Sporadic cefiderocol resistance in Escherichia coli from the United Arab Emirates involves multifactorial mechanisms reversible by novel beta-lactamase inhibitors. - Related Documents




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506101.0000Sporadic cefiderocol resistance in Escherichia coli from the United Arab Emirates involves multifactorial mechanisms reversible by novel beta-lactamase inhibitors. Cefiderocol (CFDC), a novel siderophore-cephalosporin, is effective against multidrug-resistant (MDR) pathogens, but the emergence of resistance threatens its future use in treating infections. This study reports the emergence of CFDC resistance in four E. coli strains isolated from immunocompromised and critically ill patients in the United Arab Emirates, and provides a comprehensive genomic analysis of these strains, aiming to uncover the mechanisms driving this resistance. Whole-genome sequencing with bioinformatic analysis revealed specific beta-lactamase variants (NDM-5, CMY-2/145, and OXA-181) and unique mutations in siderophore-iron transport genes (cirA, fepA, fecA, fiu, and tonB) and penicillin-binding proteins (PBPs) associated with resistance. Phylogenetic analysis showed that the strains were not clonally related, indicating the sporadic nature of resistance. To address this challenge, we evaluated the efficacy of several novel beta-lactamase inhibitors (BLIs) combined with CFDC. In vitro susceptibility testing demonstrated that these inhibitors restored the antibacterial activity of CFDC against resistant strains. Zidebactam, with intrinsic antibacterial activity, caused the most significant reduction in CFDC minimum inhibitory concentrations (MICs), while the activity of other inhibitors (taniborbactam and xeruborbactam) was dependent on the genetic makeup of the strains, especially mutations in the siderophore-iron uptake genes. Our findings underscore the importance of genomic surveillance in deciphering antibiotic resistance mechanisms. Novel BLIs and partner antibiotics could be added weapons in the fight against MDR bacteria; thus, we recommend using combinations with novel BLIs as innovative therapeutic options to combat the emerging threat of CFDC resistance, after proper validation of their in vivo efficacy.202541023121
505410.9995In vitro resistance development gives insights into molecular resistance mechanisms against cefiderocol. Cefiderocol, a novel siderophore cephalosporin, demonstrates promising in vitro activity against multidrug-resistant Gram-negative bacteria, including carbapenemase-producing strains. Nonetheless, only a few reports are available regarding the acquisition of resistance in clinical settings, primarily due to its recent usage. This study aimed to investigate cefiderocol resistance using an in vitro resistance development model to gain insights into the underlying molecular resistance mechanisms. Cefiderocol susceptible reference strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) and a clinical Acinetobacter baumannii complex isolate were exposed to increasing cefiderocol concentrations using a high-throughput resistance development model. Cefiderocol susceptibility testing was performed using broth microdilution. Whole-genome sequencing was employed to identify newly acquired resistance mutations. Our in vitro resistance development model led to several clones of strains exhibiting cefiderocol resistance, with MIC values 8-fold to 512-fold higher than initial levels. In total, we found 42 different mutations in 26 genes, of which 35 could be described for the first time. Putative loss-of-function mutations were detected in the envZ, tonB, and cirA genes in 13 out of 17 isolates, leading to a decrease in cefiderocol influx. Other potential resistance mechanisms included multidrug efflux pumps (baeS, czcS, nalC), antibiotic-inactivating enzymes (ampR, dacB), and target mutations in penicillin-binding-protein genes (mrcB). This study reveals new insights into underlying molecular resistance mechanisms against cefiderocol. While mutations leading to reduced influx via iron transporters was the most frequent resistance mechanism, we also detected several other novel resistance mutations causing cefiderocol resistance.202439080477
474420.9993Whole-Genome Sequencing of Resistance, Virulence and Regulation Genes in Extremely Resistant Strains of Pseudomonas aeruginosa. BACKGROUND/OBJECTIVES: Pseudomonas aeruginosa is a clinically significant opportunistic pathogen, renowned for its ability to acquire and develop diverse mechanisms of antibiotic resistance. This study examines the resistance, virulence, and regulatory mechanisms in extensively drug-resistant clinical strains of P. aeruginosa. METHODS: Antibiotic susceptibility was assessed using the Minimum Inhibitory Concentration (MIC) method, and whole-genome sequencing (WGS) was performed on the Illumina NovaSeq platform. RESULTS: The analysis demonstrated a higher prevalence of virulence genes compared to resistance and regulatory genes. Key virulence factors identified included secretion systems, motility, adhesion, and biofilm formation. Resistance mechanisms observed comprised efflux pumps and beta-lactamases, while regulatory systems involved two-component systems, transcriptional regulators, and sigma factors. Additionally, phenotypic profiles were found to correlate with resistance genes identified through genotypic analysis. CONCLUSIONS: This study underscores the significant resistance and virulence of the clinical P. aeruginosa strains analyzed, highlighting the urgent need for alternative strategies to address infections caused by extensively drug-resistant bacteria.202539846701
