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900900.8966Experimental Evolution of Magnetite Nanoparticle Resistance in Escherichia coli. Both ionic and nanoparticle iron have been proposed as materials to control multidrug-resistant (MDR) bacteria. However, the potential bacteria to evolve resistance to nanoparticle bacteria remains unexplored. To this end, experimental evolution was utilized to produce five magnetite nanoparticle-resistant (FeNP(1-5)) populations of Escherichia coli. The control populations were not exposed to magnetite nanoparticles. The 24-h growth of these replicates was evaluated in the presence of increasing concentrations magnetite NPs as well as other ionic metals (gallium III, iron II, iron III, and silver I) and antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline). Scanning electron microscopy was utilized to determine cell size and shape in response to magnetite nanoparticle selection. Whole genome sequencing was carried out to determine if any genomic changes resulted from magnetite nanoparticle resistance. After 25 days of selection, magnetite resistance was evident in the FeNP treatment. The FeNP populations also showed a highly significantly (p < 0.0001) greater 24-h growth as measured by optical density in metals (Fe (II), Fe (III), Ga (III), Ag, and Cu II) as well as antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline). The FeNP-resistant populations also showed a significantly greater cell length compared to controls (p < 0.001). Genomic analysis of FeNP identified both polymorphisms and hard selective sweeps in the RNA polymerase genes rpoA, rpoB, and rpoC. Collectively, our results show that E. coli can rapidly evolve resistance to magnetite nanoparticles and that this result is correlated resistances to other metals and antibiotics. There were also changes in cell morphology resulting from adaptation to magnetite NPs. Thus, the various applications of magnetite nanoparticles could result in unanticipated changes in resistance to both metal and antibiotics.202133808798
82810.8958Screening for Resistant Bacteria, Antimicrobial Resistance Genes, Sexually Transmitted Infections and Schistosoma spp. in Tissue Samples from Predominantly Vaginally Delivered Placentae in Ivory Coast and Ghana. Medical complications during pregnancy have been frequently reported from Western Africa with a particular importance of infectious complications. Placental tissue can either become the target of infectious agents itself, such as, e.g., in the case of urogenital schistosomiasis, or be subjected to contamination with colonizing or infection-associated microorganisms of the cervix or the vagina during vaginal delivery. In the retrospective cross-sectional assessment presented here, the quantitative dimension of infection or colonization with selected resistant or pathogenic bacteria and parasites was regionally assessed. To do so, 274 collected placental tissues from Ivory Coastal and Ghanaian women were subjected to selective growth of resistant bacteria, as well as to molecular screening for beta-lactamase genes, Schistosoma spp. and selected bacterial causative agents of sexually transmitted infections (STI). Panton-Valentine-negative methicillin-resistant Staphylococcus aureus (MRSA) was grown from 1.8% of the tissue samples, comprising the spa types t008 and t688, as well as the newly detected ones, t12101 (n = 2) and t12102. While the culture-based recovery of resistant Enterobacterales and nonfermentative rod-shaped Gram-negative bacteria failed, molecular assessments confirmed beta-lactamase genes in 31.0% of the samples with multiple detections of up to four resistance genes per sample and bla(CTX-M), bla(IMP), bla(GES), bla(VIM), bla(OXA-58)-like, bla(NDM), bla(OXA-23)-like, bla(OXA-48)-like and bla(KPC) occurring in descending order of frequency. The beta-lactamase genes bla(OXA-40/24)-like, bla(NMC_A/IMI), bla(BIC), bla(SME), bla(GIM) and bla(DIM) were not detected. DNA of the urogenital schistosomiasis-associated Schistosoma haematobium complex was recorded in 18.6% of the samples, but only a single positive signal for S. mansoni with a high cycle-threshold value in real-time PCR was found. Of note, higher rates of schistosomiasis were observed in Ghana (54.9% vs. 10.3% in Ivory Coast) and Cesarean section was much more frequent in schistosomiasis patients (61.9% vs. 14.8% in