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513400.9808Genomic analysis and antibiotic resistance of a multidrug-resistant bacterium isolated from pharmaceutical wastewater treatment plant sludge. Pharmaceutical wastewater treatment plants (PWWTPs) serve as reservoirs for antibiotic-resistant bacteria (ARBs) and antibiotic resistance genes (ARGs). In this study, a multiantibiotic-resistant strain of Acinetobacter lwoffii (named N4) was isolated from the dewatered sludge of a PWWTP. N4 exhibited high resistance to both antibiotics and metals, with minimum inhibitory concentrations (MICs) of chloramphenicol and cefazolin reaching 1024 mg·L(-1) and MICs of Cu(2+) and Zn(2+) reaching 512 mg·L(-1). Co-sensitization experiments revealed that when antibiotics are co-existing with heavy metal ions (such as TET and Cd(2+), AMP and Cu(2+)) could enhance the resistance of N4 to them. Whole-genome sequencing of N4 revealed a genome size of 0.37 Mb encoding 3359 genes. Among these, 23 ARGs were identified, including dfrA26, bl2be(CTXM), catB3, qnrB, rosB, tlrC, smeD, smeE, mexE, ceoB, oprN, acrB, adeF, ykkC, ksgA and sul2, which confer resistance through mechanisms such as efflux pumps, enzyme modification and target bypass. Additionally, the N4 genome contained 187 genes associated with human disease and 249 virulence factors, underscoring its potential pathogenicity. Overall, this study provides valuable insights into ARBs in PWWTPs and highlights the potential risks posed by multidrug-resistant strains such as N4.202539626482
775410.9793Deciphering the interaction impacts between antiseptic benzethonium chloride and biofilm nitrification system: Performance, resistance mechanisms and biodegradation. Benzethonium chloride (BEC) is one of emerging bacteriostatic agents. BEC-bearing wastewater generated during sanitary applications in food and medication is easily combined with other wastewater streams to flow into wastewater treatment plants. This study focused on the long-term (231 days) impacts of BEC on the sequencing moving bed biofilm nitrification system. Nitrification performance was tolerant to low concentration of BEC (≤ 0.2 mg/L), but the nitrite oxidation was severely inhibited when the concentration of BEC was 1.0-2.0 mg/L. Partial nitrification maintained about 140 days with nitrite accumulation ratio over 80%, mainly caused by the inhibition of Nitrospira, Nitrotoga and Comammox. Notably, BEC exposure in the system might cause the co-selection of antibiotic resistance genes (ARGs) and disinfectant resistance genes (DRGs), and the resistance of biofilm system to BEC was strengthened by efflux pumps mechanism (qacEdelta1 and qacH) and antibiotic deactivation mechanism (aadA, aac(6')-Ib and blaTEM). Extracellular polymeric substances secretion and BEC biodegradation were also contributed to the system microorganisms resisting BEC exposure. In addition, Klebsiella, Enterobacter, Citrobacter and Pseudomonas were isolated and identified as BEC degrading bacteria. The metabolites of N,N-dimethylbenzylamine, N-benzylmethylamine and benzoic acid were identified, and the biodegradation pathway of BEC was proposed. This study brought new knowledge about the fate of BEC in biological treatment units and laid a foundation for its elimination from wastewater.202337209516
316520.9793Metagenomic and Recombination Analyses of Antimicrobial Resistance Genes from Recreational Waters of Black Sea Coastal Areas and Other Marine Environments Unveil Extensive Evidence for Their both Intrageneric and Intergeneric Transmission across Genetically Very Diverse Microbial Communities. Microbial communities of marine coastal recreation waters have become large reservoirs of AMR genes (ARGs), contributing to the emergence and transmission of various zoonotic, foodborne and other infections that exhibit resistance to various antibiotics. Thus, it is highly imperative to determine ARGs assemblages as well as mechanisms and trajectories of their transmission across these microbial communities for our better understanding of the evolutionary trends of AMR (AMR). In this study, using metagenomics approaches, we screened for ARGs in recreation waters of the Black Sea coastal areas of the Batumi City (Georgia). Also, a large array of the recombination detection algorithms of the SplitsTree, RDP4, and GARD was applied to elucidate genetic recombination of ARGs and trajectories of their transmission across various marine microbial communities. The metagenomics analyses of sea water samples, obtained from across the above marine sites, could identify putative ARGs encoding for multidrug resistance efflux transporters mainly