Multidrug-resistant Pathogens Research Articles

Isolation and identification of a novel phage targeting clinical multidrug-resistant Corynebacterium striatum isolates.

Over the past decade, Corynebacterium striatum (C. striatum), an emerging multidrug-resistant (MDR) pathogen, has significantly challenged healthcare settings, especially those involving individuals with weakened immune systems. The rise of these superbugs necessitates innovative solutions. This study aimed to isolate and characterize bacteriophages targeting MDR-C. striatum. Utilizing 54 MDR-C. striatum isolates from a local hospital as target strains, samples were collected from restroom puddles for phage screening. Dot Plaque and Double-layer plate Assays were employed for screening. A novel temperate bacteriophage, named CSP1, was identified through a series of procedures, including purification, genome extraction, sequencing, and one-step growth curves. CSP1 possesses a 39,752 base pair circular double-stranded DNA genome with HK97-like structural proteins and potential for site-specific recombination. It represents a new species within the unclassified Caudoviricetes class, as supported by transmission electron microscopy, genomic evolutionary analysis, and collinearity studies. Notably, CSP1 infected and lysed 21 clinical MDR-C. striatum isolates, demonstrating a wide host range. The phage remained stable in conditions ranging from -40 to 55°C, pH 4 to 12, and in 0.9% NaCl buffer, showing no cytotoxicity. The identification of CSP1 as the first phage targeting clinical C. striatum strains opens new possibilities in bacteriophage therapy research, and the development of diagnostic and therapeutic tools against pathogenic bacteria.

Antibacterial Properties of Bacterial Endophytes Isolated from the Medicinal Plant Origanum heracleoticum L.

Bacterial endophytic communities associated with medicinal plants synthesize a plethora of bioactive compounds with biological activities. Their easy isolation and growth procedures make bacterial endophytes an untapped source of novel drugs, which might help to face the problem of antimicrobial resistance. This study investigates the antagonistic potential of endophytic bacteria isolated from different compartments of the medicinal plant O. heracleoticum against human opportunistic pathogens. A panel of endophytes was employed in cross-streaking tests against multidrug-resistant human pathogens, followed by high-resolution chemical profiling using headspace-gas chromatography/mass spectrometry. Endophytic bacteria exhibited the ability to antagonize the growth of opportunistic pathogens belonging to the Burkholderia cepacia complex (Bcc). The different inhibition patterns observed were related to their taxonomic attribution at the genus level; most active strains belong to the Gram-positive genera Bacillus, Arthrobacter, and Pseudarthrobacter. Bcc strains of clinical origin were more sensitive than environmental strains. Cross-streaking tests against other 36 human multidrug-resistant pathogens revealed the highest antimicrobial activity towards the Coagulase-negative staphylococci and Klebsiella pneumoniae strains. Interestingly, strains of human origin were the most inhibited, in both groups. Concerning the production of volatile organic compounds (VOCs), the strain Arthrobacter sp. OHL24 was the best producer of such compounds, while two Priestia strains were good ketones producers and so could be considered for further biotechnological applications. Overall, this study highlights the diverse antagonistic activities of O. heracleoticum-associated endophytes against both Bcc and multidrug-resistant (MDR) human pathogens. These findings hold important implications for investigating bacterial endophytes of medicinal plants as new sources of antimicrobial compounds.

Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia.

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase, while defects in divisome accessory proteins increased susceptibility to multiple β-lactams. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and β-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators.IMPORTANCEThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

Exploring actinobacteria isolated from coral originated from Tulamben Bali in inhibiting multidrug resistance bacteria

Increasing bacterial resistance has reduced the effectiveness of most clinical antibiotics. The limited option of antibiotics to treat Multi-Drug Resistance pathogen infections indicates that there is an urgency to explore new antibiotic candidates. Coral is recognized to produce bioactive compounds that have the potential to develop new medicinal materials with antibacterial properties. The use of marine invertebrates for compound production will reduce populations in nature, therefore associated microbes are preferred due to their environmental friendliness. Actinobacteria is one of the coral-associated microbes capable of producing active compounds of secondary metabolites as antibacterials. This study aimed to identify coral and obtain isolates of Actinobacteria associated with coral potential as an antibacterial against MDR bacteria. This research uses an explorative method. The method used is coral identification based on the morphology and shape of the sclerite. Screening of antibacterial activity was carried out using the agar plug method, microscopic observation using Gram staining and molecular identification of potential isolates by PCR. The results showed that the coral genera obtained were Siphonogorgia sp., Menella sp., Echinomuricea sp., Dendronephthya sp., and Epizoanthus sp. The results of antibacterial activity indicated that 19 isolates inhibited the growth of MDR pathogen bacteria. However, only the potential isolates were identified. The microscopic observations showed isolate III.ISP2.10-2.3 and III.ISP2.10-2.6 have Gram-positive coccus and bacillus-shaped, respectively. Potential isolates III.ISP2.10-2.3 (Micrococcus yunnanensis) were found to have antibacterial activity against Acinetobacter baumanii, Pseudomonas aeruginosa, and Bacillus subtilis, while isolate III.ISP2.10-2.6 (Brevibacterium pigmentatum) had the greatest activity against B. subtilis.

