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902100.9235The 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
140010.9216Comparative 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
999720.9216RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation. The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.201021209831
600630.9208Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae. NOSO-502 is a preclinical antibiotic candidate of the Odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the Colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations on genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in gene crrB were found to be resistant to NOSO-502.CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which finally induces the restructuring of the lipopolysaccharide present on the outer membrane and thus leading to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes such as H239_3063, an ABC transporter transmembrane region; H239_3064, a putative efflux pump also known as KexD; and H239_3065, a N-acetyltransferase.To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA or crrB deleted K. pneumoniae NCTC 13442 CrrB(P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but no other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.202333685902
844340.9202Large-scale bioinformatic analysis of the regulation of the disease resistance NBS gene family by microRNAs in Poaceae. In the present study, we have screened 71, 713, 525, 119 and 241 mature miRNA variants from Hordeum vulgare, Oryza sativa, Brachypodium distachyon, Triticum aestivum, and Sorghum bicolor, respectively, and classified them with respect to their conservation status and expression levels. These Poaceae non-redundant miRNA species (1,669) were distributed over a total of 625 MIR families, among which only 54 were conserved across two or more plant species, confirming the relatively recent evolutionary differentiation of miRNAs in grasses. On the other hand, we have used 257 H. vulgare, 286T. aestivum, 119 B. distachyon, 269 O. sativa, and 139 S. bicolor NBS domains, which were either mined directly from the annotated proteomes, or predicted from whole genome sequence assemblies. The hybridization potential between miRNAs and their putative NBS genes targets was analyzed, revealing that at least 454 NBS genes from all five Poaceae were potentially regulated by 265 distinct miRNA species, most of them expressed in leaves and predominantly co-expressed in additional tissues. Based on gene ontology, we could assign these probable miRNA target genes to 16 functional groups, among which three conferring resistance to bacteria (Rpm1, Xa1 and Rps2), and 13 groups of resistance to fungi (Rpp8,13, Rp3, Tsn1, Lr10, Rps1-k-1, Pm3, Rpg5, and MLA1,6,10,12,13). The results of the present analysis provide a large-scale platform for a better understanding of biological control strategies of disease resistance genes in Poaceae, and will serve as an important starting point for enhancing crop disease resistance improvement by means of transgenic lines with artificial miRNAs.201627349470
51650.9198Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence. Most bacteria possess alcohol dehydrogenase (ADH) genes (Adh genes) to mitigate alcohol toxicity, but these genes have functions beyond alcohol degradation. Previous research has shown that ADH can modulate quorum sensing in Acinetobacter baumannii, a rising opportunistic pathogen. However, the number and nature of Adh genes in A. baumannii have not yet been fully characterized. We identified seven alcohol dehydrogenases (NAD(+)-ADHs) from A. baumannii ATCC 19606, and examined the roles of three iron-containing ADHs, ADH3, ADH4, and ADH6. Marker-less mutation was used to generate Adh3, Adh4, and Adh6 single, double, and triple mutants. Disrupted Adh4 mutants failed to grow in ethanol-, 1-butanol-, or 1-propanol-containing mediums, and recombinant ADH4 exhibited strongest activity against ethanol. Stress resistance assays with inorganic and organic hydroperoxides showed that Adh3 and Adh6 were key to oxidative stress resistance. Virulence assays performed on the Galleria mellonella model organism revealed that Adh4 mutants had comparable virulence to wild-type, while Adh3 and Adh6 mutants had reduced virulence. The results suggest that ADH4 is primarily involved in alcohol metabolism, while ADH3 and ADH6 are key to stress resistance and virulence. Further investigation into the roles of other ADHs in A. baumannii is warranted.202134576087
80160.9196Redox-sensitive transcriptional regulator SoxR directly controls antibiotic production, development and thiol-oxidative stress response in Streptomyces avermitilis. The redox-sensitive transcriptional regulator SoxR is conserved in bacteria. Its role in mediating protective response to various oxidative stresses in Escherichia coli and related enteric bacteria has been well established. However, functions and regulatory mechanisms of SoxR in filamentous Streptomyces, which produce half of known antibiotics, are unclear. We report here that SoxR pleiotropically regulates antibiotic production, morphological development, primary metabolism and thiol-oxidative stress response in industrially important species Streptomyces avermitilis. SoxR stimulated avermectin production by directly activating ave structural genes. Four genes (sav_3956, sav_4018, sav_5665 and sav_7218) that are homologous to targets of S. coelicolor SoxR are targeted by S. avermitilis SoxR. A consensus 18-nt SoxR-binding site, 5'-VSYCNVVMHNKVKDGMGB-3', was identified in promoter regions of sav_3956, sav_4018, sav_5665, sav_7218 and target ave genes, leading to prediction of the SoxR regulon and confirmation of 11 new targets involved in development (ftsH), oligomycin A biosynthesis (olmRI), primary metabolism (metB, sav_1623, plcA, nirB, thiG, ndh2), transport (smoE) and regulatory function (sig57, sav_7278). SoxR also directly activated three key developmental genes (amfC, whiB and ftsZ) and promoted resistance of S. avermitilis to thiol-oxidative stress through activation of target trx and msh genes. Overexpression of soxR notably enhanced antibiotic production in S. avermitilis and S. coelicolor. Our findings expand our limited knowledge of SoxR and will facilitate improvement of methods for antibiotic overproduction in Streptomyces species.202233951287
