Kirby-Bauer Research Articles

Niosome-embedded in situ gels for root canal irrigation: preparation and optimization of a composite system for chlorhexidine delivery

Niosomes could increase the efficacy of the irrigants by providing a controlled drug release. Incorporating niosomes into an in situ gel matrix maximizes the drug retention time in the root canal system. The focus of this study was the fabrication and characterization of a niosomal in situ gel system for delivering chlorhexidine to root canals. For this aim, chlorhexidine-loaded niosomes were optimized by evaluating the impacts of surfactant type, surfactant-to-cholesterol ratio, lipid-to-drug weight ratio, and ethanol concentration on particle size and entrapment efficiency (EE) using an experimental design. The in situ gels were also optimized with the aid of an experimental design by identifying the effects of chitosan and β-glycerophosphate concentrations as well as the solution stirring time on the gelation time and gelation temperature. The optimum niosomal nanoformulation showed an average particle size of 115 nm, EE% of ∼80%, and PDI of 0.1. The optimum in situ gels showed the lowest gelation time and appropriate gelation temperature (60 s and 29 °C, respectively). The in vitro release studies showed that niosomal in situ gels successfully sustained the release of ∼97% of chlorhexidine over 144 hours with Higuchi kinetics. Furthermore, the biological evaluations revealed that the niosomal in situ gels were cytocompatible. The system also exhibited antibacterial activity against Enterococcus faecalis bacteria, Streptococcus mutants, and Aggregatibacter actinomycetemcomitans by respectively showing 18.0, 33.3, and 48.3 mm inhibition zones in the disk diffusion test. Overall, the results indicated that niosome-embedded in situ gels showed promising potential for antimicrobial delivery to root canals.

Standardization, Characterization and Potential Biological Activity of Ozonized Sunflower Vegetable Oil

Objective: The ozone molecule, composed of three oxygen atoms, possesses high oxidizing power and is widely utilized for various applications, including therapeutic treatments involving the administration of medicinal ozone to combat various diseases. The standardization of ozonized oil is essential to ensure patient safety. The objective of this study was to develop protocols for the standardization and characterization of ozonized oil using analytical techniques such as gas chromatography-mass spectrometry (GC-MS) and to evaluate its biological activity through microbiological and cytotoxic assays. Methods: Standardization and characterization were carried using gas chromatography coupled to mass spectrometry (CG-MS), and microbiological tests including agar diffusion, time-kill and MIC and cytotoxic tests were employed in bacterial cells. Results: The obtained results indicated that after 480 minutes of ozonization in 100 mL of sunflower oil (OG), a significant antimicrobial potential was observed, leading to the formation of ozonides. Antibacterial and antifungal activity assays, conducted using the ‘time kill’ method, agar diffusion, and Minimum Inhibitory Concentration (MIC) tests, demonstrated activity against Staphylococcus aureus, Escherichia coli, Salmonella choleraesuis, Pseudomonas aeruginosa, Candida albicans, Aspergillus brasiliensis, and Malassezia furfur. Conclusion: Results demonstrate the therapeutic potential of the application of ozonized oil in the treatment of topic infectious diseases. In summary, the standardization of the ozonization process of sunflower oil showed to be promising, providing a product exerting antimicrobial activity and therapeutic potential, opening perspectives for its application in various areas of medicine.

Genomic Insights Into a Strong Biofilm‐Forming Enterococcus faecalis MTR_EFS01 Strain Isolated From a Shrimp in Bangladesh

ABSTRACTEnterococcus faecalis is known for its ability to form strong biofilms and its role as an opportunistic pathogen. In this study, we screened and characterized a multidrug‐resistant (MDR) and strong biofilm‐forming E. faecalis isolate obtained from a shrimp sample to determine its genetic diversity, molecular epidemiology, and underlying factors associated with antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs). The E. faecalis MTR_EFS01 strain was isolated using culturing, staining, and biochemical tests and MALDI‐TOF methods. The MDR profile of the strain was determined through the disk diffusion test. The complete genomic sequence of E. faecalis MTR_EFS01 was obtained using the Illumina NextSeq2000 platform. The de novo assembly of the E. faecalis MTR_EFS01 genome revealed a total length of 2,862,301 bp with 80.0 × coverage. This genome comprised 38 contigs, a G + C content of 37.4%, and identified two CRISPR arrays, seven prophages, and 55 RNA genes. The E. faecalis MTR_EFS01 strain was classified as ST862 with a high pathogenicity index of 0.896. The strain harbored eight ARGs conferring resistance to tetracycline, erythromycin, trimethoprim, and MDR efflux pumps. Furthermore, 27 VFGs were identified in this strain, linked to antiphagocytosis, adherence, biofilm formation, enzymes, and immune invasion. Metabolic functional analysis revealed that our strain had 243 subsystems, with the most abundant genes associated with carbohydrate metabolism, amino acids and derivatives, and protein metabolism. The findings in this study underscore the importance of continuous monitoring, research, and collaborative efforts to address the growing threat of MDR and biofilm‐forming pathogens in diverse settings.

