Anti-biofilm activity of caffeine against uropathogenic E. coli is mediated by curli biogenesis.

Biofilms are assemblages of sessile microorganisms that form an extracellular matrix around themselves and mediate attachment to surfaces. The major component of the extracellular matrix of Uropathogenic E. coli and other Enterobacteriaceae are curli fibers, making biofilms robust and resistant to antimicrobials. It is therefore imperative to screen antibiofilm compounds that can impair biofilm formation. In the present study, we investigated the curli-dependent antibiofilm activity of caffeine against UPEC strain CFT073 and commensal strain E. coli K-12MG1655.Caffeine significantly reduced the biofilm formation of both UPEC and E. coli K-12 by 86.58% and 91.80% respectively at 48 mM caffeine as determined by Crystal Violet assay. These results were further confirmed by fluorescence microscopy and Scanning Electron Microscope (SEM). Caffeine significantly reduced the cytotoxicity and survivability of UPEC. Molecular docking analysis revealed a strong interaction between caffeine and curli regulator protein (Csg D) of E. coli. The qRT-PCR data also showed significant downregulation in the expression of CsgBA and the CsgDEFG operon at both 24 mM and 48 mM caffeine. The findings revealed that caffeine could inhibit E. coli biofilm formation by regulating curli assembly and thus may be used as an alternative therapeutic strategy for the treatment of chronic E. coli biofilm-related infections.

Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli.

Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting 11 essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e.dnaB, ftsZ and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that 9mer PNAs are generally as effective in inhibiting bacterial growth as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.

Differences in recipient ability of uropathogenic Escherichia coli strains in relation with their pathogenic potential.

Conjugation is recognized as a mechanism driving dissemination of antibacterial resistances and virulence factors among bacteria. In the presented work conjugative transfer frequency into clinical uropathogenic Escherichia coli strains (UPEC) isolated from patients with symptomatic urinary tract infections was investigated. From 93 obtained UPEC strains only 29 were suitable for conjugation experiments with the plasmid pOX38, a well-known F-plasmid derivative. The study was focused on comparison of conjugation frequencies in plankton and biofilm, including comparison of conjugation frequencies in high and low biofilm biomass with their virulence potential. It was shown that the conjugation frequency depended on the biofilm biomass and was significantly higher in thin (OD580 < 0.3) than in thick biofilm (OD580 ≥ 0.3). Nonmetric multidimensional scaling analysis revealed that higher conjugation frequencies in plankton and biofilm were directly positively correlated with the sum of virulence-associated genes of the recipient strain and presence of multidrug antibiotic resistances. On the other hand, the sum of insensitivities to different bacteriocins was negatively correlated with an increase in the conjugative transfer level. Our results obtained hence indicate that the evolution of potentially more pathogenic strains via conjugation is depended on the strains' ability to be a "good" recipient in the conjugative transfer, possibly due to the ability to form thinner biofilms.

Phenotypic and Genotypic Characteristics of Uropathogenic Escherichia coli Isolates from Kenya.

Introduction: Uropathogenic Escherichia coli (UPECs) are a significant cause of urinary tract infections (UTIs). In Kenya, UTIs are typically treated with ß-lactam antibiotics without antibiotic susceptibility testing, which could accelerate antibiotic resistance among UPEC strains. Aim: This study determined the occurrence of UPEC producing extended-spectrum ß-lactamases (ESBLs), the genes conferring resistance to ß-lactams, and the phylogenetic groups associated with ESBLs in Kenyan UPECs. Methodology: Ninety-five UPEC isolates from six Kenyan hospitals were tested for ESBL and plasmid-mediated AmpC ß-lactamase (pAmpC) production by combined disk diffusion and disk approximation tests, respectively. Real-time and conventional polymerase chain reactions (PCRs) were used to detect three ESBL and six pAmpC genes, respectively, and phylogenetic groups were assigned by a quadruplex PCR method. Results: Twenty-four percent UPEC isolates were ESBL producers with blaCTX-M (95.6%), blaTEM (95.6%), and blaSHV (21.7%) genes detected. Sixteen isolates had blaCTX-M/TEM, whereas five had blaTEM/CTX-M/SHV. A total of 5/23 ESBLs were cefoxitin resistant, but no AmpC genes were detected. The UPECs belonged predominantly to phylogenetic groups B2 (31/95; 32.6%) and D (30/95; 31.6%), while groups B2 and A had the most ESBL producers. Conclusions: ß-Lactam antibiotics have reduced utility for treating UTIs as a quarter of UPECs were ESBL producing. Single or multiple ESBL genes were present in UPECs, belonging primarily to phylogenetic groups B2 and A.

