Gram-negative Bacterial Pathogens Research Articles

Antimicrobial resistance profile and associated factors of hospital-acquired gram-negative bacterial pathogens among hospitalized patients in northeast Ethiopia

BackgroundAntimicrobial resistance is a major global public health issue. Infections caused by resistant species are associated with higher mortality rates, longer hospital stays, medication failure, and rising medical costs. The World Health Organisation has declared multidrug resistance-associated infections as an epidemic of public health concern.ObjectiveThis study aimed to evaluate the antimicrobial resistance profile and associated factors of hospital-acquired Gram-negative bacterial pathogens among hospitalized patients in Northeast Ethiopia.Materials and methodsA health facility-based cross-sectional study was conducted among hospitalized patients from March 2021 to February 2022. About 810 clinical specimens were collected, transported, and processed from admitted patients following the standard bacteriological procedures. The clinical samples were inoculated onto blood agar, MacConkey agar, and chocolate agar. Furthermore, the species identification was done using gram reactions, colony morphology, and color and biochemical tests. Antimicrobial susceptibility tests, extended-spectrum beta-lactamase, and carbapenemase production were performed as per the clinical laboratory standard institute guidelines. For analysis, the information was entered into Epi-data and exported to SPSS. A P value of < 0.05 with a 95% confidence interval was considered as a statistically significant association.ResultsOut of 810 clinical specimens, 285/810 (35.2%) developed bacterial infections. From the isolated bacteria, E. coli was the predominant bacteria accounting for 78/285 (27.4%) followed by K. pneumoniae, 69/285(24.42%), whereas P. vulgaris accounted for the least, 7/285 (2.5%). Overall, 132/285 (46.3%) and 99/285 (34.7%) of culture-positive patients were infected by extended-spectrum beta-lactamase and carbapenemase-producing bacteria. The overall multidrug resistance rate of the isolated bacteria was 89.4%. The highest antibiotic resistance rates were detected for doxycycline (92.9%), amoxicillin-clavulanic acid (83.9%), and ampicillin (93%). The least antibiotic resistance rate was observed for meropenem at 41.1% and amikacin at 1.7%, respectively.Conclusions and recommendationsIn the study area, significant health concerns include a range of hospital-acquired bacterial infections associated with elevated rates of multidrug resistance, Extended-spectrum beta-lactamase (ESBL), and carbapenemase-producing bacterial pathogens. Consequently, it is recommended to conduct drug-susceptibility testing of isolates and molecular detection at a national level to optimize antibiotic usage for treating prevalent bacterial infections in this area.

Extended-spectrum β-lactamase-producing Escherichia coli from poultry: A review

Extended-spectrum β-lactamases (ESBLs) are β-lactamase enzymes produced by Gram-negative bacterial pathogens that harbor the ESBL genes. In addition, most ESBL genes are plasmid-mediated and usually encode a broader spectrum of antimicrobial resistance, especially to penicillins, first-generation, second-generation, and third-generation cephalosporins, as well as monobactam, such as aztreonam. Escherichia coli has become an opportunistic pathogen, especially in poultry, and has been implicated in zoonotic diseases that can be transmitted to humans, resulting in public health problems. Poultry can act as carriers of ESBL-producing E. coli (ESBL-EC) bacteria to humans through poultry meat that is contaminated by waste products, feces, and excretions. The ESBL gene CTX-M type was identified as the main cause of infection in humans and was detected in poultry as a cause of infection accompanied by clinical symptoms. Several studies have also shown a link between E. coli and ESBL gene transfer from birds to humans. Controlling the spread of ESBL-EC involves maintaining the cleanliness of poultry products, especially meat, and eliminating contaminant sources from poultry. Likewise, maintaining the environmental cleanliness of poultry slaughterhouses and poultry farms must be taken as a precautionary measure to curtail the increasing spread of ESBL-EC into the environment. This review aimed to explain the spread of ESBL-producing E. coli in poultry. Keywords: Escherichia coli, extended-spectrum β-lactamase, poultry, public health, zoonosis.

VirB11, a traffic ATPase, mediated flagella assembly and type IV pilus morphogenesis to control the motility and virulence of Xanthomonas albilineans.