women without Schistosoma spp. DNA in the placenta). Nucleic acid sequences of nonlymphogranuloma-venereum-associated Chlamydia trachomatis and of Neisseria gonorrhoeae were recorded in 1.1% and 1.9% of the samples, respectively, while molecular attempts to diagnose Treponema pallidum and Mycoplasma genitalium did not lead to positive results. Molecular detection of Schistosoma spp. or STI-associated pathogens was only exceptionally associated with multiple resistance gene detections in the same sample, suggesting epidemiological distinctness. In conclusion, the assessment confirmed considerable prevalence of urogenital schistosomiasis and resistant bacterial colonization, as well as a regionally expected abundance of STI-associated pathogens. Continuous screening offers seem advisable to minimize the risks for the pregnant women and their newborns.202337623959
543820.8947Genomic Insights into Staphylococcus aureus Isolates Exhibiting Diminished Daptomycin Susceptibility. Daptomycin is one of the last therapeutic resources for multidrug-resistant gram-positive bacteria. Despite its structural similarities with glycopeptides, its mechanisms of action and resistance are different and in some respects are not completely understood. Mutations in several genes have been associated with daptomycin resistance, especially in mprF, walkR, rpoB and rpoC, but their role and importance remain to be elucidated. We have studied mutations in 11 genes, which have been previously associated with daptomycin non-susceptibility, in nine daptomycin-non-susceptible Staphylococcus aureus clinical isolates (daptomycin MIC: >1 mg/L). Susceptibility to daptomycin, vancomycin, linezolid, oxacillin, telavancin and dalbavancin was studied. walkR, agrA, cls1, cls2, fakA, pnpA, clpP, prs, rpoB, rpoC and mprF were amplified by PCR and sequenced. The sequences were compared with the S. aureus ATCC 25923 complete genome (GenBank gi: 685631213) by using BLAST(®) software. We did not find any changes in walkR, pnpA, prs and clpP. All isolates excepting isolate MSa5 showed a high number of significant mutations (between 13 and 25 amino acid changes) in mprF. Most isolates also showed mutations in the rpo genes, the cls genes and fakA. Daptomycin non-susceptibility in S. aureus clinical isolates seems to be reached through different mutation combinations when compared to S. aureus ATCC 25293. Especially mprF and cls1 showed very high polymorphism in most isolates. Meanwhile, one isolate, MSa5, showed only single mutation in mprF (P314T).202438535549
538030.8946In Vitro Screening of a 1280 FDA-Approved Drugs Library against Multidrug-Resistant and Extensively Drug-Resistant Bacteria. Alternative strategies against multidrug-resistant (MDR) bacterial infections are suggested to clinicians, such as drug repurposing, which uses rapidly available and marketed drugs. We gathered a collection of MDR bacteria from our hospital and performed a phenotypic high-throughput screening with a 1280 FDA-approved drug library. We used two Gram positive (Enterococcus faecium P5014 and Staphylococcus aureus P1943) and six Gram negative (Acinetobacter baumannii P1887, Klebsiella pneumoniae P9495, Pseudomonas aeruginosa P6540, Burkholderia multivorans P6539, Pandoraea nosoerga P8103, and Escherichia coli DSM105182 as the reference and control strain). The selected MDR strain panel carried resistance genes or displayed phenotypic resistance to last-line therapies such as carbapenems, vancomycin, or colistin. A total of 107 compounds from nine therapeutic classes inhibited >90% of the growth of the selected Gram negative and Gram positive bacteria at a drug concentration set at 10 µmol/L, and 7.5% were anticancer drugs. The common hit was the antiseptic chlorhexidine. The activity of niclosamide, carmofur, and auranofin was found against the selected methicillin-resistant S. aureus. Zidovudine was effective against colistin-resistant E. coli and carbapenem-resistant K. pneumoniae. Trifluridine, an antiviral, was effective against E. faecium. Deferoxamine mesylate inhibited the growth of XDR P. nosoerga. Drug repurposing by an in vitro screening of a drug library is a promising approach to identify effective drugs for specific bacteria.202235326755