Are Uropathogenic Bacteria Living in Multispecies Biofilm Susceptible to Active Plant Ingredient-Asiatic Acid?

Urinary tract infections (UTIs) are a serious health problem in the human population due to their chronic and recurrent nature. Bacteria causing UTIs form multispecies biofilms being resistant to the activity of the conventionally used antibiotics. Therefore, compounds of plant origin are currently being searched for, which could constitute an alternative strategy to antibiotic therapy. Our study aimed to determine the activity of asiatic acid (AA) against biofilms formed by uropathogenic Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa. The influence of AA on the survival, biofilm mass formation by bacteria living in mono-, dual-, and triple-species consortia as well as the metabolic activity and bacterial cell morphology were determined. The spectrophotometric methods were used for biofilm mass synthesis and metabolic activity determination. The survival of bacteria was established using the serial dilution assay. The decrease in survival and a weakening of the ability to create biofilms, both single and multi-species, as well as changes in the morphology of bacterial cells were noticed. As AA works best against young biofilms, the use of AA-containing formulations, especially during the initial stages of infection, seems to be reasonable. However, there is a need for further research concerning AA especially regarding its antibacterial mechanisms of action.

Resistencia antimicrobiana de uropatógenos en adultos mayores / Antimicrobial resistance of uropathogens in older adults

Introducción: En la actualidad, la resistencia antimicrobiana ha sido declarada por la Organización Mundial de la Salud como un problema de salud pública. Objetivo: Determinar el perfil de resistencia antimicrobiana de uropatógenos en adultos mayores. Métodos: Estudio descriptivo transversal de 567 urocultivos positivos de adultos mayores atendidos durante el año 2017 en una clínica privada en Lima, Perú. El análisis univariado se realizó por distribución de frecuencias, promedio y desviación estándar. Se estimó la asociación entre la producción de BLEE con respecto a las características epidemiológicas y el tipo de atención mediante Chi cuadrado con un nivel de significación de 0,05. Resultados: La edad promedio de la población fue de 74,1 años (DE:10,7). El 71,8 por ciento de los urocultivos positivos pertenecieron al sexo femenino. Los principales uropatógenos aislados en todos los niveles de atención fueron: E. coli, E. coli BLEE y K. pneumoniae BLEE. La E. coli presentó 69,3 por ciento de resistencia a ampicilina; y la E. coli BLEE tuvo el 100 por ciento de resistencia a ampicilina, ceftriaxona y ceftazidima. El 62 por ciento de microorganismos BLEE se encontraron en la atención ambulatoria. Se evidenció asociación estadísticamente significativa entre los agentes productores de BLEE y el sexo (p=0,004), mas no respecto al tipo de atención (p=0,144) ni subgrupos de edad (p=0,669). Conclusiones: La resistencia antimicrobiana es altamente prevalente en los adultos mayores. El sexo femenino fue el más afectado y el uropatógeno más frecuente la E. coli, este presenta una alta resistencia a ampicilina y mayor sensibilidad a nitrofurantoína. Se determinó un alto porcentaje de agentes productores de BLEE en la atención ambulatoria(AU)

