DRUG RESISTANCE Fitness cost of antibiotic susceptibility during bacterial infection Damien Roux, 1,2,3 * Olga Danilchanka, 4 * Thomas Guillard, 1,5† Vincent Cattoir, 4,6† Hugues Aschard, 7 Yang Fu, 4 Francois Angoulvant, 8 Jonathan Messika, 2 Jean-Damien Ricard, 2 John J. Mekalanos, 4 Stephen Lory, 4 Gerald B. Pier, 1‡ David Skurnik 1‡ Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contrib- ute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on how to best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance. Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strains during antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result in concomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of this accepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genes across many functional categories and operons that contributed to maximal in vivo fitness during lung infections in animal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivo fitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteria Acinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our results show that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitness advantages that enhance virulence in drug-resistant microbes. INTRODUCTION The challenges presented by the continued increase in antibiotic re- sistance among microbial pathogens are confounded by the complex- ity of the interplay among the microbes, drugs, and infected hosts. For many years, it has been assumed that acquisition of resistance comes with a negative fitness cost for most microorganisms (1). However, using a bank of ~300,000 different transposon (Tn) insertion mutants of Pseudomonas aeruginosa strain PA14, we showed recently (2, 3) that gastrointestinal (GI) tract colonization and bacterial dissemination into the spleen after induction of neutropenia strongly selects for mu- tant bacteria that are resistant to the antibiotic carbapenem and are unable to produce the porin OprD, an outer membrane channel re- quired for drug entry into the bacterial cell (4, 5). Both carbapenem- resistant, OprD-deficient laboratory strains and comparable clinical isolates were uniformly found to be more fit for in vivo persistence than matched, isogenic, drug-susceptible strains ( 3). In the clinic, carbapenem- resistant P. aeruginosa strains are commonly isolated from patients, and OprD deficiency is the main mechanism responsible for this resistance in patients (6). Therefore, a potentially overlooked consequence of the acquisition of antimicrobial resistance could be enhanced fitness and virulence of pathogens. This hypothesis suggests that a reduction in antibiotic use might not lead to the expected beneficial effects of having fewer drug-resistant strains that cause infections. Instead, in the ab- sence of selective pressure from antibiotics, extant drug-resistant strains might still outcompete the less resistant but also less fit strains for environmental persistence, host infectivity, and transmission. Severe and difficult to manage manifestations of microbial infec- tions often occur in the respiratory tract. Examples include P. aeruginosa infections in individuals with cystic fibrosis (7, 8) as well as immuno- compromised, neutropenic patients (9) and those hospitalized in inten- sive care units (ICUs) who also require mechanically assisted ventilation (10). One of the main obstacles to successful therapy for a substantial fraction of P. aeruginosa strains is their high level of intrinsic and ac- quired resistance to antibiotics that are active against other Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae, including amoxicillin–clavulanic acid (b-lactams), kanamycin (an aminoglycoside), nalidixic acid (a quinolone), and sulfonamide-trimethoprim (6). Because these drugs are not used clinically for the treatment of P. aeruginosa, this pathogen most likely retains the chromosomally encoded resistance mechanisms in the absence of antibiotic selective pressures. Thus, to obtain a more comprehensive view of the genetic basis of P. aeruginosa virulence during lung infections and to analyze the con- tribution of intrinsic and acquired antibiotic resistance to fitness in this setting, we used a saturated Tn insertion bank in P. aeruginosa strain PA14 to ascertain the roles of all nonessential gene products for fitness in murine lungs. Our results indicated that the loss of genes that encode intrinsic antibiotic resistance factors is associated with re- duced fitness during pulmonary infection, whereas the acquisition of drug resistance enhanced fitness in the same setting. To ascertain whether this principle also applied to other bacterial pathogens, we analyzed the effect on fitness associated with the loss or acquisition of antibiotic resistance in Acinetobacter baumannii , a problematic, multidrug-resistant organism commonly associated with hospital infections (11), and in Vibrio cholerae, an organism that is responsible for a major disease in various parts of the world and does not routinely encounter antibiotic selective pressure during treatment. Our findings consistently indicate 1 Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA. 2 INSERM, IAME, UMR 1137, F-75018 Paris, France. 3 Université Paris Diderot, Sorbonne Paris Cité, F-75018 Paris, France. 4 Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA. 5 EA 4687, Faculté de Médecine, Université de Reims Champagne-Ardenne, 51092 Reims, France. 6 EA 4655, Faculté de Médecine, Université de Caen Basse-Normandie, 14033 Caen, France. 7 Department of Epidemiology, Harvard School of Public Health, Boston, MA 02115, USA. 8 Hôpitaux de Paris (AP-HP), Pédiatrique Emergency Département, Hôpital Necker-Enfants Malades and Université Paris Descartes, 75015 Paris, France. *These authors contributed equally to this work (co–first authors). †These authors contributed equally to this work (co–second authors). ‡Corresponding author. E-mail: [email protected] (G.B.P.); [email protected]. harvard.edu (D.S.) RESEARCH ARTICLE www.ScienceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 1 by guest on May 26, 2021 http://stm.sciencemag.org/ Downloaded from
Transcript
R E S EARCH ART I C L E
DRUG RES I S TANCE
hD
ownloaded from
Fitness cost of antibiotic susceptibility duringbacterial infectionDamien Roux,1,2,3* Olga Danilchanka,4* Thomas Guillard,1,5† Vincent Cattoir,4,6†
Hugues Aschard,7 Yang Fu,4 Francois Angoulvant,8 Jonathan Messika,2 Jean-Damien Ricard,2
John J. Mekalanos,4 Stephen Lory,4 Gerald B. Pier,1‡ David Skurnik1‡
Advances in high-throughput DNA sequencing allow for a comprehensive analysis of bacterial genes that contrib-ute to virulence in a specific infectious setting. Such information can yield new insights that affect decisions on howto best manage major public health issues such as the threat posed by increasing antimicrobial drug resistance.Much of the focus has been on the consequences of the selective advantage conferred on drug-resistant strainsduring antibiotic therapy. It is thought that the genetic and phenotypic changes that confer resistance also result inconcomitant reductions in in vivo fitness, virulence, and transmission. However, experimental validation of thisaccepted paradigm is modest. Using a saturated transposon library of Pseudomonas aeruginosa, we identified genesacross many functional categories and operons that contributed to maximal in vivo fitness during lung infections inanimal models. Genes that bestowed both intrinsic and acquired antibiotic resistance provided a positive in vivofitness advantage to P. aeruginosa during infection. We confirmed these findings in the pathogenic bacteriaAcinetobacter baumannii and Vibrio cholerae using murine and rabbit infection models, respectively. Our resultsshow that efforts to confront the worldwide increase in antibiotic resistance might be exacerbated by fitnessadvantages that enhance virulence in drug-resistant microbes.
tt
by guest on M
ay 26, 2021p://stm
.sciencemag.org/
INTRODUCTION
The challenges presented by the continued increase in antibiotic re-sistance among microbial pathogens are confounded by the complex-ity of the interplay among the microbes, drugs, and infected hosts. Formany years, it has been assumed that acquisition of resistance comeswith a negative fitness cost for most microorganisms (1). However,using a bank of ~300,000 different transposon (Tn) insertion mutantsof Pseudomonas aeruginosa strain PA14, we showed recently (2, 3) thatgastrointestinal (GI) tract colonization and bacterial disseminationinto the spleen after induction of neutropenia strongly selects for mu-tant bacteria that are resistant to the antibiotic carbapenem and areunable to produce the porin OprD, an outer membrane channel re-quired for drug entry into the bacterial cell (4, 5). Both carbapenem-resistant, OprD-deficient laboratory strains and comparable clinicalisolates were uniformly found to be more fit for in vivo persistence thanmatched, isogenic, drug-susceptible strains (3). In the clinic, carbapenem-resistant P. aeruginosa strains are commonly isolated from patients, andOprD deficiency is the main mechanism responsible for this resistancein patients (6). Therefore, a potentially overlooked consequence of theacquisition of antimicrobial resistance could be enhanced fitness andvirulence of pathogens. This hypothesis suggests that a reduction inantibiotic use might not lead to the expected beneficial effects of having
1Divisionof InfectiousDiseases, Department ofMedicine, Brigham andWomen’s Hospital,HarvardMedical School, Boston, MA 02115, USA. 2INSERM, IAME, UMR 1137, F-75018 Paris,France. 3Université Paris Diderot, Sorbonne Paris Cité, F-75018 Paris, France. 4Departmentof Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.5EA 4687, Faculté de Médecine, Université de Reims Champagne-Ardenne, 51092 Reims,France. 6EA 4655, Faculté de Médecine, Université de Caen Basse-Normandie, 14033Caen, France. 7Department of Epidemiology, Harvard School of Public Health, Boston, MA02115, USA. 8Hôpitaux de Paris (AP-HP), Pédiatrique Emergency Département, HôpitalNecker-Enfants Malades and Université Paris Descartes, 75015 Paris, France.*These authors contributed equally to this work (co–first authors).†These authors contributed equally to this work (co–second authors).‡Corresponding author. E-mail: [email protected] (G.B.P.); [email protected] (D.S.)
www.Scie
fewer drug-resistant strains that cause infections. Instead, in the ab-sence of selective pressure from antibiotics, extant drug-resistantstrains might still outcompete the less resistant but also less fit strainsfor environmental persistence, host infectivity, and transmission.
