Tobramycin enhances Mycobacterium abscessus fitness through whiB7 induction

Tobramycin enhances Mycobacterium abscessus fitness through whiB7 induction | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Tobramycin enhances Mycobacterium abscessus fitness through whiB7 induction View ORCID Profile Jodi M. Corley , Jack H. Congel , Kelsey C. Haist , Alma E. Ochoa , Kenneth C. Malcolm , William J. Janssen , Jerry A. Nick , Katherine B. Hisert doi: https://doi.org/10.1101/2025.10.01.679559 Jodi M. Corley a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jodi M. Corley For correspondence: corleyj{at}njhealth.org Jack H. Congel a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kelsey C. Haist a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alma E. Ochoa a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kenneth C. Malcolm a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site William J. Janssen a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jerry A. Nick a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Katherine B. Hisert a Department of Medicine, National Jewish Health , Denver, CO 80206 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Nontuberculous mycobacteria (NTM) are opportunistic pathogens that cause pulmonary disease (PD) in people with bronchiectasis and other chronic airways diseases. Difficulty treating and eradicating NTM-PD highlights the need for improved understanding of bacterial mechanisms to establish chronic infections. People with the genetic disorder cystic fibrosis (CF) develop bronchiectasis and are the population at highest risk of NTM-PD, caused mainly by Mycobacterium avium or Mycobacterium abscessus ( Mabsc ). The majority of people with CF (pwCF) and bronchiectasis develop chronic Pseudomonas aeruginosa airway infections. We hypothesized that antibiotics used to treat P. aeruginosa infections could enhance Mabsc persistence in the CF airway. Here we demonstrate that clinically relevant concentrations of tobramycin, which does not kill Mabsc but is frequently administered to pwCF with chronic P. aeruginosa infections, induced Mabsc expression of whiB7 , a transcription factor that activates genes associated with resistance to host defenses. Tobramycin promoted Mabsc resistance to killing by hydrogen peroxide (H 2 O 2 ) and survival in both human macrophages and mice. Deletion of whiB7 increased Mabsc susceptibility to killing by H 2 O 2 , decreased ability of Mabsc to persist in macrophages, and disrupted ability of tobramycin to enhance Mabsc survival. Transcriptomic data defining the tobramycin associated WhiB7 regulon revealed differential gene expression of factors that could enhance Mabsc resistance to stress conditions such as those in found in the CF lung. Overall, our data indicate that administration of tobramycin to pwCF may have unexpected off-target effects, enhancing Mabsc whiB7 expression and promoting Mabsc persistent infection. Significance Statement Pulmonary infections caused by Mycobacterium abscessus ( Mabsc ) are increasing in prevalence, especially in people with cystic fibrosis (pwCF) and bronchiectasis, and are very challenging to treat. Risk factors that predispose to Mabsc pulmonary infections remain poorly defined. This study demonstrates that tobramycin, an antibiotic commonly used to treat chronic Pseudomonas aeruginosa infections in pwCF, promotes Mabsc persistence by inducing Mabsc expression of the transcription factor whiB7 . WhiB7 enhances Mabsc resistance to oxidative stress and survival in host macrophages. Transcriptome analysis reveals that tobramycin activates a large WhiB7 dependent regulon linked to stress tolerance and immune evasion. These findings highlight a clinically relevant off-target effect of tobramycin that may contribute to Mabsc persistence in polymicrobial infections. Introduction Airway infection with the non-tuberculous mycobacteria (NTM) species Mycobacterium abscessus complex ( Mabsc ) poses a major challenge for people with chronic airway disease, including bronchiectasis, and people with bronchiectasis caused by the genetic disease cystic fibrosis (CF) are at highest risk( 1 ). Mabsc pulmonary disease (PD) is difficult to treat, and associated with poor clinical outcomes( 1 , 2 ). Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have greatly improved health in people with CF (pwCF), but do not appear to reverse bronchiectasis( 3 ). As bronchiectasis is an independent risk factor for development of Mabsc lung disease( 4 ), Mabsc infections are likely to remain a threat to pwCF. It thus remains imperative to determine what factors promote Mabsc persistence in order to identify those at risk and inform development of more effective and targeted therapies to eradicate Mabsc infections. Prior studies have sought to identify risk factors for Mabsc -PD; however, beyond bronchiectasis being a prominent risk factor, studies have failed to detect commonalities among study cohorts( 5 – 8 ). For example, while some find that chronic P. aeruginosa airway colonization is associated with Mabsc lung infection, others do not( 7 – 10 ). Ultimately, susceptibility is likely multi-factorial, with different combinations of risk factors affecting different individuals. As pwCF are both at highest risk for Mabsc -PD and a highly studied population( 11 ), we looked to the CF literature for clues regarding potentially modifiable risk factors for Mabsc infection. We considered whether previously identified associations between CF airway co-infections and Mabsc lung infection could result from off-target effects of commonly used antibiotics. For instance, Pseudomonas aeruginosa infection is common in pwCF( 11 ), and those with chronic P. aeruginosa infections are usually subjected to many courses of antibiotics including the aminoglycoside tobramycin( 12 , 13 ). Thus, if present, Mabsc is also exposed to these therapeutics. However, because Mabsc is inherently resistant to killing by tobramycin, it is generally assumed that administration of tobramycin affects neither viability nor fitness of Mabsc ( 14 ). Recent studies have determined that sublethal doses of several ribosome targeting antibiotics used to treat Mabsc infections (the aminoglycoside