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516600.9975Illegitimate recombination: an efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis. BACKGROUND: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. RESULTS: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). CONCLUSIONS: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.201222966811
976010.9974Mutations leading to ceftolozane/tazobactam and imipenem/cilastatin/relebactam resistance during in vivo exposure to ceftazidime/avibactam in Pseudomonas aeruginosa. Identifying resistance mechanisms to novel antimicrobials informs treatment strategies during infection and antimicrobial development. Studying resistance that develops during the treatment of an infection can provide the most clinically relevant mutations conferring resistance, but cross-sectional studies frequently identify multiple candidate resistance mutations without resolving the driver mutation. We performed whole-genome sequencing of longitudinal Pseudomonas aeruginosa from a patient whose P. aeruginosa developed imipenem/cilastatin/relebactam and ceftolozane/tazobactam resistance during ceftazidime/avibactam treatment. This analysis determined new mutations that arose in isolates resistant to both imipenem/cilastatin/relebactam and ceftolozane/tazobactam. Mutations in penicillin-binding protein 3 ftsI, the MexAB-OprM repressor nalD, and a virulence regulator pvdS were found in resistant isolates. Importantly, drug efflux was not increased in the resistant isolate compared to the most closely related susceptible isolates. We conclude that mutations in peptidoglycan synthesis genes can alter the efficacy of multiple antimicrobials. IMPORTANCE: Antibiotic resistance is a significant challenge for physicians trying to treat infections. The development of novel antibiotics to treat resistant infections has not been prioritized for decades, limiting treatment options for infections caused by many high-priority pathogens. Cross-resistance, when one mutation provides resistance to multiple antibiotics, is most problematic. Mutations that cause cross-resistance need to be considered when developing new antibiotics to guide developers toward drugs with different targets, and thus a better likelihood of efficacy. This work was undertaken to determine the mutation that caused resistance to three antibiotics for highly resistant Pseudomonas aeruginosa infection treatment while the bacteria were exposed to only one of these agents. The findings provide evidence that drug developers should endeavor to find effective antibiotics with new targets and that medical providers should utilize medications with different mechanisms of action in bacteria that have become resistant to even one of these three agents.202539932323
476820.9974Attenuating the virulence of the resistant superbug Staphylococcus aureus bacteria isolated from neonatal sepsis by ascorbic acid, dexamethasone, and sodium bicarbonate. BACKGROUND: Infections affecting neonates caused by Staphylococcus aureus are widespread in healthcare facilities; hence, novel strategies are needed to fight this pathogen. In this study, we aimed to investigate the effectiveness of the FDA-approved medications ascorbic acid, dexamethasone, and sodium bicarbonate to reduce the virulence of the resistant Staphylococcus aureus bacteria that causes neonatal sepsis and seek out suitable alternatives to the problem of multi-drug resistance. METHODS: Tested drugs were assessed phenotypically and genotypically for their effects on virulence factors and virulence-encoding genes in Staphylococcus aureus. Furthermore, drugs were tested in vivo for their ability to reduce Staphylococcus aureus pathogenesis. RESULTS: Sub-inhibitory concentrations (1/8 MIC) of ascorbic acid, dexamethasone, and sodium bicarbonate reduced the production of Staphylococcus aureus virulence factors, including biofilm formation, staphyloxanthin, proteases, and hemolysin production, as well as resistance to oxidative stress. At the molecular level, qRT-PCR was used to assess the relative expression levels of crtM, sigB, sarA, agrA, hla, fnbA, and icaA genes regulating virulence factors production and showed a significant reduction in the relative expression levels of all the tested genes. CONCLUSIONS: The current findings reveal that ascorbic acid, dexamethasone, and sodium bicarbonate have strong anti-virulence effects against Staphylococcus aureus. Thus, suggesting that they might be used as adjuvants to treat infections caused by Staphylococcus aureus in combination with conventional antimicrobials or as alternative therapies.202236348266
