Gram-negative Pathogens Research Articles

Piceatannol: a renaissance in antibacterial innovation unveiling synergistic potency and virulence disruption against serious pathogens.

In the relentless battle against multi-drug resistant Gram-negative bacteria, piceatannol emerges as a beacon of hope, showcasing unparalleled antibacterial efficacy and a unique ability to disrupt virulence factors. Our study illuminates the multifaceted prowess of piceatannol against prominent pathogens-Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. Notably, piceatannol demonstrated a remarkable ability to inhibit biofilm formation, reduce bacterial mobility, and diminish extracellular enzyme synthesis.Mechanistic insights into piceatannol's activity unraveled its impact on membrane potential, proton motive force, and ATP production. Furthermore, our study delved into piceatannol's anti-quorum sensing (QS) activity, showcasing its potential to downregulate QS-encoding genes and affirming its affinity to critical QS receptors through molecular docking. Crucially, piceatannol exhibited a low propensity for resistance development, positioning it as a promising candidate for combating antibiotic-resistant strains. Its mild effect on red blood cells (RBCs) suggests safety even at higher concentrations, reinforcing its potential translational value. In an in vivo setting, piceatannol demonstrated protective capabilities, significantly reducing pathogenesis in mice infected with P. aeruginosa and P. mirabilis. This comprehensive analysis positions piceatannol as a renaissance in antibacterial innovation, offering a versatile and effective strategy to confront the evolving challenges posed by resilient Gram-negative pathogens.

Investigation of colistin utilization in the treatment of multidrug-resistant gram-negative nosocomial bloodstream ınfections in children and literature review.

This retrospective study aimed to assess the effectiveness and safety of colistin used in combination therapy for treating nosocomial bloodstream infections caused by multi-drug resistant gram-negative pathogens in pediatric patients. Patients aged between 1month and 18years consecutively hospitalized with healthcare-associated bloodstream infections necessitating the administration of intravenous colistin at Dr. Sami Ulus Training and Research Hospital between January 2015 and January 2020 were included in the study. Patient-specific detailed clinical information, prognoses, and laboratory findings on days 1, 3, and 7 of colistin treatment were obtained from medical records. The study included 45 pediatric patients receiving intravenous colistin; 26 (57.8%) were male and 19 (42.2%) were female, with a median age of 18months. While the clinical response was observed at 82.2% and microbiological response at 91.1% with colistin treatment, two patients (4.4%) discontinued treatment due to side effects without assessing treatment response. The most common adverse effect associated with the use of colistin was nephrotoxicity, which occurred in eight patients (17.8%). Among these patients, only one had pre-existing chronic kidney failure. Conclusion: Colistin used in combination therapy may be effective and safe for treating nosocomial infections caused by multi-drug resistant gram-negative bacteria in pediatric patients, who often have high mortality rates and limited treatment options. What is Known: • Colistin is an antibacterial agent used in the treatment of infections caused by multidrug-resistant Gram-negative bacteria (MDR-GNB) and is associated with significant adverse effects such as nephrotoxicity. • The increasing prevalence of hospital-acquired infections has led to the expanded use of colistin in clinical practice. What is New: • The study demonstrates a high clinical and microbiological response rate to combination therapy with colistin in the treatment of infections caused by MDR-GNB. • The study highlights the importance of monitoring nephrotoxicity in pediatric patients receiving colistin, showing that these effects can be reversible after treatment cessation.

The role of aztreonam in rational antibacterial therapy of resistant nosocomial gram-negative infections. The new life for a well-known β-lactam