537940.8945Membrane-Targeting Triphenylphosphonium Functionalized Ciprofloxacin for Methicillin-Resistant Staphylococcus aureus (MRSA). Multidrug-resistant (MDR) bacteria have become a severe problem for public health. Developing new antibiotics for MDR bacteria is difficult, from inception to the clinically approved stage. Here, we have used a new approach, modification of an antibiotic, ciprofloxacin (CFX), with triphenylphosphonium (TPP, PPh(3)) moiety via ester- (CFX-ester-PPh(3)) and amide-coupling (CFX-amide-PPh(3)) to target bacterial membranes. In this study, we have evaluated the antibacterial activities of CFX and its derivatives against 16 species of bacteria, including MDR bacteria, using minimum inhibitory concentration (MIC) assay, morphological monitoring, and expression of resistance-related genes. TPP-conjugated CFX, CFX-ester-PPh(3), and CFX-amide-PPh(3) showed significantly improved antibacterial activity against Gram-positive bacteria, Staphylococcus aureus, including MDR S. aureus (methicillin-resistant S. aureus (MRSA)) strains. The MRSA ST5 5016 strain showed high antibacterial activity, with MIC values of 11.12 µg/mL for CFX-ester-PPh(3) and 2.78 µg/mL for CFX-amide-PPh(3). The CFX derivatives inhibited biofilm formation in MRSA by more than 74.9% of CFX-amide-PPh(3). In the sub-MIC, CFX derivatives induced significant morphological changes in MRSA, including irregular deformation and membrane disruption, accompanied by a decrease in the level of resistance-related gene expression. With these promising results, this method is very likely to combat MDR bacteria through a simple TPP moiety modification of known antibiotics, which can be readily prepared at clinical sites.202033143023
517150.8938Adaptive laboratory-evolved MRSA with PPEF manifests cross-susceptibility to oxacillin and hypersensitivity to ciprofloxacin. Emerging resistance to current antibiotics is a global threat to human health. Therefore, comprehending the mechanism behind antibiotic resistance holds paramount importance. In the pursuit of finding new antibacterial agents, our group has developed a small molecule, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole), having bisbenzimidazole as a pharmacophore, targeting bacterial type IA topoisomerase, a novel drug target in bacteria. We examined the emergence of mutations leading to PPEF resistance in laboratory-evolved Staphylococcus aureus strains. The growth curve revealed that S. aureus 25923 PPEF-resistant (SA-PR) and methicillin-resistant S. aureus 43300 PPEF-resistant (MRSA-PR) attained stationary phase earlier than their respective reference strains. RNA sequencing analysis revealed that atpD (ATP synthase gene) was downregulated by 2 log(2)-fold in both SA-PR and MRSA-PR strains, whereas there was 10 to 13 log(2)-fold downregulation of mecR1 (methicillin resistance-inducing gene), ble (bleomycin resistance-inducing gene), blaZ (beta-lactamase), pbp (penicillin-binding protein gene), ermA (rRNA adenine methyltransferase gene), and kdpB (potassium-transporting ATPase) in the MRSA-PR strain. Quantitative reverse-transcriptase PCR data confirmed these results. Additionally, MRSA-PR showed a 5 log(2)-fold upregulation of comG and a 9 log(2)-fold downregulation of topB, indicating increased genomic variability and stress adaptation contributing to resistance. Genomic sequencing revealed deletions of resistance genes, including aac(6')-aph(2''), aadD, mecA, and blaZ in MRSA-PR, resulting in a gain in resistance and a diminishing returns epistasis pattern in PPEF-evolved S. aureus strains. This led to the development of an evolved MRSA-PR strain susceptible to oxacillin, ciprofloxacin, gentamicin, and imipenem. Our findings indicate that adaptation to PPEF has increased antibiotic susceptibility, thereby changing the clinical outcomes of infections.IMPORTANCEThis study investigates how Staphylococcus aureus bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) strain, develop resistance to a new candidate antibacterial compound, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole). The research found that resistant strains grew slower and showed significant changes in the activity of genes related to antibiotic resistance. Some resistance genes were deleted in the resistant MRSA strain, making it more sensitive to other antibiotics like oxacillin and ciprofloxacin. These findings highlight how resistance to PPEF leads to increased sensitivity to conventional antibiotics. This suggests that developing combination therapies of PPEF with other antibiotics could optimize treatment regimens and slow resistance evolution. This study also indicates that the antibiotic regimens could be designed to force resistant bacteria into evolutionary trade-offs, where they lose resistance to widely used antibiotics while gaining resistance to a new compound like PPEF.202540662666