Short-term effects of antimicrobial stewardship programs on antibiotics usage, clinical outcomes, and multidrug resistant organisms in the post COVID-19 era

BackgroundAntimicrobial resistance is associated with increasing mortality rates and the emergence of multidrug-resistant (MDR) organisms. There is scarcity of data on the short-term impact of Antimicrobial Stewardship Programs (ASP) on antibiotic usage, clinical outcome and MDR organisms’ pattern following the COVID-19 pandemic. This study evaluated the short-term effects of ASP on antibiotic usage, clinical outcomes and MDR organisms’ pattern in the post COVID-19 era. MethodsConducted at a tertiary academic health center, this observational study involved adult patients (≥18 years) in the general medical unit, treated with oral or intravenous antibiotics from May 1, 2021, to April 30, 2022. The applied ASP strategy was a prospective audit and feedback where a weekly meeting was held to discuss the antimicrobial therapy of admitted patient, after which recommendations were made regarding antimicrobial use. ResultsThe study included 301 patients with 166 (55.1%) pre-ASP and 135 (44.9%) post-ASP. The median (IQR) age was 69 (55−77) years with 56.1% were female. Antibiotic usage dropped by 25.2% post-ASP. The length of hospital stay (LOS) was longer post-ASP (7 days vs. 7.9 days, p = 0.001), with MDR infections being a significant predictor (OR: 0.486, p < 0.001). There were no significant differences in 28-day readmission, recurrence of infections and all-cause mortality. Post-ASP, MDR pathogens increased (17.0% vs. 6.6%, p = 0.013), however, after separating post-ASP into two three-months periods, MDROs numbers decreased slightly (13 vs. 10). ConclusionShort-term ASP implementation post COVID-19 reduced antibiotic usage while other clinical outcomes remained unchanged. Nonetheless an increase in MDR pathogens and LOS was observed. Further research is required to assess ASP’s long-term impact on MDR infections rates and specific patient group outcomes.

Kaempferol promotes wound-healing in diabetic rats through antibacterial and antioxidant effects, devoid of proliferative action

The investigation of novel phytochemicals for the prevention and treatment of infections caused by multidrug-resistant pathogens is gaining attention. The current study evaluated the in-vitro antimicrobial activity of kaempferol against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Its in-vivo efficacy in inhibiting these pathogens was determined using an excision wound model in nicotinamide-streptozocin- -induced diabetic rats. Kaempferol displayed an inhibitory effect against the tested bacteria both in vitro and in vivo. It also healed excision wounds at a 1% (w/w) concentration. An increase in antioxidant enzymes in wounded tissue was observed on kaempferol treatment. A reduction in the MRSA and P. aeruginosa counts in wounded tissue together with a reduced epithelization period was observed when compared to the infected control. A thicker epithelium, new capillaries, and a decrease in inflammatory cells were detected by hematoxylin and eosin staining. Furthermore, an increase in collagen fibers and their deposition was observed by Masson’s trichrome staining. Kaempferol at 40 µM did not display any toxicity for human keratinocytes grown in media containing high glucose and it did not affect the expression of the pro-healing cytokines genes vascular endothelial growth factor (VEGF) and transforming growth factor-b-1 (TGFβ1). Kaempferol displayed antibacterial and antioxidant actions but did not increase the expression of proliferative genes.

Genotypic and phenotypic characterization of determinants that mediate antimicrobial resistance in Escherichia coli strains of clinical origin in South-Western Nigeria.