Xanthomonas albilineans (Xal) is a gram-negative bacterial pathogen responsible for developing sugarcane leaf scald disease, which engenders significant economic losses within the sugarcane industry. In the current study, homologous recombination exchange was carried out to induce mutations within the virB/D4-like type IV secretion system (T4SS) genes of Xal. The results revealed that the virB11-deletion mutant (ΔvirB11) exhibited a loss in swimming and twitching motility. Application of transmission electron microscopy analysis further demonstrated that the ΔvirB11 failed to develop flagella formation and type IV pilus morphology and exhibited reduced swarming behaviour and virulence. However, these alterations had no discernible impact on bacterial growth. Comparative transcriptome analysis between the wild-type Xal JG43 and the deletion-mutant ΔvirB11 revealed 123 differentially expressed genes (DEGs), of which 28 and 10 DEGs were notably associated with flagellar assembly and chemotaxis, respectively. In light of these findings, we postulate that virB11 plays an indispensable role in regulating the processes related to motility and chemotaxis in Xal.

Repurposing cetylpyridinium chloride and domiphen bromide as phosphoethanolamine transferase inhibitor to combat colistin-resistant Enterobacterales

The emergence of plasmid-encoded colistin resistance mechanisms, MCR-1, a phosphoethanolamine transferase, rendered colistin ineffective as last resort antibiotic against severe infections caused by clinical Gram-negative bacterial pathogens. Through screening FDA-approved drug library, we identified two structurally similar compounds, namely cetylpyridinium chloride (CET) and domiphen bromide (DOM), which potentiated colistin activity in both colistin-resistant and susceptible Enterobacterales. These compounds were found to insert their long carbon chain to a hydrophobic pocket of bacterial phosphoethanolamine transferases including MCR-1, competitively blocking the binding of lipid A tail for substrate recognition and modification, resulting in the increase of bacterial sensitivity to colistin. In addition, these compounds were also found to dissipate bacterial membrane potential leading to the increase of bacterial sensitivity to colistin. Importantly, combinational use of DOM with colistin exhibited remarkable protection of test animals against infections by colistin-resistant bacteria in both mouse thigh infection and sepsis models. For mice infected by colistin-susceptible bacteria, the combinational use of DOM and colistin enable us to use lower dose of colistin to for efficient treatment. These properties render DOM excellent adjuvant candidates that help transform colistin into a highly potent antimicrobial agent for treatment of colistin-resistant Gram-negative bacterial infections and allowed us to use of a much lower dosage of colistin to reduce its toxicity against colistin-susceptible bacterial infection such as carbapenem-resistant Enterobacterales.

Discovery of D8-03 as an Inhibitor of Intracellular Growth of Francisella tularensis.

Francisella tularensis is a Gram-negative facultative intracellular bacterial pathogen that is classified by the Centers for Disease Control and Prevention as a Tier 1 Select Agent. F. tularensis infection causes the disease tularemia, also known as rabbit fever. Treatment of tularemia is limited to few effective antibiotics which are associated with high relapse rates, toxicity, and potential emergence of antibiotic-resistant strains. Consequently, new therapeutic options for tularemia are needed. Through screening a focused chemical library and subsequent structure-activity relationship studies, we have discovered a new and potent inhibitor of intracellular growth of Francisella tularensis, D8-03. Importantly, D8-03 effectively reduces bacterial burden in mice infected with F. tularensis. Preliminary mechanistic investigations suggest that D8-03 works through a potentially novel host-dependent mechanism and serves as a promising lead compound for further development.

Broad-Spectrum Antibacterial Activity of Antioxidant Octyl Gallate and Its Impact on Gut Microbiome.

In this study, we investigated the antibacterial activity of octyl gallate (OG), an antioxidant food additive, against both Gram-positive and Gram-negative bacterial pathogens. OG demonstrated robust bactericidal activity against Gram-positive bacterial pathogens with minimum inhibitory concentrations (MIC) of 4 to 8 µg/mL and minimum bactericidal concentrations (MBC) of 8 to 16 µg/mL in vitro. However, OG exhibited limited antibacterial activity against Gram-negative bacteria, including E. coli, although it could inhibit bacterial growth in vitro. Importantly, OG administration in mice altered the fecal microbiome, significantly reducing microbial diversity, modifying community structure, and increasing the abundance of beneficial bacteria. Additionally, OG displayed low cytotoxicity and hemolytic activity. These findings suggest that OG could be developed as a novel antibacterial agent, particularly against multi-drug-resistant MRSA. Our results provide new insights into the therapeutic potential of OG in modulating the gut microbiome, especially in conditions associated with microbial imbalance, while ensuring food safety.

