Kirby-Bauer Research Articles

Klebsiella spp. In Healthy Pigs: Reservoirs of Antimicrobial Resistance and Potential Pathogenic Threats.

The aim of this study was to isolate Klebsiella spp. from clinically healthy animals fed diets with or without antimicrobial growth promoters (AGP). Additionally, the study evaluated whether the inclusion of growth promoters affected the recovery of multi-drug-resistant isolates. A total of 144 isolates were obtained from rectal swabs on Simmons citrate agar supplemented with 1% inositol. Of these, 45 non-replicative isolates underwent extensive characterization, including molecular and phenotypic analyses. Sequencing identified that 77% were Klebsiella pneumoniae, 14.5% Klebsiella aerogenes, and 8.5% Klebsiella variicola. Isolates exhibiting the same polymorphic profiles were detected across different animals and treatments, with and without AGP. Seventy-one percent were multidrug-resistant, as determined by disk diffusion testing. The isolates harbored genes such as mcr-1, blaCTX-M-2, sul2, tetB, qnrS, and dfrA, among others. Additionally, genes encoding siderophores like enterobactin, aerobactin, and yersiniabactin were detected via PCR. Thirty-nine isolates were strong biofilm producers, 45% moderate, and 16% weak in vitro tests. The predominant genetic profiles included single, double, or triple-locus variants of ST25, ST147, and ST4691. Two novel sequence types were identified: ST7694 (K. pneumoniae) and ST7699 (K. variicola). Survival and persistence analyses in Galleria mellonella showed that these isolates exhibited a virulent phenotype and an enhanced capacity for multiplication in the early hours of infection. Clinically healthy swine act as reservoirs for multidrug-resistant Klebsiella spp. exhibiting significant virulence phenotypes. The identification of novel sequence types contributes to epidemiological surveillance and the One Health framework.

Impact of the 10-valent pneumococcal conjugate vaccine (PCV10) on pneumococcal carriage in healthy children and children with acute otitis media and pneumonia: emergence of serotypes 3, 6C and 19A in Croatia.

In 2019, the 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in the Croatian immunization programme, a first for this European PCV-naïve country. This study aimed to evaluate the impact of PCV10 on pneumococcal serotype distribution among asymptomatic children and children with pneumonia and/or acute otitis media. Cross-sectional studies were conducted before and after the PCV10 introduction, with nasopharyngeal swabs collected from 1500 healthy children under 48months of age. An additional 324 children under 18years with pneumonia and/or acute otitis media, from whom Streptococcus pneumoniae was isolated, were also included. Isolates were identified by conventional methods, serotyped by Quellung reaction, and tested for antimicrobial susceptibility using disk diffusion and gradient test methods. We report prevalence, absolute risk (prevalence) difference (RD) and relative risk (prevalence) ratio (RR) differences between exposed and control children. Carriage prevalence among healthy children increased from 19.9% to 28.7%, primarily due to a rise in non-vaccine serotypes (NVT). Adjusted probabilities for serotypes 6C (RR 3.18; 95% CI, 1.43-7.06), 11A (RR 2.8; 95% CI, 1.22-6.39), 19A (RR 4.18; 95% CI, 1.18-14.9) and 23A (RR 3.93; 95% CI, 1.87-8.24) were significantly higher in healthy exposed children. Prevalences of these serotypes were also higher in exposed children with pneumonia/acute otitis media. In this cohort, serotype 3 increased (RR 4.6; 95% CI, 2.02-10.3), becoming the leading post-PCV10 isolate in the overall studied population. Serotypes 3 and 19A were almost entirely responsible for complicated pneumonia cases for which the probability increased by 21-fold. Antimicrobial susceptibility remained similar across periods. In the early post-vaccine period significant increase of PCV10 vaccine-related serotypes (6C, 19A) was observed. Continued monitoring is also essential due to concerning rise of serotype 3 in patients with mucosal infections and a higher risk for complicated pneumonia.

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.

P-2111. Automated Bacterial Growth Detection, Bacterial Species Estimation and Antimicrobial Susceptibility Testing on Agar Plates Using a Light Transmission-based Monitoring System