amikacin; tetracyclines; and macrolide antibiotics, including azithromycin and clarithromycin) promote Mabsc antibiotic resistance by transcriptionally activating whiB7 which encodes the transcription factor WhiB7( 15 – 20 ). WhiB7 is conserved in actinomycetes including Mabsc, Mycobacterium smegmatis, Mycobacterium tuberculosis , and Streptomyces spp., among others. In addition to playing a role in antibiotic resistance, across species WhiB7 functions as a global regulator, inducing genes involved in cell division and resistance to host stresses such as amino acid starvation, oxidative stress, and phagocytosis( 20 – 22 ). Although studies have evaluated the crucial role of WhiB7 in resistance to antibiotics used to treat Mabsc infection, there is a notable deficiency in research exploring off-target effects of antibiotics aimed at other CF pathogens, such as tobramycin. Because of the frequency of co-infections of these two species ( 9 ), off-target effects of anti-pseudomonad treatment are a potential component of the complex dynamic between Mabsc and P. aeruginosa co-infections. Our study aimed to evaluate how tobramycin contributes to the risk of acquiring chronic Mabsc infections. Herein we demonstrate that administration of tobramycin to treat airway co-infections such as P. aeruginosa may inadvertently promote Mabsc persistence by inducing whiB7 expression. Results Tobramycin induces whiB7 gene expression in Mabsc Clinically relevant concentrations of antibiotics frequently used in pwCF to kill bacteria other than Mabsc were evaluated for their ability to induce whiB7 expression in Mabsc ( 12 , 23 , 24 ). Clinically relevant antibiotic concentrations were defined in two ways. First, we followed the Clinical Laboratory Standard Institute (CLSI) antibiotic breakpoints for P. aeruginosa ( 25 , 26 ). Second, the peak concentration of antibiotic in the blood (C max ) at clinically administered doses was considered (Table S1)( 27 – 33 ). Based on these values the killing capacities of amikacin, tobramycin, ciprofloxacin, levofloxacin, and azithromycin were measured against our P. aeruginosa reference strain (Table S2). To ensure that antibiotics used in our studies would not kill Mabsc , minimum inhibitory concentration (MIC) assays against Mabsc reference strains ATCC 19977 and 390S( 34 ) were performed ( Figure 1a ). Download figure Open in new tab Figure 1. Mabsc whiB7 expression is induced by tobramycin. a) Table depicting amikacin, tobramycin, ciprofloxacin, levofloxacin and azithromycin MIC breakpoints as defined by CLSI guidelines and as determined by experiment for Mabsc ATCC 19977 and 390S WT strains. (b, c) Mabsc ATCC 19977 whiB7 transcripts were measured by qRT-PCR after Mabsc treatment with b) 1 μg/ml amikacin (positive control), 4 μg/ml tobramycin, 4 μg/ml ciprofloxacin, 4 μg/ml levofloxacin, or 5 μg/ml azithromycin for 24 hours or after c) treatment with 1, 4, 15, or 25 μg/ml tobramycin compared to no treatment at 1, 4, and 24 hours post tobramycin exposure. Statistical analyses for all experiments were performed by one-way ANOVA with Dunnett’s multiple comparisons post-hoc test relative to untreated WT at the same time point; *p <0.05, **p0.002, ****p < 0.0001, comparing each condition to no treatment at the same time point. Each bar represents the average of at least 3 experiments. To test whether selected antibiotics induce whiB7 in Mabsc , cultures of the Mabsc reference strain ATCC 19977 were grown in media containing tobramycin (15 µg/ml) or in sublethal doses of the control antibiotics amikacin (1 µg/ml) or azithromycin (4 µg/ml), ciprofloxacin (4 µg/ml) or levofloxacin (4 µg/ml) for 24 hours. Consistent with our hypothesis, tobramycin significantly induced Mabsc whiB7 expression as compared to Mabsc cultures not exposed to antibiotics ( Figure 1b, c ). Induction of whib7 expression was both dose- and time-dependent ( Figure 1c ). In contrast, fluoroquinolone antibiotics ciprofloxacin and levofloxacin, which are commonly used to treat P. aeruginosa infections in pwCF, did not induce whiB7 gene expression at any tested concentration over 24 hours ( Figure 1b , S1-S3). Induction of whiB7 expression also occurred in the clinical isolate Mabsc 390S following exposure to amikacin and tobramycin (Figure S1). Since Mabsc whiB7 expression plateaued at tobramycin doses greater than 15 µg/ml, and this concentration does not inhibit Mabsc growth, 15 μg/ml tobramycin was used to induce WhiB7 in subsequent experiments. Tobramycin enhances Mabsc resistance to reactive oxygen species and survival in macrophages We next investigated how tobramycin induction of Mabsc whiB7 expression could alter host-pathogen interactions. RNAseq studies have shown that the Mabsc WhiB7 regulon includes genes predicted to be important for survival in oxidative stress environments( 20 – 22 ), such as the highly inflamed CF airway( 35 ). We hypothesized that induction of whiB7 expression by tobramycin would promote Mabsc resistance to reactive oxygen species (ROS). We investigated Mabsc ’s resistance to oxidative stress by determining the hydrogen peroxide (H 2 O 2 ) minimum inhibitory concentration (MIC) of Mabsc cultures. Pre-treatment of Mabsc with amikacin or tobramycin resulted in higher H 2 O 2 MICs compared to untreated Mabsc , suggesting that both antibiotics may promote resistance to ROS in vivo ( Figure 2a ). Download figure Open in new tab Figure 2. Tobramycin promotes Mabsc resistance to ROS and growth in human primary macrophages in a whiB7 -dependent manner. a) Mabsc ATCC 19977 treated with 15 µg/mL tobramycin or 1 µg/mL amikacin exhibited increased resistance to H 2 O 2 , as determined by minimum inhibitory concentration (MIC) assays. b) Tobramycin and amikacin treated Mabsc showed enhanced intracellular survival in human monocyte-derived macrophages (MDMs) at 72 hours post infection compared to untreated Mabsc . (c, d) Mabsc 390S WT and Δ whiB7 strains were treated with tobramycin or amikacin followed by assessment of c) H 2 O 2 MICs or d) survival in MDMs. (e, f) Δ whiB7 strains containing a plasmid constitutively expressing whiB7 (pWhiB7) or empty vector (pV) were assessed for e) whiB7 expression by qRT-PCR and f) H 2 O 2 MICs. Statistical