Bacteria Acinetobacter Baumannii Research Articles

Antibacterial activity of silver doped hydroxyapatite toward multidrug-resistant clinical isolates of Acinetobacter baumannii

Bacteria Acinetobacter baumannii is a persistent issue in hospital-acquired infections due to its fast and potent development of multi-drug resistance. To address this urgent challenge, a novel biomaterial using silver (Ag+) ions within the hydroxyapatite (HAp) lattice has been developed to prevent infections in orthopedic surgery and bone regeneration applications without relying on antibiotics. The aim of the study was to examine the antibacterial activity of mono-substituted HAp with Ag+ ions and a mixture of mono-substituted HAps with Sr2+, Zn2+, Mg2+, SeO32- and Ag+ ions against the A. baumannii.The samples were prepared in the form of powder and disc and analyzed by disc diffusion, broth microdilution method, and scanning electron microscopy. The results from the disc-diffusion method have shown a strong antibacterial efficacy of the Ag-substituted and mixture of mono-substituted HAps (Sr, Zn, Se, Mg, Ag) toward several clinical isolates. The Minimal Inhibitory Concentrations for the powdered HAp samples ranged from 32 to 42 mg/L (Ag+ substituted) and 83–167 mg/L (mixture of mono-substituted), while the Minimal Bactericidal Concentrations after 24 h of contact ranged from 62.5 (Ag+) to 187.5–292 mg/L (ion mixture). The lower substitution level of Ag+ ions in a mixture of mono-substituted HAps was the cause of lower antibacterial effects measured in suspension. However, the inhibition zones and bacterial adhesion on the biomaterial surface were comparable. Overall, the clinical isolates of A. baumannii were effectively inhibited by substituted HAp samples, probably in the same amount as by other commercially available silver-doped materials, and such materials may provide a promising alternative or supplementation to antibiotic treatment in the prevention of infections associated with bone regeneration. The antibacterial activity of prepared samples toward A. baumannii was time-dependent and should be considered in potential applications.

Bloodstream infections and antibiotic resistance at a regional hospital, Colombia, 2019-2021.

To assess antibiotic susceptibility of World Health Organization (WHO) priority bacteria (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Salmonella spp., Staphylococcus aureus, and Streptococcus pneumoniae) in blood cultures at the Orinoquía regional hospital in Colombia. This was cross-sectional study using routine laboratory data for the period 2019-2021. Data on blood samples from patients suspected of a bloodstream infection were examined. We determined: the total number of blood cultures done and the proportion with culture yield; the characteristics of patients with priority bacteria; and the type of bacteria isolated and antibiotic resistance patterns. Of 25 469 blood cultures done, 1628 (6%) yielded bacteria; 774 (48%) of these bacteria were WHO priority pathogens. Most of the priority bacteria isolated (558; 72%) were gram-negative and 216 (28%) were gram-positive organisms. Most patients with priority bacteria (666; 86%) were hospitalized in wards other than the intensive care unit, 427 (55%) were male, and 321 (42%) were ≥ 60 years of age. Of the 216 gram-positive bacteria isolated, 205 (95%) were Staphylococcus aureus. Of the 558 gram-negative priority bacteria isolated, the three most common were Escherichia coli (34%), Klebsiella pneumoniae (28%), and Acinetobacter baumannii (20%). The highest resistance of Staphylococcus aureus was to oxacillin (41%). For gram-negative bacteria, resistance to antibiotics ranged from 4% (amikacin) to 72% (ampicillin). Bacterial yield from blood cultures was low and could be improved. WHO priority bacteria were found in all hospital wards. This calls for rigorous infection prevention and control standards and continued surveillance of antibiotic resistance.

