Multidrug-resistant Clinical Strains Research Articles

Dual phage-incorporated electrospun polyvinyl alcohol-eudragit nanofiber matrix for rapid healing of diabetic wound infected by Pseudomonas aeruginosa and Staphylococcus aureus.

Diabetic wound healing remains a healthcare challenge due to co-occurring multidrug-resistant (MDR) bacterial infections and the constraints associated with sustained drug delivery. Here, we integrate two new species of phages designated as PseuPha1 and RuSa1 respectively lysing multiple clinical MDR strains of P. aeruginosa and S. aureus into a novel polyvinyl alcohol-eudragit (PVA-EU†) nanofiber matrix through electrospinning for rapid diabetic wound healing. PVA-EU† evaluated for characteristic changes that occurred due to electrospinning and subjected to elution, stability and antibacterial assays. The biocompatibility and wound healing ability of PVA-EU† were assessed through mouse fibroblast cell line NIH3T3, followed by validation through diabetic mice excision wound co-infected with P. aeruginosa and S. aureus. The electrospinning resulted in the incorporation of ~ 75% active phages at PVA-EU†, which were stable at 25°C for 30days and at 4°C for 90days. PVA-EU† showed sustained release of phages for 18h and confirmed to be detrimental to both mono- and mixed-cultures of target pathogens. The antibacterial activity of PVA-EU† remained unaltered in the presence of high amounts of glucose, whereas alkaline pH promoted the activity. The matrix exerted no cytotoxicity on NIH3T3, but showed significant (p < 0.0001) wound healing in vitro and the process was rapid as validated through a diabetic mice model. The sustained release, quick wound closure, declined abundance of target MDR bacteria in situ and histopathological signs of recovery corroborated the therapeutic efficacy of PVA-EU†. Taken together, our data signify the potential application of PVA-EU† in the rapid treatment of diabetic wounds without the aid of antibiotics.

Characterization and therapeutic potential of MRABP9, a novel lytic bacteriophage infecting multidrug-resistant Acinetobacter baumannii clinical strains

Acinetobacter baumannii is one of the most important pathogens of healthcare-associated infections. The rising prevalence of multidrug-resistant A. baumannii (MRAB) strains and biofilm formation impact the outcome of conventional treatment. Phage-related therapy is a promising strategy to tame troublesome multidrug-resistant bacteria. Here, we isolated and evaluated a highly efficient lytic phage called MRABP9 from hospital sewage. The phage was a novel species within the genus Friunavirus and exhibited lytic activity against 2 other identified MRAB strains. Genomic analysis revealed it was a safe virulent phage and a pectate lyase domain was identified within its tail spike protein. MRABP9 showed potent bactericidal and anti-biofilm activity against MRAB, significantly delaying the time point of bacterial regrowth in vitro. Phage administration could rescue the mice from acute lethal MRAB infection. Considering its features, MRABP9 has the potential as an efficient candidate for prophylactic and therapeutic use against acute infections caused by MRAB strains.

A Combination of β-Aescin and Newly Synthesized Alkylamidobetaines as Modern Components Eradicating the Biofilms of Multidrug-Resistant Clinical Strains of Candida glabrata.

The current trend in microbiological research aimed at limiting the development of biofilms of multidrug-resistant microorganisms is increasingly towards the search for possible synergistic effects between various compounds. This work presents a combination of a naturally occurring compound, β-aescin, newly synthesized alkylamidobetaines (AABs) with a general structure-CnTMDAB, and antifungal drugs. The research we conducted consists of several stages. The first stage concerns determining biological activity (antifungal) against selected multidrug-resistant strains of Candida glabrata (C. glabrata) with the highest ability to form biofilms. The second stage of this study determined the activity of β-aescin combinations with antifungal compounds and alkylamidobetaines. In the next stage of this study, the ability to eradicate a biofilm on the polystyrene surface of the combination of β-aescin with alkylamidobetaines was examined. It has been shown that the combination of β-aescin and alkylamidobetaine can firmly remove biofilms and reduce their viability. The last stage of this research was to determine the safety regarding the cytotoxicity of both β-aescin and alkylamidobetaines. Previous studies on the fibroblast cell line have shown that C9 alkylamidobetaine can be safely used as a component of anti-biofilm compounds. This research increases the level of knowledge about the practical possibilities of using anti-biofilm compounds in combined therapies against C. glabrata.

Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.

