Pyruvate-formate lyase–derived formate regulates the response of Salmonella Typhimurium to meropenem and ciprofloxacin

Pyruvate-formate lyase–derived formate regulates the response of Salmonella Typhimurium to meropenem and ciprofloxacin | 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 Pyruvate-formate lyase–derived formate regulates the response of Salmonella Typhimurium to meropenem and ciprofloxacin Debapriya Mukherjee , Sakshi Vasant Wagle , Pallab Ghosh , Abhishek Biswas , Dipshikha Chakravortty doi: https://doi.org/10.1101/2025.05.22.655487 Debapriya Mukherjee 1 Department of Microbiology and Cell Biology, Division of Biological Sciences, Indian Institute of Science , Bangalore, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sakshi Vasant Wagle 1 Department of Microbiology and Cell Biology, Division of Biological Sciences, Indian Institute of Science , Bangalore, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pallab Ghosh 1 Department of Microbiology and Cell Biology, Division of Biological Sciences, Indian Institute of Science , Bangalore, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abhishek Biswas 1 Department of Microbiology and Cell Biology, Division of Biological Sciences, Indian Institute of Science , Bangalore, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dipshikha Chakravortty 1 Department of Microbiology and Cell Biology, Division of Biological Sciences, Indian Institute of Science , Bangalore, India 2 School of Biology, Indian Institute of Science Education and Research , Thiruvananthapuram, 695551 Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: dipa{at}iisc.ac.in Abstract Full Text Info/History Metrics Preview PDF Abstract Salmonella enterica serovar Typhimurium (STM) employs various strategies to endure antibiotic stress, but the contribution of central metabolites to resistance remains insufficiently characterized. In this work, we identify intracellular formate, produced by pyruvate-formate lyase (PflB), as a key determinant of STM susceptibility to meropenem and ciprofloxacin. Deletion of pflB disrupts cellular pH homeostasis, impairing efflux pump function, increasing reactive oxygen species, and causing membrane depolarization, collectively heightening sensitivity to antibiotics. Supplementing extracellular formate reverses these phenotypes. Mechanistically, pH imbalance activates the RpoE– csrB regulatory pathway, leading to reduced expression of efflux pump genes acrB and tolC , while extracellular formate engages the BarA/SirA two-component system to further influence the CsrA/ csrB network. Disrupting the formate pool interferes with efflux-dependent resistance, revealing a previously unrecognized signalling function for intracellular metabolites. Overall, our results position formate as a metabolic driver of antibiotic outcomes in STM and highlight it as a potential target for mitigating resistance. Introduction The global escalation of antimicrobial resistance (AMR) has been driven largely by decades of antibiotic overuse and misuse ( 1 , 2 ). Among the resistant pathogens of greatest concern, multidrug-resistant (MDR) Salmonella has become a significant public health threat, contributing heavily to global illness and death. In 2019, AMR-associated Salmonella infections were estimated to cause approximately 150,000 deaths worldwide, with 23,700 of these attributed specifically to MDR strains ( 3 ). Within this landscape, Salmonella enterica serovar Typhimurium (STM) remains one of the most prevalent MDR serotypes, compromising the efficacy of frontline antibiotics and posing major challenges to healthcare systems ( 4 – 7 ). To survive antibiotic exposure, Salmonella relies on a broad array of resistance mechanisms, including enzymatic degradation of antimicrobial compounds, efflux pump–mediated drug export, structural modification of antibiotics, and alteration or protection of cellular targets ( 8 , 9 ). Although these classical pathways are well characterized, the influence of bacterial metabolism on shaping AMR in Salmonella is still poorly defined. Our previous work identified pyruvate-formate lyase (PflB), encoded by pflB , as an important contributor to STM pathogenesis. PflB regulates intracellular formate levels, a short-chain fatty acid essential for maintaining intracellular pH (ipH) homeostasis. Deletion of pflB disturbs ipH balance, resulting in membrane stress, activation of the extra cytoplasmic sigma factor RpoE, increased expression of the small RNA csrB , and repression of flagellar gene expression ( 10 ). We also observed that pflB loss increases STM sensitivity to meropenem and ciprofloxacin, pointing to a previously unrecognized relationship between formate metabolism and antibiotic resistance. In the present study, we systematically evaluated how intracellular formate pools and extracellular formate supplementation influence antibiotic susceptibility in STM. We show that the absence of pflB leads to disrupted internal pH, causing membrane damage that activates RpoE and alters expression of the efflux pump gene acrB . Additionally, extracellular formate modulates antibiotic resistance through the BarA/SirA