scholarly journals A Novel Gene Amplification Causes Upregulation of the PatAB ABC Transporter and Fluoroquinolone Resistance in Streptococcus pneumoniae

2015 ◽  
Vol 59 (6) ◽  
pp. 3098-3108 ◽  
Author(s):  
Alison J. Baylay ◽  
Alasdair Ivens ◽  
Laura J. V. Piddock
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Abc Transporter ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Gene Dosage ◽  
Fluoroquinolone Resistance ◽  
Reporter System ◽  
Content Type ◽  
Genomic Duplication

ABSTRACTOverexpression of the ABC transporter genespatAandpatBconfers efflux-mediated fluoroquinolone resistance inStreptococcus pneumoniaeand is also linked to pneumococcal stress responses. Although upregulation ofpatABhas been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression ofpatABin M184, a multidrug-resistant mutant of S.pneumoniaeR6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included thepatABgenes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels ofpatABin M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy ofpatAhad no effect on multidrug resistance. Using a green fluorescent protein reporter system, increasedpatABexpression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy ofpatAB. This is the first report of a large genomic duplication causing antibiotic resistance inS. pneumoniaeand also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

2014 ◽  
Vol 58 (9) ◽  
pp. 5191-5201 ◽  
Author(s):  
Giorgia Letizia Marcone ◽  
Elisa Binda ◽  
Lucia Carrano ◽  
Mervyn Bibb ◽  
Flavia Marinelli
Keyword(s):  
Antibiotic Resistance ◽  
Gene Dosage ◽  
Antibiotic Production ◽  
Resistance Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Penicillin G ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
High Level

ABSTRACTGlycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens.Nonomuraeasp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonicalvanHAXgenes. Consequently, we investigated the role of the β-lactam-sensitived,d-peptidase/d,d-carboxypeptidase encoded byvanYn, the onlyvan-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic inNonomuraeasp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we variedvanYngene dosage and expressedvanHatAatXatfrom the teicoplanin producerActinoplanes teichomyceticusinNonomuraeasp. ATCC 39727. Knocking outvanYn, complementing avanYnmutant, or duplicatingvanYnhad no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production.Nonomuraeasp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYnactivity. The heterologous expression ofvanHatAatXatincreased A40926 resistance inNonomuraeasp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. ThevanYn-based self-resistance inNonomuraeasp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants ofEnterococcus faeciumselectedin vitrofor high-level resistance to glycopeptides and penicillins.

scholarly journals Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Reed M. Stubbendieck ◽  
Paul D. Straight
Keyword(s):  
Antibiotic Resistance ◽  
Abc Transporter ◽  
Bacterial Communities ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Bioactive Metabolites ◽  
Signaling System ◽  
Content Type ◽  
Two Component ◽  
Analytical Approaches

ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches.

scholarly journals Novel Mechanisms of Efflux-Mediated Levofloxacin Resistance and Reduced Amikacin Susceptibility in Stenotrophomonas maltophilia

2020 ◽  
Vol 65 (1) ◽  
pp. e01284-20
Author(s):  
Punyawee Dulyayangkul ◽  
Karina Calvopiña ◽  
Kate J. Heesom ◽  
Matthew B. Avison
Keyword(s):  
Abc Transporter ◽  
Abc Transporters ◽  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Regulatory System ◽  
Fluoroquinolone Resistance ◽  
Content Type ◽  
Antimicrobial Drug Resistance ◽  
Regulatory Systems ◽  
Selection Of

ABSTRACTFluoroquinolone resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant factor is overproduction of efflux pumps, particularly SmeDEF, following mutation. Here, we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVr, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVr causing overproduction of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Overproduction of one ABC transporter, Smlt1651-4 (renamed SmaCDEF), causes levofloxacin resistance in K M6 LEVr. Overproduction of the other ABC transporter, Smlt2642/3 (renamed SmaAB), and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVr. Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

scholarly journals Refining the Pneumococcal Competence Regulon by RNA Sequencing

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Jelle Slager ◽  
Rieza Aprianto ◽  
Jan-Willem Veening
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Rna Sequencing ◽  
Genome Annotation ◽  
Stress Factors ◽  
Microarray Technology ◽  
Human Pathogen ◽  
Exogenous Dna ◽  
Content Type ◽  
Opportunistic Human Pathogen

ABSTRACTCompetence for genetic transformation allows the opportunistic human pathogenStreptococcus pneumoniaeto take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniaeis an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

scholarly journals A Low-Affinity Penicillin-Binding Protein 2x Variant Is Required for Heteroresistance in Streptococcus pneumoniae

2014 ◽  
Vol 58 (7) ◽  
pp. 3934-3941 ◽  
Author(s):  
Hansjürg Engel ◽  
Moana Mika ◽  
Dalia Denapaite ◽  
Regine Hakenbeck ◽  
Kathrin Mühlemann ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Abc Transporter ◽  
Binding Protein ◽  
Population Analysis ◽  
Resistance Testing ◽  
Penicillin Binding Protein ◽  
Secondary Resistance ◽  
Content Type ◽  
Abc Transporter Genes