518160.8935Differential Expression of fimH, ihf, upaB, and upaH Genes in Biofilms- and Suspension-Grown Bacteria From Samples of Different Uropathogenic Strains of Escherichia coli. Uropathogenic Escherichia coli (UPEC) strains are the main bacteria that cause urinary tract infections (UTIs). UPEC are a significant public health hazard due to their high proliferation, antibiotic resistance, and infection recurrence. The ability to form biofilms is a mechanism of antibiotic resistance, which requires the expression of different genes such as fimH, ihf, upaB, and upaH. Despite the relevance of biofilm formation in bacterial pathogenicity, differences in the expression level of these genes among bacterial growth conditions have been little studied. Here, we have characterized the expression of fimH, ihf, upaB, and upaH genes in biofilms and suspension-grown bacteria of different E. coli strains. These included the UPEC CFT073, the multidrug-resistant strain CDC-AR-0346, and clinical isolates obtained from UTI patients. The expression of fimH, ihf, upaB, and upaH was markedly heterogeneous in clinical isolates, both in terms of transcript levels and response to suspension or biofilm conditions. That expression pattern was distinct from the one in UPEC CFT073, where upaB and upaH were upregulated and ihf and fimH were slightly downregulated in biofilm. In conclusion, the data presented here show that the pattern of biofilm-associated genes in the clinical isolates from UTI patients is not fully related to the reference strain of UPEC CFT073. However, analysis of a larger number of samples is required.202439703715
601270.8934Metal resistance-related genes are differently expressed in response to copper and zinc ion in six Acidithiobacillus ferrooxidans strains. Metal resistance of acidophilic bacteria is very significant during bioleaching of copper ores since high concentration of metal is harmful to the growth of microorganisms. The resistance levels of six Acidithiobacillus ferrooxidans strains to 0.15 M copper and 0.2 M zinc were investigated, and eight metal resistance-related genes (afe-0022, afe-0326, afe-0329, afe-1143, afe-0602, afe-0603, afe-0604, and afe-1788) were sequenced and analyzed. The transcriptional expression levels of eight possible metal tolerance genes in six A. ferrooxidans strains exposed to 0.15 M Cu(2+) and 0.2 M Zn(2+) were determined by real-time quantitative PCR (RT-qPCR), respectively. The copper resistance levels of six A. ferrooxidans strains declined followed by DY26, DX5, DY15, GD-B, GD-0, and YTW. The zinc tolerance levels of six A. ferrooxidans strains exposed to 0.2 M Zn(2+) from high to low were YTW > GD-B > DY26 > GD-0 > DX5 > DY15. Seven metal tolerance-related genes all presented in the genome of six strains, except afe-0604. The metal resistance-related genes showed different transcriptional expression patterns in six A. ferrooxidans strains. The expression of gene afe-0326 and afe-0022 in six A. ferrooxidans strains in response to 0.15 M Cu(2+) showed the same trend with the resistance levels. The expression levels of genes afe-0602, afe-0603, afe-0604, and afe-1788 in six strains response to 0.2 M Zn(2+) did not show a clear correlation between the zinc tolerance levels of six strains. According to the results of RT-qPCR and bioinformatics analysis, the proteins encoded by afe-0022, afe-0326, afe-0329, and afe-1143 were related to Cu(2+) transport of A. ferrooxidans strains.201425023638
