Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in
                    Chlamydia trachomatis

ABSTRACT Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, including Chlamydia trachomatis . Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogen C. trachomatis lacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against a C. trachomatis genomic library using a conditional-lethal lpxH mutant Escherichia coli strain, we have identified an open reading frame (Ct461, renamed lpxG ) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function in E. coli and catalyzes the conversion of UDP-DAGn to lipid X in vitro . LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity of C. trachomatis , validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools for C. trachomatis and should be broadly applicable for the functional characterization of other essential C. trachomatis genes . IMPORTANCE Chlamydia trachomatis is a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of many Chlamydia genes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against the C. trachomatis genomic library using a conditional-lethal mutant of E. coli and identified the missing essential gene in the lipid A biosynthetic pathway, which we designated lpxG . We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activity in vitro . Overexpression of LpxG in C. trachomatis leads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating the in vivo function of LpxG and highlighting the importance of regulated lipid A biosynthesis in C. trachomatis .

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Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC

mBio ◽

10.1128/mbio.00674-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 13

Author(s):

Nadine Lemaître ◽

Xiaofei Liang ◽

Javaria Najeeb ◽

Chul-Jin Lee ◽

Marie Titecat ◽

...

Keyword(s):

Bacterial Infections ◽

Biosynthetic Pathway ◽

Lipid A ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Bubonic Plague ◽

Gram Negative ◽

New Class

ABSTRACT The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis. Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

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Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01553-05 ◽

2006 ◽

Vol 50 (7) ◽

pp. 2478-2486 ◽

Cited By ~ 47

Author(s):

Andrea Giacometti ◽

Oscar Cirioni ◽

Roberto Ghiselli ◽

Federico Mocchegiani ◽

Fiorenza Orlando ◽

...

Keyword(s):

Septic Shock ◽

Antimicrobial Peptide ◽

Gram Negative Bacteria ◽

Amphibian Skin ◽

Necrosis Factor Alpha ◽

Gram Negative ◽

Rat Models ◽

E Coli ◽

Tnf Α

ABSTRACT Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

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Diacylglycerol kinase A is essential for polymyxin resistance provided by EptA, MCR-1 and other lipid A phosphoethanolamine transferases.

Journal of Bacteriology ◽

10.1128/jb.00498-21 ◽

2021 ◽

Author(s):

Alexandria B. Purcell ◽

Bradley J. Voss ◽

M. Stephen Trent

Keyword(s):

Lipid A ◽

Cell Envelope ◽

Diacylglycerol Kinase ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Severe Reduction ◽

Bacterial Fitness ◽

Phosphoethanolamine Transferases ◽

Kinase A

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated post-transcriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis.

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Synthesis and in vitro Biological Activity of New 4,6-Disubstituted 3(2H)-Pyridazinone-acetohydrazide Derivatives

Zeitschrift für Naturforschung C ◽

10.1515/znc-2012-5-604 ◽

2012 ◽

Vol 67 (5-6) ◽

pp. 257-265

Author(s):

Murat Sukuroglu ◽

Tijen Onkol ◽

Fatma Kaynak Onurdağ ◽

Gulsen Akalın ◽

M. Fethi Şahin

Keyword(s):

Biological Activity ◽

Antimicrobial Activities ◽

Cytotoxic Activities ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Preliminary Results ◽

H Nmr ◽

Pyridazinone Derivatives

New 3(2H)-pyridazinone derivatives containing a N’-benzyliden-acetohydrazide moiety at position 2 were synthesized. The structures of these newly synthesized compounds were confi rmed by IR, 1H NMR, and MS data. These compounds were tested for their antibacterial, antifungal, antimycobacterial, and cytotoxic activities. The compounds 2-[4-(4-chlorophenyl)- 6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N’-(4-tert-butylbenzyliden)acetohydrazide and 2-[4-(4-chlorophenyl)-6-(morpholin-4-yl)-3-oxo-(2H)-pyridazin-2-yl]-N’-(4-chlorobenzyliden) acetohydrazide exhibited activity against both Gram-positive and Gram-negative bacteria. Most of the compounds were active against E. coli ATCC 35218. The preliminary results of this study revealed that some target compounds exhibited promising antimicrobial activities

