Biocide-Induced Emergence of Antibiotic Resistance in Escherichia coli. - Related Documents




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469701.0000Biocide-Induced Emergence of Antibiotic Resistance in Escherichia coli. Biocide use is essential and ubiquitous, exposing microbes to sub-inhibitory concentrations of antiseptics, disinfectants, and preservatives. This can lead to the emergence of biocide resistance, and more importantly, potential cross-resistance to antibiotics, although the degree, frequency, and mechanisms that give rise to this phenomenon are still unclear. Here, we systematically performed adaptive laboratory evolution of the gut bacteria Escherichia coli in the presence of sub-inhibitory, constant concentrations of ten widespread biocides. Our results show that 17 out of 40 evolved strains (43%) also decreased the susceptibility to medically relevant antibiotics. Through whole-genome sequencing, we identified mutations related to multidrug efflux proteins (mdfA and acrR), porins (envZ and ompR), and RNA polymerase (rpoA and rpoBC), as mechanisms behind the resulting (cross)resistance. We also report an association of several genes (yeaW, pyrE, yqhC, aes, pgpA, and yeeP-isrC) and specific mutations that induce cross-resistance, verified through mutation repairs. A greater capacity for biofilm formation with respect to the parent strain was also a common feature in 11 out of 17 (65%) cross-resistant strains. Evolution in the biocides chlorophene, benzalkonium chloride, glutaraldehyde, and chlorhexidine had the most impact in antibiotic susceptibility, while hydrogen peroxide and povidone-iodine the least. No cross-resistance to antibiotics was observed for isopropanol, ethanol, sodium hypochlorite, and peracetic acid. This work reinforces the link between exposure to biocides and the potential for cross-resistance to antibiotics, presents evidence on the underlying mechanisms of action, and provides a prioritized list of biocides that are of greater concern for public safety from the perspective of antibiotic resistance. SIGNIFICANCE STATEMENT: Bacterial resistance and decreased susceptibility to antimicrobials is of utmost concern. There is evidence that improper biocide (antiseptic and disinfectant) use and discard may select for bacteria cross-resistant to antibiotics. Understanding the cross-resistance emergence and the risks associated with each of those chemicals is relevant for proper applications and recommendations. Our work establishes that not all biocides are equal when it comes to their risk of inducing antibiotic resistance; it provides evidence on the mechanisms of cross-resistance and a risk assessment of the biocides concerning antibiotic resistance under residual sub-inhibitory concentrations.202133717036
469910.9996Exposure to DDAB disinfectants promotes antimicrobial resistance to antibiotics and collateral-sensitivity to polymyxins in Salmonella enterica. SALMONELLA: as an important food-borne zoonotic pathogen, is found in soil and processing environment by human or animal feces, causing serious public health problems. Quaternary ammonium compounds (QACs) disinfectants are widely used in hospitals, livestock farms and food processing sites because of their low toxicity and broad-spectrum disinfection. However, sub-lethal levels of QACs disinfectants can induce bacteria to develop tolerance to disinfectants and cross-resistance to other antimicrobial agents. The acquired resistance will undoubtedly pose a threat to the prevention of antimicrobial resistance. In this study, Salmonella enterica SE211 was induced by the sub-inhibitory concentration and sub-lethal concentration of dodecyl dimethyl ammonium bromide (DDAB) in vitro. Following exposure to DDAB, the strains showed increased resistance to DDAB, doxycycline, amphenicols and fluoroquinolones, and increased sensitivity to colistin drugs. Phenotypic experiments showed that the induced strains exhibited changes in efflux pump activity, biofilm formation ability, motility and membrane characterization. Next-generation sequencing revealed mutations in induced strains involved in LPS-related genes (msbA, lptDE) and cationic antimicrobial peptide (CAMP) resistance-related genes (phoQ, pmrD). Transcriptome sequencing (RNA-seq) analysis revealed up-regulation of efflux pump genes and down-regulation of CAMP resistance, LPS and peptidoglycan related genes. Our study provided a theoretical basis for the potential consequences of disinfection failures and environmental residues of QACs disinfectants on the evolution of antibiotic resistance in salmonella. Furthermore, the induction of colistin sensitivity in salmonella by DDBA resulted in the emergence of collateral sensitivity, which offered a new strategy for drug combination applications to prevent the rise of colistin-resistant superbugs.202540021029