analyses for all experiments were performed by one-way ANOVA with Dunnett’s multiple comparisons post-hoc test relative to untreated WT; *p < 0.05, ***p<0.005, ****p<0.0001. MIC values are known between experiments. Each bar represents the average of at least 3 experiments. RNAseq studies have also shown that the Mabsc WhiB7 regulon includes genes potentially important for invasion of macrophages, and whiB7 is highly induced in M. tuberculosis after ingestion by macrophages( 36 , 37 ). Macrophages in the CF airways provide host defense against NTM species( 38 – 40 ). As most macrophages found in the CF airway are monocyte derived macrophages (MDMs) recruited from the blood( 38 ), evaluated the ability of Mabsc pretreated with aminoglycosides for ability to survive in MDMs. Mabsc exposed for 24 hours to either sublethal doses of amikacin or clinically relevant doses of tobramycin exhibited enhanced growth in human primary MDMs at 72 hours compared to untreated Mabsc ( Figure 2b ). The Mabsc ΔwhiB7 mutant demonstrates impaired ROS resistance and growth in macrophages To determine if enhanced resistance to host anti-microbial mechanisms following pre-treatment of Mabsc with tobramycin is dependent on induction of whiB7 , we performed H 2 O 2 MIC assays and macrophage infection assays using an Mabsc whiB7 deletion mutant ( ΔwhiB7 ) made in the background of the Mabsc clinical isolate 390S( 41 ). As seen with Mabsc 19977, wild type (WT) 390S had a higher H 2 O 2 MIC when exposed to sublethal concentrations of amikacin or anti-pseudomonal concentrations of tobramycin ( Figure 2c ). Mabsc Δ whiB7 was more sensitive to killing by H 2 O 2 , with a significantly lower MIC compared to WT ( Figure 2c ). Furthermore, exposure of Mabsc Δ whiB7 to either amikacin or tobramycin for 24 hours prior to exposure to H 2 O 2 did not enhance resistance to H 2 O 2 ( Figure 2c ). Mabsc Δ whiB7 also showed decreased growth in human primary MDMs compared to WT after 72 hours of infection ( Figure 2d ). Additionally, growth of Mabsc Δ whiB7 was not restored to WT levels in human primary MDMs after Mabsc Δ whiB7 was primed for 24 hours with tobramycin or amikacin ( Figure 2d ). Collectively, these data demonstrate the importance of whiB7 expression for Mabsc survival in host cells, and support that the mechanism by which aminoglycosides enhance Mabsc ROS tolerance and enhanced growth in macrophages occurs via a WhiB7-mediated pathway(s). Overexpression of whiB7 leads to increased ROS resistance To confirm that enhanced ROS sensitivity of Mabsc Δ whiB7 was due to loss of whiB7 , not unmarked mutations, we complemented Mabsc Δ whiB7 by introducing a constitutively expressed whiB7 gene under control of the Hsp60 promoter on a multicopy, replicating plasmid (pWhiB7). Using qRT-PCR we found that the empty vector did not affect whiB7 expression (comparison of Mabsc WT and pV whiB7) , and the complemented mutant over-expressed whiB7 ( Figure 2e ). The Mabsc Δ whiB7 isolate containing pWhiB7 was not only restored for resistance to H 2 O 2 , but also exhibited increased H 2 O 2 MIC, demonstrating that increases in whiB7 expression correlate with increased ROS resistance. The vector alone (pV) did not affect H 2 O 2 MIC in Mabsc Δ whiB7 , with an MIC similar to vectorless Δ whiB7 ( Figure 2f ). Parenteral tobramycin improves survival of Mabsc in murine lungs during acute infection To determine if tobramycin enhances Mabsc survival in the lung, we employed an acute model of murine Mabsc pneumonia ( Figure 3 ). C57BL/6J mice were injected intraperitoneally (IP) with tobramycin (150 mg/kg)( 42 ) 1 day prior to oropharyngeal (OP) inoculation with 5×10 6 CFU Mabsc 19977, and then every day during a 7-day infection. Mice administered tobramycin demonstrated increased Mabsc colony forming units (CFUs) in the lungs at 7 days post infection (dpi) as compared to vehicle-treated mice ( Figure 3a, b ). To confirm that IP tobramycin levels reached clinically relevant concentrations in murine lungs, a second group of mice was dosed with IP tobramycin or vehicle via the same protocol, but were infected OP with 5×10 6 CFU P. aeruginosa . Lungs from P. aeruginosa infected mice were harvested 1 dpi for CFU enumeration ( Figure 3a, c ). Vehicle treated mice demonstrated a 2.4 log decrease in CFUs in the lungs with > 2 × 10 4 CFUs remaining. However, no P. aeruginosa CFUs were detected in the lungs of tobramycin treated mice, confirming that a therapeutic dose of tobramycin had been achieved ( Figure 3c ). Thus, administration of tobramycin at doses used to treat Pseudomonas infections can enhance survival of Mabsc in the lungs during acute infection. Download figure Open in new tab Figure 3. Administration of systemic tobramycin that achieves concentrations in murine lung sufficient to kill P. aeruginosa is associated with increased pulmonary Mabsc burden. a) Diagram of acute mouse model of Mabsc or P. aeruginosa infection for CFU quantification. Created with BioRender ( https://www.biorender.com/ ). Mice were administered tobramycin (150 mg/kg, I.p.) or vehicle control 1 day prior to and on each day of pulmonary infection with Mabsc ATCC 19977 or P. aeruginosa via oropharyngeal aspiration. Lungs were harvested for CFU enumeration at 7 dpi (Mabsc) (b) or 1 dpi ( P. aeruginosa ) (c) . Statistical significance was determined by Student’s t test (* p < 0.05). RNA sequencing reveals high overlap between the tobramycin and WhiB7 dependent regulons To characterize which components of the WhiB7 regulon were modulated by tobramycin, we compared gene expression profiles from Mabsc WT and Δ whiB7 strains treated with tobramycin to untreated WT ( Figure 4 , Tables S3, S4,). Strikingly, we determined that of the approximated 5,000 annotated genes in the Mabsc genome, over half of the transcripts were differentially expressed (log 2 fold change >1) when comparing tobramycin-treated to untreated (2742 genes), or when comparing the Mabsc Δ whiB7 strain compared to Mabsc WT (2671 genes; Figure 4 ). In support of our hypothesis that tobramycin promotes expression of WhiB7, there were 2384 (80.68%) overlapping genes in the tobramycin-induced and WhiB7-dependent regulons, and expression of over 80% of overlapping