Multidrug resistant bacterial pathogens employ different mechanisms in evading the action of antibiotics. Multidrug resistance is wide spread among strains of Escherichia coli implicated in several infections including urinary tract infections, gastrointestinal infections, meningitis and bacteraemia. This study investigates the antibiotic resistance profile, efflux pump activity and biofilm formation ability of E. coli strains isolated from clinical samples. A total of 32E. coli strains isolated from clinical samples were characterized and subjected to antibiotic susceptibility testing using standard methods. Isolates were screened phenotypically for biofilm formation and efflux pump activity. While molecular detection of genes encoding curli fimbriae and efflux pump activity was done by PCR. All 32 (100%) E. coli isolates were resistant to ceftazidime, cefuroxime, cefixime, amoxicillin-clavulanate, ofloxacin and ciprofloxacin. While 30 (93.8%) were resistant to gentamicin, 27 (84.4%) were resistant to cefepime and the least resistance of 15.6% was to imipenem. Efflux pump encoding gene tolC was detected in 13(40.6%) of the isolates, while 1(3.1%) harboured acrA gene. acrB gene was not detected in any of the isolates. Seven (21.9%) of the isolates were strong biofilm formers, while 5 (15.6%) and 20 (62.5%) were moderate and weak biofilm formers respectively. csgA gene was detected in all E. coli isolates. High antibiotic resistance of E. coli strains observed in this study is of public health significance. . It is therefore important to scale up efforts in regular monitoring of antibiotic resistance in both community and hospital settings.

BACTERIOLOGICAL STUDY OF VENTILATOR-ASSOCIATED PNEUMONIA AND ANTIBIOTIC SUSCEPTIBILITY OF ISOLATES

Objective: The present study determined the prevalence of various aerobic bacteria causing ventilator-associated pneumonia in adult patients. Initially the bacteria causing ventilator-associated pneumonia was isolated from ET samples and studied the antimicrobial susceptibility pattern of bacterial isolates.&#x0D; Methods: Total 250 endotracheal aspiration (ET) samples were collected from patients admitted in Medical, Respiratory and Surgical ICUs for 1 y period. ET aspirates were collected under aseptic precautions and processed as per standard operating procedure for the identification of microorganisms. The antibiotic susceptibility test was performed by using Kirby-Bauer disk diffusion method as per CLSI guidelines.&#x0D; Results: Out of the 250 samples processed, culture-positive were 34.8% (n=87) and culture-negative were 65.2% (n=163). Out of 87 culture-positive samples, polymicrobial growth was observed in 9.19% (n=8) and monomicrobial growth was observed in 90.8% (n=79). Gram negative bacilli 95.7% (n=91), and gram-positive cocci isolates are 4.2% (n=4). Among Gram-negative organisms isolated, A. baumannii is the most common isolate 33 (34.7%), followed by P. aeruginosa 28 (29.5%) and K. pneumoniae 20 (21.0%) E. coli 8 (8.4%) and E. cloacae 2 (2.1%). Out of 4 Gram-positive organisms isolated, 3 (3.1%) were MSSA, and 1(1.1%) was MRSA.&#x0D; Conclusion: VAP is increasingly associated with multidrug-resistant (MDR) pathogens due to the production of ESBL, Amp C b-lactamase, Metallo-b-lactamase. It is important to carry out aggressive surveillance to determine the prevalence of MDR organisms and to generate a local antibiogram periodically. Early and appropriate antibiotics in right doses followed by de-escalation based on microbiological culture results are essential to curtail the VAP rate. VAP bundle care shall be implemented correctly.

Enhanced antibacterial activity of bovine milk exosome-based drug formulation against bacterial pathogens

Milk is not only a source of nutrients, but also contains exosomes (Exo) that can serve as a vehicle for drug delivery. Here, we obtained bovine milk Exo using three efficient methods, demonstrating high quality for commercial production. The optimized Exo displayed a size of 105.2 nm and an entrapment efficiency of 88.4 %. The Exo has been functionalized with a combination therapy comprising isobavachalcone (IS) and polymyxin B (PB), referred to as IP-Exo. The antibacterial efficacy of IP-Exo was significantly enhanced, enabling the elimination of 99 % of multidrug-resistant (MDR) bacterial pathogens in 4 h. Furthermore, scanning electron microscopy images demonstrated that the drug combination led to the complete dismantling of the bacterial structure. IP-Exo showed nearly 100 % microbial inhibition in fresh orange juice and accelerated wound healing in mouse models. Collectively, IP-Exo provides excellent potential for application within the food industry and animal husbandry as a defense against bacterial pathogens.