992120.9996Identification of Multiple Low-Level Resistance Determinants and Coselection of Motility Impairment upon Sub-MIC Ceftriaxone Exposure in Escherichia coli. Resistance to third-generation cephalosporins among Gram-negative bacteria is a rapidly growing public health threat. Among the most commonly used third-generation cephalosporins is ceftriaxone. Bacterial exposure to sublethal or sub-MIC antibiotic concentrations occurs widely, from environmental residues to intermittently at the site of infection. Quality of ceftriaxone is also a concern, especially in low- and middle-income countries, with medicines having inappropriate active pharmaceutical ingredient (API) content or concentration. While focus has been largely on extended-spectrum β-lactamases and high-level resistance, there are limited data on specific chromosomal mutations and other pathways that contribute to ceftriaxone resistance under these conditions. In this work, Escherichia coli cells were exposed to a broad range of sub-MICs of ceftriaxone and mutants were analyzed using whole-genome sequencing. Low-level ceftriaxone resistance emerged after as low as 10% MIC exposure, with the frequency of resistance development increasing with concentration. Genomic analyses of mutants revealed multiple genetic bases. Mutations were enriched in genes associated with porins (envZ, ompF, ompC, and ompR), efflux regulation (marR), and the outer membrane and metabolism (galU and pgm), but none were associated with the ampC β-lactamase. We also observed selection of mgrB mutations. Notably, pleiotropic effects on motility and cell surface were selected for in multiple independent genes, which may have important consequences. Swift low-level resistance development after exposure to low ceftriaxone concentrations may result in reservoirs of bacteria with relevant mutations for survival and increased resistance. Thus, initiatives for broader surveillance of low-level antibiotic resistance and genomic resistance determinants should be pursued when resources are available. IMPORTANCE Ceftriaxone is a widely consumed antibiotic used to treat bacterial infections. Bacteria, however, are increasingly becoming resistant to ceftriaxone. Most work has focused on known mechanisms associated with high-level ceftriaxone resistance. However, bacteria are extensively exposed to low antibiotic concentrations, and there are limited data on the evolution of ceftriaxone resistance under these conditions. In this work, we observed that bacteria quickly developed low-level resistance due to both novel and previously described mutations in multiple different genes upon exposure to low ceftriaxone concentrations. Additionally, exposure also led to changes in motility and the cell surface, which can impact other processes associated with resistance and infection. Notably, low-level-resistant bacteria would be missed in the clinic, which uses set breakpoints. While they may require increased resources, this work supports continued initiatives for broader surveillance of low-level antibiotic resistance or their resistance determinants, which can serve as predictors of higher risk for clinical resistance.202134787446
481430.9995Increased Usage of Antiseptics Is Associated with Reduced Susceptibility in Clinical Isolates of Staphylococcus aureus. Hospital-acquired infection is a major cause of morbidity and mortality, and regimes to prevent infection are crucial in infection control. These include the decolonization of vulnerable patients with methicillin-resistant Staphylococcus aureus (MRSA) carriage using antiseptics, including chlorhexidine and octenidine. Concern has been raised, however, regarding the possible development of biocide resistance. In this study, we assembled a panel of S. aureus isolates, including isolates collected before the development of chlorhexidine and octenidine and isolates, from a major hospital trust in the United Kingdom during a period when the decolonization regimes were altered. We observed significant increases in the MIC and minimum bactericidal concentration (MBC) of chlorhexidine in isolates from periods of high usage of chlorhexidine. Isolates with increased MICs and MBCs of octenidine rapidly emerged after octenidine was introduced in the trust. There was no apparent cross-resistance between the two biocidal agents. A combination of variable-number tandem repeat (VNTR) analysis, PCR for qac genes, and whole-genome sequencing was used to type isolates and examine possible mechanisms of resistance. There was no expansion of a single strain associated with decreased biocide tolerance, and biocide susceptibility did not correlate with carriage of qac efflux pump genes. Mutations within the NorA or NorB efflux pumps, previously associated with chlorhexidine export, were identified, however, suggesting that this may be an important mechanism of biocide tolerance. We present evidence that isolates are evolving in the face of biocide challenge in patients and that changes in decolonization regimes are reflected in changes in susceptibility of isolates.IMPORTANCE Infection in hospitals remains a major cause of death and disease. One way in which we combat this is by decolonizing at-risk patients from carriage of bacteria which can cause disease such as MRSA. This is done with antiseptics, including chlorhexidine and octenidine. There is concern, however, that bacteria may be able to become resistant to these antiseptics. In this study, we looked at isolates of MRSA and found that there was a correlation between the use of antiseptics and increased resistance in the isolates. We also suggest that the mechanism by which these more tolerant isolates may become resistant to antiseptics is that of changing a transport pump that exports these agents. This information suggests that we need to study the impact of antiseptics on clinically important bacteria more closely.201829844113