Evaluation of Larrea tridentata Extracts and Their Antimicrobial Effects on Strains of Clinical Interest

The use of medicinal plants represents an alternative method for bacterial control due to their chemical compositions. This study’s objective was to determine the inhibitory capacity of Larrea tridentata extracts against microbial strains of clinical interest. Four extracts were prepared, their phytochemical profiles were determined, and their antioxidant capacities were quantified. Additionally, the minimum concentrations of hemolysis were determined using human blood erythrocytes. For the extracts’ growth inhibitory capacity, six bacterial and two fungal strains were evaluated using the disk diffusion test. Commercial medications specific for each strain were used as controls. The ethanolic extracts registered the greatest diversity of metabolites related to antibacterial activity. The inhibitory activities of the ethanolic extract and the Cedax® control were similar for Enterococcus faecalis. A principal component analysis was performed with X2 and ANOVA tests to identify the relationships and the effects of the extracts on bacterial inhibition, obtaining p > 0.05 with a confidence level of 95%. This research highlights the potential of L. tridentata extracts as an alternative treatment and to mitigate the growing problem of resistance to traditional antibiotics.

Antibacterial and photocatalytic activities of biosynthesized zinc oxide nanoparticle using novel plant P. macrosolen L. leaf extract

In this study, ZnO nanoparticles were produced by an easy and cost effective approach using P. macrosolen L. leaf extract and zinc chloride as Zn ion source. The as-biosynthesized ZnO nanoparticles were analyzed by powder-X-ray diffraction (PXRD), Fourier transform infrared (FTIR) spectroscopy, UV–visible spectroscopy, Focused ion beam-scanning electron microscopy (FIB-SEM) and energy dispersive X-ray (EDX) characterization techniques. The XRD result revealed the formations of ZnO-nanoparticles with hexagonal structure, spherical like shape and average crystalline size of 47 nm. The presence of flavonoids in P. macrosolen L. extracts was confirmed by phytochemical tests, indicating that flavonoids primarily acted as reducing agents during nanoparticle formation. The FTIR spectrum also revealed broad and strong peaks corresponding to –OH groups, confirming the high concentration of phenolic acids present in the extract. The energy band gap of the biosynthesized ZnO Nanoparticles, calculated using the Tauc relation, was found to be 3.0 eV, indicating the presence of a blue shift. FIB-SEM was used to analyze the microstructure and identify any aggregations. The EDX analysis revealed the presence of zinc and oxygen, with weights of 16.33% and 83.67%, respectively. The disk diffusion test was performed on solid agar plates and demonstrated that the substance is effective against E. coli bacteria as an antibacterial agent. The photocatalytic degradation rate constants of the optimized ZnO samples under visible light are approximately 0.06455 min⁻1 for MO and 0.04865 min⁻1 for TB, achieving 94.47% and 92.34% degradation of MO and TB, respectively. These rates are significantly higher compared to unmodified ZnO, which has a degradation rate of 0.0075 min⁻1. The improvement in visible-light photocatalytic performance can be attributed to the formation of ZnO nanoparticles, which arise from the matching of crystal lattices and energy bands in ZnO. ZnO Nanoparticles produce excited electrons under visible light irradiation. These excited electrons in the ZnO nanoparticles are directly transferred to the conduction band (CB), resulting in notable visible light photocatalytic performance.