two-component system and the downstream CsrA/ csrB regulatory network, collectively contributing to reduced acrB expression. Together, these results demonstrate that intracellular formate directly governs STM antibiotic susceptibility, while extracellular formate exerts distinct signalling effects. Overall, our findings establish formate as a key metabolic determinant of AMR and highlight formate homeostasis as a promising target for combating MDR Salmonella . Materials and Methods Bacterial strains and culture conditions The wild-type Salmonella enterica subsp. enterica serovar Typhimurium strain 14028S used in this study was kindly provided by Professor Michael Hensel (Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Germany). All bacterial strains were preserved as glycerol stocks at −80 °C and revived on Luria-Bertani (LB) agar plates (HiMedia) supplemented with the appropriate antibiotics when necessary (50 µg/ml kanamycin, 50 µg/ml ampicillin, or 25 µg/ml chloramphenicol). Single colonies were inoculated into LB broth supplemented with the appropriate antibiotics and incubated overnight at 37 °C with shaking at 170 rpm. The overnight cultures were then diluted 1:100 in fresh LB medium, with or without 40 mM sodium formate (Sigma), and grown under the same conditions for 6 hours to obtain bacteria in the logarithmic growth phase. The optical density at 600 nm (OD₆₀₀) was subsequently adjusted to 0.1 in Mueller-Hinton Broth (MHB), corresponding to approximately 10⁶ colony-forming units (CFU), for use in downstream experiments. A detailed list of bacterial strains and plasmids used in this study is provided in Table 1 . View this table: View inline View popup Table 1. Determination of MIC of meropenem, ciprofloxacin, ceftazidime Logarithmic phase bacterial cultures, adjusted to an OD₆₀₀ of 0.1, were inoculated into 96-well microtiter plates containing Mueller-Hinton Broth (MHB) supplemented with serial dilutions of antibiotics. The concentrations used were as follows: meropenem (8 to 0.0156 mg/L) (Sigma), ciprofloxacin (0.5 to 0.0009 mg/L) (Sigma), and ceftazidime (128 to 0.125 mg/L). Plates were incubated at 37 °C with shaking at 170 rpm for 14-16 hours. Bacterial growth was assessed by measuring OD₆₀₀ using a Tecan microplate reader. Estimation of bacterial viability using resazurin assay Following antibiotic exposure and incubation for 14-16 hours, 20 µL of resazurin solution (prepared from a 0.2 mg/mL stock) was added to each well and incubated for 2 hours in the dark. Thereafter, fluorescence intensity was measured using a Tecan microplate reader with excitation at 540 nm and emission at 590 nm. Cell viability was assessed by monitoring the color change of resazurin. A stronger purple coloration indicated higher cell viability. Flow cytometry mediated quantification of live/dead population, intracellular reactive oxygen species (ROS), membrane depolarization, and ipH After 14–16 hours of antibiotic treatment, bacterial cells were stained with a panel of fluorescent dyes: Propidium Iodide (PI, 1 µg/mL) for live/dead discrimination, dichlorodihydrofluorescein diacetate (DCF 2 DA, 10 µM) to assess intracellular reactive oxygen species (ROS), Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol (DiBAC 4 , 1 µg/mL) for measuring membrane depolarization, and 2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein Acetoxymethyl Ester (BCECF-AM, 20 µM) for intracellular pH (ipH) comparison. Stained samples were incubated in the dark for 15 minutes and subsequently analyzed using the BD FACSVerse flow cytometer (BD Biosciences, USA). Data including dot plots, density plots, median fluorescence intensity (MFI), and the percentage of positively stained cells were acquired and processed using BD FACSVerse software. Determination of intracellular formate and pyruvate concentration Following exposure of logarithmic-phase cultures of STM WT and passaged strains to sub-lethal antibiotic concentrations and a 16-hour incubation, the cultures were centrifuged at 6,000 rpm for 10 minutes. The resulting pellet was washed twice with 1X PBS to remove residual media and then resuspended in PBS. A 100 µl portion of this suspension was plated on LB agar to determine the CFU count. The remaining suspension was lysed by sonication, and cellular debris were cleared by centrifugation at 12,000 g for 10 minutes. Formate and pyruvate levels in the supernatant were quantified using the Abcam formate estimation kit (ab111748) and Elabscience pyruvate estimation kit (E-BC-K130-S) according to the manufacturer’s instructions, and the concentrations were interpolated from a standard curve. The concentrations were normalized with the CFU obtained from plating. RNA isolation and RT-qPCR Logarithmic-phase bacterial cultures were harvested by centrifugation at 6,000 rpm for 10 minutes at 4 °C, and the resulting cell pellets were resuspended in TRIzol reagent. Samples were stored at −80 °C until further processing. Total RNA was extracted using the chloroform–isopropanol method, and RNA pellets were washed with 70% ethanol and resuspended in 30 µL of DEPC-treated Milli-Q water. RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and