Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli.

Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention.

Identification of a copper-responsive small molecule inhibitor of uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC ΔtolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli, this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

Competitive fitness of asymptomatic bacteriuria E. coli strain 83972 against uropathogens in human urine.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. The main causative agent of UTI is uropathogenic Escherichia coli (UPEC). There is an immediate need for novel prophylactic and treatment strategies against UTI because of the increasing incidence of antimicrobial resistance among uropathogens. ABU 83972, an asymptomatic bacteriuria-causing E. coli strain, prevents UTI by suppressing the colonization of UPEC. However, the nature of competition and growth repression of UPEC by ABU 83972 is unclear and is the subject of our investigation. Here, we characterized the growth kinetics of ABU 83972 and uropathogens in human urine and laboratory media. Next, we performed a series of competitive co-culture experiments where ABU 83972 and uropathogens were inoculated at a 1:1 ratio in human urine and in various media, and their relative abundance was determined. In human urine, ABU 83972 outcompeted UPEC and additional uropathogens, reaching up to 90% of the total population after 24 hours of incubation. In contrast, UPEC outcompeted ABU 83972 in LB and M9 minimal media and exhibited superior colonization than ABU 83972 in the mouse urinary bladder. Since engineered living materials (ELMs) can be used to retain an organism of interest in a particular location, we developed ABU 83972-containing ELMs that effectively outcompeted UPEC in human urine. In summary, our work establishes that ABU 83972 outcompetes UPEC in a milieu- and cell-density-dependent manner, highlighting the importance of the metabolites and nutrients found in the human urine as determinants of the competitive fitness of ABU 83972.

A cynomolgus monkey E. coli urinary tract infection model confirms efficacy of new FimH vaccine candidates.

The increase in urinary tract infections (UTI) caused by antibiotic-resistant Escherichia coli requires the development of new therapeutic agents and prophylactic vaccines. To evaluate the efficacy of new lead candidates, we implemented a cynomolgus macaque UTI challenge model that mimics human uncomplicated cystitis in response to transurethral challenge with a multidrug-resistant (MDR) E. coli serotype O25b ST131 isolate. E. coli fimbrial adhesin FimH and O-antigens are separately under clinical evaluation by others as vaccine candidates to prevent UTI and invasive urosepsis disease, respectively. Accordingly, we assessed the protective efficacy of three 50-µg intramuscular doses of a novel recombinant FimH antigen adjuvanted with liposomal QS21/MPLA compared with saline placebo in groups of nine animals. A third group was vaccinated with this FimH formulation in combination with 1 µg each of a four-valent mixture of serotype O1a, O2, O6, and O25b O-antigen CRM197 lattice glycoconjugates. Both vaccines elicited high levels of serum FimH IgG and adhesin blocking antibodies at the time of bacterial challenge and, for the combination group, O-antigen-specific antibodies. Following bacterial challenge, both vaccinated groups showed >200- and >700-fold reduction in bacteriuria at day 2 and day 7 post-infection compared with placebo, respectively. In parallel, both vaccines significantly reduced levels of inflammatory biomarkers IL-8 and myeloperoxidase in the urine at day 2 post-infection relative to placebo. Results provide preclinical proof-of-concept for the prevention of an MDR UTI infection by these new vaccine formulations.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen.

IMPORTANCE: While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.

Suppression of virulence factors of uropathogenic Escherichia coli by Trans-resveratrol and design of nanoemulgel.

BACKGROUND: Development of multidrug resistance in Uropathogenic Escherichia coli (UPEC) makes treatment of Urinary Tract Infections (UTIs) a major challenge. This study was conducted to investigate the effect of trans-resveratrol (t-RSV) at a subinhibitory concentration (sub-MIC-t-RSV) on phenotypic and genotypic expression of virulence factors of clinical isolates of UPEC and develop a nanoformulation of t-RSV. Fifty-five clinical UPEC strains were investigated for the presence of virulence factors by phenotypic methods and PCR detection of virulence genes. The effect of sub-MIC-t-RSV was studied on the phenotypic and genotypic expression of virulence factors. t-RSV-loaded nanoemulgel formulation was prepared and characterized. RESULTS: Out of the 55 tested isolates, 50.9% were biofilm producers, 23.6% showed both mannose-sensitive and mannose-resistant hemagglutination, 21.8% were serum-resistant, 18.2% were hemolysin producers, while 36.4% showed cytotoxic effect on HEp-2 cells. A total of 25.5% of the isolates harbor one or more of hly-A, cnf-1 and papC genes, while 54.5% were positive for one or more of fimH, iss and BssS genes. A concentration of 100 µg/mL of t-RSV effectively downregulates the phenotypic and genotypic expression of the virulence factors in positive isolates. A stable t-RSV-nanaoemulgel with droplet size of 180.3 nm and Zetapotential of -46.9 mV was obtained. CONCLUSION: The study proves the effective role of t-RSV as an antivirulence agent against clinical UPEC isolates in vitro and develops a stable t-RSV-nanoemulgel formulation to be assessed in vivo. The promising antibacterial and antivirulence properties of t-RSV place this natural compound to be a better alternative in the treatment of persistent UTIs.

