Competition with Pseudomonas aeruginosa induces Staphylococcus aureus in an antibiotic-tolerant viable but non culturable state

Competition with Pseudomonas aeruginosa induces Staphylococcus aureus in an antibiotic-tolerant viable but non culturable state | 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 Competition with Pseudomonas aeruginosa induces Staphylococcus aureus in an antibiotic-tolerant viable but non culturable state View ORCID Profile Urszula Łapińska , View ORCID Profile Nicolas Oswaldo Gomez , View ORCID Profile Gayathri Chandran , View ORCID Profile Thomas Tunstall , View ORCID Profile Sophia Zborowsky , View ORCID Profile Giulia Tolle , View ORCID Profile Anthony D. Verderosa , María García-Castillo , Paul A. O’Neill , View ORCID Profile Audrey Farbos , View ORCID Profile Aaron R. Jeffries , Andrew J. Young , View ORCID Profile Rafael Canton , View ORCID Profile Pierluigi Caboni , View ORCID Profile Mark A. T. Blaskovich , Adilia Warris , View ORCID Profile Krasimira Tsaneva-Atanasova , View ORCID Profile Stefano Pagliara , The ERADIAMR consortium doi: https://doi.org/10.1101/2025.04.30.651255 Urszula Łapińska 1 Living Systems Institute, University of Exeter , Exeter, Devon, EX4 4QD, UK 2 Biosciences, University of Exeter , Exeter, Devon, EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Urszula Łapińska For correspondence: s.pagliara{at}exeter.ac.uk u.lapinska{at}exeter.ac.uk Nicolas Oswaldo Gomez 3 Department of Molecular Bacteriology, Helmholtz Center for Infection Research , 38124 Braunschweig, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicolas Oswaldo Gomez Gayathri Chandran 1 Living Systems Institute, University of Exeter , Exeter, Devon, EX4 4QD, UK 4 MRC Centre for Medical Mycology, University of Exeter , Exeter, Devon, EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gayathri Chandran Thomas Tunstall 1 Living Systems Institute, University of Exeter , Exeter, Devon, EX4 4QD, UK 5 EPSRC Hub for Quantitative Modelling in Healthcare, University of Exeter , Exeter, EX4 4QJ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas Tunstall Sophia Zborowsky 1 Living Systems Institute, University of Exeter , Exeter, Devon, EX4 4QD, UK 2 Biosciences, University of Exeter , Exeter, Devon, EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sophia Zborowsky Giulia Tolle 6 Department of Life and Environmental Sciences, University of Cagliari , Cittadella Universitaria, 09042 Monserrato, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giulia Tolle Anthony D. Verderosa 7 Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland , St. Lucia, QLD, 4072, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anthony D. Verderosa María García-Castillo 8 Servicio de Microbiología, Hospital Universitario Ramón y Cajal and Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS) , Madrid, Spain 9 CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III , Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paul A. O’Neill 2 Biosciences, University of Exeter , Exeter, Devon, EX4 4QD, UK 10 Exeter Sequencing Service, Biosciences, University of Exeter , Exeter EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Audrey Farbos 2 Biosciences, University of Exeter , Exeter, Devon, EX4 4QD, UK 10 Exeter Sequencing Service, Biosciences, University of Exeter , Exeter EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Audrey Farbos Aaron R. Jeffries 2 Biosciences, University of Exeter , Exeter, Devon, EX4 4QD, UK 10 Exeter Sequencing Service, Biosciences, University of Exeter , Exeter EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aaron R. Jeffries Andrew J. Young 11 Centre for Ecology and Conservation, University of Exeter , Penryn, Cornwall TR10 9FE, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rafael Canton 8 Servicio de Microbiología, Hospital Universitario Ramón y Cajal and Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS) , Madrid, Spain 9 CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III , Madrid, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rafael Canton Pierluigi Caboni 6 Department of Life and Environmental Sciences, University of Cagliari , Cittadella Universitaria, 09042 Monserrato, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pierluigi Caboni Mark A. T. Blaskovich 7 Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland , St. Lucia, QLD, 4072, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mark A. T. Blaskovich Adilia Warris 4 MRC Centre for Medical Mycology, University of Exeter , Exeter, Devon, EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Krasimira Tsaneva-Atanasova 1 Living Systems Institute, University of Exeter , Exeter, Devon, EX4 4QD, UK 5 EPSRC Hub for Quantitative Modelling in Healthcare, University of Exeter , Exeter, EX4 4QJ, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Krasimira Tsaneva-Atanasova Stefano Pagliara 1 Living Systems Institute, University of Exeter , Exeter, Devon, EX4 4QD, UK 2 Biosciences, University of Exeter , Exeter, Devon, EX4 4QD, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stefano Pagliara For correspondence: s.pagliara{at}exeter.ac.uk u.lapinska{at}exeter.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Interactions between different species of pathogenic microbes often affect clinical outcome by altering the virulence or antibiotic resistance of individual microbes. By investigating the interactions between Staphylococcus aureus and Pseudomonas aeruginosa at the population, single-cell and molecular level we discovered that a sub-population of S. aureus enters in a viable non-culturable state that it is not detected via standard microbiology assays. In the presence of P. aeruginosa , S. aureus adopts a survival lifestyle similar to previously described intracellular S. aureus persisters, downregulating nitrogen metabolism and amino acid biosynthesis, while upregulating protein maturation processes. Entrance in a viable but non culturable state is the primary survival strategy of S. aureus in response to vancomycin treatment, whereas only a minority of the S. aureus population survive ciprofloxacin treatment while in a viable but non culturable state. These bacterial interactions may shape the evolution of resistance traits of co-infecting pathogens. Manipulating these interspecies adaptations could provide new opportunities for early therapeutic interventions. Introduction Interspecies microbial interactions have shaped the population structure of microbial communities in ecosystems and their biodiversity ( 1 ). Microbes can engage in mutualistic, neutral or antagonistic interactions, when sharing the same environment ( 2 , 3 ). In nutrient poor environments lacking essential nutrients, including carbon, nitrogen and iron sources, scavenging is a widely used microbial strategy among bacteria. In these settings bacteria compete for resources ( 4 ) also via antibiosis, that involves the synthesis of antimicrobial molecules that inhibit growth or kill other bacterial species ( 3 ). Interactions between co-colonizing bacteria may influence the development and persistence of co-infections by altering pathogen virulence ( 5 ) and susceptibility to antibiotics ( 6 , 7 ), ultimately affecting the health of host humans, animals and plants ( 8 ). Nevertheless, antibiotic therapies are often designed against individual pathogens without consideration of how interspecies interactions may alter each pathogen susceptibility to antibiotic therapy ( 6 ). Clinical trials have often reported a lack of association between clinical response to antibiotic therapy and in vitro susceptibility testing (i.e. the measurement of an antibiotic minimum inhibitory concentration (MIC) ( 9 )), also because these tests are generally carried out against individual microbes ( 10 ) besides other important factors ( 11 )( 12 ). Therefore, antibiotic therapy often fails to eradicate co-infecting pathogens ( 6 ), especially if such pathogens are already intrinsically resistant to key clinical antibiotics ( 13 , 14 ), thus further exacerbating the current antimicrobial resistance (AMR) crisis ( 15 ). Two of the most important bacterial pathogens of humans, the gram-negative Pseudomonas aeruginosa and the gram-positive Staphylococcus aureus , are frequently co-isolated from infections of catheters, endotracheal tubes, skin, eyes, and the respiratory tract, including the airways of people with cystic fibrosis (CF) ( 16 ). Indeed, an increased trend in prevalence of S. aureus and P. aeruginosa co-infections in patients with CF from 30% to 50% was recorded in a longitudinal study between 2004 and 2016 ( 17 ), although detection frequencies of S. aureus and P. aeruginosa have decreased in more recent years thanks to the use of elexacaftor, tezacaftor and ivacaftor ( 18 ). Moreover, a number of studies have shown that co-infection is associated with diminished lung function, more rapid pulmonary decline, delayed wound repair, increased risk of death and increased resistance to antibiotics ( 8 , 19 – 22 ), which adds to S. aureus and P. aeruginosa intrinsic and acquired antibiotic resistance as individual species ( 13 , 14 ), making these infections difficult to treat ( 23 , 24 ). The interactions between P. aeruginosa and S. aureus are complex. P. aeruginosa can display antagonistic interactions with S. aureus by secreting molecules that interfere with the growth, metabolism, and cellular homeostasis of S. aureus . These molecules include siderophores, such as pyoverdine and pyochelin, that sequester extracellular iron ( 25 ), proteases such as elastase that cleaves pentaglycine bridges in the S. aureus peptidoglycan ( 26 ), surfactants such as rhamnolipids that interfere with the S. aureus cell membrane ( 6 ), and redox-active secondary metabolites that inhibit S. aureus respiration, such as 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) ( 27 ), hydrogen cyanide and the phenazine molecule pyocyanin ( 8 ). These interactions are strain-dependent ( 28 , 29 ) and often do not prevent co-colonization in vivo ( 30 ). For example, P. aeruginosa isolates from co-infected CF patients are less antagonistic towards S. aureus than isolates from mono-infected CF patients ( 23 ). Moreover, P. aeruginosa isolates displaying a mucoid phenotype are less competitive towards S. aureus compared to non-mucoid P. aeruginosa isolates ( 28 ) due to alginate overexpression that, in turn, reduces the production of siderophores, (HQNO), and rhamnolipids ( 23 ). Indeed, a large proportion of S. aureus isolates are either unaffected or only growth inhibited by mucoid P. aeruginosa (i.e. with a S. aureus population reduction smaller than 100-fold) but killed by non-mucoid P. aeruginosa (i.e. with a S. aureus population reduction larger than 100-fold). A smaller proportion of S. aureus isolates are killed by both non-mucoid and mucoid P. aeruginosa . Finally, a very small number of S. aureus isolates are not killed by either P. aeruginosa phenotypes ( 28 ). On the other hand, the growth of P. aeruginosa is generally not affected by the presence of S. aureus . These interactions dramatically alter the susceptibility of S. aureus to the killing activities of clinically relevant antibiotics such as vancomycin, ciprofloxacin and tobramycin ( 6 , 7 , 10 , 29 , 31 – 36 ). For example, in response to the presence of P. aeruginosa , S. aureus can upregulate efflux pumps belonging to the Nor family leading to an increase in resistance of S. aureus to tetracycline and ciprofloxacin ( 29 ) or enter in a small-colony-variant (SCV) state becoming resistant to aminoglycosides and beta-lactams ( 37 ). In contrast, P. aeruginosa LasA protease potentiates killing of S. aureus by vancomycin, whereas P. aeruginosa rhamnolipids facilitate proton-motive force-independent tobramycin uptake in S. aureus , thus increasing S. aureus susceptibility to these two antibiotics ( 6 ). This wealth of knowledge has been obtained by using population level studies, therefore less is known about the interactions between P. aeruginosa and S. aureus at the sub-population and single-cell level, leaving a gap in our understanding of possible heterogeneities in the strategies adopted by these two bacteria within polymicrobial communities. Here we investigate the dynamics of the interactions between S. aureus and P. aeruginosa using both laboratory strains and clinical isolates. We employed microbiology, microscopy, transcriptomic and lipidomic analyses to study the dynamics of these interactions at the population, at the single-cell and at the molecular level. We further unravelled the impact of such interactions on antibiotic treatment both at the whole population and at the sub-population level. These early bacterial interactions may shape the evolution, virulence, and resistance of pathogens co-infecting humans and animals and could provide new opportunities for early therapeutic interventions by impeding microbial community adaptations in chronic mixed infections and subsequent patient worsening. Results S. aureus population decline in the continued presence of P. aeruginosa In order to investigate the interactions between S. aureus and P. aeruginosa , we firstly measured the population dynamics of both bacteria in mono-culture and co-culture. We followed the growth of a methicillin-susceptible S. aureus strain (ATCC25923) and a non-mucoid P. aeruginosa strain (PA14) in Lysogeny broth (LB) in well-mixed flasks via the colony forming unit (CFU) assay using selective agar plates (i.e. Pseudomonas isolation agar and Trypticase soy agar) ( 28 ). We found that the growth of S. aureus was not affected by the presence of P. aeruginosa during the first 4 h of co-culture compared to mono-culture (p-values > 0.05 according to unpaired t-tests at each time point); S. aureus growth was significantly reduced between 5 h and 9 h of co-culture compared to mono-culture (p-values < 0.05); at the 9 h time point S. aureus population dynamics inverted from expansion to contraction down to a minimum after 13 h of co-culture ( Figure 1A and Data A-B in S1 File). In contrast, the growth of P. aeruginosa was not affected by the presence of S. aureus ( Figure 1B and Data C-D in S1 File). We recorded a 200- and 700-fold reduction in S. aureus ATCC25923 population after 13 h of co-culture with two different non-mucoid strains of P. aeruginosa , i.e. PA14 and PAO1, respectively (Figure S1 