from the Major Facilitator and Resistance Nodulation Division superfamilies. The data, generated by SplitsTree (fit ≥95.619; bootstrap values ≥ 95; Phi p ≤ 0.0494), RDP4 (p ≤ 0.0490), and GARD, provided strong statistical evidence not only for intrageneric recombination of these ARGs, but also for their intergeneric recombination across fairly large and diverse microbial communities of marine environment. These bacteria included both human pathogenic and nonpathogenic species, exhibiting collectively the genera of Vibrio, Aeromonas, Synechococcus, Citromicrobium, Rhodobacteraceae, Pseudoalteromonas, Altererythrobacter, Erythrobacter, Altererythrobacter, Marivivens, Xuhuaishuia, and Loktanella. The above nonpathogenic bacteria are strongly suggested to contribute to ARGs transmission in marine ecosystems.202234922301
468930.9792Abundant resistome determinants in rhizosphere soil of the wild plant Abutilon fruticosum. A metagenomic whole genome shotgun sequencing approach was used for rhizospheric soil micribiome of the wild plant Abutilon fruticosum in order to detect antibiotic resistance genes (ARGs) along with their antibiotic resistance mechanisms and to detect potential risk of these ARGs to human health upon transfer to clinical isolates. The study emphasized the potential risk to human health of such human pathogenic or commensal bacteria, being transferred via food chain or horizontally transferred to human clinical isolates. The top highly abundant rhizospheric soil non-redundant ARGs that are prevalent in bacterial human pathogens or colonizers (commensal) included mtrA, soxR, vanRO, golS, rbpA, kdpE, rpoB2, arr-1, efrA and ileS genes. Human pathogenic/colonizer bacteria existing in this soil rhizosphere included members of genera Mycobacterium, Vibrio, Klebsiella, Stenotrophomonas, Pseudomonas, Nocardia, Salmonella, Escherichia, Citrobacter, Serratia, Shigella, Cronobacter and Bifidobacterium. These bacteria belong to phyla Actinobacteria and Proteobacteria. The most highly abundant resistance mechanisms included antibiotic efflux pump, antibiotic target alteration, antibiotic target protection and antibiotic inactivation. antimicrobial resistance (AMR) families of the resistance mechanism of antibiotic efflux pump included resistance-nodulation-cell division (RND) antibiotic efflux pump (for mtrA, soxR and golS genes), major facilitator superfamily (MFS) antibiotic efflux pump (for soxR gene), the two-component regulatory kdpDE system (for kdpE gene) and ATP-binding cassette (ABC) antibiotic efflux pump (for efrA gene). AMR families of the resistance mechanism of antibiotic target alteration included glycopeptide resistance gene cluster (for vanRO gene), rifamycin-resistant beta-subunit of RNA polymerase (for rpoB2 gene) and antibiotic-resistant isoleucyl-tRNA synthetase (for ileS gene). AMR families of the resistance mechanism of antibiotic target protection included bacterial RNA polymerase-binding protein (for RbpA gene), while those of the resistance mechanism of antibiotic inactivation included rifampin ADP-ribosyltransferase (for arr-1 gene). Better agricultural and food transport practices are required especially for edible plant parts or those used in folkloric medicine.202337646836
902840.9791Efflux 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
904650.9790Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps. Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874-PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.201627702426
470160.9790Gene interaction network studies to decipher the multi-drug resistance mechanism in Salmonella enterica serovar Typhi CT18 reveal potential drug targets. Salmonella enterica subsp. enterica serovar Typhi, a human enteric pathogen causing typhoid fever, developed resistance to multiple antibiotics over the years. The current study was dedicated to understand the multi-drug resistance (MDR) mechanism of S. enterica serovar Typhi CT18 and to identify potential drug targets that could be exploited for new drug discovery. We have employed gene interaction network analysis for 44 genes which had 275 interactions. Clustering analysis resulted in three highly interconnecting clusters (C1-C3). Functional enrichment analysis revealed the presence of drug target alteration and three different multi-drug efflux pumps in the bacteria that were associated with antibiotic resistance. We found seven genes (arnA,B,C,D,E,F,T) conferring resistance to Cationic Anti-Microbial Polypeptide (CAMP) molecules by membrane Lipopolysaccharide (LPS) modification, while macB was observed to be an essential controlling hub of the network and played a crucial role in MacAB-TolC efflux pump. Further, we identified five genes (mdtH, mdtM, mdtG, emrD and mdfA) which were involved in Major Facilitator Superfamily (MFS) efflux system and acrAB contributed towards AcrAB-TolC efflux pump. All three efflux pumps were seen to be highly dependent on tolC gene. The five genes, namely tolC, macB, acrA, acrB and mdfA which were involved in multiple resistance pathways, can act as potential drug targets for successful treatment strategies. Therefore, this study has provided profound insights into the MDR mechanism in S. Typhi CT18. Our results will be useful for experimental biologists to explore new leads for S. enterica.202032097747