627640.9995A shared mechanism of multidrug resistance in laboratory-evolved uropathogenic Escherichia coli. The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low Amp(R) and High Amp(R), respectively. Whole-genome sequencing revealed that both Low and High Amp(R) strains contained mutations in the marR, acrR, and envZ genes. The High Amp(R) strain exhibited a single additional mutation in the nlpD gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the Amp(R) strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the Amp(R) strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment in vivo, implicating the increasing difficulty in treating host infections caused by the Amp(R) strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics.202438899601
440450.9995Adaptation to Biocides Cetrimide and Chlorhexidine in Bacteria from Organic Foods: Association with Tolerance to Other Antimicrobials and Physical Stresses. Chlorhexidine (CH) and quaternary ammonium compounds (QAC), such as cetrimide (CE), are widely used as disinfectants because of their broad antimicrobial spectrum. However, their frequent use for disinfection in different settings may promote bacterial drug resistance against both biocides and clinically relevant antibiotics. This study analyzes the effects of stepwise exposure to cetrimide (CE) and chlorhexidine (CH) of bacteria from organic foods and previously classified as biocide-sensitive. Gradual exposure of these strains to biocides resulted in mainly transient decreased antimicrobial susceptibility to other antibiotics and to biocides. Biocide-adapted bacteria also exhibit alterations in physiological characteristics, mainly decreased heat tolerance, or gastric acid tolerance in CE-adapted strains, while bile resistance does not seem to be influenced by biocide adaptation. Results from this study suggest that changes in membrane fluidity may be the main mechanism responsible for the acquisition of stable tolerance to biocides.201728177232
428760.9995Widely Used Benzalkonium Chloride Disinfectants Can Promote Antibiotic Resistance. While the misuse of antibiotics has clearly contributed to the emergence and proliferation of resistant bacterial pathogens, with major health consequences, it remains less clear if the widespread use of disinfectants, such as benzalkonium chlorides (BAC), a different class of biocides than antibiotics, has contributed to this problem. Here, we provide evidence that exposure to BAC coselects for antibiotic-resistant bacteria and describe the underlying genetic mechanisms. After inoculation with river sediment, BAC-fed bioreactors selected for several bacterial taxa, including the opportunistic pathogen Pseudomonas aeruginosa, that were more resistant to several antibiotics than their counterparts in a control (no BAC) bioreactor. A metagenomic analysis of the bioreactor microbial communities, confirmed by gene cloning experiments with the derived isolates, suggested that integrative and conjugative elements encoding a BAC efflux pump together with antibiotic resistance genes were responsible for these results. Furthermore, the exposure of the P. aeruginosa isolates to increasing concentrations of BAC selected for mutations in pmrB (polymyxin resistance) and physiological adaptations that contributed to a higher tolerance to polymyxin B and other antibiotics. The physiological adaptations included the overexpression of mexCD-oprJ multidrug efflux pump genes when BAC was added in the growth medium at subinhibitory concentrations. Collectively, our results demonstrated that disinfectants promote antibiotic resistance via several mechanisms and highlight the need to remediate (degrade) disinfectants in nontarget environments to further restrain the spread of antibiotic-resistant bacteria.IMPORTANCE Benzalkonium chlorides (BAC) are biocides broadly used in disinfectant solutions. Disinfectants are widely used in food processing lines, domestic households, and pharmaceutical products and are typically designed to have a different mode of action than antibiotics to avoid interfering with the use of the latter. Whether exposure to BAC makes bacteria more resistant to antibiotics remains an unresolved issue of obvious practical consequences for public health. Using an integrated approach that combines metagenomics of natural microbial communities with gene cloning experiments with isolates and experimental evolution assays, we show that the widely used benzalkonium chloride disinfectants promote clinically relevant antibiotic resistance. Therefore, more attention should be given to the usage of these disinfectants, and their fate in nontarget environments should be monitored more tightly.201829959242