580280.8931Dissecting vancomycin-intermediate resistance in staphylococcus aureus using genome-wide association. Vancomycin-intermediate Staphylococcus aureus (VISA) is currently defined as having minimal inhibitory concentration (MIC) of 4-8 µg/ml. VISA evolves through changes in multiple genetic loci with at least 16 candidate genes identified in clinical and in vitro-selected VISA strains. We report a whole-genome comparative analysis of 49 vancomycin-sensitive S. aureus and 26 VISA strains. Resistance to vancomycin was determined by broth microdilution, Etest, and population analysis profile-area under the curve (PAP-AUC). Genome-wide association studies (GWAS) of 55,977 single-nucleotide polymorphisms identified in one or more strains found one highly significant association (P = 8.78 E-08) between a nonsynonymous mutation at codon 481 (H481) of the rpoB gene and increased vancomycin MIC. Additionally, we used a database of public S. aureus genome sequences to identify rare mutations in candidate genes associated with VISA. On the basis of these data, we proposed a preliminary model called ECM+RMCG for the VISA phenotype as a benchmark for future efforts. The model predicted VISA based on the presence of a rare mutation in a set of candidate genes (walKR, vraSR, graSR, and agrA) and/or three previously experimentally verified mutations (including the rpoB H481 locus) with an accuracy of 81% and a sensitivity of 73%. Further, the level of resistance measured by both Etest and PAP-AUC regressed positively with the number of mutations present in a strain. This study demonstrated 1) the power of GWAS for identifying common genetic variants associated with antibiotic resistance in bacteria and 2) that rare mutations in candidate gene, identified using large genomic data sets, can also be associated with resistance phenotypes.201424787619
537590.8931Mechanism of Eravacycline Resistance in Clinical Enterococcus faecalis Isolates From China. Opportunistic infections caused by multidrug-resistant Enterococcus faecalis strains are a significant clinical challenge. Eravacycline (Erava) is a synthetic fluorocycline structurally similar to tigecycline (Tige) that exhibits robust antimicrobial activity against Gram-positive bacteria. This study investigated the in vitro antimicrobial activity and heteroresistance risk of Eravacycline (Erava) in clinical E. faecalis isolates from China along with the mechanism of Erava resistance. A total of 276 non-duplicate E. faecalis isolates were retrospectively collected from a tertiary care hospital in China. Heteroresistance to Erava and the influence of tetracycline (Tet) resistance genes on Erava susceptibility were examined. To clarify the molecular basis for Erava resistance, E. faecalis variants exhibiting Erava-induced resistance were selected under Erava pressure. The relative transcript levels of six candidate genes linked to Erava susceptibility were determined by quantitative reverse-transcription PCR, and their role in Erava resistance and heteroresistance was evaluated by in vitro overexpression experiments. We found that Erava minimum inhibitory concentrations (MICs) against clinical E. faecalis isolates ranged from ≤0.015 to 0.25 mg/l even in strains harboring Tet resistance genes. The detection frequency of Erava heteroresistance in isolates with MICs ≤ 0.06, 0.125, and 0.25 mg/l were 0.43% (1/231), 7.5% (3/40), and 0 (0/5), respectively. No mutations were detected in the 30S ribosomal subunit gene in Erava heteroresistance-derived clones, although mutations in this subunit conferred cross resistance to Tige in Erava-induced resistant E. faecalis. Overexpressing RS00630 (encoding a bone morphogenetic protein family ATP-binding cassette transporter substrate-binding protein) in E. faecalis increased the frequency of Erava and Tige heteroresistance, whereas RS12140, RS06145, and RS06880 overexpression conferred heteroresistance to Tige only. These results indicate that Erava has potent in vitro antimicrobial activity against clinical E. faecalis isolates from China and that Erava heteroresistance can be induced by RS00630 overexpression.202032523563
6159100.8931Gene expression profiling of Cecropin B-resistant Haemophilus parasuis. Synthetically designed antimicrobial peptides (AMPs) present the potential of replacing antibiotics in the treatment of bacterial infections. However, microbial resistance to AMPs has been reported and little is known regarding the underlying mechanism of such resistance. The naturally occurring AMP cecropin B (CB) disrupts the anionic cell membranes of Gram-negative bacteria. In this study, CB resistance (CBR) was induced in Haemophilusparasuis SH0165 by exposing it to a series of CB concentrations. The CB-resistant H.parasuis strains CBR30 and CBR30-50 were obtained. The growth curves of SH0165 and CBR30 showed that CBR30 displayed lower growth rates than SH0165. The result of transmission electron microscopy showed