Dimeric 2-aminoimidazoles are highly active adjuvants for gram-positive selective antibiotics against Acinetobacter baumannii

The Centers for Disease Control and Prevention (CDC) reports that hospital acquired infections have increased by 65% since 2019. One of the main contributors is the gram-negative bacterium Acinetobacter baumannii. Previously, we reported aryl 2-aminoimidazole (2-AI) adjuvants that potentiate macrolide antibiotics against A. baumannii. Macrolide antibiotics are typically used to treat infections caused by gram-positive bacteria, but are ineffective against most gram-negative bacteria. We describe a new class of dimeric 2-AIs that are highly active macrolide adjuvants, with lead compounds lowering minimum inhibitory concentrations (MICs) to or below the gram-positive breakpoint level against A. baumannii. The parent dimer lowers the clarithromycin (CLR) MIC against A. baumannii 5075 from 32 μg/mL to 1 μg/mL at 7.5 μM (3.4 μg/mL), and a subsequent structure activity relationship (SAR) study identified several compounds with increased activity. The lead compound lowers the CLR MIC to 2 μg/mL at 1.5 μM (0.72 μg/mL), far exceeding the activity of both the parent dimer and the previous lead aryl 2-AI. Furthermore, these dimeric 2-AIs exhibit considerably reduced mammalian cell toxicity compared to aryl-2AI adjuvants, with IC50s of the two lead compounds against HepG2 cells of >200 μg/mL, giving therapeutic indices of >250.

Essential oil of Origanum minutiflorum exhibits anti-inflammatory and antioxidative effects in human bronchial cells and antimicrobial activity on lung pathogens

IntroductionOregano essential oil (OEO) is one of the most widely used essential oils worldwide due to its huge therapeutic benefits. Nevertheless, data on the effects of the endemic species Origanum minutiflorum, also known as wild or Turkish oregano, is scarce. On the other hand, various chronic lung diseases, characterised by persistent inflammation, oxidative stress, and common bacterial infections, do not have effective pharmacological therapy. Hence, the aim of this study is to examine the effects of wild oregano essential oil (WOEO) on human bronchial epithelial cells and lung pathogens. MethodsWe provided a detailed chemical composition of WOEO using GC-MS and GC-FID analysis. Anti-inflammatory effects of WOEO were analysed using the qRT-PCR and ELISA methods, while antioxidative properties were examined by using the dichlorofluorescein assay in BEAS-2B cells. Antibacterial activity was tested on lung pathogens by using the agar diffusion assay. ResultsThe major constituents of WOEO, analysed in this study, were carvacrol, linalool, p-cymene, γ-terpinene, and (E)-caryophyllene. We found that treatment with WOEO attenuated LPS-induced IL8 gene expression and hydrogen peroxide-induced oxidative stress in BEAS-2B cells. Moreover, WOEO showed an inhibitory effect on pathogenic bacteria Acinetobacter baumannii and highly resistant Klebsiella pneumoniae commonly seen in healthcare-associated pneumonia. ConclusionOur work presents new insights into the anti-inflammatory, antioxidative, and antimicrobial properties of WOEO which may be used as a simple and local treatment in various chronic lung diseases.

Intranasal immunization with outer membrane vesicles (OMV) protects against airway colonization and systemic infection with Acinetobacter baumannii

The multidrug-resistant bacteria Acinetobacter baumannii is a major cause of hospital-associated infection; a vaccine could significantly reduce this burden. The aim was to develop a clinically relevant model of A. baumannii respiratory tract infection and to test the impact of different immunization routes on protective immunity provided by an outer membrane vesicle (OMV) vaccine. BALB/c mice were intranasally challenged with isolates of oxa23-positive global clone GC2 A. baumannii from the lungs of patients with ventilator-associated pneumonia. Mice were immunized with OMVs by the intramuscular, subcutaneous or intranasal routes; protection was determined by measuring local and systemic bacterial load. Infection with A. baumannii clinical isolates led to a more disseminated infection than the prototype A. baumannii strain ATCC17978; with bacteria detectable in upper and lower airways and the spleen. Intramuscular immunization induced an antibody response but did not protect against bacterial infection. However, intranasal immunization significantly reduced airway colonization and prevented systemic bacterial dissemination. Use of clinically relevant isolates of A. baumannii provides stringent model for vaccine development. Intranasal immunization with OMVs was an effective route for providing protection, demonstrating that local immunity is important in preventing A. baumannii infection.