Severe and difficult to manage manifestations of microbial infec-tions often occur in the respiratory tract. Examples include P. aeruginosainfections in individuals with cystic fibrosis (7, 8) as well as immuno-compromised, neutropenic patients (9) and those hospitalized in inten-sive care units (ICUs) who also require mechanically assisted ventilation(10). One of the main obstacles to successful therapy for a substantialfraction of P. aeruginosa strains is their high level of intrinsic and ac-quired resistance to antibiotics that are active against other Gram-negativebacteria such as Escherichia coli and Klebsiella pneumoniae, includingamoxicillin–clavulanic acid (b-lactams), kanamycin (an aminoglycoside),nalidixic acid (a quinolone), and sulfonamide-trimethoprim (6). Becausethese drugs are not used clinically for the treatment of P. aeruginosa, thispathogen most likely retains the chromosomally encoded resistancemechanisms in the absence of antibiotic selective pressures.
Thus, to obtain a more comprehensive view of the genetic basis ofP. aeruginosa virulence during lung infections and to analyze the con-tribution of intrinsic and acquired antibiotic resistance to fitness inthis setting, we used a saturated Tn insertion bank in P. aeruginosastrain PA14 to ascertain the roles of all nonessential gene productsfor fitness in murine lungs. Our results indicated that the loss of genesthat encode intrinsic antibiotic resistance factors is associated with re-duced fitness during pulmonary infection, whereas the acquisition of drugresistance enhanced fitness in the same setting. To ascertain whetherthis principle also applied to other bacterial pathogens, we analyzedthe effect on fitness associated with the loss or acquisition of antibioticresistance in Acinetobacter baumannii, a problematic, multidrug-resistantorganism commonly associated with hospital infections (11), and inVibrio cholerae, an organism that is responsible for a major disease invarious parts of the world and does not routinely encounter antibioticselective pressure during treatment. Our findings consistently indicate
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 1
that for each of these very different pathogens, maintenance of intrin-sic drug resistance and acquisition of new resistances promote fitnessand survival in an infected host, a finding counter to the currently pre-vailing view in the field (1) that increased antibiotic resistance has anegative fitness cost.
by guest on May 26, 2021
http://stm.sciencem
ag.org/D
ownloaded from
RESULTS
Transposon sequencing analysis in an animal model ofP. aeruginosa acute pneumoniaTo establish lung infection, C3H/HeN female mice were inoculated witha bank of ~300,000 random Tn insertion mutants of P. aeruginosaPA14 grown overnight in LB as previously described (2, 3). This in-oculum assured that a bacterial burden at least 10 times greater thanthe size of the bank [that is, >3 × 106 colony-forming units (CFU)]was recovered from the lung for each time point examined (1, 6, and24 hours, or until the mice were moribund, between 36 and 48 hoursafter infection for the last time point). Bacterial DNA was recovered,and Tn-chromosome junctions were sequenced and quantified usingthe high-throughput techniques described previously (2, 3). A compre-hensive analysis of the results was carried out using a rigorous strategyto determine the gain or loss of strains with Tn insertions in differentgenes (2). This strategy involved first grouping the disrupted genesinto functional classes, then into operons, and finally conducting aspecific gene-by-gene analysis to indicate the likely role of each indi-vidual gene product in microbial biology in vivo. Circular representa-tions of the genome at several scales were used to depict the specificgenes of interest, their locations within the P. aeruginosa genome, theirsize, and their associated phenotypes. The sequencing reads per kilo-base per million [RPKM; defined as (number of reads)/(kilobase lengthof gene) × (millions of reads in the data set)] were used to account fordifferences in gene sizes and sequencing efficiencies between differentDNA samples (2, 3).
Analysis by functional classesThe changes in in vivo fitness of mutants within each of 27 functionalgenomic classes (12, 13) (www.pseudomonas.com) are shown in fig. S1(see data files S1 and S2 for a more complete description of the anal-ysis by functional classes). In the bacterial population recovered frommouse lungs 1 hour after infection, we observed minor changes in thedistribution of Tn mutants compared to those in the original inoculumgrown in culture in LB (fig. S1A). This result confirmed the absenceof an initial bottleneck after intranasal installation of the library in aP. aeruginosa population. Loss of gene functionality after Tn insertionthat leads to enhanced fitness (that is, more reads from Tn-interruptedgenes in the output compared with the input) occurs rarely in virulencestudies. Only Tn insertions in genes in one functional class, motilityand attachment, were found to be significantly increased at all timepoints analyzed (fig. S1). A marked (>679%) increase in RPKM forthis group was observed at 24 hours after infection, whereas RPKMrecovered for all other functional classes were decreased (fig. S1C). With-in the motility and attachment functional class, only strains with Tninsertions in genes associated with the production of flagella (see datafile S2) and type IVa pili (T4aP) displayed an increased in vivo fitnessduring murine lung infection (fig. S2). This pattern for the T4aP Tninsertion mutants was similar to that previously found in bacterialstrains recovered from the mouse cecum during P. aeruginosa GI col-
www.Scie
onization (fig. S3). Strains with Tn insertions that lead to a phenotypeof preserved or even increased production of pili (hyperfimbrial) (2)did not display increased in vivo fitness. In contrast, P. aeruginosa thatcarried a Tn insertion in most of the genes needed to produce T4aPdisplayed a reduction in fitness after 48 hours in water, a natural hab-itat for P. aeruginosa (fig. S4) (14), indicating that these structures arebeneficial for survival in the environment but are detrimental to sur-vival in an infected lung, perhaps because T4aP is recognized by thehost’s immune defenses.
Analysis by operonsSignificant changes in fitness of strains with Tn insertions in virulencefactor (VF) genes defined in the VF database (15) (www.mgc.ac.cn)were observed only after 24 hours of infection (Fig. 1 and fig. S5). Tninsertions in genes that encode protein components of the quorum-sensing system and of the type 1, 2, 3, 5, and 6 secretion systems(which permit the secretion of proteins to the extracellular space) aswell as proteins that participate in the synthesis of the exopolysaccharidesalginate and Pel, lipopolysaccharide (LPS), the rhamnolipids, pyochelin,pyoverdine, and pyocyanin were all under-represented after 24 hoursof infection compared to those in the strains recovered at the 6-hourtime point. The only exceptions were Tn insertions in the algR gene,which encodes a regulator of the production of both alginate and T4aP;the fitness of strains that carried these mutations was increased after24 hours in the lung (Fig. 1), likely because of the negative impact of T4aPexpression in vivo. These observations indicate that alginate is a bonafide VF, but the absence of T4aP is dominant for survival in the host.
Impact of lung infection duration on P. aeruginosa fitnessAfter 24 hours in the lung, only one nonessential P. aeruginosa genecluster, PA14_23360-23470, which contains 12 genes associated withLPS O-antigen production, had a >10-fold decrease in RPKM (fig. S6).By comparison, in the GI tract model, after 5 days of infection, >75%of the Tn insertion mutations in genes important for bacterial coloni-zation had at least a 10-fold decrease in RPKM (Fig. 2A) (2). This notabledifference suggests that either the shorter duration of selection or con-ditions in the respiratory tract have a differential selective effect onP. aeruginosa fitness. To discriminate between these interpretations,we compared the complete RPKM data sets generated after 5 daysof GI tract colonization with those obtained from acute lung infectionat various times after inoculation. Examination of these data sets revealedvery few changes in the composition of the Tn insertion mutants after1 or 6 hours of lung infection (Fig. 2, B and C). Notably, more pro-nounced differences in fitness were seen after 24 hours of infection (Fig.2D) but still not close to the magnitude of the RPKM changes found inthe GI tract after 5 days. However, when we analyzed the impact of lunginfection at a point at which the mice were moribund (36 to 48 hours;Fig. 2E), we found that the RPKM changes were very similar to thosefound after 5 days of GI tract colonization (Fig. 2, A and E, and tableS1). This finding supports the hypothesis that P. aeruginosa uses simi-lar strategies to establish infection and overcome host defenses in theGI tract and lung but that detection of maximal differences is depen-dent on having sufficient time for selection.