Background. The situation regarding the prevalence of resistant pathogens, types of β-lactamase products, and, accordingly, the justification of rational approaches to antibacterial therapy in Ukraine remains insufficiently studied. Objective: based on the determination of the etiological structure of the causative agents of hospital infections in the Kharkiv region and the state of their resistance due to the production of β-lactamases, to propose rational approaches to antibacterial therapy and assess the feasibility of using aztreonam as a potentially effective mean of treating infections caused by resistant bacteria. Materials and methods. In 251 patients, samples of biomaterials were tested by the polymerase chain reaction for the presence of the following gram-negative pathogens: P.aeruginosa, Enterobacter, E.coli, K.pneumoniae, Proteus spp. and A.baumanii. A molecular genetic study was also conducted to identify certain types of β-lactamases. Results. In the structure of hospital pathogens, the absolute majority of gram-negative bacteria belong to the enterobacteria family. When determining the profile of β-lactamases, we found that in 59.38 % of cases, E.coli was able to produce metallo-β-lactamases, which makes the therapy with carbapenems or ceftazidime-avibactam impossible. An alternative in such cases is a combination of aztreonam and ceftazidime-avibactam. When the causative agent K.pneumoniae is detected, in some cases there may also be a need for combined therapy with the use of aztreonam. It was found that Enterobacter spp. in most cases is sensitive to carbapenems. A.baumanii is completely insensitive to carbapenems and requires alternative approaches to antibacterial therapy, including aztreonam, ceftazidime-avibactam, and polymyxins. P.aeruginosa was found only as part of polymicrobial associations, so it was impossible to analyze its β-lactamase production profile. Conclusions. The results of the conducted study demonstrate an extremely high detection of gram-negative antibiotic-resistant pathogens in patients of the intensive care units in the Kharkiv region. Microorganisms such as E.coli and A.baumanii have the most unfavorable profile of antibiotic resistance, which is due to the high frequency of production of serine carbapenemases and metallo-β-lactamases, whose presence makes the effective use of carbapenems impossible.

Antibiotic susceptibility patterns and trends of the gram-negative bacteria isolated from the patients in the emergency departments in China: results of SMART 2016–2019

BackgroundThe study aims were to evaluate the species distribution and antimicrobial resistance profile of Gram-negative pathogens isolated from specimens of intra-abdominal infections (IAI), urinary tract infections (UTI), respiratory tract infections (RTI), and blood stream infections (BSI) in emergency departments (EDs) in China.MethodsFrom 2016 to 2019, 656 isolates were collected from 18 hospitals across China. Minimum inhibitory concentrations were determined by CLSI broth microdilution and interpreted according to CLSI M100 (2021) guidelines. In addition, organ-specific weighted incidence antibiograms (OSWIAs) were constructed.ResultsEscherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) were the most common pathogens isolated from BSI, IAI and UTI, accounting for 80% of the Gram-negative clinical isolates, while Pseudomonas aeruginosa (P. aeruginosa) was mainly isolated from RTI. E. coli showed < 10% resistance rates to amikacin, colistin, ertapenem, imipenem, meropenem and piperacillin/tazobactam. K. pneumoniae exhibited low resistance rates only to colistin (6.4%) and amikacin (17.5%) with resistance rates of 25–29% to carbapenems. P. aeruginosa exhibited low resistance rates only to amikacin (13.4%), colistin (11.6%), and tobramycin (10.8%) with over 30% resistance to all traditional antipseudomonal antimicrobials including ceftazidime, cefepime, carbapenems and levofloxacin. OSWIAs were different at different infection sites. Among them, the susceptibility of RTI to conventional antibiotics was lower than for IAI, UTI or BSI.ConclusionsGram-negative bacteria collected from Chinese EDs exhibited high resistance to commonly used antibiotics. Susceptibilities were organ specific for different infection sites, knowledge which will be useful for guiding empirical therapies in the clinic.

Determining glycosyltransferase functional order via lethality due to accumulated O-antigen intermediates, exemplified with Shigella flexneri O-antigen biosynthesis.

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.