Peptidoglycan endopeptidase MepM of uropathogenic Escherichia coli contributes to competitive fitness during urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are common bacterial infections, primarily caused by uropathogenic Escherichia coli (UPEC), leading to significant health issues and economic burden. Although antibiotics have been effective in treating UPEC infections, the rise of antibiotic-resistant strains hinders their efficacy. Hence, identifying novel bacterial targets for new antimicrobial approaches is crucial. Bacterial factors required for maintaining the full virulence of UPEC are the potential target. MepM, an endopeptidase in E. coli, is involved in the biogenesis of peptidoglycan, a major structure of bacterial envelope. Given that the bacterial envelope confronts the hostile host environment during infections, MepM's function could be crucial for UPEC's virulence. This study aims to explore the role of MepM in UPEC pathogenesis. RESULTS: MepM deficiency significantly impacted UPEC's survival in urine and within macrophages. Moreover, the deficiency hindered the bacillary-to-filamentous shape switch which is known for aiding UPEC in evading phagocytosis during infections. Additionally, UPEC motility was downregulated due to MepM deficiency. As a result, the mepM mutant displayed notably reduced fitness in causing UTIs in the mouse model compared to wild-type UPEC. CONCLUSIONS: This study provides the first evidence of the vital role of peptidoglycan endopeptidase MepM in UPEC's full virulence for causing UTIs. MepM's contribution to UPEC pathogenesis may stem from its critical role in maintaining the ability to resist urine- and immune cell-mediated killing, facilitating the morphological switch, and sustaining motility. Thus, MepM is a promising candidate target for novel antimicrobial strategies.

High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Type 1 fimbria and P pili: regulatory mechanisms of the prototypical members of the chaperone-usher fimbrial family.

Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon.

Biofilm-producing and carbapenems-resistant Escherichia coli nosocomial uropathogens: a cross-sectional study.

OBJECTIVES: This cross-sectional study aims to determine the incidence and potential risk factors associated with biofilm-producing uropathogenic Escherichia coli (UPEC) nosocomial strains from a tertiary care hospital and to examine the prospective correlation between biofilm generation and antibiotic resistance phenotypes and genotypes. METHODS: A total of 130 UPEC nosocomial isolates were identified, their biofilm formation was quantified using a modified microtiter plate assay, and their antibiotic susceptibilities were assessed utilizing the disc diffusion method. Isolates were then subjected to PCR assays targeting blaKPC, blaVIM, blaIMP, and blaOXA48 genes. RESULTS: Over half of the isolates (n = 76, 58.5%) were biofilm producers. Among 17 carbapenem-resistant isolates, 6 (42.9%) isolates harbored the blaOXA48 gene, and only 1 (9.1%) isolate was positive for the blaVIM gene. Prior antibiotic therapy (aOR 15.782, p 0.000) and diabetes mellitus DM (aOR 11.222, p 0.016) were the significant risk factors associated with biofilm production, as determined by logistic regression analysis of the data. In addition, gentamicin resistance was the only statistically significant antibiotic resistance pattern associated with biofilm production (aOR 9.113, p 0.02). CONCLUSIONS: The findings of this study emphasize the significance of implementing proper infection control measures to avoid the horizontal spread of biofilm formation and associated antimicrobial resistance patterns among UPEC nosocomial strains.

Characterization of bacteriophages infecting multidrug-resistant uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.

Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress.

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that class 1a pili of uropathogenic E. coli (UPEC), as well as class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC class 5 pili. Examining structural and steered molecular dynamics simulation data, we find that this difference in class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking ß-strands in class 1 pili stabilizes subunits, allowing them to tolerate higher forces than class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche, since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of class 5 ETEC pili.