genes were inversely regulated in tobramycin exposed Mabsc WT as compared to Mabsc Δ whiB7 ( Figure 4c, d ). Additionally, only 149 genes were significantly changed in Mabsc Δ whiB7 treated with tobramycin compared to Mabsc Δ whiB7 not exposed to tobramycin ( Figure 4c, d ). All of the top 25 genes up- or down-regulated by tobramycin in Mabsc WT were inversely regulated in Mabsc Δ whiB7 compared to WT, with only 7 genes also differentially expressed in Mabsc Δ whiB7 treated with tobramycin compared to untreated Mabsc Δ whiB7 (Tables S3, S4). Download figure Open in new tab Figure 4. RNA-seq analysis of differential gene expression defining the tobramycin associated WhiB7 regulon. Volcano plots display differential expression profiles for a) Mabsc 390S WT treated with tobramycin (15 μg/ml) compared to untreated WT and b) 390S Δ whiB7 compared to WT. Each gene is represented by a point; significantly up-regulated and down-regulated genes are shown in red and blue respectively and whiB7 is represented by a circled gold star. c) Heat map of differentially expressed genes comparing the three conditions: WT treated with tobramycin compared to untreated WT, Δ whiB7 compared to WT, and Δ whiB7 treated with tobramycin compared to untreated Δ whiB7 . d) Venn diagram summarizing the overlap of significantly differentially expressed genes among the three groups with number of genes and % of significant genes listed. Consistent with the previously published WhiB7 RNAseq study( 19 ), we identified several WhiB7 regulated genes responsive to tobramycin treatment that encode factors predicted to be important for macrophage survival and antibiotic resistance factors like eis2 (MAB_4532c; S3, S4)( 19 , 43 ). The Mabsc WhiB7 and tobramycin regulons also included genes encoding factors that aid bacterial survival during oxidative stress conditions and phagocytosis, katA (MAB_0351)( 37 , 44 ) and pknE (MAB_4736)( 45 ) as well as those that modulate components the Mabsc cell envelope, fadE5 (MAB_4437)( 46 , 47 ) and mps2 (MAB_4098c) ( 48 , 49 ). Other WhiB7 and tobramycin regulated genes include those that elicit inflammatory toll like receptor (TLR)-2 responses in the host, such as lpqH (MAB_0885c; Tables S3, S4)( 50 ). The large overlap in the Mabsc WhiB7 and tobramycin-responsive regulons further suggest that the mechanism by which tobramycin regulates the majority of its regulon occurs via modulating whiB7 expression. Discussion Although pwCF and bronchiectasis are at high risk of contracting Mabsc -PD, an unresolved question is why some pwCF develop persistent Mabsc infection, while others do not. Here we show that tobramycin, a standard of care aminoglycoside used to treat P. aeruginosa infections in pwCF( 12 , 13 ), upregulates whiB7 and its downstream regulon( 21 ), enhancing Mabsc resistance to oxidative stress and promoting survival in macrophages. These phenotypes were absent in a whiB7 deletion mutant, emphasizing the importance of the WhiB7 regulon in shaping Mabsc physiology in ways that favor establishment of chronic infection. WhiB7 is a conserved actinomycete transcription factor that connects antibiotic induced ribosomal stress to mycobacterial defense( 18 , 19 , 51 ). Prior work has demonstrated that ribosome targeting antibiotics including aminoglycosides, macrolides, tetracyclines, and lincosamides induce whiB7 expression( 15 – 21 ), leading to upregulation of antibiotic resistance factors such as eis2 and erm( 41 ) ( 19 ). Extensive studies involving whiB7-mediated antibiotic resistance establish whiB7 as a central hub of intrinsic drug resistance. Stimuli that induce whiB7 , however, are not limited to antibiotics. Stressors that may be encountered in vivo can also induce whiB7 ( 21 , 52 , 53 ). For example, whiB7 is induced during M. tuberculosis infection in mouse lungs( 53 ). We found that Mabsc exposure to doses of tobramycin that induce whiB7 enhanced Mabsc survival in macrophages and in mice, indicating that aminoglycosides and other antibiotics may be more potent whiB7 inducing stimuli than host factors. Consistent with other reports, we found that ribosome targeting antibiotics ( 16 , 21 ) induced whiB7 expression; we add to these studies by demonstrating that fluoroquinolones ciprofloxacin and levofloxacin, which target bacterial DNA gyrase, did not induce whiB7 . Previous RNAseq studies in Mabsc defined antibiotic associated WhiB7 regulons, including clindamycin (a lincosamide antibiotic that targets the ribosome)( 54 ), and sub-inhibitory levels of amikacin( 16 ), further demonstrating the importance of ribosome targeting antibiotics for induction of whiB7 . Recent studies have proposed mechanisms by which ribosome stalling during translation, as would occur with ribosome targeting antibiotics, results in enhanced WhiB7 expression( 55 , 56 ). Thus, ribosomal stress activation by a variety of ribosome targeting antibiotics is a key mechanism for activation of whiB7 expression and the subsequent phenotypes associated with induction of the WhiB7 regulon. However, to the best of our knowledge, this study is the first to specifically demonstrate that tobramycin, widely used to treat P. aeruginosa but not considered effective against Mabsc , can not only induce whiB7 , but also program Mabsc to survive in vitro and in vivo stressors. Apart from antibiotic resistance, WhiB7 mediates a broader range of adaptive stress responses that may promote immune evasion and host persistence. For example, M. tuberculosis eis (homolog of Mabsc eis2 ) suppresses macrophage activation and promotes intracellular bacterial survival by modulating host signaling pathways, including JNK pathway( 57 ). Further, previous work demonstrated that amikacin treatment and overexpression of whiB7 in Mabsc promoted survival in the macrophage-like THP-1 cell line( 16 ). We have extended these data by showing that whiB7 activation by tobramycin promotes Mabsc survival in primary human macrophages, and increasing levels of WhiB7 are associated with increasing resistance to reactive oxygen species. A handful of studies have characterized the WhiB7 regulon across actinomycetes, revealing both conserved and species-specific genes( 19 – 22 , 51 , 57 , 58 ). RNAseq