Antimicrobial Natural Products from Plant Pathogenic Fungi.

Isolates of a variety of fungal plant pathogens (Alternaria radicina ICMP 5619, Cercospora beticola ICMP 15907, Dactylonectria macrodidyma ICMP 16789, D. torresensis ICMP 20542, Ilyonectria europaea ICMP 16794, and I. liriodendra ICMP 16795) were screened for antimicrobial activity against the human pathogenic bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Mycobacterium abscessus, and M. marinum and were found to have some activity. Investigation of the secondary metabolites of these fungal isolates led to the isolation of ten natural products (1-10) of which one was novel, (E)-4,7-dihydroxyoct-2-enoic acid (1). Structure elucidation of all natural products was achieved by a combination of NMR spectroscopy and mass spectrometry. We also investigated the antimicrobial activity of a number of the isolated natural products. While we did not find (E)-4,7-dihydroxyoct-2-enoic acid (1) to have any activity against the bacteria and fungi in our assays, we did find that cercosporin (7) exhibited potent activity against Methicillin resistant Staphylococcus aureus (MRSA), dehydro-curvularin (6) and radicicol (10) exhibited antimycobacterial activity against M. marinum, and brefeldin A (8) and radicicol (10) exhibited antifungal activity against Candida albicans. Investigation of the cytotoxicity and haemolytic activities of these natural products (6-8 and 10) found that only one of the four active compounds, radicicol (10), was non-cytotoxic and non-haemolytic.

Biological Properties of 12 Newly Isolated Acinetobacter baumannii-Specific Bacteriophages.

Infections with the opportunistic Gram-negative bacterium Acinetobacter baumannii pose a serious threat today, which is aggravated by the growing problem of multi-drug resistance among bacteria, caused by the overuse of antibiotics. Treatment of infections caused by antibiotic-resistant A. baumannii strains with the use of phage therapy is not only a promising alternative, but sometimes the only option. Therefore, phages specific for clinical multi-drug resistant A. baumannii were searched for in environmental, municipal, and hospital wastewater samples collected from different locations in Poland. The conducted research allowed us to determine the biological properties and morphology of the tested phages. As a result of our research, 12 phages specific for A. baumannii, 11 of which turned out to be temperate and only one lytic, were isolated. Their lytic spectra ranged from 11 to 75%. The plaques formed by most phages were small and transparent, while one of them formed relatively large plaques with a clearly marked 'halo' effect. Based on Transmission Electron Microscopy (TEM), most of our phages have been classified as siphoviruses (only one phage was classified as a podovirus). All phages have icosahedral capsid symmetry, and 11 of them have a long tail. Optimal multiplicity of infections (MOIs) and the adsorption rate were also determined. MOI values varied depending on the phage-from 0.001 to 10. Based on similarities to known bacteriophages, our A. baumannii-specific phages have been proposed to belong to the Beijerinckvirinae and Junivirinae subfamilies. This study provides an additional tool in the fight against this important pathogen and may boost the interest in phage therapy as an alternative and supplement to the current antibiotics.

Catheterization of mice triggers resurgent urinary tract infection seeded by a bladder reservoir of Acinetobacter baumannii.

The antibiotic-resistant bacterium Acinetobacter baumannii is a leading cause of hospital-associated infections. Despite surveillance and infection control efforts, new A. baumannii strains are regularly isolated from health care facilities worldwide. In a mouse model of urinary tract infection, we found that mice infected with A. baumannii displayed high bacterial burdens in urine for several weeks. Two months after the resolution of A. baumannii infection, inserting a catheter into the bladder of mice with resolved infection led to the resurgence of a same-strain urinary tract infection in ~53% of the mice within 24 hours. We identified intracellular A. baumannii bacteria in the bladder epithelial cells of mice with resolved infection, which we propose could act as a host reservoir that was activated upon insertion of a catheter, leading to a resurgent secondary infection.