Identification of Extended Spectrum Beta Lactamases Producing Pseudomonas aeruginosa from Subclinical Mastitis Milk Samples in an Organized Dairy Farm of SVVU, Andhra Pradesh

The present study was conducted during August 2021 at Buffalo Research Station, Venkatarammanagudem of West Godavari District, Andhra Pradesh, India and was aimed to detect the presence of extended-spectrum beta-lactamases (ESBL) producing P. aeruginosa from sub-clinical mastitis that are apparently healthy Murrah buffaloes. Inflammation of the udder by microbial infection is one of the leading economic disease in the dairy sector. Most of the pathogens especially Pseudomonas aeruginosa are refractory to the antibiotic theraphy. Nowadays, the antimicrobial resistance owed by the bacteria is at an alarming rate. Pseudomonas aeruginosa, a multi drug resistant pathogen carry the extended spectrum beta lactamase genes in its plasmids and thus become resistant to antibiotic therapy. A total of 276 milk samples were collected from 69 milch animals in the month of August, 2021 comprising from all the four quarters of each buffalo. Nine (n=9) (3.26%) P. aeruginosa isolates were identified from the sub-clinical mastitis milk samples from the total 276 samples collected in the study. The isolates on morphological analysis were typical to P. aeruginosa. Seven isolates were positive for phenotypical beta-lactamases production. A total of 1 and 2 isolates were found reactive of the blaSHV and blaOXA genes, and two isolates harboured both blaSHV and blaOXA genes, respectively on PCR assay. No blaTEM gene was found in the isolates. Antibiogram of the ESBL producing P. aeruginosa isolates (n=7) possessed 100% resistance against most of the commonly used antimicrobials like ampicillin, amoxycillin, clindamycin, co-trimoxazole oxytetracycline, streptomycin and ceftriaxone (85%). The P. aeruginosa isolates were sensitive to amikacin (100%), ciprofloxacin (87.4%), gentamicin (84%), norfloxacin (84.8%) and enrofloxacin (82%).

Detection of bla-AIM Metallo Beta Lactamase Gene among Stenotrophomonas Maltophilia and Carbapenem Resistant Pseudomonas Aeruginosa Isolated from Various Infections in AL- Najaf Province

Stenotrophomonas maltophilia is a "rapidly evolving pathogen of concern" that is increasingly being identified. The World Health Organization also recognizes it as one of the hospitals' most significant multi-drugresistant pathogens. Also, Pseudomonas aeruginosa is an opportunistic human pathogen that causes most healthcare-associated infections, and it is considered a paradigm of antibiotic resistance development. In many hospitals across the globe, carbapenem-resistant Pseudomonas aeruginosa has emerged as a significant source of infection. The present study aimed to study the isolation and diagnosis of S. maltophilia and P. aeruginosa from different clinical samples, evaluate the occurrence of carbapenem resistance of P. aeruginosa isolated from clinical samples, and investigate the dissemination of the bla-AIM genes between these isolates. Eight hundred fifty specimens were collected from various clinical samples between 2022 and 2023. The specimens included 220 swabs (burn), 200 (urine), 140 (stool), and 130(wound). 90 (ear),50 (throat), 10 (Cerebrospinal fluid), and 10 (blood). Represented by 680 specimens contained bacterial growth, and 170 specimens had no bacterial growth. Out of the 680 bacterial growth isolates, 410 revealed growths of Gram-negative bacteria, and 270 were Gram-positive bacteria. On MacConkey ag, ar 180/410 bacteria were lactose ferment; other isolates, es 230/410 of the isolates were lactose non-fermented bacteria. In a cross-sectional manner, Stenotrophomonas maltophilia and Pseudomonas aeruginosa isolates during this period were isolated and identified depending on the primary methods of diagnosis, then the use of the VITEK-2 compact system. The results showed 42 isolates of S. maltophilia and 80 isolates of P. aeruginosa from total Gram-negative bacteria. The results show that only five isolates contained the AIM gene, with a percentage of (10.4 %) of the 48 Carbapenem Resistant Pseudomonas aeruginosa isolates, five isolates from 42 S. maltophilia contain the AIM gene with a percentage (11.9%), based on the Polymerase chain reactions assay. Keywords: Stenotrophomonas maltophilia, Carbapenem Resistance, Pseudomonas aerginosa.