Fitness cost of antibiotic susceptibility—Analysis ofselected genesBy comparing the RPKM data for each nonessential P. aeruginosa geneat each time point of bacterial sampling during murine lung infection,
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 2
Fig. 1. In vivo fitness of P. aeruginosa mutants with Tn insertions infour classes of genes annotated as VFs after 1, 6, or 24 hours of infec-
outer circle. The VFs are depicted in four color-coded categories (green,orange, gray, and purple) in the green, pink, and yellow inner circles,
tion in the murine lung. The inner green circle represents RPKM changesbetween LB-cultured P. aeruginosa and 1 hour of lung infection; pink circledepicts changes from 1 to 6 hours of infection; and yellow circle representschanges from 6 to 24 hours of infection. The outer circle is the entireP. aeruginosa chromosome with each gene represented by one of six dif-ferent shades of blue organized into a repetitive pattern. The genes asso-ciated with the identified VFs are represented at ×20 magnification in the
www.Scie
and across a gradient of lighter to darker bars to differentiate the differentgene clusters. A decrease in RPKM (bars pointing to circle’s center indicatea decreased fitness) of Tn insertions in genes encoding for all the knownmajor VFs of P. aeruginosa was observed after 24 hours of lung infection(yellow circle) but not after 1 hour (green circle) or 6 hours (pink circle) ofinfection, except for Tn insertions in genes encoding LPS O-antigens (darkergreen).
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 3
we identified a total of 116 genes with Tn insertions that had at least100 RPKM after 24 hours of infection and with a twofold or greaterincrease in RPKM after 36 to 48 hours of infection (table S2). Thesefindings strongly suggest that these 116 genes are ones that, when dis-rupted, are likely to lead to enhanced fitness in an in vivo infection.Within this collection of 116 genes, in addition to those that encodeproducts involved in motility and attachment, we measured fitnessenhancement of P. aeruginosa strains with Tn insertions in the oprDgene (PA14_Tn-oprD), which also imparts a carbapenem resistancephenotype, and glpT (PA14_Tn-glpT), which is responsible forfosfomycin resistance (Fig. 3A). The OprD protein is a channel forentry of carbapenem antibiotics, and the glpT gene encodes a glycerol3-phosphate transporter that brings fosfomycin into E. coli andP. aeruginosa (16).
We attempted to corroborate the transposon sequencing (TnSeq)findings by using individual mutants in the murine pneumonia model.Here, we used strains with Tn insertions in the oprD and glpT genes ob-tained from the ordered Tn library of P. aeruginosa PA14 (17). Moreof the mice infected with the Tn-oprD or the Tn-glpT strains becamemoribund or died when compared with those infected with the wild-typePA14 strain or the complemented strains Tn-oprD::oprD or Tn-glpT::glpT(Fig. 3B; wild type versus Tn-oprD: P <0.0001, wild type versus Tn-glpT:
www.Scie
P < 0.0001, Tn-oprD versus Tn-oprD::oprD: P = 0.0002, and Tn-glpTversus Tn-glpT::glpT: P < 0.0001, log-rank test). These observationsindicate a specific increased virulence after loss of the OprD or GlpTproteins.
To extend our results to clinical P. aeruginosa strains, and partic-ularly to carbapenem resistance, which is a major issue in the treat-ment of P. aeruginosa infections, we used our murine pneumoniamodel to evaluate virulence of isogenic clinical strains recovered fromtwo patients at different time points. For each patient, the clinicalP. aeruginosa isolates taken at earlier time points (strains 48.1 and51.1) had intact oprD genes and were carbapenem susceptible [imipe-nem minimum inhibitory concentration (MIC) <1 mg/liter] (3); incontrast, both isolates taken from the patients at later time points(strains 48.2 and 51.2) carried mutations in the oprD genes that ceasedproduction of OprD and were carbapenem-resistant (imipenemMIC≥32 mg/liter) (3). As shown in fig. S7, carbapenem-resistant and OprD-deficient clinical strains 48.2 and 51.2 were significantly more virulent inthe murine pneumonia model than their corresponding carbapenem-susceptible strains (P = 0.0039, log-rank test) and also were morevirulent compared to the oprD mutant strains complemented withan intact oprD gene (48.2 versus 48.2::PoprD: P = 0.0016 and 51.2 ver-sus 51.2::PoprD: P = 0.0001, log-rank test).
Fig. 2. In vivo fitness of P. aeruginosa PA14 Tn library. (A) RPKMchanges that occurred after five days of GI tract colonization by P. aerugi-nosa, as previously reported (3). (B to E) Relative ranking and absolutenumber of RPKM that changed for 5977 genes of P. aeruginosa PA14 dur-ing lung infection, comparing the RPKM in the LB input with those ob-tained 1 hour after infection (B) or comparing RPKM obtained 1 hourafter infection with those obtained at 6 hours after infection, and 24 hoursafter infection with 32 to 48 hours after infection (labeled “Lung 48 h”) (C toE). Dots above the input lines indicate Tn insertions in genes with a positivefitness (increase in in vivo RPKM), whereas dots below the input line indi-cate those with a negative fitness (decrease in in vivo RPKM).
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 4
We previously showed that the increased in vivo fitness of OprD-deficient P. aeruginosa is associated with an enhanced killing of murinemacrophages (3). Therefore, we compared the survival of activatedmurine J774 macrophages after infection with wild-type or glpT-deleted(DglpT) P. aeruginosa constructed in a PA14 background that lackedthe exoU gene (to avoid the cytotoxic effects of this effector of the bacte-ria’s type 3 secretion system). The DglpT strain was able to kill macrophages,whereas there was a 40% increase in macrophage numbers infected withthe glpT-complemented strain 24 hours after infection (Fig. 3C). Toassess the role of GlpT in the resistance of P. aeruginosa to phagocytickilling, we tested wild-type, DglpT, and glpT-complemented strains forsurvival in murine J774 macrophages (Fig. 3D) and murine MH-S al-veolar macrophages (Fig. 3E). Both of these cell types were able to eitherlimit replication or survival of the wild-type and glpT-complementedstrains, whereas the DglpT strain was able to replicate intracellularly dur-ing the 24-hour experimental infection period. Thus, the increased fit-ness and virulence of the fosfomycin-resistant Tn-glpT mutant in thepneumonia model may be linked to its enhanced killing of and survivalwithin macrophages and possibly other cells of the immune system.
www.Scie
P. aeruginosa is intrinsically resistant to multiple antibiotics fromseveral different classes, and consequently, these drugs cannot be usedto treat infections. We wondered whether—similar to glpT- and oprD-deficient, antibiotic-resistant strains generated by Tn mutagenesis (Fig. 3)—chromosomal genes that encode determinants of intrinsic antibioticresistance are associated with an increased in vivo fitness (and thusevolutionarily selected) as a co-result of the expression of resistance-promoting gene products.
To this end, we evaluated the fitness of strains with Tn insertionsin genes coding for natural resistance of P. aeruginosa to several differ-ent antibiotic classes: ampC (encodes a cephalosporinase for resistanceto amoxicillin–clavulanic acid), aph (encodes an aminoglycoside phos-photransferase for high-level resistance to kanamycin), and themexAB-oprM operon (encodes the components of an efflux pump for theresistance to both nalidixic acid and trimethoprim-sulfonamide) (ta-bles S3 to S5). Analysis of the TnSeq data generated to evaluate fitnessfor murine GI colonization (3) and the current data set from murinelung infections showed that Tn insertions in genes within these threedistinct antibiotic resistance loci resulted in drug-susceptible strains
Fig. 3. Increased fitness of carbapenem (PA14_Tn-oprD)– and fosfomycin(PA14_Tn-glpT)–resistant P. aeruginosa Tn insertion mutants in murine
resistant glpT mutant compared to the WT or glpT-complemented strains.(D) Multiplication of P. aeruginosa PA14 WT, DglpT, and glpT-complemented
lung infection. (A) Increases in RPKM in Tn-glpT or Tn-oprD P. aeruginosastrains from the TnSeq analysis in the murine model of pneumonia. (B)Virulence of P. aeruginosa PA14 glpT or oprD Tn insertion mutants in lung in-fections compared to wild-type (WT) and complemented strains (n = 12 miceper group; WT versus Tn-oprD: P < 0.0001, WT versus Tn-glpT: P < 0.0001, Tn-oprD versus Tn-oprD::PoprD: P = 0.0002, and Tn-glpT versus Tn-glpT::PglpT: P <0.0001, log-rank test). (C) Killing of J774 macrophages by the fosfomycin-
mutant inside J774 macrophages. (E) Multiplication of the internalized DglpTcompared with WT and glpT-complemented strains in MH-S alveolar macro-phages after 24 hours. For (C) to (F), bars represent means of triplicate de-terminations, and error bars indicate the SD. *P < 0.05, Tukey’s post hoc testversus control. P < 0.05, overall analysis of variance (ANOVA) for each dataset. PA14 background lacking the exoU gene was used in the in vitro studiesto avoid cytotoxic effects of this effector of the type 3 secretion system.