and Data E in S1 File). Download figure Open in new tab Figure 1 A sub-population of S. aureus enters a viable but non culturable state in the presence of P. aeruginosa . (a-b) Temporal dependence of bacterial population size for (a) S. aureus ATCC25923 growing in mono-culture (filled circles) or in co-culture with P. aeruginosa PA14 in well-mixed flasks (open circles) and (b) for P. aeruginosa PA14 growing in mono-culture (filled squares) or in co-culture with S. aureus ATCC25923 in well-mixed flasks (open squares). Data points are the mean and standard deviation of colony forming unit (CFU) measurements carried out in biological triplicate each consisting of technical duplicate. Numerical values for each replicate are reported in Data A-D in S1 File. The solid lines are fitting of these data to our mathematical model with parameter estimates reported in Table S1. (c) Brightfield and fluorescence microscopy images of S. aureus SH1000 mCherry and P. aeruginosa PA14 Δ flgK introduced in microfluidic chambers after 10 h of co-culture in flasks and after regrowth in the microfluidic chambers for 0, 6 h or 9 h. One representative dividing, non-dividing or dying S. aureus SH1000 mCherry is denoted with a blue, red or magenta circle, respectively. Scale bar: 10 µm. (d) Temporal dependence of single-cell mCherry fluorescence for dividing, non-dividing or dying S. aureus (blue, red or magenta data points, respectively). Data points are the mean and standard error of ten single-cell measurements from biological triplicate experiments. Numerical values are reported in Data M in S1 File. (e) Corresponding fraction of dividing (vertically patterned bars), non-dividing (horizontally patterned bars), or dying (checkered patterned bars) S. aureus in mono-culture or in co-culture with P. aeruginosa within microfluidic chambers. (f) Corresponding dependence of doubling time on the generation number for S. aureus in mono-culture (green filled circles) or in co-culture with P. aeruginosa (open blue circles) within microfluidic chambers. Each point reports the doubling time of an individual bacterium with numerical values reported in Data Q in S1 File. *** denotes a p-value < 0.001, **** denotes a p-value < 0.0001. (g) Corresponding temporal dependence of bacterial population size for S. aureus growing in mono-culture (filled green circles) or in co-culture with P. aeruginosa (open blue circles) and for P. aeruginosa growing in co-culture (open black squares) in microfluidic chambers inoculated with bacteria after 10 h of culture in well-mixed flasks. Data points are the mean and standard error of cell count measurements carried out in biological triplicate experiments each consisting of four microfluidic chambers. Numerical values for each replicate are reported in Data N-P in S1 File. The solid lines are fitting of these data to our mathematical model with parameter estimates reported in Table S2 and Table S3. Next, we investigated the interactions between methicillin-resistant S. aureus isolate RYC157 and P. aeruginosa isolate RYC157 co-isolated from a dual-species infection in a CF patient. We recorded a slower growth of P. aeruginosa RYC157 compared to both S. aureus RYC157 and P. aeruginosa PA14 and we observed a population decline in S. aureus RYC157 only after 20 h of co-culture (Figure S2B and Data F and G in S2 File). Moreover, we observed a population decline in both S. aureus RYC157 and S. aureus RYC165 (a different methicillin-resistant S. aureus strain co-isolated from a dual-species infection with P. aeruginosa also in a CF patient) after 9 h and 10 h of co-culture with P. aeruginosa PA14 (Figure S2C and S2D and Data H-K in S1 File) consistent with the data obtained for S. aureus ATCC25923. These data suggest that P. aeruginosa PA14 kills S. aureus ATCC25923, RYC157 and RYC165, considering that previous co-culture assays ascribed to killing bacterial population reductions larger than 100-fold ( 28 , 38 ) and that we measured an inversion in the S. aureus population dynamics. However, it is also conceivable that in the continued presence of P. aeruginosa , S. aureus progressively enters a viable but non-culturable state ( 39 ) and that individual S. aureus cells do not form colonies on selective agar plates, thus leading to an underestimation of the S. aureus population. In order to rationalise these population dynamics, we developed a mathematical model based on the simplified assumptions that the growth rates of S. aureus and P. aeruginosa are modulated by the proportion of active biochemical processes which contribute towards replication, and that P. aeruginosa secretes molecules that can either kill or induce a non-culturable state in S. aureus (see Methods). By fitting this model to our population dynamics data via least-squares regression, we were able to capture the experimentally measured population dynamics for the different S. aureus and P. aeruginosa strains in both mono- and co-culture ( Figure 1A , 1B and S2) with parameter estimations reported in Table S1. Taken together, these data and model suggest that the decline observed for S. aureus in co-culture is due to either S. aureus death or entrance in a viable non-culturable state. A sub-population of S. aureus enters a viable but non-culturable state in the presence of P. aeruginosa In order to test the non-mutually exclusive hypotheses above, we employed our previously described single-cell microfluidics-based time-lapse microscopy platform( 40 – 42 ) with bacteria hosted in 1 µm high and 150 µm wide circular chambers connected to a 20 µm high and 200 µm wide main delivery chamber. To facilitate long-term time-lapse microscopy of individual bacteria hosted within these chambers, we used a P. aeruginosa deletion mutant with reduced motility due to a lack of flagella (PA14 Δ flgK ( 43 )) and a S. aureus fluorescent reporter strain (SH1000 mCherry ( 44 )) to facilitate distinguishing between viable and non-viable S. aureus cells. We verified that after 13 h of co-culture with P. aeruginosa PA14 Δ flgK in well-mixed flasks, both S. aureus ATCC 25923 and S. aureus SH1000 mCherry displayed a significant reduction in population size with respect to S. aureus mono-cultures, similar to the reduction in population size observed after co-culture with P. aeruginosa PA14 or PAO1 (Figure S1 and Data E in S1 File). These data confirm that the antagonistic interactions recorded for the pairs P. aeruginosa PA14 (or PAO1) and S. aureus ATCC 25923 are well recapitulated when using the pair P. aeruginosa PA14 Δ flgK and S. aureus SH1000 mCherry. Therefore, we harvested a 100 µL aliquot from a S. aureus SH1000 mCherry and P. aeruginosa PA14 Δ flgK co-culture after 10 h of growth in a well-mixed flask and inoculated it in four microfluidic chambers continuously supplied via the main delivery chamber with LB for 9 h. We imaged each chamber every 10 min for 9 h and repeated this experiment in biological triplicate using three independent 10 h old co-cultures as inoculum. By tracking individual S. aureus cells in each chamber in each experiment over time ( Figure 1C and 1D ), we found that 70% of S. aureus cells resumed growth in the presence of P. aeruginosa , 28% of S. aureus did not resume growth within the 9 h exposure to LB, and 2% of S. aureus cells died ( Figure 1E and Data L in S1 File). S. aureus cells that did not resume growth and division retained motility, exploring the microfluidic chambers during the 9 h exposure to LB ( Figure 1C ). Furthermore, these viable but non culturable cells displayed a significantly lower mCherry fluorescence at t = 0 compared to S. aureus cells that than resumed growth, but maintained stable mCherry fluorescence over time and did not stain with propidium iodide similarly to dividing cells ( Figure 1D and Data M in S1 File). In contrast, S. aureus cells that eventually died while in co-culture with P. aeruginosa , displayed a sharp decrease in mCherry fluorescence ( Figure 1D ) and stained with propidium iodide. The ratio of dividing over non-dividing S. aureus cells varied across the different microfluidic chambers investigated but did not depend on the initial ratio of P. aeruginosa cell number over S. aureus cell number in each chamber (Pearson correlation coefficient r = 0.03, p-value = 0.92, Figure S3 and Data O in S1 File). In contrast, 100% of S. aureus cells resumed growth upon inoculation in the microfluidic chambers after 10 h of mono-culture in well-mixed flasks ( Figure 1E and Data N in S1 File). Moreover, the lag times of individual S. aureus cells in co-culture with P. aeruginosa were significantly longer and more heterogeneous compared with the lag times of S. aureus cells in mono-culture (1 st generation in Figure 1F ). Similarly, the doubling times of the 2 nd and 3 rd generation S. aureus daughter cells in co-culture were significantly longer and more heterogeneous compared with the doubling times of S. aureus cells in mono-culture and only 4 th generation daughter cells doubled at similar rates in mono- and co-culture ( Figure 1F and Data Q in S1 File). Impact of the S. aureus viable but non-culturable state on population dynamics As a result, the population dynamics of S. aureus in co-culture with P. aeruginosa in microfluidic chambers was different with respect to that measured for S. aureus in mono-culture in microfluidic chambers. In mono-culture, the S. aureus population in each chamber readily and homogeneously expanded exponentially, variations in cell number per chamber being dictated by variant initial inoculum per chamber in range from 5 to 30 cells ( Figure 1G , Figure S4A and Data N in S1 File). In contrast, the S. aureus population started to increase only after 5 h of co-culture with P. aeruginosa and growth was heterogeneous across the different chambers ( Figure 1G , Figure S4B and Data O in S1 File). The expansion in S. aureus population was preceded by an inversion in the trend of the P. aeruginosa population dynamics that expanded during the first 2.5 h of co-culture with S. aureus and decreased from the 2.5 h time point onwards ( Figure 1G , Figure S4C and Data P in S1 File), due to escape out of the chambers, possibly linked to increased cellular motility via type IV pili ( 43 ). As a consequence, the ratio of P. aeruginosa cell number over S. aureus cell number per chamber was initially between 10 and 25 (upon inoculation after 10 h of co-culture in well-mixed flasks), it increased to a maximum of 35 after 2.5 h of co-culture in the microfluidic chambers and then decreased down to a minimum that was lower than 1 in most of the microfluidic chambers investigated (Figure S4D). These distinct populations dynamics of S. aureus and P. aeruginosa in mono- or co-culture were well described by a mathematical model that accounts for the resumption of intracellular activity and bacterial growth and the fact that P. aeruginosa cells are able to escape the microfluidic chambers (see Methods and Figure 1G ) with parameter estimations reported in Table S2 and Table S3. In order to further test whether the non-dividing S. aureus sub-population had an impact on S. aureus population dynamics, we used both LB and selective agar plates (i.e. Pseudomonas isolation agar and Trypticase soy agar) ( 28 ) to enumerate S. aureus and P. aeruginosa after co-culture in well-mixed flasks. In accordance with our microscopy data, we found that S. aureus counts on selective plates were higher than counts on LB agar plates (Figure S5 and Data B, D and R-S in S1 File), possibly due to a larger sub-population of S. aureus remaining in a non-dividing state on LB agar plates, where also P. aeruginosa can grow compared to selective plates where P. aeruginosa cannot grow. Taken together these data demonstrate that the presence of P. aeruginosa induces a sub-population of S. aureus in a viable but non culturable state that is not detectable using standard microbiology techniques, such as the colony forming unit assay, leading to a significant decrease of the culturable S. aureus population over time but to a limited decrease of the viable S. aureus population. P. aeruginosa rewires its transcriptome faster than S. aureus in co-culture In order to investigate the molecular mechanisms involved in the interactions between S. aureus and P. aeruginosa , we employed an unbiased approach to examine changes in gene expression by performing genome-wide comparative transcriptome analysis between mono-and co-cultures ( 25 , 45 ). Specifically, we extracted RNA from S. aureus ATCC25923 and P. aeruginosa PA14 from biological triplicates after 3 h of mono- or co-culture. At this time point, the population size of S. aureus from the co-culture was only 1.5-fold lower compared to the population size of S. aureus from the mono-culture, suggesting that effects of competition had not fully translated into changes in population dynamics ( 46 ), and both S. aureus and P. aeruginosa were in their exponential phase of growth ( Figure 1A - 1B ). Moreover, we also extracted RNA from S. aureus and P. aeruginosa after 10 h of mono- and co-culture. At this time point, the population size of S. aureus from the co-culture was 10-fold lower compared to the population size of S. aureus from the mono-culture, suggesting that effects of competition had fully translated into changes in population dynamics, and both S. aureus and P. aeruginosa mono-cultures were in their stationary phase of growth ( Figure 1A - 1B ). Principal component analysis revealed both major divergences across different culturing conditions and high within group reproducibility, i.e. transcriptome replicates from each condition clustered together. We found that the transcriptomes of S. aureus after 3 h in co-culture were close to those measured for S. aureus after 3 h in mono-culture confirming that by this time point P. aeruginosa did not have a significant impact on S. aureus (Figure S6A and S2 File). In contrast, the transcriptomes of S. aureus after 10 h in co-culture were very different, in both principal components, to those measured for S. aureus after 10 h in mono-culture confirming that by this time point P. aeruginosa did have a significant impact on S. aureus (Figure S6A). Moreover, the transcriptomes of P. aeruginosa after 10 h in co-culture were different to those measured for P. aeruginosa after 10 h in mono-culture in terms of principal component 2 (Figure S6B and S2 