Structure and antibacterial activity relationship of quercetin and rutin against test and clinical resistant gramm-negative strains of bacteria

Quorum quenching decreases Pseudomonas aeruginosa virulence factors and biofilm formation, alleviating infections in animal models. Nevertheless, it is usually performed in laboratory strains such as PAO1 and PA14, and studies involving clinical or environmental isolates are scarce. In this work, the effects of ZnO nanoparticles, a potent quorum and virulence quencher for the PAO1 strain, were tested in six clinical strains from cystic fibrosis patients, a furanone C-30 resistant clinical strain from urine, two PA14 gallium resistant mutants, a PA14 C-30 resistant mutant and four environmental isolates. ZnO nanoparticles effectively decreased elastase, pyocyanin, and biofilm formation for most of the strains; regardless their origin or their resistance against the canonical quorum quencher C-30 or the novel antimicrobial gallium. The data indicate ZnO nanoparticles may have a broad spectrum for the quorum quenching of relevant strains and that may be an alternative to treat Ps.aeruginosa recalcitrant infections. Virulence inhibition by quorum quenchers in Pseudomonas aeruginosa is usually tested in laboratory strains and studies of their effects in relevant clinical and environmental strains are scarce. This study is significant as the effects of ZnO nanoparticles in QS-dependent virulence factor production were tested in six clinical strains from cystic fibrosis patients, a C-30 resistant clinical strain from urine, two PA14 gallium resistant mutants, a PA14 C-30 resistant mutant, and four environmental isolates. ZnO nanoparticles decreased elastase, pyocyanin, and biofilms for most of the strains; indicating they have broad spectrum and may be an alternative to treat Ps.aeruginosa infections.

Introduction. Non-fermenting Gram-negative bacilli are known as one of the most frequent causative agents of hospital-acquired infections. Acinetobacter baumannii, as causative agent of infection complications of different localization, has obtained recently high resistance to anti-biotics and has belonged to ESKAPE group of pathogens. Antimicrobials, recommended for the prophylaxis and therapy of hospital-acquired infections, have been failing in their effectiveness and lead to selection of antibiotic resistant strains of A. baumannii. The aim of this research was to substantiate the way of overcoming of resistance in clinical strains of A. baumannii, by means of synergic antimicrobial activity of antibiotics and antiseptic decamethoxinum®. Material and methods. The research was carried out on 190 clinical strains of A. baumannii, isolated from patients with burn disease during the period 2011–2015. The sensitivity of clinical strains of A. baumannii was determined to such antibiotics as ampicillin/sulbactam, cefoperazone, cefoperazone/sulbactam, meropenem, imipenem, amikacin, ciprofloxacin, gatifloxacin and antiseptic decamethoxinum® (DCM; Registration certificate No UA/14444/01/01 since 24.06.2015. Order of the Ministry of Health of Ukraine No 373). The sensitivity of A. baumannii to antibiotics and DCM was determined by means of disk diffusion test and serial dilution (Order of the Ministry of Health of Ukraine No167 since 05.04.2007; EUCAST expert rules).The study of the influence of antiseptic DCM on the sensitivity of acinetobacteria to antibiotics was studied on 35 clinical strains of A. baumannii, drafted from the general number of isolates enrolled in the research. For this, the sensitivity of A. baumannii to antibiotics in the presence of sub-minimal inhibitory concentrations (subMIC) of DCM was identified. The received experimental data were analyzed by “Statistica 6.0”. Results and discussion. The changes of antibiotic sensitivity profile of A. baumannii for five years were shown. It was found that the sensitivity of A. baumannii to majority of antibiotics, selected for study, decreased significantly. But the only ampicillin/sulbactam was found to have vice versa tendency. We found the rising quantity of antibiotic resistant strains of A. baumannii. At the same time, high resistance of acinetobacteria to fluoroquinolones (ciprofloxacin– 96,1%; gatifloxacin– 95,8%) was found in 2015. The in vitro research of combined activity of DCM antiseptic remedy and early mentioned antibiotics against clinical strains of A. baumannii demonstrated the reveal antibiotic effectiveness. As follows, minimal inhibitory concentrations of antibiotics decreased in 1.5–4 times in the mediums which contained subMIC of DCM. Especially this tendency was found in resistant clinical strains. Conclusion. Under selective influence of antibiotics protected by β-lactamase inhibitors, carbapenems, fluoroquinolones aminoglycosides increase the antibiotic resistance in A. baumannii, causative agents of infectious complications in patients with burn disease. The antiseptic remedy decamethoxinum® helps to improve antibiotic sensitivity in resistant A. baumannii.