comparisons of the WhiB7 regulon in Mabsc and M. smegmatis revealed very limited overlap( 19 ). Our transcriptomic profiling revealed that around half of the Mabsc genome was differentially expressed in response to either WhiB7 or tobramycin, with about 80% overlap between the two transcriptional responses. Shared WhiB7 and tobramycin induced genes include stress response genes, cell envelope regulators, and virulence-associated factors suggesting a convergent mechanism through which antibiotic exposure primes Mabsc for host adaptation. This is likely an evolutionary strategy for Mabsc to survive environmental stressors such as secreted antibiotics by soil microbes and predation by amoeba ( 59 ). Our gene expression threshold (log 2 fold change >1) was more permissive than the prior Hurst-Hess et al RNAseq study defining the Mabsc WhiB7 regulon as 128 genes (log 2 fold change >4)( 19 ). We chose this more permissive threshold in order to capture physiologically relevant transcriptional changes. Most previously identified WhiB7 regulon genes were also detected in our studies (83 out of 128 genes). However, only 4 of the top 25 most regulated genes in the WhiB7 regulon identified in our studies and denoted in Table S3 were previously identified by Hurst-Hess. Furthermore, several key virulence determinants were identified in our study with log 2 fold changes of just below the >4 cutoff. For example, irtA and mmpS5 , genes important for encoding factors that regulate iron transport( 60 , 61 ), were found to be downregulated in our study only. The previous studies used the reference strain ATCC 19977 while ours used Mabsc 390S, suggesting that, besides different stringencies between our studies, strain to strain variability may account for differences observed in the WhiB7 regulon. Several limitations of our study warrant discussion. While we demonstrated tobramycin-induced whiB7 activity in two Mabsc strains, inter-strain variability in clinical isolates may influence the generalizability of whiB7- mediated responses. Future studies will assess whiB7 induction and regulon activity in clinical Mabsc isolates from pwCF. We predict that differential tobramycin-induced expression of whiB7 in clinical isolates may help explain the heterogeneity of susceptibility to Mabsc- PD in pwCF and P. aeruginosa infections. Additionally, our mouse infection model evaluates how tobramycin influences acute infection in healthy WT mice, a model that does not recapitulate the complex lung environment during chronic CF infection. Our ongoing work will explore how tobramycin and WhiB7 influence establishment of infection in murine models of chronic airway disease and during chronic Mabsc infection. Even with these limitations, these findings have important clinical implications. First, our data highlight potential unintended consequences of standard antibiotic therapy in those with polymicrobial infections. Our observations support the concept that therapeutic strategies in pwCF and airways diseases should consider not only the direct antimicrobial effects of treatment, but also their potential off-target impacts on co-infecting bacteria. Prior studies in pwCF have noted unintended effects of antibiotics to enhance survival of non-targeted microbes. Azithromycin, which does not kill Pseudomonas , was shown to reduce the efficacy of inhaled tobramycin to kill P. aeruginosa in pwCF by inducing P. aeruginosa antibiotic resistance factors including MexXY ( 62 ). Off-target effects of antibiotics that enhance survival of bystander organisms likely occurs in other instances of polymicrobial infections beyond CF airways disease, such as other chronic lung diseases, chronic wounds, otitis media infections, and urinary tract infections( 63 , 64 ). Our results highlight that antibiotics can act as environmental cues that unexpectedly alter bacterial physiology and may enhance persistence of off-target organisms. Finally, our findings suggest that WhiB7’s dual role in antibiotic resistance and immune evasion may contribute to the challenge of eradicating Mabsc infections. Targeting global regulators like WhiB7 may offer new therapeutic opportunities. Understanding how antibiotic exposure shapes pathogen behavior and persistence will be critical for designing novel therapies for chronic infections in airways diseases like CF. Materials and Methods Bacterial strain and plasmid construction Strains, plasmids, primer sequences, and cloning details are provided in supplementary Tables S5, S6. Routine cloning was performed with E. coli DH5α cultured in Luria Bertani (LB; Difco) medium with kanamycin (Research Products International) (50 μg/ml) as required. The whiB7 expression plasmid was generated using primer pairs listed in Table S6 and cloned by isothermal assembly into pSD5.Hsp60( 65 ). Mabsc Δ whiB7 was constructed and donated by Kyle Rohde, PhD (University of South Florida)( 41 ). For additional details, see SI Appendix, SI Materials and Methods . Antibiotic MIC assays For P. aeruginosa MIC assays, 18-hour cultures of P. aeruginosa PA14 were centrifuged at 13,000xg for 1 minute, washed, resuspended in PBS to an A 600 of 0.08 to 0.1 (1.5 × 10 8 CFU), and then 100 μl of bacteria were added to 100 μl of Mueller Hinton Broth (Oxoid; MHB) in 96-well plates. Plates incubated statically for 24 hours at 37°C with humidity (Table S2). For Mabsc MICs, 3 day cultures of Mabsc strains ATCC 19977 and 390S WT were washed, then diluted to 2 × 10 6 CFU/ml in 7H9-ADC-glycerol. Subsequently 100 μl of bacteria were added to 100 μl of 7H9-ADC-glycerol containing serial dilutions of amikacin, tobramycin, ciprofloxacin, levofloxacin, and azithromycin (concentrations of 0-100 μg/ml) in 96 well plates, then grown in a humidified incubator at 37°C for 5 days ( Figure 1a ). The lowest concentration of antibiotic inhibiting bacterial growth was defined as the MIC. Quantitative Reverse Transcription PCR (qRT-PCR) RNA was extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs) per the manufacturer instructions. cDNA generation and amplification were performed with PrimeTime qPCR primer and probe sets (Integrated DNA Technologies, Coralville, IA) listed in Table S6 in a one-step reaction with the Luna Probe One-Step RT-qPCR 4X Mix with UDG (New England Biolabs) per manufacturer’s protocol. Relative abundance of transcripts was quantified by determining the difference of target transcript compared to a reference gene ( rpoB ) by calculating 2 -ΔΔCT values. For additional details, see SI Appendix, SI Materials and Methods . Measurement of hydrogen peroxide MICs Mabsc cultures were diluted to a cell density of 1 × 10 6 CFU in PBS, to which varying concentrations (0-150mM) of 30% H 2 O 2 (Fisher; 9.8M) were added. After 24 hours, 100 μl was plated to 7H10-OADC-glycerol plates to assess for growth. The lowest concentration of H 2 O 2 inhibiting bacterial growth, as determined by no growth on plates, was defined as the MIC. Experiments were performed at least 3 times. Generation of monocyte derived macrophages (MDMs) Blood was obtained from healthy human donors with IRB approval. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Ficoll-Plaque Plus (Cytiva), and monocytes were purified from PBMCs via negative selection (Miltenyi Monocyte Isolation Kit II). Monocytes were plated at a density of 2.5 × 10 5 cells per well in 48 well plates, and differentiated to MDMs using recombinant murine M-CSF as described( 66 ). Monocyte and MDM culture media did not contain antibiotics to prevent confounding influences on infection assays. Macrophage infection models Mabsc were added to MDMs at a multiplicity of infection (MOI) 1:1. MDMs were centrifuged for 1 minute at 1000xg to enhance microbe and macrophage contact, then incubated at 37 °C in 5% CO 2 . After 60 minutes incubation, MDMs were washed 3X in HBSS to remove extracellular bacteria, then 1) lysed with 0.1% triton (Sigma-Aldrich) in 1X PBS and serially diluted in PBS with 0.1% triton before plating on either LB agar for ATCC 19977 isolates or 7H10-OADC-glycerol for 390S isolates to determine Mabsc uptake by quantification of intracellular, or 2) fresh media were added and cells were incubated at 37 °C in 5% CO 2 for 24 or 72 additional hours then washed and lysed with 0.1% triton in PBS as previously described. Experiments were performed at least 3 times. Mouse infection model Male and female C57BL/6J (WT) mice, 8-9 weeks old were inoculated with Mabsc 19977 (5×10 6 CFUs per mouse) or P. aeruginosa PA14 (5 × 10 6 CFU per mouse) via oropharyngeal (o.p.) aspiration. One day prior to infection, mice were treated via intraperitoneal (i.p.) injection with tobramycin (150 mg/kg, ~ 150 μl per mouse of 20mg/ml tobramycin in H 2 O). Vehicle treated mice received 150 μl of water. At noted time points, mice were euthanized and lungs were removed for homogenization and plating for CFU quantification as described( 66 ). For additional details, see SI Materials and Methods . RNA sequencing analyses Late log cultures (3 days) of Mabsc 390S WT and Δ whiB7 were back-diluted into 7H9-ADC-glycerol media with or without tobramycin (15 μg/ml) for 24 hours (3 replicates per condition), then processed for RNA as previously described using the Monarch Total RNA Extraction kit per manufacturer’s protocol. Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit (Bacteria; New England Biolabs) per manufacturer’s specifications. Quality control of RNA was performed using the TapeStation (Agilent). RNA library preparation, amplification, and sequencing were performed by the National Jewish Health Genomics Core. Library size (base pair), concentration, and quality scores were calculated via TapeStation. Libraries were sequenced using Illumina NextSeq 2000 at a sequencing depth of 50 million paired end reads per sample. For additional details, see SI Appendix, SI Materials and Methods . Statistical analyses For statistical analyses used in this study, see SI Appendix, SI Materials and Methods . Acknowledgments This work was supported by funding from the CF Foundation awards CORLEY23F0, HISERT19R3, NICK20Y2-SVC, NICK20Y2 and NIH award R01HL167956. We thank Dr. Kyle Rohde for supplying the Mabsc 390S WT and Δ whiB7 strains, Dr. William DePas providing the pSD5.Hsp60 empty vector, the National Jewish Health Genomics Core for RNA sequencing and KC Anderson for RNAseq analysis, Dr. Michael Strong for providing M. abscessus gene annotations, and Dr. Elaine Epperson for assistance with RNA extraction protocols. Funder Information Declared Cystic Fibrosis Foundation, https://ror.org/00ax59295 , CORLEY23F0 , NICK20Y2-SVC , NICK20Y2 , HISERT19R3 National Institutes of Health, https://ror.org/01cwqze88 , R01HL167956 Footnotes Competing Interest Statement: No competing interests. References 1. ↵ S. L. Martiniano , J. A. Nick , C. L. Daley , Nontuberculous Mycobacterial Infections in Cystic Fibrosis . Clin. Chest Med . 43 , 697 – 716 ( 2022 ). OpenUrl PubMed 2. ↵ A. R. Hauser , M. Jain , M. Bar-Meir , S. A. McColley , Clinical significance of microbial infection and adaptation in cystic fibrosis . Clin. Microbiol. Rev . 24 , 29 – 70 ( 2011 ). OpenUrl Abstract / FREE Full Text 3. ↵ K. B. Hisert , et al. , Understanding and addressing the needs of people with cystic fibrosis in the era of CFTR modulator therapy . Lancet Respir. Med . 11 , 916 – 931 ( 2023 ). OpenUrl CrossRef PubMed 4. ↵ L. Viviani , M. J. Harrison , A. Zolin , C. S. Haworth , R. A. Floto , Epidemiology of nontuberculous mycobacteria (NTM) amongst individuals with cystic fibrosis (CF) . J. Cyst. Fibros . 15 , 619 – 623 ( 2016 ). OpenUrl CrossRef PubMed 5. ↵ M. Verregghen , H. G. Heijerman , M. Reijers , J. van Ingen , C. K. van der Ent , Risk factors for Mycobacterium abscessus infection in cystic fibrosis patients; a case-control study . J. Cyst. Fibros. Off. J. Eur. Cyst. Fibros. Soc . 11 , 340 – 343 ( 2012 ). OpenUrl 6. S. L. Martiniano , J. A. Nick , C. L. Daley , Nontuberculous Mycobacterial Infections in Cystic Fibrosis . Thorac. Surg. Clin . 29 , 95 – 108 ( 2019 ). OpenUrl CrossRef PubMed 7. ↵ J. R. Honda , S. Alper , X. Bai , E. D. Chan , Acquired and genetic host susceptibility factors and microbial pathogenic factors that predispose to nontuberculous mycobacterial infections . Curr. Opin. Immunol . 54 , 66 – 73 ( 2018 ). OpenUrl CrossRef PubMed 8. ↵ I. K. Park , K. N. Olivier , Nontuberculous Mycobacteria in Cystic Fibrosis and Non–Cystic Fibrosis Bronchiectasis . Semin. Respir. Crit. Care Med . 