Introduction: At present, the World Health Organization as a public health problem has declared antimicrobial resistance. Objective: To determine the antimicrobial resistance profile of uropathogens in older adults. Methods: Cross-sectional descriptive study of 567 positive urine cultures from older adults treated during 2017 in a private clinic in Lima, Peru. Univariate analysis was performed by frequency distribution, mean and standard deviation. The association between ESBL production with respect to epidemiological characteristics and type of care is estimated using Chi square with a significance level of 0.05. Results: The average age of the population was 74.1 years (SD: 10.7). 71.8percent of the positive urine cultures were from female sex. The main uropathogens isolated at all levels of care were E. coli, E. coli ESBL and K. pneumoniae ESBL. E. coli showed 69.3percent resistance to ampicillin; and E. coli ESBL had 100percent resistance to ampicillin, ceftriaxone, and ceftazidime. 62percent of ESBL microorganisms were found in outpatient care. There was a statistically significant association between ESBL-producing agents and gender (p = 0.004), but not with regard to type of care (p = 0.144) or age subgroups (p = 0.669). Conclusions: Antimicrobial resistance is highly prevalent in older adults. The female sex was the most affected and the most frequent uropathogen was E. coli, highly resistant to ampicillin and greater sensitivity to nitrofurantoin. High percentage of ESBL-producing agents was determined in outpatient care(AU)

Differences of virulence factors, and antimicrobial susceptibility according to phylogenetic group in uropathogenic Escherichia coli strains isolated from Korean patients.

BACKGROUND: Escherichia coli is among the most common uropathogens. Increased antibiotic resistance in Gram negative bacilli is global concern. Alternative therapeutic options including vaccines against uropathogenic E. coli (UPEC) have been developed. In this study, we compared the genotypic characteristics and antimicrobial susceptibility of UPEC according to phylogenetic groups. METHODS: We retrospectively reviewed the medical records of pyelonephritis patients with UPEC between February 2015 and June 2018. The study was conducted at a medical center in Korea. We compared the clinical and genotypic characteristics of UPEC according to phylogenetic groups. The phylogenetic groups and 29 virulence factors were identified using multiplex polymerase chain reaction. RESULTS: Phylogenetic group analysis revealed that most uropathogenic E. coli belonged to groups B2 and D: B2 (276, 77.7%), D (62, 17.5%), B1 (12, 3.4%), and A (5, 1.4%). Among the virulence factors, fyuA, fimH, traT, iutA, papG allele II, and papC were the most frequently observed. Phylogenetic group B2 was more closely related to virulence factors, including fimH, sfa/focED, focG, hlyA, cnf1, fyuA, and PAI, than group D. Groups B2 and D showed similar clinical presentations and complications. Group B2 had mostly healthcare-associated infections and antimicrobial resistance. Group D mostly had community-acquired infections. The K1 serotype was prevalent in group B2, and K5 was the most prevalent in group D. CONCLUSIONS: Phylogenetic group B2 had more proportions and types of virulence factors than group D. Group B2 showed a high presentation of virulence factors related to adhesions and toxins. An increased presentation of antimicrobial resistance and healthcare-associated infections was also noted. Considering the genetic characteristics of UPEC, alternative therapeutic options targeting frequent virulence factors might be considered in addition to antibiotics.

Escherichia coli type-1 fimbriae are critical to overcome initial bottlenecks of infection upon low-dose inoculation in a porcine model of cystitis.

Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models' natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.

Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC.

Urinary tract infections (UTI) are the most common infectious diseases in the world. It is becoming increasingly tough to treat because of emergence of antibiotic resistance. So, there is an exigency to develop novel anti-virulence therapeutics to combat multi-drug resistance pathogenic strains. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) discovery has revolutionized the gene editing technology for targeted approach. The greatest obstacle for CRISPR/Cas9 is cargo delivery systems and both viral and plasmid methods have disadvantages. Here, we report a highly efficient novel CRISPR based gene editing strategy, CRISPR-dots for targeting virulence factor Fimbrial Adhesion (papG gene), the bacterial adhesion molecule. Carbon quantum dots (CQD) were used as a delivery vehicle for Cas9 and gRNA into CFT073, a UPEC strain. CQDs were covalently conjugated to cas9 and papG-targeted guide RNA (gRNA) forming a nanocomplex CRISPR-dots (Cri-dots) as confirmed by DLS and transmission electron microscopy. Cri-dots-papG significantly targeted papG as demonstrated by decrease in the expression of papG.Further papG deficient UPEC had significantly reduced adherence ability and biofilm forming ability as demonstrated by fluorescence microscopy and scanning electron microscopy. Also, papG deficient UPEC had reduced virulence as shown by significantly increased survival of Caenorhabditis elegans (C. elegans) worms compared to UPEC. Our findings suggest that targeting of papG gene using Cri-dots nanocomplexes significantly reduced the pathogenicity of UPEC. Thus, Cri-dots nanocomplex offer a novel anti-bacterial strategy against multi-drug resistant UPEC.

