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Membrane Permeability and Regulation of Drug “Influx and Efflux” in Enterobacterial Pathogens
Abstract and Figures
In Enterobacteriaceae, membrane permeability is a key in the level of susceptibility to antibiotics. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems has been reported. These phenomena are frequently associated with other resistance mechanisms such as alteration of antibiotics or modification of the drug targets, in various clinical isolates showing a Multi Drug Resistant phenotype (MDR). In Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of MDR isolates. These bacterial isolates exhibit a noticeable reduction of functional porins per cell due to a decrease, a complete shutdown of synthesis, or the expression of an altered porin and a high expression of efflux systems (e.g. overexpression of the pump). The combined action of these mechanisms during an infection confers a significant decrease in bacterial sensitivity to antibiotherapy ensuring dissemination and colonization of the patient and favours the acquisition of additional mechanisms of resistance. MarA and ramA are involved in a complex regulation cascade controlling membrane permeability and actively participate in the triggering of the MDR phenotype. Mutations in regulator genes have been shown to induce the overproduction of efflux and the down-regulation of porin synthesis. In addition, various compounds such as salicylate, imipenem or chloramphenicol are able to activate the MDR response. This phenomenon has been observed both in vitro during culture of bacteria in the presence of drugs and in vivo during antibiotic treatment of infected patients. These effectors activate the expression of specific global regulators, marA, ramA, or target other genes located downstream in the regulation cascade.
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... In antibiotic sensitivity testing approximately 3 from 4 strains of S. enterica were multidrug-resistant MDR, in which bacterial strains are resistant to more than three classes of antibiotics (Magiorakos et al. 2012), this was expected due to worldwide spreading of MDR bacteria (Hawkey et al. 2019;Coipan et al. 2020 andWHO 2021). As previously documented for clinical isolates, the variation between the five cities is related to reduce prescribing of particular drugs in certain cities (Gatto et al. 2006;Davin-Regli et al. 2008). The isolation of one strain of bacteria resistant to more than ten antibiotics in poultry has led us to fear that there will be no effective medicines available to treat resistant infections one day (Handayani et al., 2017). ...
... The isolation of one strain of bacteria resistant to more than ten antibiotics in poultry has led us to fear that there will be no effective medicines available to treat resistant infections one day (Handayani et al., 2017). Chloramphenicol, which was blindly supplied by veterinarians in Egypt for diarrhoea, was found to stimulate the MDR response by activating the production of certain regulatory mRNA or other genes, according to Davin-Regli et al. (2008). While point mutations in DNA gyrase genes or activation of the efflux pump may cause enhanced resistance in food-borne isolates (Meakins et al. 2008), horizontal transfer and clonal expansion of resistance genes may occur among food-producing animals and humans (Hawkey 2008). ...
... Nokhodchi et al. (2012) in their review article clarify the challenge in drug delivery to combat Salmonella spp. and fail of new antibiotic to eradicate the pathogens completely, due to difficulty of transport of antibiotic retail through membrane (Drulis-Kawa and Dorotkiewicz-Jach 2010); reduced cell membrane permeability has been dedicated as a key mechanism of resistance to antibiotic (Davin-Regli et al. 2008), while nanoparticles adhere to cell membrane of Salmonella and in some species of Salmonella can release into the interior of the bacteria as in case of Salmonella Enteritidis, thus can interfere with bacterial resistance and infection mechanism which involve low membrane permeability or efflux system (Mugabe et al. 2006). But the dosage of silver nanoparticles differs according to its concentration (Ranjan et al. 2009). ...