cell membranes of the CB-resistant CBR30 and CBR30-50 were smoother than SH0165. Microarrays detected 257 upregulated and 254 downregulated genes covering 20 clusters of orthologous groups (COGs) of the CB-resistant CBR30 compared with SH0165 (>1.5-fold change, p < 0.05). Sixty genes were affected in CBR30-50 covering 18 COGs, with 28 upregulated and 32 downregulated genes. Under the COG function classification, the majority of affected genes in the CB-resistant CBR30 and CBR30-50 belong to the category of inorganic ion transport, amino acid transport, and metabolism. The microarray results were validated by real-time quantitative reverse transcription PCR. This study may provide useful guidance for understanding the molecular mechanism underlying H.parasuis resistance to CB.201424862339
5234110.8930A Multidrug-Resistant Escherichia coli Caused the Death of the Chinese Soft-Shelled Turtle (Pelodiscus sinensis). The rapid increase in drug resistance in recent years has become a significant global public health concern. Escherichia coli are ubiquitous bacteria, widely distributed in various environments. This study isolated a bacterial strain (HD-593) from diseased Chinese soft-shelled turtles (Pelodiscus sinensis). The bacterium was identified based on morphology, biochemical tests, and 16S rRNA sequencing, confirming it as E. coli. Drug susceptibility tests revealed that the HD-593 strain was highly resistant to ceftriaxone, enrofloxacin, doxycycline, sulfadiazine, gentamicin, neomycin, florfenicol, carbenicillin, cefradine, erythromycin, penicillin, ampicillin, midecamycin, and streptomycin. Resistance gene analysis confirmed the presence of quinolone resistance genes (oqxA and oqxB), aminoglycoside resistance genes (aac(3)-II and aphA1), a β-lactam resistance gene (blaTEM), and an acylaminol resistance gene (floR) in HD-593. The median lethal dose (LD50) of HD-593 for P. sinensis was 6.53 × 10(5) CFU/g. Biochemical analysis of serum revealed that HD-593 infection caused a significant reduction in total protein, albumin, and globulin levels, while markedly increasing the levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Histopathological analysis revealed severe intestinal damage characterized by villi detachment and muscle cell necrosis. Additionally, extensive splenocyte necrosis with nuclear marginalization, glomerular swelling, and pronounced hepatic steatosis accompanied by distended sinusoids were observed. This study identified a multidrug-resistant E. coli strain from deceased P. sinensis, suggesting that drug resistance genes may circulate in aquaculture ecosystems, posing potential risks to aquaculture.202540431566
1400120.8929Comparative 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
6189130.8928Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus. Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.201323894076
9033140.8928Stress Resistance Development and Genome-Wide Transcriptional Response of Escherichia coli O157:H7 Adapted to Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde. Thymol, carvacrol, and trans-cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including Escherichia coli O157:H7. However, little is known regarding direct resistance and cross-resistance development in E. coli O157:H7 after adaptation to sublethal levels of these compounds, and information is scarce on microbial adaptive responses at a molecular level. The present study demonstrated that E. coli O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), displaying an extended lag phase duration and a lower maximum growth rate. EO-adapted cells developed direct resistance against lethal EO treatments and cross-resistance against heat (58°C) and oxidative (50 mM H(2)O(2)) stresses. However, no induction of acid resistance (simulated gastric fluid, pH 1.5) was observed. RNA sequencing revealed a large number (310 to 338) of differentially expressed (adjusted P value [P(adj) ], <0.05; fold change, ≥5) genes in 1/2T and 1/2C cells, while 1/2TC cells only showed 27 genes with altered expression. In accordance with resistance phenotypes, the genes related to membrane, heat, and oxidative stress responses and genes related to iron uptake and metabolism were upregulated. Conversely, virulence genes associated with motility, biofilm formation, and efflux pumps were repressed. This study demonstrated the development of direct