Magnitude and antimicrobial susceptibility profile of bacteria isolated from pediatric sepsis cases at University of Gondar Hospital, Northwest Ethiopia

BackgroundSepsis is one of the major causes of morbidity and mortality among pediatric patients throughout the world. The varying microbiological pattern of sepsis warrants the need for researches on the causative organisms and their antimicrobial susceptibility pattern. The epidemiology of neonatal and pediatric sepsis in Ethiopia is under-research. The objective of this study was to evaluate the burden of bacterial pathogens and their antimicrobial susceptibility patterns among children suspected of sepsis.MethodsAn institutional-based prospective cross-sectional study was conducted on 370 pediatric(age birth-15 years) patients suspected of sepsis at the University of Gondar Comprehensive Specialized hospital from December 2020 to November 2021. Blood samples were collected aseptically and inoculated into Tryptone Soya Broth for culture. The organisms grown were identified by standard microbiological methods and subjected to antibiotic susceptibility testing by modified Kirby-Bauer disk diffusion method recommended by Clinical laboratory and standard institute. Methicillin resistance was confirmed using Cefoxitin disk diffusion method. Data entry and analysis were done using Statistical Package for Social Sciences (SPSS) version 26 software. A p-value less than 0.05 at 95% confidence interval was considered statically significant.ResultsOut of the total 370 study subjects, 21.6% (80/370) of them were culture positive. Of these, 43 (53.8%) and 37 (46.3%) were Gram-positive and Gram-negative bacterial pathogens, respectively. The most prevalent Gram-positive bacterial isolate was Staphylococcus aureus (n = 24; 30%) and coagulase negative staphylococci (n = 7; 8.8%). Among the Gram-negative bacterial isolates, the leading bacteria was Klebsiella pneumoniae (n = 20; 25%) followed by Escherichia coli (n = 7; 8.8%). Clindamycin, Chloramphenicol, Gentamicin and Ciprofloxacin were the most effective antibiotics against Gram-positive bacterial isolates while Amikacin, Meropenem and Chloramphenicol were effective against Gram-negative pathogens. Methicillin resistance was detected in 45.8% of Staphylococcus aureus. Multi-drug resistance (MDR) antimicrobial susceptibility pattern was observed in 76% of the bacterial isolates.ConclusionGram positive bacteria were the predominant isolates among pediatric sepsis cases and most of the bacterial isolates showed MDR. Staphylococcus aureus and Klebsiella pneumoniae were frequently isolated bacteria. The high prevalence of drug resistance warrants rational use of antibiotics and the need for regular antibiotic susceptibility surveillance studies.

CTC-177, a novel drug-Fc conjugate, shows promise as an immunoprophylactic agent against multidrug-resistant Gram-negative bacterial infections.

The widespread emergence of antibiotic resistance including MDR in Gram-negative bacterial pathogens poses a critical challenge to the current antimicrobial armamentarium. To create a novel drug-Fc conjugate (DFC) that can be delivered at sustained and prolonged levels while simultaneously activating the host immune response to combat MDR Gram-negative infections. The Cloudbreak™ platform was used to develop DFCs consisting of a targeting moiety (TM) (a polymyxin-derived dimer) attached via a non-cleavable linker to an effector moiety (EM) (the Fc domain of human IgG1). In vitro activities of the DFCs were assessed by MIC testing. Neutropenic mouse models of thigh infection, septicaemia and pneumonia were used to evaluate in vivo efficacy. Pharmacokinetics were evaluated in mice and cynomolgus monkeys. A single prophylactic dose of our lead DFC, CTC-177, resulted in significantly decreased bacterial burdens and reduced inflammation comparable to daily treatment with colistin in septicaemia and pneumonia mouse models. Furthermore, CTC-177 prophylaxis was able to restore colistin efficacy in colistin-resistant septicaemia, reducing bacterial burdens beyond the limit of detection. Finally, CTC-177 displayed a long terminal half-life of over 24 and 65 h in mice and cynomolgus monkeys, respectively. These data support the continued development of Cloudbreak™ DFCs as broad-spectrum prophylactic agents against Gram-negative infections.