440570.9995Copper Resistance of the Emerging Pathogen Acinetobacter baumannii. Acinetobacter baumannii is an important emerging pathogen that is capable of causing many types of severe infection, especially in immunocompromised hosts. Since A. baumannii can rapidly acquire antibiotic resistance genes, many infections are on the verge of being untreatable, and novel therapies are desperately needed. To investigate the potential utility of copper-based antibacterial strategies against Acinetobacter infections, we characterized copper resistance in a panel of recent clinical A. baumannii isolates. Exposure to increasing concentrations of copper in liquid culture and on solid surfaces resulted in dose-dependent and strain-dependent effects; levels of copper resistance varied broadly across isolates, possibly resulting from identified genotypic variation among strains. Examination of the growth-phase-dependent effect of copper on A. baumannii revealed that resistance to copper increased dramatically in stationary phase. Moreover, A. baumannii biofilms were more resistant to copper than planktonic cells but were still susceptible to copper toxicity. Exposure of bacteria to subinhibitory concentrations of copper allowed them to better adapt to and grow in high concentrations of copper; this copper tolerance response is likely achieved via increased expression of copper resistance mechanisms. Indeed, genomic analysis revealed numerous putative copper resistance proteins that share amino acid homology to known proteins in Escherichia coli and Pseudomonas aeruginosa Transcriptional analysis revealed significant upregulation of these putative copper resistance genes following brief copper exposure. Future characterization of copper resistance mechanisms may aid in the search for novel antibiotics against Acinetobacter and other highly antibiotic-resistant pathogens. IMPORTANCE: Acinetobacter baumannii causes many types of severe nosocomial infections; unfortunately, some isolates have acquired resistance to almost every available antibiotic, and treatment options are incredibly limited. Copper is an essential nutrient but becomes toxic at high concentrations. The inherent antimicrobial properties of copper give it potential for use in novel therapeutics against drug-resistant pathogens. We show that A. baumannii clinical isolates are sensitive to copper in vitro, both in liquid and on solid metal surfaces. Since bacterial resistance to copper is mediated though mechanisms of efflux and detoxification, we identified genes encoding putative copper-related proteins in A. baumannii and showed that expression of some of these genes is regulated by the copper concentration. We propose that the antimicrobial effects of copper may be beneficial in the development of future therapeutics that target multidrug-resistant bacteria.201627520808
469880.9995Antidepressant 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
992290.9995De novo acquisition of antibiotic resistance in six species of bacteria. Bacteria can become resistant to antibiotics in two ways: by acquiring resistance genes through horizontal gene transfer and by de novo development of resistance upon exposure to non-lethal concentrations. The importance of the second process, de novo build-up, has not been investigated systematically over a range of species and may be underestimated as a result. To investigate the DNA mutation patterns accompanying the de novo antibiotic resistance acquisition process, six bacterial species encountered in the food chain were exposed to step-wise increasing sublethal concentrations of six antibiotics to develop high levels of resistance. Phenotypic and mutational landscapes were constructed based on whole-genome sequencing at two time points of the evolutionary trajectory. In this study, we found that (1) all of the six strains can develop high levels of resistance against most antibiotics; (2) increased resistance is accompanied by different mutations for each bacterium-antibiotic combination; (3) the number of mutations varies widely, with Y. enterocolitica having by far the most; (4) in the case of fluoroquinolone resistance, a mutational pattern of gyrA combined with parC is conserved in five of six species; and (5) mutations in genes coding for efflux pumps are widely encountered in gram-negative species. The overall conclusion is that very similar phenotypic outcomes are instigated by very different genetic changes. The outcome of this study may assist policymakers when formulating practical strategies to prevent development of antimicrobial resistance in human and veterinary health care.IMPORTANCEMost studies on de novo development of antimicrobial resistance have been performed on Escherichia coli. To examine whether the conclusions of this research can be applied to more bacterial species, six species of veterinary importance were made resistant to six antibiotics, each of a different class. The rapid build-up of resistance observed in all six species upon exposure to non-lethal concentrations of antimicrobials indicates a similar ability to adjust to the presence of antibiotics. The large differences in the number of DNA mutations accompanying de novo resistance suggest that the mechanisms and pathways involved may differ. Hence, very similar phenotypes can be the result of various genotypes. The implications of the outcome are to be considered by policymakers in the area of veterinary and human healthcare.202539907470