883930.9974Bacteriophage infection drives loss of β-lactam resistance in methicillin-resistant Staphylococcus aureus. Bacteriophage (phage) therapy is a promising means to combat drug-resistant bacterial pathogens. Infection by phage can select for mutations in bacterial populations that confer resistance against phage infection. However, resistance against phage can yield evolutionary trade-offs of biomedical relevance. Here, we report the discovery that infection by certain staphylococcal phages sensitizes different strains of methicillin-resistant Staphylococcus aureus (MRSA) to β-lactams, a class of antibiotics against which MRSA is typically resistant. MRSA cells that survive infection by these phages display significant reductions in minimal inhibitory concentration against different β-lactams compared to uninfected bacteria. Transcriptomic profiling reveals that these evolved MRSA strains possess highly modulated transcriptional profiles, where numerous genes involved in S. aureus virulence are downregulated. Phage-treated MRSA exhibited attenuated virulence phenotypes in the form of reduced hemolysis and clumping. Despite sharing similar phenotypes, whole-sequencing analysis revealed that the different MRSA strains evolved unique genetic profiles during infection. These results suggest complex evolutionary trajectories in MRSA during phage predation and open up new possibilities to reduce drug resistance and virulence in MRSA infections.202540637714
884640.9973Phage Resistance Accompanies Reduced Fitness of Uropathogenic Escherichia coli in the Urinary Environment. Urinary tract infection (UTI) is among the most common infections treated worldwide each year and is caused primarily by uropathogenic Escherichia coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred a consideration of alternative treatment strategies, such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, namely, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6 to 8 h of coincubation. Whole-genome sequencing revealed that UPEC strains resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. Phage-resistant strains displayed several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells and increased biofilm formation in some isolates. Interestingly, these phage-resistant UPEC isolates demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation and were more sensitive to several outer membrane-targeting antibiotics than parental strains. Additionally, phage-resistant UPEC isolates were attenuated in bladder colonization in a murine UTI model. In total, our findings suggest that while resistance to phages, such as HP3 and ES17, may arise readily in the urinary environment, phage resistance is accompanied by fitness costs which may render UPEC more susceptible to host immunity or antibiotics. IMPORTANCE UTI is one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control UTI unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest; however, much like with antibiotics, bacteria can readily become resistant to phages. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage resistance during bacterial infection. In our current study, we found that while phage-resistant bacteria quickly emerged in vitro, these bacteria were less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, of which each is evaded only at the cost of bacterial fitness.202235920561
375850.9973The effects of hormones on sex differences in infection: from genes to behavior. Males of many species are more susceptible than females to infections caused by parasites, fungi, bacteria, and viruses. One proximate cause of sex differences in infection is differences in endocrine-immune interactions. Specifically, males may be more susceptible to infection than females because sex steroids, specifically androgens in males and estrogens in females, modulate several aspects of host immunity. It is, however, becoming increasingly more apparent that in addition to affecting host immunity, sex steroid hormones alter genes and behaviors that influence susceptibility and resistance to infection. Thus, males may be more susceptible to infection than females not only because androgens reduce immunocompetence, but because sex steroid hormones affect disease resistance genes and behaviors that make males more susceptible to infection. Consideration of the cumulative effects of sex steroid hormones on susceptibility to infection may serve to clarify current discrepancies in the literature and offer alternative hypotheses to the view that sex steroid hormones only alter susceptibility to infection via changes in host immune function.200010940438