Abstract Background Culturing bacteria on agar plates is essential for diagnosing bacterial infections, but it usually takes overnight and is labor-intensive. We have developed a novel device using a thin-film transistor image sensor to measure light transmission through agar plates every five minutes during incubation (Nakada et al. ASM 2023 and IDweek 2023). Here we present a proof of concept study for using this device for automated early detection and disk-diffusion susceptibility testing and compare the results to those obtained by experienced microbiology technicians. Methods Chromogenic agar plates were used for early detection and subsequent species estimation based on colony color changes, i.e. Enterobacteriaceae on CHROMagar mSuper CARBA plates and Staphylococci on MDRS-K plates. Statistical background subtraction was used for early detection of bacterial growth and histogram comparison of pseudo-color images created by correcting the intensity of red, blue, and green lights for species estimation. For disk diffusion tests susceptible and resistant E. coli and S. aureus were inoculated on Mueller-Hinton plates against the known agents and incubated for 18 hours with monitoring. Transmitted light images after 18 hours were first binarized and the distance of the shortest bacterial growth pixel from the center of the corresponding antibiotic disk was measured as the inhibition zone diameter. Results The device detected Enterobacteriaceae and MRSA as early as 6 and 8 hours after incubation, respectively, when the suspensions were densely streaked (Figure 1). The device also estimated the bacterial species with sensitivity and specificity over 90% for Enterobacteriaceae, however, the specificity for MRSA against MRCNS was approximately 70% (Table 1). An example of the automated inhibition zone measurement is shown in Figure 2. All automated measurements agreed well with the manual measurements (Table 2). Conclusion The performance in the bacterial detection, species estimation and the zone diameter measurement indicated that the device can be useful in accelerating, automating, and standardizing routine testing in microbiology laboratories. Disclosures Goh Ohji, MD, Ph.D, CarbGem Inc.: Advisor/Consultant Masakazu Nakajima, B. Engineering, CarbGeM Inc.: Board Member|CarbGeM Inc.: Ownership Interest

P-2100. Comparison of Fosfomycin MIC Values between Parent and Inner Colonies (IC) of Escherichia coli Arising During Disk Diffusion (DD) Testing

Abstract Background Recent studies have documented uropathogenic E. coli displaying the presence of IC during fosfomycin DD testing. These IC often demonstrate increased minimal inhibitory concentrations (MIC) when compared to their parent isolates. Investigating the clinical relevance of these IC and the extent of their resistance is a first step in determining whether they are associated with fosfomycin failure. Methods E. coli isolates (n = 69) collected from the University of Minnesota Medical Center (UMMC) that produced ≥ 3 discrete IC during at least one DD test were included. Parent and IC (n = 69) isolates underwent broth microdilution (BMD) testing using fosfomycin concentrations of 0.5-64 µg/mL for parent isolates and 8-1024 µg/mL for IC with incubation at 37°C for 16-20 hours. All susceptibility testing was supplemented with 25 µg/mL glucose-6-phosphate and conducted in duplicate on separate days. E. faecalis ATCC 29212 and E. coli ATCC 25922 were included as quality control strains. The results were evaluated using both CLSI (susceptible [S]: 64 µg/mL) and EUCAST (S: 8 µg/mL) breakpoints. Categorical agreement between the two guidelines were calculated. Results E. coli parent isolates had an MIC range of 1 to >64 µg/mL with a MIC50/90 of 8/32 µg/mL (Table 1). The IC isolates had significantly higher MIC ranges (< 8 and >1024 µg/mL) and MIC50/90 values of 256/ >1024 µg/mL. The parent isolates had 94.2% (n = 65) and 69.6% (n = 48) susceptibility per CLSI and EUCAST breakpoints, respectively. Using the more stringent EUCAST breakpoints, all IC isolates were resistant whereas 65.2% (n = 45) were considered resistant per CLSI breakpoints. Categorical agreement among the parent collection was 75.4% and 65.2% in the IC collection (Table 2). Conclusion Our data indicate IC arising from E. coli during fosfomycin DD testing are notably more resistant than their parent isolates, with an MIC50 increase of five, 2-fold dilutions. Future directions for this work include expanding the isolate collection and examining the fitness of these IC in comparison to their parents. Improving the understanding of the frequency, resistance, and fitness of IC arising from fosfomycin DD testing will help determine an optimal interpretation of IC, which may in turn improve patient outcomes. Disclosures Elizabeth B. Hirsch, PharmD, FCCP, FIDSA, GSK: Advisor/Consultant|GSK: Honoraria

P-175. Assessing the Dynamics of Fecal Colonization with Antibiotic-Resistant Bacteria in a Chilean Cohort: A Longitudinal Study