Higher Prevalence of Extended Spectrum ß-Lactamase Producing Uropathogenic Escherichia coli Among Patients with Diabetes from a Tertiary Care Hospital of Kathmandu, Nepal.

This study aimed to determine the occurrence of antibiotic resistance genes for ß-lactamases; blaTEM and blaCTX-M in uropathogenic Escherichia coli isolates from urinary tract infection (UTI) suspected diabetic and nondiabetic patients. A hospital-based cross-sectional study was conducted in Kathmandu Model Hospital, Kathmandu, in association with the Department of Microbiology, GoldenGate International College, Kathmandu, Nepal, from June to December 2018. A total of 1,267 nonduplicate midstream urine specimens were obtained and processed immediately for isolation of uropathogens. The isolates were subjected to antibiotic susceptibility testing and extended spectrum ß-lactamase (ESBL) confirmation. In addition, blaTEM and blaCTX-M genes were detected using specific primers. The overall prevalence of UTI was 17.2% (218/1,267), of which patients with diabetes were significantly more infected; 32.3% (31/96) as compared with nonpatients with diabetes, 15.9% (187/1,171). A total of 221 bacterial isolates were obtained from 218 culture-positive specimens in which E. coli was the most predominant; 67.9% (150/221). Forty-four percent (66/150) of the total E. coli was multidrug resistant and 37.3% (56/150) were ESBL producers. Among 56 isolates, 92.3% (12/13) were from patients with diabetes, and 83.0% (44/53) were from nondiabetics. Furthermore, 84.9% of the screened ESBL producers were confirmed to possess either single or both of blaTEM and blaCTX-M genes. The blaTEM and blaCTX-M genes were detected in 53.6% and 87.5% of the phenotypically ESBL confirmed E. coli, respectively. Higher rates of ESBL producing uropathogenic E. coli are associated among patients with diabetes causing an alarming situation for disease management. However, second-line drugs with broad antimicrobial properties are still found to be effective drugs for multidrug resistance strains.

Characterization of Anti-Bacterial Effect of the Two New Phages against Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are among the events that most frequently need medical intervention. Uropathogenic Escherichia coli are frequently their causative agents and the infections are sometimes complicated by the presence of polyresistant nosocomial strains. Phage therapy is a tool that has good prospects for the treatment of these infections. In the present study, we isolated and characterized two bacteriophages with broad host specificity against a panel of local uropathogenic E. coli strains and combined them into a phage cocktail. According to genome sequencing, these phages were closely related and belonged to the Tequatrovirus genus. The newly isolated phages showed very good activity on a panel of local clinical E. coli strains from urinary tract infections. In the form of a two-phage cocktail, they were active on E. coli strains belonging to phylogroups B2 and D, with relatively lower activity in B1 and no response in phylogroup A. Our study is a preliminary step toward the establishment of a national phage bank containing local, well-characterized phages with therapeutic potential for patients in Slovakia.