RNA integrity was verified by electrophoresis on a 2% agarose gel. For removal of genomic DNA, 2 µg of total RNA was treated with TURBO DNase (Thermo Fisher Scientific) at 37 °C for 30 minutes, followed by inactivation with 5 mM Na₂EDTA at 65 °C for 10 minutes. First-strand cDNA synthesis was performed using the PrimeScript RT reagent kit (Takara, Cat. No. RR037A) according to the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) was carried out using the TB Green RT-qPCR kit (Takara) on a QuantStudio 5 Real-Time PCR System. Gene expression was analysed using intragenic primers and normalized to 16S rRNA levels. Primer sequences are provided in Table 2 . View this table: View inline View popup Table 2. Efflux pump activity by Nile Red Assay Efflux pump activity was quantified following the method outlined by Lyu et al ( 11 ). In brief, bacterial cultures in the logarithmic growth phase were adjusted to an optical density of 0.1 at 600 nm (OD₆₀₀). A volume of 100 µl from each normalized culture was dispensed into wells of a 96-well microplate and incubated with Nile Red dye at a final concentration of 48 µg/ml. The plate was incubated in the dark under shaking conditions for 3 hours. Fluorescence measurements were then obtained using a TECAN microplate reader, with excitation and emission wavelengths set at 549 nm and 628 nm, respectively. Fluorescence intensity values were normalized against absorbance readings at 600 nm. Atomic Force Microscopy After 14–16 hours of antibiotic exposure, bacterial cells were drop-cast onto sterile glass coverslips. Once air-dried, the cells were fixed by treating them with 3.5% paraformaldehyde for 10 minutes. Residual salts were removed by washing the samples three times with double-autoclaved MilliQ water. The coverslips were then dried in a vacuum desiccator prior to imaging with the NX10 Atomic Force Microscope (AFM). Image processing and measurements of cell length and height were performed using XEI software. Animal experiments All animal experiments were conducted with prior approval from the Institutional Animal Ethics Clearance Committee (IAEC) at the Indian Institute of Science, Bangalore. The institute is registered under CPCSEA with registration number 48/1999/CPCSEA. All procedures strictly adhered to the guidelines set by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The ethical approval number for this study is CAF/Ethics/853/2021. A modified protocol based on the method described by Roy Chowdhury et al. was employed to evaluate the organ burden following infection and antibiotic treatment with the wild-type (WT) and mutant bacterial strains( 12 ). Briefly, 10⁶ cells from overnight cultures were orally gavaged into 6–8-week-old C57BL/6 mice on day 0 (0 dpi). Mice received 1 mg/kg of the respective antibiotics on days 2 and 4 post-infection. On day 5 post-infection, the animals were ethically euthanized, and the intestine, liver, and spleen were collected. These organs were homogenized, and serial dilutions of the homogenates were plated on Salmonella-Shigella (SS) agar. Bacterial load was quantified and expressed as log₁₀ CFU per gram of tissue. To compare mortality rates between STM WT and STM Δ pflB under antibiotic exposure, groups of mice (n=5) were orally administered 10⁶ cells from stationary phase cultures of each strain. Starting from day 2 post-infection, 1 mg/kg of the respective antibiotics was administered every alternate day till the mice survived. Survival was monitored daily and represented as percent survival. A 125 mM solution of sodium formate was supplemented in the drinking water provided to the animals in both experimental setups. Estimation of Formate in mouse serum by Gas Chromatography-Mass Spectrometry To quantify formate levels in mouse serum following supplementation with 125 mM sodium formate in drinking water, a protocol adapted from Hughes et al was employed( 13 ). Male C57BL/6 mice (6–8 weeks old) were provided with 125 mM sodium formate in their drinking water. At five days post-infection, blood was collected via retro-orbital puncture. After clotting, serum was separated by centrifugation at 10,000 × g for 20 minutes and subsequently used for formate quantification. Sodium formate (Sigma) served as an external standard. Both standards and biological samples were derivatized prior to mass spectrometry analysis. To protonate the formate, 2 M hydrochloric acid was added to each sample in a 1:1 ratio. Liquid-liquid extraction was performed to isolate formic acid into ethyl acetate (Sigma-Aldrich). Residual water was removed by passing the ethyl acetate extract through an anhydrous sodium sulfate column (Sigma-Aldrich). The extract was then incubated at 80 °C for 1 hour with the derivatizing reagent N,O-Bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich) containing 1% chlorotrimethylsilane (Fluka). Derivatized samples were transferred to autosampler vials for analysis using Gas Chromatography-Mass Spectrometry (GC-MS; Agilent Technologies, 7890A GC, 5975C MS). Injection parameters were as follows: injection temperature of 200 °C, injection split ratio of 1:40, and injection volume of 1 µL. The oven temperature program began at 40 °C (held for 4 minutes), increased to 120 °C at a rate of 5 °C/min, and then to 200 °C at 25 °C/min, with a final hold of 3 minutes. Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min. Chromatographic separation was performed using a 30 m × 0.25 mm × 0.40 µm DB-624 column (Agilent). The interface temperature was maintained at 220 °C, and the electron impact ion source operated at 230 °C with an ionization energy of 70 eV and 1235 EM volts. For qualitative analysis, selected ion monitoring in single quadrupole mode was employed with a scan at m/z 103. The retention time for formate and its deuterated analog was approximately 5.9 minutes. The target and reference (qualifier) ions for formate were m/z = 103 and m/z = 75, respectively. Sample concentrations were determined by interpolating from a standard calibration curve generated using known concentrations. Determination of the fraction of antibiotic persister The determination of antibiotic persister was performed by modifying a protocol described by Drescher et al ( 14 ). Logarithmic phase cultures of STM WT and STM Δ pflB strains were treated with 15X and 25X the minimum inhibitory concentration (MIC) of meropenem (2 and ciprofloxacin. To determine the persister population, aliquots were collected at specified timepoints post-treatment and plated for colony-forming unit (CFU) enumeration. Similarly, 24-hour-old biofilm cultures were exposed to the same antibiotic concentrations, and persister cells were quantified via plating. The resulting CFU counts were normalized against the pre-inoculum values to calculate fold viability relative to the 0-hour timepoint. Data were expressed as log₁₀(fold decrease in CFU relative to 0 h). Statistical Analysis Each experiment was independently repeated between 1 and 12 times, as specified in the figure legends. Statistical analyses were conducted using an unpaired Student’s t-test, or two-way ANOVA, as indicated in the respective legends. Data from mouse experiments were analysed using the Mann–Whitney U test. A p-value of less than 0.05 was considered statistically significant. Results are presented as Mean ± SD or Mean ± SEM, as noted in the figure legends. All data analysis and graphical representations were performed using GraphPad Prism version 8.4.2. Results pflB was transcriptionally upregulated in ciprofloxacin resistant mutants, indicating a possible involvement of intracellular formate in regulating Salmonella AMR In our earlier work, we demonstrated that pflB plays a central role in maintaining ipH in Salmonella , which in turn governs the adhesion–invasion switch during its pathogenic lifestyle ( 10 ). Other studies have also highlighted the importance of ipH in shaping bacterial antimicrobial responses ( 15 , 16 ). These observations prompted us to investigate whether intracellular formate levels might also contribute to the regulation of AMR in Salmonella . To investigate formate-associated responses, we analysed pflB expression in ciprofloxacin-resistant mutants (Cip-R C1, C2, C3) generated by passaging STM for 160 generations under sublethal ciprofloxacin. All Cip-R strains showed increased pflB transcription relative to WT ( Figure 1A ) . In Cip-R C1, sublethal ciprofloxacin further improved pflB expression ( Figure 1B ) , which coincided with elevated intracellular formate levels under drug exposure ( Figure 1C ) . Download figure Open in new tab Figure 1: Higher pflB transcription in the presence of antibiotics was frequently observed as a characteristic of ciprofloxacin and meropenem mutants, and loss of pflB was linked to impaired acid homeostasis when exposed to sublethal concentrations of meropenem and ciprofloxacin. (A) Transcriptional upregulation of pflB in Cip-R 1, 2, 3 ciprofloxacin resistant STM strains. Data representative of N=3, n=9 and is presented as mean+/- SD. (B) Transcriptional upregulation of pflB in Cip-R C1 upon exposure to sub-lethal doses of ciprofloxacin. Data representative of N=3, n=9 and is presented as mean+/- SD. (C) Quantification of intracellular formate levels in STM WT and Cip-R C1 strains upon exposure to sub-lethal dose of ciprofloxacin. Data representative of N=2, n=4 and is presented as mean+/- SD. (D) Transcriptional levels of pflB in Mer-R C1, 2, 3 meropenem resistant STM strains. Data representative of N=3, n=9 and is presented as mean+/- SD. (E) Transcriptional upregulation of pflB in Mer-R C1, 3 upon exposure to sub-lethal doses of ciprofloxacin. Data representative of N=3, n=9 and is presented as mean+/- SD. (F) Quantification of intracellular formate levels in STM WT and Mer-R C1 strains upon exposure to sub-lethal dose of ciprofloxacin. Data representative of N=2, n=4 and is presented as mean+/- SD. (G) Bar graphs showing PI-positive population in STM WT and STM WT/pQE60: pflB upon exposure to meropenem and ciprofloxacin. Data representative of N=2, n=4 and is presented as mean+/- SD. (H) Normalized 488/405 ratio of STM WT and STM WT/pQE60: pflB in presence of sub-lethal doses of meropenem and ciprofloxacin. Data representative of N=2, n=4 and is presented as mean+/- SD. (I) Quantification