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 5
with a decreased in vivo fitness for both GI colonization and lung in-fection (fig. S8, A and B). Therefore, we tested the in vitro and in vivofitness of specific mutants with Tn insertions in the selected genes.First, we confirmed the loss of the appropriate antibiotic resistancefor each mutant (tables S3 to S5). Furthermore, we showed that therewere no growth differences between wild-type P. aeruginosa PA14strain and mutated strains with Tn insertions in the ampC, aph,mexA,or oprM genes when cultured separately in vitro in LB (fig. S9A). Tomeasure the in vitro competition index of the mutants, we mixed thevarious Tn insertion mutants with an equal amount of the wild-typePA14 strain in LB and grew the cells on agar plates to count coloniesafter 24 hours of competition in liquid culture. Wild-type PA14 wasable to outgrow only the Tn-oprM strain (fig. S9B), indicating thatthe loss of OprM decreases fitness in LB when in competition withwild-type cells (2). In contrast, we observed no comparable fitness costassociated with the loss of OrpM or any of the other antibioticresistance genes when cocultured in water with the wild-type PA14strain for up to 5 days (fig. S9C). Thus, genes that encode intrinsicantibiotic resistance factors in P. aeruginosa are not essential for invitro survival in a natural environment such as water.
Next, we tested the ampC, aph, mexA, and oprM Tn insertion mu-tants for fitness in the murine GI colonization and pneumonia models(Figs. 4 and 5). The in vivo fitness of all four Tn insertion mutants was
www.Scie
reduced when compared to wild-type PA14 in the GI tract (Fig. 4) andthe lung infection model (Fig. 5A). The magnitude of the decrease infitness in the GI colonization model ranged from near total—as deter-mined by the rapid clearance of the PA14_Tn-ampC and PA14_Tn-mexA mutants 24 hours after establishing colonization (Fig. 4, A andB)—to partial, as shown by a ~40 to 50% decrease in in vivo fitness ofthe PA14_Tn-aph strain (Fig. 4C, left panel) and the >85% decrease inrecovery of the PA14_Tn-oprM mutant (Fig. 4D, left panel). In thelung infection model (Fig. 5A), fewer mice became moribund or diedby 48 hours after challenge with P. aeruginosa strains that carried Tninsertions in the ampC (P = 0.0162), mexA (P = 0.0288), oprM (P =0.0036), or aph (P = 0.0050) genes (by log-rank test). Together,these data suggest that loss of intrinsic antibiotic resistance inP. aeruginosa is associated with a reduced in vivo fitness.
To validate the observed differences between in vitro and in vivofitness of Tn mutants in intrinsic antibiotic resistance genes, we per-formed quantitative reverse transcription polymerase chain reaction(qRT-PCR) experiments to measure expression levels of the four genesin LB, water, and the ceca of colonized mice. mRNA transcripts forthe ampC, mexA, oprM, and aph genes were found at higher levels inbacterial cells recovered from the GI tract compared with that in bac-terial cells recovered from the two in vitro conditions, indicative of animportant in vivo role for these genes and consistent with the absence
by guest on May 26, 2021
tp://stm.sciencem
ag.org/
Fig. 4. Effect of the loss of intrinsic antibiotic resistance genes inP. aeruginosa on fitness during GI tract colonization. The ability to col-
gene when P. aeruginosa was grown in LB, drinking water used for coloni-zation, or recovered from the murine GI tract. (A) Resistance to amoxicillin–
onize the murine GI tract was measured by the competitive index (CI). The CIis calculated by dividing the proportion of mutant cells at the end of the com-petition by the proportion at the start. (A to D) A CI ratio <1 in the left-handpanels indicates that the Tn insertion mutant is less fit. Right-hand panels de-pict the qRT-PCR analysis of the expression of the transcript for each indicated
clavulanic acid (Tn-ampC). (B and C) Resistance to nalidixic acid, chloramphenicol,and cotrimoxazole (Tn-mexA and Tn-oprM). (D) Resistance to kanamycin(Tn-aph). Bars for CI represent means from four mice, and error bars representthe SD. Bars for qRT-PCR represent means of three individual experiments,and error bars represent the SD. *P < 0.05, one-sample t test (default = 1).
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 6
of an in vitro growth defect of the corresponding Tn insertion mutants(Fig. 4, A to D, right panels). Furthermore, the transcript level for theoprM gene was higher in LB than in water (Fig. 4D, right panel),consistent with the in vitro defect of PA14_Tn-oprM observed in com-petition with wild type in LB but not in water (fig. S9, B and C). Inaddition, we found a progressive increase in the expression of all fourintrinsic resistance genes in wild-type P. aeruginosa after internalizationby macrophages (Fig. 5, B to E), parallel to the observed effects onbacterial survival determined after macrophage internalization for allstrains except DampC (fig. S10). Together, ampC, mexA, oprM, andaph genes exhibited increased mRNA transcript levels in vitro (macro-phages) and in vivo (GI tract and lung), supporting the conclusionthat a functional loss of these gene products and the resultant changefrom antibiotic-resistant to antibiotic-sensitive phenotypes results indecreased fitness.
Fitness cost of antibiotic susceptibility in otherpathogenic bacteriaA. baumannii is another major Gram-negative pathogen, commonlyisolated in the ICU, that has numerous intrinsic and acquired antimicro-bial resistance factors (11). A recent report (18) used the TnSeq ap-proach to describe the A. baumannii genes necessary for persistence inthe murine lung and found that A. baumanniimutants with a Tn inser-tion in the A1S_1649 and A1S_1801 genes—which are predicted to
www.Scie
encode resistance–nodulation–cell division (RND)–type efflux systems—were significantly less fit in mice. The RNDs play a major role inthe intrinsic multidrug resistance phenotypes of both A. baumanniiand P. aeruginosa (6, 19, 20). To test our hypothesis that intrinsic andacquired antibiotic resistance enhances fitness during infection, in otherclinically relevant organisms, we assessed the fitness of Tn insertionsin the A1S_1649 and A1S_1801 genes prepared in a highly virulentA. baumannii strain AB5075 (21) using a murine model of lethalityafter intraperitoneal challenge (22). As expected, mutations in both genesled to increased susceptibility to antibiotics, with the MIC for tobramycindecreasing from 48 mg/liter for AB5075 wild type to 12 and 16 mg/literfor AB5075 with Tn insertions in homologs of A1S_1649 and A1S_1801,respectively. Furthermore, both of the more antibiotic-susceptibleAB5075 strains were also less virulent, with significantly fewer mice(P = 0.01, log-rank test) entering a moribund state than when infectedwith the wild-type parental strain (Fig. 6A).
Last, we decided to extend our finding that an increase in fitnesscould be associated with antibiotic resistance by evaluating whetherantibiotic resistance might provide an increase in in vivo fitness usingV. cholerae, the causative agent of cholera (23). Care for cholera patientsis generally focused on treating signs and symptoms, and assuringadequate oral or intravenous hydration of the patients. Antibiotics arenot usually a major factor in the treatment of cholera, although theiruse can decrease the volume and duration of diarrhea by 50% and are
Fig. 5. Effect of the loss of intrinsic antibiotic resistance genes inP. aeruginosa on fitness in the murine lung infection model. (A) Survival
(B to E) mRNA transcript expression levels of ampC, aph, mexA, and oprM asdetermined by qRT-PCR for the indicated P. aeruginosa mutant strain during
curves of mice after lung infection with WT P. aeruginosa or strains carrying Tninsertions in oprM, mexA, ampC, or aph genes. A significant decrease in viru-lence was observed between WT and the various insertion mutants (oprM: P =0.0036, ampC: P = 0.0162,mexA: P = 0.0288, and aph: P = 0.0050, log-rank test).
infection of J774 macrophages for 1, 6, or 24 hours or in the murine lung for1 hour. For each sample, transcript levels of ampC, aph,mexA, and oprM wereassessed by relative quantification using the 2−DDCt method. Expression of therpsL gene was used as a housekeeping control gene.