Antibacterial activity of aqueous and methanol extracts of &lt;i&gt;Jatropha tanjorensis&lt;/i&gt; and &lt;i&gt;Adansonia digitata&lt;/i&gt; against selected clinical bacteria pathogens

The powdered plant parts (bark, stem, and leaf) were extracted with methanol, and cold and hot water using the maceration method. A modified agar well diffusion technique was used for the assessment of the antibacterial activities of the aqueous and methanol extracts of J. tanjorensis and A. digitata. The methanol extract of A. digitata bark gave the highest yield of 75 % while the lowest yield was observed with the cold water extract of A. digitata bark of 33 %. The phytochemical screening showed an abundance of alkaloids, terpernoids, saponins, flavonoids, tannin, glycoside, and phenol. The bark, leaf, and stem of J. tanjorensis and A. digitata showed varying degrees of antibacterial activities. against the Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Proteus spp, and Klebsiella spp) and Gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumonia). The bark, stem, and leaf extract of J. tanjorenis significantly (p &lt; 0.05) inhibited all the pathogens except P. aeruginosa and S. aureus.The result of the minimum bactericidal concentration MBC for the combined extracts of A. digitata and J. tanjorenis subfractions showed that the extracts have greater antibacterial activities at concentrations not lesser than 100 mg/ml. Thus, J. tanjorensis and A. digitata could be used as potent herbal remedies to mitigate the adverse effects of Gram-negative and positive clinical pathogens. Thus, the aim of this study was to evaluate the antibacterial activity of aqueous and methanol extracts of Jatropha tanjorensis and Adansonia digitata against selected Clinical bacteria pathogens.

Marine-Derived Cytosine Arabinoside (Ara-C) Inhibits Biofilm Formation by Inhibiting PEL Operon Proteins (Pel A and Pel B) of Pseudomonas aeruginosa: An In Silico Approach.

Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative biofilm-forming opportunistic human pathogen whose vital mechanism is biofilm formation for better survival. PelA and PelB proteins of the PEL operon are essential for bacterial-synthesized pellicle polysaccharide (PEL), which is a vital structural component of the biofilm. It helps in adherence of biofilm on the surface and maintenance of cell-to-cell interactions and with other matrix components. Here, in-silico molecular docking and simulation studies were performed against PelA and PelB using ten natural bioactive compounds, individually [podocarpic acids, ferruginol, scopadulcic acid B, pisiferic acid, metachromin A, Cytarabine (cytosine arabinoside; Ara-C), ursolic acid, oleanolic acid, maslinic acid, and betulinic acid], those have already been established as anti-infectious compounds. The results obtained from AutoDock and Glide-Schordinger stated that a marine-derived cytosine arabinoside(Ara-C) among the ten compounds binds active sites of PelA and PelB, exhibiting strong binding affinity [Trp224 (hydrogen), Ser219 (polar), Val234 (hydrophobic) for PelA; Leu365 and Glu389 (hydrogen), Gln366 (polar) for PelB] with high negative binding energy -5.518kcal/mol and -6.056kcal/mol, respectively. The molecular dynamic and simulation studies for 100ns showed the MMGBSA binding energy scores are -16.4kcal/mol (Ara-C with PelA), and -22.25kcal/mol (Ara-C with PelB). Further, ADME/T studies indicate the IC50 values of AraC are 6.10mM for PelA and 18.78mM for PelB, which is a comparatively very low dose. The zero violation of Lipinski's Rule of Five further established that Ara-C is a good candidate for drug development. Thus, Ara-C could be considered a potent anti-biofilm compound against PEL operon-dependent biofilm formation of P. aeruginosa.

Restoring colistin sensitivity in colistin-resistant Salmonella and Escherichia coli: combinatorial use of berberine and EDTA with colistin.

Colistin is last-resort antibiotic used to treat serious clinical infections caused by MDR bacterial pathogens. The recent emergence of transferable plasmid-mediated COL resistance gene mcr-1 has raised the specter of a rapid worldwide spread of COL resistance. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. Our research showed that berberine combined with EDTA effectively reversed COL resistance both in vivo and in vitro through multiple modes of action. The discovery of berberine in combination with EDTA as a new and safe COL adjuvant provides a therapeutic regimen for combating Gram-negative bacteria infections. Our findings provide a potential therapeutic option using existing antibiotics in combination with antibiotic adjuvants and address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

Miconazole and phenothiazine hinder the quorum sensing regulated virulence in Pseudomonas aeruginosa.

