6.6 kcal/mol) for AAF/II followed by 541 MMV687800 which had -5.7 kcal/mol again, indicating that AAF/II could be a likely target 542 for MMV687800. 543

This study has some limitations. EAEC are highly heterogeneous and the full spectrum of 544 lineages and virulence factors is not covered by the two strains used for this screen, or even 545 by the 27 strains employed to better understand activity profiles of the hits. We determined 546 the probable mechanism of action of only one hit, taking advantage of the easily-generated 547

VirulenceFinder output and the bank of mutants we had on hand. In doing so, we were able 548 to generate proof of principle and rule out Aap, AAF/II and Hra1 as targets for the other 549 compounds. However, it is conceivable that at least some of the targets of the other 550 compounds may not be unique to EAEC or may be genes of unknown function. More 551 intensive and unbiased comparative genomic approaches, currently underway, will be needed 552 to exhaustively screen for the targets of the other four hits, which could well be more 553 promising than the hit highlighted in this study. 554 555

In conclusion, this study identified five biofilm inhibiting but non-growth inhibiting 556 compounds that have not been previously described as bacterial anti-adhesins or Gram-557 negative antibacterials. Hits discovered from this screen will add to the drug discovery 558 pipeline for this neglected pathogen, improve understanding on EAEC colonization and 559 enhance EAEC-based interventions. Additionally, the understandings from our experiments 560 that some of our hits are unlikely to target known EAEC adhesins is initiating investigations Nitazoxanide -5.4 -6.5 -6.2 -5.5

Estimating diarrhea mortality among young children in low and middle income countries

Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since

Diarrheoa data: Diarrheoa as cause of death in children under 5

Institute for Health Metrics and Evaluation (IHME) Global burden of disease

Relationship between growth and illness, enteropathogens and dietary intakes in the first 2 years of life: findings from the MAL-ED birth cohort study

Enteroaggregative Escherichia coli (EAEC) impairs growth while malnutrition worsens EAEC infection: a novel murine model of the infection malnutrition cycle

Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study

Insight into Host Recognition by Aggregative Adherence Fimbriae of Enteroaggregative Escherichia coli

Pathogenesis of enteroaggregative Escherichia coli infection

Enteroaggregative Escherichia coli associated with a foodborne outbreak of gastroenteritis

Diarrheagenic Escherichia coli: Prevelence and Pathotype Distribution in Children from Peruvian Rural Communities

Colonization by Enteroaggregative Escherichia coli in travelers with and without diarrhea

Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells

Use of antibiotics in children younger than two years in eight countries: a prospective cohort study

Heterogeneous Virulence of Enteroaggregative Escherichia coli Strains Isolated from Children in Southwest Nigeria

Enteroaggregative Escherichia coli

Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition

Identification of Enterotoxigenic Escherichia coli from infants attending healthcare centers in Ibadan north

Neglected disease research and development: how much are we really spending?

Diarrheagenic Escherichia coli in sub-Saharan Africa: status, uncertainties and necessities

A novel dispersin protein in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli: an emerging enteric pathogen

Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro

Identification of enteroaggregative Escherichia coli in infants with acute diarrhea based on biofilm production in Manipal

Diarrheaassociated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili

Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers

Epidemiology and Clinical Manifestations of Enteroaggregative Escherichia coli

Nitazoxanide inhibits biofilm production and hemagglutination by enteroaggregative Escherichia coli strains by blocking assembly of AafA fimbriae

Enteroaggregative Escherichia coli pathotype: a genetically heterogeneous emerging foodborne enteropathogen

Biofilm formation as microbial development

Microtiter dish biofilm formation assay

Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli

Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Rapid screening of inhibitors of Mycobacterium tuberculosis growth using tetrazolium salts

Solution structure of the novel dispersin protein of enteroaggregative Escherichia coli