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 7
recommended for patients with moderate to severe dehydration (23).Further, antibiotic resistance is not considered a major issue forV. cholerae, despite some reports indicating more frequent isolationof antibiotic-resistant strains (24–26). On the basis of data from aTnSeq analysis that identified a number of genes required for V. choleraeGI tract colonization (27), we tested for polymyxin sensitivity as wellas fitness in an infant rabbit model of cholera, V. cholerae strains, witheither a Tn insertion in tolC—a porin homolog of OprM involved inbacterial multidrug resistance and survival of pathogens during infec-tion (28)—or a Tn insertion in lpp—which is predicted to encode themajor outer membrane lipoprotein Lpp (29) conferring susceptibilityto polymyxin B. As shown in Fig. 6 (B to D), the tolC::Tn mutant wasmore susceptible to polymyxin B and had a decreased ability to col-onize the GI tract of infant rabbits (P < 0.0001), whereas the polymyx-
www.Scie
in B–resistant lpp::Tn strain was able to significantly outcompetethe wild-type V. cholerae C6706 strain in vivo (P < 0.01, as determinedby t test).
DISCUSSION
A number of studies have suggested that phenotypic changes that con-fer antibiotic resistance are associated with a fitness cost in vivo and areduction in infectivity (for example, decreased virulence and micro-bial transmission) in pathogenic bacteria such as E. coli (30), Salmonellaenterica serovar Typhimurium (31), P. aeruginosa (32), Mycobacteriumtuberculosis (33), and Staphylococcus aureus (34). However, the data re-ported here show that a fitness cost associated with microbial antibiotic
Fig. 6. Fitness cost of antibiotic susceptibility in A. baumannii andV. cholerae. (A) Effect of Tn insertions in genes associated with constitutive
WT, tolC::Tn, and lpp::Tn were grown in liquid culture (LB) in the presenceof polymyxin (0.5 mg/liter) for 5 hours, and 10-fold serial dilutions were plat-
antibiotic resistance on the virulence of A. baumannii in a murine lethal peri-tonitis infection setting. WT A. baumannii (AB) strain 5075 and isogenicstrains with Tn insertions in genes homologous to A1S_1649 andA1S_1801 were used at a challenge dose of 5 × 109 CFU per mouse, admi-nistrated intraperitoneally. A significant decrease in virulence was observedwhen comparing the WT with either of the two Tn insertion mutants(AB5075-A1S_1649 and AB5075-A1S_A1S_1801; P = 0.01, log-rank test). (B)Effect of Tn insertions in genes tolC and lpp (tolC::Tn and lpp::Tn, respective-ly) on V. cholerae susceptibility to antibiotic polymyxin. V. cholerae C6706
ed on agarose plates (LB). (C and D) Infant rabbit competition assays usingWT, lpp::Tn, and tolC::Tn mutant V. cholerae strains. The in vivo CIs weredetermined phenotypically. V. cholerae C6706 WT strain had an insertion ofTnFGL3 into lacZ (lacZ::Tn), whereas tolC::Tn and lpp::Tn produced b-galac-tosidase for differentiation when competing against the parental DlacZ WTstrain. Inoculum used was 109 CFU per rabbit (C) or 107 CFU per rabbit (D).Significance was determined with the Student’s t test by comparing thecolonization ratios of C6706 WT lacZ::Tn versus the tolC::Tn (P < 0.0001)or lpp::Tn (P < 0.01) mutant strains. Mean plus SEM is shown.
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 8
resistance might not be a general feature of all infections in whichtherapy-associated resistance is encountered. With the use of a varietyof clinically relevant bacterial strains (P. aeruginosa, A. baumannii, andV. cholerae) and in vivo models of infection, a consistent picture emerged:acquisition of antibiotic resistance increased in vivo fitness, and intrin-sic resistance to different classes of antibiotics also enhanced in vivovirulence and fitness. This conclusion was supported not only by re-sults generated with a Tn library in P. aeruginosa but also by analysisof naturally occurring, antibiotic-resistant strains isolated from infectedpatients. Together, these results indicate the utility of Tn insertions toaccurately identify phenotypes in experimental analyses even thoughother mutations, which do not result in total loss of a gene product orits function, could plausibly produce less-pronounced or qualitativelydifferent results. Increased fitness of antibiotic-resistant strains was notspecific to P. aeruginosa, as there was also a measurable in vivo fitnesscost when genes encoding antibiotic resistance were interrupted inboth A. baumannii and V. cholerae. Increases in in vivo fitness forantibiotic-resistant strains occurred in mouse and rabbit infectionmodels, thus providing broad-based support for challenging theconcept that drug-resistant bacteria are less fit and thus less virulentduring infections.
The increased virulence of antibiotic-resistant strains in experimen-tal settings raises a serious concern that drug-resistant strains might bebetter fit to cause serious, more difficult to treat infections beyond justthe issues raised by the complexity of antibiotic treatment. These find-ings also could have an impact on the use of drugs such as fosfomycin,which is dispensed in combination with other antibiotics and whichhas been proposed as a means to avoid the emergence of drug-resistantP. aeruginosa (35). Using the same P. aeruginosa strain with the PA14background, Rodríguez-Rojas et al. (36) reported that there was nofitness cost of the Tn-glpT fosfomycin-resistant mutant strain when testedin a mouse model of pneumonia that used a mouse strain differentfrom the one used here, the C57BL/6J mouse. Although these findingsdid not demonstrate the marked increased fitness that we measuredfor the P. aeruginosa Tn-glpT strain, both studies imply that fosfomycintreatments that lead to the emergence of resistant strains will not re-sult in an in vivo fitness cost for bacterial survival and conceivablycould lead to increased in vivo virulence, which would be problematicin the ICU setting (37). Overall, infections caused by microbial strainswith natural or acquired resistance to drugs such as fosfomycin orcarbapenems could have serious clinical consequences beyond thedifficulties in choosing an effective therapeutic treatment. It is alreadyestablished that acquired carbapenem resistance in P. aeruginosa leadsto increased patient mortality (38).
The basis for the increased transcription of genes that encode anti-biotic resistance determinants in P. aeruginosa in the absence of selectivepressures from the drugs might be related to the need for organisms tocoexist with the natural microbiota of animals and humans as well aswith environmental organisms. In these settings, pathogenic organismsencounter multiple species-level phylotypes that produce antimicrobialcompounds (39). A recent report showed that methicillin-resistantS. aureus selected to resist bacteriocins secreted by other microbial strainsalso became resistant to intermediate levels of vancomycin (40), one ofthe only antibiotics available to treat multidrug-resistant S. aureus in-fections. Furthermore, the recent identification of a wide distributionof biosynthetic gene clusters for synthesis of the thiopeptide antibioticlactocillin within the organisms that make up the human microbiomesuggests that microbial interactions in the host play a major role in the
www.Scie
selection of general drug resistance factors that are also present inpathogenic bacteria (41). Most of the identified antibiotic biosyntheticclusters present in the normal microbiome (3118 total clusters werefound in 2430 reference genomes) still cannot be assigned a biologicalfunction. This vast potential for the production of antimicrobials bythe microbiota represents a potential strategy that allows human com-mensal bacteria to outcompete pathogens, with concomitant pressureplaced on pathogens to become resistant to natural antimicrobialfactors that then confer resistance to modern antimicrobial drugs.
Overall, genes in pathogenic microorganisms that confer intrinsicand acquired antibiotic resistance could provide a survival advantagewhen in competition with other microbes within a particular niche.Alternatively, gene products that mediate antibiotic resistance mightplay a role in defense against bactericidal factors produced by the host,including antimicrobial peptides (AMPs). At least several AMPs, in-cluding human cathelicidin LL-37, human b-defensins 1, 2, and 3, andCRAMP, are able to kill P. aeruginosa (42, 43). Overexpression of theMexAB-OprM efflux system in P. aeruginosa confers resistance tothe synthetic AMP polymyxin B (44), and we found that polymyxinB–resistant V. cholerae also has an increased in vivo fitness com-pared with the wild-type parental strain. Other factors, such as thehost’s diet, could also play a role in maintaining intrinsic antibioticresistance genes, because foods (45) such as sweet potatoes and coffeecontain caffeic acid, a compound that has an antimicrobial activitytoward P. aeruginosa (46). In conclusion, the findings that intrinsicand acquired antibiotic resistance genes are associated with increasedin vivo fitness of P. aeruginosa, A. baumannii, and V. cholerae infour different experimental infection settings, along with recent re-ports about the lack of fitness costs associated with antibiotic resistancein S. enterica serovar Typhimurium (47) and E. coli (48), emphasize thenecessity to effectively control the emergence of antibiotic-resistantpathogens as well as the development of alternative approaches to pre-vent and treat infections. Last, our findings point to additional potentialconsequences, wherein virulent strains of serious microbial pathogensthat are both more drug-resistant and more pathogenic may be estab-lishing themselves as the predominant organisms able to infect at-riskhumans.
MATERIALS AND METHODS
A full description of the experimental methods can be found in Sup-plementary Materials and Methods.
