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140000.9802Comparative genomic analysis of Escherichia coli strains obtained from continuous imipenem stress evolution. The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.202235147175
506110.9775Sporadic cefiderocol resistance in Escherichia coli from the United Arab Emirates involves multifactorial mechanisms reversible by novel beta-lactamase inhibitors. Cefiderocol (CFDC), a novel siderophore-cephalosporin, is effective against multidrug-resistant (MDR) pathogens, but the emergence of resistance threatens its future use in treating infections. This study reports the emergence of CFDC resistance in four E. coli strains isolated from immunocompromised and critically ill patients in the United Arab Emirates, and provides a comprehensive genomic analysis of these strains, aiming to uncover the mechanisms driving this resistance. Whole-genome sequencing with bioinformatic analysis revealed specific beta-lactamase variants (NDM-5, CMY-2/145, and OXA-181) and unique mutations in siderophore-iron transport genes (cirA, fepA, fecA, fiu, and tonB) and penicillin-binding proteins (PBPs) associated with resistance. Phylogenetic analysis showed that the strains were not clonally related, indicating the sporadic nature of resistance. To address this challenge, we evaluated the efficacy of several novel beta-lactamase inhibitors (BLIs) combined with CFDC. In vitro susceptibility testing demonstrated that these inhibitors restored the antibacterial activity of CFDC against resistant strains. Zidebactam, with intrinsic antibacterial activity, caused the most significant reduction in CFDC minimum inhibitory concentrations (MICs), while the activity of other inhibitors (taniborbactam and xeruborbactam) was dependent on the genetic makeup of the strains, especially mutations in the siderophore-iron uptake genes. Our findings underscore the importance of genomic surveillance in deciphering antibiotic resistance mechanisms. Novel BLIs and partner antibiotics could be added weapons in the fight against MDR bacteria; thus, we recommend using combinations with novel BLIs as innovative therapeutic options to combat the emerging threat of CFDC resistance, after proper validation of their in vivo efficacy.202541023121
904820.9772RNA Sequencing Elucidates Drug-Specific Mechanisms of Antibiotic Tolerance and Resistance in Mycobacterium abscessus. Mycobacterium abscessus is an opportunistic pathogen notorious for its resistance to most classes of antibiotics and low cure rates. M. abscessus carries an array of mostly unexplored defense mechanisms. A deeper understanding of antibiotic resistance and tolerance mechanisms is pivotal in development of targeted therapeutic regimens. We provide the first description of all major transcriptional mechanisms of tolerance to all antibiotics recommended in current guidelines, using RNA sequencing-guided experiments. M. abscessus ATCC 19977 bacteria were subjected to subinhibitory concentrations of clarithromycin (CLR), amikacin (AMK), tigecycline (TIG), cefoxitin (FOX), and clofazimine (CFZ) for 4 and 24 h, followed by RNA sequencing. To confirm key mechanisms of tolerance suggested by transcriptomic responses, we performed time-kill kinetic analysis using bacteria after preexposure to CLR, AMK, or TIG for 24 h and constructed isogenic knockout and knockdown strains. To assess strain specificity, pan-genome analysis of 35 strains from all three subspecies was performed. Mycobacterium abscessus shows both drug-specific and common transcriptomic responses to antibiotic exposure. Ribosome-targeting antibiotics CLR, AMK, and TIG elicit a common response characterized by upregulation of ribosome structural genes, the WhiB7 regulon and transferases, accompanied by downregulation of respiration through NuoA-N. Exposure to any of these drugs decreases susceptibility to ribosome-targeting drugs from multiple classes. The cytochrome bd-type quinol oxidase contributes to CFZ tolerance in M. abscessus, and the sigma factor sigH but not antisigma factor MAB_3542c is involved in TIG resistance. The observed transcriptomic responses are not strain-specific, as all genes involved in tolerance, except erm(41), are found in all included strains.202234633851