Molecular markers of M. tuberculosis virulence in lung tissue (experimental study)

To use a biological model, in order to determine significant molecular immunohistochemical markers characterizing the virulence of the "Buryat" and "Omsk" subtypes of the M. tuberculosis Beijing genotype in lung tissue. Lung samples of the C57BL/6 male mice were obtained during experimental infection with M. tuberculosis strains: the reference laboratory strain H37Rv, multidrug-resistant clinical strains 396 (highly lethal and hypervirulent «Buryat» genotype Beijing 14717-15) and 6691 (low-lethal and low-virulent "Omsk" genotype Beijing 1071-32) on days 14, 21, 60 and 120. They were studied by histological and immunohistochemical methods. The relative areas of expression of IL-6, IL-12A, iNOS, and TNF-α in the lung tissue of model animals were established. A study of strain 396 showed that both disease progression and damage to lung tissue are associated with a highly reactive immune response and increased synthesis of iNOS and strain characteristics that block the production of TNF-α. On the contrary, for strain 6691 a low reactivity of the immune response was revealed, with statistically significantly lower values of the relative area of expression of NOS and TNF-α during all observation periods (days 14-120). All animals that survived to day 120 showed a similar morphological picture with differences in cytokine levels, indicating a nonlinear relationship between proinflammatory factors and the damage substratum. The progression of the disease and damage of lung tissue were associated with a highly reactive immune response and increased synthesis of iNOS, strain properties that block the TNF-α production. Thus, iNOS and TNF-α can act as molecular markers characterizing the virulence of the "Buryat" and "Omsk" subtypes of M. tuberculosis in lung tissue.

Characterization of the broad-spectrum phage vB_CsaM_CBT2 and its inhibition of multidrug-resistant Cronobacter sakazakii in powered infant formula

Cronobacter is a group of foodborne pathogens linked to serious and deadly infections in newborns and babies through the consumption of contaminated powdered infant formula (PIF). Meanwhile, antimicrobial misuse has led to emergence of multi-drug resistant (MDR) Cronobacter strains, affecting food safety. Here, a lytic phage vB_CsaM_CBT2 (CBT2) was isolated from a wastewater treatment plant in Hefei as an effective antimicrobial alternative to antibiotics. Phage CBT2 has a broad host spectrum. It can target and lyse 17 strains including MDR clinical strain C. sakazakii GZcsf-1 from four species of C. sakazakii, C. turicensis, C. muytjensii and C. condimenti in the genus Cronobacter. Some Cronobacter phages have a narrow host range and have only been confirmed to infect a single strain of Cronobacter. In addition, excellent tolerance to temperature (25 °C–70 °C) and pH values (3-11) of phage CBT2 were determined. Based on genomic and phylogenetic analyses, the phage CBT2 was successfully identified belonging to the Straboviridae, and genes related with drug resistance and virulence were not observed. After treatment of the phage CBT2 in four brands of rehydrated PIF, the viable counts of host bacteria (C. sakazakii GZcsf-1) were reduced 3.0 log CFU/mL (＞99.9%) after 6 h at 37 °C. The findings in this study provided an alternative tool for prevention and control of Cronobacter contamination in PIF.

Antimicrobial activity of Pantanal macrophytes against multidrug resistant bacteria shows potential for improving nature-based solutions

The presence of macrophytes in constructed wetlands (CW) applied within the concept of nature-based solution (NBS) is crucial. Besides their role in the phytoremediation of pollutants, the cultivated plants, if properly selected, can also produce a range of bioactive compounds, including those with potential inhibitory effects against pathogenic bacteria. Bioactive compounds, such as flavonoids and ellagitannins, known for their remarkable antimicrobial properties, offer a sustainable solution to the global challenge of antibiotic resistance. In this study, we investigated macrophyte species adapted to the water variations in the Pantanal region (flood and dry seasons), focusing on those with both antimicrobial and ornamental potential, that could improve the ability of a CW system to treat greywater. Seven different macrophyte species were collected, cultivated, and evaluated for their antimicrobial activity (AA) against five standard bacterial strains and five multidrug-resistant clinical bacterial strains. Extracts from the leaf, stem, and root of Polygonum acuminatum showed the best AA against clinical multidrug-resistant strains of Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with minimum inhibitory concentration (MIC) values ranging from 78.13 to 312.5 µg.mL−1. Furthermore, the leaves of Ludwigia lagunae and P. acuminatum also showed considerable AA against standard strains of Enterococcus faecalis (MIC = 19.5 and 39.06 µg.mL−1, respectively) and Pseudomonas aeruginosa (MIC = 156.25 µg.mL−1 for both). High-Performance Liquid Chromatography with Ultraviolet and Mass Spectrometry analysis of these extracts identified important bioactive compounds, including flavonoids (quercetin 3-rhamnoside, hydropiperoside, quercetin 3-O-rutinoside, quercetin, myricetin 3-O-rhamnoside, kaempferol 3-O-rhamnoside, formononetin, and calycosin) and ellagitannins (3,3′4-tri-O-methylelagic acid, galloys-HHDP-glucose, and ellagic acid), along with other bioactive compounds. These results indicate that P. acuminatum and L. lagunae hold potential for application in CW owing to their antimicrobial property, local adaptability, and ornamental appeal.

