Phytochemicals of Aloe barbadensis miller as Potential Inhibitors of Uropathogenic Escherichia coli for Urinary Tract Infection Therapy: An in Silico Approach

Urinary tract infections (UTIs) are common infections caused by normal skin or rectum bacteria that get into the urethra and infect the urinary tract. Although the infection can affect various parts of the tract, bladder infections are the most prevalent kind. Uropathogenic Escherichia Coli (UPEC) is the most common pathogen associated with UTI development. Therefore, inhibiting the UPEC protein target (PDB ID: 8BVD) appears to be a promising therapeutic strategy. Therefore, in this study, molecular docking and dynamics were conducted to examine the antibacterial activity of Aloe barbadensis miller against UPEC bacteria. The Aloe barbadensis miller natural compounds licochalcone A, palmidin B and palmidin C were downloaded from PubChem with amoxicillin, which was used as a control drug and studied against the target molecule. The potential parameters examined were the docking scores, absorption, distribution, metabolism, excretion, toxicity (ADMET), bioavailability, root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonding, radius of gyration, and potential energy of the system. Docking scores showed that all ligands demonstrated an admirable candidature as an inhibitor to 8BVD molecule, and the score hierarchy is licochalcone A (-6.4 kcal/mol), palmidin C (-6.1 kcal/mol), palmidin B (-6.0 kcal/mol), and amoxicillin (-5.9 kcal/mol). All ligands appeared to have good drug-like properties and oral bioavailability. Molecular dynamic studies showed that all ligands exhibited an excellent nominee as inhibitors in their vicinity at 20 ns. However, there is a relatively high fluctuation of palmidin B compared with other compounds, which seems to be more stable. This work suggests that the selected phytoconstituents could be used as inhibitors of the 8BVD protein in the fight against UTIs. However, further investigation on the clinical and experimental validation of UTI treatment’s specific mechanisms and effects is still welcomed.

Regulation of fim genes in uropathogenic Escherichia coli

Uropathogenic Escherichia coli(UPEC)is the leading cause of urinary tract infections in women,causing significant morbidity and mortality in this population.Adherence to host epithelial cells is a pivotal step in the pathogenesis of UPEC.One of the most important virulence factors involved in mediating this attachment is the type 1 pilus(type 1 fimbria)encoded by a set of fim genes arranged in an operon.The expression of type 1 pili is controlled by a phenomenon known as phase variation,which reversibly switches between the expression of type 1 pili(Phase-ON)and loss of expression(Phase-OFF).Phase-ON cells have the promoter for the fimA structural gene on an invertible DNA element called fimS,which lines up to allow transcription,whereas transcription of the structural gene is silenced in Phase-OFF cells.The orientation of the fimS invertible element is controlled by two site-specific recombinases,FimB and FimE.Environmental conditions cause transcriptional and post-transcriptional changes in UPEC cells that affect the level of regulatory proteins,which in turn play vital roles in modulating this phase switching ability.The role of fim gene regulation in UPEC pathogenesis will be discussed.

Inhibitory Activities of Lactic Acid Bacteria against Multi-Drug Resistant Uropathogenic <i>Staphylococcus saprophyticus</i>Isolated from Symptomatic Women in Lagos, Nigeria

The uropathogenic Staphylococcus saprophyticus is reported severally to be resistant to the drugs often used empirically for treatment of urinary tract infections (UTIs). Their ability to exhibit resistance to multiple drugs is a great deal of threat to successes recorded in the management of UTIs caused by this pathogen. Lactic acid bacteria (LAB) have been demonstrated to exhibit antimicrobial activities but studies about their prospect against multi-drug resistant S. saprophyticus are quite few. This study therefore investigated activities of LAB against the multi-drug resistant S. saprophyticus recovered from urine samples of symptomatic women. The three different species of LAB (Lactobacillus fermentum BTA 62, Lactobacillus johnsonii BTA 86 and Weissella confusa BTA 40) previously isolated and identified by 16S rRNA sequencing were selected based on their history of antimicrobial activities. Their metabolites were employed in the antagonistic assays against six (6) multi-drug resistant test pathogens recovered from urine samples of symptomatic, non-pregnant women attending clinics in Lagos, Nigeria and the control (S. saprophyticus subs bovis strain DSM 18669) following standard procedures. The pathogens showed resistances to almost all the antibiotics except levofloxacin, ciprofloxacin, imipenem while the control showed resistance to three. The LAB, L. fermentum inhibited five (83.3%) of the pathogens with zone diameter of 12 - 17 mm, followed by W. confusa inhibiting three (50%) with 15 - 17 mm. Lactobacillus johnsonii on the other hand, inhibited a pathogen and the control with zones of 13 mm and 14 mm respectively. In conclusion, the extracted metabolites of LAB inhibited the growth of multi-drug resistant clinical isolates of uropathogenic S. saprophyticus and may therefore be potent alternatives to antibiotics.