A total no. of 65 Salmonella enterica isolates recovered from food samples, feces of diarrheic calves, poultry, and hospital patient in large five cities at Northern West Egypt were obtained from the Department of Microbiology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt. The 65 Salmonella enterica isolates had the inv A gene were grouped into 11 Salmonella enterica serovars with dominance of S. Enteritidis and S. Kentucky serovars. Their resistance pattern were characterized by using 18 antibiotics from different classes. Approximately 80% of the isolates were multidrug resistant (MDR). Enterobacterial repetitive intergenic consequences polymerase chain reaction (ERIC-PCR) typing of 7 strains of S. Enteritidis showed 5 clusters with dissimilarity 25%. S. Enteritidis clusters in 2 main groups A and B. Group A have 2 human strain (HE2 and HE3) and one food origin (FE7) with a similarity 99%. Group B divided into B1 (FE2) and B2 (FE3) with a similarity ratio ≥ 93%, while ERIC-PCR analysis of 5 strains of S. Kentucky revealed 4 ERIC types, clustered in 2 main groups A and B with similarity 75%. We studied the effect of silver nanoparticles (Ag-NPs) on 10 antibiotic resistant strains of S . Enteritidis and S. Kentucky. The broth microdilution minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were detected. Evaluation of the affection using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed different ratios of Ag-NPs and microorganism as well as at different contact time ended finally with morphological alteration of the bacteria. We submitted new method in vivo to explore the activity of nanosilver in chicken.
Key points
• Importance of ERIC-PCR to determine the relatedness between Salmonella isolates.
• Effect of silver nanoparticles to confront the antibacterial resistance .
• Studying the effect of silver nanoparticles in vivo on infected chicken with Salmonella .
... Enfin, le dernier mécanisme de résistance consiste à limiter l'entrée des composées antimicrobiens dans la cellule en modifiant la perméabilité membranaire. La diminution de l'absorption passive (via les porines) ou l'augmentation de l'excrétion active des antibiotiques sont largement étudiées car il s'agit des principaux contributeurs de ce phénotype bactérien de multirésistance (Alekshun and Levy, 2007;Davin-Regli et al., 2008;James et al., 2009;Li and Nikaido, 2009;Martinez, 2009;Nikaido, 2009;Nikaido and Pagès, 2012;Piddock, 2006;Poole, 2007). Ces processus se mettent en place via l'activation de divers types de transporteurs membranaires qui permettent l'excrétion des antibiotiques. ...
... Stenotrophomonas maltophilia qui présente une virulence atténuée lorsque la pompe SmeDEF est surexprimée. Une analyse du profil métabolique des bactéries a permis de montrer que cette diminution de la virulence résulte d'un surcharge métabolique créée par la surproduction de la pompe (Alonso et al., 2004 (Bialek et al., 2010;Bratu et al., 2009;Davin-Regli et al., 2008;Padilla et al., 2010;Pagès and Amaral, 2009 Les pompes décrites chez K. pneumoniae, en plus de fournir à la bactérie un phénotype de résistance antibiotique, peuvent intervenir dans une multitude de fonction. Les systèmes KmrA et KpnGH de la famille MFS sont à l'origine de résistances à diverses substances comprenant par exemple l'acriflavine, le DAPI (4ʹ,6-diamidino-2-phenylindole), le méthyl viologène ou encore le bromure d'éthidium (Ogawa et al., 2006;Srinivasan et al., 2014). ...
Klebsiella pneumoniae est une entérobactérie à Gram-négatif ubiquiste que l’on retrouve dans l’environnement (sols, eaux de surface, …) ainsi que dans le système digestif humain. En milieu hospitalier, cette bactérie est un pathogène opportuniste à l’origine d’infections liées aux soins, notamment pulmonaires et urinaires. Grâce à sa capacité à former des biofilms, K. pneumoniae colonise facilement les épithéliums tels que ceux des voies aériennes supérieures et du tractus digestif, ainsi que des dispositifs médicaux invasifs. Les phénomènes de colonisation et de formation de biofilm sont des éléments clés de la pathogénicité de K. pneumoniae. Ils impliquent des structures de surface, et notamment la capsule polysaccharidique et les fimbriae de type I et de type III (Paczosa et al., 2020). De précédents travaux menés au laboratoire ont permis d’identifier d’autres facteurs de pathogénicité, certains étant spécifiquement impliqués dans la colonisation du tube digestif et des surfaces (Balestrino et al., 2005, 2008; Coudeyras et al., 2008; De Araujo et al., 2010; Hennequin and Forestier, 2007, 2009; Maroncle et al., 2002, 2006). De