Development of ionic liquid-capped carbon dots derived from Tecoma stans (L.) Juss. ex Kunth: Combatting bacterial pathogens in diabetic foot ulcer pus swabs, targeting both standard and multi-drug resistant strains

Bacterial infection is rapidly growing and is a severe danger to global welfare worldwide. Consequently, there is a growing demand for novel antibacterial medications. As an outcome, the evaluation of antibacterial assets of carbon dots (CDs) including a variety of ionic liquids (ILs) were employed to reside advance notice of the structure tunability of ILs along with abundant surface functional group of designed amine-modified CDs, have recently sparked a lot of interest. In this study for the first time, highly efficient ionic-liquid decorated CDs were synthesized to assess the impact of capped-ionic liquid on the degree of antibacterial activity. Fluorescent amine-modified CDs (CDs-NH2) were obtained from the Tecoma stans (yellow elder flower) extract by a single-step carbonization method in presence of ethylenediamine, and then capped with ionic liquids; 3-(carboxymethyl)-1-methyl-1-H-imidazol-3-ium bromide (ILIM) and 3-(carboxymethyl)-1-methyl-1-H-benzo[d]imidazole-3-ium bromide (ILBIM) by a simple carbodiimide chemistry. In-vitro antibacterial activity of CDs-NH2, ILIM@CDs and ILBIM@CDs were evaluated against common pathogens, Escherichia coli (E. coli) as Gram(-ive) bacteria and Staphylococcus aureus (S. aureus) as Gram(+ive) bacteria as well as against clinical multi-drug resistant strain obtained from pus swab of diabetic foot ulcer, by microdilution method as well as well-diffusion method. The results showed that functional ILs-capped CDs have enhanced antibacterial activity against standard (E. coli, S. aureus) and clinical resistant bacterial strains, with minimal inhibitory concentrations (MICs) values range from 0 to 64 μg/mL for standard E. coli/ S. aureus and 250 μg/mL for drug resistant clinical strains, respectively. Structure activity analysis and molecular docking studies revealed that the antibacterial activity of ILBIM@CDs/ILIM@CDs is particularly better compared to CDs-NH2 and ionic-liquids.

Antibacterial and antibiofilm efficacy of colistin & meropenem conjugated silver nanoparticles against Escherichia coli and Klebsiella pneumoniae.

The progressive increase in infections caused by multidrug-resistant (MDR) Gram-negative bacteria and the emergence of resistance to last-resort antimicrobial drugs in recent years necessitate the development of new therapeutic strategies. This study was conducted to obtain nanostructured antimicrobials by conjugating colistin (COL) and meropenem (MEM) antibiotics with biosynthesized silver nanoparticles (bio-AgNPs) via the green synthesis method using Rosa damascena extract, and to investigate the antibacterial and antibiofilm activity of these nanostructures against Escherichia coli and Klebsiella pneumoniae strains. Ultraviolet-visible spectrophotometry, high-resolution-transmission electron microscopy, atomic force microscopy, X-ray diffraction, and Fourier transform-infrared spectroscopy analyses were performed to determine the physical and chemical properties of synthesized bio-AgNPs, COL@bio-AgNPs, MEM@bio-AgNPs, and COL&MEM@bio-AgNPs. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of nanoparticles were determined on standard and MDR clinical strains. The antibiofilm efficacy and cytotoxic effect of nanoparticles were evaluated by the crystal violet dye method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye method, respectively. The characterization analyses demonstrated that the synthesized nanoparticles had crystal structure and spherical morphology (5.6-30.2 nm in size). Antibiotic conjugated nanoparticles exhibited better antimicrobial activity and lower MIC values (0.125-4 µg/mL) on the tested strains compared to free antibiotics, and MIC values were decreased up to 1024-fold (p < 0.05). Antibiotic conjugated nanoparticles were found to be more effective in biofilm eradication than free antibiotics and bio-AgNPs and had a less inhibitory effect on peripheral blood mononuclear cell viability. The findings revealed that antibiotic-conjugated nanoparticles have the potential to be used as an effective antimicrobial drug against MDR E. coli and K. pneumoniae strains.