Prevalence of multi-drug resistant uropathogenic Escherichia coli in Potohar region of Pakistan

Objective:To scrutinize patterns of multi-drug-resistant uropathogenic Escherichia coli(UPEC) strains and particularly of fluoroquinolone-resistance this is an alternative choice for the treatment of urinary tract infections.Methods:Bacterial samples(n = 250) were collected from out-patients from August 2012 to August 2014 Islamabad.Antibiotic susceptibility profiling and determination of minimum inhibitory concentrations(MICs) and minimum bactericidal concentrations were performed according to the guidelines of Clinical and Laboratory Standards Institute(CLSI,2012).Genes,qnrA,qnrB and qnrS were identified by DNA amplification and sequencing.Results:The highest percentage of UPEC isolates were resistant to co-trimoxazole(82%) followed by cephalothin(80%),2nd Gen,3rd Gen and 4th Gen cephalosporins,respectively.Resistance against gentamicin,amikacin remained 29% and 4%.For other drugs including nitrofurantoin,tetracycline,carbapenem and beta-lactam inhibitors remained below 10%.Altogether,59% of the isolates were resistant to at least three antibiotics including one fluoroquinolone.Overall,MICs for ciprofloxacin remained(MIC≥256 μg/mL) and for levofloxacin(MIC≥16 μg/mL and 32 μg/mL).No significant differences were observed regarding MIC values of extended spectrumβ-lactamase(ESBL) and non-ESBL producers.For qnrS and qnrB positive isolates MICs remained above 32 μg/mL.Prevalence of UPEC was significantly higher among females and 40% of the isolates were ESBL producers.Conclusions:Higher percentages of ESBL producing UPEC were associated with urinary tract infections.Moreover,the majority of these isolates were multi-drug resistant and fluoroquinolone-resistant.

Correlation of biofilm formation of uropathogenic Escherichia coli(UPEC) and fimbriae genotypes

Objective:In the present study we try to correlate between pathogenic intrinsic factor of Escherichia coli(E.coli) presented with different fimbria genotyes and biofilm formation with host immune factor entitled interleukin-6(IL-6) secretion as defense mechanism.Methods:A total of 91 pediatrics complaining of pyuria were included in the present study.In addition,20 healthy control children were included.Full microbiological study was performed for isolated E.coli.PapC alleles were studied by multiple alleles PCR and biofilm formation was studied.IL-6 was measured in urine.Results:IL-6 had statistically significant elevation in patients’urine compared to control.From biofilm study, it was found that 19 isolated E.coli had formed biofilm in vitro.Moreover,urine samples with positive biofilm formation of E.coli had statistically significant lower IL-6 secretion than those with negative E.coli for biofilms.The distribution of fimbria genotypes showed that the frequent genotype was for alleleⅠ(34.3%) followed by mixed allelesⅠandⅡ(24.1%).There was significant correlation between mixed alleles(Ⅰ&Ⅱ)and biofilm formation.Conclusion: The present study highlights the presence of significant strains of E.coli causing urinary tract infections capable of biofilm formation.Biofilm formation is associated with less innate immunity manifested by lower urinary IL- 6.The majority of isolates had fimbria genes.It appears that mixed allelesⅠandⅡhave prominent virulence effect with tendency for biofilm formation.