plus, dans le but d’identifier de nouvelles structures d’adhésion de K. pneumoniae, une analyse in silico a permis d’identifier une structure de surface de type « chaperone-usher » (CU), appelée Kpg, impliquée dans la colonisation intestinale dans un modèle murin (Khater et al., 2015). A partir d’un biofilm mature, les bactéries peuvent se disperser sous forme de bactéries isolées ou de petits agrégats. Cette étape de dispersion a longtemps été sous-estimée dans le processus de développement des infections bactériennes, mais des données récentes indiquent qu’elle jouerait un rôle crucial dans l’apparition d’infections aiguës ou chroniques difficiles à éradiquer. Alors que l’impact de la dispersion des biofilms dans la physiopathologie des infections à K. pneumoniae est méconnue, nous avons montré que les bactéries dispersées K. pneumoniae sont dans un état physiologique spécifique et qu’elles ont des propriétés de colonisation des surfaces accrues comparativement aux formes planctoniques (Guilhen et al., 2019). Une approche globale par séquençage d’ARN (RNAseq) a identifié de nombreux gènes de K. pneumoniae surexprimés dans les bactéries dispersées comparativement aux formes sessiles ou planctoniques, et notamment les gènes mdtJI codant la pompe à efflux MdtIJ de la famille des SMR (Guilhen et al., 2016, 2019).L’objectif de mon projet de thèse était de caractériser les fimbriae Kpg et la pompe à efflux MdtJI afin de décrire leur rôle dans la pathogénicité de K. pneumoniae. Une analyse in silico a montré que le système Kpg est retrouvé au sein de plusieurs espèces d’Entérobactéries, principalement au sein d’espèces du genre Klebsiella, et que son acquisition résulte probablement de transferts horizontaux de gènes. Bien que le système Kpg soit faiblement exprimé dans la souche K. pneumoniae CH1476 en conditions de laboratoire, sa localisation membranaire a été confirmée par Western blot. L’analyse phénotypique d’un mutant déficient ∆kpgABCD a montré que le système Kpg est impliqué dans la formation de biofilm en présence de sels biliaires, une condition retrouvée au sein du tractus digestif. En effet, alors que la formation de biofilm de la souche sauvage de K. pneumoniae est stimulée par la présence de sels biliaires, aucun effet des sels biliaires n'a été observé pour le mutant isogénique. Aucun rôle du fimbriae Kpg dans l’adhésion aux cellules intestinales n’a cependant été démontré.La seconde partie de ma thèse a consisté à analyser les propriétés de la pompe à efflux MdtJI. (...)
... In fact, efflux pumps can extrude out extensive variety of various antibiotic classes. For this reason, its inhibition increases bacterial susceptibility, and mixture with multiple antimicrobials may fit .There are several methods to inhibit efflux pumps: (i) adding functional group to drug substrates to interfere with recognition ,(ii) interfering with efflux genes expression, (iii) can block the action of small molecules designed efflux pumps as substrate analogues, or (iv) can intervene with the pump's energy transfer mechanism, (v) interfering with channel protein system or (vi) block channels can be [101,102] .Consequently, it may be confirmed that inhibition of efflux results in various positive outcomes. (i) booms the action of eliminated antimicrobials, (ii) maintain drug concentration at therapeutic doses, and (iii) lessen remedy length by means of lowering MDR [103,104] . ...
This book series invites all the Specialists, Professors, Doctors, Scientists, Academicians, Healthcare professionals, Nurses, Students, Researchers, Business Delegates, and Industrialists across the globe to publish their insights and convey recent developments in the field of Nursing, Pharmaceutical Research and Innovations in Pharma Industry. Book series on Pharmacy and Nursing covers research work in a set of clinical sciences and medicine. It also provides a remarkable opportunity for the pharmacy and nursing based academic and research communities to address new challenges and share solutions and discuss future research directions.
... Therefore, the delivery of the drug to all cells is currently a big challenge to clinicians. Moreover, the reduced membrane permeability of microorganisms has been cited as a key mechanism of resistance to antibiotics (Davin-Regli et al., 2008). Unfortunately, it is a universal fact that new antibiotics ultimately develop resistance . ...
Background
Avian salmonellosis is a group of diseases caused by bacteria from the genus Salmonella with a negative impact on poultry, particularly chickens. In addition, salmonellosis is a global food-borne infection.