File). Next, we examined changes in gene expression between S. aureus and P. aeruginosa co-cultures and mono-cultures at both time points by using a conservative cut-off of 2-fold change (i.e. log 2 fold change > 1 or log 2 fold change < -1) and adjusted p-value (via Benjamini-Hochberg) smaller than 0.05 to identify differentially expressed genes ( 46 , 47 ). This analysis revealed that P. aeruginosa reacted faster to the presence of the other species, whereas S. aureus responded at a later stage: the number of significantly differentially regulated P. aeruginosa genes decreased from 397 to 250 (i.e. from 7% to 4% of the genome) from 3 h to 10 h of co-culture with respect to mono-culture; in striking contrast, the number of significantly differentially regulated S. aureus genes dramatically increased from 153 to 814 (i.e. from 6% to 33% of the genome) from 3 h to 10 h of co-culture with respect to mono-culture (Figure S7 and S3-S6 Files, RNA transcript counts for all genes and individual replica are reported in S2 File). P. aeruginosa upregulates the expression of the pyochelin pathway and DNA-templated transcription initiation in the continued presence of S. aureus Next, we performed gene ontology enrichment analysis on each subset of differentially regulated genes between mono- and co-culture and identified biological processes that were significantly upregulated or downregulated across each comparison (i.e. with an enrichment score larger or smaller than zero, respectively ( 47 – 49 )). We found that quinone biosynthesis, cellular respiration and molybdopterin cofactor metabolism processes were upregulated in P. aeruginosa in co-culture with respect to mono-culture at t = 3 h, whereas membrane transport, carbohydrate and fatty acid biosynthesis and DNA-templated transcription initiation processes were downregulated ( Figure 2A and S7 File). At t = 10 h instead iron transport, DNA-templated transcription initiation, protein folding, peptide and dicarboxylic acid metabolism processes were upregulated, whereas biosynthesis regulation, homeostasis and carbohydrate metabolism processes were downregulated in P. aeruginosa in co-culture with respect to mono-culture ( Figure 2B and S8 File). Download figure Open in new tab Figure 2. Molecular interactions between P. aeruginosa and S. aureus . (a and b) Biological processes that are significantly enriched in the comparisons of the transcriptomes of P. aeruginosa in mono-culture vs co-culture at (a) t = 3 h or (b) at t = 10 h. (d and e). Biological processes that are significantly enriched in the comparisons of the transcriptomes of S. aureus in mono-culture vs co-culture at (d) t = 3 h or (e) at t = 10 h. The colour coding indicates the calculated adjusted p-values reported in S7 and S8 File, red indicating the lowest and blue indicating the highest adjusted p-value. (c and f) Differential expression of (c) P. aeruginosa and (f) S. aureus biological processes at t = 10 h compared to t = 3 h in mono-culture (filled bars) or co-culture (open bars). The bars and error bars report the mean and standard error of the transcript log 2 fold change (calculated from biological triplicate transcriptomic measurements) of five representative genes for each pathway. log 2 fold change data for all genes in each pathway are reported in Figure S8 and Figure S12 with numerical values reported in S9-S10 and S14-S15 Files. Next, we compared the temporal regulation of these processes in P. aeruginosa in mono-culture and co-culture. Genes in the quinone biosynthesis, cellular respiration and molybdopterin cofactor metabolism processes were significantly more downregulated at t =10 h vs t = 3 h in co-culture compared to mono-culture. In contrast, genes in the membrane transport and peptide metabolism processes were significantly more downregulated at t = 10 h in mono-culture compared to co-culture ( Figure 2C , Figure S8 and S9-S10 Files). Genes in the regulation of nitrogen metabolism, carbohydrate metabolism and homeostasis were significantly more upregulated at t =10 h in mono-culture compared to co-culture. In contrast, genes in the carbohydrate biosynthesis and protein folding processes were significantly more upregulated at t =10 h in co-culture compared to mono-culture ( Figure 2C , Figure S8 and S9-S10 Files). Strikingly, genes in the DNA-templated transcription initiation (including the sigma factor pvdS ), iron transport and dicarboxylic acid metabolism processes displayed opposite regulation when P. aeruginosa was in co-culture compared to mono-culture: in mono-culture these genes were downregulated at t = 10 h compared to t = 3 h, whereas in co-culture these genes were upregulated at t = 10 h compared to t = 3h ( Figure 2C , Figure S8 and S9-S10 Files). Notably, P. aeruginosa employs two siderophores, pyochelin and pyoverdine, for the uptake of iron and other metals. The pyoverdine synthesis pathway was upregulated at t = 10 h compared to t = 3 h both in mono-culture and co-culture (Figure S9A); in striking contrast, the pyochelin synthesis pathway was strongly downregulated at t = 10 h compared to t = 3 h in mono-culture and strongly upregulated at t = 10 h compared to t = 3 h in co-culture (Figure S9B and S9-S10 Files), suggesting that secretion of pyochelin, but not of pyoverdine, is a P. aeruginosa response to the presence of S. aureus . We assessed the effect of pyochelin on S. aureus viability by co-culturing S. aureus in the presence of a P. aeruginosa siderophore-negative knockout mutant (PAO1 Δ pvdD Δ pchEF ( 50 )). A similar reduction in S. aureus population was seen as with the P. aeruginosa PAO1 parental strain, (Figure S1), suggesting that siderophore secretion alone was not responsible for the observed inversion in the population dynamics of S. aureus . Pathways previously implicated in the response of P. aeruginosa to the presence of S. aureus , i.e. the phenazine, HQNO, hydrogen cyanide, alginate, rhamnolipid, exopolysaccharide, or elastase pathways and the quorum sensing master regulators lasR , rhlR and mvfR ( 6 , 8 , 26 , 27 , 46 , 51 ) displayed a similar temporal regulation in co-culture and mono-culture (Figure S10 and S9-S10 Files), suggesting that these pathways are not induced by the presence of S. aureus . These RNA-seq data were further validated by quantitative real-time PCR for twenty genes (including genes in the P. aeruginosa pyoverdine, pyochelin and HQNO pathways), showing a strong correlation with the transcriptome data (Figure S11 and S11 File). Taken together, these data suggest that after prolonged exposure to S. aureus , P. aeruginosa transports more substrates, particularly iron, performs more transcription initiation, protein repair and carbohydrate biosynthesis, while reducing respiration and control over biosynthesis and homeostasis. S. aureus increases the expression of cellular catabolism and chaperone genes in the continued presence of P. aeruginosa Gene ontology enrichment analysis revealed that biological regulation was the only process upregulated in S. aureus in co-culture with respect to mono-culture at t = 3 h, whereas nitrogen metabolism and biosynthesis, organic substance transport and cellular organization processes were downregulated ( Figure 2D and S12 File). At t = 10 h instead cellular catabolism and protein maturation processes were upregulated, whereas alpha-amino acid biosynthesis, nucleoside metabolism, biogenesis and nitrogen transport processes were downregulated in S. aureus in co-culture with respect to mono-culture ( Figure 2E and S13 File). Next, we compared the temporal regulation of these processes in S. aureus in mono-culture and co-culture. Genes in the protein maturation process were significantly more downregulated at t = 10 h in mono-culture than in co-culture. In contrast, genes in the nucleoside monophosphate metabolism and cellular biogenesis processes were significantly more downregulated at t = 10 h in co-culture than in mono-culture ( Figure 2E , Figure S12 and S14-S15 Files). Genes in the cellular catabolism process displayed a remarkably opposite regulation in co-culture with respect to mono-culture: these genes were downregulated at t = 10 h compared to t = 3h in mono-culture, whereas were upregulated in co-culture. The opposite was true for genes in the alpha-amino acids biosynthesis process that were upregulated at t = 10 h compared to t = 3h in mono-culture, whereas were downregulated in co-culture ( Figure 2E , Figure S12 and S14-S15 Files). These RNA-seq data were further validated by quantitative real-time PCR for twenty genes, showing a strong correlation with the transcriptome data (Figure S11 and S11 File). Taken together, these data suggest that after prolonged exposure to P. aeruginosa , S. aureus transports less nitrogen, produces less nucleosides and amino acids and reduces biogenesis, while increasing its catabolic rates and chaperone activity, possibly to reduce protein aggregation that is linked to bacterial dormancy ( 42 , 52 ). These features are consistent with our data showing that sub-populations of S. aureus enter in a viable but non-culturable state during co-culture with P. aeruginosa ( Figure 1 ). Moreover, our transcriptomic analysis also revealed that in the presence of P. aeruginosa , S. aureus downregulated the biosynthesis of key amino acids, such as valine, leucine, isoleucine, lysine and tryptophan, and aminoacyl-tRNA synthetases, whereas S. aureus upregulated the heat shock stimulon, the codY regulon and the ribosome recycling gene frr (S6 File), similarly to gene expression changes previously reported for intracellular persisters ( 53 ), a comparison on which we expand in the Discussion. During co-culture P. aeruginosa and S. aureus upregulate phosphatidylglycerol lipids Next, we used ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry ( 47 , 54 ) to quantify the relative abundance of membrane lipids in P. aeruginosa PA14 and S. aureus ATCC25923 after 10 h of co-culture with respect to after 10 h mono-cultures, all comparisons having being performed in biological triplicates. Within the lipidomes extracted from the co-cultures, lipids could not be assigned unambiguously to P. aeruginosa or S. aureus ; therefore, Table 1 reports quantitative information only on lipids that were detected in either species in mono-cultures and in co-cultures. View this table: View inline View popup Table 1. Lipids differentially regulated in co-cultures compared with mono-cultures . Lipid class, fatty acid composition, adduct type, retention time (RT), theoretical and experimental mass-to-charge ratio (m/z), molecular formula, variable importance in projection (VIP) score, and mass error (Δppm) measured via quantitative time-of-flight liquid chromatography mass spectrometry. We found that after 10 h of co-culture, the expression of phosphatidylglycerol (PG) and monogalactosyl diacylglycerol (MGDG) lipids was up regulated and the expression of phosphatidylcholine (PC) lipids was downregulated with respect to 10 h of P. aeruginosa mono-culture. The regulation of phosphatidylethanolamine (PE) lipids was instead mixed with PE 32:0 and PE34:0 being upregulated in co-culture and PE 33:1, PE 36:1 and PE 36:2 being downregulated in co-culture ( Table 1 ). Furthermore, the expression of a large subset of PG lipids and some fatty acids was upregulated after 10 h of co-culture with respect to 10 h of S. aureus mono-culture, whereas the expression of glycosyldiacylglycerol (DGDG) and lysophosphatidylcholine lipids (LPC) was downregulated in co-culture with respect to 10 h of S. aureus mono-culture ( Table 1 ). Taken together, these data suggest that phosphatidylglycerol lipids are upregulated in both P. aeruginosa and S. aureus after 10 h of co-culture, whereas phosphatidylcholine lipids are downregulated in both P. aeruginosa and S. aureus after 10 h of co-culture with respect to the corresponding mono-cultures. The presence of P. aeruginosa decreases the bactericidal efficacy of ciprofloxacin against S. aureus when used at supra-inhibitory concentrations Next, we set out to determine the impact of the interactions between S. aureus and P. aeruginosa on antibiotic treatment. We employed two antibiotics that are routinely used for the treatment of cystic fibrosis patients: ciprofloxacin, a quinolone that has bactericidal activity against both S. aureus and P. aeruginosa and is commonly used to treat P. aeruginosa during coinfection; vancomycin, a frontline glycopeptide that is used to treat methicillin-resistant S. aureus in cystic fibrosis patients, but it is not effective against P. aeruginosa ( 6 ). We performed time-killing assays ( 49 ) trying to reflect an infection-like condition wherein the microbial community is already established prior to the application of any treatment ( 31 ). Therefore, we added ciprofloxacin or vancomycin to 3 h old mono- or co-cultures, i.e. in the exponential phase of growth and before we recorded an impact of P. aeruginosa PA14 presence on S. aureus ATCC25923 growth, or to 10 h old mono- or co-cultures, i.e. in the stationary phase of growth and after we recorded an impact of P. aeruginosa presence on S. aureus growth. The minimum inhibitory concentration (MIC) of ciprofloxacin against P. aeruginosa and S. aureus was 0.125 µg mL -1 and 0.5 µg mL -1 , respectively. Therefore, when we performed time-killing assays using ciprofloxacin at the MIC value measured for S. aureus (i.e. 0.5 µg mL -1 ), we found that ciprofloxacin had a three or one orders of magnitude lower efficacy against S. aureus compared to P. aeruginosa mono-cultures in the exponential (with a 3-log vs a 6-log reduction) or stationary (with a 2-log vs a 3-log) phase of growth ( Figure 3A-B and S16 File). Consistently, when ciprofloxacin was employed at 10× or 25× its MIC (against S. aureus ) it had a weaker efficacy against S. aureus compared to P. aeruginosa mono-cultures in the stationary phase of growth (with a 4-log vs a 6-log reduction, respectively, Figure S13E-H). Download figure Open in new tab Figure 3 The presence of P. aeruginosa does not affect the bactericidal efficacy of ciprofloxacin against S. aureus when used at inhibitory concentrations. (a-b) Temporal dependence of bacterial population size for S. aureus (filled circles) or P. aeruginosa (filled squares) in mono-culture, S. aureus (open circles) or P. aeruginosa (open squares) in co-culture in well-mixed flasks treated