scholarly journals Serratia marcescens RamA expression is under PhoP-dependent control and modulates lipid A-related genes transcription and antibiotic resistance phenotypes

2021 ◽  
Author(s):  
Javier F. Mariscotti ◽  
Eleonora García Véscovi
Keyword(s):  
Antibiotic Resistance ◽  
Serratia Marcescens ◽  
Nalidixic Acid ◽  
Lipid A ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Opportunistic Pathogen ◽  
Binding Motif ◽  
Promoter Regions ◽  
Wide Range

Serratia marcescens is an enteric bacterium that can function as an opportunistic pathogen with with increasing incidence in clinical settings. This is mainly due to the ability of express a wide range of virulence factors and the acquisition of antibiotic resistance mechanisms. For these reasons, S. marcescens has been declared by the WHO as a research priority to develop alternative antimicrobial strategies. In this work, we found a PhoP-binding motif in the promoter region of transcriptional regulator RamA of the S. marcescens RM66262. We demonstrated that the expression of ramA is autoregulated and that ramA is also part of the PhoP/PhoQ regulon. We have also shown that PhoP binds directly and specifically to ramA, mgtE1, mgtE2, lpxO1 and lpxO2 promoter regions and that RamA binds to ramA and lpxO1 but not to mgtE1 and lpxO2, suggesting an indirect control for these latter genes. Finally, we have demonstrated that, in S. marcescens, the RamA overexpression induces the AcrAB-TolC efflux pump required to reduce the susceptibility of the bacteria to tetracycline and nalidixic acid. In sum, we herein show the first report describing the regulation of ramA under the PhoP/PhoQ regulon, and the regulatory role of RamA in S. marcescens. Importance We demonstrate that, in S. marcescens, the transcriptional regulator RamA is autoregulated and also controlled by the PhoP/PhoQ signal transduction system. We have shown that PhoP is able to directly and specifically bind to ramA, mgtE1, mgtE2, lpxO1 and lpxO2 promoter regions. In addition, RamA is able to directly interact with the promoter regions of ramA, lpxO1 but indirectly regulates mgtE1 and lpxO2. Finally, we found that, in S. marcescens, RamA overexpression induces the AcrAB-TolC efflux pump required to reduce susceptibility to tetracycline and nalidixic acid. Collectively, these results further our understanding of PhoP/PhoQ regulon in S. marcescens and demonstrate the involvement of RamA in the protection against antibiotic challenges.

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis

2008 ◽  
Vol 54 (5) ◽  
pp. 411-416 ◽  
Author(s):  
Sanela Begic ◽  
Elizabeth A. Worobec
Keyword(s):  
Antibiotic Resistance ◽  
Substrate Specificity ◽  
Serratia Marcescens ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Active Efflux ◽  
Major Mechanism

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance–Nodulation–Cell Division family pump with limited substrate specificity.

scholarly journals Antibiotic Resistance in Staphylococcus aureus in Patients Hospitalized in Imam Reza Hospital of Kermanshah, Iran (2016 - 2018)

2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Mohammad Hossein Zamanian ◽  
Marya Shirvani ◽  
Alireza Janbakhsh ◽  
Babak Sayad ◽  
Siavash Vaziri ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Opportunistic Pathogen ◽  
Cross Sectional Study ◽  
Penicillin G ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
Cross Sectional ◽  
Healthcare Settings ◽  
Wide Range