Anti-uropathogenic activity, drug likeness, physicochemical and molecular docking assessment of(E-)-N'-(substituted-benzylidene)-2-(quinolin-8-yloxy) acetohydrazide

Objective: To deal with the anti-uropathogenic and in silico screening of(E-)-N'-(substitutedbenzylidene)-2-(quinolin-8-yloxy)acetohydrazide analogues in order to search the potential anti-uropathogenic agents.Methods: Three(E-)-N'-(substituted-benzylidene)-2-(quinolin-8-yloxy)acetohydrazide analogues were synthesized. Structure elucidation was done using various spectroscopic techniques including infrared radiation, 1hydrogen-nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, etc. Physicochemical score, bioactivity score and molecular docking studies were carried out using Lipinski's rule of five, Molinspiration(web based software), Autodock 4.2 tools. In vitro anti-uropathogenic activity was carried out against four pathogens named as Staphylococcus aureus(S. aureus), Staphylococcus epidermidis, Proteus mirabilis and Escherichia coli by disc diffusion method and macro-dilution test following their morphological and biochemical characterization.Results: The formation of(E-)-N'-(substituted-benz ylidene)-2-(quinolin-8-yloxy)acetohydrazide is confirmed from the spectroscopic results. All the compounds were found in compliance with Lipinski's rule of five and exhibited bioactivity score from-0.50 to 0.00. Docking results revealed that compound-1 is forming one hydrogen bond with TYR 576 and two hydrogen bond with GLU 569, while compound-2 is forming one hydrogen bond with ARG 599, and compound-3 forming 0 hydrogen bond. The anti-uropathogenic evaluation exhibited that compound one exhibited better activity against S. aureus, while it was found to possess moderate to good activity against both Gram-positive bacteria and Gram-negative bacteria excluding S. aureus.Conclusions: Our study revealed that compound one exhibited better activity than the standard in case of S. aureus and moderate to good activity against rest of the pathogens. Molecular docking, physicochemical and bioactivity studies strongly supported the experimental results. From the well obtained results it was concluded that compound-1 can lead as potential antiuropathogenic agents.

Bacterial Uropathogen among Benign Prostatic Hyperplasia Patients at a Tertiary Hospital in Nigeria

As the lumen of the prostatic urethra becomes compromised by fibroadenomatous growth in the periurethral region of the prostate of men with Benign prostatic hyperplasia (BPH), urine outflow is obstructed progressively resulting in incomplete bladder empting causing stasis and may predispose patients to infection. Mid stream urine samples were collected from 94 BPH patients. Macroscopy, microscopy, culture and antibiotic susceptibility test were carried out on isolated uropathogens. Isolated bacteria were characterized using biochemical tests. Isolated bacterial pathogens include Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Morgarella morgani, K. pneumonia and Enterococcus faecalis in order of frequency. The isolates show highest resistance of 87.1% to Ciprofloxacin and least resistance of 12.9% to Imipenem. The isolates were found to be multi-drug resistant and the 12.9% resistance to Imipenem suggests presence of Carbapenemase producing bacteria among the isolates.

Epidemiological Study of Rapidly Emerging Uropathogens Isolated from Urinary Catheter and Its Influential Demographic Factors Responsible for Contamination

Urinary tract infections (UTI) are very common in all the developed as well as developing countries in which the majority of infections are catheter associated. Catheter associated urinary tract infections (CAUTI) is one of the major causes of hospital acquired infections. The aim of this study is to investigate influential demographic factors responsible for contamination associated with the rate of CAUTI, while taking into account type of urinary catheter used, the most common organisms found, patient diagnosis, age, gender, and comparison with other studies. During the study, 22 uropathogenic species were isolated from the different segments of urinary catheter samples of the patients collected from 12 different hospitals of Amravati city, Maharashtra, India from January 2015 to 2018. Gram negative organisms were the most frequent isolates, with Pseudomonas aeruginosa (16.08%) being the most common followed by Escherichia coli (10%) and Candida albicans accounted for almost 11% of all the uropathogens. It was found that the majority of uropathogens were isolated from the section A (Catheter segment inside the bladder) and section E (Catheter segment connected to drainage tube) of the urinary catheter. The duration of the catheterization plays the major role in the contamination and further infection to continue. It was observed that the female catheterized patients are more prone to the contamination and infection as compared to male catheterized patient. The antibiotic sensitivity pattern indicates that MAR (Multiple Antibiotic Resistance) index was more than 0.2 for almost all the uropathogens tested concluding that there is antibiotic stress on uropathogens and rate of resistance increased rapidly. Also it was found that there was a statistically significant association between the duration of catheterization, type of disease, age of patient and type of catheter with respect to gender.