506270.9196sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51. The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.202438718038
82780.9194Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient. Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6 % G + C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66 % with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, bla(OXA-61), tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.202439208964
80590.9190LexR 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
8440100.9189A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection. A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13.IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.201931594818
806110.9189A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069
4452120.9186Whole-Genome Analysis of Acinetobacter baumannii Strain AB43 Containing a Type I-Fb CRISPR-Cas System: Insights into the Relationship with Drug Resistance. The CRISPR-Cas system is a bacterial and archaea adaptive immune system and is a newly recognized mechanism for controlling antibiotic resistance gene transfer. Acinetobacter baumannii (A. baumannii) is an important organism responsible for a variety of nosocomial infections. A. baumannii infections have become problematic worldwide because of the resistance of A. baumannii to multiple antibiotics. Thus, it is clinically significant to explore the relationship between the CRISPR-Cas system and drug resistance in A. baumannii. This study aimed to analyze the genomic characteristics of the A. baumannii strain AB3 containing the type I-Fb CRISPR-Cas system, which was isolated from a tertiary care hospital in China, and to investigate the relationship between the CRISPR-Cas system and antibiotic resistance in this strain. The whole-genome sequencing (WGS) of the AB43 strain was performed using Illumina and PacBio sequencing. The complete genome of AB43 consisted of a 3,854,806 bp chromosome and a 104,309 bp plasmid. The specific characteristics of the CRISPR-Cas system in AB43 are described as follows: (1) The strain AB43 carries a complete type I-Fb CRISPR-Cas system; (2) Homology analysis confirmed that the cas genes in AB43 share high sequence similarity with the same subtype cas genes; (3) A total of 28 of 105 A. baumannii AB43 CRISPR spacers matched genes in the bacteriophage genome database and the plasmid database, implying that the CRISPR-Cas system in AB43 provides immunity against invasive bacteriophage and plasmids; (4) None of the CRISPR spacers in A. baumannii AB43 were matched with antimicrobial resistance genes in the NCBI database. In addition, we analyzed the presence of antibiotic resistance genes and insertion sequences in the AB43 strain and found that the number of antibiotic resistance genes was not lower than in the "no CRISPR-Cas system" strain. This study supports the idea that the CRISPR-Cas system may inhibit drug-resistance gene expression via endogenous gene regulation, except to the published mechanism that the CRISPR-Cas system efficiently limits the acquisition of antibiotic resistance genes that make bacteria sensitive to antibiotics.202236080431
6173130.9186Mutation 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
9048140.9185RNA 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
5170150.9183Synergistic 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
5192160.9182Genome Sequencing Analysis of a Rare Case of Blood Infection Caused by Flavonifractor plautii. BACKGROUND Flavonifractor plautii belongs to the clostridium family, which can lead to local infections as well as the bloodstream infections. Flavonifractor plautii caused infection is rarely few in the clinic. To understand better Flavonifractor plautii, we investigated the drug sensitivity and perform genome sequencing of Flavonifractor plautii isolated from blood samples in China and explored the drug resistance and pathogenic mechanism of the bacteria. CASE REPORT The Epsilometer test method was used to detect the sensitivity of flavonoid bacteria to antimicrobial agents. PacBio sequencing technology was employed to sequence the whole genome of Flavonifractor plautii, and gene prediction and functional annotation were also analyzed. Flavonifractor plautii displayed sensitivity to most drugs but resistance to fluoroquinolones and tetracycline, potentially mediated by tet (W/N/W). The total genome size of Flavonifractor plautii was 4,573,303 bp, and the GC content was 59.78%. Genome prediction identified 4,506 open reading frames, including 9 ribosomal RNAs and 66 transfer RNAs. It was detected that the main virulence factor-coding genes of the bacteria were the capsule, polar flagella and FbpABC, which may be associated with bacterial movement, adhesion, and biofilm formation. CONCLUSIONS The results of whole-genome sequencing could provide relevant information about the drug resistance mechanism and pathogenic mechanism of bacteria and offer a basis for clinical diagnosis and treatment.202438881048