with ciprofloxacin at the MIC value measured against S. aureus , i.e. 0.5 µg mL -1 . Bacteria were subjected to a 5 h ciprofloxacin treatment after being harvested from either (a) 3 h or (b) 10 h of mono-culture or co-culture. Data points are the mean and standard deviation of colony forming unit (CFU) measurements carried out in biological triplicate each consisting of technical duplicate. Numerical values for each replicate are reported in S16 File. (c) Microscopy images of S. aureus and P. aeruginosa introduced in microfluidic chambers after 10 h of co-culture in flasks and after exposure to ciprofloxacin (0-4 h) and LB (4-7 h) in the microfluidic chambers. Scale bar: 10 µm. (d) Corresponding temporal dependence of bacterial population size for S. aureus growing in mono-culture (filled green circles) or in co-culture with P. aeruginosa (open blue circles) and for P. aeruginosa growing in mono-culture (filled black squares) or co-culture (open black squares). Data points are the mean and standard error of cell count measurements carried out in biological triplicate experiments each consisting of four microfluidic chambers. Numerical values for each replicate are reported in S17 File. (e) Corresponding fraction of dividing (vertically patterned bars), non-dividing (horizontally patterned bars) or lysing (diagonally patterned bars) S. aureus in mono-culture or in co-culture with P. aeruginosa within microfluidic chambers. After 3 h or 10 h of co-culture, S. aureus displayed similar survival to ciprofloxacin treatment compared to that measured for S. aureus after 3 h or 10 h of monoculture when ciprofloxacin was employed at its MIC ( Figure 3A and Figure 3B , respectively). In contrast, when ciprofloxacin was employed at 10× or 25× its MIC, S. aureus displayed increased survival to ciprofloxacin after both 3 h or 10 h of co-culture compared to the corresponding mono-cultures. Indeed, persisters constituted 0.01% of the S. aureus population in 3 h old co-cultures, whereas 3 h old S. aureus mono-cultures did not display detectable persisters (Figure S13E and S13G). Furthermore, persisters constituted 1% of S. aureus population in 10 h old co-cultures and only 0.01% of S. aureus population in 10 h old mono-cultures (Figure S13F and S13H and S16 File). After 3 h of co-culture, P. aeruginosa displayed increased survival to ciprofloxacin treatment compared to that measured for P. aeruginosa after 3 h of mono-culture when ciprofloxacin was employed at its MIC (with a 4-log vs a 6-log reduction, Figure S13C). However, we did not observe a difference in terms of ciprofloxacin efficacy against P. aeruginosa between 10 h mono- and co-cultures (Figure S13D). Moreover, when ciprofloxacin was employed at 10× or 25× its MIC (against S. aureus ), P. aeruginosa did not display any detectable persisters after 3 h or 10 h of mono- or co-culture (Figure S13E-H and S16 File). Taken together, these data demonstrate that the presence of a competitor species does not affect the efficacy of ciprofloxacin against S. aureus or P. aeruginosa when this antibiotic is used at its minimum inhibitory concentration; in contrast, when ciprofloxacin is used above its inhibitory concentration its efficacy against S. aureus decreases in the presence of P. aeruginosa with the emergence of persisters also in the exponential phase of growth. Next, we set out to determine whether the decline in the S. aureus and P. aeruginosa populations during ciprofloxacin treatment was due to cell death or entrance in a viable but non-culturable state. We employed the above described single-cell microfluidics-based platform to image and track the fate of individual S. aureus and P. aeruginosa cells harvested from 10 h old well-mixed mono-cultures or 10 h old well-mixed co-cultures, while exposing them first to ciprofloxacin dissolved in LB medium for 4 h (at its MIC value against S. aureus , i.e. 0.5 µg mL -1 ) and then to LB medium for 3 h within microfluidic chambers ( Figure 3C ). These experiments were performed in biological triplicates. S. aureus harvested from 10 h old well-mixed mono-cultures grew and doubled during the 4 h exposure to ciprofloxacin in all the twelve microfluidic chambers imaged across three biological replicates; during the subsequent exposure to LB medium S. aureus continued to double in some chambers, while cells stopped doubling or lysed in other chambers. However, by the end of the experiments, S. aureus lysis was observed in all the imaged chambers (Figure S14A and S17 File) which led to a significant decrease in S. aureus population ( Figure 3D ). By the end of the experiments, 5 S. aureus cells were in a dividing state, 9 S. aureus cells were in a viable but non culturable state, whereas 252 S. aureus cells had lysed, i.e. 2%, 3% and 95% of the population, respectively ( Figure 3E ). S. aureus populations harvested from 10 h old well-mixed co-cultures displayed an overall similar response to ciprofloxacin to the one described above for mono-cultures ( Figure 3D ) in accordance with our time-killing assay data ( Figure 3B ). However, cell lysis ( Figure 3C ) and population decline started at an earlier time point in co-culture compared to mono-culture ( Figure 3D , Figure S14B and S17 File) and by the end of the experiment fewer S. aureus cells were in a dividing or viable but non culturable state (i.e. 1.5% and 0.8%, respectively) compared to mono-culture ( Figure 3E ). P. aeruginosa harvested from 10 h old well-mixed mono-cultures displayed a slower growth compared to S. aureus mono-cultures during the 4 h exposure to ciprofloxacin, followed by population decline due to both cell lysis and cell escape from the microfluidic chambers. P. aeruginosa harvested from 10 h old well-mixed co-cultures displayed a similar population dynamics, but with a slightly higher surviving fraction ( Figure 3D , Figure S14 and S17 File). Taken together, these data demonstrate that the decline in the S. aureus and P. aeruginosa populations during ciprofloxacin treatment is primarily due to cell death rather than entrance in a viable but non-culturable state, that ciprofloxacin has a bactericidal effect against both S. aureus and P. aeruginosa and that the presence of a competitor species does not significantly affect the efficacy of ciprofloxacin against either S. aureus or P. aeruginosa when this antibiotic is used at its minimum inhibitory concentration. Synergistic growth inhibitory effect of vancomycin and P. aeruginosa against S. aureus Next, we performed time-killing assays to evaluate the impact of the interactions between P. aeruginosa and S. aureus on the efficacy of vancomycin, an antibiotic that is only effective against S. aureus . We found that both at its MIC value against S. aureus (i.e. 1 µg mL -1 ), or at 10× or 25× its MIC, vancomycin had a moderate efficacy when added for 5 h to S. aureus mono-cultures in the exponential or stationary phase (i.e. less than 1-log reduction in bacterial counts, Figure 4AB , Figure S15 and S18 File). As expected, P. aeruginosa was not affected by vancomycin at any of the antibiotic concentrations employed (Figure S15 and S18 File). Download figure Open in new tab Figure 4 Synergistic growth inhibitory effect of vancomycin and P. aeruginosa against S. aureus . (a-b) Temporal dependence of bacterial population size for S. aureus in mono-culture (filled circles), S. aureus (open circles) or P. aeruginosa (open squares) in co-culture in well-mixed flasks treated with vancomycin at its MIC value against S. aureus , i.e. 1 µg mL -1 . Bacteria were subjected to a 5 h vancomycin treatment after being harvested from either (a) 3 h or (b) 10 h of mono-culture or co-culture. Data points are the mean and standard deviation of colony forming unit (CFU) measurements carried out in biological triplicate each consisting of technical duplicate. Numerical values for each replicate are reported in S18 File. (c) Distribution of accumulation of vancomycin-NBD in individual S. aureus cells harvested from 10 h old mono- (green histogram) or co-culture (blue histogram), exposed to 16 µg mL -1 vancomycin-NBD for 1 h and measured via flow cytometry. The data presented are representative of biological triplicate measurements with numerical values reported in Data A in S19 File. (d) Corresponding mean values of vancomycin-NBD fluorescence distributions in S. aureus harvested from 10 h old mono- (filled circles) or co-culture (open circles), exposed to vancomycin-NBD for 20 min, 40 min, or 60 min. The data reported are the mean and standard deviation of mean values extracted from fluorescence distributions from biological triplicate each constituted of technical triplicate. (e) Microscopy images of S. aureus and P. aeruginosa introduced in microfluidic chambers after 10 h of co-culture in flasks and after exposure to vancomycin (0-4 h) and LB (4-7 h) in the microfluidic chambers. Representative non-dividing S. aureus are denoted with red circles. Scale bar: 10 µm. (f) Corresponding fraction of dividing (vertically patterned bars) or non-dividing (horizontally patterned bars) S. aureus in mono-culture or in co-culture with P. aeruginosa within microfluidic chambers. (g) Corresponding temporal dependence of bacterial population size for S. aureus in mono-culture (filled green circles) or in co-culture with P. aeruginosa (open blue circles) and for P. aeruginosa in co-culture (open black squares). Data points are the mean and standard error of cell count measurements carried out in biological triplicate experiments each consisting of four microfluidic chambers. Numerical values for each replicate are reported in S20 File. After 3 h of co-culture, S. aureus displayed similar survival to vancomycin treatment compared to that measured for S. aureus after 3 h of mono-culture when vancomycin was employed either at its MIC ( Figure 4A ), at 10×, or 25× its MIC (Figure S15E and S15G). In striking contrast, after 10 h of co-culture, S. aureus displayed a 5-log reduction in survival to vancomycin treatment at its MIC compared to S. aureus mono-culture ( Figure 4B and S18 File). These data suggest a synergistic bactericidal or growth inhibitory effect between vancomycin, that when used against S. aureus mono-cultures had a negligible efficacy (Figure S11D), and P. aeruginosa that caused only a 2-log reduction of S. aureus counts in co-culture compared to mono-culture in the absence of vancomycin (Figure S15B). This synergistic effect was possibly due to the effect of the P. aeruginosa extracellular protease, LasA, that cleaves pentaglycine bridges in S. aureus peptidoglycan ( 26 ) and was strongly upregulated in 10 h old P. aeruginosa mono-cultures and co-cultures compared to 3 h old P. aeruginosa mono-cultures and co-cultures (8.7 and 8.8 log 2 fold change, respectively, Figure S10B). Moreover, we recorded a 3-log reduction of S. aureus counts for vancomycin treatment at 10×, or 25× its MIC in co-culture compared to that measured for S. aureus after 10 h of mono-culture (Figure S15F and S15H). It is also important to observe the differences in the population dynamics, in response to vancomycin treatment, between S. aureus mono-cultures and co-cultures. After 3 h mono-culture, the S. aureus population gradually decreased during vancomycin at all concentrations tested. This population dynamics suggests that S. aureus is tolerant to vancomycin at the population level, rather than at the sub-population level with the presence of persisters that would instead lead to a bi-phasic killing dynamics ( 55 , 56 ). Similar population dynamics were measured for S. aureus after 3 h of co-culture, except for a sharp decrease in cell counts after 5 h treatment with vancomycin ( Figure 4A , S15C, S15E and S15G). These data suggest that after 3 h of co-culture and 5 h of vancomycin treatment in co-culture, a different type of growth inhibition of S. aureus cells becomes evident because P. aeruginosa starts to exert its negative effect on S. aureus in accordance with the data presented in Figure 1 . After 10 h mono-culture, the S. aureus population did not decrease during vancomycin treatment neither at 1×, 10×, nor at 25× its MIC indicating complete tolerance to vancomycin at the population level. In contrast, after 10 co-culture, the S. aureus population displayed a bi-phasic dynamics with a gradual decrease during the first 3 h of vancomycin treatment followed by a sharp decrease in cell counts ( Figure 4B , S15D, S15F and S15H), i.e. the opposite trend observed when a bacterial population harbours persisters ( 55 , 56 ). Taken together, these data suggest that the presence of P. aeruginosa enhances either killing or growth inhibition of S. aureus via vancomycin that has an otherwise negligible efficacy against stationary phase S. aureus . Vancomycin accumulation in S. aureus is lower in the presence of P. aeruginosa Next, we set out to test the hypothesis that the accumulation of vancomycin in S. aureus could be altered by the continued presence of P. aeruginosa . In fact, we observed an increase in PG lipids in co-culture with respect to S. aureus mono-culture ( Table 1 ). PG lipids are negatively charged and could therefore bind and sequester positively charged vancomycin molecules( 57 ) hindering their translocation through the membrane and their binding to the D-alanyl-D-alanine (D-Ala-D-Ala) peptide motif of the peptidoglycan precursor ( 58 ). In order to test this hypothesis, we used flow cytometry and a fluorescent derivative of vancomycin, vancomycin-nitrobenzofuran (NBD) ( 47 , 59 ). Upon incubation in vancomycin-NBD at a concentration of 16 µg mL -1 (i.e. 8× the MIC value of vancomycin-NBD against S. aureus ) in M9 minimal medium for 1 h, we found similar distributions of vancomycin-NBD single-cell fluorescence for 10 h old mono-cultures of S. aureus ATCC 25923 and S. aureus SH1000 mCherry; vancomycin-NBD did not accumulate instead in 10 h old mono-cultures of P. aeruginosa PA14 (Figure S16A and Data A in S19 File). As expected, S. aureus SH1000 mCherry displayed ECD fluorescence levels distinguishable from M9 minimal medium signal, whereas neither S. aureus ATCC25923 nor P. aeruginosa PA14 displayed ECD fluorescence levels distinguishable from M9 minimal medium (Figure S16B). Next, we incubated 10 h old co-cultures of S. aureus SH1000 mCherry and P. aeruginosa PA14 in vancomycin-NBD at a concentration of 16 µg mL -1 in M9 minimal medium. By