Prevalence and Associated Risk Factors of Uropathogenic Klebsiella Species in Port Harcourt

Background: Urinary tract infection (UTI) is a severe public health issue that affects a wide range of people around the world with Klebsiella pneumoniae accounting for up to 25% of all urinary tract infections. The higher rate of UTI associated with uropathogenic Klebsiella species has been associated with the emergence of hypervirulent and antibiotic-resistant strains facilitated by the misuse and overuse of antibiotics as well as other sociodemographic and behavioural practices of susceptible individuals. This study was aimed at investigating the prevalence and associated risk factors of uropathogenic Klebsiella species in Port Harcourt, Nigeria. Methodology: The study employed a descriptive cross-sectional study design comprising 300 subjects clinically suspected of having urinary tract infections attending the Rivers State University Medical Centre and the Rivers State University Teaching Hospital between March to August 2022. A standard urine culture procedure was used to ascertain significant bacteriuria after which Klebsiella colonies were isolated and identified using standard bacteriological techniques. The data generated from this study was represented as frequency and percentages, and inferential statistics were carried out using Chi-square with the aid of GraphPad Prism Software Version 9. Statistical significance was defined as a p-value of less than 0.05 at a 95% confidence interval. Result: The prevalence of uropathogenic Klebsiella sp. was 16%, with sex, pregnancy status, and religion of the individuals substantially linked (p p Klebsiella species. Conclusion: This study reports a relatively high prevalence of uropathogenic Klebsiella species at 16%, with the sex and pregnancy status of the subjects being significantly associated (p Klebsiella species in the current study. Health promotion and awareness efforts should be prioritised to inform susceptible demographics about their risks for urinary tract infections associated with uropathogenic Klebsiella species via targeted educational campaigns, collaboration with healthcare providers, use of social media and online platforms, workplace wellness programs, and community outreach programs amongst others. Antimicrobial susceptibility testing before prescriptions and treatment should be emphasized and upheld in all clinical settings.

Prevalence of Uropathogenic Escherichia coli among Adult Male Patients 40 Years and above with Haematuria and Impaired Kidney Attending General Hospitals in Benue State

Haematuria is the presence of red blood cells in urine. It is most often caused by urinary tract infections of which Escherichia coli is frequently implicated. Impairment of kidney functions could occur as a result of infection or other complications of the kidney. The aim of the study was to determine the prevalence of uropathogenic Escherichia coli among adult male patients with haematuria and impaired kidneys attending a general hospital in Benue state. Three hundred and sixty-eight (368) samples of urine were collected from 368 male patients (≥ 40 years) attending the 23 general hospitals in Benue state. Each of the urine samples was divided into two parts for haematuria and isolation and identification of Escherichia coli. Blood samples (368) were also collected from the patients and used for quantitative determination of creatinine and estimation of glomerular filtration rate. The presence of haematuria was 45.1% and ranges from 12.5% to 100%. Prevalence of haematuria with respect to age shows that patients within the age group of 90 - 99 years had the highest rate (100%) and the least were those within the ages of 40 - 49 years (20.0%). Isolation rate of uropathogenic Escherichia coli was 16.3% and ranged from 6.3 to 37.5%. Patients within the age group of 90 - 99 years had the highest elevated impaired renal function of 4 (80%), followed by patients within the ages of 80 - 89 years [17 (77.3%)] and the lowest were those within the ages of 40 - 49 [6 (10.0%)]. The overall presence of haematuria in the patients was high (45.1%) with similar high Escherichia coli isolation rate and impaired renal function which could mean that acute or chronic kidney disease may set in.