4406100.9995A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa. The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa's outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic.IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.202031871033
3801110.9995Macrophage Cell Lines and Murine Infection by Salmonella enterica Serovar Typhi L-Form Bacteria. Antibiotic resistance of pathogenic bacteria has emerged as a major threat to public health worldwide. While stable resistance due to the acquisition of genomic mutations or plasmids carrying antibiotic resistance genes is well established, much less is known about the temporary and reversible resistance induced by antibiotic treatment, such as that due to treatment with bacterial cell wall-inhibiting antibiotics such as ampicillin. Typically, ampicillin concentration in the blood and other tissues gradually increases over time after initiation of the treatment. As a result, the bacterial population is exposed to a concentration gradient of ampicillin during the treatment of infectious diseases. This is different from in vitro drug testing, where the organism is exposed to fixed drug concentrations from the beginning until the end. To mimic the mode of antibiotic exposure of microorganisms within host tissues, we cultured the wild-type, ampicillin-sensitive Salmonella enterica serovar Typhi Ty2 strain (S. Typhi Ty2) in the presence of increasing concentrations of ampicillin over a period of 14 days. This resulted in the development of a strain that displayed several features of the so-called L-form of bacteria, including the absence of the cell wall, altered shape, and lower growth rate compared with the parental form. Studies of the pathogenesis of S. Typhi L-form showed efficient infection of the murine and human macrophage cell lines. More importantly, S. Typhi L-form was also able to establish infection in a mouse model to the extent comparable to its parental form. These results suggested that L-form generation following the initiation of treatment with antibiotics could lead to drug escape of S. Typhi and cell to cell (macrophages) spread of the bacteria, which sustain the infection. Oral infection by the L-form bacteria underscores the potential of rapid disease transmission through the fecal-oral route, highlighting the need for new approaches to decrease the reservoir of infection.202235587200
4700120.9994Mechanisms of Salmonella typhimurium Resistance to Cannabidiol. The emergence of multi-drug resistance (MDR) poses a huge risk to public health globally. Yet these recalcitrant pathogens continue to rise in incidence rate with resistance rates significantly outpacing the speed of antibiotic development. This therefore presents related health issues such as untreatable nosocomial infections arising from organ transplants and surgeries, as well as community-acquired infections that are related to people with compromised immunity, e.g., diabetic and HIV patients, etc. There is a global effort to fight MRD pathogens spearheaded by the World Health Organization, thus calling for research into novel antimicrobial agents to fight multiple drug resistance. Previously, our laboratory demonstrated that Cannabidiol (CBD) is an effective antimicrobial against Salmonella typhimurium (S. typhimurium). However, we observed resistance development over time. To understand the mechanisms S. typhimurium uses to develop resistance to CBD, we studied the abundance of bacteria lipopolysaccharide (LPS) and membrane sterols of both CBD-susceptible and CBD-resistant S. typhimurium strains. Using real-time quantitative polymerase chain reaction (rt qPCR), we also analyzed the expression of selected genes known for aiding resistance development in S. typhimurium. We found a significantly higher expression of blaTEM (over 150 mRNA expression) representing over 55% of all the genes considered in the study, fimA (over 12 mRNA expression), fimZ (over 55 mRNA expression), and integron 2 (over 1.5 mRNA expression) in the CBD-resistant bacteria, and these were also accompanied by a shift in abundance in cell surface molecules such as LPS at 1.76 nm, ergosterols at 1.03 nm, oleic acid at 0.10 nm and MPPSE at 2.25nm. For the first time, we demonstrated that CBD-resistance development in S. typhimurium might be caused by several structural and genetic factors. These structural factors demonstrated here include LPS and cell membrane sterols, which showed significant differences in abundances on the bacterial cell surfaces between the CBD-resistant and CBD-susceptible strains of S. typhimurium. Specific key genetic elements implicated for the resistance development investigated included fimA, fimZ, int2, ompC, blaTEM, DNA recombinase (STM0716), leucine-responsive transcriptional regulator (lrp/STM0959), and the spy gene of S. typhimurium. In this study, we revealed that blaTEM might be the highest contributor to CBD-resistance, indicating the potential gene to target in developing agents against CBD-resistant S. typhimurium strains.202540142444
