Primary tuberculous mycobacterial granulomas provide a niche for superinfecting Mycobacterium abscessus

Primary tuberculous mycobacterial granulomas provide a niche for superinfecting Mycobacterium abscessus | 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 Primary tuberculous mycobacterial granulomas provide a niche for superinfecting Mycobacterium abscessus Denise Wee , Manitosh Pandey , Yao Chen , Paolo A Lorenzini , Eve WL Chow , Yue Wang , Amit Singhal , View ORCID Profile Stefan H Oehlers doi: https://doi.org/10.1101/2025.05.05.652332 Denise Wee 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Manitosh Pandey 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yao Chen 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paolo A Lorenzini 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eve WL Chow 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yue Wang 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amit Singhal 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stefan H Oehlers 1 * * * , Singapore, Singapore Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefan H Oehlers For correspondence: stefan_oehlers{at}idlabs.a-star.edu.sg Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Prior and concurrent tuberculosis infection are among the most important susceptibility factors for nontuberculous mycobacterial infection in Asia. Here we model this process in zebrafish with a primary Mycobacterium marinum infection followed by a secondary M. abscessus infection. We demonstrate preferential growth of secondary M. abscessus infection inside primary M. marinum granulomas. Granuloma-resident secondary M. abscessus is protected from macrophage-mediated immune control and antibiotic therapy. Successful colonization is driven by expansion of M. abscessus feeding on caseum produced by the primary M. marinum ESX-1 virulence program in a nutritionally separate niche from M. marinum . Our data suggest tuberculous granulomas may provide a long-lasting niche for the growth of the opportunistic pathogen Mycobacterium abscessus . Introduction Infections by Mycobacterium abscessus are an emerging global health problem, and the rising rates of infection by similar non-tuberculous mycobacteria (NTM) threaten to undermine the progress made in tuberculosis (TB) control in many countries ( 1 ). Post-TB lung disease is an important but understudied chronic respiratory disease that contributes to mortality and morbidity, including raising the risk of subsequent NTM and TB infections in successfully treated TB patients ( 2 , 3 ). In countries with endemic TB, NTM-TB coinfection accounts for up to 10% of the NTM patient population (Shandong Province, China) and 3% of the TB patient population (Taiwan) ( 4 , 5 ). This association contrasts with the known protective effect of homologous M. tuberculosis infection against M. tuberculosis superinfection and the heterologous protection conferred by the M. bovis BCG vaccine against subsequent mycobacterial infections. The granuloma is the histological hallmark of TB infection and serves as the primary immunological interface between pathogenic mycobacteria and the host immune system. These long-lasting structures persist despite immune and antibiotic containment of M. tuberculosis , with varying estimates of their role in maintaining a reservoir for subclinical TB. In the case of calcified granulomas, they can remain lifelong ( 6 , 7 ). Evidence that granulomas can harbor superinfecting tuberculous mycobacteria spans both experimental and clinical datasets, including key milestone studies demonstrating homing of superinfecting Mycobacterium marinum into zebrafish and frog granulomas, M. tuberculosis and M. bovis into granulomas in mice and humans, respectively, and a high rate of cutaneous granulomas containing multiple mycobacterial species ( 8 - 12 ). Intriguingly, NTM have been detected in IS 6110 positive TB granuloma biopsy tissues, demonstrating infiltration of NTM into M. tuberculosis granulomas ( 13 ). However, the role of tuberculous granulomas in driving susceptibility to NTM infection has yet to be examined. We hypothesized organized caseated granulomas may act as a protective niche for superinfecting mycobacteria, shielding them from cross-protective immune control and providing a readily available source of nutrients for rapid NTM growth. Here, we demonstrate that colonization with the natural mycobacterial pathogen M. marinum increases the susceptibility of zebrafish to M. abscessus . This susceptibility can be directly attributed to macrophage-mediated, ESX1-dependent carriage of M. abscessus into pre-existing caseous granulomas, where it is shielded from containment by the host immune system. We find that M. abscessus adapts to growth in the necrotic granuloma, enabling occupancy of a distinct nutritional niche. Results Primary M. marinum infection predisposes adult zebrafish to secondary M. abscessus infection Previous studies using the frog- M. marinum and macaque- M. tuberculosis models have demonstrated protection