Evaluation of possible occurrence of mutation in MMR repair system genes in resistant and sensitiveclinical strains of Mycobacterium tuberculosisby using sequencing method AmirPoyan Afzali1, Mohammad Arjomndzadegan2, Azam Ahmadi 3, Sayead Hossein Hosseini1, Manijeh Kahbazi4 , Mojtaba Tousheh5 1- Department of Microbiology, Faculty of Science, Islamic Azad University of Tehran Research 2- Infectious Diseases Research center (IDRC) and Department of Microbiology and Immunology, School of medicine, Arak University of Medical Sciences, Arak, Iran 3- Department of molecular genetics, Tarbiat Modarres University, Tehran and Infectious Diseases Research center (IDRC), Arak University of Medical Sciences, Arak , Iran , azam.ahmadi@modares.ac.ir 4- Infectious Diseases Research center (IDRC), Department of Pediatric Infectious, Arak University of Medical Sciences, Arak, Iran 5- Department of Cellular and Molecular Biology, Faculty of Science, University of Isfahan Abstract: Background:during recent years, the incidence and spread of drug resistance in Mycobacterium tuberculosis, the bacterium causing tuberculosis, has set this disease in World Health Organizationpriorities alignment of diseases like AIDS and hepatitis. Study of close examination of resistant and susceptible clinical strains genotypes is necessary to overcome drug resistance. Among the numerous repair systems, only there are limited number of encoding genes of DNA repair enzymes in Mycobacterium tuberculosis. Commonly these genes have been conserved and any changes among them likely increasethe mutation occurance due to the impossibility of correctionof spontaneous mutations insensitive strains of this bacteria.mut genes encodeDNA repairable enzymes.This study investigated the mutations in these genes and the effect of these mutations on tuberculosis drug resistance. Materials&Methods: In this study,of 29 available specimens,we were selected 8 susceptible strains and 21 resistantstrains andafter ordering appropriate primers and performing the proliferation reaction two types of amplicons produced which includingfragments of genes mut T2 and mut T4 and they were sent inorder to sequencing. Results:The results of chain reactionprimer represents an appropriate choice of primerswhich were investigated. Sequencing results showed that overall 73% of resistant strains that had been selected for study of mutT4gene, have no mutations in codons 48of mutT4 gene, and 70% of resistant strains have no GGA >>> CGA mutation at codon 58 of mutT2 gene. Conclusion: One of the strategies to overcome tuberculosis drug resistance is a close examination of genotypes of resistant and susceptible clinical strains. Results of this study was performedby examining changes in mut T2 and mut T4 gene sequence. The mutation in mut T2 always associated with mutation in mut T4, in this way, the first mutation may occurs in mut T4and after that, the second mutationmay occurs in mut T2. Importance of this is determined by study of encoded proteins by these two genes and position of mut t4thanmut T2 in the MUT HLS spatial structure of protein complex. Results of comparison of drug resistance and occurrence of mutations in hot-spots of mut T2 and mut T4 genes illustrated that these genes are conserved in resistant strains. However, there is no significant relation in susceptible strains. Keywords: Mycobacterium tuberculosis, mut T2, PCR, sequencing

Introduction.Candida glabrata is a pathogenic yeast in humans, recognized for its genomic plasticity and increasing prevalence of antifungal resistance, including multidrug-resistant phenotypes, especially in the US and European countries.Hypothesis. This study hypothesizes that the resistance mechanisms in clinically resistant strains of C. glabrata differ from laboratory-generated resistant strains.Aim. This study aims to understand the resistance mechanism in Indian clinical isolates of C. glabrata.Methodology. A total of 240 clinical isolates of C. glabrata were tested for antifungal susceptibility and one resistant strain was artificially synthesized in the laboratory. Both clinical and lab-generated resistant strains were analysed for antifungal resistance using methods such as phenotypic assays, real-time quantitative PCR, Fluorescence-activated cell sorting (FACS) analysis and targeted gene sequencing. Mechanisms involving drug efflux pumps, mismatch repair pathways, ergosterol biosynthesis pathway and biofilm formation were systematically studied.Results. Among clinical isolates, one susceptible-dose dependent strain and three fluconazole-resistant strains were identified. Both clinical and lab-generated resistant strains demonstrated antifungal resistance phenotypically, with increased expression of CDR1. Targeted gene sequencing revealed novel mutations in PDR1, while mutations in MSH2 served as genotypic markers for resistance. Overexpression of ERG11 was seen in a lab-generated resistant strain where a specific mutation was identified. Biofilm activity contributed to resistance in one of the clinical strains.Conclusion. This study reports for the first time the fluconazole resistance mechanism in C. glabrata from India. The findings underscore the diversity of resistance mechanisms among clinical and lab-generated isolates, emphasizing the need for novel antifungal therapies to address these emerging resistance profiles effectively.