75130.9768Global transcriptomics and targeted metabolite analysis reveal the involvement of the AcrAB efflux pump in physiological functions by exporting signaling molecules in Photorhabdus laumondii. In Gram-negative bacteria, resistance-nodulation-division (RND)-type efflux pumps, particularly AcrAB-TolC, play a critical role in mediating resistance to antimicrobial agents and toxic metabolites, contributing to multidrug resistance. Photorhabdus laumondii is an entomopathogenic bacterium that has garnered significant interest due to its production of bioactive specialized metabolites with anti-inflammatory, antimicrobial, and scavenger deterrent properties. In previous work, we demonstrated that AcrAB confers self-resistance to stilbenes in P. laumondii TT01. Here, we explore the pleiotropic effects of AcrAB in this bacterium. RNA sequencing of ∆acrA compared to wild type revealed growth-phase-specific gene regulation, with stationary-phase cultures showing significant downregulation of genes involved in stilbene, fatty acid, and anthraquinone pigment biosynthesis, as well as genes related to cellular clumping and fimbrial pilin formation. Genes encoding putative LuxR regulators, type VI secretion systems, two-partner secretion systems, and contact-dependent growth inhibition systems were upregulated in ∆acrA. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in ∆acrA. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the ∆acrA mutant had altered bioluminescence and defective orange pigmentation due to disrupted anthraquinone production. These findings confirm the role of stilbenes as signaling molecules involved in gene expression, thereby shaping these phenotypes. Furthermore, we showed that AcrAB contributes to swarming and swimming motilities independently of stilbenes. Collectively, these results highlight that disrupting acrAB causes transcriptional and metabolic dysregulation in P. laumondii, likely by impeding the export of key signaling molecules such as stilbenes, which may serve as a ligand for global transcriptional regulators.IMPORTANCERecent discoveries have highlighted Photorhabdus laumondii as a promising source of novel anti-infective compounds, including non-ribosomal peptides and polyketides. One key player in the self-resistance of this bacterium to stilbene derivatives is the AcrAB-TolC complex, which is also a well-known contributor to multidrug resistance. Here, we demonstrate the pleiotropic effects of the AcrAB efflux pump in P. laumondii TT01, impacting secondary metabolite biosynthesis, motility, and bioluminescence. These effects are evident at transcriptional, metabolic, and phenotypic levels and are likely mediated by the efflux of signaling molecules such as stilbenes. These findings shed light on the multifaceted roles of efflux pumps and open avenues to better explore the complexity of resistance-nodulation-division (RND) pump-mediated signaling pathways in bacteria, thereby aiding in combating multidrug-resistant infections.202540920493
636440.9761Characterization of clumpy adhesion of Escherichia coli to human cells and associated factors influencing antibiotic sensitivity. Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. IMPORTANCE: The study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.202438530058
517050.9760Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
998860.9760Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites. Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T. atroviride exudates. In contrast, P. simiae and P. putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δtmk3) gene of T. atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T. atroviride, bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δtmk3 mutant. Unexpectedly, exudates from wild-type T. atroviride and the Δtmk3 mutant rescued purine auxotrophic mutants of H. seropedicae, K. michiganensis and P. simiae. Metabolomic analysis on exudates from wild-type T. atroviride and the Δtmk3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma-metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.202337651474
902170.9759The Involvement of the csy1 Gene in the Antimicrobial Resistance of Acinetobacter baumannii. Acinetobacter baumannii is an important, opportunistic nosocomial pathogen that causes a variety of nosocomial infections, and whose drug resistance rate has increased in recent years. The CRISPR-Cas system exists in several bacteria, providing adaptive immunity to foreign nucleic acid invasion. This study explores whether CRISPR-Cas is related to drug resistance. Antibiotics were used to treat strains ATCC19606 and AB43, and the expression of CRISPR-related genes was found to be changed. The Csy proteins (Csy1-4) were previously detected to promote target recognition; however, the potential function of csy1 gene is still unknown. Thus, the Rec(Ab) homologous recombination system was utilized to knock out the csy1 gene from A. baumannii AB43, which carries the Type I-Fb CRISPR-Cas system, and to observe the drug resistance changes