The increasing demand for biocompatible and infection-resistant surgical implants has driven research towards surface modifications of polymeric materials. This thesis investigates the functionalization of polyvinylidene fluoride (PVDF) surgical meshes with bentonite-based nanocomposites to enhance their antimicrobial efficacy and drug delivery potential. The modified meshes were prepared using combinations of bentonite nanoparticles with silver nanoparticles, Levofloxacin, and propolis. Comprehensive characterization was performed using scanning electron microscopy (SEM) and antimicrobial activity assays against various bacterial strains. Additionally, the diffusion behavior of the therapeutic agents through the bacterial membranes was modeled using the COMSOL Multiphysics software. The results demonstrated that the incorporation of bentonite-based composites significantly improved the antimicrobial properties of the PVDF meshes while maintaining their structural integrity and biocompatibility. This study provides insights into the development of advanced surgical materials with sustained drug release capabilities, offering potential applications in infection-prone medical procedures. Antibiotic resistance increases each year, and for this reason, researchers have focused on creating new types of materials able to combat bacteria and fungi. The use of systemic antimicrobials has increased in recent years for the treatment of skin infections; however, these drugs come with a risk of side effects such as diarrhea, stomach cramps, and the development of antibiotic resistance in the microbiota of the intestines. The main aim of this work was to integrate these four composites and study their antimicrobial activity properties. The panel of bacterial cultures included Staphylococcus aureus 1199, Staphylococcus aureus 1199B, and Staphylococcus epidermidis ATCC 14990) and a clinical isolate (Pseudomonas aeruginosa), and they were used to evaluate the antibacterial activity of the prepared samples. The designed nanocomposites showed high activity against all of the test cultures compared with that of the non-modified meshes. A high inhibition zone was exhibited for NB@LVF@AgNP@P (55 ± 0.5 mm) when it was tested with Staphylococcus aureus 1199 and Staphylococcus epidermidis ATCC 14990.

Background/Objectives: Antimicrobial consumption (AMC) and stewardship (AMS) are effective strategies to combat increasing antimicrobial resistance rates worldwide. Post-Soviet countries (Armenia, Azerbaijan, Belarus, Estonia, Georgia, Kazakhstan, Kyrgyzstan, Latvia, Lithuania, Moldova, the Russian Federation, Tajikistan, Turkmenistan, Ukraine, and Uzbekistan) have implemented essential components for AMC and AMS to different extents to inform and guide decision-making and amend their policies. This review assesses and characterizes the existing systems in post-Soviet countries.

Methods: Papers were searched in PubMed, Google Scholar, Embase, CINAHL, CyberLeninka, and Scopus according to the inclusion criteria. This search also included gray literature covering the official sites of international and national health organizations. Three reviewers screened and then carefully assessed the articles using the JBI and AACODS appraisal checklists.

Results: Eleven (73.3%) countries with updated national action plans for combatting antimicrobial resistance defined AMC and AMS as strategic directions or objectives. Fifteen (100%) countries submit antimicrobial consumption data to international networks. Yet considerable disparities exist in the complexity of the monitoring systems and data sources employed across the region, except for in Georgia, which does not use AMC data for policy adjustments. Across these nations, disparities exist in the implementation of key elements supporting AMS, with no related policies reported in Tajikistan; well-established guidelines and feedback mechanisms are reported in Belarus, Estonia, Kazakhstan, and the Russian Federation; ten countries (66.7%) document only the partial implementation of stewardship practices.

Conclusions: The review provides key insights into the existing AMC and AMS implementation in the former USSR countries. Further efforts are needed to strengthen and expand antimicrobial stewardship programs, improve surveillance systems, and address key challenges in these regions.

Antimicrobial resistance (AMR) poses a significant threat to public health by compromising the effectiveness of infection treatments. Consuming food or water contaminated with AMR bacteria can lead to the transmission of resistant strains within the gut microbiota. However, the impact of intermittent clinical doses and prolonged sub-clinical exposure to antimicrobials on the persistence and transfer of AMR genes remains unclear.