Staphylococcus aureus is a major pathogen in clinical microbiology. It is known to cause infections at various body sites and can be life-threatening. The development of resistance to many well-established antibiotic treatments and the prevalence of methicillin-resistant S. aureus (MRAS) among hospital patients and the general community pose challenges in treating the pathogen. The antimicrobial effect of photodynamic therapy (PDT) has been a subject of study for a long time and can offer new strategies for dealing with resistant strains. In our study, we searched for a positive synergistic relationship between PDT and the standard antibiotics used to treat S. aureus and MRSA infections. The phototoxic profile of deuteroporphyrin (DP) in both resistant and susceptible clinical strains of S. aureus was determined by plating of treated and untreated broth cultures. Electron microscopy imaging was done to explore possible sites of damage and free-radical accumulation in the cells during DP-PDT. Minimal inhibitory concentration (MIC) of oxacillin, gentamicin, vancomycin, rifampin, and fusidic acid was determined using the broth dilution method, and the checkerboard method was used to detect and evaluate the synergistic potential of DP-PDT and antibiotic combinations. A synergistic combination was further characterized using broth cultures and plating. DP-PDT using a light dose of 15 J/cm2 showed a bactericidal effect even with a small concentration of 17 μM DP. Transmission electron microscopy indicated profound damage in the cell wall and cell membrane, and the appearance of mesosome-like structures. Free radicals tend to localize in the cell membrane and inside the mesosome. No synergistic effect was detected by combining PDT with gentamicin, vancomycin, rifampin, and fusidic acid treatments. A positive synergistic effect was observed only in DP-PDT-oxacillin combined treatment using the checkerboard method. The effect was observed in clinical antibiotic-resistant isolates after DP-PDT using a light dose of 46 J/cm2 and small concentrations of DP. Oxacillin MIC decreased below 2 μg/ml in resistant strains under such conditions. Cultures which did not undergo new cycles of DP-PDT recovered their original oxacillin resistance after a few generations. PDT with porphyrins shows possible new therapeutic options in treating drug-resistant S. aureus at body sites suitable for irradiation. The synergistic effect of DP-PDT with oxacillin on clinical strains illustrates the potential of PDT to augment traditional antibiotic treatment based on cell wall inhibitors. Lasers Surg. Med. 50:535-551, 2018. © 2018 Wiley Periodicals, Inc.

The effect of King's Vegetable oil, a fixed non-mineral oil, on the antibacterial activity of ampicillin trihydrate, a water- insoluble form of ampicillin, was investigated against resistant clinical strains of Staphylococcus aureus. In the agardiffusion method employed, 40% of the resistant clinical strains tested showed sensitivity to different oil-dispersed concentrations of ampicillin trihydrate, which ranged from 0.06ìg/ml to 1.25ìg/ml. The resistant strains were among the clinicalstrains detected with Beta-lactamase. This finding is presented as a preliminary report on the potentiality of employing an oil medium to effect a "cure" of antibiotic resistance in staphylococci besides the use of acridine dyes, ethidium bromide, ultraviolet radiation and other measures.

Pseudomonas aeruginosa is a ubiquitous bacterium, which is able to change its physiological characteristics in response to different habitats. Environmental strains are presumably less pathogenic than clinical strains and whether or not the clinical strains originate from the environment or through inter-host transmission remains poorly understood. To minimize the risk of infection, a better understanding of proteomic profiling of P. aeruginosa is necessary for elucidating the correlation between environmental and clinical strains. Based on antimicrobial susceptibility and patterns of virulence, we selected 12 clinical and environmental strains: (i) environmental, (ii) multidrug resistant (MDR) clinical and (iii) susceptible clinical strains. Whole-cell protein was extracted from each strain and subjected to two-dimensional differential gel electrophoresis (2-D DIGE) and liquid chromatography tandem mass spectrometry quadrupole time-of-flight (LC-MS QTOF). All 12 strains were clustered into 3 distinct groups based on their variance in protein expression. A total of 526 matched spots were detected and four differentially expressed protein spots (p < 0.05) were identified and all differential spots were downregulated in MDR strain J3. Upregulation of chitin binding and BON domain proteins was present in the environmental and some MDR strains, whereas the clinical strains exhibited distinct proteomic profiles with increased expression of serine protein kinase and arginine/ornithine transport ATP-binding proteins. Significant difference in expression was observed between susceptible clinical and MDR strains, as well as susceptible clinical and environmental strains. Transition from an environmental saprophyte to a clinical strain could alter its physiological characteristics to further increase its adaptation.