in wild-type and csy1-deleted strains. The AB43Δcsy1 mutant strain was found to become resistant to antibiotics, while the wild-type strain was sensitive to antibiotics. Moreover, transcriptome analysis revealed that the csy1 gene regulates genes encoding CRISPR-Cas-related proteins, drug-resistant efflux pumps, membrane proteins, and oxidative phosphorylation-related proteins, inhibiting antimicrobial resistance in A. baumannii. The in vitro resistance development assay revealed that the complete CRISPR-Cas system could inhibit the development of bacterial resistance. Our findings expand our understanding of the role of CRISPR-Cas csy1 gene in A. baumannii and link the CRISPR-Cas system to the biogenesis of bacterial drug-resistant structures.202235155494
901980.9759Deleting qseC downregulates virulence and promotes cross-protection in Pasteurella multocida. QseC, a histidine sensor kinase of the QseBC two-component system, acts as a global regulator of bacterial stress resistance, biofilm formation, and virulence. The function of QseC in some bacteria is well understood, but not in Pasteurella multocida. We found that deleting qseC in P. multocida serotype A:L3 significantly down-regulated bacterial virulence. The mutant had significantly reduced capsule production but increased resistance to oxidative stress and osmotic pressure. Deleting qseC led to a significant increase in qseB expression. Transcriptome sequencing analysis showed that 1245 genes were regulated by qseC, primarily those genes involved in capsule and LPS biosynthesis and export, biofilm formation, and iron uptake/utilization, as well as several immuno-protection related genes including ompA, ptfA, plpB, vacJ, and sodA. In addition to presenting strong immune protection against P. multocida serotypes A:L1 and A:L3 infection, live ΔqseC also exhibited protection against P. multocida serotype B:L2 and serotype F:L3 infection in a mouse model. The results indicate that QseC regulates capsular production and virulence in P. multocida. Furthermore, the qseC mutant can be used as an attenuated vaccine against P. multocida strains of multiple serotypes.202134801081
617390.9759Mutation in crrB encoding a sensor kinase increases expression of the RND-type multidrug efflux pump KexD in Klebsiella pneumoniae. BACKGROUND: RND-type multidrug efflux systems in Gram-negative bacteria protect them against antimicrobial agents. Gram-negative bacteria generally possess several genes which encode such efflux pumps, but these pumps sometimes fail to show expression. Generally, some multidrug efflux pumps are silent or expressed only at low levels. However, genome mutations often increase the expression of such genes, conferring the bacteria with multidrug-resistant phenotypes. We previously reported mutants with increased expression of the multidrug efflux pump KexD. We aimed to identify the cause of KexD overexpression in our isolates. Furthermore, we also examined the colistin resistant levels in our mutants. METHODS: A transposon (Tn) was inserted into the genome of Klebsiella pneumoniae Em16-1, a KexD-overexpressing mutant, to identify the gene(s) responsible for KexD overexpression. RESULTS: Thirty-two strains with decreased kexD expression after Tn insertion were isolated. In 12 of these 32 strains, Tn was identified in crrB, which encodes a sensor kinase of a two-component regulatory system. DNA sequencing of crrB in Em16-1 showed that the 452nd cytosine on crrB was replaced by thymine, and this mutation changed the 151st proline into leucine. The same mutation was found in all other KexD-overexpressing mutants. The expression of crrA increased in the mutant overexpressing kexD, and the strains in which crrA was complemented by a plasmid showed elevated expression of kexD and crrB from the genome. The complementation of the mutant-type crrB also increased the expression of kexD and crrA from the genome, but the complementation of the wild-type crrB did not. Deletion of crrB decreased antibiotic resistance levels and KexD expression. CrrB was reported as a factor of colistin resistance, and the colistin resistance of our strains was tested. However, our mutants and strains carrying kexD on a plasmid did not show increased colistin resistance. CONCLUSION: Mutation in crrB is important for KexD overexpression. Increased CrrA may also be associated with KexD overexpression.202337331490