Synergistic Antibiofilm Action of Cinnamomum verum and Brazilian Green Propolis Hydroethanolic Extracts against Multidrug-Resistant Strains of Acinetobacter baumannii and Pseudomonas aeruginosa and Their Biocompatibility on Human Keratinocytes.

The accumulated dental biofilm can be a source of oral bacteria that are aspirated into the lower respiratory tract causing ventilator-associated pneumonia in hospitalized patients. The aim of this study was to evaluate the synergistic antibiofilm action of the produced and phytochemically characterized extracts of Cinnamomum verum and Brazilian green propolis (BGP) hydroethanolic extracts against multidrug-resistant clinical strains of Acinetobacter baumannii and Pseudomonas aeruginosa, in addition to their biocompatibility on human keratinocyte cell lines (HaCaT). For this, High-performance liquid chromatography analysis of the plant extracts was performed; then the minimum inhibitory and minimum bactericidal concentrations of the extracts were determined; and antibiofilm activity was evaluated with MTT assay to prevent biofilm formation and to reduce the mature biofilms. The cytotoxicity of the extracts was verified using the MTT colorimetric test, evaluating the cellular enzymatic activity. The data were analyzed with one-way ANOVA and Tukey's tests as well as Kruskal-Wallis and Dunn's tests, considering a significance level of 5%. It was possible to identify the cinnamic aldehyde in C. verum and p-coumaric, caffeic, and caffeoylquinic acids as well as flavonoids such as kaempferol and kaempferide and Artepillin-C in BGP. The combined extracts were effective in preventing biofilm formation and reducing the mature biofilms of A. baumannii and P. aeruginosa. Moreover, both extracts were biocompatible in different concentrations. Therefore, C. verum and BGP hydroethanolic extracts have bactericidal and antibiofilm action against multidrug resistant strains of A. baumannii and P. aeruginosa. In addition, the combined extracts were capable of expressively inhibiting the formation of A. baumannii and P. aeruginosa biofilms (prophylactic effect) acting similarly to 0.12% chlorhexidine gluconate.

Phenotypic and Genotypic Characterization of Recently Isolated Multidrug-Resistant Acinetobacter baumannii Clinical and Aquatic Strains and Demonstration of Silver Nanoparticle Potency.

This study aims to demonstrate the effectiveness of silver nanoparticles (Ag NPs) on multidrug-resistant (MDR) Acinetobacter baumannii (AB) strains isolated from the clinical and aquatic environment. Three types of Ag NPs were investigated for their antimicrobial, antibiofilm, and antivirulence properties on a total number of 132 AB strains isolated in the same temporal sequence from intra-hospital infections (IHIs), wastewater (WW), and surface water (SW) samples between 2019 and 2022 from different Romanian locations and characterized at the phenotypic and genotypic levels. The comparative analysis of the antimicrobial resistance (AR) profiles according to the isolation source and the geographical location demonstrated a decrease in MDR level in AB recovered from WW samples in 2022 from north-eastern/central/southern regions (N-E/C-W/analyzed strains S): 87.5/60/32.5%. The AB strains were lecithinase, caseinase, amylase, and lipase producers, had variable biofilm formation ability, and belonged to six genotypes associated with the presence of different virulence genes (ompA, csuE, bap, and bfmS). The Ag NPs synthesized with the solvothermal method exhibited an inhibitory effect on microbial growth, the adherence capacity to the inert substratum, and on the production of soluble virulence factors. We report here the first description of a powerful antibacterial agent against MDR AB strains circulating between hospitals and anthropically polluted water in Romania.

Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-β-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems.

Carbapenem resistance mediated by metallo-β-lactamases (MBL) such as New Delhi metallo-β-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential β-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Isolation and characterization of novel lytic bacteriophages that infect multi drug resistant clinical strains of Escherichia coli.