against homologous superinfection ( 8, 14, 15 ). The adult zebrafish platform provides a unique intersection of tuberculous granuloma formation when challenged with M. marinum and an immunologically-intact permissive host for M. abscessus ( 16 , 17 ). We performed a sequential infection experiment in adult zebrafish to determine if primary tuberculous infection promotes resistance or susceptibility to secondary M. abscessus infection. A high-dose secondary infection with of 10 6 CFU of M. abscessus caused unexpected mortality in animals with a primary M. marinum infection ( Figure 1A ). Animals infected with a primary M. marinum infection and a lower-dose secondary infection with 5x10 5 CFU of M. abscessus exhibited a higher M. abscessus burden than naïve animals ( Figure 1B ). Download figure Open in new tab Figure 1. Pre-existing M. marinum infection worsens secondary M. abscessus infection but protects from secondary M. marinum infection A.Survival curve of adult zebrafish infected with combinations of primary M. marinum (Mm) and secondary M. abscessus (Mabs) at 2 weeks post primary infection. Data is from a single experiment with 45 animals. Statistical analysis reported is a Log-rank test comparing the PBS/Mabs group with the Mm/Mabs group. B.Quantification of secondary M. abscessus burden at 2 weeks post secondary infection in naïve and primary M. marinum- infected adult zebrafish infected at 2 weeks post primary infection. C.Quantification of secondary M. marinum engraftment at 2 weeks post secondary infection in naïve and primary M. marinum- infected adult zebrafish infected at 2 weeks post primary infection. Data is pooled from two biological replicates. Statistical analysis reported is a Fisher’s Exact Test. D.Quantification of secondary M. marinum burden at 2 weeks post secondary infection in naïve and primary M. marinum- infected adult zebrafish infected at 2 weeks post primary infection. E.Representative image of multilobed granuloma in 2 week post secondary infection adult zebrafish infected with secondary M. abscessus at 2 weeks post primary M. marinum infection. Scale bar indicates 10 μm. Mammalian models of M. tuberculosis sequential infections have demonstrated significant protection from infection ( 8 , 14 , 15 , 18 ). We performed similar homotypic experiments with sequential M. marinum infection to determine if our M. abscessus phenotype was an artifact of an exhausted immune system. We found that primary M. marinum infection protected against a homotypic secondary M. marinum infection both by preventing the engraftment of a low-dose M. marinum infection ( Figure 1C ) and reducing the recovered M. marinum burden from a higher dose infection ( Figure 1D ). Cryosectioning of sequentially infected fish was performed to determine the spatial localization of superinfecting M. abscessus relative to primary M. marinum granulomas. Consistent with the homologous superinfection literature ( 8 ), M. marinum granulomas were found to be colonized by secondary M. abscessus ( Figure 1E ). Colonization of primary M. marinum granulomas accelerates the growth of superinfecting M. abscessus While the adult zebrafish experimental system facilitates the study of classic hypoxic caseous necrotic granulomas ( 19 - 21 ), it does not readily facilitate real-time imaging of host-microbe interactions, as can be achieved with zebrafish embryos. Furthermore, sequential intraperitoneal injections likely bath the primary granuloma in secondary M. abscessus facilitating ready uptake. For these reasons we switched to the use of the zebrafish embryo- M. marinum / M. abscessus infection system to study the mechanisms of primary M. marinum infection-mediated susceptibility to secondary M. abscessus infection. We established an experimental sequential infection system in zebrafish embryos by performing the primary injection into the neural tube at the previously characterized “trunk” injection site ( 21 ), then performing secondary injection into the circulation at 3 days post primary infection (dppi) ( Figure 2A ). Timelapse imaging revealed uptake of M. abscessus from the circulation into established M. marinum granulomas ( Figure 2B and Supplementary Video 1). Download figure Open in new tab Figure 2. Ingress into M. marinum granulomas protects M. abscessus from innate immunity A.Schematic illustrating embryo superinfection experimental set up. Abbreviation: Days post secondary infection (dpsi). B.Still images extracted 900 minutes apart from Supplementary Video 1. Site of M. abscessus (purple) injection is indicated within Tg(kdrl:egfp)- positive vasculature (green) below primary M. marinum granuloma (yellow). Scale bars indicate 100 μm. C. Quantification of secondary M. abscessus burden in 3 dpsi embryos infected with primary M. marinum . D. Quantification of secondary M. abscessus burden in 3 dpsi embryos infected with primary M. marinum grouped by quartile of M. abscessus found within M. marinum granulomas. E. Quantification of secondary M. abscessus burden in 3 dpsi embryos infected with primary