Synthesis and antibacterial activity of 2-benzylidene-3-oxobutanamide derivatives against resistant pathogens.

Antibiotic resistance evolves naturally through random mutation. Resistance to antimicrobials is an urgent public health crisis that requires coordinated global action. The ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are primarily responsible for the rise in resistant pathogens. There is an immediate requirement to identify a novel molecular scaffold with potent anti-microbial properties. We developed an efficient one-step synthesis of 2-benzylidene-3-oxobutanamide and its derivatives, which allowed the introduction of an α,β-unsaturated ketone moiety in the quest to identify a new molecular scaffold. Seven compounds exhibited very good antibacterial activity in vitro against WHO priority drug-resistant bacteria such as methicillin resistant Staphyloccus aureus (MRSA) and Acinetobacter baumannii-Multi drug resistant (MDR-AB). In cultured human embryonic kidney cells and hemolysis assays, the potent compounds displayed minimal toxicity. These findings suggest that these small molecules with excellent diversity have the potential to combat antibacterial resistance.

Enhancing the antimicrobial activity of silver nanoparticles against ESKAPE bacteria and emerging fungal pathogens by using tea extracts.

The sale of antibiotics and antifungals has skyrocketed since 2020. The increasing threat of pathogens like ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which are effective in evading existing antibiotics, and yeasts like Candida auris or Cryptococcus neoformans is pressing to develop efficient antimicrobial alternatives. Nanoparticles, especially silver nanoparticles (AgNPs), are believed to be promising candidates to supplement or even replace antibiotics in some applications. Here, we propose a way to increase the antimicrobial efficiency of silver nanoparticles by using tea extracts (black, green, or red) for their synthesis. This allows for using lower concentrations of nanoparticles and obtaining the antimicrobial effect in a short time. We found that AgNPs synthesized using green tea extract (G-TeaNPs) are the most effective, causing approximately 80% bacterial cell death in Gram-negative bacteria within only 3 hours at a concentration of 0.1 mg mL-1, which is better than antibiotics. Ampicillin at the same concentration (0.1 mg mL-1) and within the same duration (3 h) causes only up to 40% decrease in the number of S. aureus and E. cloacae cells (non-resistant strains). The tested silver nanoparticles also have antifungal properties and are effective against C. auris and C. neoformans, which are difficult to eradicate using other means. We established that silver nanoparticles synthesized with tea extracts have higher antibacterial properties than silver nanoparticles alone. Such formulations using inexpensive tea extracts and lower concentrations of silver nanoparticles show a promising solution to fight various pathogens.

Investigation of plant metabolites as potential inhibitors of Acinetobacter baumannii: An In-Silico approach