883860.9972Dual RNA-seq analysis reveals the interaction between multidrug-resistant Klebsiella pneumoniae and host in a mouse model of pneumonia. BACKGROUND: Multidrug-resistant Klebsiella pneumoniae (MDR-KP) poses a significant global health threat, associated with high morbidity and mortality rates among hospitalized patients. The interaction between MDR-KP and its host is highly complex, and few studies have investigated these interactions from both the pathogen and host perspectives. Here, we explored these interactions in a mouse model of pneumonia using dual RNA-seq analysis. METHODS: PCR identification and antimicrobial susceptibility test were employed to screen for MDR-KP strains. A mouse model of pneumonia was established through aerosolized intratracheal inoculation with high-dose or low-dose bacteria. Bacterial loads, pathological changes, inflammatory cytokine expression, and immune cell infiltration were assessed post-challenge. Dual RNA-seq analysis was conducted on lung tissues following infection. RESULTS: NY13307 was identified as an MDR-KP strain with minimal virulence factor genes and broad-spectrum drug resistance. High-dose bacteria induced more severe pulmonary pathological changes, a significant increase in bacterial load, and notably elevated secretion of inflammatory cytokines compared to low-dose bacteria. Alveolar macrophages and resident interstitial macrophages were identified as the primary sources of these cytokines. Further RNA-seq analysis revealed that, compared to the low-dose group, the high-dose group significantly upregulated hypoxia and pro-inflammatory cytokine-related genes in the host, and siderophore-related genes in the bacteria. Correlation analysis demonstrated a significant association between siderophore-related genes and clusters of genes related to pro-inflammatory cytokines and hypoxia. CONCLUSIONS: In this mouse model of bacterial pneumonia, excessive siderophore expression may trigger the activation of hypoxia signaling pathways and the release of pro-inflammatory cytokines, ultimately reducing survival rates.202540702458
840370.9972Uncovering virulence factors in Cronobacter sakazakii: insights from genetic screening and proteomic profiling. The increasing problem of antibiotic resistance has driven the search for virulence factors in pathogenic bacteria, which can serve as targets for the development of new antibiotics. Although whole-genome Tn5 transposon mutagenesis combined with phenotypic assays has been a widely used approach, its efficiency remains low due to labor-intensive processes. In this study, we aimed to identify specific genes and proteins associated with the virulence of Cronobacter sakazakii, a pathogenic bacterium known for causing severe infections, particularly in infants and immunocompromised individuals. By employing a combination of genetic screening, comparative proteomics, and in vivo validation using zebrafish and rat models, we rapidly screened highly virulent strains and identified two genes, rcsA and treR, as potential regulators of C. sakazakii toxicity toward zebrafish and rats. Proteomic profiling revealed upregulated proteins upon knockout of rcsA and treR, including FabH, GshA, GppA, GcvH, IhfB, RfaC, MsyB, and three unknown proteins. Knockout of their genes significantly weakened bacterial virulence, confirming their role as potential virulence factors. Our findings contribute to understanding the pathogenicity of C. sakazakii and provide insights into the development of targeted interventions and therapies against this bacterium.IMPORTANCEThe emergence of antibiotic resistance in pathogenic bacteria has become a critical global health concern, necessitating the identification of virulence factors as potential targets for the development of new antibiotics. This study addresses the limitations of conventional approaches by employing a combination of genetic screening, comparative proteomics, and in vivo validation to rapidly identify specific genes and proteins associated with the virulence of Cronobacter sakazakii, a highly pathogenic bacterium responsible for severe infections in vulnerable populations. The identification of two genes, rcsA and treR, as potential regulators of C. sakazakii toxicity toward zebrafish and rats and the proteomic profiling upon knockout of rcsA and treR provides novel insights into the mechanisms underlying bacterial virulence. The findings contribute to our understanding of C. sakazakii's pathogenicity, shed light on the regulatory pathways involved in bacterial virulence, and offer potential targets for the development of novel interventions against this highly virulent bacterium.202337750707