976030.9993Mutations leading to ceftolozane/tazobactam and imipenem/cilastatin/relebactam resistance during in vivo exposure to ceftazidime/avibactam in Pseudomonas aeruginosa. Identifying resistance mechanisms to novel antimicrobials informs treatment strategies during infection and antimicrobial development. Studying resistance that develops during the treatment of an infection can provide the most clinically relevant mutations conferring resistance, but cross-sectional studies frequently identify multiple candidate resistance mutations without resolving the driver mutation. We performed whole-genome sequencing of longitudinal Pseudomonas aeruginosa from a patient whose P. aeruginosa developed imipenem/cilastatin/relebactam and ceftolozane/tazobactam resistance during ceftazidime/avibactam treatment. This analysis determined new mutations that arose in isolates resistant to both imipenem/cilastatin/relebactam and ceftolozane/tazobactam. Mutations in penicillin-binding protein 3 ftsI, the MexAB-OprM repressor nalD, and a virulence regulator pvdS were found in resistant isolates. Importantly, drug efflux was not increased in the resistant isolate compared to the most closely related susceptible isolates. We conclude that mutations in peptidoglycan synthesis genes can alter the efficacy of multiple antimicrobials. IMPORTANCE: Antibiotic resistance is a significant challenge for physicians trying to treat infections. The development of novel antibiotics to treat resistant infections has not been prioritized for decades, limiting treatment options for infections caused by many high-priority pathogens. Cross-resistance, when one mutation provides resistance to multiple antibiotics, is most problematic. Mutations that cause cross-resistance need to be considered when developing new antibiotics to guide developers toward drugs with different targets, and thus a better likelihood of efficacy. This work was undertaken to determine the mutation that caused resistance to three antibiotics for highly resistant Pseudomonas aeruginosa infection treatment while the bacteria were exposed to only one of these agents. The findings provide evidence that drug developers should endeavor to find effective antibiotics with new targets and that medical providers should utilize medications with different mechanisms of action in bacteria that have become resistant to even one of these three agents.202539932323
482140.9993Enterobacter hormaechei replaces virulence with carbapenem resistance via porin loss. Pathogenic Enterobacter species are of increasing clinical concern due to the multidrug-resistant nature of these bacteria, including resistance to carbapenem antibiotics. Our understanding of Enterobacter virulence is limited, hindering the development of new prophylactics and therapeutics targeting infections caused by Enterobacter species. In this study, we assessed the virulence of contemporary clinical Enterobacter hormaechei isolates in a mouse model of intraperitoneal infection and used comparative genomics to identify genes promoting virulence. Through mutagenesis and complementation studies, we found two porin-encoding genes, ompC and ompD, to be required for E. hormaechei virulence. These porins imported clinically relevant carbapenems into the bacteria, and thus loss of OmpC and OmpD desensitized E. hormaechei to the antibiotics. Our genomic analyses suggest porin-related genes are frequently mutated in E. hormaechei, perhaps due to the selective pressure of antibiotic therapy during infection. Despite the importance of OmpC and OmpD during infection of immunocompetent hosts, we found the two porins to be dispensable for virulence in a neutropenic mouse model. Moreover, porin loss provided a fitness advantage during carbapenem treatment in an ex vivo human whole blood model of bacteremia. Our data provide experimental evidence of pathogenic Enterobacter species gaining antibiotic resistance via loss of porins and argue antibiotic therapy during infection of immunocompromised patients is a conducive environment for the selection of porin mutations enhancing the multidrug-resistant profile of these pathogens.202539977318