78170.9788Efflux as a mechanism of resistance to antimicrobials in Pseudomonas aeruginosa and related bacteria: unanswered questions. Pseudomonas aeruginosa is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents. This intrinsic multidrug resistance is caused by synergy between a low-permeability outer membrane and expression of a number of broadly-specific multidrug efflux (Mex) systems, including MexAB-OprM and MexXY-OprM. In addition to this intrinsic resistance, these and three additional systems, MexCD-OprJ, MexEF-OprN and MexJK-OprM promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes by mutational events. In addition to antibiotics, these pumps export biocides, dyes, detergents, metabolic inhibitors, organic solvents and molecules involved in bacterial cell-cell communication. Homologues of the resistance-nodulation-division systems of P. aeruginosa have been found in Burkholderia cepacia, B. pseudomallei, Stenotrophomonas maltophilia, and the nonpathogen P. putida, where they play roles in resistance to antimicrobials and/or organic solvents. Despite intensive studies of these multidrug efflux systems over the past several years, their precise molecular architectures, their modes of regulation of expression and their natural functions remain largely unknown.200312917802
902580.9787BING, a novel antimicrobial peptide isolated from Japanese medaka plasma, targets bacterial envelope stress response by suppressing cpxR expression. Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.202134108601
903990.9787Resistance of the Burkholderia cepacia complex to fosmidomycin and fosmidomycin derivatives. The Burkholderia cepacia complex (BCC) is a group of 17 closely related opportunistic pathogens that are able to infect the respiratory tract of cystic fibrosis patients. BCC bacteria are intrinsically resistant to many antibiotics and are therefore difficult to eradicate. Fosmidomycin could be a new therapeutic agent to treat BCC infections as it inhibits 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in the non-mevalonate pathway essential in BCC bacteria for isoprenoid synthesis. In this study, the antimicrobial activity of fosmidomycin and eight fosmidomycin derivatives towards 40 BCC strains was investigated. All BCC strains were resistant to fosmidomycin, although addition of glucose-6-phosphate reduced the minimum inhibitory concentration values of FR900098, the fosmidomycin acetyl derivative, from 512 mg/L to 64 mg/L for Burkholderia multivorans and B. cepacia. This enhanced activity was linked to increased expression of the genes involved in glycerol-3-phosphate transport, which appears to be the only route for fosmidomycin import in BCC bacteria. Furthermore, upregulation of a fosmidomycin resistance gene (fsr) encoding an efflux pump was observed during fosmidomycin and FR900098 treatment. These results strongly suggest that the observed resistance in BCC bacteria is due to insufficient uptake accompanied by fosmidomycin and FR900098 efflux.201121724375
3070100.9785Analysis of Antibiotic Resistance Genes in Water Reservoirs and Related Wastewater from Animal Farms in Central China. This study aimed to explore the phenotype and relationship of drug resistance genes in livestock and poultry farm wastewater and drinking water reservoirs to provide evidence for the transmission mechanisms of drug resistance genes, in order to reveal the spread of drug resistance genes in wastewater from intensive farms in Central China to urban reservoirs that serve as drinking water sources and provide preliminary data for the treatment of wastewater from animal farms to reduce the threat to human beings. DNA extraction and metagenomic sequencing were performed on eight groups of samples collected from four water reservoirs and four related wastewaters from animal farms in Central China. Metagenomic sequencing showed that the top 20 AROs with the highest abundance were