of intracellular formate levels in STM WT upon exposure to sub-lethal dose of ciprofloxacin and meropenem. Data representative of N=3, n=3 and is presented as mean+/-SD. (J) Normalized 488/405 ratio of STM WT and STM Δ pflB in presence of sub-lethal doses of meropenem and ciprofloxacin. Data representative of N=2, n=4 and is presented as mean+/-SD. (Unpaired two-tailed Student’s t-test for column graphs, Two-way ANOVA for grouped data, Mann-Whitney U-test for animal experiment data (*** p < 0.0001, *** p < 0.001, ** p<0.01, * p<0.05)) In contrast, meropenem-resistant strains (Mer-R C1, C2, C3), obtained via sublethal meropenem passaging, displayed reduced pflB transcription ( Figure 1D ) . However, Mer-R C1 and Mer-R C3 upregulated pflB upon sublethal meropenem exposure ( Figure 1E ) . Consistently, Mer-R C1 had a higher intracellular concentration formate than the WT strain ( Figure 1F ) . Together, these results indicate that elevated intracellular formate is a recurring feature of strains evolved under ciprofloxacin or meropenem pressure. Since pflB encodes pyruvate-formate lyase, the major source of intracellular formate in Salmonella , we used cloned pflB in the plasmid pQE60 and overexpressed it using Isopropyl β-D-1-thiogalactopyranoside (IPTG) to assess the functional impact of enhanced pflB ( 17 ). In STM WT, pflB overexpression reduced the Propidium Iodide (PI)–positive population—signifying lower cell death—after meropenem treatment, with no effect observed under ciprofloxacin exposure ( Figure 1G ) . pflB overexpression also decreased intracellular pH in both untreated and meropenem-treated cells ( Figure 1H ) , consistent with our earlier observation that pflB deletion disturbs pH homeostasis. Aligned with these observations, meropenem and ciprofloxacin exposure continued to increase intracellular formate in our laboratory WT strain ( Figure 1I ) . To probe the role of ipH in antibiotic responses, we monitored ipH changes in STM WT and STM Δ pflB following sublethal meropenem or ciprofloxacin treatment using the ratiometric dye BCECF-AM. STM WT exhibited a reduced 488/405 ratio under antibiotic stress, indicating cytoplasmic acidification. STM Δ pflB , which shows a higher basal ratio, did not acidify upon antibiotic exposure, suggesting resistance to pH drops. Formate supplementation partially rescued the Δ pflB phenotype by decreasing the 488/405 ratio under antibiotic stress. Although formate alone had little effect on WT ipH, antibiotic-treated WT displayed an increased 488/405 ratio with formate ( Figure 1J ) . Since PflB converts pyruvate into formate, we found that pflB deletion resulted in elevated intracellular pyruvate levels under untreated conditions. These higher pyruvate levels in STM Δ pflB persisted during meropenem and ciprofloxacin exposure. In contrast, STM WT strain showed an overall increase in pyruvate upon antibiotic treatment, suggesting that the cell may upregulate pflB to channel pyruvate toward formate production as a strategy to counter antibiotic stress by lowering intracellular pH ( Figure S1A ) . Download figure Open in new tab Figure S1: Deletion of pflB increased pyruvate levels in STM—a pattern also observed under antibiotic treatment—and additionally heightened STM’s susceptibility to the third-generation cephalosporin ceftazidime, with a comparable sensitivity seen upon extracellular formate supplementation. (A) Bar graphs showing fold change in intracellular pyruvate concentrations in STM WT, STM Δ pflB , STM Δ pflB /pQE60: pflB upon exposure to meropenem and ciprofloxacin. Data representative of N=2, n=4 and is presented as mean+/- SD. (B) Ceftazidime susceptibility in STM WT, STM Δ pflB , and STM Δ pflB /pQE60: pflB , with or without formate, was evaluated using the microfold dilution method. Data is presented as mean+/-SEM of N=10. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Together, these findings indicate that formate is a key metabolite whose intracellular levels commonly rise during antibiotic exposure, and whose depletion disrupts pH homeostasis under antibiotic stress. Deletion of pflB enhances susceptibility of STM to meropenem and ciprofloxacin, whereas supplementation of extracellular formate also heightens susceptibility of STM to these antibiotics To investigate the impact of deleting pflB , which was consistently upregulated during antibiotic exposure, we examined how this gene deletion affected the antibiotic susceptibility of STM. To assess the sensitivity of the STM Δ pflB strain to meropenem and ciprofloxacin, both the wild-type and mutant strains were treated with a range of antibiotic concentrations. The STM Δ pflB mutant showed significantly increased sensitivity to meropenem, particularly at concentrations of 1 mg/L and 0.5 mg/L ( Figure 2A ) . While the partial restoration of bacterial growth upon sodium formate supplementation was not statistically significant, it suggests that intracellular formate deficiency may contribute to the heightened sensitivity to meropenem. Interestingly, when formate was added to wild-type cells—despite their intact intracellular formate levels—a significant reduction in survivability