Growth Kinetics Assay Research Articles

Integration of ZnO‐Ag Nanocomposites into PMMA Thin Film for Enhanced Antibacterial Applications

AbstractThe exploration of advanced materials has become imperative for addressing challenges in diverse fields, including healthcare and infection prevention. This research contributes to this domain by focusing on the synthesis, characterization, and evaluation of ZnO‐Ag nanocomposites and embedding them in PMMA matrix to obtain free‐standing flexible membrane with enhanced antibacterial potential and reusability of material. The study employs a solvothermal method for the decoration of silver nanoparticles onto zinc oxide nanorods, with the objective of harnessing the synergistic antibacterial properties of both materials. The study evaluates the antibacterial potential of the nanocomposites through well diffusion and growth kinetic assays, revealing their enhanced efficacy with a zone of inhibition (ZOI) of 21 ± 1.05 mm against Escherichia coli (E. coli). The implications of this research extend to the realm of biomedical applications, positioning the nanocomposite‐polymer membrane as promising candidates for infection prevention in healthcare premises.

Genetic and pathogenic potential of highly pathogenic avian influenza H5N8 viruses from live bird markets in Egypt in avian and mammalian models

Since its first isolation from migratory birds in Egypt in 2016, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks among domestic poultry in various areas of the country affecting poultry health and production systems. However, the genetic and biological properties of the H5N8 HPAI viruses have not been fully elucidated yet. In this study, we aimed to monitor the evolution of circulating H5N8 viruses and identify the pathogenicity and mammalian adaptation in vitro and in vivo. Three H5N8 HPAI viruses were used in this study and were isolated in 2021–2022 from poultry and wild birds during our routine surveillance. RNA extracts were subjected to full genome sequencing. Genetic, phylogenetic, and antigenic analyses were performed to assess viral characteristics and similarities to previously isolated viruses. Phylogenetic analysis showed that the hemagglutinin genes of the three isolates belonged to clade 2.3.4.4b and grouped with the 2019 viruses from G3 with high similarity to Russian and European lineages. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, several mutations associated with increased virulence and polymerase activity in mammals were observed. Growth kinetics assays showed that the H5N8 isolate is capable of replicating efficiently in mammalian cells lines. In vivo studies were conducted in SPF chickens (White Leghorn), mice, and hamsters to compare the virological characteristics of the 2022 H5N8 isolates with previous H5N8 viruses isolated in 2016 from the first introduction. The H5N8 viruses caused lethal infection in all tested chickens and transmitted by direct contact. However, we showed that the 2016 H5N8 virus causes a higher mortality in chickens compared to 2022 H5N8 virus. Moreover, the 2022 virus can replicate efficiently in hamsters and mice without preadaptation causing systemic infection. These findings underscore the need for continued surveillance of H5 viruses to identify circulating strains, determine the commercial vaccine’s effectiveness, and identify zoonotic potential.

Phage susceptibility testing methods or 'phagograms': where do we stand and where should we go?

Phage therapy is a highly promising approach to address the challenge that is presented by the global burden of antimicrobial resistance. Given the natural specificity of phages, phage susceptibility testing (PST) is a prerequisite for successful personalized therapy, allowing the selection of active phages from large and diverse collections. However, the issue of an easy-to-use and standardized technique remains. In this review, we describe the principles, advantages and drawbacks of two routinely used PST techniques: plaque and growth kinetic assays. These are labour-intensive and time-consuming methods that require automation of one or more steps, including preparation of test panels, incubation, reading and analysis of results. In addition to automation, there is an urgent need to establish a reference method to enable efficient of PST techniques selection of therapeutic phages. We discuss knowledge gaps and parameters that need to be investigated to work towards this goal.

Deciphering the Antibiofilm, Antibacterial, and Antioxidant Potential of Essential Oil from Indian Garlic and its Phytocompounds Against Foodborne Pathogens.