ANALYSIS OF HOSPITAL-ACQUIRED MRSA INFECTIONS IN PUS SAMPLES: A COMPREHENSIVE DISCUSSION

Background: Hospital-acquired infections (HAIs) represent a significant healthcare challenge globally, with Staphylococcus aureus (S. aureus) being a leading cause of skin and soft tissue infections. Methicillin-resistant Staphylococcus aureus (MRSA) poses an even greater threat due to its resistance to commonly used antibiotics. Understanding the prevalence and resistance patterns of MRSA is crucial to developing effective infection control strategies, especially in resource-limited settings like Peshawar, Pakistan. Objective: To determine the frequency of hospital-acquired infections caused by S. aureus in skin abscesses and evaluate the antibiotic resistance patterns of MRSA in patients from District Peshawar. Methods: A total of 525 pus samples were collected from patients with skin abscesses during hospital stays in Peshawar. Samples were processed in a microbiology laboratory using standard protocols, including Gram staining, colony morphology assessment, and biochemical tests to identify S. aureus. Methicillin resistance was confirmed using cefoxitin and oxacillin disc diffusion tests, and the presence of the mecA gene was validated through PCR. Antibiotic susceptibility testing was performed for 14 commonly prescribed antibiotics following Clinical and Laboratory Standards Institute (CLSI) guidelines. Statistical analysis was conducted using SPSS version 26. Results: Out of 525 pus samples, 100 isolates were confirmed as MRSA. The prevalence of MRSA was higher in males (54%) than females (46%). The highest infection rate was observed in the 21–40 years age group (35%), followed by 1–20 years (27%), 41–60 years (25%), and above 60 years (11%). Resistance was highest to cefoxitin (100%), oxacillin (94%), ciprofloxacin (81%), and amoxicillin (79%). However, isolates showed high susceptibility to vancomycin (93%), linezolid (95%), and teicoplanin (90%). Conclusion: The study highlights the alarming prevalence of MRSA in hospital-acquired skin abscesses in Peshawar, coupled with significant antibiotic resistance. Strengthened infection control measures, antibiotic stewardship programs, and ongoing surveillance are essential to curb the spread of resistant pathogens.

Resistance Profile of Strains of Escherichia coli and Salmonella sp. Isolated from Raw Cow Milk Sold in the Department of Vina in Cameroon

Aims: Antimicrobial resistance, defined by the WHO as “resistance of a micro-organism to an antibiotic to which it was previously sensitive”, results from the ability of the bacterium to withstand the attack of antibiotics. This resistance is a major factor in the risk of therapeutic failure and the spread of multidrug-resistant strains from animals to humans. The present study was aimed to assess the antibiotic resistance of strains of Escherichia coli and Salmonella sp. Isolated from cattle farms in the Department of Vina, Cameroon. Place and Duration of Study: The study involved 30 strains of Salmonella (05) and Escherichia coli (25) isolated from 60 samples of raw cow milk, collected from July to August 2021 in Vina, Adamawa Province. The samples were each made up of 15 ml of milk, taken aseptically from cattle farms and sales depots. Methodology: Antibiotic susceptibility of 25 strains of Escherichia coli and 5 Salmonella sp. strains was wanted, according to the standard Mueller-Hinton agar diffusion method 16 antibiotic discs. Results: The diameters of the inhibition zones formed showed fairly high resistance rates, 87.5% for Salmonella sp. and 75% for E. coli. Bacterial strains are all resistant to the Penicillin, Macrolides and Diaminopyrimidines tested. Good sensitivity was observed with levofloxacin (100%), cefixime (82.14%) and gentamycin (78.60%). Conclusion: The importance of these resistances reflects the intensive previous use of antibiotics in livestock farming as growth-promoting additives.

Phytochemical Screening, Antioxidant and Antibacterial Properties of Alcoholic Extract of Saussurea costus Roots

Background: Diseases in their essence are non-curable and the underlying pathogenesis is bidirectional of inflammation and oxidative stress. Fighting oxidative stress and inflammation should be the target for any first-line therapy, perhaps via chemical or herbal therapy. In the present study, we aimed to identify the antioxidant and antibacterial activities of Saussurea costus roots. The study aimed to harness S. costus plant as antimicrobial via assessment of its antibacterial efficacy against pathogens by disc diffusion and minimum inhibitory concentration (MIC) tests. Methods: Alcoholic extract of S. costus roots was tested for antioxidant activity using diphenyl picrylhydrazyl (DPPH) and antibacterial effects on a few common bacterial species (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus spp.). Result: The contents of S. costus extracts revealed of the presence of therapeutic principle components with variety of medicinal effects, including alkaloids, terpenoid, phenols and others. Different concentration of extracts have comparable antioxidant activity to ascorbic acid (81.96%). Similarly, different concentrations of the extract have confirmed antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli with no impacts on Proteus spp.