was observed under antibiotic stress at the same concentrations. Comparable results were obtained with ciprofloxacin, where statistically significant effects were noted at 0.03125, 0.015625, 0.0078, and 0.0039 mg/L ( Figure 2B ) . These findings were further supported by resazurin assay data ( Figure 2C , D) . The enhanced susceptibility of STM Δ pflB and its recovery in the presence of sodium formate was also true for ceftazidime, a third generation cephalosporin ( 18 ) ( Figure S1B ) . Download figure Open in new tab Figure 2: Deletion of pflB increases STM’s sensitivity to meropenem and ciprofloxacin, while extracellular formate supplementation similarly enhances antibiotic susceptibility. (A) Meropenem susceptibility of STM WT, STM Δ pflB , and STM Δ pflB /pQE60: pflB , with or without formate treatment, was assessed using the microfold dilution method. Data presented as mean+/- SEM of N=12. (B) Ciprofloxacin susceptibility of STM WT, STM Δ pflB , and STM Δ pflB /pQE60: pflB , with or without formate treatment, was assessed using the microfold dilution method. Data presented as mean+/- SEM of N=10. (C) Resazurin assay of STM WT, STM Δ pflB , and STM Δ pflB /pQE60: pflB , with or without formate treatment, in increasing concentrations of meropenem. (D) Resazurin assay of STM WT, STM Δ pflB , and STM Δ pflB /pQE60: pflB , with or without formate treatment, in increasing concentrations of ciprofloxacin. (Unpaired two-tailed Student’s t-test for column graphs, Two-way ANOVA for grouped data, Mann-Whitney U-test for animal experiment data (*** p < 0.0001, *** p < 0.001, ** p<0.01, * p<0.05)) To investigate how deletion of pflB affects STM’s antibiotic susceptibility, we treated logarithmic-phase cultures overnight with sub-lethal doses of meropenem (0.5 mg/L) and ciprofloxacin (0.015625 mg/L)and analyzed PI uptake via flow cytometry. STM Δ pflB showed a higher PI-positive population than WT, which was significantly reduced with sodium formate supplementation. In contrast, formate supplementation increased PI-positive cells in WT upon antibiotic exposure ( Figure 3A , B) . Download figure Open in new tab Figure 3: Increased antibiotic sensitivity following pflB deletion was linked to transcriptional downregulation of efflux pumps, a pattern also observed in formate-treated wild-type cells. (A) Dot plots (SSC-A vs PI-A) and bar graph illustrate increased bacterial killing with meropenem and ciprofloxacin following pflB deletion and formate supplementation in STM WT. Data representative of N=3, n≥3. (B) Bar graphs depict the PI-positive population after exposure to meropenem and ciprofloxacin following pflB deletion and formate supplementation in STM WT. Data represent N=3, n≥3 and are shown as mean ± SD. (C) Bar graphs display the DiBAC 4 -positive population under meropenem and ciprofloxacin treatment after pflB deletion and formate supplementation in STM WT. Data represent N=3, n≥3 and are shown as mean ± SD. (D) Bar graphs present the DCF 2 DA-positive population upon exposure to meropenem and ciprofloxacin following pflB deletion and formate supplementation in STM WT. Data represent N=3, n≥3 and are shown as mean ± SD. (E) Nile red assay quantifies efflux pump activity in STM WT, STM Δ pflB, and STM Δ pflB /pQE60: pflB . Data representative of N=3, n=5 and is presented as mean+/- SD. (F-H) Transcript levels of acrB , tolC , and ompF measured in STM WT and STM Δ pflB , with or without formate treatment. Data representative of N=3, n≥3 and is presented as mean+/- SD. (Unpaired two-tailed Student’s t-test for column graphs, Two-way ANOVA for grouped data, Mann-Whitney U-test for animal experiment data (*** p < 0.0001, *** p < 0.001, ** p<0.01, * p<0.05)) We next assessed whether pflB deletion and formate supplementation affected susceptibility to additional antibiotics. STM Δ pflB displayed increased sensitivity to sub-lethal doses of the fluoroquinolone levofloxacin (1 µg/ml), the carbapenem doripenem (1 µg/ml), and the β-lactam ampicillin (64 µg/ml), with partial restoration of resistance following formate supplementation ( 19 – 21 ). Formate addition to STM WT also heightened susceptibility to these antibiotics ( Figure S2A –C) . These observations suggest that the increased antimicrobial sensitivity caused by pflB deletion or by external formate supplementation may extend beyond meropenem and ciprofloxacin. Nevertheless, to maintain focus and clarity, we concentrated on meropenem and ciprofloxacin in the subsequent analyses. Download figure Open in new tab Figure S2: Deletion of pflB increased STM susceptibility to the antibiotics levofloxacin, doripenem, and ampicillin, and this effect was partially reversed by formate supplementation. Formate addition to STM WT also heightened its sensitivity to these antibiotics. Bar graphs show the log₁₀ fold reduction in CFU relative to untreated controls for STM WT, STM Δ pflB , and STM Δ pflB /pQE60: pflB , with or without formate, following exposure to sub-lethal concentrations of levofloxacin (A), doripenem (B), and ampicillin (C). Data representative of N=3, n=4 and is presented as mean+/- SD. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Given that bactericidal antibiotics induce reactive oxygen species (ROS) irrespective of their mechanisms, we assessed intracellular ROS using DCF 2 DA staining( 22 – 24 ). Consistent with PI staining results, STM Δ pflB exhibited elevated DCF 2 DA-positive populations under antibiotic stress, which was mitigated by formate. Similarly, formate supplementation increased ROS levels in WT cells exposed to antibiotics ( Figure 3C , S3 A) . These findings suggest that loss of pflB , and thus intracellular formate, increases STM’s susceptibility to meropenem and ciprofloxacin, while exogenous formate to wild-type cells also enhances antibiotic sensitivity. Deletion of pflB was associated with a transcriptional downregulation of antibiotic efflux pumps, which was also common for formate supplemented in wild-type cells Our previous work demonstrated increased membrane depolarization in STM Δ pflB compared to WT cells ( 10 ). Previous studies have shown that outer membrane of Gram-negative pathogens forms a robust permeability barrier which can prevent antibiotics to reach their target ( 25 ). Given that antimicrobial agents like antimicrobial peptides can exert bactericidal effects via membrane depolarization we hypothesized that the enhanced antibiotic susceptibility of STM Δ pflB may be attributable, at least in part, to its depolarized membrane state( 26 – 30 ). Notably, membrane depolarization was further exacerbated upon exposure to sub-lethal concentrations of meropenem and ciprofloxacin, while sodium formate partially alleviated this effect in STM Δ pflB . In contrast, formate supplementation had no impact on membrane potential in WT cells under unstressed conditions, although increased depolarization was observed upon antibiotic exposure ( Figure 3D , S3 B) . Efflux-mediated resistance represents a major mechanism of antibiotic failure in Gram-negative bacteria. Salmonella harbors multiple efflux systems, among which the TolC-dependent resistance-nodulation-division (RND) family plays a central role in expelling a broad range of clinically relevant antibiotics( 31 – 34 ). As proton motive force (PMF) and pH homeostasis are critical for the functioning of these pumps, and membrane depolarization can disrupt PMF, we hypothesized that the reduced antibiotic efflux in STM Δ pflB may stem from impaired PMF( 11 ). To test this, we employed a nile red efflux assay. STM Δ pflB retained significantly higher levels of nile red fluorescence, indicating decreased efflux activity. Formate supplementation reduced nile red retention, suggesting partial restoration of efflux capacity. Similarly, formate-treated STM WT cells exhibited increased nile red fluorescence upon antibiotic exposure, consistent with reduced efflux function ( Figure 3E ) . To determine whether reduced efflux activity was due solely to membrane depolarization or also involved transcriptional regulation, we measured mRNA levels of acrB and tolC , two key components of the RND efflux system in Salmonella ( 35 ). Deletion of pflB resulted in transcriptional downregulation of both genes, with partial recovery upon formate supplementation. Interestingly, formate supplementation in STM WT also led to reduced acrB and tolC expression ( Figure 3F , G) . These findings suggest that while membrane depolarization may directly impair efflux pump activity, additional transcriptional regulatory mechanisms may also contribute to reduced efflux and heightened antibiotic susceptibility. Outer membrane proteins (Omps) influence antibiotic susceptibility in Salmonella , with reduced OmpF expression typically linked to resistance ( 36 – 38 ). Notably, ompF was downregulated in STM Δ pflB ( Figure 3H ) , even though this strain showed increased antibiotic sensitivity. This indicates that the heightened susceptibility of STM Δ pflB occurs independently of Omps, as decreased OmpF would normally correlate with resistance rather than greater sensitivity. Formate supplementation in drinking water and intrabacterial formate deficiency both enhanced bacterial clearance by antibiotics in-vivo We tested whether our in-vitro results also applied in living animals by giving C57BL/6 mice an oral dose of 10⁶ CFU of STM WT or STM Δ pflB . The mice also received 125 mM sodium formate in their drinking water ( Figure 4A ) . Earlier work suggests that mice need about 40 mmol of formate per day. Since C57BL/6 mice usually drink around 30 ml of water daily, they would need about a 1 M solution to reach that amount. However, previous studies showed that 500 mM sodium formate can cause weight loss, so we selected a safer dose of 125 mM, representing about 10% of the estimated daily requirement ( 39 ). In our pilot tests, this amount was enough to significantly raise serum formate levels by 5 days post-infection ( Figure S4A ) . Our findings showed that adding formate to the drinking water together with meropenem treatment lowered bacterial loads more than meropenem alone in the intestine, liver, and spleen of C57BL/6 mice. As expected, the STM Δ pflB mutant showed reduced organ colonization ( 10 ); but formate supplementation did not restore its numbers. Meropenem further reduced