628080.9972Genomic variation in Pseudomonas aeruginosa clinical respiratory isolates with de novo resistance to a bacteriophage cocktail. Pseudomonas aeruginosa is an opportunistic pathogen that can cause sinus infections and pneumonia in cystic fibrosis (CF) patients. Bacteriophage therapy is being investigated as a treatment for antibiotic-resistant P. aeruginosa infections. Although virulent bacteriophages have shown promise in treating P. aeruginosa infections, the development of bacteriophage-insensitive mutants (BIMs) in the presence of bacteriophages has been described. The aim of this study was to examine the genetic changes associated with the BIM phenotype. Biofilms of three genetically distinct P. aeruginosa strains, including PAO1 (ATCC 15692), and two clinical respiratory isolates (one CF and one non-CF) were grown for 7 days and treated with either a cocktail of four bacteriophages or a vehicle control for 7 consecutive days. BIMs isolated from the biofilms were detected by streak assays, and resistance to the phage cocktail was confirmed using spot test assays. Comparison of whole genome sequencing between the recovered BIMs and their respective vehicle control-treated phage-sensitive isolates revealed structural variants in two strains, and several small variants in all three strains. These variations involved a TonB-dependent outer membrane receptor in one strain, and mutations in lipopolysaccharide synthesis genes in two strains. Prophage deletion and induction were also noted in two strains, as well as mutations in several genes associated with virulence factors. Mutations in genes involved in susceptibility to conventional antibiotics were also identified in BIMs, with both decreased and increased antibiotic sensitivity to various antibiotics being observed. These findings may have implications for future applications of lytic phage therapy.IMPORTANCELytic bacteriophages are viruses that infect and kill bacteria and can be used to treat difficult-to-treat bacterial infections, including biofilm-associated infections and multidrug-resistant bacteria. Pseudomonas aeruginosa is a bacterium that can cause life-threatening infections. Lytic bacteriophage therapy has been trialed in the treatment of P. aeruginosa infections; however, sometimes bacteria develop resistance to the bacteriophages. This study sheds light on the genetic mechanisms of such resistance, and how this might be harnessed to restore the sensitivity of multidrug-resistant P. aeruginosa to conventional antibiotics.202540162801
884890.9972Harnessing the effect of iron deprivation to attenuate the growth of opportunistic pathogen Acinetobacter baumannii. Acinetobacter baumannii is an opportunistic pathogen having high infectivity among immunocompromised patients. The bacteria are resistant to major first-line antibiotics and have become a serious concern in the aspect of nosocomial and community-acquired infections. To overcome this dire situation, the necessity of introducing new approaches is undeniable, which can bypass the need for conventional antibiotic therapy. In this article, we have pinpointed the importance of iron in A. baumannii. Iron is an essential micronutrient in all bacteria. Loss of iron acquisition leads to membrane destabilization, and change in the expression of iron-transporting or -metabolizing genes causes death of the bacteria. Iron scavenging was primarily mediated by different chelators, and β-thujaplicin showed the best antibacterial efficacy with respect to time killing assay and CFU analysis. When iron (Fe(2+)) was supplemented after initial deficiency, the growth of the bacteria was seen to be restored. Iron deprivation also disintegrates the biofilm matrix, a major cause of bacterial resistance against different types of antibiotics. Moreover, iron scavenging promotes inhibition of biofilm sessile persister cells, the root cause of recalcitrant and chronic infection. As a part of antimicrobial therapy, β-thujaplicin was treated alongside colistin and chloramphenicol at an amount significantly lower than its MIC value. Our results indicated that β-thujaplicin nicely complemented those antibiotics to potentiate their antimicrobial action. In a nutshell, iron chelating agents are potential alternative therapeutics that can be used alongside different antibiotics to circumvent the resistance of different nosocomial pathogens.202540202344