Aim
The aim of this study was to evaluate the effect of nano-emulsion difloxacin (NED) and commercial difloxacin (CD) water supplement on broiler's growth, feed intake, and body weight, weight gain, growth rate, feed conversion ratio (FCR), and mortality rate (MR). The antibiotic sensitivity was determined both in-vivo and in-vitro for NED against Salmonella enterica Serovar enteritidis in chickens.
Methods
1500 one-day of age chicks were grouped into five groups as follows: group 1 (G1) control negative group, G2 control positive group (infected and not treated), G3 (infected and treated with CD, and G4 and G5 (infected and treated with NED at different doses). Samples, including the intestine, liver, and spleen were collected. Agar well diffusion test and minimum inhibitory concentrations were adopted. Histopathological lesions on different tissues were studied. During 35 days of the experiment, the feed intake, growth rate, growth gain, FCR, and MR were recorded daily. In addition, a variety of analytical techniques including transmission electron microscopic analysis, dynamic light scattering, UV-visible spectroscopy, and zeta-potential analysis were applied to characterize NED.
Results
The agar well diffusion test indicated that NED was in-vitro effective against S. enteritidis isolates than CD. The minimum inhibitory concentration was recorded as NED inhibited bacterial growth till well 8 at a concentration of 0.78 µg/ml; on the other hand, the CD inhibited bacterial growth till well 6 at a concentration of 0.62 µg/ml. Growth performance and MRs in the groups treated with NED are significantly reduced.
Conclusion
Treatment of broiler's drinking water with NED at doses of 0.5 and 1 ml instead of pure CD was able to enforce a new perspective, antibacterial efficacy, enhancing the productive performance, and reducing the MRs of broilers.
... As for β-lactams and others, the Enterobacteriaceae were all resistant (100%). Previous work corroborates our results on the sensitivity of Enterobacteriaceae strains isolated to the antibiotics tested [53,54]. In addition, some work has shown that vancomycin, penicillin G, and macrolides are effective against food-borne S. aureus strains [52,55]. ...
This study aimed to characterize the pathogenicity of bacteria isolated from the starter of two traditional beers produced and consumed in Benin. After standard microbial identification, species were identified by specific biochemical tests such as catalase, coagulase, and API 20 E. Antibiotic sensitivity was tested according to the French Society of Microbiology Antibiogram Committee. The crystal violet microplate technique evaluated the biofilm production and conventional PCR was used to identify genes encoding virulence and macrolide resistance. According to our data, the traditional starter known as kpètè-kpètè that is used to produce beer is contaminated by Enterobacteriaceae and staphylococci species. Thus, 28.43% of the isolated bacteria were coagulase-negative staphylococci (CNS), and 10.93% coagulase-positive staphylococci (CPS). Six species such as Klebsiella terrigena (1.38%), Enterobacter aerogens (4.14%), Providencia rettgeri (5.51%), Chryseomonas luteola (6.89%), Serratia rubidae (15.16%), and Enterobacter cloacae (27.56%) were identified among Enterobacteriaceae. Those bacterial strains are multi-resistant to conventional antibiotics. The hight capability of produced biofilms was recorded with Enterobacter aerogens, Klebsiella terrigena (100%), Providencia rettgeri (75%), and Staphylococcus spp (60%). Enterobacter cloacae (4%) and coagulase-negative Staphylococcus (5.55%) harbor the macrolide resistance gene. For other strains, these genes were not detected. Foods contaminated with bacteria resistant to antibiotics and carrying a virulence gene could constitute a potential public health problem. There is a need to increase awareness campaigns on hygiene rules in preparing and selling these traditional beers.
... As for β-lactams and others, the Enterobacteriaceae were all resistant (100%). Previous work corroborates our results on the sensitivity of Enterobacteriaceae strains isolated to the antibiotics tested [53,54]. In addition, some work has shown that vancomycin, penicillin G, and macrolides are effective against food-borne S. aureus strains [52,55]. ...