Detection of bla-AIM Metallo Beta Lactamase Gene among Stenotrophomonas Maltophilia and Carbapenem Resistant Pseudomonas Aeruginosa Isolated from Various Infections in AL- Najaf Province

Stenotrophomonas maltophilia is a "rapidly evolving pathogen of concern" that is increasingly being identified. The World Health Organization also recognizes it as one of the hospitals' most significant multi-drugresistant pathogens. Also, Pseudomonas aeruginosa is an opportunistic human pathogen that causes most healthcare-associated infections, and it is considered a paradigm of antibiotic resistance development. In many hospitals across the globe, carbapenem-resistant Pseudomonas aeruginosa has emerged as a significant source of infection. The present study aimed to study the isolation and diagnosis of S. maltophilia and P. aeruginosa from different clinical samples, Evaluate the occurrence of carbapenem resistance of P. aeruginosa isolated from clinical samples and investigate the dissemination of the bla-AIM genes between these isolates. A total of 850 specimens were collected from various clinical samples between 2022 and 2023. The specimens included 220 swabs (burn), 200 (urine), 140 (stool), and 130(wound). 90 (ear),50 (throat), 10 (Cerebrospinal fluid), and 10 (blood). Represented by 680 specimens contained bacterial growth, and 170 specimens had no bacterial growth. Out of the 680 bacterial growth isolates, 410 revealed growths of Gram-negative bacteria, and 270 were Gram-positive bacteria. On MacConkey ag, ar 180/410 bacteria were lactose ferment; other isolates, es 230/410 of the isolates were lactose non-fermented bacteria. In a cross-sectional manner, Stenotrophomonas maltophilia and Pseudomonas aeruginosa isolates during this period were isolated and identified depending on the primary methods of diagnosis, then the use of the VITEK-2 compact system. The results showed 42 isolates of S. maltophilia and 80 isolates of P. aeruginosa from total Gram-negative bacteria. The results show that only five isolates contained the AIM gene, with a percentage of (10.4 %) of the 48 Carbapenem Resistant Pseudomonas aeruginosa isolates, five isolates from 42 S. maltophilia contain the AIM gene with a percentage (11.9%), based on the Polymerase chain reactions assay. Keywords: Stenotrophomonas maltophilia, Carbapenem Resistance, Pseudomonas aerginosa.

Deciphering the molecular and functional basis of TMexCD1: the plasmid-encoded efflux pump of resistance-nodulation-division superfamily.

Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623TMexD1) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675TMexD1) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.

Recent Advances in the Development of Polymyxin Antibiotics: 2010-2023.

The polymyxins are nonribosomal lipopeptides produced by Paenibacillus polymyxa and are potent antibiotics with activity specifically directed against Gram-negative bacteria. While the clinical use of polymyxins has historically been limited due to their toxicity, their use is on the rise given the lack of alternative treatment options for infections due to multidrug resistant Gram-negative pathogens. The Gram-negative specificity of the polymyxins is due to their ability to target lipid A, the membrane embedded LPS anchor that decorates the cell surface of Gram-negative bacteria. Notably, the mechanisms responsible for polymyxin toxicity, and in particular their nephrotoxicity, are only partially understood with most insights coming from studies carried out in the past decade. In parallel, many synthetic and semisynthetic polymyxin analogues have been developed in recent years in an attempt to mitigate the nephrotoxicity of the natural products. Despite these efforts, to date, no polymyxin analogues have gained clinical approval. This may soon change, however, as at the moment there are three novel polymyxin analogues in clinical trials. In this context, this review provides an update of the most recent insights with regard to the structure-activity relationships and nephrotoxicity of new polymyxin variants reported since 2010. We also discuss advances in the synthetic methods used to generate new polymyxin analogues, both via total synthesis and semisynthesis.

Tobramycin Reduces Pulmonary Toxicity of Polymyxin B via Inhibiting the Megalin-Mediated Drug Uptake in the Human Lung Epithelial Cells.