simultaneously using the FITC and ECD flow cytometer detectors, we gated only cells that expressed mCherry at levels distinguishable from M9 minimal medium, i.e. S. aureus SH1000 mCherry cells ( Figure 4C and S16). We compared the distributions of vancomycin-NBD single-cell fluorescence for gated S. aureus SH1000 mCherry cells from 10 h old co-cultures and 10 h old mono-cultures after 20 min, 40 min and 60 min incubation in vancomycin-NBD at a concentration of 16 µg mL -1 in M9 minimal medium. According to our hypothesis above, we found that S. aureus SH1000 mCherry cells from 10 h old co-cultures accumulated significantly lower levels of vancomycin-NBD compared to S. aureus SH1000 mCherry cells from 10 h old co-cultures ( Figure 4C , Figure 4D , Figure S17 and S19 File). Moreover, using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry we found a further increase in S. aureus PG lipid expression in both mono-cultures and co-cultures treated with vancomycin ( Table 2 ). These data suggest that the upregulation of charged PG lipids is a strategy employed by S. aureus to counteract exposure to either P. aeruginosa or vancomycin, indirectly suggesting that both stressors elicit a similar response in S. aureus in terms of membrane lipid composition. View this table: View inline View popup Download powerpoint Table 2. Lipids differentially regulated after vancomycin treatment . Lipid class, fatty acid composition, adduct type, retention time (RT), theoretical and experimental mass-to-charge ratio (m/z), molecular formula, variable importance in projection (VIP) score, and mass error (Δppm) measured via quantitative time-of-flight liquid chromatography mass spectrometry. Taken together, these data demonstrate that the synergistic killing or growth inhibitory effect of vancomycin and P. aeruginosa is not due to increased accumulation of vancomycin in S. aureus in the presence of P. aeruginosa . The combined effect of vancomycin and P. aeruginosa induces S. aureus in a viable but non culturable state Next, we set out to determine whether the decline in the S. aureus population measured in the presence of P. aeruginosa and vancomycin is due to killing of S. aureus cells or S. aureus cells entering in a viable but non-culturable state. As above, we employed our single-cell microfluidics-based platform to image and track the fate of individual S. aureus harvested from 10 h old well-mixed mono-cultures or individual S. aureus and P. aeruginosa cells harvested from 10 h old well-mixed co-cultures. We exposed these bacteria, while hosted within microfluidic chambers, first to vancomycin dissolved in LB medium at its MIC value against S. aureus , i.e. 1 µg mL -1 , for 4 h and then to LB medium for 3 h. The growth of S. aureus harvested from 10 h old well-mixed mono-cultures was highly heterogeneous: in two of the twelve microfluidic chambers imaged across three biological replicates, we recorded sustained bacterial doubling both during exposure to vancomycin and during exposure to LB medium; in three chambers we recorded sustained doubling only during exposure to LB medium; in three chambers we recorded only sporadic bacterial doubling or no doubling at all; in four chambers we recorded sporadic growth during exposure to vancomycin followed by cell lysis during exposure to LB medium or cells stained by PI at the end of the experiment (Figure S18A and Data A in S20 File). Overall, 11% of the cells within the initial S. aureus population were growing and dividing by the end of the experiment, 52% of the S. aureus cells were in a motile but non-dividing state and 37% of the S. aureus cells either lysed or stained with PI at the end of the experiments ( Figure 4F and Data B in S20 File). As a result, the S. aureus population dynamics was characterised by very slow and sporadic growth during exposure to vancomycin from 24 ± 13 bacteria per chamber at t = 0 to 33 ± 17 bacteria per chamber at t = 4 h ( Figure 4G and Data A S20 File) with an average lag time of 44 min followed by an average doubling time of 180 min (Figure S19). During the subsequent exposure to LB, the S. aureus population dynamics was characterised by faster but heterogeneous growth up to 87 ± 149 bacteria per chamber at t = 7 h ( Figure 4G and Data A S20 File) and an average doubling time of 32 min (Figure S19). This population dynamics is in accordance with the one we recorded via bulk CFU assays ( Figure 4B ) that did not allow us, however, to capture the non-dividing S. aureus phenotype and the large heterogeneity in growth during and after treatment with vancomycin. S. aureus harvested from 10 h old well-mixed co-cultures did not display any bacterial doubling across the twelve imaged microfluidic chambers during exposure to vancomycin; sustained doubling during the following exposure to LB medium was recorded only in three chambers, whereas the dominant phenotype observed in the remaining chambers was a motile but non-dividing state of S. aureus that did not stain with PI (Figure S18 B and Data C in S20 File). Overall, 9% of the cells within the initial S. aureus population were growing and dividing by the end of the experiment, 79% of the S. aureus cells were in a motile but non-dividing state and 12% of the S. aureus cells either lysed or stained with PI at the end of the experiments ( Figure 4F and Data B in S20 File). As a result, S. aureus population dynamics was characterised by no growth during exposure to vancomycin ( Figure 4G and Data C in S20 File) with an average lag time of around 6 h (hence regrowth started only after vancomycin had been removed, Figure S19). During the subsequent exposure to LB, the S. aureus population dynamics was characterised by limited growth with an overall increase from 9 ± 5 bacteria per chamber at t = 0 to 12 ± 8 bacteria per chamber at t = 7 h ( Figure 4G and Data C in S20 File) and an average doubling time of 34 min (Figure S19). This population dynamics is different with respect to the one we recorded via bulk CFU assays showing that in the presence of P. aeruginosa a 4 h treatment with vancomycin at its MIC value against S. aureus , causes a 1-log decline in the S. aureus population ( Figure 4B ). It is conceivable that the decline observed in our bulk assays is not due cell death but to a proportion of the population entering a viable but non-culturable state that we can capture via microfluidics-microscopy but that is undetectable via CFU assays. However, it is also conceivable that these differences are due to decreased concentrations of LasA, or other molecules secreted by P. aeruginosa , in the open microfluidic chambers (where fresh nutrients are constantly supplied, secreted bacterial molecules are washed away and P. aeruginosa can escape, see below) compared to the closed well-mixed flasks (where secreted bacterial molecules and P. aeruginosa cannot escape the flasks). During exposure to vancomycin, P. aeruginosa harvested from 10 h old well-mixed co-cultures displayed growth comparable to that recorded in the absence of vancomycin ( Figure 4G and Figure 1G , respectively). P. aeruginosa population expanded during the first 2 h of exposure to vancomycin and decreased from the 2 h time point onwards in all imaged chambers (Figure S18C and Data E in S20 File) due to increased cellular motility, possibly via type IV pili( 43 ), and escape out of the chambers. As a consequence, the ratio of P. aeruginosa cell number over S. aureus cell number per microfluidic chamber was 27 ± 23 at t = 0, it increased to a maximum of 58 ± 49 after 2 h and then decreased down to a minimum of 15 ± 15 at t = 7 h (Figure S18D). Discussion S. aureus population decline in the presence of P. aeruginosa is conserved across a diverse set of S. aureus and P. aeruginosa strains S. aureus population initial expansion followed by a steady decline in the continued presence of P. aeruginosa had been previously reported. Specifically, planktonic and biofilm S. aureus 8325-4 displayed a population decline after 6 h or 14 h co-culture with P. aeruginosa PA14 on monolayers of cystic fibrosis bronchial epithelial airway cells or plastic, respectively ( 25 ). Furthermore, planktonic S. aureus Newman displayed a population decline after 4 h co-culture with P. aeruginosa PA14 in microtiter plates ( 46 ). S. aureus JE2 also displayed a population decline after 12 h co-culture with P. aeruginosa PAO1 on agar plates ( 28 ), whereas S. aureus SA2597 displayed a population decline after 4 h co-culture with P. aeruginosa PA2596 on agar plates ( 29 ). We further demonstrate the generality of these population dynamics by showing that planktonic S. aureus ATCC 25923, RYC157 and RYC165 displayed a population decline after 9 or 10 h co-culture with P. aeruginosa PA14. However, S. aureus RYC157 displayed a population decline only after 20 h of co-culture with P. aeruginosa RYC157, in accordance with previous data suggesting that P. aeruginosa isolates from co-infected CF patients are less antagonistic towards S. aureus than isolates from mono-infected CF patients ( 23 ). The dynamics of these microbial interactions have been generally rationalised using Lotka-Volterra pairwise models which are based on the fitness effect of such interactions ( 60 , 61 ). However, these pairwise models often perform less well than mechanistic models in terms of capturing diverse microbial interactions ( 62 ). We introduce a simplified mechanistic model that allows us to capture the population dynamics of S. aureus and P. aeruginosa both in mono- and co-culture and to output parameter estimates that might allow, in future studies, to explain the time difference in the onset of S. aureus decline across different strains, media and environmental setups. S. aureus enters a viable but non-culturable state in the presence of P. aeruginosa The observed S. aureus population decline could be due either to killing of S. aureus by P. aeruginosa or S. aureus entering a viable but non-culturable state or a persister state with very long lag time ( 63 – 65 ) and therefore becoming undetectable via colony forming unit assays. The viable but non-culturable state is a ubiquitous survival strategy adopted by many bacteria ( 66 , 67 ), including S. aureus ( 68 – 70 ), in response to adverse environmental conditions such as starvation ( 39 , 71 ), cold stress ( 72 ), host-induced oxidative stress and ATP depletion ( 73 ), or antibiotic treatment ( 69 , 74 ). Intriguingly, S. aureus in a viable but non-culturable state has been recently detected via qPCR in the sputum of cystic fibrosis patients concurrently with poor lung functionality ( 75 ) and in biofilm samples from central venous catheters ( 76 ). Furthermore, recent data suggested that populations of S. aureus ATCC 25923 and of S. aureus clinical isolates entered a viable but non-culturable state after 48 h co-culture in dual-species biofilms with P. aeruginosa PA14 ( 77 ). Here we show that a subpopulation of S. aureus enters a viable but non-culturable state already after 10 h of planktonic co-culture with P. aeruginosa and that individual viable but non-culturable S. aureus cells remain in a motile non-dividing state for at least a further 9 h, whereas other clonal S. aureus cells resume growth. Consistent with these data we also observed that S. aureus cells that resumed growth displayed a longer lag time compared to S. aureus cells harvested from mono-culture and displayed an overall more heterogeneous growth. These findings are consistent with previously observed heterogeneity in the activation of S. aureus stationary markers and persister formation ( 78 ) and previous data suggesting that several S. aureus isolates are growth inhibited but not killed by P. aeruginosa ( 28 ). Our single-cell microscopy data suggest that S. aureus cell death and lysis plays a limited role in the S. aureus population decline observed in the presence of P. aeruginosa in our study and possibly also in previous studies using bulk microbiological assays ( 25 , 28 , 46 ). The observed population decline in the presence of P. aeruginosa is instead primarily due to S. aureus entering a viable but non-culturable state leading to a possible underestimation or non-detection in clinical samples which could constitute a serious risk to human health. Future studies should investigate whether the presence of P. aeruginosa cells is necessary to induce S. aureus in a viable but non-culturable state or whether P. aeruginosa supernatants are sufficient for this, considering that a previous study at the population-level showed that P. aeruginosa supernatants have an impact on the growth of S. aureus ( 31 ). Similarities between S. aureus viable but non-culturable cells in the presence of P. aeruginosa and S. aureus intracellular persisters Consistent with our data showing that S. aureus enters a viable but non-culturable state, our RNA-sequencing analysis demonstrates that in the presence of P. aeruginosa , S. aureus drastically rewires its transcriptome and downregulates key metabolic processes including nucleoside metabolism, nitrogen metabolism and transport, amino acid biosynthesis, biogenesis and cellular organization, while upregulating cellular catabolism and protein maturation. These molecular processes were reprogrammed in response to an increase in molecular transport, particularly iron, DNA-templated transcription initiation, protein repair and carbohydrate biosynthesis in P. aeruginosa , with a downregulation of carbon and nitrogen metabolism. The observed downregulation of nitrogen metabolism in both S. aureus and P. aeruginosa is consistent with previous transcriptomic data highlighting nitrogen starvation as a co-culture specific response in both P. aeruginosa and S. aureus with the upregulation of the master nitrogen regulator ntrC and the downregulation of gltB and gltD , coding for glutamine to glutamate conversion ( 46 ). Dysregulation of genes involved in major metabolism pathways of carbohydrates and amino acids, such as acsA which encodes the acetyl-coenzyme A synthetase, was also reported for S. aureus SA2597 co-cultured with P. aeruginosa PA2596 ( 29 ). We found some important similarities between the transcriptomic profile of S. aureus in co-culture with P. aeruginosa and S. aureus intracellular persisters treated with oxacillin within macrophages. Intracellular persisters down regulated the biosynthesis of key amino acids, such as