Background: Staphylococcus aureus is an opportunistic pathogen, which often causes asymptomatic diseases. This bacterium could cause several disorders, ranging from skin infections to life-threatening diseases. S. aureus could also develop resistance to a wide range of antibiotics. Objectives: The present study aimed to investigate the prevalence and pattern of antibiotic resistance in isolated S. aureus in Imam Reza Hospital of Kermanshah, Iran. Methods: This descriptive cross-sectional study was conducted on 2,228 patients with a positive S. aureus culture who were admitted to Imam Reza Hospital during 2016 - 2018. In accordance with the CLSI protocol, bacterial isolates were separated using laboratory tests, and antibiotic susceptibility was assessed using the standard disk-diffusion method. Data analysis was performed in SPSS version 24. Results: Most of the S. aureus-positive cases were isolated from the emergency ward (43.7%), blood samples (40.1%), and urine samples (23.9%). The highest antibiotic resistance was observed against erythromycin, penicillin G, ofloxacin, cefoxitin, clindamycin, and piperacillin. With 93.3% and 81.8% sensitivity, vancomycin and teicoplanin were respectively the most effective antibiotics against S. aureus. Conclusions: According to the results, the prevalence of resistant S. aureus strains could be due to the long-term hospitalization of patients and the overuse of antibiotics in infection treatment. Therefore, proper monitoring and development of effective infection control methods are essential in these healthcare settings.

scholarly journals The Transcriptional Repressor FarR Is Not Involved in Meningococcal Fatty Acid Resistance Mediated by the FarAB Efflux Pump and Dependent on Lipopolysaccharide Structure

2010 ◽  
Vol 76 (10) ◽  
pp. 3160-3169 ◽  
Author(s):  
Stephanie Schielke ◽  
Corinna Schmitt ◽  
Carolin Spatz ◽  
Matthias Frosch ◽  
Alexandra Schubert-Unkmeir ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipid A ◽  
Efflux Pump ◽  
Acyl Chain ◽  
Acid Resistance ◽  
Transcriptional Regulator ◽  
Permeability Barrier ◽  
Membrane Anchor ◽  
Pump System

ABSTRACT Free fatty acids are important antimicrobial substances regulating the homeostasis of colonizing bacteria on epithelial surfaces. Here, we show that meningococci express a functional farAB efflux pump, which is indispensable for fatty acid resistance. However, other than in Neisseria gonorrhoeae, the transcriptional regulator FarR is not involved in regulation of this operon in Neisseria meningitidis. We tested the susceptibility of 23 meningococcal isolates against saturated and unsaturated long-chain fatty acids, proving that meningococci are generally highly resistant, with the exception of serogroup Y strains belonging to sequence type 23. Using genetically determined lipopolysaccharide (LPS)-truncated mutant strains, we show that addition of the LPS core oligosaccharide and hexa-acylation of its membrane anchor lipid A are imperative for fatty acid resistance of meningococci. The sensitivity of the serogroup Y strains is due to naturally occurring mutations within the lpxL1 gene, which is responsible for addition of the sixth acyl chain on the LPS membrane anchor lipid A. Therefore, fatty acid resistance in meningococci is provided by both the active efflux pump FarAB and by the natural permeability barrier of the Gram-negative outer membrane. The transcriptional regulator FarR is not implicated in fatty acid resistance in meningococci, possibly giving rise to a constitutively active FarAB efflux pump system and thus revealing diverse mechanisms of niche adaptation in the two closely related Neisseria species.

scholarly journals Effect of Two Unique Nanoparticle Formulations on the Efficacy of a Broadly Protective Vaccine Against Pseudomonas Aeruginosa

2021 ◽  
Vol 12 ◽  
Author(s):  
Debaki R. Howlader ◽  
Sayan Das ◽  
Ti Lu ◽  
Gang Hu ◽  
David J. Varisco ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Protective Efficacy ◽  
Initial Step ◽  
Opportunistic Pathogen ◽  
Multi Drug Resistance ◽  
Successful Vaccine ◽  
Wide Range ◽  
Tlr4 Agonist ◽  
Opsonophagocytic Killing