Accumulation of polymyxins in the lung epithelial cells can lead to increased mitochondrial oxidative stress and pulmonary toxicity. Aminoglycosides and polymyxins are used, via intravenous and pulmonary delivery, against multidrug-resistant Gram-negative pathogens. Our recent in vitro and animal studies demonstrated that the co-administration of polymyxins with aminoglycosides decreases polymyxin-induced pulmonary toxicity. The aim of this study was to investigate the in vitro transport and uptake of polymyxin B and tobramycin in human lung epithelial Calu-3 cells and the mechanism of reduced pulmonary toxicity resulting from this combination. Transport, intracellular localization, and accumulation of polymyxin B and tobramycin were investigated using doses of 30 mg/L polymyxin B, 70 mg/L tobramycin, and the combination of both. Adding tobramycin significantly (p < 0.05) decreased the polymyxin B-induced cytotoxicity in Calu-3 cells. The combination treatment significantly reduced the transport and uptake of polymyxin B and tobramycin in Calu-3 cells, compared to each drug alone, which supported the reduced pulmonary toxicity. We hypothesized that cellular uptake of polymyxin B and tobramycin shared a common transporter, megalin. We further investigated the megalin expression of Calu-3 cells using confocal microscopy and evaluated megalin activity using a megalin substrate, FITC-BSA, and a megalin inhibitor, sodium maleate. Both polymyxin B and tobramycin significantly inhibited FITC-BSA uptake by Calu-3 cells in a concentration-dependent manner. Sodium maleate substantially inhibited polymyxin B and tobramycin transport and cellular accumulation in the Calu-3 cell monolayer. Our study demonstrated that the significantly reduced uptake of polymyxin B and tobramycin in Calu-3 cells is attributed to the mechanism of action that determines that polymyxin B and tobramycin share a common transporter, megalin.

Efficient biogenesis of calcium oxide nanoparticles using the extract of Eleusine coracana seeds and their application against multidrug-resistant ocular bacterial pathogens

Visual impairment due to corneal keratitis-causing bacteria is becoming a matter of health concern. The bacterial colonization and their resistance to multiple drugs need imperative attention. To overcome the issue of alternative remedial therapeutic agents, particularly for topical application, a study was carried out to synthesize calcium oxide nanoparticles (CaO NPs) using the biomaterial Eleusine coracana seed aqueous extract. The biosynthesized calcium oxide nanoparticles (CaO NPs) are non-toxic or less-toxic chemical precursors. Moreover, CaO NPs are eco-friendly and are used for several industrial, biomedical, and environmental applications. Biosynthesized CaO NPs were characterized using ultraviolet–visible spectroscopy, Fourier transform-infrared spectroscopy, scanning electron microscopy, and dynamic light scattering study. The synthesized CaO NPs exhibit with good anti-inflammatory activities with dose dependant (50–250 μg/mL). Moreover, Eleusine coracana-mediated CaO NPs significantly inhibited the multiple drug-resistant Gram-positive Staphylococci epidermidis and Enterococcus faecalis and Gram-negative Escherichia coli and Klebsiella pneumoniae that were isolated from the corneal ulcer. This study provides a potential therapeutic option for multiple drug-resistant corneal pathogens that cause vision impairment.

Effects of Phomopsidione on the Viability, Virulence, and Metabolites Profile of Methicillin-Resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) infections have become one of the most threatening multidrug-resistant pathogens. Thus, an ongoing search for anti-MRSA compounds remains an urgent need to effectively treating MRSA infections. Phomopsidione, a novel antibiotic isolated from Diaporthe fraxini, has previously demonstrated potent anti-candidal activity. The present study aimed to investigate the effects of phomopsidione on the viability, virulence, and metabolites profile of MRSA. MRSA was sensitive to phomopsidione in a concentration-dependent manner. Phomopsidione exhibited minimum inhibitory concentration and minimum bactericidal concentration of 62.5 and 500.00µg/mL against MRSA on broth microdilution assay. The compound showed significant reduction in virulence factors production including extracellular polymeric substances quantification, catalase, and lipase. An untargeted metabolomics analysis using liquid chromatography-high resolution mass spectrometry revealed a significant difference in the metabolites profile of MRSA with 13 putatively identified discriminant metabolites. The present study suggested the potential of phomopsidione as a promising anti-MRSA agent.