Garlic (Allium sativum L.), particularly its volatile essential oil, is widely recognized for medicinal properties. We have evaluated the efficacy of Indian Garlic Essential Oil (GEO) for antimicrobial and antibiofilm activity and its bioactive constituents. Allyl sulfur-rich compounds were identified as predominant phytochemicals in GEO, constituting 96.51% of total volatile oils, with 38% Diallyl trisulphide (DTS) as most abundant. GEO exhibited significant antibacterial activity against eleven bacteria, including three drug-resistant strains with minimum inhibitory concentrations (MICs) ranging from 78 to 1250µg/mL. In bacterial growth kinetic assay GEO effectively inhibited growth of all tested strains at its ½ MIC. Antibiofilm activity was evident against two important human pathogens, S. aureus and P. aeruginosa. Mechanistic studies demonstrated that GEO disrupts bacterial cell membranes, leading to the release of nucleic acids, proteins, and reactive oxygen species. Additionally, GEO demonstrated potent antioxidant activity at IC50 31.18mg/mL, while its isolated constituents, Diallyl disulphide (DDS) and Diallyl trisulphide (DTS), showed effective antibacterial activity ranging from 125 to 500µg/mL and 250-1000µg/mL respectively. Overall, GEO displayed promising antimicrobial and antibiofilm activity against enteric bacteria, suggesting its potential application in the food industry.

Residue K28 of Zika Virus NS5 Protein Is Implicated in Virus Replication and Antagonism of STAT2.

The identification of four potential nonstructural 5 (NS5) residues-K28, K45, V335, and S749-that share the same amino acid preference in STAT2-interacting flaviviruses [Dengue virus (DENV) and Zika virus (ZIKV)], but not in STAT2-non-interacting flaviviruses [West Nile virus (WNV) and/or Yellow fever virus (YFV)] from an alignment of multiple flavivirus NS5 sequences, implied a possible association with the efficiency of ZIKV to antagonize the human signal transducer and activator of transcription factor 2 (STAT2). Through site-directed mutagenesis and reverse genetics, mutational impacts of these residues on ZIKV growth in vitro and STAT2 antagonism were assessed using virus growth kinetics assays and STAT2 immunoblotting. The results showed that mutations at the residue K28 significantly reduced the efficiency of ZIKV to antagonize STAT2. Further investigation involving residue K28 demonstrated its additional effects on the phenotypes of ZIKV-NS5 nuclear bodies. These findings demonstrate that K28, identified from sequence alignment, is an important determinant of replication and STAT2 antagonism by ZIKV.

In vitro Antifungal Efficacy of Some Plant Extracts against Fungal Pathogens Causing Diseases in Solanaceous Crops

Background: This research aimed to assess the antifungal potential of oil and organic leaf extracts obtained from Ocimum gratissimum, Kopsia fruticosa and Hibiscus rosa-sinensis against fungal pathogens affecting solanaceous crops. Methods: Oil of O. gratissimum was obtained by hydro-distilled method using Clevenger apparatus. Leaf extracts of K. fruticosa and H. rosa-sinensis were extracted using three organic solvents namely hexane, ethanol and methanol. The antifungal activity of the oil and organic leaf extracts against the test fungal pathogens was determined by the agar well diffusion method. The efficacy of the oil and organic leaf extracts against the pathogens was evaluated by their minimum inhibitory concentration (MIC), ability to inhibit spore germination and growth kinetic assay. Result: The oil of O. gratissimum (1000 ppm) significantly inhibited the growth of Cercospora capsici (81.4%), Alternaria alternata (73.7%) and Fusarium oxysporum (68.7%). Hexane extract of K. fruticosa and methanol extract of H. rosa-sinensis (1500 µg/well) displayed strong antifungal activity against Colletotrichum acutatum with inhibition percentages of 75.9% and 82.4% respectively. Similarly, ethanol extract of K. fruticosa and methanol extract of H. rosa-sinensis showed effective inhibition against C. capsici and A. alternata. The oil of O. gratissimum showed MIC values of 50, 100 and 200 µg/mL against C. capsici, A. alternata and F. oxysporum respectively. Ethanol extract of K. fruticosa showed MIC values ranging from 100 to 350 µg/mL, while H. rosa-sinensis methanol extract displayed MIC values ranging from 50 to 200 µg/mL against the fungal pathogens. The oil extract also demonstrated strong inhibition of spore germination of the test fungi. Our study suggests the potential of plant-derived essential oils and extracts as effective antifungal agents for crop protection.

Antifungal activity of Annona crassiflora Mart. dichloromethane fraction against strains of C. albicans.