Effect of Fecal Microbiota Transplant on Antibiotic Resistance Genes Among Patients with Chronic Pouchitis.

Pouchitis is common among patients with ulcerative colitis (UC) who have had colectomy with ileal pouch-anal anastomosis. Antibiotics are first-line therapy for pouch inflammation, increasing the potential for gut colonization with multi-drug resistant organisms (MDRO). Fecal microbial transplant (FMT) is being studied in the treatment of pouchitis and in the eradication of MDRO. Prior work using aerobic antibiotic culture disks suggests that some patients with chronic pouchitis may regain fluoroquinolone sensitivity after FMT. However, gut MDRO include anaerobic, fastidious organisms that are difficult to culture using traditional methods. We aimed to assess whether FMT reduced the abundance of antibiotic resistance genes (ARG) or affected resistome diversity, evenness, or richness in patients with chronic pouchitis. We collected clinical characteristics regarding infections and antibiotic exposures for 18 patients who had previously been enrolled in an observational study investigating FMT as a treatment for pouchitis. Twenty-six pre- and post-FMT stool samples were analyzed using FLASH (Finding Low Abundance Sequences by Hybridization), a CRISPR/Cas9-based shotgun metagenomic sequence enrichment technique that detects acquired and chromosomal bacterial ARGs. Wilcoxon rank sum tests were used to assess differences in clinical characteristics, ARG counts, resistome diversity and ARG richness, pre- and post-FMT. All 13 of the patients with sufficient stool samples for analysis had recently received antibiotics for pouchitis prior to a single endoscopic FMT. Fecal microbiomes of all patients had evidence of multi-drug resistance genes and ESBL resistance genes at baseline; 62% encoded fluoroquinolone resistance genes. A numerical decrease in overall ARG counts was noted post-FMT, but no statistically significant differences were noted (P = 0.19). Richness and diversity were not significantly altered. Three patients developed infections during the 5-year follow-up period, none of which were associated with MDRO. Antibiotic resistance genes are prevalent among antibiotic-exposed patients with chronic pouchitis. FMT led to a numerical decrease, but no statistically significant change in ARG, nor were there significant changes in the diversity, richness, or evenness of ARGs. Further investigations to improve FMT engraftment and to optimize FMT delivery in patients with inflammatory pouch disorders are warranted.

Loop-mediated isothermal amplification assay coupled with lateral flow dipstick for the rapid detection of methicillin-resistant Staphylococcus pseudintermedius from dogs.

Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.

Salvadora persica nanoparticles effect on the specific properties of a bio-composite dental material

The aim of this study is to evaluate the antibacterial activity of dental material containing nanoparticles of the extract of S. persica against Streptococcus pneumonia, Streptococcus mutans and Haemophilus influenza, and to examine their triboligical and mechanical properties. The specific properties of flowable dental material containing different percentage of the extract are investigated using the below different kinds of tests: - Antibacterial activity (including agar diffusion test), - Tribological behavior (including a pin-on-disk tribometer, surface profilometer and scanning electron microscopy (SEM)), - Mechanical behavior (including compressive strength, bending strength and bending modulus). The agar diffusion test reveals significant difference between the samples, whereas the antibacterial one demonstrates that the bacterial growth is diminished by increasing the nanoparticle content. The bending strength and the bending modulus remain unchanged by a 10% incorporation of the nanoparticles while the compressive strength increases significantly. The extract containing resins show higher wear resistance. The production of dental material with antibacterial activity, with suitable wear resistance, and particularly without any significant sacrificing effect on the mechanical properties is practically desirable in dental material science.

ANTIBIOTIC SUSCEPTIBILITY PATTERN OF E. COLI ISOLATED FROM THE URINE SAMPLES OF PATIENTS VISITED CIVIL HOSPITAL MADYAN SWAT