This study aimed to determine the pathogenicity of the bacteria isolated and characterized from the kpètè kpètè used to produce two fermented beers in Benin. Species were identified by specific biochemical tests such as catalase, coagulase, and API 20 E. Antibiotic sensitivity was tested according to the French Society of Microbiology Antibiogram Committee. The crystal violet microplate technique and conventional PCR evaluated biofilm production to identify genes encoding virulence and macrolide resistance. Our data shows that Kpètè Kpètè used to produce beers are contaminated by Enterobacteriaceae species (Klebsiella terrigena, Enterobacter aerogens, Providencia rettgeri, Chryseomonas luteola, Serratia rubidae, and Enterobacter cloacae) and Staphylococcus spp. These multidrug-resistant strains can produce biofilms with a strong predominance of Enterobacter aerogens, Klebsiella terrigena (100%), and Staphylococcus spp (60%). Enterobacter cloacae (4%) and Coagulase negative Staphylococcus (5.55%) harbor the macrolide resistance gene. For other strains, these genes were not detected. Foods contaminated with bacteria resistant to antibiotics and carrying a virulence gene could constitute a potential public health problem. There is a need to increase awareness campaigns on hygiene rules in preparing and selling these traditional beers.
... Bacteria engage a variety of mechanisms to resist the action of antibiotics. For some bacteria, the balance in membrane permeability/impermeability plays a key role in limiting the uptake of antimicrobial compounds and the subsequent diffusion and efflux that reduces the intracellular concentration [48]. We found 26 chromosomal genes in all the SG strains, with most encoding efflux pumps, associated with resistance to 16 classes of antimicrobial agents as well as antiseptics and disinfectants. ...
Salmonella Gallinarum (SG) is the causative agent of fowl typhoid (FT), a disease that is harmful to the poultry industry. Despite sanitation and prophylactic measures, this pathogen is associated with frequent disease outbreaks in developing countries, causing high morbidity and mortality. We characterized the complete genome sequence of Colombian SG strains and then performed a comparative genome analysis with other SG strains found in different regions worldwide. Eight field strains of SG plus a 9R-derived vaccine were subjected to whole-genome sequencing (WGS) and bioinformatics analysis, and the results were used for subsequent molecular typing; virulome, resistome, and mobilome characterization; and a comparative genome study. We identified 26 chromosome-located resistance genes that mostly encode efflux pumps, and point mutations were found in gyrase genes (gyrA and gyrB), with the gyrB mutation S464T frequently found in the Colombian strains. Moreover, we detected 135 virulence genes, mainly in 15 different Salmonella pathogenicity islands (SPIs). We generated an SPI profile for SG, including C63PI, CS54, ssaD, SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-6, SPI-9, SPI-10, SPI-11, SPI-12, SPI-13, and SPI-14. Regarding mobile genetic elements, we found the plasmids Col(pHAD28) and IncFII(S) in most of the strains and 13 different prophage sequences, indicating a frequently obtained profile that included the complete phage Gifsy_2 and incomplete phage sequences resembling Escher_500465_2, Shigel_SfIV, Entero_mEp237, and Salmon_SJ46. This study presents, for the first time, the genomic content of Colombian SG strains and a profile of the genetic elements frequently found in SG, which can be further studied to clarify the pathogenicity and evolutionary characteristics of this serotype.
... A widely used method is the measurement of the fluorescence of several molecules [480][481][482]. The fluorescent agent must be a component with high permeability capacity, as membrane permeability can be regulated by reducing the number of porins or by increasing the lipopolysaccharide layer of the cell envelope [483,484]. β-lactams, fluoroquinolones such as levofloxacin, macrolides, tetracyclines, trimethoprim, sulfonamides, chloramphenicol, glycylcyclines, and ethidium bromide. [200,234,328,431,[474][475][476] As an example of these agents, EtBr stands out as a DNAintercalating fluorochrome, a common substrate of MDR efflux pumps, including almost all S. aureus efflux pumps studied [485,486]. ...