The gram-negative bacteria Acinetobacter baumannii is responsible for a broad spectrum of dangerous nosocomial infections. Acinetobacter baumannii is easily transmitted into the body via mechanical ventilators, open wounds, and intravascular catheters. Bloodstream infections and ventilator-associated pneumonia caused by A. baumannii have the greatest mortality rates. Due to its multidrug resistance (MDR), broad drug resistance, and pan-drug resistance phenotypes, A. baumannii is now recognized as a particularly challenging pathogen to control. In order to develop novel therapeutics for the existing condition, an in-silico approach was used. Based on the review of the literature, five drug target proteins including OmpA (Peptidoglycan-binding domain), CarO, DcaP, OmpW, and PBP were identified to be vital for the pathogen's survival, infection to the host, and multidrug resistance. A total of 20 potential plant metabolites with antibacterial properties were docked against these proteins. Among them, Corilagin, (+)-Lyoniresinol-3 alpha-O-beta-d-glucopyranoside, Epsilon-Viniferin, and Epigallocatechin gallate (EGCG) showed superior binding energy compared to the reference drug carbapenem. Corilagin and -(+)-Lyoniresinol-3 alpha-O-beta-d-glucopyranoside showed the best binding energy with DcaP protein having a docking score of about −261.74, and −238.19 respectively. Those two best protein-ligand complexes were assessed for MD simulation where the average RMSD value of 3.5 A° for Dcap-Corilagin and 3.0 A° for Dcap-(+)-Lyoniresinol-3 alpha-O-beta-d-glucopyranoside indicated their excellent stability. In-silico ADME analysis showed all four metabolites had a good estimated solubility (ESOL) between −3.56 and −6.32, a Log P range of 1.87–2.71 and they responded negatively in Blood-Brain Barrier, and CYP inhibition. Toxicity study showed that the expected new drugs, particularly Corilagin, are completely non-carcinogenic, non-toxic, and safe to use as therapeutic treatments against Acinetobacter baumannii. Therefore, all the predicted four metabolites could be used as a medication against Acinetobacter baumannii. As a result of the promising outcomes from our present study, we strongly recommend more in vivo analysis for experimental confirmation.

97. A Cross-kingdom Vaccine Protects against Multiple Healthcare-associated Infections

Abstract Background Rise in multi-drug-resistance (MDR) among pathogens and increasing populations at risk for these infections raises the threat of nearly untreatable infectious diseases. Thus, novel vaccine strategies to prevent and/or treat MDR pathogens would benefit global health immensely. Candida albicans (CA) cell wall proteins Als3p and Hyr1p protect against candidemia due to CA and MDR C. auris (CAU). Further, Hyr1p shares structural homology with conserved hemagglutinin (FhaB) and OmpA proteins in many Gram-negative bacteria (GNB) Acinetobacter baumannii (AB), Klebsiella pneumoniae (KP), and Pseudomonas aeruginosa (PA). Thus, Hyr1p antigen-based active and passive vaccinations protect against AB and KP pneumonia. We hypothesized that a dual Als3p/Hyr1p antigen vaccine formulated with CAF01 (a clinical stage adjuvant) that can promote a balanced Th1/Th2/Th17 immune response is likely to protect against multiple healthcare-associated infections caused by Candida species and MDR GNB. Methods To formulate Als3p/Hyr1p antigen, we mixed CAF01 with Als3p and Hyr1p antigens in the following ratios: 0/0, 10/10, 10/30, 30/10 and 30/3 µg for each dose. CD-1 mice with each dose subcutaneously, followed by booster immunization on day 21. For efficacy evaluation, vaccinated mice (n=20 mice/group) were immunosuppressed by administration of cyclophosphamide and cortisone acetate on day -2 and +3, relative to infection with either CAU, AB, KP or PA. Infection occurred two weeks following the booster immunization. For, CA infection, immunocompetent mice were infected intravenously two weeks following a primary booster or after a second booster given on day 35. Results All Als3p/Hyr1p formulations induced robust immunity against both antigens. For CA infection, two booster immunizations provide superior protection over one booster immunization. For CAU and GNB, one booster dose was enough to provide significant protection. Further, both 30/10 and 10/10 vaccine formulations protected significantly against all five infections. Specifically, for fungal infections, 30/10 and 10/10 formulations showed 30-40% and 40-50% survival efficacies (vs. 0% for placebo), respectively. TABLE 1. Survival efficacy of Als3p/Hyr1p formulations against Candida species and Gram-negative bacterial infections. Conclusion Our study shows that the Als3p/Hyr1p induced a robust protective immunity against CA, MDR CAU and GNB. Disclosures All Authors: No reported disclosures.