Background: Antibiotic resistance is a global challenge, particularly in urinary tract infections (UTIs) caused by Escherichia coli (E. coli), which is a leading pathogen in both nosocomial and community-acquired infections. Regular monitoring of antibiotic susceptibility profiles is essential due to the rising prevalence of drug-resistant bacteria. This study focused on evaluating the antibiotic susceptibility pattern of E. coli isolated from urine samples of patients attending Civil Hospital Madyan, Swat, to guide effective empirical therapy. Objective: To determine the antibiotic susceptibility profile of E. coli isolated from urine samples of UTI patients visiting Civil Hospital Madyan, Swat. Methods: A cross-sectional study was conducted from December 2022 to July 2023 in the Department of Microbiology, Government Degree College Madyan, Swat. A total of 450 mid-stream urine samples were collected from patients with suspected UTIs. The samples were cultured on MacConkey agar and incubated at 37°C for 24–48 hours under aerobic conditions. Isolated bacterial colonies were identified using gram staining, microscopy, and biochemical tests. Antibiotic susceptibility testing was performed on Mueller-Hinton agar using the Kirby-Bauer disc diffusion method with multiple antibiotic discs. The bacterial isolates were categorized into resistant (R), sensitive (S), or intermediate (I) groups by measuring inhibition zone diameters. Data were statistically analyzed and presented in figures and tables. Results: Out of 450 urine samples, 125 (27.0%) were culture-positive, with E. coli being the most frequently isolated bacterium (60%). Culture positivity was higher in females (70%) than males (30%). Among hospital areas, samples from the outpatient department (70%) showed the highest culture positivity, followed by wards (25%) and the emergency department (5%). Antibiotic susceptibility testing revealed that E. coli exhibited the highest resistance to ampicillin (97.1%), ceftriaxone (92%), moxifloxacin (88.2%), cefixime (86%), and ceftazidime (80.1%). Conversely, the highest sensitivity was observed to fosfomycin (95.3%), sulzone (85.2%), imipenem (85.0%), and amikacin (78%). Conclusion: This study confirmed that E. coli is the predominant pathogen causing UTIs and displayed significant resistance to commonly used antibiotics such as ampicillin, ceftriaxone, moxifloxacin, cefixime, and ceftazidime. However, fosfomycin, imipenem, and amikacin were found to be the most effective antibiotics against E. coli. These findings underscore the importance of conducting antibiotic susceptibility testing to guide effective therapy and combat antimicrobial resistance.

Effect of ozone oil and non-surgical periodontal treatment in patients with type 2 diabetes. In-vivo and in-vitro studies with fibroblasts and Candida albicans.

To evaluate the clinical effectiveness of ozonated sunflower oil (Oz) as an adjunctive of non-surgical periodontal therapy in patients with type 2 diabetes mellitus (DM2), on fibroblast cell viability and migration and the effectiveness of Oz on a Candida albicans (C. albicans) culture. In total, 32 sites in 16 DM2 with moderate to advanced periodontal disease with periodontal pocket depths ≥5mm were selected. The treatments were divided into two groups: control, saline solution (SS) as an adjunctive of scaling and root planing (SRP+SS), and test, Oz as an adjunctive of SRP (SRP+Oz). Hematological [fasting glucose level (FGL) and hemoglobin A1c (HbA1c)] and microbiological samples were collected from the participants at baseline and three months after periodontal treatment and the microbiological samples were analyzed by PCR. C. albicans was previously tested by the agar diffusion test. The effect of Oz was tested on cell viability and fibroblast migration. The groups showed no statistically significant differences (paired t-test-p>0.05) regarding hematological parameters, FGL (median - baseline 171.41, 3 months 164mg/dL), and HbA1c (baseline 8%, 3 months 7.5%) (Kruskal-Wallis One-Way Nonparametric-p>0.05) after periodontal therapy. The groups showed statistical differences for periodontal parameters between baseline and three months (paired t-test-p<0.05). PCR analysis showed a reduction in the percentage of C. albicans in the SRP+Oz group after three months (McNemar's test-p=0.002). Cell viability was lower in the high glucose Dulbecco's modified Eagle's medium (4500 mg/L) than in low glucose (1000 mg/L) (RM-ANOVA-p<0.0001). The wound healing test showed reduced fibroblast migration (one-way ANOVA with Dunnett's post-test-p<0.01). Oz showed high C. albicans antifungal inhibition (Kruskal-Wallis test-p=0.0001). SRP+Oz effectively reduced C. albicans in-vitro and in-vivo but showed no clinical improvements compared to the control. Cell viability and wound healing of fibroblasts showed no improvements.

Keratin nanoparticles derived from feather waste for novel antibacterial delivery.