Antibiotic resistance is a major health problem worldwide. Pseudomonas aeruginosa is a Gram-negative pathogen with an arsenal of virulence factors and elevated antimicrobial resistance. It is a leading cause of nosocomial infections with high morbidity and mortality. The significant time and effort required to develop new antibiotics can be circumvented using alternative therapeutic strategies, including anti-virulence targets. This study aimed to investigate the anti-virulence activity of the FDA-approved drugs miconazole and phenothiazine against P. aeruginosa. The phenotypic effect of sub-inhibitory concentrations of miconazole and phenothiazine on biofilm, pyocyanin, protease, rhamnolipid and hemolysin activities in PAO1 strain was examined. qRT-PCR was used to assess the effect of drugs on quorum-sensing genes that regulate virulence. Further, the anti-virulence potential of miconazole and phenothiazine was evaluated in silico and in vivo. Miconazole showed significant inhibition of Pseudomonas virulence by reducing biofilm-formation approximately 45-48%, hemolytic-activity by 59%, pyocyanin-production by 47-49%, rhamnolipid-activity by approximately 42-47% and protease activity by 36-40%. While, phenothiazine showed lower anti-virulence activity, it inhibited biofilm (31-35%), pyocyanin (37-39%), protease (32-40%), rhamnolipid (35-40%) and hemolytic activity (47-56%). Similarly, there was significantly reduced expression of RhlR, PqsR, LasI and LasR following treatment with miconazole, but less so with phenothiazine. In-silico analysis revealed that miconazole had higher binding affinity than phenothiazine to LasR, RhlR, and PqsR QS-proteins. Furthermore, there was 100% survival in mice injected with PAO1 treated with miconazole. In conclusion, miconazole and phenothiazine are promising anti-virulence agents for P. aeruginosa.

Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using 3-Hydroxypyridin-4(1H)-one-Fluorescein Probes.

The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.

Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

Nanomedicine: Patuletin-conjugated with zinc oxide exhibit potent effects against Gram-negative and Gram-positive bacterial pathogens.

With the emergence of drug-resistance, there is a need for novel anti-bacterials or to enhance the efficacy of existing drugs. In this study, Patuletin (PA), a flavanoid was loaded onto Gallic acid modified Zinc oxide nanoparticles (PA-GA-ZnO), and evaluated for antibacterial properties against Gram-positive (Bacilluscereus andStreptococcus pneumoniae)and Gram-negative (Samonella entericaand Escherichia coli) bacteria. Characterization of PA, GA-ZnO and PA-GA-ZnO' nanoparticles was accomplished utilizing fourier-transform infrared spectroscopy, efficiency of drug entrapment, polydispersity index, zeta potential, size, and surface morphology analysis through atomic force microscopy. Using bactericidal assays, the results revealed that ZnO conjugation displayed remarkable effects and enhanced Patuletin's effects against both Gram-positive and Gram-negative bacteria, with the minimum inhibitory concentration observed at micromolar concentrations. Cytopathogenicity assays exhibited that the drug-nanoconjugates reduced bacterial-mediated human cell death with minimal side effects to human cells. When tested alone, drug-nanoconjugates tested in this study showed limited toxic effects against human cells in vitro. These are promising findings, but future work is needed to understand the molecular mechanisms of effects of drug-nanoconjugates against bacterial pathogens, in addition to in vivo testing to determine their translational value. This study suggests that Patuletin-loaded nano-formulation (PA-GA-ZnO) may be implicated in a multi-target mechanism that affects both Gram-positive and Gram-negative pathogen cell structures, however this needs to be ascertained in future work.

186 Evaluation of the effects of a Lactobacillus postbiotic product (LumensaM) in medicated and non-medicated nursery pig diets