M. marinum, M. abscessus was delivered by co-injection with clodronate microsomes. F. Quantification of fold change in M. abscessus burden during treatment with 10 μM clarithromycin from 2 dpsi to 4 dpsi. Consistent with our adult zebrafish data, fluorescent pixel count revealed an increased M. abscessus burden in embryos 3 days post secondary infection (dpsi), when sequentially infected at 3 dppi with M. marinum , compared to M. abscessus infection of naïve embryos ( Figure 2C ). Furthermore, analysis of M. abscessus burden in sequentially infected embryos grouped by quartile of colonization revealed a higher overall M. abscessus burden in embryos with a high rate of colonization ( Figure 2D ). Together, these data suggested colonization of primary granulomas accelerated secondary M. abscessus growth. To examine the contribution of prior M. marinum granuloma formation relative to more general M. marinum -mediated immune subversion, we compared M. abscessus growth following mono-and co-injection with M. marinum . Unlike our sequential injection infection data, co-injection of M. marinum did not affect the growth of M. abscessus in zebrafish embryos, demonstrating the importance of granuloma formation in driving susceptibility to sequential M. abscessus infection ( Supplementary Figure 1A ). Interestingly, we found a contrasting antagonistic effect of M. abscessus infection on M. marinum growth, reminiscent of a trained immunity effect ( Supplementary Figure 1B ). Download figure Open in new tab Supplementary Figure 1. A. Quantification of M. abscessus burden in 4 days post infection embryos co-injected with M. abscessus and M. marinum . B. Quantification of M. marinum burden in 4 days post infection embryos co-injected with M. abscessus or M. smegmatis and M. marinum . C. Quantification of M. smegmatis burden in 4 days post infection embryos co-injected with M. smegmatis and M. marinum . To determine if immune pressure restricts M. abscessus growth, resulting in preferential growth within primary M. marinum granulomas, we co-injected clodronate with secondary M. abscessus to deplete macrophages and dexamethasone immune suppression to alleviate immune pressure. Total M. abscessus burden increased with clodronate co-injection, driven entirely by an increase in the extra-granuloma compartment. This finding is consistent with our hypothesis that granulomas provide M. abscessus with a physical sanctuary from the host immune control ( Figure 2F ). Caseum increases mycobacterial antibiotic tolerance by excluding antibiotics and rewiring bacterial physiology ( 22 , 23 ). Specifically, stationary phase M. abscessus in caseum is markedly more resistant to frontline antibiotics such as bedaquiline, clarithromycin, imipenem, clofazimine, and moxifloxacin compared to actively growing broth cultures ( 23 ).. Clarithromycin, an example of a current front line antibiotic, was effective at restraining the growth of M. abscessus in naïve but not M. marinum infected animals when treatment as initiated after granuloma colonization ( Figure 2F ). Opportunistic pathogens passively colonize primary M. marinum granulomas in zebrafish embryos We examined the role of secondary mycobacterial infection in directing the colonization of primary granulomas by comparing the M. abscessus parental “low virulence” smooth colony morphotype to the more virulent rough colony morphotype used in our previous experiments. There was no difference in colonization by either colony morphotype compared to each other or relative to the lower rate of colonization seen in M. marinum superinfection ( Supplementary Figure 2A ). Interestingly, the rate of granuloma colonization was similar between WT and ΔESX1 M. marinum secondary infections, suggesting the differentiating factor between M. marinum and M. abscessus granuloma colonization potential is species-rather than virulence factor-specific. Furthermore, we did not observe a growth advantage for secondary M. marinum infection compared to infection into naïve embryos ( Figure 3A ). Download figure Open in new tab Supplementary Figure 2. A.Quantification of M . marinum WT, ΔESX1 M. marinum , R and S M. abscessus colonization of primary M. marinum granulomas at 3 dpsi. B.Representative image of secondary infection UPEC distribution in zebrafish embryos with primary M. marinum infection at 3 dpsi. Arrows indicate locations of UPEC fluorescent signals, scale bar represents 100 μm. C.Representative image of secondary infection C. auris distribution in a zebrafish embryo with primary M. marinum infection at 3 dpsi. Arrows indicate locations of C. auris fluorescent signals within M. marinum granulomas, scale bar represents 100 μm. Download figure Open in new tab Supplementary Figure 3. A.Still image extracted from Supplementary Video 1. White box demarcates area of local Tg(kdrl:egfp)- positive vasculature (green) around primary M. marinum granuloma (yellow). Extravasating M. abscessus (purple) are indicated by white arrow for local vasculature or green arrow for distal vasculature. B.Quantification