Genetic Diversity of Uropathogenic Escherichia coli Isolated from Human T Lymphotropic Virus Type I (HTLV-I) Infected Individuals

Urinary tract infection (UTI) is a frequent pathology among HTLV-I+ individuals being capable of severely compromising the kidneys and bladder. Molecular characteristics of uropathogenic Escherichia coli (UPEC) from HTLV-I+ infected individuals are unknown. UPEC isolates from HTVL-I+ individuals, with and without clinical symptoms of myelopathy, were submitted to genetic typing seeking to infer bacterial diversity and potential virulence. 71 bacterial isolates were characterized according to random amplified polymorphic DNA and phylotypes. Phylotyping classified E. coli into four phylogenetic groups: A (18.3%), B1 (16.9%), B2 (39.4%), and D (25.3%) and 8 phylotypes according to the presence of the genetic sequences chuA, yjaA and the DNA fragment TSPE4.C2: ﹣﹣﹣ (5.6%) and ﹣+﹣ (12.6%) in phylogroup A, ﹣﹣+ (7.0%) and ﹣++ (9.8%) in B1, +++ (32.3%) and ++﹣ (7.0%) in B2, +﹣﹣ (15.4%) and +﹣+ (9.8%) in D. The B2 phylogroup was the most prevalent among HTLV﹣ associated myelopathy and asymptomatic individuals. RAPD-PCR typing revealed a high degree of bacterial polymorphism indicating a non-clonal origin. Genotypes were not found to be distributed according to clinical status or epidemiological features. Our results lead us to suggest that the neurological impairment in HTLV-I+ individuals can be a risk factor for urinary infections due E. coli which are caused by distinct bacterial lineages.

Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli （UPEC） to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium （K-SFM） with epidermal growth factor （EGF） and bovine pituitary extract （BPE）. Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678- 54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnfl of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

Multi-drug Resistant Uropathogenic Escherichia coli and Its Treatment by Chinese Medicine

Objectives： To investigate the resistance and virulence profiles of uropathogenic Escherichia coli（UPEC） and its treatment by Chinese medicine（CM） Fuzheng Qingre Lishi Formula（扶正清热利湿方, FQLF）. Methods： UPEC strains were isolated from recurrent urinary tract infections（UTIs） patients. Patient sensitivities to 17 antibiotics were tested by the disk diffusion method. Virulence genes were screened by plolymerase chain reaction. A mouse model was constructed using a multi-drug resistant and virulent UPEC strain and treated with FQLF or the antibiotic imipenem. The treatment efficacy was evaluated by bacterial clearance from urine and the urinary organs. Results： A total of 90 UPEC strains were collected, and 94.4% of the isolates were resistant to at least 1 antibiotic. Approximately 66.7% of the UPEC strains were multi-drug resistant. More than one virulence gene was found in 85.6% of the isolates. The extended-spectrum β-lactamases（ESBL）-positive strains were more resistant than the negative ones. The virulence gene number was positively correlated with the resistance number（P〈0.05）. A mouse model was successful y constructed using UPEC10. Treatment with either FQLF or antibiotics significantly cleared bacteria from the mouse urine after 14 days. In the untreated control, the bacteria lasted for 28 days. FQLF treatment of the UTI mouse model greatly reduced the bacterial number in the kidney and bladder, but could not completely clear the bacteria. Conclusions： Multi-drug resistance is common among UPEC isolates, and the resistance is positively related with virulence. FQLF could treat UPEC UTIs, but could not completely clear the bacteria from the host.

Prevalence of ESBL phenotype,bla_(CTX-M-1),bla_(SHV)and bla_(TEM)genes among uropathogenic Escherichia coli isolates from 3 military hospitals of Tehran,Iran

Objective:To determine the extended-spectrum beta-lactamase(ESBL)production and prevalence of bla_(CTX-M-1),bla_(SHV)and bla_(TEM)genes among uropathogenic Escherichia coli(UPEC)isolates from 3 military hospitals of Tehran during 2015-2016.Methods:One-hundred and eleven isolates were adopted.The antibiotic susceptibility testing was conducted according to Clinical and Laboratory Standards Institute guidelines.The combine disk was used for phenotypic ESBL production.The ceftazidime MIC was conducted with the micro-broth dilution test.The PCR assay was used to detect the bla_(CTX-M-1),bla_(SHV)and bla_(TEM)genes.Results:In the broth microdilution method,103(92.7%)isolates showed minimal inhibitory concentration(MIC)≥1μg/mL,and also in the combined disk method,89(80.1%of all)were ESBL positive.On the other hand,among 91 ceftazidime resistant isolates,86(77.4%of all)were ESBL positive.The difference between the two methods for ESBL confirmation was not significant.The result of MIC was similar to the disk diffusion method in the detection of phenotypic ESBL production.Among ESBL producer isolates,the prevalence of bla_(CTX-M-1),bla_(SHV)and bla_(TEM)was 77.4%(n=86),47.4%(n=53)and 2.4%(n=2),respectively.These genes were amplified in a wide range MIC of ceftazidime.Conclusions:The prevalence of multi-drug resistant UPEC and ESBL positive isolates was high in military hospitals.The majority of UPEC isolates amplified bla_(CTX-M-I)and bla_(SHV)typeβ-lactamase genes.One-third of isolates were positive in presence of both these genes.There was no relation between ceftazidime MIC and presence of beta-lactamase genes.