valine, leucine, isoleucine, lysine and tryptophan ( 53 ). Accordingly, we found down regulation of genes encoding lysine biosynthesis, dapA-D , glutamine biosynthesis, glnA , valine and isoleucine biosynthesis, ilvD , in S. aureus co-cultured with P. aeruginosa . Moreover, intracellular persisters induced the heat shock stimulon, including genes dnaK , grpE , clpB , groEL and groES ( 53 ) that participate in the degradation of defective proteins ( 79 ). Accordingly, we found upregulation of these and other protein maturation genes in S. aureus co-cultured with P. aeruginosa but we did not find evidence of dark foci induced by protein aggregation ( 42 , 52 , 73 , 80 ). Intracellular S. aureus persisters also displayed ATP levels comparable to control S. aureus cells ( 53 ). Similarly, we did not find evidence of strong downregulation of genes involved in energetic metabolism and redox reactions (e.g. the L-lactate dehydrogenase or the NADP-dependent isocitrate dehydrogenase) in S. aureus in co-culture with P. aeruginosa , in accordance with a previous study ( 29 ). These data are instead different from previous findings that P. aeruginosa inhibits S. aureus respiration inducing a low ATP state ( 6 ) and that persister formation in S. aureus is associated with ATP depletion ( 78 ). We also found upregulation of the codY regulon that is de-repressed under amino acid starvation ( 81 ) and upon S. aureus uptake in macrophages, playing a role in intracellular persistence through ppGpp regulation ( 53 ). Intracellular S. aureus persisters also displayed dysregulated but active protein synthesis: aminoacyl-tRNA synthetase encoding genes were downregulated, whereas members of the protein synthesis machinery and ribosome recycling were upregulated( 53 ). Similarly, we found a downregulation of aminoacyl-tRNA synthetase encoding genes such as thiL , mnmA or trnB in co-culture with P. aeruginosa and a slight upregulation of elongation factors infAB and ribosome recycling gene frr . Moreover, we observed reduced but active expression of mCherry in viable but non-culturable S. aureus in co-culture with P. aeruginosa , similarly to reduced but active expression of GFP in intracellular persisters ( 53 ). Taken together these data suggest that viable but non-culturable S. aureus in co-culture with P. aeruginosa adopt a survival lifestyle that resembles the one adopted by intracellular S. aureus persisters ( 53 ). Entrance in a viable but non-culturable state could also be partly due to inhibition of the electron transport chain of S. aureus via the downregulation of qoxA-D encoding the cytochrome aa3 quinol oxidase and the upregulation of the narK1 antiporter gene, in accordance with a previous study on the early responses between P. aeruginosa PA14 and S. aureus Newman ( 46 ) and a previous study on S. aureus intracellular persisters( 53 ). However, we also found upregulation of cytochrome C oxidase genes coxAB , in contrast with a previous study ( 46 ). We also found increased expression of the lactose operon lacA-G in S. aureus co-cultured with P. aeruginosa , similarly to what observed for intracellular S. aureus persisters ( 53 ); however, we did not find a significant downregulation of glucose metabolism enzyme encoding genes (a part from gap that was strongly downregulated), whereas previous work suggested that a carbon source shift between glucose and lactose is a trigger factor for intracellular persistence ( 82 ). Moreover, we did not find strong evidence of increased expression of genes encoding enzymes for fermentation in S. aureus such as pflAB , ldh , adhEP , gap , tpiA . In contrast, two previous studies that investigated genome-wide gene expression profile changes in S. aureus in coculture for 6 h or 3 h with P. aeruginosa found increased expression of genes encoding enzymes for fermentation in S. aureus ( 25 , 46 ). Similarly, L-lactate dehydrogenase lldA was the most upregulated gene of P. aeruginosa in response to S. aureus Newman lactate secretion ( 46 ), whereas according to our transcriptomic data it was only slightly upregulated after both 3 h and 10 h co-culture, consistent with reduced lactate secretion from S. aureus . Therefore, our transcriptomic data do not fully support that S. aureus entrance in a viable but non-culturable state in the presence of P. aeruginosa is linked to inhibition of the electron transport chain of S. aureus or a switch from glucose to lactose metabolism or a switch from respiration to fermentation in S. aureus . Finally, our data suggest that P. aeruginosa -produced HQNO, rhamnolipids and siderophores might not underlie the S. aureus population decline that we observe in our culture conditions. In fact, coculture with Δ pvdA Δ pchE or Δ pqsA P. aeruginosa strains (that lack synthesis of pyoverdine and pyochelin or the production of HQNO, respectively) markedly reduced P. aeruginosa -mediated killing of S. aureus 8325-4 biofilms ( 25 ). In contrast, we found that coculture with the Δ pvdA Δ pchEF strain did not reduce S. aureus population decline. Moreover, we found that the pyoverdine, rhamnolipid and HQNO pathways were upregulated to similar levels after 10 h of mono- or coculture. In contrast, the pyochelin pathway, DNA-templated transcription initiation, including the sigma factors pvdS and rpoD , and dicarboxylic acid metabolism processes were strongly upregulated in co-culture with S. aureus but strongly downregulated in mono-culture, thus suggesting that the continued presence of S. aureus induces these processes in P. aeruginosa . Our data also suggest downregulation of genes involved in intracellular accumulation of purine in S. aureus , such as the uracil transporter PyrP, the pyrimidine de novo synthesis operon pyrPEBCAA - carB - pyrF , and the purine de novo synthesis gene purA . In contrast, a previous study suggested that these genes were among the most highly induced in S. aureus in co-culture compared to mono-culture ( 46 ). Taken together these data demonstrate that multiple pathways contribute to a different extent to the generation and maintenance of S. aureus viable but non-culturable cells in the presence of P. aeruginosa and that S. aureus adopts a survival lifestyle that resembles the one adopted by intracellular S. aureus persisters ( 53 ). However, we acknowledge that our transcriptomic data are acquired from S. aureus populations that contain a mixture of viable but non-culturable cells and growing cells and that our data do not take into account post-translational modifications that represent a second control point to survive external stressors. Differential efflux regulation contributes to ciprofloxacin decreased efficacy against S. aureus in the presence of P. aeruginosa Ciprofloxacin treatment was less effective against stationary phase compared to exponential phase S. aureus or P. aeruginosa mono-cultures in line with previous findings and possibly due to a larger subset of bacteria being in a non-replicating and low metabolically active state in the stationary phase of growth ( 49 , 78 , 83 – 86 ). In line with this hypothesis, ciprofloxacin treatment became even less effective against S. aureus in co-culture with P. aeruginosa compared to S. aureus mono-culture, due to a subpopulation of S. aureus entering a viable but non-culturable state. Reduced ciprofloxacin efficacy against S. aureus while in co-culture with P. aeruginosa is in accordance with a previous study reporting that the supernatants of several P. aeruginosa isolates were antagonistic towards ciprofloxacin killing ( 6 ). Increased S. aureus survival to ciprofloxacin was due to the downregulation of the cellular targets of ciprofloxacin, i.e. the DNA gyrase GyrAB and the topoisomerase IV ParCE; the downregulation of genes that were previously found to be downregulated in S. aureus persisters hosted by high-oxidative-stress cells ( 73 ) or induced by reactive oxygen species ( 87 ), i.e. rplA , rplM , atpA , prfA and sdhAB ; the upregulation of the NorA, NorB LmrS, SepA, Tet( 38 ) and SbnD efflux pumps which are known to extrude ciprofloxacin and other quinolones ( 88 ) in S. aureus , while in co-culture with P. aeruginosa compared to mono-culture as recorded in our transcriptomic data. These data were also consistent with previous findings that while in co-culture with P. aeruginosa , S. aureus upregulates efflux pumps belonging to the Nor family leading to an increase in resistance of S. aureus to tetracycline and ciprofloxacin ( 29 ). Alternatively, HQNO secreted by P. aeruginosa could inhibit S. aureus respiration inducing a low ATP, multidrug-tolerant state in S. aureus ( 6 ). However, we observed limited induction of the HQNO pathway in P. aeruginosa both in mono- and co-cultures and we did not observe HQNO-induced inhibition of S. aureus respiration ( 6 ). Finally, our single-cell analysis revealed that both S. aureus and P. aeruginosa grew and doubled during exposure to ciprofloxacin and started to lyse only during the subsequent exposure to LB medium, leading to a significant decrease in both bacterial populations which was primarily due to cell death rather than entrance in a viable but non-culturable state. Vancomycin reduced accumulation in viable but non culturable S. aureus in the presence of P. aeruginosa Vancomycin had a moderate efficacy against exponential phase S. aureus and was not effective against stationary phase S. aureus in accordance with previous clinical data( 89 ). In contrast, S. aureus displayed a 1-log or a 5-log reduction in survival to vancomycin treatment at its MIC after co-culture with P. aeruginosa PA14 for 3 h or 10 h, respectively, according to our time-killing assays. These data are in agreement with a previous study that investigated vancomycin efficacy against exponential phase S. aureus HG003 exposed to the supernatants of several P. aeruginosa strains and found a 2-log reduction in survival to vancomycin treatment after exposure to P. aeruginosa PA14 supernatant ( 6 ). This study also found that vancomycin killing of S. aureus was mediated by P. aeruginosa extracellular lytic enzyme, LasA, that cleaves pentaglycine cross bridges in S. aureus peptidoglycan and has been shown to attack the cell wall of S. aureus during in vivo competition ( 90 ). In line with these data we found that lasAB were strongly upregulated at t = 10 h compared to t = 3 h both in mono- and co-culture. Our data therefore further corroborate the hypothesis that LasA mediates population decline of S. aureus in co-culture with P. aeruginosa and demonstrate that LasA secretion is not induced by the presence of S. aureus . Our single-cell analyses, however, favour the hypothesis that the observed population decline of S. aureus under vancomycin treatment in co-culture is due to S. aureus entering a viable but non-culturable state rather than being killed, since we observed limited S. aureus cell death in co-culture in the presence of vancomycin within the timeframe of our experiments. We also found that vancomycin accumulation in S. aureus is reduced in co-culture compared to mono-culture, which is consistent with S. aureus entrance in a viable but non-culturable state, with a possible downregulation of cell wall turnover which is targeted by vancomycin. Accordingly, our transcriptomic analysis revealed upregulation of cell wall induction stimulon in co-culture compared to mono-culture, including the two-component regulatory system VraS/VraR involved in the control of the cell wall peptidoglycan biosynthesis ( 91 ), as previously observed for intracellular persisters ( 53 ). We also measured downregulation of penicillin-binding protein 4 (Pbp4) that has transpeptidase and carboxypeptidase activities and catalyzes the final step in peptidoglycan synthesis ( 92 ). Moreover, our lipidomic analysis revealed changes in the S. aureus membrane, with an increase in PG lipids and a decrease in PC lipids, in co-culture compared to mono-culture. PG lipids were further upregulated in co-cultures treated with vancomycin, sequestering positively charged vancomycin molecules ( 57 ) and hindering their translocation through the membrane and their binding to the D-alanyl-D-alanine peptide motif of the peptidoglycan precursor ( 58 ). These alterations in membrane lipid composition may play a critical role in modulating membrane rigidity and permeability, ultimately shaping S. aureus response to antibiotic treatment. Additionally, HQNO, pyoverdine and pyochelin secreted by P. aeruginosa could also decrease vancomycin accumulation in S. aureus by slowing down cellular growth. However, we observed limited induction of the HQNO pathway in P. aeruginosa both in mono- and co-cultures and we observed neither HQNO-induced S. aureus shift to fermentative growth ( 31 ) nor HQNO-induced inhibition of S. aureus respiration ( 6 ). Our findings demonstrate that S. aureus population decline in the presence of P. aeruginosa is primarily due to a subpopulation of S. aureus entering in a viable but non-culturable state that cannot be detected via traditional microbiology and antibiotic susceptibility assays. Failure to detect these subpopulations could lead to an underestimation or non-detection of S. aureus in clinical samples, which would constitute a serious risk to human health, particularly in chronic infections, since entrance in a viable but non culturable state is the primary survival strategy of S. aureus in response to vancomycin treatment. Materials and Methods Chemicals and bacterial strains All chemicals were purchased from Merck unless otherwise stated. LB medium (10 g L -1 tryptone, 5 g L -1 yeast extract and 10 g L -1 NaCl) and LB agar plates (LB with 15 g L -1 agar) were used for planktonic growth and streak plates as previously described ( 93 ). Selective media for P. aeruginosa was Pseudomonas isolation agar (PIA; Merck product number: 17208), while selective medium for S. aureus was Trypticase soy agar (TSA; 22091) with added 7.5% NaCl, called Staphylococcus isolation agar (SIA) ( 28 ). Both PIA and SIA were prepared following the protocols provided by the producer. Carbon-free M9-minimal medium (M6030) used for flow cytometry was prepared using 100 mL of 5× M9 minimal salts, with an additional 1 mL of 1 M MgSO 4 and 50 μL of 1 M CaCl 2 and make up to a volume of 500 mL using Milli-Q water. M9 was then filtered through a 0.22 μm Minisart® Syringe Filter (Sartorius, Germany). Phosphate