Abstract Two studies in weanling pigs were conducted to evaluate the effects of a Lactobacillus postbiotic product (LFP; Lumensa, Verdesian Life Sciences, Salt Lake City, UT) in medicated and non-medicated nursery pig diets on performance, daily fecal scores and some fecal microbiome measures. A 2 X 3 factorial factorial design with main effects of in-feed medication (0 vs 55 ppm of Carbadox) and LFP inclusion rate (0, 0.075, or 0.15%) was used in each study. In study 1, LFP in liquid form was added to the treatment diets, and in study 2 LFP in dry form was used in the treatment diets. Each study used 36 pens containing 10 weanling pigs (PIC 800 x 1050; 21 d of age) per pen, and there were 6 pens for each treatment group. A pelleted three-phase commercial nursery feeding program was used. Dry LFP product increased final body weight (BW) linearly (P &amp;lt; 0.05) and tended to improve gain to feed ratio (G:F; P = 0.07) in study 2. Final BW was increased (P = 0.006) in nursery pigs fed medicated diets in study 2 but not in study 1. Feeding the dry LFP in study 2 improved growth performance (P &amp;lt; 0.05) in nursery pigs when they were fed non-medicated diets. In study 1, it was found that feeding LFP tended (P = 0.10) to decrease final BW variability (pen BW CV) on d 42. Daily fecal scores were recorded during both studies, and no treatment effects were observed. During study 2, fresh fecal samples were collected from each pen on d 21 and d 42 for microbial analysis. The Shannon Index was found to be moderately correlated (P &amp;lt; 0.05) to BW results on d 21 and 42 (Study 1, r = -0.31 and 0.19; Study 2, r= -0.49 and 0.27, d 21 and d 42, respectively). It was found that feeding LFP decreased the proportion of Gammaproteobacteria, which includes several Gram-negative pathogens, on d 21 (P &amp;lt; 0.05). In conclusion, feeding LFP to nursery pigs as dry product may increase average daily gain in pigs fed non-medicated diets and decrease BW variability (pen BW CV) when pigs are fed nursery diets containing carbadox. Also, feeding LFP may lead to changes in the fecal microbiome.

First report of KPC variants conferring ceftazidime-avibactam resistance in Colombia: introducing KPC-197.

Latin America and the Caribbean is an endemic region for carbapenemases. Increasingly high rates of Klebsiella pneumoniae carbapenemase (KPC) have established ceftazidime-avibactam (CZA) as an essential antimicrobial for the treatment of infections due to MDR Gram-negative pathogens. Although other countries in the region have reported the emergence of CZA-resistant KPC variants, this is the first description of such enzymes in Colombia. This finding warrants active surveillance, as dissemination of these variants could have devastating public health consequences.

Polyamine-Mediated Sensitization of Klebsiella pneumoniae to Macrolides through a Dual Mode of Action.

Chemicals bacteria encounter at the infection site could shape their stress and antibiotic responses; such effects are typically undetected under standard lab conditions. Polyamines are small molecules typically overproduced by the host during infection and have been shown to alter bacterial stress responses. We sought to determine the effect of polyamines on the antibiotic response of Klebsiella pneumoniae, a Gram-negative priority pathogen. Interestingly, putrescine and other natural polyamines sensitized K. pneumoniae to azithromycin, a macrolide protein translation inhibitor typically used for Gram-positive bacteria. This synergy was further potentiated in the physiological buffer, bicarbonate. Chemical genomic screens suggested a dual mechanism, whereby putrescine acts at the membrane and ribosome levels. Putrescine permeabilized the outer membrane of K. pneumoniae (NPN and β-lactamase assays) and the inner membrane (Escherichia coli β-galactosidase assays). Chemically and genetically perturbing membranes led to a loss of putrescine-azithromycin synergy. Putrescine also inhibited protein synthesis in an E. coli-derived cell-free protein expression assay simultaneously monitoring transcription and translation. Profiling the putrescine-azithromycin synergy against a combinatorial array of antibiotics targeting various ribosomal sites suggested that putrescine acts as tetracyclines targeting the 30S ribosomal acceptor site. Next, exploiting the natural polyamine-azithromycin synergy, we screened a polyamine analogue library for azithromycin adjuvants, discovering four azithromycin synergists with activity starting from the low micromolar range and mechanisms similar to putrescine. This work sheds light on the bacterial antibiotic responses under conditions more reflective of those at the infection site and provides a new strategy to extend the macrolide spectrum to drug-resistant K. pneumoniae.