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Population Reductions of Gram-Negative Pathogens Following Treatments with Nisin and Chelators under Various Conditions

Journal of Food Protection ◽

10.4315/0362-028x-58.9.977 ◽

1995 ◽

Vol 58 (9) ◽

pp. 977-983 ◽

Cited By ~ 94

Author(s):

CATHERINE N. CUTTER ◽

GREGORY R. SIRAGUSA

Keyword(s):

Divalent Cations ◽

Escherichia Coli O157 ◽

Gram Negative Bacteria ◽

Bacterial Populations ◽

Gram Negative ◽

E Coli ◽

Transmission Electron ◽

Colony Forming Units ◽

Citrate Phosphate

When used in combination with chelating agents (EDTA, EGTA, citrate, phosphate), the bacteriocin nisin is effective for reducing populations of gram-negative bacteria in vitro. This study examined parameters (buffers, temperature presence of divalent cations) that affect nisin inhibition of Escherichia coli O157:H7 and Salmonella typhimurium. Approximately 7 log10 colony-forming units (CFU) per ml of E. coli and S. typhimurium were treated in PBS or MOPS buffers containing 50 μg/ml of purified nisin, alone or in combination with 500 mM lactate, 100 mM citrate, 50 mM EDTA, and 1% (wt/vol) sodium hexametaphosphate (pH 7.0) at 37°C for 60 min or 5°C for 30 min. Surviving bacterial populations were compared to untreated controls (buffers without nisin). Data indicated that treatments with nisin in buffers resulted in reductions of 4.30 and 2.30 log10 CFU/ml of E. coli and S. typhimurium, respectively, as compared to untreated controls. Population reductions ranging from 2.29 to 5.49 log10 CFU/ml were observed when cells were treated with nisin and chelator combinations at either 37°C for 60 min or 5°C for 30 min. The addition of magnesium and calcium to buffers with nisin decreased inhibition. Data obtained from spectrophotometric experiments indicated that treatments were causing the release of cellular constituents. However, transmission electron microscopy (TEM) analyses were inconclusive, since cellular membranes did not appear to be disrupted.

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Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽

10.1128/msphere.00143-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Axel B. Janssen ◽

Toby L. Bartholomew ◽

Natalia P. Marciszewska ◽

Marc J. M. Bonten ◽

Rob J. L. Willems ◽

...

Keyword(s):

Escherichia Coli ◽

Lipid A ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Anionic Lipid ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Two Component ◽

Resistant Strains

ABSTRACT 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.

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Identification of LpxL, a Late Acyltransferase of Francisella tularensis

Infection and Immunity ◽

10.1128/iai.01288-06 ◽

2007 ◽

Vol 75 (11) ◽

pp. 5518-5531 ◽

Cited By ~ 16

Author(s):

Molly K. McLendon ◽

Birgit Schilling ◽

Jason R. Hunt ◽

Michael A. Apicella ◽

Bradford W. Gibson

Keyword(s):

Francisella Tularensis ◽

Lipid A ◽

Structural Features ◽

Spectrometric Analysis ◽

Temperature Sensitive ◽

Gram Negative Bacteria ◽

Dependent Manner ◽

Gram Negative ◽

Acyl Chains

ABSTRACT Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria, and the lipid A region of LPS mediates stimulation of the immune system in a structure-dependent manner. Unlike the LPS of many other gram-negative bacteria, the LPS of Francisella tularensis isolated from in vitro cultures is not proinflammatory. This observed lack of proinflammatory prowess may reflect structural features of the lipid A, such as the number and length of the acyl chains and the single-phosphate group. To better understand this phenotype, we have begun to elucidate LPS biosynthesis in F. tularensis. We present complementation, mutational, and chemical data demonstrating that F. tularensis FTT0232c encodes a functional late acyltransferase enzyme with specificity similar to that of the Escherichia coli LpxL ortholog. Expression of this late acyltransferase complemented the temperature-sensitive and hypoacylated lipid A phenotypes of an E. coli lpxL mutant, expression of FTT0232c is increased during intracellular growth relative to that during in vitro growth, and finally, LPS obtained from a mutant of F. tularensis lacking FTT0232c showed an abundant triacyl lipid A species after mass spectrometric analysis, consistent with the loss of an LpxL late acyltransferase.