Study designThe aim of this study was to explore the concept that antibiotic re-sistance could be associated with increased in vivo fitness and increasedvirulence during bacterial infections. We did an initial screening byusing a next- generation, high-throughput sequencing approach, termedTnSeq, on three different saturated banks of mutants of the pathogenicbacteria: P. aeruginosa, A. baumannii, and V. cholera. The TnSeq wasapplied in vivo using mouse and rabbit models of bacterial infections.A comprehensive approach was used to manage the millions of se-quencing reads generated by the TnSeq: an initial analysis by functionalclasses, then an analysis by operon, and finally a gene-by-gene analysis.The signals detected by TnSeq in favor of the in vivo association be-tween antibiotic resistance and increased fitness and virulence wereconfirmed using individual strains of each bacterial species. Individual
nceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 9
mutants were tested in vivo to confirm the results of the TnSeq screen-ing. In vitro experiments were designed to remove potential bias,complete the in vivo finding, raise hypotheses, and reveal mechanisms.
Predefined study components: The primary pathogenic agent se-lected was P. aeruginosa because of the severity of P. aeruginosa infec-tions and the major intrinsic and acquired antibiotic resistance of thisspecies. The second pathogen selected was A. baumannii to confirmthe initial results in another antibiotic-resistant pathogenic bacterium.The last pathogen, V. cholerae, was chosen for its medical impact. Themain mouse model selected was a pneumonia model because of theclinical relevance of P. aeruginosa lung infections. The antibiotics se-lected belonged to the main classes used to treat P. aeruginosa infec-tions: b-lactams, aminoglycosides, and fluoroquinolones. The cell lineselected was macrophages, to explore the role of the host immune de-fenses, including alveolar macrophages, to gain a more completeunderstanding of the interplay of antibiotic resistance and in vivo fit-ness in a murine model of pneumonia. Mice were housed under spe-cific pathogen–free conditions, and all animal experiments compliedwith institutional and federal guidelines regarding the use of animalsin research.
Statistical methodsTwo-sample comparisons used t tests, either unpaired for normallydistributed data or the Mann-Whitney test for nonparametric data.Survival was analyzed by log-rank tests. For analysis of TnSeq results,the fold changes under all conditions were determined, and the resultsfor all genes were analyzed for statistically significant differences intheir occurrence using the On Proportions function of CLC withcorrected P values calculated by the Bonferroni false discovery ratemethod.
by guest on May 26, 2021
rg/
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/7/297/297ra114/DC1Materials and MethodsFig. S1. Evolution of the RPKM sequencing reads for Tn insertions in genes in each of 27 func-tional classes from LB to the lung after 1 hour (A), from 1 to 6 hours in the lung (B), and from 6to 24 hours in the lung (C).Fig. S2. In vivo fitness of P. aeruginosa mutants with Tn insertions in genes from the functionalclass “motility and attachment” after 1, 6, or 24 hours of infection in the lung.Fig. S3. Comparative in vivo fitness (in the GI tract and lung) of bacterial strains with Tn inser-tions in genes needed to produce T4aP components.Fig. S4. Comparative in vitro fitness in LB and water of bacterial strains with Tn insertion mu-tants in genes needed to produce T4aP components.Fig. S5. Comparative in vivo fitness of bacterial strains with Tn insertions in genes that encodeall of the annotated VFs of P. aeruginosa (after 1, 6, or 24 hours in the lung).Fig. S6. Evolution over time of the RPKM for Tn-interrupted genes in the LPS O-antigen locus.Fig. S7. Increased virulence of oprD mutant carbapenem-resistant clinical strains of P. aeruginosa(48.2 and 51.2) in lung infection compared to isogenic (48.1 and 51.1) and complemented strains(48.2::PoprD and 51.2::PoprD).Fig. S8. Evolution over time of the changes in the RPKM for Tn-interrupted genes associatedwith constitutive antibiotic resistance in P. aeruginosa in the GI tract, spleen, and lung.Fig. S9. Analysis of in vitro growth and survival of Tn mutants deficient in genes associatedwith constitutive antibiotic resistance in P. aeruginosa.Fig. S10. Effect of deletion of intrinsic antibiotic resistance genes on survival of P. aeruginosa inJ774 macrophages.Table S1. Analysis of selective pressures detected by RPKM reads during P. aeruginosa lunginfection.Table S2. Genes (116) whose loss shows an increased fitness for lung infection based on having Tninsertions with at least 100 reads after 24 hours and with reads further increasing more thantwofold between 24 and 48 hours of infection.
www.Scien
Table S3. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted forgenes encoding intrinsic antibiotic resistance.Table S4. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted forgenes encoding intrinsic antibiotic resistance (inhibition diameter).Table S5. Antibiotic sensitivity results for wild-type P. aeruginosa PA14 and strains deleted forgenes encoding intrinsic antibiotic resistance (MIC as measured by E test).Table S6. Primers for genomic amplification used in this study.Table S7. Bacterial strains used in this study.Table S8. Plasmids used in this study.Data file S1. Analysis of the TnSeq data by functional classes of P. aeruginosa PA14.Data file S2. Fitness of flagellin mutants identified in the study.References (49–59)
REFERENCES AND NOTES
1. D. I. Andersson, D. Hughes, Antibiotic resistance and its cost: Is it possible to reverseresistance? Nat. Rev. Microbiol. 8, 260–271 (2010).
2. D. Skurnik, D. Roux, H. Aschard, V. Cattoir, D. Yoder-Himes, S. Lory, G. B. Pier, A comprehensiveanalysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries. PLOS Pathog. 9, e1003582 (2013).
3. D. Skurnik, D. Roux, V. Cattoir, O. Danilchanka, X. Lu, D. R. Yoder-Himes, K. Han, T. Guillard,D. Jiang, C. Gaultier, F. Guerin, H. Aschard, R. Leclercq, J. J. Mekalanos, S. Lory, G. B. Pier, Enhancedin vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealedthrough high-throughput sequencing. Proc. Natl. Acad. Sci. U.S.A. 110, 20747–20752 (2013).
4. Y. Carmeli, N. Troillet, G. M. Eliopoulos, M. H. Samore, Emergence of antibiotic-resistantPseudomonas aeruginosa: Comparison of risks associated with different antipseudomonalagents. Antimicrob. Agents Chemother. 43, 1379–1382 (1999).
5. B. Georges, J. M. Conil, A. Dubouix, M. Archambaud, E. Bonnet, S. Saivin, V. Lauwers-Cancès,C. Cristini, P. Cougot, J. F. Decun, O. Mathe, G. Chabanon, N. Marty, T. Seguin, G. Houin, Riskof emergence of Pseudomonas aeruginosa resistance to b-lactam antibiotics in intensive careunits. Crit. Care Med. 34, 1636–1641 (2006).
6. P. D. Lister, D. J. Wolter, N. D. Hanson, Antibacterial-resistant Pseudomonas aeruginosa:Clinical impact and complex regulation of chromosomally encoded resistance mechanisms.Clin. Microbiol. Rev. 22, 582–610 (2009).
7. M. C. Gaspar, W. Couet, J. C. Olivier, A. A. Pais, J. J. Sousa, Pseudomonas aeruginosa infectionin cystic fibrosis lung disease and new perspectives of treatment: A review. Eur. J. Clin.Microbiol. Infect. Dis. 32, 1231–1252 (2013).
8. A. Folkesson, L. Jelsbak, L. Yang, H. K. Johansen, O. Ciofu, N. Høiby, S. Molin, Adaptation ofPseudomonas aeruginosa to the cystic fibrosis airway: An evolutionary perspective. Nat.Rev. Microbiol. 10, 841–851 (2012).
9. G. Maschmeyer, I. Braveny, Review of the incidence and prognosis of Pseudomonas aeruginosainfections in cancer patients in the 1990s. Eur. J. Clin. Microbiol. Infect. Dis. 19, 915–925 (2000).
10. S. Fujitani, H. Y. Sun, V. L. Yu, J. A. Weingarten, Pneumonia due to Pseudomonas aeruginosa:Part I: Epidemiology, clinical diagnosis, and source. Chest 139, 909–919 (2011).
11. L. S.Munoz-Price, R.A.Weinstein, Acinetobacter infection.N. Engl. J.Med.358, 1271–1281 (2008).12. C. K. Stover, X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman,
W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman,Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer,G. K. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. Hancock, S. Lory, M. V. Olson,Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.Nature 406, 959–964 (2000).
13. G. L. Winsor, D. K. Lam, L. Fleming, R. Lo, M. D. Whiteside, N. Y. Yu, R. E. Hancock, F. S. Brinkman,Pseudomonas Genome Database: Improved comparative analysis and population genomicscapability for Pseudomonas genomes. Nucleic Acids Res. 39, D596–D600 (2011).