Comparison of infection of different cell lines by uropathogenic Escherichia coli

Studying the interaction between uropathogenic Escherichia coli (UPEC) and uroepithelial cells is important in elucidating the pathogenesis of urinary tract infection. In this study, the African green monkey kidney cells (Vero), human kidney carcinoma cells (Ketr-3) and bladder carcinoma cells (EJ) were infected by UPEC132, a clinical strain isolated from Tianjin, China, and were compared for their capacities to allow the adherence and invasion by this strain. The results revealed that all these cell lines could be attached and invaded by UPEC132. The adherence rates for Vero, Ketr-3 and EJ cells were (49.20 ± 7.55)%, (55.22 ± 4.09)% and (73.20 ± 5.26)%, respectively, and invasion frequencies were (2.61 ± 0.32)×10-3, (3.00 ± 0.34)×10-3 and (3.25 ± 0.20)×10-3, respectively. The statistical analysis showed that the adherence rate for EJ cells was significantly higher than those for the other two cell lines (P<0.05), and the invasion frequencies for EJ and Ketr-3 cells had no statistical differences (P>0.05) but were higher than that for Vero cells (P<0.05). Three cell lines were detected for the receptors for P pili of UPEC by using indirect immunofluorescence. The results showed that receptors existed on the surfaces of all cell lines, and the highest distribution was found on the surface of EJ cells. Additionally, the invasion of EJ cells by recombinant UPEC132/pSELECT-GFP could be directly visualized using confocal microscopy. These data strongly implicated that EJ cells could be more easily infected by UPEC132 than the other cells, and thus could serve as a good experimental target for further investigation of UPEC infection.

A novel gene R049 identified in uropathogenic Escherichia coli provides partial protection in mice from colonization

Uropathogenic Escherichia coli(UPEC) is the most common causative organism of human urinary tract infection(UTI).Several UPEC virulence factors have been identified,but more are yet to be found.We previously identified a novel 789-bp-long DNA fragment(named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique.In the present study,we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity.Through bioinformatic analysis,we predicted that this gene is a 1311-bp open reading frame(ORF),which we designated ORFR049(GenBank accession No.:EF488001).We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography.Using mouse immunosera generated by the purified protein,we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting.To test the potential of this protein as a vaccine candidate,we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract.The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group.However,the degree of renal pathological damage was not significantly improved in the immunized mice.Our study has identified a novel gene of UPEC which can generate protective immunity against UTI.This novel gene provides a promising new vaccine candidate.

尿路感染细胞实验中庆大霉素对尿路致病大肠杆菌的杀伤作用及细胞毒性研究

目的在尿路感染体外细胞实验中,比较不同浓度庆大霉素(0、10、20、50、100、200μg/mL)对尿路致病大肠杆菌(UPEC)的杀伤作用,对尿路上皮细胞及炎性细胞如巨噬细胞的毒性作用。方法比较不同浓度庆大霉素对不同量UPEC J96菌株(10^(8)、10^(7)、10^(6))的杀伤情况;CCK-8实验检测不同浓度庆大霉素在不同时间内(2 h或24 h)对原代培养的C57BL/6雄鼠肾小管上皮细胞、腹腔巨噬细胞、人膀胱上皮细胞系J82的毒性作用;根据实验选择合适的庆大霉素浓度和作用时间,检测J96对小鼠肾小管上皮细胞的黏附、入侵以及小鼠巨噬细胞对J96的吞噬、清除作用。结果庆大霉素(≥10μg/mL)对J96的杀伤作用均强于1%的青霉素/链霉素双抗(P<0.0001),高浓度庆大霉素(≥100μg/mL)可在30 min内完全杀伤高达10^(8)的J96。50μg/mL庆大霉素作用2 h可对人膀胱上皮细胞系J82产生毒性作用(P<0.05)。结论本文明确了不同细胞体外实验时适宜的庆大霉素处理浓度及时间,人膀胱上皮细胞系J82对庆大霉素更为敏感。