Products derived from medicinal plants with antimicrobial activity are considered a promising alternative in the treatment of fungal infections. In this perspective, this study proposed to evaluate the antifungal activity of the dichloromethane fraction of Annona crassiflora Mart. against C. albicans strains. Tests were carried out to determine Minimum Inhibitory Concentration (MIC), Minimum Fungicide Concentration (MFC), microbial growth kinetics, fungal cell wall and membrane mechanisms of action, antifungal biofilm activity, and cytotoxic effects on human erythrocytes. The extract presented MIC and MFC values that ranged from 256 µg/mL to 1,024 µg/mL, with fungicidal activity in the microbial growth kinetics assay. The mechanism of action did not occur through damage to the cell wall or via binding to ergosterol in the membrane, though the fraction presents activity against biofilm and is not cytotoxic in human erythrocytes. The dichloromethane fraction of Annona crassiflora Mart. presented antifungal activity and reduced biofilm growth, without toxicity against human erythrocytes; however, further studies are needed to define its mechanism of action.

2780. Factors associated with Staphylococcus aureus susceptibility to two lytic bacteriophages; Comparison of phage host susceptibility using a reference set of S. aureus

Abstract Background Timely access is a major concern when considering phage therapy. Using susceptible phage is critical for the outcomes of phage therapy. For timely access to susceptible phages, identifying factors associated with phage susceptibility and building a reference set (RS) to compare host ranges is needed. Methods Phage K, which was a well-documented lytic S. aureus phage, and phage PBSA02, which was a new S. aureus phage from the Korean phage biobank, were used. The efficiency of plating (EOP), the relative activity of phage against specific strain/host strain, and growth kinetics assays were used to determine phage susceptibility. The isolates with high lytic activity (HLA) were defined as susceptible (EOP > 1%). The phage PBSA02 and phage K susceptible groups were compared to the non-susceptible groups to determine the associating factors of phage susceptibility. Results We constructed a methicillin-resistant S. aureus (MRSA) RS using the Australian Group on Antimicrobial Resistance (AGAR) repository. We determined the predominant sequence types (ST) of MRSA in Australia in 2021; ST22 accounted for 90.1% of HA-MRSA and 5 STs for 67.9% of CA-MRSA (figure 1). Then, we randomly selected 39 AGAR MRSA isolates corresponding to each ST from each region in Australia (figure 1). Phage PBSA02 exhibited HLA against 56.4% of the 39 RS isolates, while phage K exhibited HLA against 30.8%. Phage PBSA02 showed HLA against 100% of ST93 and ST30 isolates following ST1 (62.5%), ST22 (37.5%), ST5 (20%) and ST45 (16.7%); phage K showed HLA against 100% of ST30 and 62.5% of ST22 isolates but against none of the ST93 isolates. Phage PBSA02 susceptibility correlated with the ST of MRSA isolates and ciprofloxacin susceptibility (table 1). Isolates from pneumonia/empyema tended to show phage PBSA02 susceptibility (table 1). Phage K susceptibility was also significantly associated with the ST (table 2). Conclusion Our study demonstrated that phage susceptibility was associated with the STs of MRSA, and the RS could be useful for a standardised comparison of the phage host range. Phage PBSA02 with broad susceptibility to the RS could be useful for phage therapy. Further work includes antibiotic synergy testing and resistance mechanism identification in these isolates. Disclosures All Authors: No reported disclosures

Functionalized chitosan based nanotherapeutics to combat emerging antimicrobial resistance in bacterial pathogen

Ineffectiveness of conventional antibiotics due to antimicrobial resistance and microbial adaptations has led to the exploration of alternative or complementary therapeutics. In the present study, surface engineered nanosystems (SENS) were developed by conjugation of chitosan with rhamnolipids (RL) through amide linkage. Subsequently, FTIR and NMR analyses affirmed covalent immobilization of RL. SENS exhibited an increase in the particle size and reduction in zeta potential as compared to chitosan nanosystems (CNS). In parallel, first-line antibiotic (ciprofloxacin) was encapsulated in SENS and CNS for augmenting their antimicrobial potential against drug-resistant Salmonella enterica subsp. enterica serotype Typhi. Drug entrapment efficiency for AB-SENS (64 ± 2 %) was higher than AB-CNS. Furthermore, bacterial growth kinetics assay along with colony forming unit (CFU) assay revealed that AB-SENS inhibited the growth of Salmonella sp. even at ½ MIC with restoration of the antibiotic activity. Due to enhanced biofilm penetration ability, the diffusion of entrapped drug from AB-SENS resulted in an effective biofilm disruption to control drug-resistant Salmonella sp. Release kinetics study demonstrated a pH dependent drug release from AB-SENS in simulated gastric fluid (14.2 ± 2 %) and simulated intestinal fluid (28.4 ± 3 %). Hence, our findings suggest that void or drug-loaded SENS can be a versatile synergistic tool to revive the conventional antibiotics for improved treatment outcomes against resistant pathogens.