In Silico Drug Repurposing Endorse Amprenavir, Darunavir and Saquinavir to Target Enzymes of Multidrug Resistant Uropathogenic E. Coli.

Multidrug resistance is a paramount impediment to successful treatment of most hospital acquired bacterial infections. A plethora of bacterial genera exhibit differential levels of resistance to the existing antibiotics. Prevalent Uropathogenic Escherichia coli or UPEC conduce high mortality among them. Multi-Drug Resistant bacterial strains utilize precise mechanisms to bypass effects of antibiotics. This is probably due to their familiar genomic origin. In this article drug repositioning method have been utilised to target 23 enzymes of UPEC strains viz. CFT073, 536 and UTI89. 3-D drug binding motifs have been predicted using SPRITE and ASSAM servers that compare amino acid side chain similarities. From the hit results anti-viral drugs have been considered for their uniqueness and specificity. Out of 14 anti-viral drugs 3 anti-HIV drugs viz. Amprenavir, Darunavir and Saquinavir have selected for maximum binding score or drug targetability. Finally, active sites of the enzymes were analyzed using GASS-WEB for eloquent drug interference. Further analyses with the active sites of all the enzymes showed that the three selected anti-HIV drugs were very much potent to inhibit their active sites. Combination or sole application of Amprenavir, Darunavir and Saquinavir to MDR-UPEC infections may leads to cure and inhibition of mortality. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01282-x.

Bladder-draining lymph nodes support germinal center B cell responses during urinary tract infection in mice.

Bacterial urinary tract infections (UTIs) are both common and exhibit high recurrence rates in women. UTI healthcare costs are increasing due to the rise of multidrug-resistant (MDR) bacteria, necessitating alternative approaches for infection control. Here, we directly observed host adaptive immune responses in acute UTI. We employed a mouse model in which wild-type C57BL/6J mice were transurethrally inoculated with a clinically relevant MDR UTI strain of uropathogenic Escherichia coli (UPEC). Firstly, we noted that rag1-/- C57BL/6J mice harbored larger bacterial burdens than wild-type counterparts, consistent with a role for adaptive immunity in UTI control. Consistent with this, UTI triggered in the bladders of wild-type mice early increases of myeloid cells, including CD11chi conventional dendritic cells, suggesting possible involvement of these professional antigen-presenting cells. Importantly, germinal center B cell responses developed by 4 weeks post-infection in bladder-draining lymph nodes of wild-type mice and, although modest in magnitude and transient in nature, could not be boosted with a second UTI. Thus, our data reveal for the first time in a mouse model that UPEC UTI induces local B cell immune responses in bladder-draining lymph nodes, which could potentially serve to control infection.

OXA-48-Producing Uropathogenic Escherichia coli Sequence Type 127, the Netherlands, 2015-2022.

During 2015-2022, a genetic cluster of OXA-48-producing uropathogenic Escherichia coli sequence type 127 spread throughout the Netherlands. The 20 isolates we investigated originated mainly from urine, belonged to Clermont phylotype B2, and carried 18 genes encoding putative uropathogenicity factors. The isolates were susceptible to first-choice antimicrobial drugs for urinary tract infections.

Comparative analysis of outer membrane vesicles from uropathogenic Escherichia coli reveal the role of aromatic amino acids synthesis proteins in motility.

Uropathogenic Escherichia coli (UPEC) are causative agent that causes urinary tract infections (UTIs) and the recent emergence of multidrug resistance (MDR) of UPEC increases the burden on the community. Recent studies of bacterial outer membrane vesicles (OMV) identified various factors including proteins, nucleic acids, and small molecules which provided inter-cellular communication within the bacterial population. However, the components of UPEC-specific OMVs and their functional role remain unclear. Here, we systematically determined the proteomes of UPEC-OMVs and identified the specific components that provide functions to the recipient bacteria. Based on the functional network of OMVs' proteomes, a group of signaling peptides was found in all OMVs which provide communication among bacteria. Moreover, we demonstrated that treatment with UPEC-OMVs affected the motility and biofilm formation of the recipient bacteria, and further identified aromatic amino acid (AAA) biosynthesis proteins as the key factors to provide their movement.

Effect of bovine lactoferrin on recurrent urinary tract infections: in vitro and in vivo evidences.