4819100.9972Gram-Negative Bacteria. Gram-negative bacteria (GNB) are among the world's most significant public health problems due to their high resistance to antibiotics. These microorganisms have significant clinical importance in hospitals because they put patients in the intensive care unit (ICU) at high risk and lead to high morbidity and mortality. Two large groups, Enterobacteriaceae and the non-fermenters, are responsible for most clinical isolates; nevertheless, other clinically concerning gram-negative organisms exist, including but not limited to Neisseria, Haemophilus spp., Helicobacter pylori, and Chlamydia trachomatis. Enterobacteriaceae   Enterobacteriaceae are a heterogeneous group widely dispersed in nature. They account for about 80% of gram-negative isolates with a myriad of disease-causing general/species in humans, including urinary tract infections, pneumonia, diarrhea, meningitis, sepsis, endotoxic shock, and many others. The general/species that frequently affect humans are Escherichia, Proteus, Enterobacter, Klebsiella, Citrobacter, Yersinia, Shigella, and Salmonella, among others. Laboratory characterization is an essential component when it comes to microorganisms; therefore, it is imperative to expose characteristics of Enterobacteriaceae, which are bacilli, non-sporulated, have variable motility, grow in the presence and absence of oxygen, ferment organisms of glucose, are cytochrome oxidase negative, and can reduce nitrate to nitrite. Non-Fermenters The non-fermenter, gram-negative bacilli (BNF) have a lower frequency of isolation when compared to Enterobacteriaceae; however, they are a relevant group since they cause severe, fatal infections, especially in the hospital environment. They also cause opportunistic diseases in ICU patients who undergo invasive procedures. The main BNF microorganisms that cause human disease are Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia pseudomallei, Stenotrophomonas., Alcaligenes, and Moraxella. These stand out for being aerobic and non-sporulated; they are incapable of fermenting sugars, using them through the oxidative route. The critical issue regarding BNF, when it comes to the antimicrobial sensitivity profile, is undoubtedly their intrinsic resistance since they produce a variety of genes with multiple mechanisms capable of mitigating the microbicidal action. Thus, it stands out in P. aeruginosa, cephalosporinase of type AmpC, and efflux systems that confer resistance to b-lactams. The most frequent are MexAB-OprM; and loss of OprD (which gives impermeability to the bacterial cell due to the loss of porin.) Acinetobacter baumannii naturally produces AmpC cephalosporinase and oxacillinase (OXA), leaving it spontaneously immune to many drugs. The genetic ingenuity of this microorganism goes further, and it combines high impermeability with genetic plasticity, combining with the resistance of mechanisms such as extended-spectrum b-lactamases (ESBL). The Stenotrophomonas exhibit a pattern of intrinsic multi-resistance, especially in patients who have had contact with carbapenems. Thus, Stenotrophomonas present several efflux pumps and produce two carbapenemases – L1 (resistance to all carbapenems) and L2 (cephalosporinase).  These mechanisms, associated or separate, restrict the treatment options to an alarming level. Sulfamethoxazole-trimethoprim remains the mainstay of treatment.  Antibiotic Resistance These organisms have a range of mechanisms to prevent the action of many antimicrobials used in clinical medicine. Some of the mechanisms of resistance include efflux pumps, alteration of the drug binding site and membrane permeability, degradation enzymes, and the conformational change of the drug culminating in its inactivation. GNB have two membranes, an external and an internal. The external membrane expresses a potent immune response inducer, lipopolysaccharide (LPS), which is composed of three units: a hydrophilic polysaccharide, O antigen, and a hydrophobic domain known as lipid A. Lipid A are responsible for the higher endotoxic activity of these bacteria. However, the LPS is heterogeneous in the various bacterial groups, and some bacteria manifest this antigen weakly due to genetic changes and are not recognized by Toll-like receptors. In contrast, there are BGN groups that can trigger such a response in large proportions. Thus, LPS can trigger the innate immune response through Toll-like receptors 4 (TLR4), which occurs in many immune cells such as monocytes, macrophages, dendritic cells, and neutrophils. The resulting activation of the innate immune response mediated by LPS together with