Abstract Background In low- and middle-income countries, the community burden of antibiotic-resistant Gram-negative bacteria (AR-GNB) colonization is high. Understanding factors influencing AR-GNB colonization is crucial for controlling antimicrobial resistance. Methods We conducted a cohort study from December 2019 to November 2020 with community-dwelling adults in Molina, an agricultural town in Central Chile. We collected questionnaires and fecal samples every two weeks for six months to identify colonization by extended-spectrum cephalosporin-resistant Enterobacterales (ESCRE). ESCRE detection involved selective media screening, MALDI-TOF identification, and disk-diffusion antibiotic-susceptibility testing. We assessed the impact of epidemiological and clinical features and exposure to various water sources, foods, and animals on i) baseline ESCRE fecal colonization, ii) ESCRE acquisition during follow-up, and iii) ESCRE clearance. The study included 198 subjects, using multiple imputations for missing predictor values. We used two hidden Markov models for each dataset, with Model 1 assuming perfect sensitivity and specificity and Model 2 estimating sensitivity. A Bayesian approach with Markov chain Monte Carlo algorithms estimated odds ratios (OR) and credible intervals (CI). Results The sample consisted of 198 residents with a mean age of 60.9 and a predominance of women (75%). Model 1 showed that treating drinking water before consumption reduced the risk of baseline ESCRE colonization (OR: 0.38; 95% CI: 0.02 – 0.89). No significant associations were found for ESCRE acquisition during follow-up. Males and individuals with comorbidities had a lower risk of clearing ESCRE (ORs: 0.58, 0.54; CIs: 0.24 – 0.97, 0.258 – 0.88, respectively). Model 2, with imperfect sensitivity, showed similar results. Conclusion This study highlights the significance of environmental and individual health factors in ESCRE colonization. Interventions like treating drinking water could reduce baseline ESCRE colonization risk. The varied impacts of gender and comorbidities on ESCRE clearance provide insights for targeted AR-GNB management strategies. Further research is needed to understand these associations and develop effective community-level interventions against AMR. Disclosures All Authors: No reported disclosures

Identifikasi Methicillin-Resistant Staphylococcus aureus pada Tenaga Kesehatan di Intensive Care Unit dan Instalasi Gawat Darurat RSU GMIM Pancaran Kasih Manado

Staphylococcus aureus is normal flora colonizing the anterior nares but may become pathogenic when the immune system is compromised. The increase and inappropriate use of antibiotics has resulted in Staphylococcus aureus becoming resistant to methicillin and other β-lactam antibiotics, known as Methicillin-Resistant Staphylococcus aureus (MRSA). MRSA-colonized healthcare workers can be a primary reservoir of MRSA, leading to transmission between healthcare workers and patients.This study aims to identify the presence of MRSA in healthcare workers in the ICU and emergency department of GMIM Pancaran Kasih General Hospital, Manado. Descriptive cross sectional study and total sampling, involving 20 healthcare workers. Nasal swab samples were cultured on MSA media, followed by bacterial identification and sensitivity testing with 30 μg cefoxitin antibiotic discs. A total of 11 samples (55%) of health workers were colonized with Staphylococcus aureus, and 4 samples (20%) were positive for MRSA. The conclusion of this study is that MRSA was found in health workers in the ICU and emergency room of RSU GMIM Pancaran Kasih Manado.

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.

Antibiogram of Borderline Oxacillin-Resistant Staphylococcus aureus (BORSA) and Modified Staphylococcus aureus (MODSA) Isolates From Human and Animals in Assam, India

Objectives: Detection of borderline oxacillin-resistant Staphylococcus aureus (BORSA) and modified Staphylococcus aureus (MODSA) strains with clinical significance are rare events. They have clinical, community, and livestock-associated significance. We aimed to screen these strains and evaluate the antibiogram through an array of antibiotics in Assam, a northeastern state of India. Method: S. aureus was confirmed biochemically and at the molecular level. Oxacillin and cefoxitin resistance were screened through the disc diffusion test. Hyperproduction of β-lactamase was screened using ticarcillin-clavulanic acid. PBP2a and PBP expressions were screened through mecA, mecC, and blaZ1, blaZ2 genes. MIC, MAR, MDR, XDR, and PDR were calculated. Findings: A total of 141 S. aureus isolates were screened. Altogether, 13 (9.22%) S. aureus isolates were mecA and/or mecC negative with oxacillin and/or cefoxitin resistance. Among the only oxacillin-resistant isolates, 33.33% (2/6) of the community-associated and 66.67% (2/3) of the livestock-associated isolates showed multiple drug resistance (MDR) with MAR index > 0.2. All the community-associated isolates showed resistance to oxacillin, while 83.33% showed resistance to ciprofloxacin & ticarcillin-clavulanic acid and 66.67% to fusidic acid. The livestock-associated isolates were 100% resistant to oxacillin, methicillin, and ticarcillin-clavulanic acid. All the oxacillin with cefoxitin-resistant isolates were MDR. Only one XDR was detected. One community-associated isolate was found to be BORSA, whereas the rest were MODSA. Altogether 61.54% had MAR index >0.2. Fusidic acid (0.064-0.125 µg/ml) had the lowest range of MIC, whereas trimethoprim, rifampicin, penicillin, and cefoxitin had higher ranges of MIC. Novelty: To the best of the authors’ knowledge, BORSA and MODSA from community and livestock-associated samples are not reported from this region. We assessed the antibiogram against 28 antibiotics (19 classes) and MIC against 12 antibiotics (11 classes). Reporting of MODSA strain from human community samples and livestock samples has great clinical importance from both human and veterinary perspectives. Keywords: MODSA, MDR, XDR, MAR, MIC, BORSA

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.