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Antibacterial Activities and Characterization of Novel Inhibitors of LpxC

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.6.1793-1799.2002 ◽

2002 ◽

Vol 46 (6) ◽

pp. 1793-1799 ◽

Cited By ~ 118

Author(s):

John M. Clements ◽

Fanny Coignard ◽

Ian Johnson ◽

Stephen Chandler ◽

Shilpa Palan ◽

...

Keyword(s):

Burkholderia Cepacia ◽

Lipid A ◽

Antibacterial Activities ◽

Vitro Assay ◽

Gram Negative ◽

E Coli ◽

Antibacterial Target ◽

Derivatives Of

ABSTRACT Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) and forms the major lipid component of the outer monolayer of the outer membrane of gram-negative bacteria. Lipid A is required for bacterial growth and virulence, and inhibition of its biosynthesis is lethal to bacteria. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A. Inhibitors of LpxC have previously been shown to have antibiotic activities. We have screened a metalloenzyme inhibitor library for antibacterial activities against an Escherichia coli strain with reduced LpxC activity. From this screen, a series of sulfonamide derivatives of the α-(R)-amino hydroxamic acids, exemplified by BB-78484 and BB-78485, have been identified as having potent inhibitory activities against LpxC in an in vitro assay. Leads from this series showed gram-negative selective activities against members of the Enterobacteriaceae, Serratia marcescens, Morganella morganii, Haemophilus influenzae, Moraxella catarrhalis, and Burkholderia cepacia. BB-78484 was bactericidal against E. coli, achieving 3-log killing in 4 h at a concentration 4 times above the MIC, as would be predicted for an inhibitor of lipid A biosynthesis. E. coli mutants with decreased susceptibility to BB-78484 were selected. Analysis of these mutants revealed that resistance arose as a consequence of mutations in the fabZ or lpxC genes. These data confirm the antibacterial target of BB-78484 and BB-78485 and validate LpxC as a target for gram-negative selective antibacterials.

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LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

Frontiers in Microbiology ◽

10.3389/fmicb.2021.676596 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xudong Tian ◽

Guillaume Manat ◽

Elise Gasiorowski ◽

Rodolphe Auger ◽

Samia Hicham ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Lipid A ◽

Negative Charge ◽

Polymyxin B ◽

Gram Negative Bacteria ◽

Bacterial Cells ◽

Gram Negative ◽

E Coli ◽

Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

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Synthesis and antimicrobial activity of some new N-(aryl-oxo-alkyl)-5-arylidene-thiazolidine-2,4-diones

Journal of the Serbian Chemical Society ◽

10.2298/jsc130114078s ◽

2014 ◽

Vol 79 (2) ◽

pp. 115-123 ◽

Cited By ~ 2

Author(s):

Anca Stana ◽

Brînduşa Tiperciuc ◽

Mihaela Duma ◽

Adrian Pîrnău ◽

Philippe Verité ◽

...

Keyword(s):

Antimicrobial Activities ◽

Fungal Strain ◽

Gram Negative Bacteria ◽

Bacterial Strains ◽

Gram Positive ◽

Reference Drug ◽

Gram Negative ◽

E Coli ◽

Inhibitory Activities

A series of new 5-(2,6-dichlorobenzylidene)thiazolidine-2,4-dione and 5-(4-methoxy-benzylidene)thiazolidine-2,4-dione derivatives (3a-h and 5a-h) were synthesized starting from 5-arylidene-thiazolidine-2,4-dione and ?-halo-ketones. The structural elucidation of the newly synthesized compounds was based on elemental analysis and spectroscopic data (MS, 1H-NMR, 13C-NMR). The synthesized compounds were screened for their antimicrobial activities against several pathogenic strains of Gram-positive and Gram-negative bacteria and one fungal strain (Candida albicans), assessed in vitro as growth inhibition diameters. Some of them displayed better inhibitory activities than that of the reference drug against the Gram-positive S. aureus, B. cereus, L. monocytogenes bacterial strains, and showed good antifungal activity against C. albicans, while the antibacterial activity against Gram-negative E. coli and S. typhimurium bacterial strains was moderate.

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