In Vitro and In Vivo Synergistic Antibacterial Functions of the Recombinant Ib-AMP4 and E50-52 Antimicrobial Peptides against Multidrug-Resistant Acinetobacter baumannii

Background: Most Acinetobacter baumannii species have become resistant to all common antibiotics. Many antimicrobial peptides (AMPs) have been identified with efficient functions in infection management. E50-52 (UniProtKB: P85148) and Ib-AMP4 (UniProtKB: O24006) AMPs have shown marked antibacterial functions. Objectives: This investigation was designed to produce E50-52 and Ib-AMP4 AMPs through recombinant protein production. Subsequently, the synergistic antimicrobial functions of these two peptides were assessed under in vitro and in vivo circumstances on multidrug-resistant A. baumannii to investigate the antimicrobial effects of Ib-AMP4 and E50-52 AMPs on drug-resistant Acinetobacter. Methods: The gene sequence of E50-52 and Ib-AMP4 AMPs were codon optimized and separately inserted into the pET-32α vector. The recombinant structures were expressed in host bacteria. The antibacterial functions of the individual and combined application of the purified refolded E50-52 and Ib-AMP4 AMPs against multidrug-resistant A. baumannii were evaluated through the time-kill, minimum inhibitory concentration (MIC), growth kinetic assays, and in vivo (mouse body) systemic infection. Results: The minimum concentrations of the produced refolded E50-52 and Ib-AMP4 AMPs against A. baumannii were 0.325 and 0.0625 mg/mL, respectively. Moreover, the checkerboard procedure confirmed the synergic effects of the produced AMPs. The use of E50-52 and Ib-AMP4 AMPs in combination resulted in an over five times reduction in log10 CFU/mL of alive cells during the first 240-min exposure. The antibacterial efficiency of the produced AMPs was confirmed by growth kinetic assays, scanning electron microscopy (SEM) results, and in vivo evaluation tests. The in vivo assay on rats confirmed the significant antibacterial functions of the produced recombinant proteins on A. baumannii systemic infection. Conclusions: The results proved the considerable synergistic antibacterial functions of the produced recombinant Ib-AMP4 and E50-52 AMPs to treat A. baumannii systemic infection effectively.

Effect of Escin Alone or in Combination with Antifungal Agents on Resistant Candida glabrata Biofilms: Mechanisms of Action.

Nowadays, the increase in antimicrobial-resistant fungi (AMR) is certainly a major health concern, and the development of alternative therapeutic strategies has become crucial. Natural products have been used to treat various infections, and their chemical properties contribute to the performance of their biological activities, such as antifungal action. The various virulence factors and mechanisms of resistance to antifungals contribute to making Candida glabrata one of the most frequent agents of candidiasis. Here we investigate the in vitro and in vivo activity of β-escin against Candida glabrata. The β-escin MICs were determined for a reference strain and two clinical isolates of C. glabrata. Furthermore, growth kinetics assays and biofilm inhibition/eradication assays (crystal violet) were performed. The differences in the expression of some anti-biofilm-associated genes were analyzed during biofilm inhibition treatment so that reactive oxygen species could be detected. The efficacy of β-escin was evaluated in combination with fluconazole, ketoconazole, and itraconazole. In addition, a Galleria mellonella infection model was used for in vivo treatment assays. Results have shown that β-escin had no toxicity in vitro or in vivo and was able to inhibit or destroy biofilm formation by downregulating some important genes, inducing ROS activity and affecting the membrane integrity of C. glabrata cells. Furthermore, our study suggests that the combination with azoles can have synergistic effects against C. glabrata biofilm. In summary, the discovery of new antifungal drugs against these resistant fungi is crucial and could potentially lead to the development of future treatment strategies.