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections in humans. In addition to its innate antibiotic resistance, P. aeruginosa is very effective in acquiring resistance resulting in the emergence of multi-drug resistance strains and a licensed vaccine is not yet available. We have previously demonstrated the protective efficacy of a novel antigen PaF (Pa Fusion), a fusion of the type III secretion system (T3SS) needle tip protein, PcrV, and the first of two translocator proteins, PopB. PaF was modified to provide a self-adjuvanting activity by fusing the A1 subunit of the heat-labile enterotoxin from Enterotoxigenic E. coli to its N-terminus to give L-PaF. In addition to providing protection against 04 and 06 serotypes of P. aeruginosa, L-PaF elicited opsonophagocytic killing and stimulated IL-17A secretion, which have been predicted to be required for a successful vaccine. While monomeric recombinant subunit vaccines can be protective in mice, this protection often does not transfer to humans where multimeric formulations perform better. Here, we use two unique formulations, an oil-in-water (o/w) emulsion and a chitosan particle, as well as the addition of a unique TLR4 agonist, BECC438 (a detoxified lipid A analogue designated Bacterial Enzymatic Combinatorial Chemistry 438), as an initial step in optimizing L-PaF for use in humans. The o/w emulsion together with BECC438 provided the best protective efficacy, which correlated with high levels of opsonophagocytic killing and IL-17A secretion, thereby reducing the lung burden among all the vaccinated groups tested.

scholarly journals Antibiotic resistance, virulence-associated genes analysis and molecular typing of Klebsiella pneumoniae strains recovered from clinical samples

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amir Mirzaie ◽  
Reza Ranjbar
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Molecular Typing ◽  
Efflux Pump ◽  
Opportunistic Pathogen ◽  
Relevant Information ◽  
Multidrug Resistant ◽  
Clinical Samples ◽  
Microbiological Methods ◽  
High Level

AbstractKlebsiella pneumoniae is a multidrug-resistant (MDR) opportunistic pathogen that causes nosocomial infections. Virulence analysis and molecular typing as powerful approaches can provide relevant information on K. pneumoniae infection. In the current study, antibiotic resistance, virulence-associated genes analysis, as well as molecular typing of K. pneumoniae strains were investigated. Out of 505 clinical samples collected from hospitalized patients, 100 K. pneumoniae strains were isolated by standard microbiological methods and subjected to the phenotypic and genotyping analysis. The highest prevalence of resistance was observed against ciprofloxacin (75%), trimethoprim–sulfamethoxazole (73%) and nitrofurantoin (68%). Virulence associated genes including entB, traT, ybts, magA, iucC, htrA and rmpA were found in 80%, 62%, 75%, 5%, 30%, 72% and 48%, of the isolates, respectively. The prevalence of biofilm-associated genes including mrkA, fimH, and mrkD were equally 88% for all tested isolates. Moreover, the efflux pump genes including AcrAB, TolC and mdtK were observed in 41 (41%), 33 (33%) and 26 (26%) of the strains respectively. A significant statistical association was observed between MDR strains and high expression of efflux pump and biofilm genes. The K. pneumoniae strains were differentiated into 11 different genetic patterns using the repetitive element sequence-based PCR (rep-PCR) technique. High prevalence of resistance, presence of various virulence factors, high level of efflux pump, and biofilm gene expression in diverse clones of K. pneumoniae strains pose an important health issue in clinical settings.

scholarly journals Natural Antibiotic Resistance of Bacteria Isolated from Larvae of the Oil Fly, Helaeomyia petrolei

2000 ◽  
Vol 66 (11) ◽  
pp. 4615-4619 ◽  
Author(s):  
Dana R. Kadavy ◽  
Jacob M. Hornby ◽  
Terry Haverkost ◽  
Kenneth W. Nickerson
Keyword(s):  
Antibiotic Resistance ◽  
Los Angeles ◽  
Nalidixic Acid ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Gut Contents ◽  
Solvent Tolerance ◽  
Bacterial Strains ◽  
Active Efflux ◽  
Mutator Strain

ABSTRACT Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nineProvidencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 108cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 μg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene–50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.

scholarly journals Genetic Variations in MexAB-OprM Efflux Pump Regulators and Their Association with Antibiotic Resistance and Sequence type in Clinical and Epidemiologically High-risk Clones of Pseudomonas Aeruginosa.