buffered saline solution (PBS; 4417) was prepared by dissolving one tablet in 200 mL of Milli-Q water, autoclaved and used for growth and killing assays. Ciprofloxacin (17850) stock solution was obtained by dissolving antibiotic powder in 0.1 M HCl at a concentration of 10 mg mL -1 and then diluted with LB to the required experimental concentration. Vancomycin (94747) stock solution was obtained by dissolving powder in Milli-Q water at a concentration of 10 mg mL -1 and then diluted with LB to required experimental concentration. Vancomycin fluorescent analogue was designed and synthesised based on structure-activity-relationship studies and protocols reported in prior publications ( 59 , 94 ), introducing an azidolysine residue for the subsequent ‘click’ reaction with nitrobenzoxadiazole (NBD)-alkyne ( 95 ). S. aureus ATCC 25923 and P. aeruginosa PA14 were purchased from Dharmacon™ (Horizon Discovery, UK). S. aureus SH1000 mCherry was kindly provided by Prof Simon Foster, University of Sheffield, P. aeruginosa PA14 flgK ::Tn5B30(Tc) was kindly provided by Prof George O’Toole, Dartmouth College, USA, P. aeruginosa PAO1 and PAO1 Δ pvdD Δ pchEF were kindly provided by Prof Pierre Cornelis, Universiteit Brussels, S. aureus and P. aeruginosa clinical isolates RYC157 and RYC165 were provided by Dr Maria Garcia and Prof Rafael Canton, IRYCIS, Madrid. All strains were stored in a 50% glycerol, 50% Milli-Q water stock at -70 °C. Streak plates for each strain were produced by thawing a small aliquot of the corresponding glycerol stock every week and plated onto LB agar plates. Mono- and co-culture growth assays Overnight S. aureus (strain ATCC25923, RYC157 or RYC165) or P. aeruginosa (strain PA14 or RYC157) mono-cultures were prepared by inoculating 100 mL fresh LB medium with a single bacterial colony from a streak plate and incubated on a shaking platform at 200 rpm and 37 °C for 17 h. S. aureus and P. aeruginosa mono-cultures and co-cultures for growth assays were prepared by transferring 100 μL of S. aureus or 100 μL P. aeruginosa (or both together for co-culture) overnight mono-cultures into 100 mL LB and incubated on a shaking platform at 200 rpm and 37 °C for 13 h while monitoring bacterial growth hourly. To do so, each culture was sampled every hour in triplicate by taking three 500 µL aliquots, these aliquots were centrifuged at 13000 rpm for 5 min, the supernatant was removed, each pellet was resuspended in PBS and serial dilutions were plated using 10 uL droplets on LB agar plates or PIA, TSA agar plates for the isolation of P. aeruginosa and S. aureus , respectively ( 28 ) and colony forming units (CFU) counted. For both mono-cultures and co-cultures growth assays all the plates after bacterial plating were dried and incubate for 16 h at 37 °C, before counting CFU. Determination of minimum inhibitory concentration Single S. aureus or P. aeruginosa bacterial colonies were picked and cultured overnight in LB at 37 °C for 17 h, then diluted 40-fold (by adding 1 mL of overnight culture to 39 mL of LB) and grown at 37 °C on a shaking platform at 200 rpm to an OD 600 of 0.5. The mid-log phase cultures (i.e. OD 600 = 0.5) were diluted to 10 6 CFU by removing from 15 mL of LB 30 µL and replacing with 30 µL of exponential phase bacterial solution. 90 µL of LB were added to the first column, and 50 µL to all other wells of a 96-well plate. 10 µL of 1.28 mg mL -1 of each unlabelled antibiotic or fluorescent antibiotic derivative stocks were added to the first column of the 96 well plate. 50 µL were then withdrawn from the first column and serially transferred to the next column until 50 µL withdrawn from the last column was discharged. After this procedure all wells contained 50 µL of antibiotic in LB solution. 50 µL of exponential phase bacterial solution was then added to each well, to give a final concentration of 5×10 5 CFU mL -1 . Each antibiotic in LB was tested in triplicates (three rows of a 96 well plate). Each plate also contained two rows of 12 positive control experiments (i.e. bacteria in LB without antibiotics added) and two rows of 12 negative control experiments (i.e LB only). Plates were covered with aluminium foil and incubated at 37 °C overnight. The minimum inhibitory concentration (MIC) of each antibiotic was determined visually, with the MIC being the lowest concentration well with no visible growth compared to the positive control experiments ( 96 ). Time-kill assays Exponential (i.e. 3 h) or stationary phase (i.e. 10 h) S. aureus (strain ATCC25923) or P. aeruginosa (strain PA14) mono- or co-cultures were prepared as described above. Four aliquots of each mono- or co-culture were diluted to an OD of 0.03 in 50 mL of LB in four separate flasks. The four flasks were challenged by using four different ciprofloxacin or vancomycin concentrations, i.e. 0×, 1×, 10×, or 25× the MIC value measured against S. aureus . In order to perform CFU counting we withdrew three 500 µL aliquots from each flask at t = 0, 1, 2, 3, 4 and 5 h after the addition of the antibiotic and added them to Eppendorf tubes that were incubated at 200 rpm at 37 °C. At each time point three aliquots were centrifuged at 13000 rpm for 5 min, the supernatant was then removed, each pellet was resuspended in PBS and serial dilutions (made in the 96-well plate) were plated using 10 uL droplets on LB agar plates or PIA, TSA agar plates for the isolation of P. aeruginosa and S. aureus , respectively ( 28 ). Determination of CFU was obtained after overnight incubation of each plate at 37 °C. Means and standard deviations of measurements carried out in biological triplicate were calculated and graphs plotted using GraphPad Prism 10. Fabrication of microfluidic devices The mould for the mother machine microfluidic device was fabricated using previously established multi-layer photolithography processes ( 97 , 98 ). This mould is equipped with twenty 1 µm high circular chambers with a diameter of 150 µm. These chambers are connected to a main delivery chamber via channels of different width in range from 20 to 50 µm. The main delivery chamber has a height of 20 µm and a width of 200 µm. Polydimethylsiloxane (PDMS) replicas of this device were realised as previously described ( 99 ). Briefly, a 10:1 (base:curing agent) PDMS mixture was cast on the mould and cured at 70 °C for 120 min in an oven ( 100 ). The cured PDMS was peeled from the epoxy mould and fluidic accesses were created by using a 0.75 mm biopsy punch (Harris Uni-Core, WPI). The PDMS chip was washed with absolute ethanol and dried with a nitrogen stream the day before the experiment. The chip was then irreversibly sealed on a glass coverslip (22×50 mm, 0.13-0.17 mm thick, Fisher) by exposing both the chip and glass surfaces to oxygen plasma treatment (10 s exposure to 30 W plasma power, Plasma etcher, Diener, Royal Oak, MI, USA) on the day of the experiment as previously described ( 101 ). We have also made available a step-by-step experimental protocol for the fabrication and handling of microfluidic devices for investigating the interactions between antibiotics and individual bacteria ( 102 ). Microfluidics-based time-lapse microscopy Overnight S. aureus ATCC25923 or SH1000 mCherry and P. aeruginosa PA14 Δ flgK mono-cultures or co-cultures were prepared as described above. A 50 mL aliquot of each culture was centrifuged for 10 min at 4000 rpm and 20 °C. The supernatant was filtered twice (Medical Millex-GS Filter, 0.22 μm, Millipore Corp.) to remove bacterial debris from the solution and used to resuspend the bacteria in their spent LB to an OD 600 of 10 as previously described( 103 ). A 10 μL aliquot of this suspension was injected by using pipette in the above described microfluidic device. The high bacterial concentration favours bacteria entering the lateral chambers from the main delivery chamber of the microfluidic device ( 39 ). The lateral chambers were filled seconds after the bacterial injection. The microfluidic device was completed by the integration of fluorinated ethylene propylene tubing (P68514, 1/32” × 0.20mm ID, Restek). The inlet tubing was connected to the inlet reservoir which was connected to a computerised pressure-based flow control system (MFCS-4C, Fluigent). This instrumentation was controlled by MAESFLO software (Fluigent) ( 104 ). The chip was mounted on an inverted microscope (IX73 Olympus, Tokyo, Japan) and the bacteria remaining in the delivery chamber of the microfluidic device were washed into the outlet tubing and into the waste reservoir by flowing LB medium or LB medium containing ciprofloxacin or vancomycin at 200 μL h -1 for 2 min. In the unchallenged mono- and co-culture experiments LB was supplied to the bacteria at 100 μL h -1 for S. aureus mono-cultures and 40 μL h -1 for P. aeruginosa mono-cultures or co-cultures—for 9 h. The lower flow rate for P. aeruginosa mono-cultures or co-cultures was used due to their higher motility compared to S. aureus . In the mono- and co-culture experiments with ciprofloxacin or vancomycin challenge, LB medium containing either ciprofloxacin at a concentration of 0.5 µg mL -1 or vancomycin at a concentration of 1 µg mL -1 was supplied for 4 h at a flow rate of 100 or 40 μL h -1 followed by delivery of LB medium only for 3 h at a flow rate of 100 or 40 μL h -1 . During the change of medium from LB containing antibiotic to LB only the main channel was flushed for 8 min at 200 μL h -1 to ensure that the antibiotic was fully removed from the device. Bright-field images were acquired every 10 min during entire experiment. Images were collected via a 60×, 1.2 N.A. objective (UPLSAPO60XW, Olympus) and a sCMOS camera (Zyla 4.2, Andor, Belfast, UK). The region of interest of the camera was adjusted to visualise one of the lateral chambers in the device and images of four different lateral chambers of the microfluidic device were acquired at each time point. The device was moved by two automated stages (M-545.USC and P-545.3C7, Physik Instrumente, Karlsruhe, Germany, for coarse and fine movements, respectively). A corresponding fluorescence image was acquired by exposing the bacteria for 0.03 s to the green excitation band of a broad-spectrum LED (CoolLED pE300white, maximal power = 200 mW Andover, UK) at 30% of its intensity (with a power associated with the beam light of 8 mW at the sample plane) and a TEXAS RED filter to quantify mCherry fluorescence from each bacterium. Fluorescence images of SH1000 mCherry were acquired every 30 min during the entire experiment, to avoid overexposing the bacteria to the LED light. In the end of all experiments propidium iodide (P1304MP, Invitrogen), was added to the main delivery chamber in order to detect cells with compromised membranes as previously described ( 39 ). The entire assay was carried out at 37 °C in an environmental chamber (Solent Scientific, Portsmouth, UK) surrounding the microscope and microfluidics equipment. Image and data analysis Images were processed using ImageJ software as previously described ( 105 , 106 ), counting and tracking individual S. aureus bacteria throughout each experiment, whereas individual P. aeruginosa bacteria were counted but not tracked at each time point in each experiment due to their higher motility. In order to estimate mCherry fluorescence from individual S. aureus bacteria, a circle was drawn around each bacterium in each bright-field image at every time point. The same circle was then used in the corresponding fluorescence image to measure the mean fluorescence intensity for each bacterium. The same circle was then moved to a section of the same chamber that did not host any bacteria in order to measure the background fluorescence. This mean background fluorescence value was subtracted from the bacterium’s fluorescence value. All data were then analysed and plotted using GraphPad Prism 10. Statistical significance was tested using either paired or unpaired, two-tailed, Welch’s t -test. Pearson correlation, means, standard deviations, coefficients of variation and medians were also calculated using GraphPad Prism 10. Flow cytometric accumulation assays The accumulation of vancomycin-NBD in individual cells from S. aureus ATCC25923 or SH1000 mCherry and P. aeruginosa PA14 Δ flgK mono- or co-cultures were carried out as previously described ( 47 , 95 ). Briefly, overnight S. aureus ATCC25923, SH1000 mCherry and P. aeruginosa PA14 ΔflgK mono-cultures and 10 h subcultures were prepared as described above. 10 h old mono-cultures and co-cultures were then adjusted to an OD 600 of 5 for S. aureus mono-cultures and 2 for co-cultures and P. aeruginosa mono-cultures. 10 h old mono-cultures and co-cultures were transferred to a microcentrifuge tube for centrifugation in a SLS4600 microcentrifuge (Scientific Laboratory Supplies, UK) at 4,000 × g and room temperature for 5 min. The supernatant was removed, and pellets were resuspended in M9 medium (control; samples without any antibiotic) or vancomycin-NBD at a concentration of 16 µg mL -1 by diluting in M9 medium at a final volume of 130 μL. Each sample containing antibiotic was incubated at 37 °C and shaken at 1,000 rpm on a ThermoMixer ® C (Eppendorf, Germany) for 1 h. For control samples (bacterial solutions without any antibiotic), pellets were dissolved in 130 μL of M9 and were then diluted ten times in M9. 