6281130.9994Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in Pseudomonas aeruginosa. While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.IMPORTANCE Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with Pseudomonas aeruginosa airway infections. As for all antimicrobial agents, bacteria can evolve resistance that decreases the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Here, using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects. We found that a diverse collection of genes contributes to aztreonam resistance, each with a small but cumulative effect. Surprisingly, we found that selection for aztreonam resistance mutations could confer increased resistance to other antibiotics and promote hypervirulence in a mouse infection model. Our study reveals inherent mechanisms of aztreonam resistance and indicates that aztreonam exposure can have unintended secondary effects.201729089424
9757140.9994Effects of different mechanisms on antimicrobial resistance in Pseudomonas aeruginosa: a strategic system for evaluating antibiotics against gram-negative bacteria. Our previous studies constructed a strategic system for testing antibiotics against specific resistance mechanisms using Klebsiella pneumoniae and Acinetobacter baumannii. However, it lacked resistance mechanisms specifically expressed only in Pseudomonas species. In this study, we constructed this system using Pseudomonas aeruginosa. In-frame deletion, site-directed mutagenesis, and plasmid transformation were used to generate genetically engineered strains with various resistance mechanisms from two fully susceptible P. aeruginosa strains. Antimicrobial susceptibility testing was used to test the efficacy of antibiotics against these strains in vitro. A total of 31 engineered strains with various antimicrobial resistance mechanisms from P. aeruginosa KPA888 and ATCC 27853 were constructed, and the same antibiotic resistance mechanism showed a similar effect on the MICs of the two strains. Compared to the parental strains, the engineered strains lacking porin OprD or lacking the regulator genes of efflux pumps all showed a ≥4-fold increase on the MICs of some of the 19 antibiotics tested. Mechanisms due to GyrA/ParC mutations and β-lactamases also contributed to their corresponding resistance as previously published. The strains constructed in this study possess well-defined resistance mechanisms and can be used to screen and evaluate the effectiveness of antibiotics against specific resistance mechanisms in P. aeruginosa. Building upon our previous studies on K. pneumoniae and A. baumannii, this strategic system, including a P. aeruginosa panel, has been expanded to cover almost all the important antibiotic resistance mechanisms of gram-negative bacteria that are in urgent need of new antibiotics.IMPORTANCEIn this study, an antibiotic assessment system for P. aeruginosa was developed, and the system can be expanded to include other key pathogens and resistance mechanisms. This system offers several benefits: (i) compound design: aid in the development of compounds that can bypass or counteract resistance mechanisms, leading to more effective treatments against specific resistant strains; (ii) combination therapies: facilitate the exploration of combination therapies, where multiple antibiotics may work synergistically to overcome resistance and enhance treatment efficacy; and (iii) targeted treatments: enable healthcare providers to prescribe more targeted treatments, reducing unnecessary antibiotic use and helping to slow the spread of antibiotic resistance. In summary, this system could streamline the development process, reduce costs, increase the success rate of new antibiotics, and help prevent and control antimicrobial resistance.202540042282
8957150.9994Transcriptome Profiling Reveals Interplay of Multifaceted Stress Response in Escherichia coli on Exposure to Glutathione and Ciprofloxacin. We have previously reported that supplementation of exogenous glutathione (GSH) promotes ciprofloxacin resistance in Escherichia coli by neutralizing antibiotic-induced oxidative stress and by enhancing the efflux of antibiotic. In the present study, we used a whole-genome microarray as a tool to analyze the system-level transcriptomic changes of E. coli on exposure to GSH and/or ciprofloxacin. The microarray data revealed that GSH supplementation affects redox function, transport, acid shock, and virulence genes of E. coli. The data further highlighted the interplay of multiple underlying stress response pathways (including