Design of primers for evaluation of some gram-negative bacteria isolated from intensive care unit patients in Wasit province, Iraq

Intensive care unit (ICU) patients frequently have consequences from healthcare-associated infections (HAIs), which include bacteremia, pneumonia, urinary tract, skin, or soft tissue infections. . In this study, a total of 100 clinical specimens (urine, sputum and pus) were collected from patients admitted in the ICU. Results showed eighty two were positive growth culture ‎and admitted to the intensive care unit distributed 1 (1.2%) Klebsiella oxytoca, 5(6.25%)‎Proteus mirabilis, 2 (2.4%)‎ Acinetobacter baumannii , 1 (1.2%)‎ Serratia marcescens and 1 (1.2%)‎ Burkholderia cepacia group bacteria . The results of this study showed that the diagnostic test for 16S rRNA ‎bacteria (Proteus mirabilis), which numbered 5 (100%), bacteria ‎‎(Acinetobacter baumannii), which numbered 2 (100%), bacteria ‎‎(Burkholderia cepacia ), which numbered 1 (100%), bacteria ‎‎(Serratia marcescens ), which numbered 1 (100%), and Bacteria ‎‎(klebsiella oxytoca ), which numbered 1 (100%) after completing the ‎phenotypic and biochemical diagnosis diagnostic test (16S rRNA).

Formic acid, an organic acid food preservative, induces viable-but-non-culturable state, and triggers new Antimicrobial Resistance traits in Acinetobacter baumannii and Klebsiella pneumoniae.

Numerous human pathogens, especially Gram-negative bacteria, are able to enter the viable-but-non-culturable (VBNC) state when they are exposed to environmental stressors and pose the risk of being resuscitated and causing infection after the removal of the trigger. Widely used food preservatives like weak organic acids are potential VBNC inducers in food processing and packaging facilities but have only been reported for food-borne pathogens. In the present study, it is demonstrated for the first time that one such agent, formic acid (FA), can induce a VBNC state at food processing, storage, and distribution temperatures (4, 25, and 37°C) with a varied time of treatment (days 4-10) in pathogenic Gram-negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. The use of hospital-associated pathogens is critical based on the earlier reports that demonstrated the presence of these bacteria in hospital kitchens and commonly consumed foods. VBNC induction was validated by multiple parameters, e.g., non-culturability, metabolic activity as energy production, respiratory markers, and membrane integrity. Furthermore, it was demonstrated that the removal of FA was able to resuscitate VBNC with an increased expression of multiple virulence and Antimicrobial Resistance (AMR) genes in both pathogens. Since food additives/preservatives are significantly used in most food manufacturing facilities supplying to hospitals, contamination of these packaged foods with pathogenic bacteria and the consequence of exposure to food additives emerge as pertinent issues for infection control, and control of antimicrobial resistance in the hospital setting.

Antibacterial Potential Analysis of Novel α-Helix Peptides in the Chinese Wolf Spider Lycosa sinensis.

The spider Lycosa sinensis represents a burrowing wolf spider (family Lycosidae) widely distributed in the cotton region of northern China, whose venom is rich in various bioactive peptides. In previous study, we used a combination strategy of peptidomic and transcriptomic analyses to systematically screen and identify potential antimicrobial peptides (AMPs) in Lycosa sinensis venom that matched the α-helix structures. In this work, the three peptides (LS-AMP-E1, LS-AMP-F1, and LS-AMP-G1) were subjected to sequence analysis of the physicochemical properties and helical wheel projection, and then six common clinical pathogenic bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) with multiple drug-resistance were isolated and cultured for the evaluation and analysis of antimicrobial activity of these peptides. The results showed that two peptides (LS-AMP-E1 and LS-AMP-F1) had different inhibitory activity against six clinical drug-resistant bacteria; they can effectively inhibit the formation of biofilm and have no obvious hemolytic effect. Moreover, both LS-AMP-E1 and LS-AMP-F1 exhibited varying degrees of synergistic therapeutic effects with traditional antibiotics (azithromycin, erythromycin, and doxycycline), significantly reducing the working concentration of antibiotics and AMPs. In terms of antimicrobial mechanisms, LS-AMP-E1 and LS-AMP-F1 destroyed the integrity of bacterial cell membranes in a short period of time and completely inhibited bacterial growth within 10 min of action. Meanwhile, high concentrations of Mg2+ effectively reduced the antibacterial activity of LS-AMP-E1 and LS-AMP-F1. Together, it suggested that the two peptides interact directly on bacterial cell membranes. Taken together, bioinformatic and functional analyses in the present work sheds light on the structure-function relationships of LS-AMPs, and facilitates the discovery and clinical application of novel AMPs.