Cholesterol-lowering effect of Pediococcus strains isolated from mother’s milk in mice

PurposeThe Pediococcus strains belong to the group of lactic acid bacteria (LAB). This study aims to isolate and identify the Pediococcus spp. from mother’s milk, and investigate their tolerance to low pH and bile salts, antibacterial activity, attachment to HT-29 cells and effect on cholesterol-lowering and digestive enzymes in mice.Design/methodology/approachPediococcus species were isolated from mother’s milk, then tested for tolerance to acid and bile salts, their antibacterial effect and attachment to HT-29 cell line. In mice experiment, the levels of triglyceride (TG), total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C) and digestive enzymes such as amylase and lipase in serum of mice were measured after administration of 108 CFU/mL bacterial suspension.FindingsA total of 24 Pediococcus spp. including 16 isolates of Pediococcus pentosaceus and 8 isolates of Pediococcus acidilactici were isolated. Fifteen isolates (62.5%) were able to survive in low pH and bile salt concentration. Ten isolates (41.6%) exhibited the most powerful inhibitory effects against both Gram-positive and Gram-negative bacterial pathogens. A total of four Pediococcus isolates (16.67%) showed strong attachment to HT-29 cells. In contrast to HDL-C and amylase, the serum levels of TC, TG and LDL-C were reduced and lipase was elevated significantly following administration of mother milk-derived Pediococcus isolates.Originality/valueThe Pediococcus isolates demonstrated good probiotic properties in particular cholesterol-lowering ability in mice. Thus, after more studies they can be considered as probiotic strain(s).

Quorum Quenching Approaches against Bacterial-Biofilm-Induced Antibiotic Resistance.

With the widespread phenomenon of antibiotic resistance and the diffusion of multiple drug-resistant bacterial strains, enormous efforts are being conducted to identify suitable alternative agents against pathogenic microorganisms. Since an association between biofilm formation and antibiotic resistance phenotype has been observed, a promising strategy pursued in recent years focuses on controlling and preventing this formation by targeting and inhibiting the Quorum Sensing (QS) system, whose central role in biofilm has been extensively demonstrated. Therefore, the research and development of Quorum Quenching (QQ) compounds, which inhibit QS, has gradually attracted the attention of researchers and has become a new strategy for controlling harmful microorganisms. Among these, a number of both natural and synthetic compounds have been progressively identified as able to interrupt the intercellular communication within a microbial community and the adhesion to a surface, thus disintegrating mature/preformed biofilms. This review describes the role played by QS in the formation of bacterial biofilms and then focuses on the mechanisms of different natural and synthetic QS inhibitors (QSIs) exhibiting promising antibiofilm ability against Gram-positive and Gram-negative bacterial pathogens and on their applications as biocontrol strategies in various fields.

IMD-mediated innate immune priming increases Drosophila survival and reduces pathogen transmission.

Invertebrates lack the immune machinery underlying vertebrate-like acquired immunity. However, in many insects past infection by the same pathogen can 'prime' the immune response, resulting in improved survival upon reinfection. Here, we investigated the mechanistic basis and epidemiological consequences of innate immune priming in the fruit fly Drosophila melanogaster when infected with the gram-negative bacterial pathogen Providencia rettgeri. We find that priming in response to P. rettgeri infection is a long-lasting and sexually dimorphic response. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding and spread. The enhanced survival of individuals previously exposed to a non-lethal bacterial inoculum coincided with a transient decrease in bacterial loads, and we provide strong evidence that the effect of priming requires the IMD-responsive antimicrobial-peptide Diptericin-B in the fat body. Further, we show that while Diptericin B is the main effector of bacterial clearance, it is not sufficient for immune priming, which requires regulation of IMD by peptidoglycan recognition proteins. This work underscores the plasticity and complexity of invertebrate responses to infection, providing novel experimental evidence for the effects of innate immune priming on population-level epidemiological outcomes.

Identification of Inhibitors for Flagellar Assembly Protein FliN of Uropathogenic Escherichia coli using Virtual Screening and Molecular Dynamics Simulation Study.

Escherichia coli (E. coli) is a gram-negative bacterial pathogen that poses a significant clinical and epidemiologic challenge. The selection pressure brought by the insufficient use of antibiotics has resulted in the emergence of multi-drug-resistant E. coli in the past ten years. Computational and bioinformatics methods for screening inhibitors have significantly contributed to discovering novel antibacterial agents. One possible target for novel anti-virulence drugs is motility. Motility inhibitors are generally effective at concentrations lower than those required for the antibacterial properties of traditional antibiotics, and they are likely to exert less selective pressure than current medicines. Motility may be essential for bacteria to survive, find nutrients, and escape unfavorable environments and biofilm formation. The FliN is a protein forming the bulk of the C ring of the flagella and is present in multiple copies (more than 100) in bacteria. Its absence in mammals makes it an attractive drug target for drug discovery. Two-thousand seven hundred seventy-eight natural compounds from the ZINC library were screened against FliN (PDB ID: 4YXB) using PyRx AutoDock Vina, and the top compounds were selected for secondary screening after sorting the results based on their binding energy. Based on interactional analysis, binding energy (- 7.78kcal/mol), and inhibition constant (1.98µM), ZINC000000619481 was the best inhibitor. This compound binds exactly as per the defined active site residues of the receptor protein. Also, molecular dynamics was performed. The eigenvalue of the selected complex was 1.241657e-05. There were no ADME properties outside of the specified range for the identified hit; it fitted exactly to the binding site of the FliN receptor well and was found to be stable in MD simulation studies. Further in vitro and in vivo studies are needed to confirm its anti-bacterial activity and use as a potential antimicrobial drug against urinary tract infections caused by E. coli.