659170.9180Generic and specific adaptive responses of Streptococcus pneumoniae to challenge with three distinct antimicrobial peptides, bacitracin, LL-37, and nisin. To investigate the response of Streptococcus pneumoniae to three distinct antimicrobial peptides (AMPs), bacitracin, nisin, and LL-37, transcriptome analysis of challenged bacteria was performed. Only a limited number of genes were found to be up- or downregulated in all cases. Several of these common highly induced genes were chosen for further analysis, i.e., SP0385-SP0387 (SP0385-0387 herein), SP0912-0913, SP0785-0787, SP1714-1715, and the blp gene cluster. Deletion of these genes in combination with MIC determinations showed that several putative transporters, i.e., SP0785-0787 and SP0912-0913, were indeed involved in resistance to lincomycin and LL-37 and to bacitracin, nisin, and lincomycin, respectively. Mutation of the blp bacteriocin immunity genes resulted in an increased sensitivity to LL-37. Interestingly, a putative ABC transporter (SP1715) protected against bacitracin and Hoechst 33342 but conferred sensitivity to LL-37. A GntR-like regulator, SP1714, was identified as a negative regulator of itself and two of the putative transporters. In conclusion, we show that resistance to three different AMPs in S. pneumoniae is mediated by several putative ABC transporters, some of which have not been associated with antimicrobial resistance in this organism before. In addition, a GntR-like regulator that regulates two of these transporters was identified. Our findings extend the understanding of defense mechanisms of this important human pathogen against antimicrobial compounds and point toward novel proteins, i.e., putative ABC transporters, which can be used as targets for the development of new antimicrobials.201019917758
9020180.9178Transcriptome Analysis Reveals the Resistance Mechanism of Pseudomonas aeruginosa to Tachyplesin I. BACKGROUND: Tachyplesin I is a cationic antimicrobial peptide with a typical cyclic antiparallel β-sheet structure. We previously demonstrated that long-term continuous exposure to increased concentration of tachyplesin I can induce resistant Gram-negative bacteria. However, no significant information is available about the resistance mechanism of Pseudomonas aeruginosa (P. aeruginosa) to tachyplesin I. MATERIALS AND METHODS: In this study, the global gene expression profiling of P. aeruginosa strain PA-99 and P. aeruginosa CGMCC1.2620 (PA1.2620) was conducted using transcriptome sequencing. For this purpose, outer membrane permeability and outer membrane proteins (OMPs) were further analyzed. RESULTS: Transcriptome sequencing detected 672 upregulated and 787 downregulated genes, covering Clusters of Orthologous Groups (COGs) of P. aeruginosa strain PA-99 compared with PA1.2620. Totally, 749 differentially expressed genes (DEGs) were assigned to 98 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and among them, a two-component regulatory system, a beta-lactam resistance system, etc. were involved in some known genes resistant to drugs. Additionally, we further attempted to indicate whether the resistance mechanism of P. aeruginosa to tachyplesin I was associated with the changes of outer membrane permeability and OMPs. CONCLUSION: Our results indicated that P. aeruginosa resistant to tachyplesin I was mainly related to reduced entry of tachyplesin I into the bacterial cell due to overexpression of efflux pump, in addition to a decrease of outer membrane permeability. Our findings were also validated by pathway enrichment analysis and quantitative reverse transcription polymerase chain reaction (RT-qPCR). This study may provide a promising guidance for understanding the resistance mechanism of P. aeruginosa to tachyplesin I.202032021330
5883190.9178Genome-wide analysis reveals the emergence of multidrug resistant Stenotrophomonas acidaminiphila strain SINDOREI isolated from a patient with sepsis. Stenotrophomonas acidaminiphila, the most recent reported species in genus Stenotrophomonas, is a relatively rare bacteria and is an aerobic, glucose non-fermentative, Gram-negative bacterium. However, little information of S. acidaminiphila is known to cause human infections. In this research, we firstly reported a multidrug-resistant strain S. acidaminiphila SINDOREI isolated from the blood of a patient with sepsis, who was dead of infection eventually. The whole genome of strain SINDOREI was sequenced, and genome comparisons were performed among six closely related S. acidaminiphila strains. The core genes (2,506 genes) and strain-specific genes were identified, respectively, to know about the strain-level diversity in six S. acidaminiphila stains. The presence of a unique gene (narG) and essential genes involved in biofilm formation in strain SINDOREI are important for the pathogenesis of infections. Strain SINDOREI was resistant to trimethoprim/sulfamethoxazole, ciprofloxacin, ofloxacin, cefepime, ceftazidime, and aztreonam. Several common and specific antibiotic resistance genes were identified in strain SINDOREI. The presence of two sul genes and exclusive determinants GES-1, aadA3, qacL, and cmlA5 is responsible for the resistance to multidrug. The virulence factors and resistance determinants can show the relationship between the phenotype and genotype and afford potential therapeutic strategies for infections.202236212813