Extensive use of beta-lactam antibiotics has led to the selection of pathogenic streptococci resistant to beta-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 beta-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S(443)SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.

Downregulation of an NfsA-like nitroreductase causes metronidazole resistance in Gardnerella vaginalis.

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains

Objective: This study aims to determine the frequency of bacterial co-infections in COVID-19-positive patients.&#x0D; Methodology: A prospective cross-sectional study was conducted in the Department of Microbiology, Pakistan, from November 15, 2021, to April 15, 2022. Blood and respiratory tract samples were collected, including sputum, bronchial lavage, and tracheal aspirate. Clinical specimens were inoculated onto a Sheep blood agar plate, Chocolate agar plate (aerobic with 5% CO2), and MacConkey’s agar. Identification was followed by specific and standard microbiological protocols. COVID-19 was confirmed by qualitative PCR. Antimicrobial susceptibility testing was performed using the Kirby Bauer disc diffusion method.&#x0D; Results: A total of 202 clinical samples, including blood, sputum, tracheal aspirates, and bronchial lavage, were collected from COVID-19-positive patients. Male patients were more common in sputum and tracheal aspirates, while female patients were more common in blood cultures. The majority of patients were over 60 years of age. Acinetobacter baumannii was predominantly isolated from blood and tracheal aspirates, exhibiting multidrug resistance, but showing complete sensitivity towards Colistin. Klebsiella pneumonia exhibited high prevalence in sputum, with complete resistance observed in Cephalosporins and Co-trimoxazole.&#x0D; Conclusion: The study concludes a high frequency of superadded bacterial co-infections, caused most prominently by Acinetobacter baumannii, Klebsiella pneumonia, and Pseudomonas aeruginosa. The majority of these are multidrug-resistant pathogens, therefore, urgent action is required to control the spread of nosocomial infections by resistant strains, which are responsible for the high mortality rate among COVID-19 critical patients.

The isolation and characterization from the sand fly Phlebotomus perniciosus of a Wickerhamomyces anomalus yeast strain (Wa1F1) displaying the killer phenotype was recently reported. In the present work, the killer toxin (KT) produced by Wa1F1 was purified and characterized, and its antimicrobial activity in vitro was investigated against fluconazole- susceptible and -resistant clinical isolates and laboratory strains of Candida albicans and C. glabrata displaying known mutations. Wa1F1-KT showed a differential killing ability against different mutant strains of the same species. The results may be useful for the design of therapeutic molecules based on Wa1F1-KT and the study of yeast resistance mechanisms.

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of polymyxin resistance (MICs of 8 to 64 mg/liter) in laboratory and clinical strains of this organism. To explore the role of PhoPQ in high-level clinical polymyxin resistance, P. aeruginosa strains with colistin MICs > 512 mg/liter that had been isolated from cystic fibrosis patients treated with inhaled colistin (polymyxin E) were analyzed. Probable loss-of-function phoQ alleles found in these cystic fibrosis strains conferred resistance to polymyxin. Partial and complete suppressor mutations in phoP were identified in some cystic fibrosis strains with resistance-conferring phoQ mutations, suggesting that additional loci can be involved in polymyxin resistance in P. aeruginosa. Disruption of chromosomal phoQ in the presence of an intact phoP allele stimulated 4-amino-l-arabinose addition to lipid A and induced transcription from the promoter of the pmrH (arnB) operon, consistent with the known role of this lipid A modification in polymyxin resistance. These results indicate that phoQ loss-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

Currently antimicrobial resistance is increasing at an alarming rate. Exposure to resistant strains hinders treatment outcomes both in rural and hospital settings. Thus, the aim of this study was to investigate five frequently used South African medicinal plants (Artemisia afra, Lippia javanica, Osmitopsis asteriscoides, Croton gratissimus and Tetradenia riparia) and test these against resistant bacterial strains (Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Serratia marcescens) and comparatively evaluate efficacy with a reference strain. The conventional antibiotic ciprofloxacin was used as a positive control to also compare susceptibility of the various strains. Most plant samples demonstrated similar or better activity against the resistant strains. A general trend demonstrated that the organic extracts followed by the essential oils were able to withstand resistant strains better than the antibiotics which showed reduced susceptibility. This demonstrates great promise as natural products provide an alternative to fighting the onslaught of antibiotic resistance.