14. K. Selezska, M. Kazmierczak, M. Müsken, J. Garbe, M. Schobert, S. Häussler, L. Wiehlmann,C. Rohde, J. Sikorski, Pseudomonas aeruginosa population structure revisited under environmentalfocus: Impact of water quality and phage pressure. Environ. Microbiol. 14, 1952–1967 (2012).
15. L. Chen, Z. Xiong, L. Sun, J. Yang, Q. Jin, VFDB 2012 update: Toward the genetic diversity andmolecular evolution of bacterial virulence factors. Nucleic Acids Res. 40, D641–D645 (2012).
16. A. Castañeda-García, A. Rodríguez-Rojas, J. R. Guelfo, J. Blázquez, The glycerol-3-phosphatepermease GlpT is the only fosfomycin transporter in Pseudomonas aeruginosa. J. Bacteriol.191, 6968–6974 (2009).
17. N. T. Liberati, J. M. Urbach, S. Miyata, D. G. Lee, E. Drenkard, G. Wu, J. Villanueva, T. Wei,F. M. Ausubel, An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14transposon insertion mutants. Proc. Natl. Acad. Sci. U.S.A. 103, 2833–2838 (2006).
18. N. Wang, E. A. Ozer, M. J. Mandel, A. R. Hauser, Genome-wide identification of Acinetobacterbaumannii genes necessary for persistence in the lung. mBio 5, e01163-14 (2014).
19. S. Coyne, P. Courvalin, B. Périchon, Efflux-mediated antibiotic resistance in Acinetobacterspp. Antimicrob. Agents Chemother. 55, 947–953 (2011).
ceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 10
20. E. J. Yoon, P. Courvalin, C. Grillot-Courvalin, RND-type efflux pumps in multidrug-resistantclinical isolates of Acinetobacter baumannii: Major role for AdeABC overexpression andAdeRS mutations. Antimicrob. Agents Chemother. 57, 2989–2995 (2013).
21. A. C. Jacobs, M. G. Thompson, C. C. Black, J. L. Kessler, L. P. Clark, C. N. McQueary, H. Y. Gancz,B. W. Corey, J. K. Moon, Y. Si, M. T. Owen, J. D. Hallock, Y. I. Kwak, A. Summers, C. Z. Li, D. A. Rasko,W. F. Penwell, C. L. Honnold, M. C. Wise, P. E. Waterman, E. P. Lesho, R. L. Stewart, L. A. Actis,T. J. Palys, D. W. Craft, D. V. Zurawski, AB5075, a highly virulent isolate of Acinetobacter baumannii,as a model strain for the evaluation of pathogenesis and antimicrobial treatments. mBio 5,e01076-14 (2014).
22. D. Skurnik, M. R. Davis Jr., D. Benedetti, K. L. Moravec, C. Cywes-Bentley, D. Roux, D. C. Traficante,R. L. Walsh, T. Maira-Litràn, S. K. Cassidy, C. R. Hermos, T. R. Martin, E. L. Thakkallapalli, S. O. Vargas,A. J. McAdam, T. D. Lieberman, R. Kishony, J. J. Lipuma, G. B. Pier, J. B. Goldberg, G. P. Priebe,Targeting pan-resistant bacteria with antibodies to a broadly conserved surface polysaccharideexpressed during infection. J. Infect. Dis. 205, 1709–1718 (2012).
23. J. B. Harris, R. C. LaRocque, F. Qadri, E. T. Ryan, S. B. Calderwood, Cholera. Lancet 379,2466–2476 (2012).
24. J. Mandal, V. Sangeetha, V. Ganesan, M. Parveen, V. Preethi, B. N. Harish, S. Srinivasan, S. C. Parija,Third-generation cephalosporin–resistant Vibrio cholerae, India. Emerg. Infect. Dis. 18, 1326–1328(2012).
25. L. Yu, Y. Zhou, R. Wang, J. Lou, L. Zhang, J. Li, Z. Bi, B. Kan, Multiple antibiotic resistance ofVibrio cholerae serogroup O139 in China from 1993 to 2009. PLOS One 7, e38633 (2012).
26. S. M. Rashed, A. Iqbal, S. B. Mannan, T. Islam, M. U. Rashid, F. T. Johura, H. Watanabe, N. A. Hasan,A. Huq, O. C. Stine, R. B. Sack, R. R. Colwell, M. Alam, Vibrio cholerae O1 El Tor and O139 Bengalstrains carrying ctxBET, Bangladesh. Emerg. Infect. Dis. 19, 1713–1715 (2013).
27. Y. Fu, M. K. Waldor, J. J. Mekalanos, Tn-Seq analysis of Vibrio cholerae intestinal colonization revealsa role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe 14, 652–663 (2013).
28. V. Koronakis, J. Eswaran, C. Hughes, Structure and function of TolC: The bacterial exit ductfor proteins and drugs. Annu. Rev. Biochem. 73, 467–489 (2004).
29. S. Matsuyama, T. Tajima, H. Tokuda, A novel periplasmic carrier protein involved in thesorting and transport of Escherichia coli lipoproteins destined for the outer membrane.EMBO J. 14, 3365–3372 (1995).
30. P. Komp Lindgren, L. L. Marcusson, D. Sandvang, N. Frimodt-Møller, D. Hughes, Biologicalcost of single and multiple norfloxacin resistance mutations in Escherichia coli implicatedin urinary tract infections. Antimicrob. Agents Chemother. 49, 2343–2351 (2005).
31. J. Björkman, D. Hughes, D. I. Andersson, Virulence of antibiotic-resistant Salmonella typhimurium.Proc. Natl. Acad. Sci. U.S.A. 95, 3949–3953 (1998).
32. E. Kugelberg, S. Löfmark, B. Wretlind, D. I. Andersson, Reduction of the fitness burden of quinoloneresistance in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 55, 22–30 (2005).
33. E. C. Bottger, M. Pletschette, D. Andersson, Drug resistance and fitness in Mycobacteriumtuberculosis infection. J. Infect. Dis. 191, 823–824 (2005).
34. I. Nagaev, J. Björkman, D. I. Andersson, D. Hughes, Biological cost and compensatory evo-lution in fusidic acid-resistant Staphylococcus aureus. Mol. Microbiol. 40, 433–439 (2001).
35. H. Giamarellou, G. Poulakou, Multidrug-resistant Gram-negative infections: What are thetreatment options? Drugs 69, 1879–1901 (2009).
36. A. Rodríguez-Rojas, M. D. Maciá, A. Couce, C. Gómez, A. Castañeda-Garcia, A. Oliver, J. Blázquez,Assessing the emergence of resistance: The absence of biological cost in vivo maycompromise fosfomycin treatments for P. aeruginosa infections. PLOS One 5, e10193 (2010).
37. F. Álvarez-Lerma, S. Grau, Management of antimicrobial use in the intensive care unit.Drugs 72, 447–470 (2012).
38. C. Pena, C. Suarez, M. Gozalo, J. Murillas, B. Almirante, V. Pomar, M. Aguilar, A. Granados, E. Calbo,J. Rodríguez-Baño, F. Rodríguez, F. Tubau, L. Martínez-Martínez, A. Oliver; Spanish Networkfor Research in Infectious Diseases REIPI, Prospective multicenter study of the impact ofcarbapenem resistance on mortality in Pseudomonas aeruginosa bloodstream infections.Antimicrob. Agents Chemother. 56, 1265–1272 (2012).
39. J. Qin, R. Li, J. Raes, M. Arumugam, K. S. Burgdorf, C. Manichanh, T. Nielsen, N. Pons, F. Levenez,T. Yamada, D. R. Mende, J. Li, J. Xu, S. Li, D. Li, J. Cao, B. Wang, H. Liang, H. Zheng, Y. Xie, J. Tap,P. Lepage, M. Bertalan, J. M. Batto, T. Hansen, D. Le Paslier, A. Linneberg, H. B. Nielsen,E. Pelletier, P. Renault, T. Sicheritz-Ponten, K. Turner, H. Zhu, C. Yu, S. Li, M. Jian, Y. Zhou,Y. Li, X. Zhang, S. Li, N. Qin, H. Yang, J. Wang, S. Brunak, J. Doré, F. Guarner, K. Kristiansen,O. Pedersen, J. Parkhill, J. Weissenbach, P. Bork, S. D. Ehrlich, J. Wang; MetaHIT Consortium,A human gut microbial gene catalogue established by metagenomic sequencing. Nature464, 59–65 (2010).
40. G. Koch, A. Yepes, K. U. Förstner, C. Wermser, S. T. Stengel, J. Modamio, K. Ohlsen, K. R. Foster,D. Lopez, Evolution of resistance to a last-resort antibiotic in Staphylococcus aureus via bacterialcompetition. Cell 158, 1060–1071 (2014).