of M. abscessus colonization of primary M. marinum granulomas at 3 dpsi treated with pazopanib. Download figure Open in new tab Figure 3. Colonization of primary granulomas provides a growth advantage to opportunistic pathogens A.Quantification of secondary M. marinum burden in 3 dpsi embryos infected with primary M. marinum . B.Quantification of secondary M. smegmatis burden in 3 dpsi embryos infected with primary M. marinum . C. Quantification of secondary M. smegmatis burden in 3 dpsi embryos infected with primary M. marinum grouped by quartile of M. smegmatis found within M. marinum granulomas. D. Images extracted from Supplementary Video 2 tracking ingress of PFA-fixed M. abscessus into a primary M. marinum granuloma from 260-276 minutes post tracking and residency of PFA-fixed M. abscessus inside granuloma for over duration of video. Arrow indicates location of PFA-fixed M. abscessus fluorescent signal, scale bar represents 100 μm. E. Quantification of secondary C. auris burden in 3 dpsi embryos infected with primary M. marinum . We next performed super infection with fluorescent Mycobacterium smegmatis , typically considered avirulent. Similar to M. abscessus , superinfecting M. smegmatis had a significant growth advantage compared to infection into age-matched naïve embryos ( Figure 3B ), and total M. smegmatis burden was higher in the embryos in the highest quartile of granuloma colonization compared to the other three quartiles ( Figure 3C ). In co-injection studies, we observed a similar lack of protective effect of M. marinum on M. smegmatis burden in the absence of pre-existing granulomas and an antagonistic effect of M. smegmatis on M. marinum burden ( Supplementary Figure 1B and 1C ). We also injected embryos with paraformaldehyde-killed M. abscessus and observed colonization of granulomas, suggesting the delivery of NTM to primary tuberculous granulomas is a passive feature of NTM species ( Figure 3D , Supplementary Video 2). This led us to test the bounds of the colonization phenotype by non-mycobacterial secondary infections. First, we used uropathogenic Escherichia coli (UPEC) as the secondary infection. This typically acute infection is capable of persisting in zebrafish embryos but we did not observe cross-genus interaction, consistent with data from a natural pathogen Salmonella experiment suggesting a different niche from NTMs ( 8 ) ( Supplementary Figure 2B ). Next, as fungal infections are a common consequence of post-TB lung disease and opportunistic yeast have the ability to survive within macrophages ( 24 ), we tested the ability of the Candida auris to colonize granulomas. We observed C. auris colonization of primary M. marinum granulomas and a growth advantage for C. auris in animals with an existing M. marinum infection ( Figure 3E , Supplementary Figure 2C ). Together, these data suggest that granuloma colonization depends on the ability of secondary infections to be recognized by macrophages, survive intracellular killing mechanisms, but avoid overstimulating the macrophage to stop and form aggregates. ESX-1 directed maturation of primary M. marinum granulomas permits growth of superinfecting M. abscessus The reproduction of the caseating granulomas seen in TB is a key advantage of the zebrafish- M. marinum infection model, and granulomas formed following trunk injection of M. marinum into embryos undergo stereotypical progression from cellular to necrotic granuloma from 3-5 dppi ( 17 , 21 ). Reducing the primary inoculum and comparing the start of secondary infection from 3 to 4 dppi revealed a granuloma maturation-dependent increase in primary granuloma colonization by superinfecting M. abscessus ( Figure 4A ). Download figure Open in new tab Figure 4. Secondary M. abscessus growth in primary granulomas is proportional to M. marinum ESX1-dependent granuloma maturation A.Quantification of M. abscessus colonization of primary M. marinum granulomas at 3 dpsi when infected at 3 or 4 dppi. B.Quantification of M. abscessus colonization of primary M. marinum granulomas at 3 dpsi treated with isoniazid. C.Quantification of animals with at least one M. abscessus colonization event of primary WT and ΔESX1 M. marinum granulomas at 1 dpsi. Data is pooled from two biological replicates. Statistical analysis reported is a Fisher’s Exact Test. D.Quantification of macrophage recruitment to WT vs ΔESX1 M. marinum granulomas across 4-22 hpsi. E.Quantification of M. abscessus colonization of primary WT and ΔESX1 M. marinum granulomas at 3 dpsi. Conversely, arresting the maturation of primary granulomas with M. marinum- bacteriostatic isoniazid treatment at the time of secondary infection reduced the rate of primary granuloma colonization by superinfecting M. abscessus ( Figure 4B ). The ESX1 type VII secretion system is the most important pro-granulomatous virulence factor in M. marinum and M. tuberculosis responsible for directly subverting phagocyte function, and driving necrosis and the recruitment of permissive macrophages to feed the granuloma ( 25 ). We