The pathogenic strains of Escherichia coli (E. coli) are frequent cause of urinary tract infections including catheter-associated, soft tissue infections and sepsis. The growing antibiotic resistance in E. coli is a major health concern. Bacteriophages are specific for their bacterial host, thus providing a novel and effective alternatives. This study focuses on isolation of bacteriophages from urban sewage treatment plants. Initially 50 different bacteriophages have been isolated against non-resistant reference E. coli strain and fifty multidrug resistant clinical isolates of extraintestinal infections. Out of which only thirty-one lytic phages which gave clear plaques were further analysed for different physico-chemical aspects such as thermal inactivation, pH, effect oforganic solvents and detergents. Two bacteriophages, ASEC2201 and ASEC2202, were selected for their ability to withstand temperature fluctuation from -20 to 62 °C and a pH range from 4 to 10. They also showed good survival (40-94%) in the presence of organic solvents like ethanol, acetone, DMSO and chloroform or ability to form plaques even after the treatment with detergents like SDS, CTAB and sarkosyl. Both efficiently killed reference strain and 40-44% of multidrug resistant clinical isolates of E. coli. Later ASEC2201 and ASEC2202 were subjected to morphological characterisation through transmission electron microscopy, which revealed them to be tailed phages. The genomic analysis confirmed them to be Escherichia phages which belonged to family Drexlerviridae of Caudovirales.

Dimeric Cholic Acid Peptide Conjugates Act as Effective Antibiotic Adjuvants against Multidrug Resistance (MDR) Gram‐Negative Bacterial Infections

AbstractMacrolides, glycopeptides, lipopeptides, and oxazolidinones class of antibiotics are used only for Gram‐positive bacteria, and are ineffective against Gram‐negative bacteria due to inability of these antibiotics to cross the Gram‐negative bacterial membranes. Here, dimeric cholic acid peptide conjugates as effective adjuvants that allow penetration of erythromycin across Gram‐negative bacteria are presented, making it effective antimicrobial. Here, it is shown that synthesized dimers2and3,having tetramethylene and pentamethylene spacers respectively between cholic acid‐triglycine conjugates, in combination with erythromycin cause synergistic antimicrobial effect against Gram‐negative bacteria. Biochemical studies reveal that dimer3causes outer and inner membrane permeabilization by interacting with lipopolysaccharide and displacing Mg2+ions, thereby allowing the erythromycin to cross bacterial membranes. Dimer3in combination with erythromycin can clear the preformed biofilms ofE. coliandP. aeruginosaeffectively. It is further demonstrated that combination of dimer3and erythromycin can clear wound infections ofE. coliandP. aeruginosa. Additionally, activities against multidrug resistant clinical strains ofE. coliandP. aeruginosawitness that combination of dimer3and erythromycin is an ideal combination to mitigate drug‐resistant infections.

Properties of Pyocin S9 from Pseudomonas aeruginosa UCM В-333

The deposited by us highly active bacteriocin producer Pseudomonas aeruginosa UСM B-333 synthesizes pyocins, which intensively inhibit phytopathogenic strains of Pseudomonas syringae — plant pathogens. This strain produces pyocins S1 and S5, as well as microcin-II-like bacteriocins. The aim of this work was to check the presence of other pyocins in P. aeruginosa UCM B-333. Methods. The concentrated bacterial lysate of P. aeruginosa UCM B-333 was separated by ion-exchange chromatography on DEAE cellulose. The fraction with studied bacteriocin was further purified by gel filtration on Sephadex G-75. To determine the belonging of investigated pyocin to a certain subtype, its molecular weight, antimicrobial activity, kinetics of the effect on sensitive microorganisms, and serological homology with carotovoricins of Pectobacterium carotovorum were studied as well as the ability to interact with siderophore receptors and nuclease activity were tested. Results. The isolated pyocin is a protein with a molecular weight of the active part of pyocin of 43.4 kDa and an immune protein — of 9 kDa. Th is substance is characterized by nonspecific DNase activity and affects sensitive cells by the single-hit response kinetics of influence through binding to receptors that are not concerned with iron transport. The revealed pyocin is not related to carotovoricins, its activity spectrum is close to other pyocins’ activities, and it affects clinical multidrug-resistant strains of Pseudomonas aeruginosa. The induction mechanism of this bacteriocin may be different from that described for other pyocins and not concerned with the RecA system. The determination of factors that stimulate the expression of pyocin S9 requires further study. Conclusions. According to the established properties, the studied substance is the closest to the foreseen pyocin S9.