Antibiotic resistance can be characterized, in biochemical terms, as an antibiotic’s inability to reach its bacterial target at a concentration that was previously effective. Microbial resistance to different agents can be intrinsic or acquired. Intrinsic resistance occurs due to inherent functional or structural characteristics of the bacteria, such as antibiotic-inactivating enzymes, nonspecific efflux pumps, and permeability barriers. On the other hand, bacteria can acquire resistance mechanisms via horizontal gene transfer in mobile genetic elements such as plasmids. Acquired resistance mechanisms include another category of efflux pumps with more specific substrates, which are plasmid-encoded. Efflux pumps are considered one of the main mechanisms of bacterial resistance to antibiotics and biocides, presenting themselves as integral membrane transporters. They are essential in both bacterial physiology and defense and are responsible for exporting structurally diverse substrates, falling into the following main families: ATP-binding cassette (ABC), multidrug and toxic compound extrusion (MATE), major facilitator superfamily (MFS), small multidrug resistance (SMR) and resistance-nodulation-cell division (RND). The Efflux pumps NorA and Tet(K) of the MFS family, MepA of the MATE family, and MsrA of the ABC family are some examples of specific efflux pumps that act in the extrusion of antibiotics. In this review, we address bacterial efflux pump inhibitors (EPIs), including 1,8-naphthyridine sulfonamide derivatives, given the pre-existing knowledge about the chemical characteristics that favor their biological activity. The modification and emergence of resistance to new EPIs justify further research on this theme, aiming to develop efficient compounds for clinical use.
... Bacteria also use a drug efflux mechanism to increase the expression of active efflux pumps or decrease the drug permeability from the cell surface, which results in low-level susceptibility to antibiotics. Certain bacteria have shown this type of resistance against fluoroquinolones prior to drug penetration by the inbuilt pump present within the bacterial cellular membrane [13]. ...
There are several bacteria called superbugs that are resistant to multiple antibiotics which can be life threatening specially for critically ill and hospitalized patients. This article provides up-to-date treatment strategies employed against some major superbugs, like methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, vancomycin-resistant Enterococcus, multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Escherichia coli. The pathogen-directed therapeutics decrease the toxicity of bacteria by altering their virulence factors by specific processes. On the other hand, the host-directed therapeutics limits these superbugs by modulating immune cells, enhancing host cell functions, and modifying disease pathology. Several new antibiotics against the global priority superbugs are coming to the market or are in the clinical development phase. Medicinal plants possessing potent secondary metabolites can play a key role in the treatment against these superbugs. Nanotechnology has also emerged as a promising option for combatting them. There is urgent need to continuously figure out the best possible treatment strategy against these superbugs as resistance can also be developed against the new and upcoming antibiotics in future. Rational use of antibiotics and maintenance of proper hygiene must be practiced among patients.
The present chapter highlights the antibacterial potential of nine highly active compounds isolated from the African medicinal plants: 8,8-bis-(dihydroconiferyl)-diferulate, buesgenine, candidone, diospyrone, ferruginin A, isobavachalcone, neobavaisoflavone, neocyclomorusin, and plumbagin. Their botanical source as well as their hypothetical biosynthetic pathways have also been discussed. Regarding their inhibitory potential towards the Gram-negative and Gram-positive bacteria, and mycobacteria, the above phytochemicals constitute potential pharmaceuticals that deserve clinical studies to develop antibacterial drugs to combat bacterial infections involving both drug-sensitive and drug-resistant phenotypes.
DNA microarrays and two-dimensional (2-D) gel electrophoresis were utilized to analyze the global effect of bile on transcription and protein synthesis in Salmonella enterica serovar Typhimurium. Two bile-regulated proteins, YciF and PagC, were identified by 2-D gel electrophoresis and mass spectrometry fingerprinting. The operon yciGFE-katN demonstrated increased transcriptional activity in the presence of bile. While this operon has previously been shown to be RpoS-regulated, data from this study suggested that yciGFE-katN is regulated by bile independent of RpoS. The PhoP–PhoQ-regulated PagC is decreased in the presence of bile. Characterization of the untranslated leader of pagC demonstrated that a 97-bp region is necessary for the bile-mediated repression of this promoter. Analysis of data from the DNA microarray revealed an effect of bile on important global mechanistic pathways in S. enterica serovar Typhimurium. Genes involved in type III secretion-mediated invasion of epithelial cells demonstrated an overall repression of transcription in the presence of bile, corroborating previously reported data from this laboratory [Infect. Immun. 68 (2000) 6763]. In addition, bile-mediated transcriptional repression of genes involved in flagellar biosynthesis and motility was observed. These data further demonstrate that bile is an important environmental signal sensed by Salmonella spp. and that bile plays a role in regulating bacterial gene expression in multiple virulence-associated pathways.