To investigate this, five groups of mice were treated with ampicillin (Amp) administered in four intermittent clinical doses, with intervals of 20, 48, and 27 days between sequential treatments. During the intervals, the mice received additional tetracycline treatment in drinking water at concentrations ranging from 0 to 100 mg/L. The mice were orally inoculated with beta-lactam-resistant Salmonella Heidelberg carrying a mobile AMR plasmid (donor) following the first Amp treatment, E. coli labeled with green fluorescent protein (EC-GFP) as a recipient after the second Amp treatment, and beta-lactam-susceptible S. Heidelberg as another recipient after the third Amp treatment. Fecal samples were collected to enumerate donor, recipient, and transconjugant bacteria on selective agar media. Transconjugants were confirmed using qPCR targeting AMR genes.

The donor S. Heidelberg persisted throughout the experiment in all mice. Following each Amp treatment, the donor population reached a high density, but gradually declined until it became undetectable. The AMR plasmid was successfully transferred to commensal E. coli, EC-GFP, and the recipient S. Heidelberg after the first, second, and third Amp treatment, respectively. Tetracycline exposure enhanced both the persistence of the donor and the transfer of the AMR plasmid.

These findings underscore the significant impact of intermittent selective pressure on the persistence and transfer of AMR genes within the gut microbiota, reinforcing the need for more judicious antibiotic use to mitigate the spread of resistance.

Background: This study investigates bacterial etiology and antibiotic resistance in pediatric leukemia patients to determine the impact of chronic pathology on treatment efficacy. The study was made in Romania.

Methods: Thirty cases of children aged 1–16 years (18 boys, 12 girls) were analyzed, identifying 13 pathogens, including 8 Gram-positive and 5 Gram-negative bacteria.

Results: Among the patients, 11 girls presented with acute lymphoblastic leukemia (ALL) type B, while one boy and one girl had acute myeloid leukemia, and, as for boys, three had ALL type T and two had pre-B ALL. The most common pathogens were methicillin-resistant Staphylococcus aureus (MRSA, 11 patients), methicillin-sensitive Staphylococcus aureus (MSSA, 6 patients), Klebsiella spp., and Staphylococcus epidermidis. Due to the patients’ compromised health, most required intensive care and strong antibiotic regimens, including linezolid, vancomycin, and ertapenem, which showed limited resistance.

Conclusions: These findings highlight the critical importance of understanding bacterial resistance patterns to guide effective treatments in vulnerable populations. Knowing specific resistance profiles can be lifesaving, allowing for tailored therapies that improve survival rates in children with leukemia facing serious bacterial infections. Focusing on the dual aspects of pediatric patients and multidrug-resistant bacterial infections, this study aims to highlight the importance of addressing these factors together to enhance therapeutic approaches in vulnerable populations.

Discovered in the 1990s as a product of the metabolism of the so-called ‘rare’ filamentous actinomycete Actinomadura lepetitiana DSM 10909 [1], GE23077 is a non-ribosomally synthesised cyclic heptapeptide antibiotic that inhibits a wide range of bacterial RNA polymerases (RNAP) in vitro while not affecting mammalian enzymes [2]. After its discovery, the molecule was later overlooked due to its more limited in vivo antimicrobial activity, which has mainly been exerted against Moraxella catarrhalis, Neisseria gonorrhoeae, and Mycobacterium smegmatis isolates [3]. However, with an increasing number of bacteria becoming resistant to antibiotics, including the major currently approved RNAP inhibitors (i.e., fidaxomicin and rifamycin), the development of new drugs to fight bacterial infections is essential. In this context, GE23077 might prove promising, as its target includes functionally critical residues of the RNAP that are different from those targeted by other inhibitors [4].

Therefore, our collaborative project aims to better understand the production of GE23077 using multiple approaches as an essential step for its further development. A high-quality genome sequence of A. lepetitiana was recently obtained using Illumina MiSeq and MinION, followed by the identification and annotation of a putative biosynthetic gene cluster (BGC) for the production of GE23077. Following the development of new methods for the genetic manipulation of this recalcitrant actinomycete, knock-out studies of key genes in the BGC and overexpression studies of two putative regulatory genes are currently ongoing. In parallel, in vitro studies are being performed on purified A. lepetitiana RNAP to evaluate its susceptibility to GE23077.

Elucidating the mechanisms behind the synthesis of this promising RNA inhibitor may also open the way to combinatorial biochemistry approaches to modify the molecule and potentially improve its in vivo antimicrobial activity.