Impact of sepses caused by three Multi-Drug Resistant bacteria in a tertiary hospital in 2018-2020

Abstract Background Healthcare-associated infections represent a major health issue, particularly if caused by multi-drug resistant (MDR) microorganisms, with additional hospitalisation days and relevant costs besides illness and deaths. This study aimed at evaluating the economic impact of healthcare-related sepses caused by three MDR Gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) in a tertiary hospital. Methods An observational, retrospective-cohort analysis was based on data related to patients admitted to the “SS. Antonio e Biagio e Cesare Arrigo” Hospital in Alessandria (Italy) between 2018 and 2020. Starting from the microbiology laboratory database, the set of patients developing sepses from the examined species was selected, by also checking that the relevant antibiogram provided evidence of resistance to ≥ 2 antimicrobial classes indicated by the European Centre for Disease Prevention and Control. Data were retrieved from patients’ medical records and from the hospital's computer-based application. Results In years 2018-2020, 174 patients developed septic infections from the examined species, the majority of which (88, i.e. 50.6%) in 2020 only. Altogether, these cases were responsible for 3,295 additional hospitalisation days (median 14 days, mean 19 days), with 901 imaging and 7,365 laboratory exams being reasonably ascribable to the septic episodes. Sepsis-related costs could be estimated in around 3 million Euros, about 85% of which (≈2.5 million Euros) were due to the actual cost of extra hospital stay. Specific antimicrobial therapy accounted for around 11.2% of the total (≈336,000 €). Conclusions The results of this study pointed out that MDR sepses were responsible for conspicuous costs, largely grounded on the extra length of hospital stays but also due to the use of highly-expensive new-generation antimicrobials. Further research is needed to explore costs of septic episodes in different contexts. Key messages • MDR sepses entailed high costs (around 3 million Euros in three years only in a single hospital), due to extra length of hospital stays and use of highly-expensive new-generation antimicrobials. • In order to provide wider-scale estimates of the economic impact of sepses, further research is needed to explore costs of septic episodes in different sanitary systems and contexts.

Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA.

Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.

Ventilator-Associated Pneumonia Caused by Multidrug-Resistant Gram-Negative Bacteria in Vietnam: Antibiotic Resistance, Treatment Outcomes, and Colistin-Associated Adverse Effects.

Background: Ventilator-associated pneumonia (VAP) caused by multidrug-resistant (MDR) gram-negative bacteria (GNB) presents a serious clinical scenario, and there is disagreement regarding the role of colistin in treatment. This study aimed to characterize the antibiotic resistance of MDR GNB and evaluate the treatment outcomes and side effects of colistin in VAP patients caused by MDR GNB, particularly in Vietnam. Methods: A prospective cohort research was undertaken. We enrolled 136 intubated patients diagnosed with VAP according to the Centers for Disease Control and Prevention (CDC) 2019. Sixty-six individuals with an isolated gram-negative bacterium (Acinetobacter baumannii, Klebsiella pneumoniae, or Pseudomonas aeruginosa) satisfied the European Centre for Disease Prevention and Control (ECDC)’s criteria for multi-antibiotic resistance. Results: GNB resistance was categorized as 10.6% MDR, 63.6% XDR, and 25.8% PDR. GNB were resistant to β-lactams 80–100%, aminoglycosides 50–86.7%, fluoroquinolones 100% and colistin 2.8–20%. The 28-day mortality rate was 54.5%, and acute kidney injury occurred at 12.1%. There was no statistically significant difference in mortality rate between groups receiving regimens with or without colistin (58.3% and 73.3%, respectively; OR = 1.964; 95%CI 0.483–7.989). Neither was there a statistically significant difference in acute renal damage rate between groups receiving regimens with or without colistin (14.3% and 9.7%, respectively; OR = 1.556; 95%CI 0.34–7.121). Conclusions: GNB had a high rate of antibiotic resistance to most antibiotics. The addition of colistin to the medication did not show significant differences in renal toxicity or mortality, while colistin resistance was relatively low; larger studies need to be conducted.