41. M. S. Donia, P. Cimermancic, C. J. Schulze, L. C. Wieland Brown, J. Martin, M. Mitreva, J. Clardy,R. G. Linington, M. A. Fischbach, A systematic analysis of biosynthetic gene clusters in thehuman microbiome reveals a common family of antibiotics. Cell 158, 1402–1414 (2014).
42. D. H. Limoli, A. B. Rockel, K. M. Host, A. Jha, B. T. Kopp, T. Hollis, D. J. Wozniak, Cationicantimicrobial peptides promote microbial mutagenesis and pathoadaptation in chronicinfections. PLOS Pathog. 10, e1004083 (2014).
www.Scien
43. E. A. Berger, S. A. McClellan, K. S. Vistisen, L. D. Hazlett, HIF-1a is essential for effective PMNbacterial killing, antimicrobial peptide production and apoptosis in pseudomonas aeruginosakeratitis. PLOS Pathog. 9, e1003457 (2013).
44. S. J. Pamp, M. Gjermansen, H. K. Johansen, T. Tolker-Nielsen, Tolerance to the antimicrobialpeptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells,and depends on the pmr and mexAB-oprM genes. Mol. Microbiol. 68, 223–240 (2008).
45. R. N. Carmody, G. K. Gerber, J. M. Luevano Jr., D. M. Gatti, L. Somes, K. L. Svenson, P. J. Turnbaugh,Diet dominates host genotype in shaping the murine gut microbiota. Cell Host Microbe 17,72–84 (2015).
46. M. K. Sakharkar, P. Jayaraman, W. M. Soe, V. T. Chow, L. C. Sing, K. R. Sakharkar, In vitrocombinations of antibiotics and phytochemicals against Pseudomonas aeruginosa. J. Microbiol.Immunol. Infect. 42, 364–370 (2009).
47. S. Baker, P. T. Duy, T. V. Nga, T. T. Dung, V. V. Phat, T. T. Chau, A. K. Turner, J. Farrar, M. F. Boni,Fitness benefits in fluoroquinolone-resistant Salmonella Typhi in the absence of antimicrobialpressure. eLife 2, e01229 (2013).
48. V. de Lastours, A. Bleibtreu, F. Chau, C. Burdet, X. Duval, E. Denamur, B. Fantin, Quinolone-resistantEscherichia coli from the faecal microbiota of healthy volunteers after ciprofloxacin exposure arehighly adapted to a commensal lifestyle. J. Antimicrob. Chemother. 69, 761–768 (2014).
49. T. T. Hoang, A. J. Kutchma, A. Becher, H. P. Schweizer, Integration-proficient plasmids forPseudomonas aeruginosa: Site-specific integration and use for engineering of reporter andexpression strains. Plasmid 43, 59–72 (2000).
50. A. Y. Koh, G. P. Priebe, G. B. Pier, Virulence of Pseudomonas aeruginosa in a murine model ofgastrointestinal colonization and dissemination in neutropenia. Infect. Immun. 73, 2262–2272 (2005).
51. D. E. Cameron, J. M. Urbach, J. J. Mekalanos, A defined transposon mutant library and its use inidentifying motility genes in Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 105, 8736–8741 (2008).
52. G. Miller, M. Feiss, The bacteriophage l cohesive end site: Isolation of spacing/substitution mu-tations that result in dependence on Escherichia coli integration host factor.Mol. Gen. Genet. 212,157–165 (1988).
53. L. G. Rahme, E. J. Stevens, S. F. Wolfort, J. Shao, R. G. Tompkins, F. M. Ausubel, Common virulencefactors for bacterial pathogenicity in plants and animals. Science 268, 1899–1902 (1995).
54. A. Rietsch, I. Vallet-Gely, S. L. Dove, J. J. Mekalanos, ExsE, a secreted regulator of type III secre-tion genes in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. 102, 8006–8011 (2005).
55. M. R. Hirvonen, B. Brüne, E. G. Lapetina, Heat shock proteins and macrophage resistance tothe toxic effects of nitric oxide. Biochem. J. 315, 845–849 (1996).
56. K. Bellmann, M. Jäättelä, D. Wissing, V. Burkart, H. Kolb, Heat shock protein hsp70 over-expression confers resistance against nitric oxide. FEBS Lett. 391, 185–188 (1996).
57. B. W. Bardoel, S. van der Ent, M. J. Pel, J. Tommassen, C. M. Pieterse, K. P. van Kessel, J. A. van Strijp,Pseudomonas evades immune recognition of flagellin in both mammals and plants. PLOS Pathog.7, e1002206 (2011).
58. N. Dasgupta, R. Ramphal, Interaction of the antiactivator FleN with the transcriptional activatorFleQ regulates flagellar number in Pseudomonas aeruginosa. J. Bacteriol. 183, 6636–6644 (2001).
59. T. S. Murray, B. I. Kazmierczak, FlhF is required for swimming and swarming in Pseudomonasaeruginosa. J. Bacteriol. 188, 6995–7004 (2006).
Acknowledgments: We thank C. Manoil at the University of Washington for providing theA. baumannii mutant strains. Funding: This work was supported by a grant from the Cystic FibrosisFoundation (PIER14GO). D.R. received grants from the AXA Research Fund and the Société de Réani-mation de Langue Française. T.G. received grants from the Conseil Régional de Champagne-Ardenne,the Philipp Fondation, and the Association pour le Développement de la Microbiologie et de l’Immu-nologie Rémoises. J.J.M. is supported by grant AI-026289 and O.D. is supported by grant AI-115962,both from the U.S. National Institute of Allergy and Infectious Diseases. D.S. was supported by agrant from the Seedlings Foundation. Author contributions: D.R. performed and was the leaderof the in vivo experiments, analyzed data, worked on the manuscript, and contributed to thestudy concept. O.D. performed and was the leader of the in vitro experiments, analyzed data,and worked on the manuscript. T.G. and V.C. performed P. aeruginosa experiments. Y.F. performedV. cholerae experiments. J.M. performed P. aeruginosa experiments. T.G., V.C., Y.F., and J.J.M. ana-lyzed data and edited the manuscript. H.A., F.A., and J.-D.R. analyzed data and performed statisticalanalyses. Seedlings Foundation and S.L. contributed to TnSeq and in vitro study design, discussion,and the manuscript. G.B.P supervised the project, developed the study concept, and edited themanuscript. D.S. supervised the project, performed experiments, analyzed data, wrote the manu-script, and contributed to the study concept. Competing interests: The authors declare that theyhave no competing interests.
Submitted 19 March 2015Accepted 27 May 2015Published 22 July 201510.1126/scitranslmed.aab1621
Citation: D. Roux, O. Danilchanka, T. Guillard, V. Cattoir, H. Aschard, Y. Fu, F. Angoulvant,J. Messika, J.-D. Ricard, J. J. Mekalanos, S. Lory, G. B. Pier, D. Skurnik, Fitness cost ofantibiotic susceptibility during bacterial infection. Sci. Transl. Med. 7, 297ra114 (2015).
ceTranslationalMedicine.org 22 July 2015 Vol 7 Issue 297 297ra114 11
Fitness cost of antibiotic susceptibility during bacterial infection
Jonathan Messika, Jean-Damien Ricard, John J. Mekalanos, Stephen Lory, Gerald B. Pier and David SkurnikDamien Roux, Olga Danilchanka, Thomas Guillard, Vincent Cattoir, Hugues Aschard, Yang Fu, Francois Angoulvant,
DOI: 10.1126/scitranslmed.aab1621, 297ra114297ra114.7Sci Transl Med
adversaries.resistance might be harder than we thought, given the enhanced fitness and virulence of our drug-resistant
in mouse and rabbit infection models. Together, these findings warn that the fight against antibiotic−−cholerae Vibrio and Acinetobacter baumannii−−authors confirmed their findings in two additional pathogenic bacteria
cquired antibiotic resistance also imparted an in vivo fitness advantage to Pseudomonas during infection. Thein vivo fitness during lung infections in animal models and found that genes that conferred both intrinsic and a
to to pinpoint genes that contributed Pseudomonas aeruginosathe authors used a saturated transposon library of to be accompanied by the dampening of in vivo fitness, virulence, and transmission. In the new work,−−validation
without experimental−−the therapeutic arena, the phenotypic changes that confer drug resistance are thought investigated the selective advantage sported by drug-resistant strains during antibiotic treatment. But outside of
drugs, antibiotic resistance also appears to enhance microbial fitness and virulence. Most studies have in addition to limiting our repertoire of curative−− add another unwelcome wrinkle to the scenarioet al.Now, Roux
drugs over time make clear the damper resistance puts on our ability to keep infectious diseases under control.is antibiotic resistance. Scary survival statistics and gloomy graphs that depict decreases in new antimicrobial
Myriad publications from Consumer Reports to Mother Jones have warned us about the pending peril thatFit foes