Epidemiologic Profile Urinary Tract Infections: Experience of the Microbiology Laboratory of the University Hospital Mohamed VI in Tangier

Urinary tract infection is one of the most common bacterial infections in humans. The urine cytobacteriological examination is the key test for its diagnosis. This work aims to Evaluate the prevalence of urinary tract infection at the Microbiology Laboratory of the University Hospital Mohamed VI in Tangier and to highlight its epidemiological and bacteriological characteristics. It was a descriptive study with retrospective data collection which took place from January 2021 to June 2022, at the Microbiology Laboratory of the university hospital Mohamed VI in Tangier. It covered all urine cytobacteriological examinations (ECBU) during the study period. We identified 77 cases of urinary tract infections out of 300 requests for (ECBU), that is a positivity rate of 25 %. The mean age was 55 years. The male gender was predominant. The epidemiological profile of the isolated strains was dominated by Enterobacteriaceae 81 %, followed by Gram-positive cocci 11 % and non-fermenting gram-negative bacteria 8 %. The main bacteria responsible for urinary tract infections in order of frequency: Escherichia coli 42 %, Klebsiella pneumoniae 20 %, Enterobacter cloacae 9 %, Staphylococcus aureus 6 %, Pseudomonas aeruginosa 5 % and Acinetobacter baumannii 3%. Most of the analyzed organisms showed resistance, especially to the beta-lactam antibiotic;the enterobacteria strains isolated had revealed resistance to amoxicillin: 74%, to amoxicillin-clavulanic acid in 40% of cases, and to third-generation cephalosporins in 24%. In terms of resistance mechanisms, 11 % of the Enterobacteriaceae were extended-spectrum β-lactamase producers and 9 % of the specimens were identified as carbapenemase producers. Of the Staphylococcus aureus strains isolated, 75% were resistant to meticillin. The Glycopeptides and linezolid were the most active molecules on these isolated strains. 15% of Enterococcus species isolated in our laboratory were resistant to glycopeptides (vancomycin and teicoplanin).

P菌毛粘附素在致肾盂肾炎大肠埃希菌对小鼠尿道上行感染中的作用

目的 比较2株具有不同P菌毛粘附素(PapG)的同血清型UPEC和1株无菌毛大肠埃希菌对小鼠泌尿道上行感染性的差异,探讨粘附素在UPEC尿道感染中的作用。方法 通过17PEC尿道内接种形成BALB/c小鼠尿道上行感染;观察尿液、肾脏剖面的菌落计数和肾组织的病理改变。结果 具有P菌毛的UPEC菌株感染之小鼠,在其尿液和肾剖面培养出大量原感染菌,肾组织呈现中、重度急性肾盂肾炎的病理改变;不同P菌毛粘附素的UPEC感染的菌落计数、肾脏病理改变严重度无显著性差异;无菌毛大肠埃希菌感染之小鼠,其尿液和肾剖面只有少量原感染茵生长,肾组织为轻至中度急性炎症改变。结论具有不同粘附素之P菌毛在介导UPEC致小鼠上行性急性肾盂肾炎中均发挥重要作用;无菌毛大肠埃希菌亦可导致小鼠肾脏炎性改变。

中草药对致肾盂肾炎大肠杆菌粘附特性的抑制作用

采用平皿打孔法测定中草药大黄和黄柏对临床分离的致肾盂肾炎大肠杆菌132和136的抗菌活性,结果均为耐药。继而,分别取不同浓度的大黄和黄柏处理上述菌株,利用血凝试验检测这两种药物对致肾盂肾炎大肠杆菌粘附特性的抑制作用。结果表明两种中草药对其均有抑制作用,大黄稍强于黄柏,提示中草药抗菌作用的机制可能与其抑制粘附有关。