Declining Trend in Antimicrobial Sensitivity in Respiratory Tract Bacterial Pathogens against Commonly Used Drugs at a Tertiary Care Hospital.

Antimicrobial resistance (AMR) is a major health issue. To determine trends in bacterial organisms in respiratory tract infections (RTIs) and their antibiotic sensitivity at a tertiary care center in India, we performed this study. Successive samples received from January 2017 to December 2021 from the respiratory tract (sputum, endotracheal secretion, and bronchoalveolar lavage) from intensive care units and medical inpatients were processed for bacterial growth. The identification of isolates and antibiotic sensitivity patterns was performed using an automated VITEK-2 system. Descriptive statistics are reported. We received 7,204 respiratory samples. Significant bacterial growth was in 3,000 (41.6%), and 2,992 (41.5%) were gram-negative. Klebsiella pneumoniae was the most prevalent, followed by Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, and Enterobacter aerogenes. Increasing secular trends were observed for Klebsiella and Pseudomonas and declining trends for Acinetobacter and Escherichia (p < 0.05). Antimicrobial sensitivity patterns showed that Klebsiella, Pseudomonas, Acinetobacter, E. coli, and Enterobacter had a high sensitivity with colistin and polymyxin (99-100%). Moderate sensitivity was observed with carbapenems (Acinetobacter: 47.5%, Enterobacter: 62.0%, Escherichia: 76.5%, Klebsiella: 72.3%, Pseudomonas: 66.7%) and tigecycline (Acinetobacter: 50.4%, Enterobacter: 68.0%, Escherichia: 81.1%, Klebsiella: 66.6%, Pseudomonas: 0%). Aminoglycosides had <50% sensitivity for various organisms, and <25% sensitivity was observed with third-generation cephalosporins and quinolones. Trend analysis showed persistent sensitivity of various pathogenic bacteria to colistin and polymyxin and declining pharmacological sensitivity in Acinetobacter (carbapenems and tigecycline), Escherichia (carbapenems, quinolones, and tigecycline), Klebsiella (carbapenems, quinolones, aminoglycosides, and tigecycline), and Pseudomonas (carbapenems and aminoglycosides) species (p < 0.05). Common respiratory tract gram-negative bacterial pathogens at a tertiary care hospital are K. pneumoniae, P. aeruginosa, A. baumannii, and E. coli. All these bacteria demonstrate high sensitivity only with colistin and polymyxin. Significant AMR is observed to carbapenems, tigecycline, aminoglycosides, and third-generation cephalosporins. Secular trends show declining antimicrobial sensitivity among various bacterial pathogens.

Membrane-targeting, ultrashort lipopeptide acts as an antibiotic adjuvant and sensitizes MDR gram-negative pathogens toward narrow-spectrum antibiotics

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide ‘SVAP9I’ that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4–8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli's outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I's synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I's distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections.

Phytofabrication of biocompatible zinc oxide nanoparticle using Gymnema sylvestre and its potent in vitro antibacterial, antibiofilm, and cytotoxicity against human breast cancer cells (MDA-MB-231).