Membrane permeability is the first step involved in resistance of bacteria to an antibiotic. The number and activity of efflux pumps and outer membrane proteins that constitute porins play major roles in the definition of intrinsic resistance in Gram-negative bacteria that is altered under antibiotic exposure.
Here we describe the genetic regulation of porins and efflux pumps of Escherichia coli during prolonged exposure to increasing concentrations of tetracycline and demonstrate, with the aid of quantitative real-time reverse transcriptase-polymerase chain reaction methodology and western blot detection, the sequence order of genetic expression of regulatory genes, their relationship to each other, and the ensuing increased activity of genes that code for transporter proteins of efflux pumps and down-regulation of porin expression.
This study demonstrates that, in addition to the transcriptional regulation of genes coding for membrane proteins, the post-translational regulation of proteins involved in the permeability of Gram-negative bacteria also plays a major role in the physiological adaptation to antibiotic exposure. A model is presented that summarizes events during the physiological adaptation of E. coli to tetracycline exposure.
The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex. In this paper, we show that motile behavior was affected in Escherichia coli hns mutants. The loss of motility resulted from a complete lack of flagella. A decrease in the level of transcription of the flhD and fliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation. Furthermore, the Fla- phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex. These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway.
The mar locus and related systems confer multiple antibiotic resistance and control expression of virulence factors and genes for metabolizing small molecules.
Despite many years of intense work investigating the function of nucleoid-associated proteins in prokaryotes, their role in bacterial physiology remains largely unknown. The two-dimensional protein patterns were compared and expression profiling was carried out on H-NS-deficient and wild-type strains of Escherichia coli K-12. The expression of approximately 5% of the genes and/or the accumulation of their protein was directly or indirectly altered in the hns mutant strain. About one-fifth of these genes encode proteins that are involved in transcription or translation and one-third are known to or were in silico predicted to encode cell envelope components or proteins that are usually involved in bacterial adaptation to changes in environmental conditions. The increased expression of several genes in the mutant resulted in a better ability of this strain to survive at low pH and high osmolarity than the wild-type strain. In particular, the putative regulator, YhiX, plays a central role in the H-NS control of genes required in the glutamate-dependent acid stress response. These results suggest that there is a strong relationship between the H-NS regulon and the maintenance of intracellular homeostasis.
We have described a gene coding for an Enterobacter cloacae protein, provisionally called OmpX (J. Stoorvogel, M. J. A. W. M. van Bussel, J. Tommassen, and J. A. M. van de Klundert, J. Bacteriol. 173:156-160, 1991). In the work reported here, OmpX was localized in the cell envelope by means of sucrose gradient fractionation of membrane vesicles. Overproduction of OmpX in Escherichia coli from a multicopy plasmid resulted in a reduction in the amount of OmpF. No accumulation of OmpF, of its uncleft precursor, or of its degradation products could be detected in various cell fractions by Western immunoblot analysis using monoclonal antibodies produced in response to OmpF. A decrease in the rate of synthesis of ompF mRNA was indicated by a beta-galactosidase assay in an ompF-lacZ fusion strain containing the cloned ompX gene and by Northern (RNA) blot analysis. These results indicate that the inhibition is at the level of transcription. Colony hybridization, using an internal ompX fragment as a probe, showed a widespread distribution of the ompX gene among clinical isolates of members of the family Enterobacteriaceae. To study the function of the OmpX protein and its role in the regulation of porin protein synthesis, the ompX gene was deleted from the Enterobacter cloacae chromosome and replaced by the aphA gene. The absence of the ompX gene had no apparent effect on cell growth or on the regulation of the porin proteins.
Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K-12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.
Chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, selected on agar containing low concentrations of tetracycline or chloramphenicol, were 6- to 18-fold less susceptible to the fluoroquinolones than were their wild-type E. coli K-12 or E. coli C parental strains. The frequency of emergence of such mutants was at least 1,000-fold higher than that of those selected by the fluoroquinolone norfloxacin directly. When Mar mutants, but not wild-type cells, were plated on norfloxacin, mutants resistant to high levels of norfloxacin (2 micrograms/ml) appeared at a relatively high (approximately 10(-7] frequency. In addition to decreased amounts of OmpF, Mar mutants had other outer membrane protein changes and were four- to eightfold less susceptible to fluoroquinolones than was an ompF::Tn5 mutant lacking only OmpF. Accumulation of [3H]norfloxacin was more than threefold lower in the Mar mutants than in wild-type cells and twofold lower than in the OmpF-deficient derivative. These differences were not attributable to a change in the endogenous active efflux system for norfloxacin in E. coli. Norfloxacin-induced inhibition of DNA synthesis was threefold lower in intact cells of a Mar mutant than in susceptible cells, but this difference was not seen in toluene-permeabilized cells. Insertion of Tn5 into marA (min 34.05 on the chromosome) led to a return of the wild-type patterns of norfloxacin accumulation, fluoroquinolone and other antimicrobial agent susceptibilities, and outer membrane protein profile, including partial restoration of OmpF. These findings together suggest that marA-dependent fluoroquinolone resistance is linked to decreased cell permeability, only part of which can be accounted for by the reduction in OmpF. Once mutated to marA, cells can achieve high levels of quinolone resistance at a relatively high frequency.
The multiple antibiotic resistance gene pqrA was cloned from the chromosomal DNA of a clinical isolate of Proteus vulgaris 881051 into Escherichia coli KY2563. The MICs of quinolones tetracycline, cephalosporin, and chloramphenicol for transformant strain DNS7020 were from 8 to 32 times higher than those for the parent strain, KY2563. The level of expression of outer membrane protein F (OmpF) by DNS7020 was lower than that of KY2563 but not as low as that of an OmpF-deficient control strain. The 1.4-kb fragment containing the pqrA gene had an open reading frame encoding a polypeptide of 122 amino acid residues with a molecular weight of about 14,000, which was consistent with the experimental value identified by the Maxicell method. The putative PqrA polypeptide showed significant amino acid sequence similarity to the E. coli proteins SoxS and MarA. These polypeptides are strongly conserved in predicted helix-turn-helix DNA binding domains. The MarA protein, which is responsible for multiple antibiotic resistance in E. coli, also decreases OmpF expression. Moreover, the SoxS protein, which is characterized as a superoxide response regulon of E. coli, has also been shown to increase resistance to many structurally unrelated antibiotics. The soxS gene increases superoxide dismutase levels in addition to decreasing OmpF expression. The expression level of superoxide dismutase with DNS7020 was about 1.5 times higher than that with KY2563. These findings suggest that the pqrA gene in P. vulgaris confers multidrug resistance in a way similar to that of the soxS and marA genes in E. coli.
The marRAB operon is one of two operons in the mar locus of Escherichia coli that are divergently transcribed from a central regulatory region, marO. The marRAB operon, transcribed from marOII, controls intrinsic resistance or susceptibility to multiple antibiotics and is inducible by structurally unrelated compounds such as tetracycline and chloramphenicol (S. P. Cohen, H. Hachler, and S. B. Levy, J. Bacteriol. 175:1484-1492, 1993). To clarify the role of the operon in response to environmental signals, its transcription was studied under different conditions, using a marOII-lacZ transcriptional fusion introduced into the chromosome of wild-type or mar-deleted cells. In wild-type cells, uncoupling agents (such as carbonyl cyanide m-chlorophenylhydrazone) and different redox-cycling compounds (e.g., menadione and plumbagin) induced expression from the marOII-lacZ fusion two- to sevenfold. In the mar-deleted strain, LacZ expression from the fusion was 10-fold higher than in wild-type cells. This activity was temperature sensitive (3-fold lower at 42 than at 30 degrees C) and decreased 20-fold with the introduction of the gene for MarR. Structurally different compounds which induce the mar operon in wild-type cells reversed the MarR repression of marOII-lacZ expression. To determine the size of MarR, it was fused to MalE as a MarR fusion protein of 144 amino acids [MarR(144)] or of 125 amino acids (deleted of 19 amino acids at the N terminus) [MarR(125)]. Only the MarR(144) fusion showed repressor ability. The purified MarR(144) fusion, but not the MarR(125) fusion, bound specifically to marO in vitro, as revealed by gel retardation, with an apparent dissociation constant of 5 x 10(-9) M. MarR, therefore, controls expression of the marRAB operon presumably by binding to marO. MarR repression in cells can be reversed by different compounds, facilitating the response of bacteria to multiple environmental stress conditions.