found impaired colonization of primary ΔESX1 M. marinum granulomas compared to primary WT M. marinum granulomas early in infection even when similar burdens are reached with a higher initial inoculum ( Figure 4C ). Interestingly, we observed similar rate of macrophage recruitment to 5 dppi primary ΔESX1 M. marinum granulomas which contrasts to the reduced rate of macrophage recruitment early in ΔESX1 M. marinum infection compared to WT M. marinum infection ( 26 ), following secondary M. abscessus infection suggesting reduced macrophage migration is not the only factor accounting for reduced colonization of primary ΔESX1 M. marinum granulomas by secondary M. abscessus ( Figure 4D ). The small difference in initial colonization rate at 1 dpsi was compounded during later stages of M. abscessus superinfection with primary ΔESX1 M. marinum granulomas carrying only a very small minority of M. abscessus by 3 dpsi suggesting a requirement for ESX1-mediated necrosis in creating primary granulomas that attract and then further support the growth of superinfecting M. abscessus ( Figure 4E ) We next investigated the mode of M. abscessus ingress into primary M. marinum granulomas by live imaging. As our sequential infection system introduced M. abscessus into the circulation, we first tracked the vascular source of M. abscessus ingression into M. marinum granulomas in the Tg(kdrl:egfp) s843 line, where vascular endothelial cells are labelled by EGFP expression ( 27 ). We observed extravasation of M. abscessus from blood vessels adjacent and distal to the granuloma followed by directional migration and ingress (Supplementary Video 1). Ingress events originating from adjacent intersegmental and dorsal longitudinal anastomotic vessels were classed as local vasculature and all other vessels were classed as distal vasculature ( Supplementary Figure 2A ). Quantification of 114 extravasation events leading to M. abscessus ingress in 11animals revealed a mix of 60 local and 54 distal extravasation events, demonstrating the possibility of both hematogenous spread and macrophage carriage. We have previously used vascular normalization as a host-directed therapy to reduce the extravasation of neutrophils around M. marinum granulomas. Here, we hypothesized that preventing vascular pathology would reduce the extravasation of M. abscessus towards granulomas in our superinfection model ( 28 ). We treated M. marinum -infected embryos with pazopanib, an FDA-approved VEGFR inhibitor with host-directed activity against M. marinum infection-induced vascular pathologies ( 21 ), starting one day prior to secondary M. abscessus infection and continuing until 3 dpsi. Treatment with pazopanib reduced the rate of primary granuloma colonization by superinfecting M. abscessus when assayed 3 dpsi ( Supplementary Figure 2B ). Previous live imaging and histological studies have implicated ingress of infected macrophages as the primary mode of M. abscessus and M. marinum dissemination in zebrafish. Our extravasation imaging demonstrated the direct migration of M. abscessus into granulomas, suggesting macrophage carriage ( 29 - 32 ). To confirm this hypothesis, we performed live imaging of granuloma ingress in the Tg(acod1:tdtomato) xt40 line, where macrophages are labelled with TdTomato (Supplementary Video 3), and carriage of M. abscessus by GFP positive and negative cells with similar DIC morphologies in the alternative TgBAC(mpeg1 . 1:egfp) vcc7 where a subset of macrophages are labelled with EGFP (Supplementary Video 4) ( 33 ). Neutrophils are abundant immune cells that play a crucial role in controlling M. abscessus and M. marinum in zebrafish embryos ( 32, 34, 35 ). To determine the potential contribution of neutrophil carriage, we performed live imaging of granuloma ingress in the Tg(lyzc:egfp) nz117 line, where neutrophils are labelled with EGFP ( 36 ). As expected, we observed neutrophil recruitment to primary granulomas and interactions with M. abscessus . However, GFP-positive neutrophil carriage of M. abscessus was rare (Supplementary Video 5). Quantification of 75 M. abscessus ingress events in 15 animals revealed only 6 events, demonstrating a low utilization of neutrophils for carriage of M. abscessus . M. abscessus adaptation to primary granuloma residency To understand how M. abscessus takes advantage of the necrotic granuloma niche we first examined the transcriptome of M. abscessus grown in complete 7H9 media with M. abscessus in the zebrafish embryo infection model. Examination of known stress response gene families: Esx-3, mycobactins, oxidative stress, and cell wall remodeling confirmed the zebrafish embryo infection model induces the expression of a virulence-associated M. abscessus gene program ( Figure 5A ). Further comparison of the M. abscessus lipid metabolism gene expression program revealed an upregulation of lipid metabolism during adaptation to infection suggesting M. abscessus may utilize the lipid-rich caseum to fuel accelerated growth during superinfection ( Figure 5B ). Download figure Open in new