resistance and cross-resistance and characterized whole-genome transcriptional responses in E. coli O157:H7 adapted to sublethal thymol, carvacrol, or trans-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in E. coli O157:H7.IMPORTANCE The present study was designed to understand transcriptomic changes and the potential development of direct and cross-resistance in essential oil (EO)-adapted Escherichia coli O157:H7. The results demonstrated altered growth behaviors of E. coli O157:H7 during adaptation in sublethal thymol, carvacrol, and trans-cinnamaldehyde. Generally, EO-adapted bacteria showed enhanced resistance against subsequent lethal EO, heat, and oxidative stresses, with no induction of acid resistance in simulated gastric fluid. A transcriptomic analysis revealed the upregulation of related stress resistance genes and a downregulation of various virulence genes in EO-adapted cells. This study provides new insights into microbial EO adaptation behaviors and highlights the risk of resistance development in adapted bacteria.201830217837
6376150.8927Mechanisms of mepA Overexpression and Membrane Potential Reduction Leading to Ciprofloxacin Heteroresistance in a Staphylococcus aureus Isolate. Heteroresistance has seriously affected the evaluation of antibiotic efficacy against pathogenic bacteria, causing misjudgment of antibiotics' sensitivity in clinical therapy, leading to treatment failure, and posing a serious threat to current medical health. However, the mechanism of Staphylococcus aureus heteroresistance to ciprofloxacin remains unclear. In this study, heteroresistance to ciprofloxacin in S. aureus strain 529 was confirmed by antimicrobial susceptibility testing and population analysis profiling (PAP), with the resistance of subclonal 529_HR based on MIC being 8-fold that of the original bacteria. A 7-day serial MIC evaluation and growth curves demonstrate that their phenotype was stable, with 529_HR growing more slowly than 529, but reaching a plateau in a similar proportion. WGS analysis showed that there were 11 nonsynonymous mutations and one deletion gene between the two bacteria, but none of these SNPs were directly associated with ciprofloxacin resistance. Transcriptome data analysis showed that the expression of membrane potential related genes (qoxA, qoxB, qoxC, qoxD, mprF) was downregulated, and the expression of multidrug resistance efflux pump gene mepA was upregulated. The combination of ciprofloxacin and limonene restored the 529_HR MIC from 1 mg/L to 0.125 mg/L. Measurement of the membrane potential found that 529_HR had a lower potential, which may enable it to withstand the ciprofloxacin-induced decrease in membrane potential. In summary, we demonstrated that upregulation of mepA gene expression and a reduction in membrane potential are the main heteroresistance mechanisms of S. aureus to ciprofloxacin. Additionally, limonene may be a potentially effective agent to inhibit ciprofloxacin heteroresistance phenotypes.202540076991
9054160.8926Clinically Relevant Concentrations of Polymyxin B and Meropenem Synergistically Kill Multidrug-Resistant Pseudomonas aeruginosa and Minimize Biofilm Formation. The emergence of antibiotic resistance has severely impaired the treatment of chronic respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. Since the reintroduction of polymyxins as a last-line therapy against MDR Gram-negative bacteria, resistance to its monotherapy and recurrent infections continue to be reported and synergistic antibiotic combinations have been investigated. In this study, comprehensive in vitro microbiological evaluations including synergy panel screening, population analysis profiling, time-kill kinetics, anti-biofilm formation and membrane damage analysis studies were conducted to evaluate the combination of polymyxin B and meropenem against biofilm-producing, polymyxin-resistant MDR P. aeruginosa. Two phylogenetically unrelated MDR P. aeruginosa strains, FADDI-PA060 (MIC of polymyxin B [MIC(polymyxin B)], 64 mg/L; MIC(meropenem), 64 mg/L) and FADDI-PA107 (MIC(polymyxin B), 32 mg/L; MIC(meropenem), 4 mg/L) were investigated. Genome sequencing identified 57 (FADDI-PA060) and 50 (FADDI-PA107) genes predicted to confer resistance to a variety of antimicrobials, as well as multiple virulence factors in each strain. The presence of resistance genes to a particular antibiotic class