Uropathogenic Escherichia coli (UPEC) strains are the primary cause of urinary tract infections (UTIs). UPEC strains are able to invade, multiply and persisting in host cells. Therefore, UPEC strains are associated to recurrent UTIs requiring long-term antibiotic therapy. However, this therapy is suboptimal due to the increase of multidrug-resistant UPEC. The use of non-antibiotic treatments for managing UTIs is required. Among these, bovine lactoferrin (bLf), a multifunctional cationic glycoprotein, could be a promising tool because inhibits the entry into the host cells of several intracellular bacteria. Here, we demonstrate that 100 µg/ml bLf hinders the invasion of 2.0 ± 0.5 × 104 CFU/ml E. coli CFT073, prototype of UPEC, infecting 2.0 ± 0.5 × 105 cells/ml urinary bladder T24 epithelial cells. The highest protection (100%) is due to the bLf binding with host surface components even if an additional binding to bacterial surface components cannot be excluded. Of note, in the absence of bLf, UPEC survives and multiplies, while bLf significantly decreases bacterial intracellular survival. After these encouraging results, an observational survey on thirty-three patients affected by recurrent cystitis was performed. The treatment consisted in the oral administration of bLf alone or in combination with antibiotics and/or probiotics. After the observation period, a marked reduction of cystitis episodes was observed (p < 0.001) in all patients compared to the episodes occurred during the 6 months preceding the bLf-treatment. Twenty-nine patients did not report cystitis episodes (87.9%) whereas the remaining four (12.1%) experienced only one episode, indicating that bLf could be a worthwhile and safe treatment in counteracting recurrent cystitis.

Synergistic Antimicrobial Activity of Eugenol in Combination with Fosfomycin to Combat Escherichia coli and Potential Effect on Plasmid-Mediated Fosfomycin Resistance Genes.

The presence of multidrug-resistant pathogenic microorganisms makes it challenging to cure bacterial illnesses. Syzygium aromaticum has been used for medicinal purposes since ancient times. The objective of this study was to investigate the potential synergistic effect of the combination of Eugenol and Fosfomycin against clinically Uropathogenic Escherichia coli (UPEC) and their possible co-treatment as well as their contribution to plasmid-mediated Fosfomycin resistance (fosA3 and fosA4) genes using molecular assays. Eugenol was extracted from clove (Syzygium aromaticum) plants using steam distillation by Clevenger and analyzed by high-performance liquid chromatography (HPLC). UPEC accounted for 63.6 % of all isolates. Specifically, 99.3 % of the UPEC isolates exhibited resistance to multiple types of antibiotics [multidrug-resistant (MDR)]. The MIC for Eugenol was 1.25-5 µg/mL, and Fosfomycin was 512-1024 µg/mL, while the MBC for Eugenol was 5-10 µg/mL and Fosfomycin was 2048 µg/mL. The synergistic effects were considerable, with 1/4 MIC of Eugenol resulting in 1/8 MIC Fosfomycin. Eugenol inhibited most of the UPEC isolates at 4-8 hours, Fosfomycin at 8-12 hours, and co-treatment at 4-8 hours. The fosA3 and fosA4 genes were detected in 5.7 % and 2.9 % of the isolates, respectively. The results showed variable gene expression changes in response to the different treatments.

Increased Levels of (p)ppGpp Correlate with Virulence and Biofilm Formation, but Not with Growth, in Strains of Uropathogenic Escherichia coli.

Urinary tract infections are one of the most frequent bacterial diseases worldwide. UPECs are the most prominent group of bacterial strains among pathogens responsible for prompting such infections. As a group, these extra-intestinal infection-causing bacteria have developed specific features that allow them to sustain and develop in their inhabited niche of the urinary tract. In this study, we examined 118 UPEC isolates to determine their genetic background and antibiotic resistance. Moreover, we investigated correlations of these characteristics with the ability to form biofilm and to induce a general stress response. We showed that this strain collection expressed unique UPEC attributes, with the highest representation of FimH, SitA, Aer, and Sfa factors (100%, 92.5%, 75%, and 70%, respectively). According to CRA (Congo red agar) analysis, the strains particularly predisposed to biofilm formation represented 32.5% of the isolates. Those biofilm forming strains presented a significant ability to accumulate multi-resistance traits. Most notably, these strains presented a puzzling metabolic phenotype-they showed elevated basal levels of (p)ppGpp in the planktonic phase and simultaneously exhibited a shorter generation time when compared to non-biofilm-forming strains. Moreover, our virulence analysis showed these phenotypes to be crucial for the development of severe infections in the Galleria mellonella model.