The global rise of bacterial resistance demands innovative strategies to enhance antibiotic efficacy. This study investigates keratin nanoparticles (KNPs) derived from waste chicken feathers as sustainable drug carriers. Antibacterial activity of KNPs was evaluated against Staphylococcus aureus and Escherichia coli using antibacterial sensitivity assays, including disc diffusion and minimum inhibitory concentration tests, while cytotoxicity was evaluated on human lymphoma cells. KNPs exhibited excellent biocompatibility, showing no cytotoxic effects on human cells or bacteria. Penicillin and vancomycin were successfully loaded onto KNPs at 4 °C, 25 °C, and 50 °C temperatures for 2 and 20-hour. Loading onto KNPs enhanced the antibacterial efficacy of penicillin and vancomycin by 4-fold and 3.8-fold, respectively, against S. aureus at 37 °C. Enhanced antibacterial efficacy was attributed to molecular interactions between keratin and penicillin, as demonstrated by molecular docking analysis. The analysis revealed that the β-lactam ring of penicillin was encapsulated within the keratin matrix, potentially shielding it from enzymatic degradation by penicillinase. This protective mechanism preserves the antibiotic's structural integrity and antibacterial activity. These findings highlight the potential of KNPs as effective drug carriers in combating resistance mechanisms. This research underscores the transformative role of sustainable biological macromolecules in modern medicine, offering a promising approach to combat antibiotic resistance.

Outbreak of carbapenem resistant Klebsiella pneumoniae in a neurorehabilitation unit: genomic epidemiology reveals complex transmission pattern in a tertiary care hospital.

Infections by Carbapenem-Resistant Enterobacterales in hospitals represent a severe threat but little is known on outbreaks in rehabilitation wards caused by Klebsiella pneumoniae producing Klebsiella pneumoniae Carbapenemase (KPC-Kp). We report an outbreak by KPC-Kp, in a Neurorehabilitation Unit in Italy, analysed through Whole-Genome Sequencing (WGS) for transmission routes reconstruction to improve management of KPC-Kp infections in rehabilitation units. We investigated cases and KPC-Kp isolates collected from February to October 2022 from hospital surveillance. Carbapenem resistance was identified with disk diffusion and minimal inhibitory concentration tests, carbapenemase production through immunochromatographic lateral flow assays. All isolates were genotyped through WGS to highlight possible phylogenetic relationships. The most likely transmission networks of KPC-Kp, were reconstructed with Bayesian discrete-time stochastic models. 19 patients were colonized by KPC-Kp. Two isolates belonged to sporadic STs (ST348 and ST219) while 9/19 and 8/19 isolates belonged to ST307 and ST716, respectively. Among ST307 isolates, we identified two genomically distinct clusters of five and two cases. All ST716 isolates were highly similar. Genotyping and the reconstruction of transmission networks of KPC-Kp based on genomic data identified seven independent introductions into the Neurorehabilitation Unit rather than a single outbreak as initially hypothesized before genomic investigation. Three of the seven introductions generated chains of secondary infections, while the remaining four remained single cases. The outbreak root-causes were identified in temporary staff shortage and insufficient microbiological surveillance. Measures were adopted accordingly and the outbreak ended. The study highlights the critical role of genomic epidemiology in hospital outbreaks.

Synthesis of cationic N-acylated thiazolidine for selective activity against Gram-positive bacteria and evaluation of N-acylation's role in membrane-disrupting activity.

The evolution of antimicrobial-resistant strains jeopardizes the existing clinical drugs and demands new therapeutic interventions. Herein, we report the synthesis of cationic thiazolidine bearing a quaternary pyridinium group, in which thiazolidine was N-acylated with fatty acid to establish a hydrophilic-lipophilic balance that disrupts bacterial membranes. The bacterial growth inhibition assays and hemolytic activity against human red blood cells indicate that the N-acylated cationic thiazolidine (QPyNATh) inhibits Gram-positive bacteria at lower minimum inhibitory concentrations (MIC) and is selective for bacteria over mammalian cells. N-Acylation modulates MIC, and it is found that the N-palmitoylated compound, QPyN16Th, had the lowest MIC (1.95 μM) against Gram-positive, Enterococcus faecalis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). In contrast, the N-myristoylated compound, QPyN14Th, showed the lowest MIC (31.25 μM) against Gram-negative, Escherichia coli, uropathogenic Escherichia coli, and Pseudomonas aeruginosa. At 1× MIC, QPyNATh permeabilizes the bacterial membrane, depolarizes the cytoplasmic membranes, and produces excess reactive oxygen species to kill the bacteria, as evidenced by live and dead staining. Interestingly, only QPyNATh containing a palmitoyl acyl chain demonstrated membrane-damaging activity at 2 μM concentrations, suggesting that the optimal hydrophilic-lipophilic balance enables QPyN16Th to selectively kill Gram-positive bacteria at lower doses. S. aureus develops resistance to ciprofloxacin quickly; however, no resistance to QPyN16Th is observed after several passages. As a proof of concept, the animal study revealed that QPyN16Th treatment reduced the bacterial burden in MRSA-infected zebrafish, allowing them to recover from infection and resume normal life. The results imply that lipidation and derivatizing thiazolidine with cationic charge offer an antimicrobial that is selective to treat Gram-positive bacterial infections, biocompatible, and less prone to develop resistance.