generally aligned with MIC results. For both strains, all monotherapies of polymyxin B failed with substantial regrowth and biofilm formation. The combination of polymyxin B (16 mg/L)/meropenem (16 mg/L) was most effective, enhancing initial bacterial killing of FADDI-PA060 by ~3 log(10) CFU/mL, followed by a prolonged inhibition of regrowth for up to 24 h with a significant reduction in biofilm formation (* p < 0.05). Membrane integrity studies revealed a substantial increase in membrane depolarization and membrane permeability in the surviving cells. Against FADDI-PA107, planktonic and biofilm bacteria were completely eradicated. In summary, the combination of polymyxin B and meropenem demonstrated synergistic bacterial killing while reinstating the efficacy of two previously ineffective antibiotics against difficult-to-treat polymyxin-resistant MDR P. aeruginosa.202133918040
6190170.8925Identifying Escherichia coli genes involved in intrinsic multidrug resistance. Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.200818807027
5231180.8925Prediction of nitrofurantoin resistance among Enterobacteriaceae and mutational landscape of in vitro selected resistant Escherichia coli. Nitrofurantoin (NIT) has long been a drug of choice in the treatment of lower urinary tract infections. Recent emergence of NIT resistant Enterobacteriaceae is a global concern. An ordinal logistic regression model based on PCR amplification patterns of five genes associated with NIT resistance (nfsA, nfsB, ribE, oqxA, and oqxB) among 100 clinical Enterobacteriaceae suggested that a combination of oqxB, nfsB, ribE, and oqxA is ideal for NIT resistance prediction. In addition, four Escherichia coli NIT-resistant mutants were in vitro generated by exposing an NIT-susceptible E. coli to varying concentrations of NIT. The in vitro selected NIT resistant mutants (NI2, NI3, NI4 and NI5) were found to have mutations resulting in frameshifts, premature/lost stop codons or failed amplification of nfsA and/or nfsB genes. The in vitro selected NI5 and the transductant colonies with reconstructed NI5 genotype exhibited reduced fitness compared to their parent strain NS30, while growth of a resistant clinical isolate (NR42) was found to be unaffected in the absence of NIT. These results emphasize the importance of strict adherence to prescribed antibiotic treatment regimens and dosage duration. If left unchecked, these resistant bacteria may thrive at sub-therapeutic concentrations of NIT and spread in the community.202234718096
13190.8924Streptomyces sp. JCK-6131 Protects Plants Against Bacterial and Fungal Diseases via Two Mechanisms. Plant bacterial and fungal diseases cause significant agricultural losses and need to be controlled. Beneficial bacteria are promising candidates for controlling these diseases. In this study, Streptomyces sp. JCK-6131 exhibited broad-spectrum antagonistic activity against various phytopathogenic bacteria and fungi. In vitro assays showed that the fermentation filtrate of JCK-6131 inhibited the growth of bacteria and fungi with minimum concentration inhibitory (MIC) values of 0.31-10% and 0.31-1.25%, respectively. In the in vivo experiments, treatment with JCK-6131 effectively suppressed the development of apple fire blight, tomato bacterial wilt, and cucumber Fusarium wilt in a dose-dependent manner. RP-HPLC and ESI-MS/MS analyses indicated that JCK-6131 can produce several antimicrobial compounds, three of which were identified as streptothricin E acid, streptothricin D, and 12-carbamoyl streptothricin D. In addition, the disease control efficacy of the foliar application of JCK-6131 against tomato bacterial wilt was similar to that of the soil drench application, indicating that JCK-6131 could enhance defense resistance in plants. Molecular studies on tomato plants showed that JCK-6131 treatment induced the expression of the pathogenesis-related (PR) genes PR1, PR3, PR5, and PR12, suggesting the simultaneous activation of the salicylate (SA) and jasmonate (JA) signaling pathways. The transcription levels of PR genes increased earlier and were higher in treated plants than in untreated plants following Ralstonia solanacearum infection. These results indicate that Streptomyces sp. JCK-6131 can effectively control various plant bacterial and fungal diseases via two distinct mechanisms of antibiosis and induced resistance.202134603354