Automated docking of flexible ligands: Applications of AutoDock

AutoDock4 Zn: An Improved AutoDock Force Field for Small-Molecule Docking to Zinc Metalloproteins

AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading

Aggregative Adherence and Intestinal Colonization by Enteroaggregative Escherichia coli Are Produced by Interactions among Multiple Surface Factors

Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli

Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli

Release of 50 new, drug-like compounds and their computational target predictions for open source anti-tubercular drug discovery

Cell-Based High-Throughput Screening Identifies Rifapentine as an Inhibitor of Amyloid and Biofilm Formation in Escherichia coli

Nitazoxanide Inhibits Pilus Biogenesis by Interfering with Folding of the Usher Protein in the Outer Membrane

The small molecule nitazoxanide selectively disrupts BAM-mediated folding of the outer membrane usher protein

Microbiology of chronic rhinosinusitis. I

The pathophysiological role of bacterial biofilms in chronic sinusitis

New is old, and old is new: recent advances in antibiotic-based, antibiotic-free and ethnomedical treatments against methicillinresistant Staphylococcus aureus wound infections

Chronic obstructive pulmonary disease (COPD): evaluation from clinical, immunological and bacterial pathogenesis perspectives

Microenvironment and microbiology of skin wounds: the role of bacterial biofilms and related factors

Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins

Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries

Role of Enteroaggregative Escherichia coli Virulence Factors in Uropathogenesis Infection and Immunity p

Enteroaggregative Escherichia coli strain in a novel weaned mouse model: exacerbation by malnutrition, biofilm as a virulence factor and treatment by nitazoxanide

Anti-virulence strategies to combat bacteria-mediated disease

Targeting bacterial integration host factor to disrupt biofilms associated with cystic fibrosis

Antibodies directed against integration host factor mediate biofilm clearance from nasopore

Structural stability of Burkholderia cenocepacia biofilms is reliant on eDNA structure and presence of a bacterial nucleic acid binding protein

Evaluation of the kinetics and mechanism of action of anti-integration host factor-mediated disruption of bacterial biofilms

DNABII proteins play a central role in UPEC biofilm structure

Anti-biofilm Activities from Resveratrol against Fusobacterium nucleatum Frontiers in Microbiology

Aberrant community architecture and attenuated persistence of uropathogenic Escherichia coli in the absence of individual IHF subunits

Antibodies against the majority subunit of type IV Pili disperse nontypeable Haemophilus influenzae biofilms in a LuxS-dependent manner and confer therapeutic resolution of experimental otitis media

Safety and availability of clofazimine in the treatment of multidrug and extensively drug-resistant tuberculosis: analysis of published guidance and meta-analysis of cohort studies

Clofazimine: current status and future prospects

Clofazimine broadly inhibits coronaviruses including SARS-CoV-2

Why miltefosine-a life-saving drug for leishmaniasis-is unavailable to people who need it the most

A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia lamblia

Primaquine: the risks and the benefits

In Silico Screening and Analysis of Broad-Spectrum Molecular Targets and Lead Compounds for Diarrhea Therapy

Screening of TB Actives for Activity against Nontuberculous Mycobacteria Delivers High Hit Rates

Screening of the Pathogen Box for inhibitors with dual efficacy against Giardia lamblia and Cryptosporidium parvum

Identification of Promising Antifungal Drugs against Scedosporium and Lomentospora Species after Screening of Pathogen Box Library

The heat resistant agglutinin 1 is an accessory enteroaggregative Escherichia coli colonization factor

Association of putative pathogenicity genes with adherence characteristics and fimbrial genotypes in typical enteroaggregative Escherichia coli from patients with and without diarrhoea in the United Kingdom

Molecular Koch's postulates applied to bacterial pathogenicity--a personal recollection 15 years later

Self-association motifs in the enteroaggregative Escherichia coli heat-resistant agglutinin 1. Microbiology (Reading)

Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in Enteroaggregative Escherichia coli