Microbiological Profile and Susceptibility Pattern of Bacterial Blood Culture Isolates in COVID-19 Patients with Septicemia from a Designated COVID Hospital in Pune

In severe Coronavirus disease 2019 (COVID-19), bloodstream infections (BSIs) are an increasing cause of morbidity and mortality. In critically ill patients with COVID-19, we aimed to evaluate the prevalence, clinical profiles, and outcomes of BSIs. This single-center prospective investigation was conducted at a tertiary care hospital in Western India. All patients (&gt;18 years of age) hospitalized in the intensive care unit (ICU) or ward with RT-PCR-confirmed COVID-19 were included. Demographic information, clinical proficiency, and antibiotic resistance patterns were assessed. Of the 550 patients admitted to the COVID ICU, subsequent BSIs occurred in 7.45% of patients. Gram-negative pathogens comprised a significant proportion of BSIs (53/73, 72.6%). The most frequent isolates were Klebsiella pneumoniae (22/73, 30.1%), Acinetobacter baumannii (11/73,15.06%), and Escherichia coli (7/23, 9.58%). In 57.8% of the cases, multidrug-resistant organisms (MDRO) were discovered. The Enterococcus and K. pneumoniae families comprise the majority of MDRO. Gram-negative bacteria (30.18% [16/53]) were resistant to carbapenems. Increased total leukocyte count, mechanical ventilation, and the presence of comorbidities were significantly associated with the incidence of BSIs. In COVID-19-linked BSIs, we discovered a high frequency of A. baumannii. Clinicians should be aware of potential BSIs in the presence of comorbidities, elevated leukocyte count, and mechanical ventilation. To improve the results, empirical antibiotics must be started promptly, and the situation must be de-escalated quickly. The most frequent isolates were A. baumannii and K. pneumoniae ([11/73, 15.06%] and [22/73, 30.1%], respectively). To reduce the incidence of MDRO, infection control procedures should be strictly followed in patients with multidrug resistance.

Role of the NtrC family response regulator in nitrogen metabolism of Acinetobacter baumannii

Acinetobacter baumannii is an important Gram-negative nosocomial pathogen that causes opportunistic infections and employs different mechanisms to survive in the presence of antibiotics in the host. Nutrient limitation is one of the important defense mechanisms of the mammalian immune system to fight against the colonization of pathogens like A. baumannii. The present study describes an NtrC-type Response Regulator (RR) A1S_1978 involved in modulating the metabolism and cell morphology of A. baumannii via a two-component system. This RR was found to be highly conserved in the Acinetobacter and other important Gram-negative pathogens. Sequence analysis reveals that this RR contains an HTH_8 DNA-binding domain. It is also observed that deletion of this RR resulted in elongated cell phenotype and altered colony morphology of A. baumannii. We showed that the ability of A. baumannii to form biofilm and pellicle is partly abolished upon deletion of this response regulator. We showed that mutant strains lacking RR A1S_1978 have diminished growth in the absence of the nitrogen source. The transcriptome analysis of the A1S_1978 deletion mutant revealed that 253 genes were differentially expressed, including 80 genes that were upregulated by at least 2-fold and 173 genes that were down regulated in the ΔA1S_1978 strain. The transcriptome data showed an association between the A1S_1978 RR and key genes related to various nitrogen and amino acid metabolism processes, which was further confirmed by real time PCR analysis. The deletion of this RR leads to a reduction in persister cell formation against ciprofloxacin antibiotic. Taken together the results of this investigation provide significant evidence that the RR A1S_1978 is a global regulator in A. baumannii.

Marine Microalgae Schizochytrium sp. S31: Potential Source for New Antimicrobial and Antibiofilm Agent.