In silico and in vitro evaluation of extract derived from Dunaliella salina, a halotolerant microalga for its antifungal and antibacterial activity

In the present study little explored halotolerant wall-less green alga Dunaliella salina was found to be a potent source of antibacterial and antifungal biomolecules. Both the target pathogens, bacteria (Escherischia coli, Klebsiella pneumoniae, and Acinetobacter baumannii) and fungi (Candida albicans, C. tropicalis, and Cryptococus sp.) were WHO prioritized. The bioassay guided approach led us to evaluate antibacterial and antifungal lead molecule(s) from an array of compounds using spectroscopic and in silico studies. The methanol derived crude extract was purified via thin layer chromatography (TLC) using solvent system methanol: chloroform (1:19). Maximum antimicrobial activity was observed in fractions D5, D6 and D7, the components of which were then recognized using high resolution-liquid chromatography/mass spectroscopy (Orbitrap) (HR-LC/MS). The screened compounds were then docked with target enzymes sterol-14-alpha demethylase and OmpF porin protein. The energy scores revealed that amongst all, lariciresinol-4-O-glucoside showed better binding affinity, in silico, using the Schrödinger Maestro 2018-1 platform. The 3-dimensional crystal structures of both the proteins were retrieved from the protein data bank (PDB), and showed binding energies of −14.35 kcal/mol, and −11.0 kcal/mol against respective drug targets. The molecular dynamics (MD) simulations were performed for 100 ns, using Desmond package, Schrödinger to evaluate the conformational stability and alteration of protein-ligand complexes during the simulation. Thus, our findings confirmed that lariciresinol-4-O-glucoside, a lignan derivative and known strong antioxidant, may be used as an important “lead” molecule to be developed as antibacterial and antifungal drugs in the future. Communicated by Ramaswamy H. Sarma

Immunoinformatics-guided designing of epitope-based subunit vaccine from Pilus assembly protein of Acinetobacter baumannii bacteria

Acinetobacter baumannii, a prominent pathogen responsible for chronic infections in the blood, urinary tract, and lungs, has a high mortality due to its virulence and limited preventive methods. The present study aims to characterize the pilus assembly protein of A. baumannii to offer leads for epitope-based vaccine development. FilF is the putative pilus assembly protein that reportedly plays a supreme character in the virulence of this WHO-listed ESKAPE bacterium. Implementing various bioinformatics tools, led to the recognition of many antigenic B and T cell epitopes. Most promising B and T-cell epitopes were selected based on their binding efficiency with commonly occurring MHC alleles. Finally, we stepped down to fourteen protective antigenic peptides. These epitopes were also revealed to be non-allergenic and non-toxic. As a result, a vaccine chimera was created by linking these epitopes with appropriate linkers and adjuvant such as β-defensins. Furthermore, homology modeling and validation were carried out, with the modeled structure being employed for molecular docking with the immunological receptor (TLR-4) found on lymphocyte cells. As a result of the molecular dynamics simulation, the interaction between human TLR-4 and the multi-epitope vaccine sequence was stable. Finally, in silico cloning and immune simulation were carried out to see the efficacy of the construct vaccine. This is the first study targeting the pilus assembly protein from A. baumannii to identify novel epitopes that hold potential for further experimental design of multi-peptide vaccine construct against the pathogen.