The increasing incidence of breast cancer and bacterial biofilm in medical devices significantly heightens global mortality and morbidity, challenging synthetic drugs. Consequently, greener-synthesized nanomaterials have emerged as a versatile alternative for various biomedical applications, offering new therapeutic avenues. This study explores the synthesis of biocompatible zinc oxide (ZnONPs) nanoparticles using Gymnema sylvestre and its antibacterial, antibiofilm, and cytotoxic properties. Characterization of ZnONPs inferred that UV-Vis spectra exhibited a sharp peak at 370nm. Fourier transform infrared spectroscopical analysis revealed the presence of active functional groups such as aldehyde, alkyne, cyclic alkene, sulfate, alkyl aryl ether, and Zn-O bonds. X-ray diffraction analysis results confirmed the crystalline nature of the nanoparticle. Scanning electron microscope analysis evidenced hexagonal morphology, and energy-dispersive X-ray analysis confirmed zinc content. High-resolution transmission electron microscope analysis showed hexagonal and rod-shaped ZnONPs with a size of 5nm. Zeta potential results affirmed the stability of nanoparticles. The ZnONPs effectively inhibited gram-positive (18-20mm) than gram-negative (12-18mm) bacterial pathogens with lower bacteriostatic and higher bactericidal values. Biofilm inhibitory property inferred ZnONPs were more effective against gram-positive (38-94%) than gram-negative bacteria (27-86%). The concentration of ZnONPs to exert 50% biofilm-inhibitory is lower against gram-positive bacteria (179.26-203.95μg/mL) than gram-negative bacteria (201.46-236.19μg/mL). Microscopic visualization inferred that at 250μg/mL, ZnONPs strongly disrupted biofilm formation, as evidenced by decreased biofilm density and altered architecture. The cytotoxicity of ZnONPs against breast cancer cells showed a dose-dependent reduction in cell viability with an IC50 value of 19.4µg/mL. AO/EB staining indicated early and late apoptotic cell death of breast cancer cells under fluorescence microscopy. The results of hemolytic activity validated the biocompatibility of the ZnONPs. Thus, the unique properties of the green-synthesized ZnONPs suggest their potential as effective drug carriers for targeted delivery in cancer therapy and the treatment of biofilm-related infections.

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.

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 indicated that supplementation of the enrichment broths 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.

Prevalence of polymyxin-resistant bacterial strains in India: a systematic review and meta-analysis.

Polymyxins, the cationic lipopeptide antibiotics, are the last line of therapeutics against the MDR Gram-negative bacterial (GNB) pathogens. Unfortunately, the rising cases of polymyxin-resistant strains from across the globe have adversely impacted their utility. While the molecular mechanisms responsible for developing polymyxin resistance (PolR) are largely understood, the prevalence of PolR strains in India has not been investigated systematically. The current study was undertaken to primarily determine the prevalence of PolR strains in India. Moreover, the extent of the spread of mobile colistin resistance (mcr) genes among the GNB strains in India was also determined. A systematic search for articles using the relevant inclusion and exclusion criteria was performed in the applicable databases for the period January 2015 to December 2023. The included 41 studies were subjected to a meta-analysis using the Comprehensive Meta-Analysis software (V4.0). Publication biases were assessed using funnel plots and Egger's regression analysis. Considering a total of 41 studies including 24 589 bacterial isolates the present meta-analysis found the rate of PolR bacteria in India to be at 15.0% (95% CI: 11.2 to 19.8). Among the Indian States, Tamil Nadu topped with the highest prevalence of PolR at 28.3%. Investigating the contribution of the mcr genes, it was observed that among the PolR strains, 8.4% (95% CI: 4.8 to 14.3) were mcr positive. The study determined the prevalence of PolR strains in India at 15.0%, which is higher than that of the global average at 10%. The study also determined that 8.4% of the PolR strains carried the mcr genes. The mcr-positive strains reported from India could be an underestimation of the actual numbers due to the non-inclusion of mcr screening in many previous studies. This study provides insight into the state of the PolR situation in India, which may be useful to develop a monitoring strategy to contain the spread of such strains and preserve the efficacy of the polymyxins.

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.

Novel antimony-based antimicrobial drug targets membranes of Gram-positive and Gram-negative bacterial pathogens.

Antibiotic resistance presents a critical global public health crisis that threatens our ability to combat bacterial infections. In light of the declining efficacy of traditional antibiotics, the use of alternative solutions, such as metal-based antimicrobial compounds, has gained renewed interest. Based on the previously synthesized innovative organoantimony(V) compounds, we selected and further characterized the antibacterial efficacy of five of them against three important Gram-positive and Gram-negative bacterial pathogens. Among these compounds, SbPh4ACO showed broad-spectrum bactericidal activity, with membrane-disrupting effects against all three pathogens. Furthermore, we revealed the synergistic potential of SbPh4ACO when combined with antibiotics, such as cefoxitin, at concentrations that exert no cytotoxic effects tested on three mammalian cell lines. This study offers the first report on the mechanisms of action of novel antimony-based antimicrobial and presents the therapeutic potential of SbPh4ACO in combating both Gram-positive and Gram-negative bacterial pathogens while enhancing the efficacy of existing antibiotics.