482350.9993A Review of Resistance to Polymyxins and Evolving Mobile Colistin Resistance Gene (mcr) among Pathogens of Clinical Significance. The global rise in antibiotic resistance in bacteria poses a major challenge in treating infectious diseases. Polymyxins (e.g., polymyxin B and colistin) are last-resort antibiotics against resistant Gram-negative bacteria, but the effectiveness of polymyxins is decreasing due to widespread resistance among clinical isolates. The aim of this literature review was to decipher the evolving mechanisms of resistance to polymyxins among pathogens of clinical significance. We deciphered the molecular determinants of polymyxin resistance, including distinct intrinsic molecular pathways of resistance as well as evolutionary characteristics of mobile colistin resistance. Among clinical isolates, Acinetobacter stains represent a diversified evolution of resistance, with distinct molecular mechanisms of intrinsic resistance including naxD, lpxACD, and stkR gene deletion. On the other hand, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa are usually resistant via the PhoP-PhoQ and PmrA-PmrB pathways. Molecular evolutionary analysis of mcr genes was undertaken to show relative relatedness across the ten main lineages. Understanding the molecular determinants of resistance to polymyxins may help develop suitable and effective methods for detecting polymyxin resistance determinants and the development of novel antimicrobial molecules.202337998799
474560.9992Determination of Antimicrobial Resistance and the Impact of Imipenem + Cilastatin Synergy with Tetracycline in Pseudomonas aeruginosa Isolates from Sepsis. Pseudomonas aeruginosa is among the most ubiquitous bacteria in the natural world, exhibiting metabolic and physiological versatility, which makes it highly adaptable. Imipenem + cilastatin and tetracycline are antibiotic combinations commonly used to treat infections caused by P. aeruginosa, including serious infections such as sepsis. In the context of bacterial infections, biofilm, formed by bacterial cells surrounded by extracellular substances forming a matrix, plays a pivotal role in the resistance of P. aeruginosa to antibiotics. This study aimed to characterize a representative panel of P. aeruginosa isolates from septicemias, assessing their susceptibility to various antibiotics, specifically, imipenem + cilastatin and tetracycline, and the impact of these treatments on biofilm formation. Results from antibiotic susceptibility tests revealed sensitivity in most isolates to six antibiotics, with four showing near or equal to 100% sensitivity. However, resistance was observed in some antibiotics, albeit at minimal levels. Notably, tetracycline showed a 100% resistance phenotype, while imipenem + cilastatin predominantly displayed an intermediate phenotype (85.72%), with some resistance (38.1%). Microdilution susceptibility testing identified effective combinations against different isolates. Regarding biofilm formation, P. aeruginosa demonstrated the ability to produce biofilms. The staining of microtiter plates confirmed that specific concentrations of imipenem + cilastatin and tetracycline could inhibit biofilm production. A significant proportion of isolates exhibited resistance to aminoglycoside antibiotics because of the presence of modifying genes (aac(3)-II and aac(3)-III), reducing their effectiveness. This study also explored various resistance genes, unveiling diverse resistance mechanisms among P. aeruginosa isolates. Several virulence genes were detected, including the las quorum-sensing system genes (lasI and lasR) in a significant proportion of isolates, contributing to virulence factor activation. However, genes related to the type IV pili (T4P) system (pilB and pilA) were found in limited isolates. In conclusion, this comprehensive study sheds light on the intricate dynamics of P. aeruginosa, a remarkably adaptable bacterium with a widespread presence in the natural world. Our findings provide valuable insights into the ongoing battle against P. aeruginosa infections, highlighting the need for tailored antibiotic therapies and innovative approaches to combat biofilm-related resistance.202338004699
505770.9992Genomic investigation unveils colistin resistance mechanism in carbapenem-resistant Acinetobacter baumannii clinical isolates. Colistin resistance in Acinetobacter baumannii is mediated by multiple mechanisms. Recently, mutations within pmrABC two-component system and overexpression of eptA gene due to upstream insertion of ISAba1 have been shown to play a major role. Thus, the aim of our study is to characterize colistin resistance mechanisms among the clinical isolates of A. baumannii in India. A total of 207 clinical isolates of A. baumannii collected from 2016 to 2019 were included in this study. Mutations within lipid A biosynthesis and pmrABC genes were characterized by whole-genome shotgun sequencing. Twenty-eight complete genomes were further characterized by hybrid assembly approach to study insertional inactivation of lpx genes and the association of ISAba1-eptA. Several single point mutations (SNPs), like M12I in pmrA, A138T and A444V in pmrB, and E117K in lpxD, were identified. We are the