tab Figure 5. M. abscessus occupies a separate niche to M. marinum within the necrotic granuloma A.Heat map of M. abscessus stress gene expression derived from RNAseq of M. abscessus at 6 dpi in zebrafish embryos compared to baseline in vitro 7H9 broth cultured M. abscessus . B.Heat map of in vivo M. abscessus lipid metabolism gene expression compared to in vitro 7H9 broth cultured M. abscessus . C.Quantification of M. abscessus growth in 7H9 media supplemented with carbon sources. D.Quantification of M. abscessus and M. marinum growth in 7H9 media diluted to 10% with PBS and supplemented with in vitro caseum. M. marinum culture was performed for 4 days prior to Day 00 when M. abscessus is added to the co-culture conditions. E.Quantification of M. marinum growth in 163 individual embryos from 0 to 3 dpsi with M. abscessus . R squared value calculated by linear regression, slope equation Y = -0.07066*X + 32.79, P=0.0481. To test the hypothesis that M. abscessus could adapt to the caseum in the necrotic core as a nutrient source, we next switched to an in vitro system. We analyzed the growth of M. abscessus in diluted 7H9 media supplemented with OADC as a positive control, RAW 264.7 cell-derived in vitro caseum ( 37 ), and the individual lipid substrates cholesterol and stearic acid. Growth curves demonstrated that M. abscessus is able to grow rapidly with a range of lipid substrates, specifically being able to utilize in vitro caseum as a substrate ( Figure 5C ). To model our in vivo sequential infections, we first cultured M. marinum in in vitro caseum, facilitating pre-conditioning of the caseum. After 4 days when M. marinum growth had plateaued, we introduced M. abscessus to the culture system. The addition of M. abscessus to the established M. marinum culture resulted in lower overall growth of M. abscessus compared to its monoculture in sterile caseum consistent with the depletion of some nutrients by M. marinum pre-conditioning ( Figure 5D ). This growth defect was ameliorated when new caseum was added to the established M. marinum culture system, suggesting that the optimal M. abscessus growth conditions overlap with nutrients utilized by M. marinum ( Figure 5D ). We observed similar stability of M. marinum CFU levels across all experiments, regardless of whether M. abscessus was added to the culture, suggesting a neutral interpretation of interaction between the two species by M. marinum . To study this interaction in vivo , we analyzed the growth of M. marinum in individual embryos with and without sequential M. abscessus infection. Analysis of embryos infected with only M. marinum revealed a wide range (11-101x) of M. marinum growth across days 3-6 post primary infection, equivalent to 0-3 dpsi in the sequential infection experiment, and all sequential infection values fell within this range ( Figure 5E ). Further analysis of M. marinum fold change from 0-3 dpsi infection demonstrated no correlation between M. marinum growth and the rate of M. abscessus colonization of primary M. marinum granulomas ( Figure 5E ). Together, these data demonstrate that M. abscessus occupies a unique niche in necrotic granulomas that is not at the expense of M. marinum . Discussion In Singapore, a country that has effectively eliminated local transmission but retains the epidemiological “scar” of historically high prevalence of TB, M. abscessus ais one of the most prevalent NTM infections, with prior TB being the most common predisposing factor ( 38 ). Evidence from mammalian models suggests that increasing TB severity drives a shift from leukocyte-mediated heterologous protection against secondary mycobacterial infection afforded by a contained primary infection toward systemic reprogramming of hematopoiesis, leading to increased susceptibility to secondary mycobacterial infection following disseminated disease ( 14 , 15 , 39 ). These observations suggest that compromised leukocyte immunity could drive increased NTM susceptibility in treated TB patients Tuberculous granulomas are a predisposing factor for opportunistic NTM infection Our data provide compelling evidence that granulomas formed during TB infection provide a niche for the growth of M. abscessus in otherwise immunologically intact resistant hosts. This finding has significant implications for explaining the increased risk of NTM infection in patients with prior or ongoing pulmonary TB. Post TB lung disease encompasses a wide range of histological lung remodeling, leading to a loss of respiratory function, which is hypothesized to impair the physical clearance of NTM following inoculation from environmental reservoirs ( 3 ). Although our models are unable to replicate the physical clearance of inhaled NTM, they faithfully produce TB-like granulomas with caseous necrosis. We posit that unresolved TB granulomas should be considered a susceptibility factor for infection by opportunistic pathogens. The rapid growth of M. abscessus and M. marinum in co-culture experiments are most likely due to the relative growth rates of the organisms rather