vanT_gene, vanY_gene, adeF, qacG, Mtub_rpsL_STR, vanY_gene_, vanW_gene, Mtub_murA_FOF, vanY_gene, vanH_gene, FosG, rsmA, qacJ, RbpA, vanW_gene, aadA6, vanY_gene, sul4, sul1, and InuF. The resistance genes mentioned above belong to the following categories of drug resistance mechanisms: antibiotic target replacement, antibiotic target protection, antibiotic inactivation, and antibiotic efflux. The resistomes that match the top 20 genes are Streptococcus agalactiae and Streptococcus anginosus; Enterococcus faecalis; Enterococcus faecium; Actinomyces viscosus and Bacillus cereus. Enterococcus faecium; Clostridium tetani; Streptococcus agalactiae and Streptococcus anginosus; Streptococcus agalactiae and Streptococcus anginosus; Acinetobacter baumannii, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Corynebacterium jeikeium, Corynebacterium urealyticum, Mycobacterium kansasii, Mycobacterium tuberculosis, Schaalia odontolytica, and Trueperella pyogenes; Mycobacterium avium and Mycobacterium tuberculosis; Aeromonas caviae, Enterobacter hormaechei, Vibrio cholerae, Vibrio metoecus, Vibrio parahaemolyticus, and Vibrio vulnificus; Pseudomonas aeruginosa and Pseudomonas fluorescens; Staphylococcus aureus and Staphylococcus equorum; M. avium, Achromobacter xylosoxidans, and Acinetobacter baumannii; Sphingobium yanoikuyae, Acinetobacter indicus, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii. Unreported drug resistance genes and drug-resistant bacteria in Central China were identified in 2023. In the transmission path of drug resistance genes, the transmission path from aquaculture wastewater to human drinking water sources cannot be ignored. For the sake of human health and ecological balance, the treatment of aquaculture wastewater needs to be further strengthened, and the effective blocking of drug resistance gene transmission needs to be considered.202438399800
2481110.9785Gene expressions of clinical Pseudomonas aeruginosa harboring RND efflux pumps on chromosome and involving a novel integron on a plasmid. The clinical strain of Pseudomonas aeruginosa XM8 harbored multiple RND-type antibiotic efflux pump genes and a novel integron In4881 on its plasmid pXM8-2, rendering it resistant to nearly all conventional antibiotics except colistin. The resistance was primarily attributed to the inactivation of the oprD gene and overexpression of several efflux pump genes, including mexAB-oprM, mexCD-oprJ, oprN-mexFE, and mexXY. In this study, the XM8 strain was comprehensively characterized using various methods. Antimicrobial susceptibility testing was performed using the BioMerieux VITEK2 system and manual double dilution methods. Gene expression levels of efflux pump-related genes were analyzed via quantitative real-time PCR. The bacterial chromosome and plasmid were sequenced using both Illumina and Nanopore platforms, and bioinformatics tools were employed to analyze mobile genetic elements associated with antibiotic resistance. The pXM8-2 plasmid containsed multiple mobile genetic elements, including integrons (In4881, In334, In413) and transposons (Tn3, TnAs1, TnAs3). Notably, In4881 was reported for the first time in this study. The presence of these elements highlights the potential for horizontal gene transfer and further spread of antibiotic resistance. Given the strong resistance profile of the XM8 strain, effective measures should be implemented to prevent the dissemination and prevalence of such multidrug-resistant bacteria.202540154852
752120.9784Screening 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
8461130.9784Complete genome sequence provides information on quorum sensing related spoilage and virulence of Aeromonas salmonicida GMT3 isolated from spoiled sturgeon. Foodborne bacteria can pose a threat to the public health due to their spoilage and virulence potential, which can be regulated by quorum sensing (QS) system. In the study, we isolated a spoilage bacteria strain Aeromonas salmonicida GMT3 from refrigerated sturgeon. The complete genome of A. salmonicida GMT3 was sequenced, and the QS related genes were assigned. QS signal molecules N-acyl-homoserine lactones (AHLs) and AI-2 were detected. Genes regulating the spoilage-related metabolic pathways, including protease and lipase secretion, amines metabolism, sulfur metabolism, motility and biofilm formation were analyzed. Furthermore, genes encoding for several virulence factors, e.g. hemolysin, aerolysin, type II secretion system (T2SS), type VI secretion system (T6SS), antibiotic and multidrug resistance were also identified. In addition, the spoilage and virulence phenotypes associated with QS including protease, swimming and swarming activity, biofilm and hemolytic activity were detected. This study provided new insights into spoilage and virulence mechanisms correlated with QS of A. salmonicida GMT3, which might promote development of new approaches for spoilage and virulence control based on QS target.202439614553