first to report two novel SNPs (T7I and V383I) in the pmrC gene. Among the five colistin-resistant A. baumannii isolates where complete genome was available, the analysis showed that three of the five isolates had ISAba1 insertion upstream of eptA. No mcr genes were identified among the isolates. We mapped the SNPs on the respective protein structures to understand the effect on the protein activity. We found that majority of the SNPs had little effect on the putative protein function; however, some SNPs might destabilize the local structure. Our study highlights the diversity of colistin resistance mechanisms occurring in A. baumannii, and ISAba1-driven eptA overexpression is responsible for colistin resistance among the Indian isolates.IMPORTANCEAcinetobacter baumannii is a Gram-negative, emerging and opportunistic bacterial pathogen that is often associated with a wide range of nosocomial infections. The treatment of these infections is hindered by increase in the occurrence of A. baumannii strains that are resistant to most of the existing antibiotics. The current drug of choice to treat the infection caused by A. baumannii is colistin, but unfortunately, the bacteria started to show resistance to the last-resort antibiotic. The loss of lipopolysaccharides and mutations in lipid A biosynthesis genes are the main reasons for the colistin resistance. The present study characterized 207 A. baumannii clinical isolates and constructed complete genomes of 28 isolates to recognize the mechanisms of colistin resistance. We showed the mutations in the colistin-resistant variants within genes essential for lipid A biosynthesis and that cause these isolates to lose the ability to produce lipopolysaccharides.202438214512
505980.9992Site-selective modifications by lipid A phosphoethanolamine transferases linked to colistin resistance and bacterial fitness. Genes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance (mcr) alleles (mcr-1, -3, -9), one intrinsic pet (eptA), and two mcr-like genes (petB, petC) in Escherichia coli. Using an isogenic expression system, we show that mcr-1 and mcr-3 confer similar phenotypes of decreased colistin susceptibility with low fitness costs. mcr-9, which is phylogenetically closely related to mcr-3, and eptA only provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media without colistin. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither impacted colistin susceptibility nor caused considerable fitness cost. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A; MCR-1, MCR-3, and PET-C selectively modify the 4'-phosphate, whereas MCR-9 and EptA modify the 1-phosphate. However, 4'-phosphate modifications facilitated by MCR-1 and -3 are associated with lowered colistin susceptibility and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that increased colistin resistance is associated with specific lipid A modification patterns that have been largely unexplored thus far. IMPORTANCE: Rising levels of resistance to increasing numbers of antimicrobials have led to the revival of last resort antibiotic colistin. Unfortunately, resistance to colistin is also spreading in the form of mcr genes, making it essential to (i) improve the identification of resistant bacteria to allow clinicians to prescribe effective drug regimens and (ii) develop new combination therapies effective at targeting resistant bacteria. Our results demonstrate that PETs, including MCR variants, are site-selective in Escherichia coli and that site-selectivity correlates with the level of susceptibility and fitness costs conferred by certain PETs. Site selectivity associated with a given PET may not only help predict colistin resistance phenotypes but may also provide an avenue to (i) improve drug regimens and (ii) develop new combination therapies to better combat colistin-resistant bacteria.202439611852
470190.9992Gene 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
5053100.9992Effects of different carbapenemase and siderophore production on cefiderocol susceptibility in Klebsiella pneumoniae. The resistance mechanism of Gram-negative bacteria to the siderophore antibiotic cefiderocol is primarily attributed to carbapenemase and siderophore uptake pathways; however, specific factors and their relationships remain to be fully elucidated. Here, we constructed cefiderocol-resistant Klebsiella pneumoniae (CRKP) strains carrying different carbapenemases and knocked out siderophore genes to investigate the roles of various carbapenemases and siderophores in the development of cefiderocol resistance. Antimicrobial susceptibility testing revealed that both bla(NDM) and bla(KPC) significantly increased the minimum inhibitory concentration (MIC) of Klebsiella pneumoniae (KP) to cefiderocol, while bla(OXA-48) showed a modest increase. Notably, KP expressing NDM exhibited a higher cefiderocol MIC compared to KP expressing KPC, although expression of NDM alone did not induce cefiderocol resistance. Laboratory evolutionary experiments demonstrated that