Virulence characterization and clonal analysis of uropathogenic Escherichia coli metallo-beta-lactamase-producing isolates.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infection (UTI); however, treatment of UTI has been challenging due to increased antimicrobial resistance (AMR). One of the most important types of AMR is carbapenem resistance (CR). CR bacteria are known as an important threat to global public health today. Class B metallo-beta-lactamases (MBLs) are one of the major factors for resistance against carbapenems. We aimed to investigate the characteristics of UPEC isolates producing MBL. METHODS: A cross-sectional study was conducted from October 2018 to December 2019 in Ahvaz; Iran. UPEC isolates were identified by biochemical and molecular methods. Metallo-beta-lactamase-producing isolates were detected using modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) tests. MBL genes, phylogenetic group, and virulence genes profile of carbapenem resistant isolates were determined. Conjugation assay and plasmid profiling were conducted to evaluate the ability of transferring of CR to other E. coli isolates. Clonal similarity of isolates were assessed using Enterobacterial intergenic repetitive element sequence (ERIC)-PCR. RESULTS: Among 406 UPEC isolates, 12 (2.95%) carbapenem-resistant were detected of which 11 were phenotypically MBL-producing strains. Four isolates were resistant to all investigated antimicrobial agents and were considered possible pandrug-resistant (PDR). blaNDM, blaOXA-48, blaIMP-1, and blaIMP-2 genes were found in 9, 5, 1, and 1 isolates, respectively. Among 30 virulence genes investigated, the traT, fyuA followed by fimH, and iutA with the frequency of 8 (66.7%), 8 (66.7%), 7 (58.3%), and 7 (58.3%) were the most identified genes, respectively. Siderophore production was the main virulence trait among carbapenem-resistant UPEC isolates. Except for two, all other isolates showed weak to moderate virulence index. In all recovered isolates, CR was readily transmitted via plasmids to other isolates during conjugation experiments. CONCLUSION: MBL and carbapenemase genes, especially blaNDM and blaOXA-48 are spreading rapidly among bacteria, which can be a threat to global public health. Therefore monitoring the emergence and dissemination of new AMR is necessary to continuously refine guidelines for empiric antimicrobial therapy. Understanding the mechanisms of resistance and virulence in this group of bacteria can play an effective role in providing new therapeutic methods.

Toxin Induction or Inhibition of Transcription or Translation Posttreatment Increases Persistence to Fluoroquinolones.

Toxin-antitoxin modules are widespread in prokaryotes, and the capacity of toxin accumulation to increase the tolerances of bacteria to antibiotics has been well documented. The conventional model for this functionality implies that an overabundance of toxin arrests bacterial growth, which inhibits processes targeted by antibiotics and thereby limits their corruption and the lethal damage that would ensue. Implicit in this model is that toxins exert their influence on antibiotic lethality before and/or during treatment, even though they are also present and functional after treatment concludes. Given recent evidence establishing that the period following antibiotic treatment (recovery) is important for the survival of nongrowing bacterial populations treated with fluoroquinolones (FQs), we assayed to what extent toxins influence bacterial survival during the recovery period. With both LdrD and MazF, toxins of type I and II systems, respectively, controlling accumulation to occur only after FQ treatment of nongrowing cultures resulted in significant increases in persisters. Further genetic investigation revealed important roles for homologous recombination and nucleotide excision repair machinery. Focusing on the wild type, we did not observe any SOS-induced toxin functioning in this manner; however, an analogous phenomenon was observed for wild-type Escherichia coli as well as uropathogenic E. coli (UPEC) when transcription or translation was inhibited during the post-FQ recovery period. Collectively, these data reveal the capacity of toxins to thwart FQ killing even after the treatment has concluded and show that FQ treatment of nongrowing bacteria can be rendered largely ineffective if bacteria cannot readily resume translation and growth at the conclusion of treatment. IMPORTANCE Overabundances of toxins have been shown to increase the antibiotic tolerances of bacteria. Largely, these effects have been attributed to the abilities of toxins to inhibit bacterial growth before and during antibiotic exposure. In this study, we assessed to what extent toxins can influence bacterial survival following antibiotic treatment, rather than before or during. Using two mechanistically distinct toxins, we show that their accumulations after antibiotic exposure have the capacity to increase the abundances of fluoroquinolone persisters from nongrowing populations. Further, we show with wild-type and uropathogenic E. coli that chemical inhibition of growth, not just that induced by toxins, produces analogous results. These observations reveal another dimension of how toxins influence antibiotic tolerance and highlight the importance of postantibiotic physiology on bacterial survival.

Diverse anti-inflammation and anti-cancer polyketides isolated from the endophytic fungi Alternaria sp. MG1.