9038140.9783Molecular mechanisms of chlorhexidine tolerance in Burkholderia cenocepacia biofilms. The high tolerance of biofilm-grown Burkholderia cepacia complex bacteria against antimicrobial agents presents considerable problems for the treatment of infected cystic fibrosis patients and the implementation of infection control guidelines. In the present study, we analyzed the tolerance of planktonic and sessile Burkholderia cenocepacia J2315 cultures and examined the transcriptional response of sessile cells to treatment with chlorhexidine. At low (0.0005%) and high (0.05%) concentrations, chlorhexidine had a similar effect on both populations, but at intermediate concentrations (0.015%) the antimicrobial activity was more pronounced in planktonic cultures. The exposure of sessile cells to chlorhexidine resulted in an upregulation of the transcription of 469 (6.56%) and the downregulation of 257 (3.59%) protein-coding genes. A major group of upregulated genes in the treated biofilms encoded membrane-related and regulatory proteins. In addition, several genes coding for drug resistance determinants also were upregulated. The phenotypic analysis of RND (resistance-nodulation-division) efflux pump mutants suggests the presence of lifestyle-specific chlorhexidine tolerance mechanisms; efflux system RND-4 (BCAL2820-BCAL2822) was more responsible for chlorhexidine tolerance in planktonic cells, while other systems (RND-3 [BCAL1672-BCAL1676] and RND-9 [BCAM1945-BCAM1947]) were linked to resistance in sessile cells. After sessile cell exposure, multiple genes encoding chemotaxis and motility-related proteins were upregulated in concert with the downregulation of an adhesin-encoding gene (BCAM2143), suggesting that sessile cells tried to escape the biofilm. We also observed the differential expression of 19 genes carrying putative small RNA molecules, indicating a novel role for these regulatory elements in chlorhexidine tolerance.201121357299
9037150.9781Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance. BACKGROUND: Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. RESULTS: To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. CONCLUSION: Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.200919761586
8532160.9780Simultaneous volatile fatty acids promotion and antibiotic resistance genes reduction in fluoranthene-induced sludge alkaline fermentation: Regulation of microbial consortia and cell functions. The impact and mechanism of fluoranthene (Flr), a typical polycyclic aromatic hydrocarbon highly detected in sludge, on alkaline fermentation for volatile fatty acids (VFAs) recovery and antibiotic resistance genes (ARGs) transfer were studied. The results demonstrated that VFAs production increased from 2189 to 4272 mg COD/L with a simultaneous reduction of ARGs with Flr. The hydrolytic enzymes and genes related to glucose and amino acid metabolism were provoked. Also, Flr benefited for the enrichment of hydrolytic-acidifying consortia (i.e., Parabacteroides and Alkalibaculum) while reduced VFAs consumers (i.e., Rubrivivax) and ARGs potential hosts (i.e., Rubrivivax and Pseudomonas). Metagenomic analysis indicated that the genes related to cell wall synthesis, biofilm formation and substrate transporters to maintain high VFAs-producer activities were upregulated. Moreover, cell functions of efflux pump and Type IV secretion system were suppressed to inhibit ARGs proliferation. This study provided intrinsic mechanisms of Flr-induced VFAs promotion and ARGs reduction during alkaline fermentation.202438266788
6373170.9780Antibiotic resistance and multidrug-resistant efflux pumps expression in lactic acid bacteria isolated from pozol, a nonalcoholic Mayan maize fermented beverage. Pozol is a handcrafted nonalcoholic Mayan beverage produced by the spontaneous fermentation of maize dough by lactic acid bacteria. Lactic acid bacteria (LAB) are carriers of chromosomal encoded multidrug-resistant efflux pumps genes that can be transferred to pathogens and/or confer resistance to compounds released during the fermentation process causing food spoiling. The aim of this study was to evaluate the antibiotic sensibility and the transcriptional expression of ABC-type efflux pumps in LAB isolated from pozol that contributes to multidrug resistance. Analysis of LAB and Staphylococcus (S.) aureus ATCC 29213 and ATCC 6538 control strains to antibiotic