combining pNDM with mutations in the siderophore uptake receptor gene cirA and pKPC with a mutation in the two-component system gene envZ led to KP reaching a high level of cefiderocol resistance. Although combining pOXA with mutations in the two-component system gene baeS did not induce cefiderocol resistance, it significantly reduced susceptibility. Moreover, siderophores could influence the development of cefiderocol resistance. Strains deficient in enterobactin exhibited increased susceptibility to cefiderocol, while deficiencies in yersiniabactin and salmochelin showed no significant alterations. In conclusion, carbapenemase gene expression facilitates cefiderocol resistance, but its presence alone is insufficient. Cefiderocol resistance in CRKP typically involves abnormal expression of certain genes and other factors, such as mutations in siderophore uptake receptor genes and two-component system genes. The enterobactin siderophore synthesis gene entB may also contribute to resistance.202439470196
4814110.9992Increased 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
6273120.9992Burkholderia multivorans Exhibits Antibiotic Collateral Sensitivity. Burkholderia multivorans is a member of the Burkholderia cepacia complex whose members are inherently resistant to many antibiotics and can cause chronic lung infections in patients with cystic fibrosis. A possible treatment for chronic infections arises from the existence of collateral sensitivity (CS)-acquired resistance to a treatment antibiotic results in a decreased resistance to a nontreatment antibiotic. Determining CS patterns for bacteria involved in chronic infections may lead to sustainable treatment regimens that reduce development of multidrug-resistant bacterial strains. CS has been found to occur in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Here, we report that B. multivorans exhibits antibiotic CS, as well as cross-resistance (CR), describe CS and CR networks for six antibiotics (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole), and identify candidate genes involved in CS. Characterization of CS and CR patterns allows antibiotics to be separated into two clusters based on the treatment drug to which the evolved strain developed primary resistance, suggesting an antibiotic therapy strategy of switching between members of these two clusters.202031393205
6280130.9992Genomic variation in Pseudomonas aeruginosa clinical respiratory isolates with de novo resistance to a bacteriophage cocktail. Pseudomonas aeruginosa is an opportunistic pathogen that can cause sinus infections and pneumonia in cystic fibrosis (CF) patients. Bacteriophage therapy is being investigated as a treatment for antibiotic-resistant P. aeruginosa infections. Although virulent bacteriophages have shown promise in treating P. aeruginosa infections, the development of bacteriophage-insensitive mutants (BIMs) in the presence of bacteriophages has been described. The aim of this study was to examine the genetic changes associated with the BIM phenotype. Biofilms of three genetically distinct P. aeruginosa strains, including PAO1 (ATCC 15692), and two clinical respiratory isolates (one CF and one non-CF) were grown for 7 days and treated with either a cocktail of four bacteriophages or a vehicle control for 7 consecutive days. BIMs isolated from the biofilms were detected by streak assays, and resistance to the phage cocktail was confirmed using spot test assays. Comparison of whole genome sequencing between the recovered BIMs and their respective vehicle control-treated phage-sensitive isolates revealed structural variants in two strains, and several small variants in all three strains. These variations involved a TonB-dependent outer membrane receptor in one strain, and mutations in lipopolysaccharide synthesis genes in two strains. Prophage deletion and induction were also noted in two strains, as well as mutations in several genes associated with virulence factors. Mutations in genes involved in susceptibility to conventional antibiotics were also identified in BIMs, with both decreased and increased antibiotic sensitivity to various antibiotics being observed. These findings may have implications for future applications of lytic phage therapy.IMPORTANCELytic bacteriophages are viruses that infect and kill bacteria and can be used to treat difficult-to-treat bacterial infections, including biofilm-associated infections and multidrug-resistant bacteria. Pseudomonas aeruginosa is a bacterium that can cause life-threatening infections. Lytic bacteriophage therapy has been trialed in the treatment of P. aeruginosa infections; however, sometimes bacteria develop resistance to the bacteriophages. This study sheds light on the genetic mechanisms of such resistance, and how this might be harnessed to restore the sensitivity of multidrug-resistant P. aeruginosa to conventional antibiotics.202540162801
5060140.9992Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates. Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen.IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.202032161146