those associated with the genes mentioned above and DNA damage repair genes) at the core of GSH, offsetting the effect of ciprofloxacin in E. coli. The results of a large-scale validation of the transcriptomic data using reverse transcription-quantitative PCR (RT-qPCR) analysis for 40 different genes were mostly in agreement with the microarray results. The altered growth profiles of 12 different E. coli strains carrying deletions in the specific genes mentioned above with GSH and/or ciprofloxacin supplementation implicate these genes in the GSH-mediated phenotype not only at the molecular level but also at the functional level. We further associated GSH supplementation with increased acid shock survival of E. coli on the basis of our transcriptomic data. Taking the data together, it can be concluded that GSH supplementation influences the expression of genes of multiple stress response pathways apart from its effect(s) at the physiological level to counter the action of ciprofloxacin in E. coli. IMPORTANCE The emergence and spread of multidrug-resistant bacterial strains have serious medical and clinical consequences. In addition, the rate of discovery of new therapeutic antibiotics has been inadequate in last few decades. Fluoroquinolone antibiotics such as ciprofloxacin represent a precious therapeutic resource in the fight against bacterial pathogens. However, these antibiotics have been gradually losing their appeal due to the emergence and buildup of resistance to them. In this report, we shed light on the genome-level expression changes in bacteria with respect to glutathione (GSH) exposure which act as a trigger for fluoroquinolone antibiotic resistance. The knowledge about different bacterial stress response pathways under conditions of exposure to the conditions described above and potential points of cross talk between them could help us in understanding and formulating the conditions under which buildup and spread of antibiotic resistance could be minimized. Our findings are also relevant because GSH-induced genome-level expression changes have not been reported previously for E. coli.201829468195
4408160.9994Multidrug resistant Acinetobacter baumannii--the role of AdeABC (RND family) efflux pump in resistance to antibiotics. Acinetobacter baumannii is an opportunistic pathogen which play the more and more greater role in the pathogenicity of the human. It is often attached with the hospital environment, in which is able easily to survive for many days even in adverse conditions. Acinetobacter baumannii is the species responsible for a serious nosocomial infections, especially in the intensive care units. Option of surviving in natural niches, and in the hospital environment could also be associated with the efflux pump mechanisms. Mechanisms of efflux universally appear in all cells (eukaryotic and prokaryotic) and play the physiological important role. In prokaryote, the main functions are evasion of such naturally produced molecules, removal of metabolic products and toxins. These pumps could also be involved in an early stage of infection, such as adhesion to host cells and the colonization. Importantly, they remove commonly used antibiotics from the cell in therapy of infections caused by these bacteria. Efflux pumps exemplify a unique phenomenon in drug resistance: a single mechanism causing resistance against several different classes of antibiotics. In Acinetobacter baumannii, the AdeABC efflux pump, a member of the resistance-nodulation-cell division family (RND), has been well characterized. Aminoglicosides, tetracyclines, erythromycin, chloramphenicol, trimethoprim, fluoroquinolones, some beta-lactams, and also recently tigecycline, were found to be substrates for this pump. Drugs, as substrates for the AdeABC pump, can increase the expression of the AdeABC genes, leading to multidrug resistance (MDR). From this reason, treatment failure and death caused by Acinetobacter baumannii infections or underlying diseases are common. Because the AdeABC pump is widespread in Acinetobacter baumannii, similarly to other pumps in Gram-negative and Gram-positive bacteria, exists a need of searching a new therapeutic solutions. Specific efflux inhibitors of pumps (EPIs), including AdeABC inhibitors, could be suppress the activity of pumps and restore the sensitivity of such important bacteria as Acinetobacter baumannii to commonly used antibiotic.200819056528