than a true virulence advantage. Dual-species granulomas are usually bounded by M. marinum or exhibit a mixed fluorescent signal, suggesting M. abscessus does not expand these histopathological structures in our assays. Furthermore, naïve embryos were largely able to control M. abscessus when infected at 5 dpf but had early granuloma formation when infected with M. marinum , consistent with M. marinum having a much higher relative growth potential per unit of inoculum across zebrafish infection assays ( 16 , 21 ). Further experiments in appropriate model systems will be required to determine the effect of primary tuberculous infection clearance from granulomas on the survival of super infecting NTM who can no longer “hide” behind ESX1 immune subversion driven by the primary species. Our findings highlight the importance of efforts to find preventative and interventional treatments for TB pathologies, including granuloma resolution, as the risks of post-infection sequalae will persist long after TB transmission is eradicated. Many NTM cases are initially diagnosed as TB, resulting in empirical anti-TB therapy to which M. abscessus is insensitive. These cases can be variably defined as recent, prior, or concomitant TB with M. abscessus infection, depending on the level of specificity in the initial diagnosis. In cases where there is evidence of M. tuberculosis at the first diagnosis, our findings suggest that these patients may be at risk of severe M. abscessus infection phenotypes, such as fibrocavitary disease compared to the bronchial nodular form. When does primary TB increase resistance to secondary mycobacterial infection? While there is mixed evidence from preclinical infection models for and against a protective effect of primary M. tuberculosis infection on reinfection, epidemiological evidence clearly demonstrates that prior M. tuberculosis or NTM infection is a risk factor for future tuberculous and non-tuberculous mycobacterial infection ( 38 , 40 , 41 ). While some of this risk can be attributed to patients remaining in physical environments with high mycobacterial exposure levels, countries such as Singapore, where TB transmission was effectively eliminated within a generation, manifest a long, lingering tail of post-TB susceptibility to NTM infection at a population level ( 38 , 42 ). The severity of primary infection in preclinical models appears to be the decisive factor for determining if primary mycobacterial infection is protective against future challenge. Naturally controlled murine and non-human primate M. tuberculosis infections clearly protect against sequential M. tuberculosis infection ( 15 , 18 ). Poor initial control of infection by highly virulent strains, systemic administration of live M. tuberculosis ( 39 , 43 ), or the converse clearing of infection ( 14 , 15 ) can compromise immunological control of the second infection by a range of mechanisms from the hematopoietic stem cell through to the pulmonary microenvironment. This suggests a Goldilocks principle whereby ongoing inflammatory and antigenic stimulation is the primary driver of protective anti-mycobacterial immune responses. It will be important to adapt these models to investigate the relative contributions of hematopoietic reprogramming and pulmonary adaptive immunity to the restriction of NTM in models that recapitulate human-like granulomas. Methods Zebrafish Zebrafish experiments were carried out under the A*STAR IACUC approvals 211667 and 221694. Adults were housed under 14 hour light / 10 hour dark cycles in 28°C recirculating systems. Embryos were produced by natural spawning and raised in E3 media supplemented with PTU at 28°C. Growth of microbes M. abscessus, M. marinum , and M. smegmatis were cultured in 7H9 or on 7H10 supplemented with OADC and hygromycin to select for pTEC fluorescent protein plasmids (L. Ramakrishnan, plasmids are available through Addgene https://www.addgene.org/Lalita_Ramakrishnan/ ). C. auris expressing mCherry was prepared as previously described and is available upon request from EWLC ( 44 ). Uropathogenic E. coli was prepared as previously described ( 45 ). PFA killing of M. abscessus was carried out by resuspending a midlog culture of M. abscessus in 4% PFA in PBS for 30 minutes at room temperature. Fixed bacteria were rinsed prior to injection. Validation of killing was performed by plating on 7H10 supplemented with OADC and incubation at 37°C for 7 days. Growth curves of M. abscessus and M. marinum were performed in 10% 7H9 media diluted with PBS at 30°C in a static incubator. In vitro caseum was produced as previously described ( 37 ). Adult zebrafish infections Adult zebrafish were injected with approximately 100 CFU M. marinum or 5×10 5 to 10 6 CFU of M. abscessus as indicated. Animals were housed under 14 hour light / 10 hour dark cycles in an isolated 28°C recirculating system and fed once daily with a nutritionally complete dry feed. Bacterial enumeration by CFU recovery was performed by bead beating individual adult zebrafish in a Tomy Micro Smash