9759150.9992Rapid emergence of resistance to broad-spectrum direct antimicrobial activity of avibactam. Avibactam (AVI) is a diazabicyclooctane (DBO) β-lactamase inhibitor used clinically in combination with ceftazidime. At concentrations higher than those typically achieved in vivo, it also has broad-spectrum direct antibacterial activity against Enterobacterales strains, including metallo-β-lactamase-producing isolates, mediated by inhibition of penicillin-binding protein 2 (PBP2). This activity has some mechanistic similarities to that of more potent novel DBOs (zidebactam and nacubactam) in late clinical development. We found that resistance to AVI emerged readily, with a mutation frequency of 2 × 10(-6) to 8 × 10(-5). Whole-genome sequencing of resistant isolates revealed a heterogeneous mutational target that permitted bacterial survival and replication despite PBP2 inhibition, in line with prior studies of PBP2-targeting drugs. While such mutations are believed to act by upregulating the bacterial stringent response, we found a similarly high mutation frequency in bacteria deficient in components of the stringent response, although we observed a different set of mutations in these strains. Although avibactam-resistant strains had increased lag time, suggesting a fitness cost that might render them less problematic in clinical infections, there was no statistically significant difference in growth rates between susceptible and resistant strains. The finding of rapid emergence of resistance to avibactam as the result of a large and complex mutational target adds to our understanding of resistance to PBP2-targeting drugs and has potential implications for novel DBOs with potent direct antibacterial activity, which are being developed with the goal of expanding cell wall-active treatment options for multidrug-resistant gram-negative infections.IMPORTANCEAvibactam (AVI) is the first in a class of novel β-lactamase inhibitor antibiotics called diazabicyclooctanes (DBOs). In addition to its ability to inhibit bacterial β-lactamase enzymes that can destroy β-lactam antibiotics, we found that AVI had direct antibacterial activity, at concentrations higher than those used clinically, against even highly multidrug-resistant bacteria. This activity is the result of inhibition of the bacterial enzyme penicillin-binding protein 2 (PBP2). Resistance to other drugs that inhibit PBP2 occurs through mutations that involve upregulation of the bacterial "stringent response" to stress. We found that bacteria developed resistance to AVI at a high rate, as a result of mutations in stringent response genes. We also found that bacteria with impairments in the stringent response could still develop resistance to AVI through different mutations. Our findings indicate the importance of studying how resistance will emerge to newer, more potent DBOs in development and early clinical use.202540503840
6276160.9992A 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
4816170.9992Sub-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
6281180.9992Evolved 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
5754190.9992Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated colistin-resistant strains and Gram-negative bacteria. OBJECTIVES: Efflux in bacteria is a ubiquitous mechanism associated with resistance to antimicrobials agents. Efflux pump inhibitors (EPIs) have been developed to inhibit efflux mechanisms and could be a good alternative to reverse colistin resistance, but only CCCP has shown good activity. The aim of our study was to identify CCCP activity in a collection of 93 Gram-negative bacteria with known and unknown colistin resistance mechanisms including isolates with mcr-1 plasmid-mediated colistin resistance. METHODS: Colistin MIC was evaluated with and without CCCP and the fold decrease of colistin MIC was calculated for each strain. In order to evaluate the effect of this combination, a time-kill study was performed on five strains carrying different colistin resistance mechanisms. RESULTS: Overall, CCCP was able to reverse colistin resistance for all strains tested. The effect of CCCP was significantly greater on intrinsically colistin-resistant bacteria (i.e. Proteus spp., Serratia marcescens, Morganella morganii and Providencia spp.) than on other Enterobacteriaceae (P < 0.0001). The same was true for bacteria with a heteroresistance mechanism compared to bacteria with other colistin resistance mechanisms (P < 0.0001). A time-kill study showed the combination was bacteriostatic on strains tested. CONCLUSIONS: These results suggest an efflux mechanism, especially on intrinsically resistant bacteria and Enterobacter spp., but further analysis is needed to identify the molecular support of this mechanism. EPIs could be an alternative for restoring colistin activity in Gram-negative bacteria. Further work is necessary to identify new EPIs that could be used in humans.201829718423