5054100.9758In vitro resistance development gives insights into molecular resistance mechanisms against cefiderocol. Cefiderocol, a novel siderophore cephalosporin, demonstrates promising in vitro activity against multidrug-resistant Gram-negative bacteria, including carbapenemase-producing strains. Nonetheless, only a few reports are available regarding the acquisition of resistance in clinical settings, primarily due to its recent usage. This study aimed to investigate cefiderocol resistance using an in vitro resistance development model to gain insights into the underlying molecular resistance mechanisms. Cefiderocol susceptible reference strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and a clinical Acinetobacter baumannii complex isolate were exposed to increasing cefiderocol concentrations using a high-throughput resistance development model. Cefiderocol susceptibility testing was performed using broth microdilution. Whole-genome sequencing was employed to identify newly acquired resistance mutations. Our in vitro resistance development model led to several clones of strains exhibiting cefiderocol resistance, with MIC values 8-fold to 512-fold higher than initial levels. In total, we found 42 different mutations in 26 genes, of which 35 could be described for the first time. Putative loss-of-function mutations were detected in the envZ, tonB, and cirA genes in 13 out of 17 isolates, leading to a decrease in cefiderocol influx. Other potential resistance mechanisms included multidrug efflux pumps (baeS, czcS, nalC), antibiotic-inactivating enzymes (ampR, dacB), and target mutations in penicillin-binding-protein genes (mrcB). This study reveals new insights into underlying molecular resistance mechanisms against cefiderocol. While mutations leading to reduced influx via iron transporters was the most frequent resistance mechanism, we also detected several other novel resistance mutations causing cefiderocol resistance.202439080477
2461110.9758In Vitro Activity of Cefiderocol on Multiresistant Bacterial Strains and Genomic Analysis of Two Cefiderocol Resistant Strains. Cefiderocol is a new siderophore cephalosporin that is effective against multidrug-resistant Gram-negative bacteria, including carbapenem-resistant strains. The aim of this study was to evaluate the activity of this new antimicrobial agent against a collection of pathogens using broth microdilution assays and to analyze the possible mechanism of cefiderocol resistance in two resistant Klebsiella pneumoniae isolates. One hundred and ten isolates were tested, comprising 67 Enterobacterales, two Acinetobacter baumannii, one Achromobacter xylosoxidans, 33 Pseudomonas aeruginosa and seven Stenotrophomonas maltophilia. Cefiderocol showed good in vitro activity, with an MIC < 2 μg/mL, and was able to inhibit 94% of the tested isolates. We observed a resistance rate of 6%. The resistant isolates consisted of six Klebsiella pneumoniae and one Escherichia coli, leading to a resistance rate of 10.4% among the Enterobacterales. Whole-genome sequencing analysis was performed on two cefiderocol-resistant Klebsiella pneumoniae isolates to investigate the possible mutations responsible for the observed resistance. Both strains belonged to ST383 and harbored different resistant and virulence genes. The analysis of genes involved in iron uptake and transport showed the presence of different mutations located in fhuA, fepA, iutA, cirA, sitC, apbC, fepG, fepC, fetB, yicI, yicJ, and yicL. Furthermore, for the first time, to the best of our knowledge, we described two Klebsiella pneumoniae isolates that synthesize a truncated fecA protein due to the transition from G to A, leading to a premature stop codon in the amino acid position 569, and a TonB protein carrying a 4-amino acid insertion (PKPK) after Lysine 103. In conclusion, our data show that cefiderocol is an effective drug against multidrug-resistant Gram-negative bacteria. However, the higher resistance rate observed in Enterobacterales underlines the need for active surveillance to limit the spread of these pathogens and to avoid the risks associated with the emergence of resistance to new drugs.202337107147
9028120.9757Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model. Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.201931324628
5163130.9756Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep. BACKGROUND: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control. METHODS: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content. RESULTS: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep. CONCLUSIONS: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.202438429820
805140.9756LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus. Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.202337166326