Six new polyketides, alternaritins A-D [(±)-1-4] and isoxanalteric acid I (8), and 25 known Alternaria toxins were isolated from the culture of an endophytic fungi Alternaria sp. MG1. 3 is a rare fungal metabolite. 6 is a new natural product, and 5, 7, and 9 are known previously but their absolute configurations have not been determined. Three enantiomers [(±)-1, (±)-7, and (±)-15] were separated via chiral HPLC resolution. The structures of those polyketides (1-9) were elucidated by spectrometric analysis using MS and NMR. The absolute configurations were established using X-ray diffraction analysis and statistical comparative analysis of the experimental ECD and OR data, in conjunction with quantum mechanical calculations. All of the compounds were evaluated for their bioactivities. Known compound 27 exerted the most potent cytotoxic activities against HT-1080 and NCI-H1299 cell lines. The new compounds, 2 and 3, showed moderate inhibition on COX-2, while a pair of isomers, 8 and 9, exhibited medium activity on COX-2 and uropathogenic Escherichia coli.

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model.

Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.

Two Novel Mutations Associated with Polymyxin-B Resistance in a Pandemic Lineage of Uropathogenic Escherichia coli of the Sequence Type 69.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are frequent pathogens worldwide, impacting on the morbidity and economic costs associated with antimicrobial treatment. OBJECTIVES: We report two novel mutations associated with polymyxin-B resistance in an UPEC isolate collected in 2019. METHODS: Isolate was submitted to antimicrobial susceptibility testing including broth microdilution for polymyxin B. Whole genome was sequenced and analyzed. RESULTS: Polymyxin-B total inhibition occurred at 16 mg/L (resistant). UPEC isolate was assigned to the phylogroup D, serotype O117:H4, and Sequence Type 69. mcr genes were not detected, but two novel mutations in the pmrA/basS (A80S) and pmrB/basR (D149N) genes were identified. CONCLUSIONS: The occurrence of non-mcr polymyxin resistance in E. coli from extraintestinal infections underscores the need of a continuous surveillance of this evolving pathogen.

Genome profiling of fluoroquinolone-resistant uropathogenic Escherichia coli isolates from Brazil.

Urinary tract infections (UTIs) are a major public health concern in both community and hospital settings worldwide. Uropathogenic Escherichia coli (UPEC) is the main causative agent of UTI and increasingly associated with antibiotic resistance. Herein, we report the draft genome sequence of 9 fluoroquinolone-resistant UPEC isolates from Brazil and examine selected major phenotypic features, such as antimicrobial resistance profile, phylogroup, serotype, sequence type (ST), virulence genes, and resistance marks. Besides the quinolone resistance, beta-lactams, ESBL production, aminoglycosides, and tetracycline resistance were observed. High prevalence of 20 virulence genes was detected in all isolates, such as those encoding type 1 fimbriae, acid tolerance system, and hemolysin E, particularly within E. coli B2 phylogroup, as ST131 and ST1193 strains, among other genomic analyses as genomic islands, resistance plasmids, and integron identification.

Dual Function Antimicrobial Loaded Lectin Carrier: A Strategy to Overcome Biomolecular Interference without Detectable Resistance.

The disposition of a drug in a biological system may be altered by complex biological fluids; especially, protein binding to drugs influences their activity. Herein, we demonstrated a convenient method involving the noncovalent formulation of butea monosperma seed lectin (BMSL) with an antimicrobial lipid, cationic N-acylethanolamine (cNAE) to mitigate the serum protein interference. Fluorescence spectroscopy and molecular docking study revealed that cNAEs readily formed noncovalent complexes with serum protein, bovine serum albumin. The resulting complexes interfered with the antimicrobial activity of cNAEs. Strikingly, the noncovalent conjugates developed with BMSL and cNAEs (BcNAE) overcame the interference from serum protein and displayed remarkable antimicrobial activity against uropathogenic Escherichia coli (UPEC). Strikingly, the minimum inhibitory concentration (MIC) of the lectin conjugates (7.81 µM) was 4-fold lower than the MIC of pure cNAE. Mechanistic studies showed that BcNAE depolarized the bacterial membrane and affected the integrity to exert the antimicrobial activity. The membrane directed activities of BcNAE on UPEC efficiently eliminated the development of resistance even after 25 passages. The hemocompatibility results and the biosafety assessed in a zebrafish model suggested that BcNAE was nontoxic with good selectivity to bacteria. While testing the therapeutic efficacy against UPEC infected zebrafish, we found that 1× MIC cNAE is ineffective due to interference from biological fluids, which is in agreement with in vitro studies. Remarkably, the infected fish treated with 1× MIC BcNAE conjugates were rescued from infection and restored to the normal life in less than 9 h. Bacterial colony count assay revealed that BcNAE was more efficient in overcoming the biological fluid interference and eliminated the bacterial burden in infected zebrafish. Histopathology analysis supported that BcNAE treatment restored the pathological changes induced by UPEC and, thus, increased survival. The high antimicrobial intensity with limited chance for resistance development and potential to overcome biomolecular interference with a lack of toxicity enhance the merits of exploring lectin conjugates against infectious pathogens.