4510170.9994Environmental concentrations of antibiotics, biocides, and heavy metals fail to induce phenotypic antimicrobial resistance in Escherichia coli. Most anthropogenically affected environments contain mixtures of pollutants from different sources. The impact of these pollutants is usually the combined effect of the individual polluting constituents. However, how these stressors contribute to the development of antimicrobial resistance in environmental microorganisms is poorly understood. Thus, a 30-day exposure experiment to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC (benzalkonium chloride) 10 and DADMAC (diallyldimethylammonium chloride) 12, was conducted using fully susceptible E. coli ATCC 25922 to ascertain any development of phenotypic or genotypic resistance. Furthermore, wild-type isolates were collected from the same aquatic environment as the stressors, analysed for phenotypic resistance using the disk diffusion method and genotypically through whole genome sequencing. Exposure to the various concentrations and combinations of the stressors did not trigger phenotypic resistance in the experimental bacteria. Furthermore, genotypic analysis of the WGS on the exposed isolates only found the macrolide resistance mdf(A) gene (also present in the control strain) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes that encoded for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations except for the robA, a transcription protein subset of the ArcC/XylS family of proteins, which confer multidrug resistance in E. coli. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues in the aquatic environmental concentrations of the stressors identified in the current study could not induce phenotypic or genotypic resistance but encoded for genes responsible for the development of persistence and tolerance in bacteria, which could be a precursor to the development of resistance in environmental bacteria.202337482346
4270180.9994Antibiotic resistant bacteria survive treatment by doubling while shrinking. Many antibiotics that are used in healthcare, farming, and aquaculture end up in environments with different spatial structures that might promote heterogeneity in the emergence of antibiotic resistance. However, the experimental evolution of microbes at sub-inhibitory concentrations of antibiotics has been mainly carried out at the population level which does not allow capturing single-cell responses to antibiotics. Here, we investigate and compare the emergence of resistance to ciprofloxacin in Escherichia coli in well-mixed and structured environments using experimental evolution, genomics, and microfluidics-based time-lapse microscopy. We discover that resistance to ciprofloxacin and cross-resistance to other antibiotics is stronger in the well-mixed environment due to the emergence of target mutations, whereas efflux regulator mutations emerge in the structured environment. The latter mutants also harbor sub-populations of persisters that survive high concentrations of ciprofloxacin that inhibit bacterial growth at the population level. In contrast, genetically resistant bacteria that display target mutations also survive high concentrations of ciprofloxacin that inhibit their growth via population-level antibiotic tolerance. These resistant and tolerant bacteria keep doubling while shrinking in size in the presence of ciprofloxacin and regain their original size after antibiotic removal, which constitutes a newly discovered phenotypic response. This new knowledge sheds light on the diversity of strategies employed by bacteria to survive antibiotics and poses a stepping stone for understanding the link between mutations at the population level and phenotypic single-cell responses. IMPORTANCE: The evolution of antimicrobial resistance poses a pressing challenge to global health with an estimated 5 million deaths associated with antimicrobial resistance every year globally. Here, we investigate the diversity of strategies employed by bacteria to survive antibiotics. We discovered that bacteria evolve genetic resistance to antibiotics while simultaneously displaying tolerance to very high doses of antibiotics by doubling while shrinking in size.202439565111
4816190.9994Sub-inhibitory concentrations of colistin and imipenem impact the expression of biofilm-associated genes in Acinetobacter baumannii. Acinetobacter baumannii is an opportunistic pathogen that is responsible for nosocomial infections. Imipenem and colistin are drugs that are commonly used to treat severe infections caused by A. baumannii, such as sepsis, ventilator-associated pneumonia, and bacteremia. However, some strains of A. baumannii have become resistant to these drugs, which is a concern for public health. Biofilms produced by A. baumannii increase their resistance to antibiotics and the cells within the inner layers of biofilm are exposed to sub-inhibitory concentrations (sub-MICs) of antibiotics. There is limited information available regarding how the genes of A. baumannii are linked to biofilm formation when the bacteria are exposed to sub-MICs of imipenem and colistin. Thus, this study's objective was to explore this relationship by examining the genes involved in biofilm formation in A. baumannii when exposed to low levels of imipenem and colistin. The study found that exposing an isolate of A. baumannii to low levels of these drugs caused changes in their drug susceptibility pattern. The relative gene expression profiles of the biofilm-associated genes exhibited a change in their expression profile during short-term and long-term exposure. This study highlights the potential consequences of overuse and misuse of antibiotics, which can help bacteria become resistant to these drugs.202438489041