A Novel Triple Reassortment H3N8 Avian Influenza Virus: Characteristics, Pathogenicity, and Transmissibility

An increasing number of new subtypes of avian influenza viruses (AIVs) are reported to be infecting humans, including H3N2, H5N1, H7N9, H10N8, and the recently emerged H3N8 virus in China in 2022. However, the genetic and biological properties of the currently prevalent H3N8 AIVs are not yet fully understood. This study reports the isolation of a novel triple reassortment H3N8 virus (GD-H3N8) from chicken flocks in Guangdong province, China, in 2022. The GD-H3N8 virus contains the Eurasian avian duck-origin H3 gene, the North American avian N8 gene, and dynamic internal genes of the H9N2 virus, and shows high homology with human H3N8 strains. The GD-H3N8 isolate has multiple mammalian adaptive mutations associated with receptor binding and virulence. Growth kinetics assays demonstrate that the GD-H3N8 isolate is capable of efficient replication in avian, mammalian, and human cells in vitro. In vivo, the GD-H3N8 isolate can replicate efficiently in mice without preadaptation, in addition to establishing systemic infection and transmission by direct contact in chickens. These findings underscore the need for continued surveillance of H3N8 viruses to identify circulating strains that may potentially threaten human health.

Antiviral effect of Favipiravir against Chandipura virus in vitro and in vivo.

Chandipura virus (CHPV) is an emerging encephalitic virus with outbreak potential in a pediatric population. It causes acute encephalitis, with clinical symptoms leading to death within 48-72 h and an alarmingly high case fatality rate up to 55%-78%. Despite the high mortality rate in children, no vaccines or antivirals are currently available; thus, repurposing licensed drugs seems to be one of the attractive therapeutic approaches. Among the various options available, Favipiravir emerged as a promising candidate, and its unique characteristics and clinical efficacy have garnered significant attention and demonstrated considerable potential in the fight against viral diseases. In the current study, we have evaluated the antiviral effect of Favipiravir against CHPV by Plaque reduction assay and viral growth kinetics assay in Vero cells and in vivo effect of drug treatment against lethal viral challenge was analysed in 10-day-old CD1 mice. A dose-dependent reduction in CHPV plaque size and number was observed in Vero cells treated with Favipiravir, with an EC50 of 92.26 μM. Complete inhibition of CHPV replication was observed at 320 μM drug concentration and 50% cytotoxicity (CC50 ) at 4774 μM, indicating a high selectivity index 51.24. In vivo, studies in mice showed 100% survival with 300 mg/kg/day of Favipiravir given orally till seventh-day postinfection. The study provides evidence of the antiviral activity of Favipiravir against CHPV infection, and further clinical evaluation may alleviate the associated mortality.

Evaluation of the Synergistic Effect of LL-37 and Oncorhyncin II Recombinant Proteins on Staphylococcus aureus Under In Vitro Conditions

Background: The treatment of Staphylococcus aureus infections has become a public health crisis due to the extensive development of antimicrobial resistance. Antimicrobial peptides (AMPs) have been introduced as promising naturally-derived antimicrobial alternatives to antibiotics. LL-37 and oncorhyncin II are 2 AMPs with notable proven antibacterial effects. Objectives: This study aimed to produce recombinant LL-37 and oncorhyncin II and investigate their synergistic effects on S. aureus (ATCC25923). Methods: The synthetic genes of LL-37 and oncorhyncin II were individually ligated into the pET32a expression vector. Transformed pET32a was introduced into Escherichia coli BL21 as an expression host. The protein expression and purification steps were optimized, and the biological effectiveness of the peptides was evaluated by assessing the minimum inhibitory concentration (MIC), time-kill, and growth kinetic tests against S. aureus. Results: The MIC assay confirmed the effective antibacterial performances of LL-37 and oncorhyncin II against S. aureus at 30.6 and 47.93 µg/mL, respectively. The peptides’ synergistic activity was validated by the checkerboard method. A combination of LL-37 and oncorhyncin II at 2 × MIC showed a sharp decline of the viable cells with over 3-time reductions in log 10 colony-forming units (CFU)/mL within the first 5 hours. The growth kinetic results confirmed the high effectiveness of the peptides’ combination in eliminating the bacterial inoculum turbidity by 50% reduction during the first hour of exposure. Conclusions: The produced recombinant LL-37 and oncorhyncin II showed effective antimicrobial function against S. aureus. The synergistic performance of the peptides was repeatedly confirmed through checkerboard, time-kill, and growth kinetic assays.