[1] Dalmastri et al. Int J Syst Evol Microbiol 2020, 70(8):4782-90. [2] Ciciliato et al. J Antibiot 2004, 57(3):210-17. [3] Sarubbi et al. Eur J Biochem 2004, 271(15):3146-54. [4] Zhang et al. eLife 2014, 3:e02450.

Introduction: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that demonstrates significant resistance to multiple antibiotics of different classes. Bacteriophages, viruses that kill bacteria, have shown great potential against multidrug-resistant (MDR) infections. The phage PaFZ4 was isolated in this study, and its capability to infect MDR Pseudomonas aeruginosa strains was characterized. The whole genome sequencing analysis of the phage was performed to understand its potential as an anti-microbial agent.

Methods: A total of 10 bacteriophages (PaFZ 1-10) were isolated from sewage samples from different areas of Dhaka city. The host range of the bacteriophages was tested against 10 different MDR P. aeruginosa strains. The most effective PaFZ4 phage was characterized at different temperatures and pH levels. The whole genome sequencing of PaFZ4 and annotation was performed using the Phagenomics server. BLASTP was used to analyze the putative products of the ORFs, and phylogenetic analysis was performed using MEGA and iTOL. Proksee was used for sequence alignment and the visualization of the coding genes.

Results: The bacteriophage PaFZ4 has a broad host range and can kill all 10 MDR strains of P. aeruginosa studied. The characterizations of the phage indicated that the optimum pH was 8 and the temperature was 50°C. However, at higher temperatures, phage stability decreased significantly. The phage PaFZ4 has a genome sequence length of 43,208 bp with 62.3% GC content. A total of 147 ORFs, along with 59 coding sequences, were detected.

Conclusion: The phage PaFZ4 shows significant potential to fight against MDR P. aeruginosa, with its broad host range and stability under optimal conditions. In the future, an in-vivo analysis will be performed to understand the effectiveness of the isolated phage.

Finding antibiotic substitutes through ecological synthesis methods based on natural compounds is becoming a necessity for the treatment and prevention of microbial infections. In the present study, three essential-oil-loaded nanoemulsions (NEs) were prepared using the high-energy method. The obtained oil-in-water emulsions were characterized by determining the droplet size, PDI, zeta potential, and transmittance. Their stability against thermodynamic conditions and conservation was also investigated. Subsequently, the NE's antibacterial activities were assessed against Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes) and Gram-negative (Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa) pathogenic bacterial strains. Our results revealed that NEs were successfully formed, with mean sizes ranging from 13.12 to 24.74 nm, confirming the nanometric size of the droplets. PDI values ranged from 0.278 to 0.317 and the percentages of transmittance exceeded 96.40%, indicating the optical clarity of the formulation. The thermodynamic stability study revealed a homogeneous size dispersion of the prepared NE1 and NE2, confirming their stabilities. Regarding NE3, an instability to centrifugal forces was registered, with phase separation observed, suggesting a heterogeneous dispersion of this preparation. The results of the antibacterial activity revealed that all tested NEs exerted a bactericidal effect against the majority of the tested strains (5 out of 6). However, a bacteriostatic effect by the tested substances against the E. coli strain was registered. NE3 exhibited the highest effect with MIC values ranging between 6.25 and 25% (v/v). The obtained NEs remain an efficient natural tool to fight against pathogenic bacteria and could be valorized as a potent drug delivery system.

INTRODUCTION: With the increasing prevalence of carbapenem-resistant isolates, it is imperative to identify and evaluate alternative treatment options to improve patient outcomes. Cefiderocol, a novel siderophore cephalosporin, is highly effective against these organisms and studies advocate its preferred use in clinical practice. A lack of local published material necessitated the present study, which is aimed to determine the antimicrobial susceptibility of cefiderocol against carbapenem-resistant Gram-negative bacilli isolated from various clinically significant specimens.

METHODS: This cross-sectional study was carried out in the Microbiology Laboratory of the Combined Military Hospital, Lahore, Pakistan over a duration of 6 months. The clinical samples from 145 patients with carbapenem-resistant isolates were tested for susceptibility against cefiderocol (CLSI 2022). The results were then compared across various subgroups.