TLR4 receptors culminates in an exacerbated response with the production of cytokines, chemokines, and interferons and their suppression. Enterobacteriaceae diffuse their plasmids by conjugation, which gives rise to resistance to almost all existing antibiotics. The family of enzymes carbapenemase – KPC, NDM-1, IMP, VIM, OXA-48 – is undoubtedly one of the most significant health challenges of the century, given the potential for dissemination between species and mortality rates due to infections caused by bacteria with such plasmids. Colistin, one of the few antibiotics that still treat multiresistant infections, already has a mobile resistance gene, mcr-1, and Enterobacteriaceae has a crucial role in the spread of this gene, with worldwide reports. Moreover, a further concern is that they usually associate these genes with other resistance genes (CTX-M, NDM, IMP), producing resistance to cephalosporins and carbapenems, enhancing the deleterious effects caused by these microorganisms.202530855801
6249110.9972Genome-Wide Identification of Antimicrobial Intrinsic Resistance Determinants in Staphylococcus aureus. The emergence of antimicrobial resistance severely threatens our ability to treat bacterial infections. While acquired resistance has received considerable attention, relatively little is known of intrinsic resistance that allows bacteria to naturally withstand antimicrobials. Gene products that confer intrinsic resistance to antimicrobial agents may be explored for alternative antimicrobial therapies, by potentiating the efficacy of existing antimicrobials. In this study, we identified the intrinsic resistome to a broad spectrum of antimicrobials in the human pathogen, Staphylococcus aureus. We screened the Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2, for increased susceptibility to the anti-staphylococcal antimicrobials (ciprofloxacin, oxacillin, linezolid, fosfomycin, daptomycin, mupirocin, vancomycin, and gentamicin). Sixty-eight mutants were confirmed by E-test to display at least twofold increased susceptibility to one or more antimicrobial agents. The majority of the identified genes have not previously been associated with antimicrobial susceptibility in S. aureus. For example, inactivation of genes encoding for subunits of the ATP synthase, atpA, atpB, atpG and atpH, reduced the minimum inhibitory concentration (MIC) of gentamicin 16-fold. To elucidate the potential of the screen, we examined treatment efficacy in the Galleria mellonella infection model. Gentamicin efficacy was significantly improved, when treating larvae infected with the atpA mutant compared to wild type cells with gentamicin at a clinically relevant concentration. Our results demonstrate that many gene products contribute to the intrinsic antimicrobial resistance of S. aureus. Knowledge of these intrinsic resistance determinants provides alternative targets for compounds that may potentiate the efficacy of existing antimicrobial agents against this important pathogen.201628066345
8843120.9972Dual RNA-seq in Streptococcus pneumoniae Infection Reveals Compartmentalized Neutrophil Responses in Lung and Pleural Space. Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease.IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.201931409659
9747130.9972A Novel Enterococcus Phage Endolysin Lys22 with a Wide Host Range Against Mixed Biofilm of Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii. The global surge in multidrug-resistant (MDR) bacterial pathogens has created an urgent imperative for innovative antimicrobial strategies. Enterococcus faecalis, Staphylococcus aureus, and Acinetobacter baumannii demonstrate remarkable antibiotic resistance and dominate hospital-acquired infections. These bacteria often form biofilms, a complex community structure that shields them from immune system phagocytosis, resists antibiotic penetration, and enhances their survival in hostile environments. In clinical cases, these bacteria often form mixed biofilms and lead to treatment failures. Phages and their derivatives have emerged as promising candidates in the fight against drug-resistant bacteria. Lys22, an endolysin derived from an enterococcus phage, has been cloned and demonstrated to possess a broad host range, effectively targeting E. faecalis, various Staphylococcus species, and A. baumannii. When applied to the biofilms formed by these bacteria, Lys22 was found to significantly inhibit both simple and complex biofilms in vitro. Virulent genes, including agrA, sarA, and icaA in S. aureus; asa1, cylA, and gelE in E. faecalis; and OmpA and lpsB in A. baumannii were also downregulated by Lys22. Notably, Lys22 also exhibited a robust protective effect against dual or triple infections involving E. faecalis, S. aureus, and A. baumannii in a zebrafish embryos model, highlighting its potential as a therapeutic agent in combatting multi-bacterial infections.202541156670