36 , 217 – 224 ( 2015 ). OpenUrl CrossRef PubMed 9. ↵ L. J. Caverly , M. Zimbric , M. Azar , K. Opron , J. J. LiPuma , Cystic fibrosis airway microbiota associated with outcomes of nontuberculous mycobacterial infection . ERJ Open Res . 7 ( 2021 ). 10. ↵ Q. Reynaud , et al. , Risk factors for nontuberculous mycobacterial isolation in patients with cystic fibrosis: A meta-analysis . Pediatr. Pulmonol . 55 , 2653 – 2661 ( 2020 ). OpenUrl PubMed 11. ↵ Cystic Fibrosis Foundation Patient Registry , “2020 Annual Data Report” (Cystic Fibrosis Foundation, 2021 ). 12. ↵ P. J. Mogayzel , et al. , Cystic fibrosis pulmonary guidelines. Chronic medications for maintenance of lung health . Am. J. Respir. Crit. Care Med . 187 , 680 – 689 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 13. ↵ P. J. Mogayzel , et al. , Cystic Fibrosis Foundation pulmonary guideline. pharmacologic approaches to prevention and eradication of initial Pseudomonas aeruginosa infection . Ann. Am. Thorac. Soc . 11 , 1640 – 1650 ( 2014 ). OpenUrl CrossRef PubMed 14. ↵ R. Nessar , E. Cambau , J. M. Reyrat , A. Murray , B. Gicquel , Mycobacterium abscessus: a new antibiotic nightmare . J. Antimicrob. Chemother . 67 , 810 – 818 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 15. ↵ M. Richard , A. V. Gutiérrez , L. Kremer , Dissecting erm(41)-Mediated Macrolide-Inducible Resistance in Mycobacterium abscessus . Antimicrob. Agents Chemother . 64 , e01879 – 19 ( 2020 ). OpenUrl PubMed 16. ↵ S.-H. Tsai , H.-C. Lai , S.-T. Hu , Subinhibitory Doses of Aminoglycoside Antibiotics Induce Changes in the Phenotype of Mycobacterium abscessus . Antimicrob. Agents Chemother . 59 , 6161 – 6169 ( 2015 ). OpenUrl Abstract / FREE Full Text 17. M. Pryjma , J. Burian , K. Kuchinski , C. J. Thompson , Antagonism between Front-Line Antibiotics Clarithromycin and Amikacin in the Treatment of Mycobacterium abscessus Infections Is Mediated by the whiB7 Gene . Antimicrob. Agents Chemother . 61 , e01353 – 17 ( 2017 ). OpenUrl PubMed 18. ↵ P. Rudra , K. Hurst-Hess , P. Lappierre , P. Ghosh , High Levels of Intrinsic Tetracycline Resistance in Mycobacterium abscessus Are Conferred by a Tetracycline-Modifying Monooxygenase . Antimicrob. Agents Chemother . 62 , e00119 – 18 ( 2018 ). OpenUrl CrossRef PubMed 19. ↵ K. Hurst-Hess , P. Rudra , P. Ghosh , Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance . Antimicrob. Agents Chemother . 61 , e01347 – 17 ( 2017 ). OpenUrl 20. ↵ K. Hurst-Hess , C. McManaman , Y. Yang , S. Gupta , P. Ghosh , Hierarchy and interconnected networks in the WhiB7 mediated transcriptional response to antibiotic stress in Mycobacterium abscessus . PLoS Genet . 19 , e1011060 ( 2023 ). OpenUrl CrossRef PubMed 21. ↵ J. Burian , et al. , The mycobacterial transcriptional regulator whiB7 gene links redox homeostasis and intrinsic antibiotic resistance . J. Biol. Chem . 287 , 299 – 310 ( 2012 ). OpenUrl Abstract / FREE Full Text 22. ↵ S. Ramón-García , et al. , WhiB7, an Fe-S-dependent Transcription Factor That Activates Species-specific Repertoires of Drug Resistance Determinants in Actinobacteria . J. Biol. Chem . 288 , 34514 – 34528 ( 2013 ). OpenUrl Abstract / FREE Full Text 23. ↵ D. P. Nichols , et al. , Developing Inhaled Antibiotics in Cystic Fibrosis: Current Challenges and Opportunities . Ann. Am. Thorac. Soc . 16 , 534 – 539 ( 2019 ). OpenUrl PubMed 24. ↵ J. D. Cogen , D. P. Nichols , C. H. Goss , R. Somayaji , Drugs, Drugs, Drugs: Current Treatment Paradigms in Cystic Fibrosis Airway Infections . J. Pediatr. Infect. Dis. Soc . 11 , S32 – S39 ( 2022 ). OpenUrl PubMed 25. ↵ CLSI , Fluoroquinolone Breakpoints for Enterobacteriaceae and Pseudomonas aeruginosa . Clin. Lab. Stand. Inst . 1st Edition ( 2019 ). 26. ↵ CLSI , AST News Update June 2023: New! CLSI M100-Ed33: Updated Aminoglycoside Breakpoints for Enterobacterales and Pseudomonas aeruginosa . Clin. Lab. Stand. Inst . Available at: https://clsi.org/about/blog/ast-news-update-june-2023-new-clsi-m100-ed33-updated-aminoglycoside-breakpoints-for-enterobacterales-and-pseudomonas-aeruginosa/ [Accessed 26 February 2025 ]. 27. ↵ R. Urso , P. Blardi , G. Giorgi , A short introduction to pharmacokinetics . Eur. Rev. Med. Pharmacol. Sci . 6 , 33 – 44 ( 2002 ). OpenUrl PubMed 28. E. Logre , et al. , Amikacin pharmacokinetic/pharmacodynamic in intensive care unit: a prospective database . Ann. Intensive Care 10 , 75 ( 2020 ). OpenUrl PubMed 29. O. Burkhardt , et al. , Once-daily tobramycin in cystic fibrosis: better for clinical outcome than thrice-daily tobramycin but more resistance development? J. Antimicrob. Chemother . 58 , 822 – 829 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 30. V. Master , et al. , Efficacy of once-daily tobramycin monotherapy for acute pulmonary exacerbations of cystic fibrosis: a preliminary study . Pediatr. Pulmonol . 31 , 367 – 376 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 31. M. LeBel , et al. , Pharmacokinetics and pharmacodynamics of ciprofloxacin in cystic fibrosis patients . Antimicrob. Agents Chemother . 30 , 260 – 266 ( 1986 ). OpenUrl Abstract / FREE Full Text 32. D. N. Fish , A. T. Chow , The clinical pharmacokinetics of levofloxacin . Clin. Pharmacokinet . 32 , 101 – 119 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 33. ↵ E. B. Wilms , D. J. Touw , H. G. M. Heijerman , Pharmacokinetics of azithromycin in plasma, blood, polymorphonuclear neutrophils and sputum during long-term therapy in patients with cystic fibrosis . Ther. Drug Monit . 28 , 219 – 225 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 34. ↵ T. F. Byrd , C. R. Lyons , Preliminary characterization of a Mycobacterium abscessus mutant in human and murine models of infection . Infect. Immun . 67 , 4700 – 4707 ( 1999 ). OpenUrl Abstract / FREE Full Text 35. ↵ A. M. Cantin , D. Hartl , M. W. Konstan , J. F. Chmiel , Inflammation in cystic fibrosis lung disease: Pathogenesis and therapy . J. Cyst. Fibros. Off. J. Eur. Cyst. Fibros. Soc . 14 , 419 – 430 ( 2015 ). OpenUrl 36. ↵ K. H. Rohde , R. B. Abramovitch , D. G. Russell , Mycobacterium tuberculosis Invasion of Macrophages: Linking Bacterial Gene Expression to Environmental Cues . Cell Host Microbe 2 , 352 – 364 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 37. ↵ V. Dubois , et al. , Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages . PLoS Pathog . 