4706150.9756Characterization of the Role of Two-Component Systems in Antibiotic Resistance Formation in Salmonella enterica Serovar Enteritidis. The two-component system (TCS) is one of the primary pathways by which bacteria adapt to environmental stresses such as antibiotics. This study aimed to systematically explore the role of TCSs in the development of multidrug resistance (MDR) in Salmonella enterica serovar Enteritidis. Twenty-six in-frame deletion mutants of TCSs were generated from S. Enteritidis SJTUF12367 (the wild type [WT]). Antimicrobial susceptibility tests with these mutants revealed that 10 TCSs were involved in the development of antibiotic resistance in S. Enteritidis. In these 10 pairs of TCSs, functional defects in CpxAR, PhoPQ, and GlnGL in various S. Enteritidis isolates led to a frequent decrease in MIC values against at least three classes of clinically important antibiotics, including cephalosporins and quinolones, which indicated the importance of these TCSs to the formation of MDR. Interaction network analysis via STRING revealed that the genes cpxA, cpxR, phoP, and phoQ played important roles in the direct interaction with global regulatory genes and the relevant genes of efflux pumps and outer membrane porins. Quantitative reverse transcription-PCR analysis further demonstrated that the increased susceptibility to cephalosporins and quinolones in ΔphoP and ΔcpxR mutant cells was accompanied by increased expression of membrane porin genes (ompC, ompD, and ompF) and reduced expression of efflux pump genes (acrA, macB, and mdtK), as well as an adverse transcription of the global regulatory genes (ramA and crp). These results indicated that CpxAR and PhoPQ played an important role in the development of MDR in S. Enteritidis through regulation of cell membrane permeability and efflux pump activity. IMPORTANCE S. Enteritidis is a predominant Salmonella serotype that causes human salmonellosis and frequently exhibits high-level resistance to commonly used antibiotics, including cephalosporins and quinolones. Although TCSs are known as regulators for bacterial adaptation to stressful conditions, which modulates β-lactam resistance in Vibrio parahaemolyticus and colistin resistance in Salmonella enterica serovar Typhimurium, there is little knowledge of their functional mechanisms underlying the development of antibiotic resistance in S. Enteritidis. Here, we systematically identified the TCS elements in S. Enteritidis SJTUF12367, revealed that the three TCSs CpxAR, PhoPQ, and GlnGL were crucial for the MDR formation in S. Enteritidis, and preliminarily illustrated the regulatory functions of CpxAR and PhoPQ for antimicrobial resistance genes. Our work provides the basis to understand the important TCSs that regulate formation of antibiotic resistance in S. Enteritidis.202236286534
752160.9755Screening of a Leptospira biflexa mutant library to identify genes involved in ethidium bromide tolerance. Leptospira spp. are spirochete bacteria comprising both pathogenic and free-living species. The saprophyte L. biflexa is a model bacterium for studying leptospiral biology due to relative ease of culturing and genetic manipulation. In this study, we constructed a library of 4,996 random transposon mutants in L. biflexa. We screened the library for increased susceptibility to the DNA intercalating agent, ethidium bromide (EtBr), in order to identify genetic determinants that reduce L. biflexa susceptibility to antimicrobial agents. By phenotypic screening, using subinhibitory EtBr concentrations, we identified 29 genes that, when disrupted via transposon insertion, led to increased sensitivity of the bacteria to EtBr. At the functional level, these genes could be categorized by function as follows: regulation and signaling (n=11), transport (n=6), membrane structure (n=5), stress response (n=2), DNA damage repair (n=1), and other processes (n=3), while 1 gene had no predicted function. Genes involved in transport (including efflux pumps) and regulation (two-component systems, anti-sigma factor antagonists, etc.) were overrepresented, demonstrating that these genes are major contributors to EtBr tolerance. This finding suggests that transport genes which would prevent EtBr to enter the cell cytoplasm are critical for EtBr resistance. We identified genes required for the growth of L. biflexa in the presence of sublethal EtBr concentration and characterized their potential as antibiotic resistance determinants. This study will help to delineate mechanisms of adaptation to toxic compounds, as well as potential mechanisms of antibiotic resistance development in pathogenic L. interrogans.201425063661