RESULTS: The mean age of the patients was 45.8±15.8 years. There were 114 (78.6%) male and 31 (21.4%) female patients with a male-to-female ratio of 3.7:1. The majority of the samples comprised blood (n=40, 27.6%) followed by pus in 35 (24.1%) and non-directed bronchial lavage in 21 (14.5%) cases. Among the carbapenem-resistant isolates, carbapenem-resistant enterobacteriacae were more common and comprised 119 (82.1%) isolates, followed by pseudomonas aeruginosa in 26 (17.9%) cases. Cefiderocol susceptibility was observed in 133 (91.7%) carbapenem-resistant isolates. When compared, there was no statistically significant difference in cefiderocol susceptibility across various subgroups based on age (p-value=1.000), gender (p-value=0.720), type of clinical specimen (p-value=0.992), or carbapenem-resistant isolate (p-value=0.694).

CONCLUSION: Cefiderocol demonstrated a high susceptibility rate against carbapenem-resistant isolates, regardless of patient demographics and microbial organism, which underscores the potential of cefiderocol as an effective alternative treatment option for such cases in future clinical practice.

Introduction: While some aspects of the host–pathogen interface are common to all infection sites, localized environments can have unique factors specific to the body site. Chronic infections are often polymicrobial in nature, with great variation in microbial communities between patients. Additionally, nutrient compositions vary at infection sites across the body. Our research demonstrates that the antibiotic susceptibilities of pathogens can dramatically shift depending on nutritional composition and the presence of other microorganisms.

Methods: Antimicrobial susceptibility testing (AST) was performed for Enterococcus faecalis in Cation-Adjusted Mueller–Hinton Broth (CAMHB), both for the organism alone and the organism grown in a polymicrobial community containing three other common wound pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii). We then performed the same AST in an anaerobic chamber to create an environment without oxygen. Further AST was then performed in media mimicking different body sites (Lubbock Chronic Wound Media (LCWM) and Synthetic Cystic Fibrosis Media 2 (SCFM2)).

Results: Our results demonstrate that Enterococcus faecalis grown in a polymicrobial community in CAMHB displayed increased susceptibility to gentamicin. We believe this is due to heme cross-feeding allowing more gentamicin to enter the cell via altered proton motive force. However, performing the same AST in anaerobic conditions reversed this phenotype. When gentamicin AST was performed in media more representative of different body sites (LCWM and SCFM2), the AST results differed from the CAMHB results. In SCFM2, E. faecalis no longer demonstrated increased susceptibility in the community. However, in LCWM, E. faecalis displayed increased susceptibility to gentamicin regardless of community presence.

Conclusions: Overall, our results demonstrate that environmental conditions play a role in determining an individual bacterium’s antibiotic susceptibility. By accounting for the infection environment when determining susceptibilities, we can prescribe more effective treatments and improve patient outcomes.

Antibiotic resistance is posing a serious threat to human health. Therefore, innovative therapeutic tools are needed to efficiently treat resistant bacteria. Aptamers are small synthetic oligonucleotides which show high specificity and affinity for a designated target. They evolve from random DNA oligonucleotide pools by means of an in vitro selection process called Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers have multiple applications in the biotechnological field and can be considered as biological drugs to target resistant microorganisms. In this work, we selected DNA aptamers to target the hemophore of the haem assimilation system (HasA) from Pseudomonas aeruginosa. The selection was performed with a DNA library with a random region of 40 nucleotides, and with HasA, which was produced in house. Twelve rounds of SELEX were performed, and the selection was monitored using qPCR and melting curve analysis. From round 1 to 12, the main fluorescence signal peak at 70°C, typical of the library, and then gradually shifted to 84°C. Selected oligonucleotides were sequenced by means of Next-Generation Sequencing, and one single sequence was found to be enriched at 75% in the pool. The most abundant candidates are currently being tested for their binding affinity and specificity. Meanwhile, our group is working on the aptamer selection towards the receptor of the haem assimilation system (HasR). Since iron is an essential micronutrient for P. aeruginosa, as for many bacteria, targeting and potentially blocking elements of the bacterium’s iron acquisition mechanisms, such as HasR and HasA, can put the bacterium at a significant metabolic disadvantage.