15 , e1008069 ( 2019 ). OpenUrl CrossRef PubMed 38. ↵ J. C. Schupp , et al. , Single-Cell Transcriptional Archetypes of Airway Inflammation in Cystic Fibrosis . Am. J. Respir. Crit. Care Med . 202 , 1419 – 1429 ( 2020 ). OpenUrl CrossRef PubMed 39. K. B. Hisert , W. C. Liles , A. M. Manicone , A Flow Cytometric Method for Isolating Cystic Fibrosis Airway Macrophages from Expectorated Sputum . Am. J. Respir. Cell Mol. Biol . 61 , 42 – 50 ( 2019 ). OpenUrl PubMed 40. ↵ Z. Prasla , R. L. Sutliff , R. T. Sadikot , Macrophage Signaling Pathways in Pulmonary Nontuberculous Mycobacteria Infections . Am. J. Respir. Cell Mol. Biol . 63 , 144 – 151 ( 2020 ). OpenUrl PubMed 41. ↵ R. Gupta , K. H. Rohde , Implementation of a mycobacterial CRISPRi platform in Mycobacterium abscessus and demonstration of the essentiality of ftsZMab . Tuberc. Edinb. Scotl . 138 , 102292 ( 2023 ). OpenUrl 42. ↵ A. Louie , W. Liu , S. Fikes , D. Brown , G. L. Drusano , Impact of Meropenem in Combination with Tobramycin in a Murine Model of Pseudomonas aeruginosa Pneumonia . Antimicrob. Agents Chemother . 57 , 2788 – 2792 ( 2013 ). OpenUrl Abstract / FREE Full Text 43. ↵ A. Rominski , et al. , Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes . J. Antimicrob. Chemother . 72 , 2191 – 2200 ( 2017 ). OpenUrl CrossRef PubMed 44. ↵ C. Manca , S. Paul , C. E. Barry , V. H. Freedman , G. Kaplan , Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro . Infect. Immun . 67 , 74 – 79 ( 1999 ). OpenUrl Abstract / FREE Full Text 45. ↵ D. Kumar , K. Palaniyandi , V. K. Challu , P. Kumar , S. Narayanan , PknE, a serine/threonine protein kinase from Mycobacterium tuberculosis has a role in adaptive responses . Arch. Microbiol . 195 , 75 – 80 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 46. ↵ F. Ripoll , et al. , Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae . BMC Genomics 8 , 114 ( 2007 ). OpenUrl CrossRef PubMed 47. ↵ S. Burbaud , et al. , Trehalose Polyphleates Are Produced by a Glycolipid Biosynthetic Pathway Conserved across Phylogenetically Distant Mycobacteria . Cell Chem. Biol . 23 , 278 – 289 ( 2016 ). OpenUrl PubMed 48. ↵ A. Pawlik , et al. , Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent mycobacterium abscessus . Mol. Microbiol . 90 , 612 – 629 ( 2013 ). OpenUrl CrossRef PubMed 49. ↵ K. Rüger , et al. , Characterization of Rough and Smooth Morphotypes of Mycobacterium abscessus Isolates from Clinical Specimens . J. Clin. Microbiol . 52 , 244 – 250 ( 2014 ). OpenUrl Abstract / FREE Full Text 50. ↵ A.-L. Roux , et al. , Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants . Cell. Microbiol . 13 , 692 – 704 ( 2011 ). OpenUrl CrossRef PubMed 51. ↵ J.-H. Lee , E.-J. Lee , J.-H. Roe , uORF-mediated riboregulation controls transcription of whiB7/wblC antibiotic resistance gene . Mol. Microbiol . 117 , 179 – 192 ( 2022 ). OpenUrl CrossRef PubMed 52. ↵ C. Larsson , et al. , Gene expression of Mycobacterium tuberculosis putative transcription factors whiB1-7 in redox environments . PloS One 7 , e37516 ( 2012 ). OpenUrl CrossRef PubMed 53. ↵ S. Homolka , S. Niemann , D. G. Russell , K. H. Rohde , Functional Genetic Diversity among Mycobacterium tuberculosis Complex Clinical Isolates: Delineation of Conserved Core and Lineage-Specific Transcriptomes during Intracellular Survival . PLoS Pathog . 6 , e1000988 ( 2010 ). OpenUrl CrossRef PubMed 54. ↵ K. R. Hurst-Hess , P. Rudra , P. Ghosh , Ribosome Protection as a Mechanism of Lincosamide Resistance in Mycobacterium abscessus . Antimicrob. Agents Chemother . 65 , e0118421 ( 2021 ). OpenUrl CrossRef PubMed 55. ↵ N. C. Poulton , et al. , Beyond antibiotic resistance: the whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria . Cell Chem. Biol . 31 , 669 - 682 .e7 ( 2024 ). OpenUrl PubMed 56. ↵ G. Baniulyte , J. T. Wade , A bacterial regulatory uORF senses multiple classes of ribosome-targeting antibiotics . eLife 13 , RP101217 ( 2025 ). OpenUrl CrossRef PubMed 57. ↵ R. P. Morris , et al. , Ancestral antibiotic resistance in Mycobacterium tuberculosis . Proc. Natl. Acad. Sci . 102 , 12200 – 12205 ( 2005 ). OpenUrl Abstract / FREE Full Text 58. ↵ J.-H. Lee , et al. , The WblC/WhiB7 Transcription Factor Controls Intrinsic Resistance to Translation-Targeting Antibiotics by Altering Ribosome Composition . mBio 11 , e00625 – 20 ( 2020 ). OpenUrl PubMed 59. ↵ V. Delafont , et al. , First Evidence of Amoebae–Mycobacteria Association in Drinking Water Network . Environ. Sci. Technol . 48 , 11872 – 11882 ( 2014 ). OpenUrl CrossRef PubMed 60. ↵ M. S. Siegrist , et al. , Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition . Proc. Natl. Acad. Sci. U. S. A . 106 , 18792 – 18797 ( 2009 ). OpenUrl Abstract / FREE Full Text 61. ↵ R. M. Wells , et al. , Discovery of a Siderophore Export System Essential for Virulence of Mycobacterium tuberculosis . PLOS Pathog . 9 , e1003120 ( 2013 ). OpenUrl CrossRef PubMed 62. ↵ D. P. Nichols , et al. , Impact of azithromycin on the clinical and antimicrobial effectiveness of tobramycin in the treatment of cystic fibrosis . J. Cyst. Fibros. Off. J. Eur. Cyst. Fibros. Soc . 16 , 358 – 366 ( 2017 ). OpenUrl 63. ↵ R. N. Brogden , R. M. Pinder , P. R. Sawyer , T. M. Speight , G. S. Avery , Tobramycin: a review of its antibacterial and pharmacokinetic properties and therapeutic use . Drugs 12 , 166 – 200 ( 1976 ). OpenUrl PubMed 64. ↵ B. M. Peters , M. A. Jabra-Rizk , G. A. O’May , J. W. Costerton , M. E. Shirtliff , Polymicrobial Interactions: Impact on Pathogenesis and Human Disease . Clin. Microbiol. Rev . 25 , 193 – 213 ( 2012 ). OpenUrl Abstract / FREE Full Text 65. ↵ D. G. Gibson , et al. , Enzymatic assembly of DNA molecules up to several hundred kilobases . Nat. Methods 6 , 343 – 345 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 66. ↵ A. E. Ochoa , et al. , Dectin-1-Independent Macrophage Phagocytosis of Mycobacterium abscessus . Int. J. Mol. Sci . 24 , 11062 ( 2023 ). OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted October 01, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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