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12mmol cell/h) in SF and for glutamine (30.8 × 10-12mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Thermally-exfoliated graphene Oxide/ZnO nanocomposite catalysts for photocatalytic hydrogen evolution and antibacterial activities

Carbon nanomaterials are of great interest due to their enhanced charge separation in hydrogen evolution reactions. Herein, heterostructured TEGO/ZnO nanocomposite catalyst was obtained by combining 3D multilayer graphene-like nanomaterial obtained by green process from recycled carbon source with ZnO to improve photocatalytic activity. Here, photodeposition of MoSx and Pt in aqueous solution is performed on TEGO/ZnO nanocomposite to obtain TEGO/ZnO/MoSx and TEGO/ZnO/Pt by reducing (NH4)2MoS4 and H2PtCl6.6H2O, respectively. Photodeposited MoSx and Pt on TEGO/ZnO nanocomposite resulted in enhanced photocatalytic activity and stability due to increased active sites and enhanced electron transfer ability due to cocatalyst effect. In addition, antibacterial activity of TEGO/ZnO nanocomposite against Escherichia coli and Staphylococcus aureus was investigated by bacterial growth kinetic assay. This work could open up a new nanomaterial for chemical and biological applications.

In Vitro and In Vivo Effect of the Imidazole Luliconazole against Lomentospora prolificans and Scedosporium spp.

Infections with Scedosporium spp. and Lomentospora prolificans have become a serious threat in clinical settings. The high mortality rates associated with these infections can be correlated with their multidrug resistance. The development of alternative treatment strategies has become crucial. Here, we investigate the in vitro and in vivo activity of luliconazole (LLCZ) against Scedosporium apiospermum (including its teleomorph Pseudallescheria boydii) and Lomentospora prolificans. The LLCZ MICs were determined for a total of 37 isolates (31 L. prolificans isolates, 6 Scedosporium apiospermum/P. boydii strains) according to EUCAST. Furthermore, the LLCZ antifungal activity was tested in vitro, using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] growth kinetics assay and biofilm assays (crystal violet and XTT assay). In addition, a Galleria mellonella infection model was used for in vivo treatment assays. The MIC90 of LLCZ was determined to be 0.25 mg/L for all tested pathogens. Growth was inhibited within 6 to 48 h of the start of incubation. LLCZ inhibited biofilm formation in both preadhesion stages and late-stage adhesion. In vivo, a single dose of LLCZ increased the survival rate of the larvae by 40% and 20% for L. prolificans and Scedosporium spp., respectively. This is the first study demonstrating LLCZ activity against Lomentospora prolificans in vitro and in vivo and the first study showing the antibiofilm effect of LLCZ in Scedosporium spp. IMPORTANCE Lomentospora prolificans and S. apiospermum/P. boydii are opportunistic, multidrug-resistant pathogens causing invasive infections in immunosuppressed patients and sometimes in healthy persons. Lomentospora prolificans is panresistant against the currently available antifungals, and both species are associated with high mortality rates. Thus, the discovery of novel antifungal drugs exhibiting an effect against these resistant fungi is crucial. Our study shows the effect of luliconazole (LLCZ) against L. prolificans and Scedosporium spp. in vitro, as well as in an in vivo infection model. These data reveal the previously unknown inhibitory effect of LLCZ against L. prolificans and its antibiofilm effect in Scedosporium spp. It represents an extension of the literature regarding azole-resistant fungi and could potentially lead to the development of future treatment strategies against these opportunistic fungal pathogens.

Microstructural, physico-chemical, antibacterial and antibiofilm efficacy of imipenem loaded chitosan nano-carrier systems to eradicate multidrug resistant Acinetobacter baumannii