Effects of Calendula officinalis and Capsicum annum glycolic extracts on planktonic cells and biofilms of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa

Empirical knowledge of natural plant extracts is increasingly proving to be a promising field. The effect of Calendula officinalis L. (CO) and Capsicum annum (CA) glycolic extracts (GlExt) have potential that should be further developed in microbial tests. The effect of CO-GlExt and CA-GlExt was evaluated on eight multidrug-resistant clinical strains of Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as collection strains for each bacterial. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract were determined in comparison with 0.12% chlorhexidine. The tests were performed on single species biofilms, at 5 min and 24 h, using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. The MIC and MBC of the extract ranged from 1.56 to 50 mg mL−1 for all strains evaluated. Analysis of the MTT assay revealed a strong antimicrobial potential of CA-GlExt, comparable to chlorhexidine. The findings suggest that CA-GlExt is effective against multidrug-resistant strains of K. pneumoniae and P. aeruginosa in planktonic state and biofilms.

Association of antibiotics and heavy metal arsenic to horizontal gene transfer from multidrug-resistant clinical strains to antibiotic-sensitive environmental strains.

The emergence of multidrug-resistant bacteria is currently posing a significant threat to global public health. By testing for resistance to different antibiotic classes, we discovered that the majority of clinical bacteria are multidrug-resistant. These clinical multidrug-resistant species have antibiotic resistance genes on their plasmids that can be horizontally transferred to various antibiotic susceptible environmental bacterial species, resulting in antibiotic-resistant transconjugates. Furthermore, we discovered that the presence of an optimal concentration of antibiotics or heavy metal (arsenic) facilitates horizontal gene transfer through the formation of transconjugants. Notably, the addition of a conjugation inhibitor (2-hexadecynoic acid, a synthetic fatty acid) completely blocked the formation of antibiotic or arsenic-induced transconjugants. We discovered a high level of arsenic in water from the Shukratal region, Uttarakhand, India, which corresponded to a high serum level of arsenic in clinically infected individuals from the Shukratal region compared to other locations in Uttarakhand. Importantly, bacterial strains isolated from infected people who drink water from the Shukratal region with high arsenic levels were found to be more antibiotic-resistant than strains isolated from other sites. We discovered that bacterial strains isolated from individuals with high serum arsenic levels are significantly more resistant to antibiotics than individuals with low serum arsenic levels within the Shurkratal. This research sheds light on imminent threats to global health in which improper clinical, industrial, and other waste disposal, increased antibiotic concentrations in the environment, and increased human interference can easily transform commensal and pathogenic bacteria found in environmental niches into life-threatening multidrug-resistant superbugs.

Genomic Characteristics and Phylogenetic Analyses of a Multiple Drug-Resistant Klebsiella pneumoniae Harboring Plasmid-Mediated MCR-1 Isolated from Tai’an City, China

Klebsiella pneumoniae is a clinically common opportunistic pathogen that causes pneumonia and upper respiratory tract infection in humans as well as community-and hospital-acquired infections, posing significant threats to public health. Moreover, the insertion of a plasmid carrying the mobile colistin resistance (MCR) genes brings obstacles to the clinical treatment of K. pneumoniae infection. In this study, a strain of colistin-resistant K. pneumoniae (CRKP) was isolated from sputum samples of a patient who was admitted to a tertiary hospital in Tai'an city, China, and tested for drug sensitivity. The results showed that KPTA-2108 was multidrug-resistant (MDR), being resistant to 21 of 26 selected antibiotics, such as cefazolin, amikacin, tigecycline and colistin but sensitive to carbapenems via antibiotic resistance assays. The chromosome and plasmid sequences of the isolated strain KPTA-2108 were obtained using whole-genome sequencing technology and then were analyzed deeply using bioinformatics methods. The whole-genome sequencing analysis showed that the length of KPTA-2108 was 5,306,347 bp and carried four plasmids, pMJ4-1, pMJ4-2, pMJ4-3, and pMJ4-4-MCR. The plasmid pMJ4-4-MCR contained 30,124 bp and was found to be an IncX4 type. It was the smallest plasmid in the KPTA-2108 strain and carried only one resistance gene MCR-1. Successful conjugation tests demonstrated that pMJ4-4-MCR carrying MCR-1 could be horizontally transmitted through conjugation between bacteria. In conclusion, the acquisition and genome-wide characterization of a clinical MDR strain of CRKP may provide a scientific basis for the treatment of K. pneumoniae infection and epidemiological data for the surveillance of CRKP.