2459170.9755In vitro antimicrobial activity and resistance mechanisms of cefiderocol against clinical carbapenem-resistant gram-negative bacteria. BACKGROUND: The rise of carbapenem-resistant gram-negative bacteria (CRGNB) necessitates new therapeutic options such as cefiderocol. OBJECTIVE: To evaluate the in vitro efficacy of cefiderocol against clinical CRGNB and investigate associated resistance mechanisms. METHODS: A total of 370 CRGNB isolates were analyzed. Minimum inhibitory concentration (MIC) values were determined, and whole genome sequencing, efflux pump inhibition assays, and RT-qPCR were conducted to assess resistance-related mutations, gene loss, and expression changes. RESULTS: Cefiderocol demonstrated potent in vitro activity, with high susceptibility rates in C. freundii (100%), K. pneumoniae (93.3%), and E. hormaechei (92.2%), and notable activity against P. aeruginosa (80.0%) and Escherichia coli (76.8%). Efflux pump inhibition by Carbonyl Cyanide m-Chlorophenyl Hydrazone (CCCP) significantly reduced MICs in resistant strains. Key resistance mechanisms included β-lactamase gene variants (bla (OXA-66), bla (OXA-23), bla (SHV-12)), mutations in envZ, cirA, nuoC, ampC, and loss or altered expression of iron transporter genes (piuA, pirA, fepA). CONCLUSION: Cefiderocol is highly effective against CRGNB; however, resistance may arise through diverse mechanisms, including efflux pump activity. Continued surveillance of emerging resistance is essential to guide its optimal clinical use.202541113641
9041180.9755Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis. BACKGROUND: Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. RESULTS: Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. CONCLUSIONS: Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.201121781329
8472190.9755Genetic architecture of resistance to plant secondary metabolites in Photorhabdus entomopathogenic bacteria. BACKGROUND: Entomopathogenic nematodes of the genus Heterorhabditis establish a symbiotic association with Photorhabdus bacteria. Together, they colonize and rapidly kill insects, making them important biological control agents against agricultural pests. Improving their biocontrol traits by engineering resistance to plant secondary metabolites (benzoxazinoids) in Photorhabdus symbiotic bacteria through experimental evolution has been shown to increase their lethality towards benzoxazinoid-defended larvae of the western corn rootworm, a serious crop pest of maize, and it is therefore a promising approach to develop more efficient biocontrol agents to manage this pest. To enhance our understanding of the genetic bases of benzoxazinoid resistance in Photorhabdus bacteria, we conducted an experimental evolution experiment with a phylogenetically diverse collection of Photorhabdus strains from different geographic origins. We cultured 27 different strains in medium containing 6-methoxy-2-benzoxazolinone (MBOA), a highly active benzoxazinoid breakdown product, for 35 24 h-cycles to select for benzoxazinoid-resistant strains. Then, we carried out genome-wide sequence comparisons to uncover the genetic alterations associated with benzoxazinoid resistance. Lastly, we evaluated the resistance of the newly isolated resistant Photorhabdus strains to eight additional bioactive compounds, including 2-benzoxazolinone (BOA), nicotine, caffeine, 6-chloroacetyl-2-benzoxazolinone (CABOA), digitoxin, fenitrothion, ampicillin, and kanamycin. RESULTS: We found that benzoxazinoid resistance evolves rapidly in Photorhabdus in a strain-specific manner. Across the different Photorhabdus strains, a total of nineteen nonsynonymous point mutations, two stop codon gains, and one frameshift were associated with higher benzoxazinoid resistance. The different genetic alterations were polygenic and occurred in genes coding for the EnvZ/OmpR two-component regulatory system, the different subunits of the DNA-directed RNA polymerase, and the AcrABZ-TolC multidrug efflux pump. Apart from increasing MBOA resistance, the different mutations were also associated with cross-resistance to 2-benzoxazolinone (BOA), nicotine, caffeine, and 6-chloroacetyl-2-benzoxazolinone (CABOA) and with collateral sensitivity to fenitrothion, ampicillin, and kanamycin. Targeted mutagenesis will provide a deeper mechanistic understanding, including the relative contribution of the different mutation types. CONCLUSIONS: Our study reveals several genomic features that are associated with resistance to xenobiotics in this important group of biological control agents and enhances the availability of molecular tools to develop better biological control agents, which is essential for more sustainable and ecologically friendly agricultural practices.202541168779