Acinetobacter baumannii, being labelled as “critical priority pathogen” is a major threat faced by humanity nowadays as it is responsible for numerous life-threatening nosocomial infections. A. baumannii is a highly resistant microbe that forms biofilms at infection site thus interfering with the permeability of antibiotics. Therefore, intrinsically antibacterial biopolymer, chitosan, is utilized to synthesize imipenem (IPM) encapsulated chitosan nano-carrier system (CS-IPM-NCS) against multidrug resistant A. baumannii (MDRAB). CS-IPM-NCS were fabricated using ionic gelation method. The developed CS-IPM-NCSs were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), zeta potential, average size and Fourier transform infrared (FTIR) spectroscopy. It was observed that SEM and AFM analyses revealed smooth, regular and homogeneously distributed nano-carrier systems. The mean size of CS-IPM-NCS was found to be < 100 nm and the zeta potential was 38.9 ± 5.52 mV that indicated the stability of the nano-carrier systems. Encapsulation efficiency was optimized to ensure high encapsulation (87 ± 3%) of IPM in 0.3% (w/v) CS-NCS. Changes in characteristic peaks in FTIR spectra demonstrated the successful encapsulation of IPM inside the CS-NCS. The antibacterial activity of CS-IPM-NCS was evaluated by growth kinetics assay and the results exhibited potent (100%) inhibition of MDRAB. The outcome of CS-IPM-NCS on the pre-formed biofilms by MDRAB strains were ∼90%, due to the cationic-anionic interaction between NCS and biofilm structures. Thus, the present study findings suggest that CS-IPM-NCS might be an alternative strategy to conventional (failing) antibiotics to eradicate MDRAB.

Chemical Characterization of Honey and Its Effect (Alone as well as with Synthesized Silver Nanoparticles) on Microbial Pathogens' and Human Cancer Cell Lines' Growth.

The antibacterial, anticancer, and wound-healing effects of honey can vary according to the type, geographical region, honey bee species, and source of the flowers. Nanotechnology is an innovative and emerging field of science with an enormous potential role in medical, cosmetics, and industrial usages globally. Metal nanoparticles that derived from silver and range between 1 nm and 100 nm in size are called silver nanoparticles (AgNPs). Much advanced research AgNPs has been conducted due to their potential antibacterial and anticancer activity, chemical stability, and ease of synthesis. The purpose of the present study was to explore the physicochemical properties of honey and the potential to use forest honey to synthesize AgNPs as well as to appraise the nanoparticles' antimicrobial and anticancer effects. Here, we used three different percentages of forest honey (20%, 40%, and 80%) as biogenic mediators to synthesize AgNPs at room temperature. The development of AgNPs was confirmed by color change (to the naked eye) and ultraviolet-visible spectroscopy studies, respectively. The absorbance peak obtained between 464 to 4720 nm validated both the surface plasmon resonance (SPR) band and the formation of AgNPs. Regarding the sugar profile, the contents of maltose and glucose were lower than the content of fructose. In addition, the results showed that the SPR band of AgNPs increased as the percentage of forest honey increased due to the elevation of the concentration of the bio-reducing agent. A bacterial growth kinetic assay indicated the strong antibacterial efficacy of honey with silver nanoparticles against each tested bacterial strain. Honey with nanotherapy was the most effective against hepatocellular carcinoma (HepG2) and colon cancer (HCT 116) cells, with IC50s of 23.9 and 27.4 µg/mL, respectively, while being less effective against breast adenocarcinoma cells (MCF-7), with an IC50 of 32.5 µg/mL.

Antibiofilm agents with therapeutic potential against enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is a predominant but neglected enteric pathogen implicated in infantile diarrhoea and nutrient malabsorption. There are no non-antibiotic approaches to dealing with persistent infection by these exceptional colonizers, which form copious biofilms. We screened the Medicines for Malaria Venture Pathogen Box for chemical entities that inhibit EAEC biofilm formation. We used EAEC strains, 042 and MND005E in a medium-throughput crystal violet-based antibiofilm screen. Hits were confirmed in concentration-dependence, growth kinetic and time course assays and activity spectra were determined against a panel of 25 other EAEC strains. Antibiofilm activity against isogenic EAEC mutants, molecular docking simulations and comparative genomic analysis were used to identify the mechanism of action of one hit. In all, five compounds (1.25%) reproducibly inhibited biofilm accumulation by at least one strain by 30-85% while inhibiting growth by under 10%. Hits exhibited potent antibiofilm activity at concentrations at least 10-fold lower than those reported for nitazoxanide, the only known EAEC biofilm inhibitor. Reflective of known EAEC heterogeneity, only one hit was active against both screen isolates, but three hits showed broad antibiofilm activity against a larger panel of strains. Mechanism of action studies point to the EAEC anti-aggregation protein (Aap), dispersin, as the target of compound MMV687800. This study identified five compounds, not previously described as anti-adhesins or Gram-negative antibacterials, with significant EAEC antibiofilm activity. Molecule, MMV687800 targets the EAEC Aap. In vitro small-molecule inhibition of EAEC colonization opens a way to new therapeutic approaches against EAEC infection.