MS-100 and plating of homogenate on 7H10 for M. marinum or LB Agar for M. abscessus supplemented with hygromycin. Growth of M. marinum was carried out at 30°C for 7 days and growth of M. abscessus was carried out at 37°C for 5 days. Histology was carried out using a Leica CM1520 to cut 10 μm thick cryosections of PFA-fixed adults and counterstaining with DAPI. Fluorescent images of preserved bacterial fluorescence were acquired using a Nikon Ni-E upright microscope. Zebrafish embryo infections Zebrafish embryos were infected by microinjection with approximately 200 CFU M. marinum , 1000 CFU ∧1ESX1 M. marinum or M. abscessus . Primary infection was performed by injection into the neural tube above the yolk sac extension at 2 dpf and secondary infection was performed by intravascular injection into the dorsal aorta or caudal vein at 5 dpf/3 dpi, unless otherwise described. Clodronate microsomes were co-injected with secondary M. abscessus infection by intravascular injection into the dorsal aorta or caudal vein at 5 dpf/3 dpi. Clarithromycin (10 μg/ml final concentration), isoniazid (50 μM final concentration), pazopanib (500 nM final concentration) were added directly to embryo media. Microscopy of zebrafish embryos All microscopy was carried out stereomicroscopy of immobilized zebrafish embryos. Static imaging was performed on a Nikon SMZ25 stereoscope or an inverted Nikon Eclipse Ti2. Time lapse imaging was performed on inverted Thermofisher EVOS7000 or Olympus IX-83 microscopes. Image analysis was performed in ImageJ/FIJI with bacterial fluorescent pixel count (FPC)carried out as previously described ( 46 ). Percentage of M. abscessus in granulomas was calculated by the formula ( M. abscessus FPC overlap with M. marinum / total M. abscessus FPC) x 100. RNA sequencing of M. abscessus Mycobacterial RNA was harvested from 3 mid-log 7H9 cultures and 3 pools of trizol-pre lysed 7 dpi M. abscessus- infected zebrafish embryos by bead beating in Lysis buffer and further processed according to manufactures protocol (MN-NucleoSpin RNA kit). Total RNA was subjected to 150 bp paired end sequencing on an Illumina Novaseq 6000 (NovogeneAIT Genomics Singapore). Sequencing quality check was carried out by FASTQC. Alignment of read pairs to Mycobacteroides abscessus genome (ASM6918v1, GenBank) was performed by STAR. The raw read count matrix was generated by featureCounts. DESeq2 analysis was performed to identify differentially expressed genes (DEGs). DEGs were manually annotated to stress response and lipid metabolism pathways for display. Statistics All analyses of infection experiments were carried out with Graphpad Prism using T-tests for pairwise comparisons or ANOVA for multiple comparisons. All data are representative of at least 3 biological replicates unless otherwise stated in the captions. Error bars on graphs represent standard deviation. Supplementary Video 1 Time lapse imaging of magenta M. abscessus ingress into a yellow M. marinum granuloma in a 5 dppi Tg(kdrl:egfp) zebrafish larva with green blood vessels. Supplementary Video 2 Time lapse imaging of magenta PFA-fixed M. abscessus ingress into a green M. marinum granuloma in a 5 dppi zebrafish larva. Supplementary Video 3 Time lapse imaging of cyan M. abscessus ingress into a yellow M. marinum granuloma in a 5 dppi Tg(acod1:tdtomato) xt40 zebrafish larva with red macrophages. Supplementary Video 4 Time lapse DIC imaging merged with fluorescent imaging of magenta M. abscessus being carried by green macrophages in a 5 dppi Tg(mpeg1 . 1:egfp) zebrafish larva with cyan M. marinum granulomas. Supplementary Video 5 Time lapse imaging of magenta M. abscessus ingress into a blue M. marinum granuloma in a 5 dppi Tg(lyzC:gfp) zebrafish larva with green neutrophils. Acknowledgements This study was funded by the Singapore Ministry of Health’s National Medical Research Council under its individual research grant scheme (OFIRG22jul-0081) to S.H.O. A*STAR IMCB Aquarium Platform for expert zebrafish husbandry. Dr Eloise Ma and the A*STAR Microscopy Platform for microscopy assistance. Dr J Muse Davis for helpful discussion of live imaging techniques. Professors Lalita Ramakrishnan and Paul Edelstein for discussion of co-infection and sequential infections. Members of A*STAR ID Labs and the SG BUG community for discussion. 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Share Primary tuberculous mycobacterial granulomas provide a niche for superinfecting Mycobacterium abscessus Denise Wee , Manitosh Pandey , Yao Chen , Paolo A Lorenzini , Eve WL Chow , Yue Wang , Amit Singhal , Stefan H Oehlers bioRxiv 2025.05.05.652332; doi: https://doi.org/10.1101/2025.05.05.652332 Share This Article: Copy Citation Tools Primary tuberculous mycobacterial granulomas provide a niche for superinfecting Mycobacterium abscessus Denise Wee , Manitosh Pandey , Yao Chen , Paolo A Lorenzini , Eve WL Chow , Yue Wang , Amit Singhal , Stefan H Oehlers bioRxiv 2025.05.05.652332; doi: https://doi.org/10.1101/2025.05.05.652332 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)