9103140.9972Development of cannabidiol derivatives as potent broad-spectrum antibacterial agents with membrane-disruptive mechanism. The emergence of antibiotic resistance has brought a significant burden to public health. Here, we designed and synthesized a series of cannabidiol derivatives by biomimicking the structure and function of cationic antibacterial peptides. This is the first report on the design of cannabidiol derivatives as broad-spectrum antibacterial agents. Through the structure-activity relationship (SAR) study, we found a lead compound 23 that killed both Gram-negative and Gram-positive bacteria via a membrane-targeting mechanism of action with low resistance frequencies. Compound 23 also exhibited very weak hemolytic activity, low toxicity toward mammalian cells, and rapid bactericidal properties. To further validate the membrane action mechanism of compound 23, we performed transcriptomic analysis using RNA-seq, which revealed that treatment with compound 23 altered many cell wall/membrane/envelope biogenesis-related genes in Gram-positive and Gram-negative bacteria. More importantly, compound 23 showed potent in vivo antibacterial efficacy in murine corneal infection models caused by Staphylococcus aureus or Pseudomonas aeruginosa. These findings would provide a new design idea for the discovery of novel broad-spectrum antibacterial agents to overcome the antibiotic resistance crisis.202438266554
5164150.9972Genome sequencing analysis of the pncA, rpsA and panD genes responsible for pyrazinamide resistance of Mycobacterium tuberculosis from Indonesian isolates. BACKGROUND: Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole-genome sequencing is a molecular method that allows for the rapid and cost-effective detection of mutations in multiple genes associated with anti-tuberculosis drug resistance. This sequencing approach addresses the limitations of culture-based methods, which may not apply to certain anti-TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results. METHODS: Thirty-four M. tuberculosis isolates were subcultured on a Lowenstein-Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD. RESULTS: The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in-depth study to understand their relationship to the phenotypic profile. CONCLUSIONS: Compared to the conventional culture method, the whole-genome sequencing method has advantages in determining anti-tuberculosis resistance profiles, especially in reduced time and bias.202439397216
5168160.9972Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and the Type IX Secretion System. Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.202134106011
633170.9972The sensor kinase PhoQ mediates virulence in Pseudomonas aeruginosa. Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. PhoP-PhoQ is a two-component regulatory system that has been identified as essential for virulence and cationic antimicrobial peptide resistance in several other Gram-negative bacteria. This study demonstrated that mutation of phoQ caused reduced twitching motility, biofilm formation and rapid attachment to surfaces, 2.2-fold reduced cytotoxicity to human lung epithelial cells, substantially reduced lettuce leaf virulence, and a major, 10 000-fold reduction in competitiveness in chronic rat lung infections. Microarray analysis revealed that PhoQ controlled the expression of many genes consistent with these phenotypes and with its known role in polymyxin B resistance. It was also demonstrated that PhoQ controls the expression of many genes outside the known PhoP regulon.200919246741