2 μL of these samples were then transferred to 96-well plates containing 98 μL of double filtered M9, giving a final dilution of 500 times. For the samples containing vancomycin, 30 μL of such samples were then transferred to microcentrifuge tubes at appropriate time points (20 min, 40 min and 60 min). A washing step was immediately performed on the vancomycin containing aliquots to eliminate any unaccumulated drug via centrifugation at 4000 × g for 5 min and its supernatant removed. Then we added 300 μL of filtered M9 to the pellets to perform a 10 times dilution and added 2 μL of these samples to 98 μL of M9 in wells of a 96 well plate. Flow cytometric measurements were performed on the CytoFLEX S Flow Cytometer (Beckman Coulter, USA) equipped with a 488 nm (50 mW) and a 405 nm (80 mW) laser. Vancomycin-NBD and mCherry fluorescence of individual bacteria was measured using the FITC-A channel (488 nm, 525/40BP) and the ECD channel (488 nm, 610/20BP), respectively. Cell size was measured using SSC-A (forward scatter) and Violet SSC-A (side scatter) channels. Avalanche photodiode gains of FSC: 1,000, SSC: 500, Violet SSC: 1, FITC: 250, ECD 250 and a threshold value of SSC-A: 10,000 to limit background noise was used. Bacteria were gated to separate cells from background noise by plotting SSC-A and Violet SSC-A. Background noise was then further separated based on cellular autofluorescence measured on the FITC-A channel for cells not treated with fluorescent-peptides. An additional gate on the ECD-A channel was then used for cells treated with vancomycin-NBD to further separate background noise from mCherry expressing S. aureus . CytoFLEX Sheath Fluid (Beckman Coulter, USA) was used as sheath fluid. Data was collected using CytExpert software (Beckman Coulter, USA) and exported to FlowJo™ version 10.9 software (BD Biosciences, USA) for analysis. Transcriptomic analysis Exponential (i.e. 3 h) and stationary phase (i.e. 10 h) S. aureus ATCC25923 and P. aeruginosa PA14 Δ flgK mono- or co-cultures were prepared as described above. RNA extractions were performed on biological triplicate of each culture condition as previously described ( 48 ), RNA extractions for stationary phase co-cultures were performed in six biological replicates to compensate for the relative low content of S. aureus with respect to P. aeruginosa cells. Briefly, to make an RNA extraction from one biological replicate, 100 mL of 3 h or 10 h old bacterial cultures were centrifuged at 4000 rpm for 15 min. The supernatant was then removed. 100 μL of bacterial solution was then transferred into an RNase free tube and 200 μL of RNA-protect bacteria reagent was added, vortexed for 5 s and then incubated for 5 min at room temperature. The sample was then centrifuged at 5000 g for 10 min and the supernatant was removed. Enzymatic lysis of S. aureus bacteria was carried out using lysostaphin stock (2 mg mL -1 ) in TE buffer. Briefly, 200 μL of 100 μg mL -1 lysostaphin was added to the bacterial pellet, vortexed and incubated for 1 h at 37 °C. Then the sample was vortexed again, proteinase K was added and the sample was incubated for 10 min at 37 °C, while vortexing every 2 min. Enzymatic lysis of P. aeruginosa bacteria was carried out using lysozyme at a concentration of 15 mg mL -1 in TE buffer and proteinase K digestion of bacteria was performed following protocol 4 in the RNAprotect ® Bacteria Reagent Handbook (Qiagen, Germany). Purification of total RNA from bacterial lysate using the RNeasy ® Mini Kit (Qiagen, Germany) was carried out following protocol 7 in the handbook. A further on-column DNase digestion using the RNase-Free DNase set (Qiagen, Germany) was performed following appendix B in the handbook. RNA concentration and quality were assessed using the Agilent High Sensitivity RNA ScreenTape System (Agilent, USA) following the provided protocol. Samples for the analysis were normalised to a mean RNA concentration of 40 ng μL -1 and the measured mean RNA integrity number equivalent (RIN e ) score was 9.1. To start the library preparation, we used 400 ng total RNA. rRNA depletion was performed with the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Microbiome kit (Illumina, USA) following the manufacturer’s instructions. cDNA libraries were compiled using as well the Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus Microbiome kit (Illumina, USA). Transcript abundance was quantified using Salmon for each gene in all samples. Differential gene expression was performed with DESeq2 in R software to quantify the log 2 fold-change in transcript reads ( 49 ) for each gene and subsequent principal component analysis using DESeq2 and a built-in R method (prcomp) ( 45 ). Gene ontology enrichment analysis was performed using the clusterProfiler package (version 4.10.0) for R ( 107 , 108 ). Enrichment in terms belonging to the “Biological Process” ontology was calculated for each gene cluster, relative to the set of all genes quantified in the experiment, via a one-sided Fisher exact test (hypergeometric test). P values were adjusted for false discovery by using the method of Benjamini and Hochberg. Finally, the lists of significantly enriched terms were simplified to remove redundant terms, as assessed via their semantic similarity to other enriched terms, using clusterProfiler’s simplify function. RT and qPCR One hundred nanograms of RNA were used for reverse transcription (RT) with random hexamer primers, following the protocol of the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). No-RT controls were included for all samples to confirm that transcript levels were not due to residual DNA. The RT reaction was carried out at 25 °C for 10 minutes for primer annealing, followed by cDNA synthesis at 37 °C for 120 minutes and termination at 85 °C for 5 minutes in a final volume of 20 µL. The resulting cDNA samples were diluted 5-fold in 10 mM Tris-HCl (pH 8) and stored at −20 °C prior to qPCR analysis. Real-time qPCR reactions were performed in a total volume of 20 µL containing 1X PowerUp™ SYBR Green Master Mix (Applied Biosystems), 500 nM of each forward and reverse primer (Supplementary Table X), and 5 ng of template cDNA. Amplification was conducted on a QuantStudio 1 Real-Time PCR system (Thermo Fisher Scientific) using the following cycling conditions: an initial uracil-DNA glycosylase (UDG) activation step at 50 °C for 2 minutes, followed by DNA polymerase activation at 95 °C for 2 minutes. PCR cycling consisted of 35 cycles of denaturation at 95 °C for 15 seconds and combined annealing/extension at 60 °C for 1 minute. At the end of amplification, a melt curve analysis was conducted by increasing the temperature from 60 °C to 95 °C at a rate of 0.1 °C/second, with continuous fluorescence data acquisition. Threshold cycle (Ct) values were determined using the Design & Analysis (DA2) software v2.6.0 (Thermo Fisher Scientific). Changes in gene expression levels (log2 fold change) were calculated using the ΔΔCt method, with an internal control gene selected for normalization for each bacterial strain. Lipid extraction and lipidomic analysis Lipidomic analysis of S. aureus ATCC25923 and P. aeruginosa PA14 mono- or co-cultures untreated or treated with vancomycin at a concentration of 1 µg mL -1 were carried out as previously described ( 47 ). Briefly, lipid extractions were performed following a modified version of the Folch extraction ( 54 , 109 ): 100 μL of sample was added to 250 μL of methanol and 125 μL of chloroform in a microcentrifuge tube; samples were incubated for 60 min and vortexed every 15 min. Then, 380 μL of chloroform and 90 μL of 0.2 M potassium chloride was added to each sample. Cells were centrifuged at 14,000 × g for 10 min to obtain a lipophilic phase which was transferred to a glass vial and dried under a nitrogen stream. The lipophilic phase was then resuspended in 20 μL of a methanol:chloroform solution (1:1 v/v ), then 980 μL of an isopropanol:acetonitrile:water solution (2:1:1, v/v/v ) was added. Samples were analysed on a Agilent 6560 Q-TOF-MS (Agilent, USA) coupled with a Agilent 1290 Infinity II LC system (Agilent, USA). An aliquot of 0.5 μL for positive ionisation mode and 2 μL for negative ionisation mode from each sample was injected into a Kinetex ® 5 μm EVO C18 100 A, 150 mm × 2.1 μm column (Phenomenex, USA). The column was maintained at 50 °C at a flow rate of 0.3 mL min -1 . For the positive ionisation mode, the mobile phases consisted of acetonitrile:water (2:3 v/v ) with ammonium formate (10 mM) and acetonitrile:isopropanol (1:9, v/v ) with ammonium formate (10 mM). For the negative ionisation mode, the mobile phases consisted of (A) acetonitrile:water (2:3 v/v ) with ammonium acetate (10 mM) and acetonitrile:isopropanol (1:9, v/v ) with ammonium acetate (10 mM). The chromatographic separation was obtained with the following gradient: 0-1 min 70% B; 1-3.5 min 86% B; 3.5-10 min 86% B; 10.1-17 min 100% B; 17.1-10 min 70% B. The mass spectrometry platform was equipped with an Agilent Jet Stream Technology Ion Source (Agilent, USA), which was operated in both positive and negative ion modes with the following parameters: gas temperature (200 °C); gas flow (nitrogen), 10 L min -1 ; nebuliser gas (nitrogen), 50 psig; sheath gas temperature (300 °C); sheath gas flow, 12 L min -1 ; capillary voltage 3500 V for positive, and 3000 V for negative; nozzle voltage 0 V; fragmentor 150 V; skimmer 65 V, octupole RF 7550 V; mass range, 40-1,700 m/z ; capillary voltage, 3.5 kV; collision energy 20 eV in positive, and 25 eV in negative mode. MassHunter software (Agilent, USA) was used for instrument control. Mathematical model to describe S. aureus and P. aeruginosa population dynamics In order to model the population dynamics recorded for mono- and co-cultures of different strains of S. aureus and P. aeruginosa in well-mixed flasks, where nutrients become limiting, we used the following set of differential equations: where X is the number of S. aureus ( S ) and P. aeruginosa ( P ) cell counts,β, is a constant of biochemical process activation when nutrients are in abundance, 2 is the rate of biochemical process inactivation when nutrients are limiting, L is the concentration of compound(s) secreted by P. aeruginosa and affecting the growth of S. aureus , is a constant representing inactive, γ 1 is the time at which nutrients become limiting. In the microfluidic chambers where nutrients do not become limiting (i.e. an open system), the growth of S. aureus in mono-culture or in co-culture with P. aeruginosa can be described by the following differential equation: Whereas the growth of P. aeruginosa in co-culture with S. aureus in the microfluidic chambers can be described by the following differential equations: Where ε X is the coefficient of the rate of escape of P. aeruginosa from microfluidic chambers, is the coefficient of the rate of escape of quorum-sensing molecules secreted by P . aeruginosa , ε Q is the concentration of quorum-sensing molecules secreted by P. aeruginosa . The equations above were fitted to our corresponding population dynamics data via least-squares regression with parameter estimations reported in Tables S1-S3. A full description of the model can be found at https://github.com/tt386/GeometricBacterialGrowth/tree/main . Supplemetary Materials This document includes: Figs. S1 to S19 Tables S1 to S3 Legends for Datasets S1 to S20 Other Supplementary Material for this manuscript includes the following: Datasets S1 to S20 Funding This work was supported by the BBSRC and the EPSRC through two grants awarded to S.P. and U.L. (BB/V008021/1, EP/Y023528/1). This work was further supported via the JPIAMR project ERADIAMR (MRC reference MR/Y033892/1) awarded to S.P. and R.C. GC and AW are supported by the Medical Research Council Centre for Medical Mycology (MR/N006364/2 and MR/V003417/1), the MRC Doctoral Training Grant (MR/W502649/1) and the NIHR Exeter Biomedical Research Centre (NIHR 203320). K.T.A. and T.T. gratefully acknowledge the financial support of the EPSRC (EP/T017856/1). A.D.V. and M.A.T.B. were supported by the NHMRC (APP2004367). R.C and M.G-C research was supported by CIBER de Enfermedades Infecciosas and through the JPIAMR project ERADIAMR. This project also utilised equipment funded by the Wellcome Trust (Multi-User Equipment Grant award number 218247/Z/19/Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the authors have applied a ‘Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. Author contributions Conceptualization, S.P.; methodology, U.L., N.O.G., G.C., T.T., S.Z., G.T., A.D.V., M.G.-C., P.A.O.N., A.F., A.R.J., A.J.Y., R.C., P.C., M.A.T.B., A.W., K.T.A. and S.P.; formal analysis, U.L., N.O.G., G.C., T.T., S.Z., G.T., P.A.O.N., P.C., A.W., K.T.A. and S.P.; generation of figures, U.L. and S.P.; investigation, U.L., N.O.G., G.C., T.T., S.Z., G.T. and S.P.; resources, A.J.Y., R.C., P.C., M.A.T.B., A.W., K.T.A. and S.P.; data curation, U.L. and S.P., writing – original draft, U.L. and S.P.; writing – review & editing, U.L., N.O.G., G.C., T.T., S.Z., G.T., A.D.V., M.G.-C, P.A.O.N., A.F., A.R.J., A.J.Y., R.C., P.C., M.A.T.B., A.W., K.T.A. and S.P.; visualization, U.L. and S.P.; supervision, A.J.Y., P.C., A.W., K.T.A. and S.P.; project administration, S.P.; funding acquisition, U.L., A.J., A.J.Y., M.A.T.B., A.W., K.T.A. and S.P.. Competing interests The authors declare they have no competing interests. Data and materials availability All data are available in the main text or the supplementary materials. Acknowledgments We thank Dr Seana Duggan and Prof Simon Foster for providing the S. aureus SH1000 mCherry strain. Funder Information Declared BBSRC , BB/V008021/1 EPSRC , EP/Y023528/1 MRC , MR/Y033892/1 Footnotes ↵ § The ERADIAMR (Effective RApid DIagnostics and treatment of AntiMicrobial Resistant bacteria) is a European project on antimicrobial resistance part of the JPI-AMR action. The ERADIAMR consortium is composed of the following persons: - Christèle Aubry, Amanda Luraschi-Eggemann, Maria Georgevia & Gilbert Greub, Lausanne, Switzerland - Gino Cathomen, Danuta Cichocka & Alexander Sturm, Muttenz, Switzerland - Maria Gracia & Rafael Canton, Madrid, Spain - Nicola Oswaldo Trinler & Susanne Häußler, Helmholtz, Germany - Niilo Kaldalu, Kristiina Vind & Tanel Tenson, Tartu, Estonia - Tailise Rodrigues, Maureen Micaletto, Urszula Łapińska & Stefano Pagliara, Exeter, UK Change in authors' list: Nicolas Oswaldo Gomez has been added to the authors list. This author has contributed to the manuscript with the construction of the mutants employed in this manuscript References 1. ↵ P. Geesink , J. ter Horst , T. J. G. Ettema , More than the sum of its parts: uncovering emerging effects of microbial interactions in complex communities . FEMS Microbiol. Ecol . 100 , 1 – 7 ( 2024 ). OpenUrl 2. ↵ S. A. West , S. P. Diggle , A. Buckling , A. Gardner , A. S. 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Ascoli , M. Lees , J. A. Meath , N. LeBaron , Preparation of lipide extracts from brain tissue . J. Biol. Chem . 191 , 833 – 841 ( 1951 ). OpenUrl FREE Full Text View the discussion thread. Back to top Previous Next Posted December 05, 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. You are going to email the following Competition with Pseudomonas aeruginosa induces Staphylococcus aureus in an antibiotic-tolerant viable but non culturable state Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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