2020 ◽  
Author(s):  
Pamela Aguilar-Rodea ◽  
Gerardo Zúñiga ◽  
René Cerritos ◽  
Benjamín Antonio Rodríguez-Espino ◽  
Gerardo E. Rodea ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
High Risk ◽  
Efflux Pump ◽  
Phylogenetic Network ◽  
Principal Component ◽  
Sequence Type ◽  
Efflux Pump Inhibitor ◽  
Wide Range ◽  
The Relationship

Abstract Background: Pseudomonas aeruginosa is a major opportunistic pathogen involved in healthcare-associated infections with high mortality rates. This bacterium exhibits elevated resistance to a wide range of antibiotics, resulting in part from the overexpression of efflux pumps, among which MexAB-OprM stands out as constitutive. Antibiotic resistance in clinical isolates is associated with mutations in the mexR, nalC, and nalD repressors that modulate the expression of this efflux pump. This study identifies point mutations in the mexR, nalC, and nalD genes and investigates their associations with antibiotic resistance and sequence type in clinical and epidemiologically high-risk clones of P. aeruginosa. Results: A total of 91 P. aeruginosa strains isolated at a pediatric hospital in Mexico (2007–2015) were classified according to their resistance to antibiotics. The strains were typed by multilocus sequencing of 7 genes. The MexAB-OprM efflux pump phenotype was determined using the minimal inhibitory concentration for the reporter antibiotic carbenicillin in the presence/absence of the efflux pump inhibitor Phe-Arg-β-naphthylamine. Sequencing of the mexR, nalC, and nalD genes to identify mutations was performed. Genetic relationship among the strains was evaluated by a phylogenetic inference analysis using maximum likelihood to construct a phylogenetic network. The relationship between variables was determined by a principal component analysis. STs revealed six main complexes. Mutations in the mexR, nalC, and nalD genes revealed 27 different haplotypes. Pan-drug and extensive drug resistant profiles were associated with specific STs with haplotypes 1 (ST1725, endemic clone), 8, 12 (ST233, epidemiologically high-risk clone), and 5 [related to dead when compared to ST1725 and ST233 (RRR 23.34; p=0.009 and RRR 32.01; p=0.025)], however the resistance in these strains was not mainly attributed to the MexAB-OprM phenotype. Strains with the same haplotype and resistant profile showed different pump behavior.Conclusions: A significant relationship between ST and resistant profiles was observed; on one hand, the mexR-nalC-nalD haplotypes were not related to the MexAB-OprM efflux pump phenotypic behavior. On the other hand, the relationship between mexR-nalC-nalD haplotypes and phylogenetically related ST, suggest mutations in these repressors are highly maintained within these STs.

scholarly journals Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in the elongation factor-encoding gene, fusA1

2019 ◽  
Author(s):  
Eve A. Maunders ◽  
Rory C. Triniman ◽  
Taufiq Rahman ◽  
Martin Welch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Large Scale ◽  
Efflux Pump ◽  
Elongation Factor ◽  
Virulence Gene ◽  
Mutant Gene ◽  
Opportunistic Pathogen ◽  
Aminoglycoside Resistance ◽  
Virulence Gene Expression

AbstractPseudomonas aeruginosa is a common opportunistic pathogen. The organism displays elevated intrinsic antibiotic resistance and can cause life-threatening infections. The gene encoding an elongation factor, FusA1, is frequently mutated in clinical isolates of P. aeruginosa from patients with cystic fibrosis (CF). Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the aminoglycoside efflux pump, MexXY. In the current work, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Through a combination of proteomic and transcriptomic analyses, we found that the fusA1 mutant also exhibited large-scale but discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III Secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI Secretion (T6S) machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6S system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosomal protein synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable to that in the PAO1-derived progenitor strain by expression of the wild-type fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (although we also identify additional contributory resistance mechanisms), mutations in fusA1 can lead to highly-selective dysregulation of virulence gene expression.