Antibiotic-resistant Pseudomonas aeruginosa from abattoir and aquaculture environment in Abakaliki, Ebonyi State, southeast Nigeria

Background: Pseudomonas aeruginosa is frequently identified as the predominant bacterial pathogen in abattoir and aquaculture settings. In Ebonyi State, Nigeria there has been a lack of thorough investigation on the impact of the organism in the environment on public health. Therefore, it was necessary to investigate the occurrence of P. aeruginosa and determine its resistance characteristics to antimicrobial agents in selected abattoirs and aquaculture facilities in Abakaliki, Ebonyi State, southeast Nigeria.Methodology: Wastewater samples from randomly selected abattoirs (n=25) and aquaculture (n=25) sites in various locations in Abakaliki, Ebonyi State, Nigeria, were collected into sterile universal bottles and transported to the microbiology laboratory of Ebonyi State University, Abakaliki, for microbiological analysis. For heterotrophic colony count, measured in colony forming unit/ml (CFU/ml), to estimate the microbial load of the samples, a 1- in-10 dilution of the samples were prepared and cultured on nutrient agar, incubated at 37oC for 24 hours. Colonies from the culture plate were then sub-cultured on Pseudomonas agar and incubated aerobically at 37oC for 24 hours. Pseudomonas aeruginosa was phenotypically confirmed on the culture plate by conventional morphological characteristics and biochemical tests. Antibiotic susceptibility testing of the P. aeruginosa isolates was performed by the disk diffusion test and results interpreted using the Clinical and Laboratory Standards Institute (CLSI) zone diameter breakpoints. Multiple antibiotic resistance index (MARI) was calculated for each isolate.Results: The microbial load varied from 3.0±2.8 to 33.4±23.5 x 105 CFU/ml for abattoir samples and 0.00 to 26.0±2.8 x 105 CFU/ml for aquaculture samples. For the abattoir, wastewater samples from the butcher table had the highest frequency of P. aeruginosa (50.0%) isolation, followed by wastewater from the drainage (26.7%), while the lowest frequency was wash water (23.3%). For the aquaculture, wastewater from earth pond had a higher frequency (63.6%) of P. aeruginosa isolation than concrete pond (36.4%). The antibiotic susceptibility result showed that P. aeruginosa exhibited high resistance rate to amoxicillin-clavulanic acid (80.0%) and cefotaxime (80.0%). Additionally, the bacteria showed resistance rate of 50.0% to tobramycin. On the other hand, the isolates demonstrated high sensitivity rates of 90.0% to imipenem and cefepime, while sensitivity rates of 60.0% were observed for meropenem and ceftazidime. The multiple antibiotic resistance index (MARI) ranged from 0.2 to 0.7, with a mean MARI of 0.6.Conclusion: The results of this study highlight the importance of close monitoring of abattoir and aquaculture settings, as they may serve as major sources for the environmental dissemination of antibiotic resistant bacteria such as P. aeruginosa.

Detection of multidrug-resistant methicillin-resistant Staphylococcus aureus from healthy black Bengal goat in Bangladesh.

The emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a growing public health concern. The objective of this study was to determine the prevalence and multi-drug resistant (MDR) profiles of MRSA in goats in Bangladesh. A total of 150 samples from goats comprised of rectal swab (n = 50), nasal swab (n = 50), and milk (n = 50) were collected. Isolation of S. aureus from samples was conducted onto mannitol salt agar (MSA). Identification of S. aureus was performed by cultural characteristics, Gram staining, biochemical tests (catalase, coagulase, indole, methyl red, and Voges-Proskaur), and nuc gene-specific PCR assay. The MRSA was identified by cefoxitin disc diffusion test and mecA gene-specific PCR assay. The MDR profiles of MRSA were performed against ampicillin, amoxicillin, gentamicin, cefoxitin, vancomycin, azithromycin, cefotaxime, ciprofloxacin and nalidixic acid by disc diffusion method. The overall prevalence of S. aureus was 35.3% and MRSA was 7.3%. The prevalence of MRSA was 12% in rectal swabs, 8% in nasal swabs, and 2% in milk. The highest resistance of MRSA was against ampicillin (91%) followed by azithromycin (55%), amoxycillin (36%), nalidixic acid (27%), ciprofloxacin (18%) and cefotaxime (9%). Most MRSA isolates (90.9%) exhibited resistance to at least three classes of antibiotics and were MDR. This study shows that goats may harbor MDR-MRSA, posing a risk to public health.

Phytochemical and Biological Investigations of Crude Extracts of Astragalus pisidicus.

Background/Objectives: Astragalus L. is a genus of the Fabaceae family, encompassing over 3000 species globally, with 380 species found in Turkey. This is the inaugural examination of the phytochemical, antioxidant, antibacterial, and cytotoxic properties of Astragalus pisidicus. Methods: The water and methanolic fractions of four parts (stems, flowers, leaves, root) as well as the whole plant were quantified and identified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) analysis. Cell death was assessed using the WST-1 assay, while apoptosis was identified by colorimetric protease assay for caspase 2, -3, -6, -8, and -9, as well as cellular DNA fragmentation assay. Antioxidant activity of A. pisidicus water and methanolic extracts was investigated with eight different assays. Antimicrobial activities of the extracts were evaluated against 16 bacterial strains by disc diffusion and broth microdilution methods. Results: A total of 13 phytochemicals were detected in the extracts at various concentrations. Hesperidin (147-40,174 µg/g extract) and hyperoside (363-2677 µg/g extract) comprised the principal constituents among the extracts. Fm (IC50 = 9.57 µg/mL), Rm (IC50 = 14.89 µg/mL), and Sm (IC50 = 9.57 µg/mL) were evaluated as active crude extracts on H1299, HT-29, and Panc-1 cells, while Rm (IC50 = 32.057 µg/mL) and Fm (IC50 = 64.25 µg/mL) were assessed as moderately active on MCF-7 and 22RV1 cells, respectively. The elevation of caspase 2, 3, 6, 8, and 9 enzyme activities, along with DNA fragmentation, signifies that the mode of cell death is apoptosis. According to the disc diffusion test results, Fm, Lm, Sm, and WPm extracts exhibited antimicrobial activity against gram (+) bacteria. Conclusions: A. pisidicus elicited apoptotic cell death in cancer cells selectively by the activation of caspases and subsequent DNA fragmentation and may serve as a novel source of an apoptosis-inducing anticancer drug.

Molecular identification and antimicrobial resistance profiling of pathogenic E. coli isolates from smallholder livestock households in Central Ethiopia.

Escherichia coli of different pathotypes are frequently involved in morbidity and mortality in animals and humans. The study aimed to identify E. coli pathotypes and determine antimicrobial resistance (AMR) profiles in Ethiopian smallholder livestock households. The pathotyping included 198 E. coli isolates identified from human and environmental samples collected from 98 households. AMR profiling was conducted on selected E. coli pathotypes from 89 households, along with known isolates from calf samples obtained from the same households. Morphological and biochemical tests were used to identify presumptive E. coli isolates. DNA was extracted and then singleplex PCR was used to amplify virulence genes. A disc diffusion test was applied for AMR profilings in E. coli pathotypes. Data were evaluated using chi-square tests and logistic regression. Calf (79.8%) and human (73.7%) samples were more likely to contain pathotypes (OR 3.2; 95% CI: 1.7, 5.9; p = 0.001 and OR 2.3; 95% CI: 1.2, 4.1; p =0.008, respectively) than the environmental samples (55.6%). ETEC (32.3%) and STEC (15.2%) were the most common pathotypes detected in the study samples. Out of the 176 isolates selected for AMR profiling, 85% were resistant to at least one drug and 36% were multi-drug resistant (MDR). The MDR isolates were found in 44 households, with 11 sharing identical pathotypes and resistance profiles among the different samples. Thus, E. coli strains were likely circulated among humans, animals, and the environment. This in turn calls for a One-health approach to improve antimicrobial usage standards and promote proper waste disposal practices.