Phytochemical analysis of medicinal plants of Nepal and their antibacterial and antibiofilm activities against uropathogenic Escherichia coli.

BACKGROUND: A biofilm is an extracellular polymeric substance (EPS) composed of polysaccharides, proteins, nucleic acids, and lipids that impede antibiotics and immune cells, thus providing a shielded environment for bacterial growth. Due to biofilm formation, some microbes can show up to 1000 fold increased resistance towards the antibiotics than the normal planktonic forms. The study was conducted to screen the crude extracts of medicinal plants used in Nepal for their in vitro antibiofilm activities. METHODS: Total phenolic and total flavonoid contents were determined by using a Folin-Ciocalteau reagent and aluminium trichloride method, respectively. Resazurin assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The initial antibiofilm activities and their inhibitory concentration (IC50) values were determined by the microtiter based modified crystal violet staining method. RESULTS: Out of 25 different plant extracts were used for the study, methanolic extracts of 20 plants showed a biofilm inhibition activity against five different strong biofilm producing Escherichia coli strains. Calotropis gigantea exhibited inhibition against all five different E. coli strains with IC50 values ranging from 299.7 ± 20.5 to 427.4 ± 2.7 µg/mL. Apart from that, Eclipta prostrata also showed biofilm formation inhibition, followed by Eupatorium adenophorum, Moringa oleifera, Ocimum tenuifolium, Oxalis lantifolia, Prunus persica, and Urtica parviflora. The extracts of C. gigantea, E. prostrata, Mangifera indica, O. tenuifolium, P. persica, and U. parviflora exhibited a moderate to poor MIC value ranging from 625 to 2500 µg/mL. The highest amount of phenolic content (TPC) was found in Acacia catechu followed by Morus alba, which was 38.9 and 25.1 mg gallic acid equivalents, respectively. The highest amount of flavonoid content was found in A. catechu followed by M. indica, which was 27.1 and 20.8 mg quercetin equivalents, respectively. CONCLUSION: Extracts of C. gigantea, E. prostrata, P. persica, U. parviflora, and O. tenuifolium showed antibacterial as well as antibiofilm activity against pathogenic and strong biofilm producing E. coli. Thus, extracts or the pure compound from these medicinal plants could be used as antibiotics in the future.

Cytochrome bd promotes Escherichia coli biofilm antibiotic tolerance by regulating accumulation of noxious chemicals.

Nutrient gradients in biofilms cause bacteria to organize into metabolically versatile communities capable of withstanding threats from external agents including bacteriophages, phagocytes, and antibiotics. We previously determined that oxygen availability spatially organizes respiration in uropathogenic Escherichia coli biofilms, and that the high-affinity respiratory quinol oxidase cytochrome bd is necessary for extracellular matrix production and biofilm development. In this study we investigate the physiologic consequences of cytochrome bd deficiency in biofilms and determine that loss of cytochrome bd induces a biofilm-specific increase in expression of general diffusion porins, leading to elevated outer membrane permeability. In addition, loss of cytochrome bd impedes the proton mediated efflux of noxious chemicals by diminishing respiratory flux. As a result, loss of cytochrome bd enhances cellular accumulation of noxious chemicals and increases biofilm susceptibility to antibiotics. These results identify an undescribed link between E. coli biofilm respiration and stress tolerance, while suggesting the possibility of inhibiting cytochrome bd as an antibiofilm therapeutic approach.