scholarly journals The multidrug resistance efflux pump MexCD-OprJ is a switcher of thePseudomonas aeruginosaquorum sensing response

2018 ◽  
Author(s):  
Manuel Alcalde-Rico ◽  
Jorge Olivares-Pacheco ◽  
Carolina Alvarez-Ortega ◽  
Miguel Cámara ◽  
José Luis Martínez
Keyword(s):  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Antibiotic Resistance Genes ◽  
Opportunistic Pathogen ◽  
Point Of View ◽  
Signal Molecules ◽  
Human Pathogens ◽  
Chronic Infections ◽  
Intrinsic Resistance ◽  
Multidrug Efflux Pump

AbstractMost antibiotic resistance genes acquired by human pathogens originate from environmental microorganisms. Therefore, understanding the additional functions of these genes, other than conferring antibiotic resistance, is relevant from an ecological point of view. We examined the effect that overexpression of the MexCD-OprJ multidrug efflux pump has in the physiology of the environmental opportunistic pathogenPseudomonas aeruginosa. Overexpression of this intrinsic resistance determinant shuts down theP. aeruginosaquorum sensing (QS) response. Impaired QS response is due to the extrusion of 4-hydroxy-2-heptylquinoline (HHQ), the precursor of thePseudomonasQuinolone Signal (PQS), leading to low PQS intracellular levels and reduced production of QS signal molecules. TheP. aeruginosaQS response induces the expression of hundreds of genes, which can be costly unless such activation becomes beneficial for the bacterial population. While it is known that the QS response is modulated by population density, information on additional signals/cues that may alert the cells about the benefits of mounting the response is still scarce. It is possible that MexCD-OprJ plays a role in this particular aspect; our results indicate that, upon overexpression, MexCD-OprJ can act as a switcher in the QS population response. If MexCD-OprJ alleviate the cost associated to trigger the QS response when un-needed, it could be possible that MexCD-OprJ overproducer strains might be eventually selected even in the absence of antibiotic selective pressure, acting as antibiotic resistant cheaters in heterogeneousP. aeruginosapopulations. This possibility may have potential implications for the treatment ofP. aeruginosachronic infections.

scholarly journals Regulation of Serratia marcescens ompF and ompC porin genes in response to osmotic stress, salicylate, temperature and pH

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 485-491 ◽  
Author(s):  
Sanela Begic ◽  
Elizabeth A. Worobec
Keyword(s):  
Antibiotic Resistance ◽  
Serratia Marcescens ◽  
Growth Temperature ◽  
High Growth ◽  
Opportunistic Pathogen ◽  
Translation Regulation ◽  
Lower Growth ◽  
Porin Proteins ◽  
Outer Membrane Permeability ◽  
High Degree

Serratia marcescens is a Gram-negative enterobacterium that has become an important opportunistic pathogen, largely due to its high degree of natural antibiotic resistance. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, which is controlled in part by OmpC and OmpF porin proteins. OmpF expression is regulated by micF, an RNA transcript encoded upstream of the ompC gene, which hybridizes with the ompF transcript to inhibit its translation. Regulation of S. marcescens porin gene expression, as well as that of micF, was investigated using β-galactosidase reporter gene fusions in response to 5, 8 and 10 % sucrose, 1, 5 and 8 mM salicylate, and different pH and temperature values. β-Galactosidase activity assays revealed that a lower growth temperature (28 °C), a more basic pH (pH 8), and an absence of sucrose and salicylate induce the transcription of the ompF gene, whereas the induction of ompC is stimulated at a higher growth temperature (42 °C), acidic pH (pH 6), and maximum concentrations of sucrose (10 %) and salicylate (8 mM). In addition, when multiple conditions were tested, temperature had the predominant effect, followed by pH. In this study, it was found that the MicF regulatory mechanism does not play a role in the osmoregulation of the ompF and ompC genes, whereas MicF does repress OmpF expression in the presence of salicylate and high growth temperature, and under low pH conditions.

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