ABSTRACTHeteroresistance to penicillin inStreptococcus pneumoniaeis the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain23F2349in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found thatpbp2x, but notpbp2borpbp1aalone, conferred heteroresistance to R6. However, a change ofpbp2xexpression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP onciaRdisruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinitypbp2xundergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded bypstS,phoU,pstB, andpstCin these highly resistant subpopulations. In conclusion, the presence of low-affinitypbp2xenables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.

scholarly journals DprA-Dependent Exit from the Competent State Regulates Multifaceted Streptococcus pneumoniae Virulence

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Jingjun Lin ◽  
Gee W. Lau
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Cell Separation ◽  
Protein A ◽  
Wild Type ◽  
Content Type ◽  
Cellular Processes ◽  
Virulence Attenuation ◽  
Specific Induction ◽  
Competence System

ABSTRACT Streptococcus pneumoniae (pneumococcus) causes multiple infectious diseases. The pneumococcal competence system facilitates genetic transformation, spreads antibiotic resistance, and contributes to virulence. DNA-processing protein A (DprA) regulates the exit of pneumococcus from the competent state. Previously, we have shown that DprA is important in both bacteremia and pneumonia infections. Here, we examined the mechanisms of virulence attenuation in a ΔdprA mutant. Compared to the parental wild-type D39, the ΔdprA mutant enters the competent state when exposed to lower concentrations of the competence-stimulating peptide CSP1. The ΔdprA mutant overexpresses ComM, which delays cell separation after division. Additionally, the ΔdprA mutant overexpresses allolytic factors LytA, CbpD, and CibAB and is more susceptible to detergent-triggered lysis. Disabling of the competent-state-specific induction of ComM and allolytic factors compensated for the virulence loss in the ΔdprA mutant, suggesting that overexpression of these factors contributes to virulence attenuation. Finally, the ΔdprA mutant fails to downregulate the expression of multiple competence-regulated genes, leading to the excessive energy consumption. Collectively, these results indicate that an inability to properly exit the competent state disrupts multiple cellular processes that cause virulence attenuation in the ΔdprA mutant.

scholarly journals Ingestibility, Digestibility, and Engineered Biological Control Potential of Flavobacteriumhibernum, Isolated from Larval Mosquito Habitats

2013 ◽  
Vol 80 (3) ◽  
pp. 1150-1158 ◽  
Author(s):  
Shicheng Chen ◽  
Michael G. Kaufman ◽  
Michelle L. Korir ◽  
Edward D. Walker
Keyword(s):  
Fluorescent Protein ◽  
Ingestion Rate ◽  
Expression System ◽  
Luciferase Reporter ◽  
Larval Feeding ◽  
Protein Markers ◽  
Reporter System ◽  
Content Type ◽  
Larval Habitats ◽  
Tree Hole

ABSTRACTFlavobacteriumhibernum, isolated from larval habitats of the eastern tree hole mosquito,A. triseriatus, remained suspended in the larval feeding zone much longer (8 days) than other bacteria. Autofluorescent protein markers were developed for the labeling ofF. hibernumwith a strong flavobacterial expression system. Green fluorescent protein (GFP)-taggedF. hibernumcells were quickly consumed by larval mosquitoes at an ingestion rate of 9.5 × 104/larva/h. The ingestedF. hibernumcells were observed mostly in the foregut and midgut and rarely in the hindgut, suggesting that cells were digested and did not pass the gut viably. The NanoLuc luciferase reporter system was validated for quantitative larval ingestion rate and bacterial fate analyses. Larvae digested 1.87 × 105cells/larva/h, and fewF. hibernumcells were excreted intact. Expression of the GFP::Cry11A fusion protein with the P20 chaperone protein fromBacillus thuringiensisH-14 was successfully achieved inF. hibernum. Whole-cell bioassays of recombinantF. hibernumexhibited high larvicidal activity againstA. triseriatusin microplates and in microcosms simulating tree holes.F. hibernumcells persisted in microcosms at 100, 59, 30, and 10% of the initial densities at days 1, 2, 3, and 6, respectively, when larvae were absent, while larvae consumed nearly all of theF. hibernumcells within 3 days of their addition to microcosms.

scholarly journals SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

2013 ◽  
Vol 57 (7) ◽  
pp. 3430-3433 ◽  
Author(s):  
Jose Antonio Escudero ◽  
Alvaro San Millan ◽  
Natalia Montero ◽  
Belen Gutierrez ◽  
Cristina Martinez Ovejero ◽  
...  
Keyword(s):  
Abc Transporter ◽  
High Mortality ◽  
Human Population ◽  
Efflux Pump ◽  
Streptococcus Suis ◽  
Open Reading Frame ◽  
Fluoroquinolone Resistance ◽  
Content Type ◽  
Reading Frame

ABSTRACTStreptococcus suisis an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterizesatR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor ofsatAB. satRis cotranscribed withsatAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones.

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