4769180.9972Human breast milk isolated lactic acid bacteria: antimicrobial and immunomodulatory activity on the Galleria mellonella burn wound model. INTRODUCTION: Managing burn injuries is a challenge in healthcare. Due to the alarming increase in antibiotic resistance, new prophylactic and therapeutic strategies are being sought. This study aimed to evaluate the potential of live Lactic Acid Bacteria for managing burn infections, using Galleria mellonella larvae as an alternative preclinical animal model and comparing the outcomes with a common antibiotic. METHODS: The antimicrobial activity of LAB isolated from human breast milk was assessed in vitro against Pseudomonas aeruginosa ATCC 27853. Additionally, the immunomodulatory effects of LAB were evaluated in vivo using the G. mellonella burn wound infection model. RESULTS AND DISCUSSION: In vitro results demonstrated the antimicrobial activity of Lactic Acid Bacteria against P. aeruginosa. In vivo results show that their prophylactic treatment improves, statistically significant, larval survival and modulates the expression of immunity-related genes, Gallerimycin and Relish/NF-κB, strain-dependently. These findings lay the foundation and suggest a promising alternative for burn wound prevention and management, reducing the risk of antibiotic resistance, enhancing immune modulation, and validating the potential G. mellonella as a skin burn wound model.202439310784
4788190.9971Acinetobacter baumannii Clinical Isolates Resist Complement-Mediated Lysis by Inhibiting the Complement Cascade and Improperly Depositing MAC. INTRODUCTION: Acinetobacter baumannii is a gram-negative opportunistic bacterium that causes life-threatening infections in immunocompromised hosts. The complement system is a critical mechanism of innate immunity that protects the human body from bacterial infections. Complement activation leads to the deposition of the membrane attack complex (MAC), which can directly lyse gram-negative bacteria. However, A. baumannii has developed evasion mechanisms to protect itself from complement. METHODS: Complement deposition was investigated by flow cytometry and Western blotting. Soluble MAC formation was assessed by ELISA. Bacterial serum resistance was determined by the SYTOX Green Assay. Galleria mellonella was used as an infection model. Genome sequencing revealed virulence genes carried by isolates. RESULTS: We examined clinical isolates of A. baumannii and found 11 isolates with MAC deposition and 5 isolates without deposition. Trypsinization of MAC-positive isolates significantly reduced MAC, indicating incorrect insertion, consistent with a lack of lysis of these strains. MAC-negative isolates inhibited alternative pathway activation and were significantly more serum-resistant. These strains were also more virulent in a G. mellonella infection model. Whole genome sequencing revealed that MAC-negative isolates carried more virulence genes, and both MAC-negative and MAC-positive A. baumannii significantly differed in capsule type. Importantly, a correlation was observed between complement inhibition and capsule type (e.g., capsule locus KL171) of MAC-negative bacteria, while the capsule type (e.g., KL230) of MAC-positive A. baumannii was associated with increased sensitivity to MAC-mediated lysis. CONCLUSION: Our findings suggest a relationship between capsule type, complement resistance, and host virulence in A. baumannii. INTRODUCTION: Acinetobacter baumannii is a gram-negative opportunistic bacterium that causes life-threatening infections in immunocompromised hosts. The complement system is a critical mechanism of innate immunity that protects the human body from bacterial infections. Complement activation leads to the deposition of the membrane attack complex (MAC), which can directly lyse gram-negative bacteria. However, A. baumannii has developed evasion mechanisms to protect itself from complement. METHODS: Complement deposition was investigated by flow cytometry and Western blotting. Soluble MAC formation was assessed by ELISA. Bacterial serum resistance was determined by the SYTOX Green Assay. Galleria mellonella was used as an infection model. Genome sequencing revealed virulence genes carried by isolates. RESULTS: We examined clinical isolates of A. baumannii and found 11 isolates with MAC deposition and 5 isolates without deposition. Trypsinization of MAC-positive isolates significantly reduced MAC, indicating incorrect insertion, consistent with a lack of lysis of these strains. MAC-negative isolates inhibited alternative pathway activation and were significantly more serum-resistant. These strains were also more virulent in a G. mellonella infection model. Whole genome sequencing revealed that MAC-negative isolates carried more virulence genes, and both MAC-negative and MAC-positive A. baumannii significantly differed in capsule type. Importantly, a correlation was observed between complement inhibition and capsule type (e.g., capsule locus KL171) of MAC-negative bacteria, while the capsule type (e.g., KL230) of MAC-positive A. baumannii was associated with increased sensitivity to MAC-mediated lysis. CONCLUSION: Our findings suggest a relationship between capsule type, complement resistance, and host virulence in A. baumannii.202539842423