Rapid diagnostic of multidrug-resistant sepsis pathogens directly from blood culture bottles using MALDI-TOF and the EUCAST RAST

In this study, rapid diagnostic of multidrug-resistant (MDR) sepsis pathogens, directly from positive blood culture (BC) bottles, was evaluated by combining MALDI-TOF and the EUCAST Rapid Antimicrobial Susceptibility Testing (RAST). Carbapenemase production in Escherichia coli and Klebsiella pneumoniae isolates was also evaluated by RAST. From 171 positive BC bottles analyzed, 79 (46 %) MDR species, including E. coli (4/34, 12 %), K. pneumoniae (33/48, 69 %), Pseudomonas aeruginosa (12/12, 100 %), Acinetobacter baumannii (15/15, 100 %), and Staphylococcus aureus (14/37, 38 %) displaying resistance to beta-lactams, fluoroquinolones, aminoglycosides, and/or trimethoprim/sulphamethoxazole, were identified. In this regard, turnaround time of direct MALDI-TOF identification and RAST was < 7 h, which was significantly (p< 0.05) lower than our routine method. Carbapenemase detection by RAST displayed 100% sensitivity and 88.7 % specificity at 8 h. This protocol could offer advantages for the treatment and clinical outcomes of septic patients, improving the rapid diagnostic of sepsis by MDR pathogens.

Characterization of ES10 lytic bacteriophage isolated from hospital waste against multidrug-resistant uropathogenic E. coli.

Escherichia coli is the major causative agent of urinary tract infections worldwide and the emergence of multi-drug resistant determinants among clinical isolates necessitates the development of novel therapeutic agents. Lytic bacteriophages efficiently kill specific bacteria and seems promising approach in controlling infections caused by multi-drug resistant pathogens. This study aimed the isolation and detailed characterization of lytic bacteriophage designated as ES10 capable of lysing multidrug-resistant uropathogenic E. coli. ES10 had icosahedral head and non-contractile tail and genome size was 48,315 base pairs long encoding 74 proteins. Antibiotics resistance, virulence and lysogenic cycle associated genes were not found in ES10 phage genome. Morphological and whole genome analysis of ES10 phage showed that ES10 is the member of Drexlerviridae. Latent time of ES10 was 30 min, burst size was 90, and optimal multiplicity of infection was 1. ES10 was stable in human blood and subsequently caused 99.34% reduction of host bacteria. Calcium chloride shortened the adsorption time and latency period of ES10 and significantly inhibited biofilm formation of host bacteria. ES10 caused 99.84% reduction of host bacteria from contaminated fomites. ES10 phage possesses potential to be utilized in standard phage therapy.

Polydispersity-mediated high efficacy of an in-situ aqueous nanosuspension of PPEF.3HCl in methicillin resistant Staphylococcus aureus sepsis model

Recently, World Health Organization declared antimicrobial resistance as the third greatest threat to human health. Absence of known cross-resistance, new class, new target, and a new mode of action are few major strategies being undertaken by researches to combat multidrug resistant pathogen. PPEF.3HCl, a bisbenzimidazole was developed as highly potent antibacterial agent against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, targeting topoisomerase IA. The present work encompasses a radical on-site generation of In-situ nanosuspension of PPEF.3HCl with enhanced efficacy against methicillin resistant S. aureus in septicemia model. We have generated instantaneously a PPEF.3HCl nanosuspension (IsPPEF.3HCl-NS) by mixing optimized monophasic PPEF.3HCl preconcentrate in propylene glycol into an aqueous medium comprising tween 80 as stabilizer. The IsPPEF.3HCl-NS showed precipitation efficiency of > 90 %, average particle size < 500 nm, retained upto 5 h, a negative zeta potential and bi/trimodal particle size distribution. Differential scanning calorimetry, X-ray diffraction confirmed partial amorphization and transmission electron microscopy revealed spherical particles. IsPPEF.3HCl-NS was non-hemolytic and exhibited good stability in serum. More significantly, it exhibited a ∼ 1.6-fold increase in macrophage uptake compared to free PPEF.3HCl in the RAW 264.7 macrophage cell line. Confocal microscopy revealed accumulation of IsPPEF.3HCl-NS within the lysosomal compartment and cell cytosol, proposing high efficacy. In terms of antimicrobial efficacy, IsPPEF.3HCl-NS outperforms free PPEF.3HCl against clinical methicillin sensitive and resistant S. aureus strains. In a pivotal experiment, IsPPEF.3HCl-NS exhibited over 83 % survival at 8 mg/kg.bw and an impressive reduction of ∼ 4–5 log-fold in bacterial load, primarily in the kidney, liver and spleen of septicemia mice. IsPPEF.3HCl-NS prepared by the In-situ approach, coupled with enhanced intramacrophage delivery and superior efficacy, positions IsPPEF.3HCl-NS as a pioneering and highly promising formulation in the battle against antimicrobial resistance.