susceptibility, minimal inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) to ethidium bromide were based in "standard methods" whereas the ethidium bromide efflux assay was done by fluorometric assay. Transcriptional expression of efflux pumps was analyzed by RT-PCR. LAB showed antibiotic multiresistance profiles, moreover, Lactococcus (L.) lactis and Lactobacillus (L.) plantarum displayed higher ethidium bromide efflux phenotype than S. aureus control strains. Ethidium bromide resistance and ethidium bromide efflux phenotypes were unrelated with the overexpression of lmrD in L. lactics, or the underexpression of lmrA in L. plantarum and norA in S. aureus. These findings suggest that, moreover, the analyzed efflux pumps genes, other unknown redundant mechanisms may underlie the antibiotic resistance and the ethidium bromide efflux phenotype in L. lactis and L. plantarum. Phenotypic and molecular drug multiresistance assessment in LAB may improve a better selection of the fermentation starter cultures used in pozol, and to control the antibiotic resistance widespread and food spoiling for health safety.201627247772
7712180.9779Metagenomic analysis of microbial communities and antibiotic resistance genes in spoiled household chemicals. Numerous attempts have been utilized to unveil the occurrences of antibiotic resistance genes (ARGs) in human-associated and non-human-associated samples. However, spoiled household chemicals, which are usually neglected by the public, may be also a reservoir of ARGs because of the excessive and inappropriate uses of industrial drugs. Based upon the Comprehensive Antibiotic Research Database, a metagenomic sequencing method was utilized to detect and quantify Antibiotic Resistance Ontology (AROs) in six spoiled household chemicals, including hair conditioner, dishwashing detergent, bath shampoo, hand sanitizer, and laundry detergent. Proteobacteria was found to be the dominant phylum in all the samples. Functional annotation of the unigenes obtained against the KEGG pathway, eggNOG and CAZy databases demonstrated a diversity of their functions. Moreover, 186 types of AROs that were members of 72 drug classes were identified. Multidrug resistance genes were the most dominant types, and there were 17 AROs whose resistance mechanisms were categorized into the resistance-nodulation-cell division antibiotic efflux pump among the top 20 AROs. Moreover, Proteobacteria was the dominant carrier of AROs with the primary resistance mechanism of antibiotic efflux. The maximum temperature of the months of collection significantly affected the distributions of AROs. Additionally, the isolated individual bacterium from spoiled household chemicals and artificial mixed communities of isolated bacteria demonstrated diverse resistant abilities to different biocides. This study demonstrated that there are abundant microorganisms and a broad spectrum profile of AROs in spoiled household chemicals that might induce a severe threat to public healthy securities and merit particular attention.202234740703
4698190.9778Antidepressant exposure as a source of disinfectant resistance in waterborne bacteria. The emergence of disinfectant-resistant pathogens in water is a major threat to public health. However, whether human-consumed pharmaceuticals can induce bacterial resistance to disinfectants remains unclear. Herein, Escherichia coli was exposed to 12 antidepressants, and susceptibility of antidepressant-induced chloramphenicol (CHL)-resistant mutants to disinfectants was tested. Whole genome sequencing, global transcriptomic sequencing, and real-time quantitative polymerase chain reaction were used to elucidate the underlying mechanisms. We observed that duloxetine, fluoxetine, amitriptyline, and sertraline significantly increased the mutation frequency of E. coli against CHL by 15- to 2948-fold. The resultant mutants increased the average MIC(50) of sodium hypochlorite, benzalkonium bromide, and triclosan roughly 2- to 8-fold. Consistently, marRAB and acrAB-tolC genes, together with ABC transporter genes (e.g., yddA, yadG, yojI, and mdlA), were triggered to increase the efflux of disinfectants out of the cell, while ompF was inhibited, reducing disinfectant penetration into the cell. Additionally, the occurrence of DNA mutations in marR and acrR in the mutants was observed, potentially resulting in increased synthesis of the AcrAB-TolC pump. This study indicates that pharmaceutical exposure may create disinfectant-resistant bacteria, which may then be released into water systems, providing novel insights into the potential source of water-borne disinfectant-resistant pathogens.202337030229