1691. Bacteriophage and Protein Synthesis Pathway Inhibitor Combinations: Antibiotic mechanism of action drives antagonistic interactions

Abstract Background Bacteriophage (phage) to augment antibiotic efficacy is a possible therapeutic option in the era of antimicrobial resistance. Studies to date have assessed phage-antibiotic synergy (PAS), however, its efficacy may be dependent upon antibiotic mechanism of action. Here, we report our in-vitro evaluation of phage-antibiotic antagonism (PAA) among phage and protein synthesis inhibitor combinations in multidrug-resistant clinical strains of P. aeruginosa, S. aureus, and E. faecium. Methods The following bacteria (phage) regimens were evaluated: 10266 (EM) and R9010 (14207) (P. aeruginosa) against gentamicin (GEN), azithromycin (AZM), and ciprofloxacin (CIP); N315 and 494 (Intesti) (S. aureus), and R497 and HOU503 (NV-497) (E. faecium) against linezolid (LNZ), minocycline (MIN), and daptomycin (DAP). Modified checkerboard (CB) MIC assays were used for preliminary screening followed by 24h time kill analyses (TKA). For CB, synergy, additive activity, and antagonism were defined as an FIC index of ≤0.5, 1–4, and &amp;gt;4, respectively. In 24h TKA, synergy and additivity were defined as a ≥2 and ≥1 log10 CFU/mL reduction from baseline, while antagonism was defined as phage-antibiotic combinations with CFU/mL higher than the most effective single treatment at 24h. Data were compared by one-way ANOVA and Tukey (HSD) test (P&amp;lt; 0.05). Results In CB analyses and 24h TKA of S. aureus and E. faecium isolates, phage-LZD and phage-MIN combinations were antagonistic (FIC &amp;gt;4) while phage-DAP was synergistic (FIC 0.5) (ANOVA range of mean differences 0.52 to 2.59 log10 CFU/mL; P&amp;lt; 0.001). For P. aeruginosa, phage-AZM and phage-GEN were antagonistic (FIC &amp;gt;4) and additive (FIC=1), respectively (ANOVA range of mean differences 1.04 to 1.95 log10 CFU/mL; P&amp;lt; 0.001). Conclusion Our results suggest that antibiotics that act on the protein synthesis pathway may lead to PAA, however, PAA interactions may be highly dependent upon antibiotic mechanism of bacterial inhibition (i.e., location of ribosomal protein synthesis inhibition, bactericidal vs. bacteriostatic) protein synthesis inhibit. Studies assessing PAA in a wider array of phage-antibiotic combinations are warranted. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support.

Synthesis and Characterization of Calcium Alginate-Based Microspheres Entrapped with TiO2 Nanoparticles and Cinnamon Essential Oil Targeting Clinical Staphylococcus aureus.

It is important to create new generations of materials that can destroy multidrug-resistant bacterial strains, which are a serious public health concern. This study focused on the biosynthesis of an essential oil entrapped in titanium dioxide (TiO2) calcium alginate-based microspheres. In this research, calcium alginate-based microspheres with entrapped TiO2 nanoparticles and cinnamon essential oil (CI-TiO2-MSs) were synthesized, using an aqueous extract of Nigella sativa seeds for TiO2 nanoparticle preparation, and the ionotropic gelation method for microsphere preparation. The microspheres obtained were spherical, uniformly sized, microporous, and rough surfaced, and they were fully loaded with cinnamon essential oil and TiO2 nanoparticles. The synthesized microspheres were analyzed for antibacterial activity against the clinical multidrug-resistant strain of Staphylococcus aureus. Disc diffusion and flow cytometry analysis revealed strong antibacterial activity by CI-TiO2-MSs. The synthesized CI-TiO2-MSs were characterized by the SEM/EDX, X-ray diffraction, and FTIR techniques. Results showed that the TiO2 nanoparticles were spherical and 99 to 150 nm in size, whereas the CI-TiO2-MSs were spherical and rough surfaced. Apoptosis analysis and SEM micrography revealed that the CI-TiO2-MSs had strong bactericidal activity against S. aureus. The in vitro antibacterial experiments proved that the encapsulated CI-TiO2-MSs had strong potential for use as a prolonged controlled release system against multidrug-resistant clinical S. aureus.