The rise of antibiotic-resistant bacteria necessitates the discovery of new, safe, and bioactive antimicrobial compounds. The antibacterial and antibiofilm activity of microalgae makes them a potential candidate for developing natural antibiotics to limit microbial infection in various fields. This study aimed to analyze the antibacterial effect of the methanolic extract of Schizochytrium sp. S31 microalgae by broth microdilution and spot plate assays. The antibacterial effects of Schizochytrium sp. S31 extract was studied on gramnegative pathogens, Pseudomonas aeruginosa, Escherichia coli 35218, Klebsiella pneumonia, which cause many different human infections, and the gram-positive pathogen Streptococcus mutans. At the same time, the antibiofilm activity of the Schizochytrium sp. S31 extract on Pseudomonas aeruginosa and Escherichia coli 35218 bacteria were investigated by crystal violet staining method. Schizochytrium sp. S31 extract at a 60% concentration for 8 hours displayed the highest antibacterial activity against P. aeruginosa, E. coli 35218, and K. pneumonia, with a decrease of 87%, 92%, and 98% in cell viability, respectively. The experiment with Streptococcus mutans revealed a remarkable antibacterial effect at a 60% extract concentration for 24 hours, leading to a notable 93% reduction in cell viability. Furthermore, the extract exhibited a dose-dependent inhibition of biofilm formation in P. aeruginosa and E. coli 35218. The concentration of 60% extract was identified as the most effective dosage in terms of inhibition. This research emphasizes the potential of Schizochytrium sp. S31 as a natural antibacterial and antibiofilm agent with promising applications in the pharmaceutical sectors. This is the first study to examine the antibacterial activity of Schizochytrium sp. S31 microalgae using broth microdilution, spot plate assays, and the antibiofilm activity by a crystal staining method. The findings of this study show that Schizochytrium sp. S31 has antibacterial and antibiofilm activities against critical bacterial pathogens.

Strain-specific Recovery of S. sonnei from Artificially Contaminated Baby Carrots: Enhancing Food-safety Investigations with a Customized Shigella Detection Method Based on Genomically predicted Antibiotic Resistance Traits

Shigella spp. are Gram-negative gastrointestinal bacterial pathogens that cause bacillary dysentery or shigellosis in humans. Isolation of Shigella from outbreak-associated foods is often problematic due to the lack of selectivity of cultural enrichment broths. To facilitate Shigella recovery from foods, we have developed strain-specific enrichment media based on the genomically predicted antimicrobial resistance (AMR) features of an outbreak-associated Shigella sonnei strain harboring resistance genes for streptomycin (STR) and trimethoprim (TMP). To assess performance of the method, baby carrots were artificially contaminated with the S. sonnei strain at low (2.4 CFU), medium (23.5 CFU), and high levels (235 CFU) along with 10-fold higher levels of a Shigella-inhibiting Escherichia coli strain. The target S. sonnei strain was successfully recovered from artificially contaminated baby carrots when enriched in modified Tryptone Soya Broth (mTSB) supplemented with TMP, whereas Shigella was not recovered from Shigella broth (SB) or SB supplemented with STR. Quantitative PCR analysis of the enrichment culture indicated that supplementation of the enrichment cultures with TMP or STR increased the relative proportion of S. sonnei in enrichment cultures, except at the lowest inoculation level for STR. Microbiome profiling of the baby carrot enrichment cultures conducted by 16S rRNA gene sequencing indicated that both SB-STR and mTSB-TMP repressed the growth of competing Enterobacteriaceae in the enrichment cultures, relative to SB without supplementation. Overall, improved Shigella recovery was achieved with the addition of the appropriate custom selective agent during cultural enrichments demonstrating that genomically informed custom selective enrichment of Shigella could be a valuable tool for supporting future foodborne shigellosis outbreak investigations.

Exploring Beta-Lactamase Inhibitory Potential of Common Medicinal Plants

The use of ?-Lactam antibiotics improperly has significant negative effects on public safety and health around the world. It causes the disturbance of gut flora, the emergence of multi-drug-resistant bacteria, and environmental contamination. The focus of this investigation was the Gram-negative pathogens Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). Potent beta-lactamase inhibitors were found among phytochemicals from medicinal plants by in-silico analysis, with Gallocatechin gallate from Green tea and Glycyrrhetinic acid from Licorice being particularly effective against TEM-1 and IMP-1 ?-Lactamases, respectively. In-vitro analysis such as D-zone assay and time-kill assay showed that the methanolic plant extracts improved the effectiveness of antibiotics against these Gram-negative pathogens. This research emphasizes the potential of medicinal plants in combating ?-Lactam antibiotic resistance, offering alternative solutions to address this global health challenge.