the bacterial load, and—unlike our in-vitro results—formate did not rescue the decreased bacterial counts. Interestingly, combining formate with meropenem did not lower organ colonization in STM Δ pflB the way it did in STM WT, suggesting that STM Δ pflB may use extracellular formate to survive antibiotic stress ( Figure 4B ). The same trends were seen with ciprofloxacin ( Figure 4C ) and ceftazidime ( Figure 4D ) in mice infected with STM WT or STM Δ pflB under formate-supplemented conditions. Download figure Open in new tab Figure S3: Enhanced antibiotic sensitivity due to pflB deletion correlates with increased DCF 2 DA and DiBAC4 staining, indicating elevated ROS levels and membrane depolarization—effects also seen in formate-treated STM WT. (A) Dot plots (SSC-A vs DCF 2 DA) showing intracellular ROS following meropenem and ciprofloxacin treatment in STM WT and STM Δ pflB with or without formate supplementation. Data representative of N=3, n≥3. (B) Dot plots (SSC-A vs DiBAC-A) showing membrane depolarization following meropenem and ciprofloxacin treatment in STM WT and STM Δ pflB with or without formate supplementation. Data representative of N=3, n≥3. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Download figure Open in new tab Figure S4: Formate supplementation increased formate levels in serum of treated mice, whereas rpoE deletion can also revert the enhanced ipH in STM Δ pflB in presence of meropenem and ciprofloxacin. (A) Bar graph showing formate levels in mice serum after exposure to formate through drinking water. (B) Normalized 488/405 ratio of STM WT, STM Δ pflB , STM Δ rpoE , STM Δ pflB Δ rpoE in presence of sub-lethal doses of meropenem and ciprofloxacin. Data representative of N=2, n=4 and is presented as mean+/- SD. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Download figure Open in new tab Figure 4: Both formate supplementation via drinking water and internal formate deficiency enhanced antibiotic-mediated bacterial clearance in vivo . (A) Schematic outlining the experimental design. (B-D) Role of formate supplementation in STM WT and STM Δ pflB clearance from the intestine, liver, and spleen following treatment with meropenem, ciprofloxacin, and ceftazidime. Data representative of N≥8 and is presented as mean+/- SD. (Unpaired two-tailed Student’s t-test for column graphs, Two-way ANOVA for grouped data, Mann-Whitney U-test for animal experiment data (*** p < 0.0001, *** p < 0.001, ** p<0.01, * p<0.05)) Deletion of rpoE from STM Δ pflB reversed the enhanced antibiotic susceptibility, associated with a recovery in the transcript levels of acrB and tolC In E. coli , RpoE (σ E ) functions as an extra cytoplasmic stress sigma factor essential for viability and for coordinating the cellular response to membrane stress ( 40 ). The membrane protein RseA, together with the periplasmic factor RseB, binds and sequesters σ E under non-stress conditions, preventing its interaction with RNA polymerase ( 41 , 42 ). When envelope stress occurs, RseA—which is intrinsically unstable—is rapidly degraded, releasing σ E to activate its target genes. These genes support the synthesis, transport, and assembly of lipopolysaccharides, phospholipids, and outer membrane proteins (OMPs), as well as proteases and chaperones required to preserve or restore outer membrane integrity ( 42 – 45 ). Recent studies further demonstrate that σ E indirectly increases the transcription of the sRNAs csrB and csrC through σ⁷⁰-dependent promoters ( 46 , 47 ) In our previous work, we demonstrated that deletion of pflB disrupts intracellular pH homeostasis, leading to membrane depolarization. This perturbation activated the extracytoplasmic sigma factor RpoE, which in turn upregulated transcription of the small RNA csrB . The csrB RNA can antagonize the activity of the global regulator CsrA by direct binding, thereby impairing processes such as flagellar biosynthesis ( 10 ). Notably, CsrA has been shown to stabilize the acrAB transcript by interacting with its 5′ untranslated region ( 48 ). Given the increased membrane depolarization observed in STM Δ pflB upon antibiotic exposure, we hypothesized that RpoE may regulate acrB expression indirectly through modulation of the CsrA– csrB regulatory circuit. To test this, we exposed the STM Δ pflB Δ rpoE strain to sublethal concentrations of meropenem and ciprofloxacin. Compared to STM Δ pflB , the double knockout exhibited a significantly reduced proportion of PI-positive cells ( Figure 5A , B) . Interestingly, deletion of rpoE in the STM Δ pflB background did not restore membrane potential under basal conditions. However, upon antibiotic challenge, we observed a significant reduction in DiBAC₄-positive cells, indicative of partial recovery in membrane polarization ( Figure 5C , S5A) . However STM Δ pflB Δ rpoE had a decreased ROS positive cells compared to STM Δ pflB , as assessed using DCF 2 DA staining ( Figure 5D , S5B) . This was consistent with a decrease in the heightened ipH of STM Δ pflB following treatment with ciprofloxacin and meropenem when rpoE was simultaneously deleted ( Figure S4B ) . Download figure Open in new tab Figure 5: Deletion of rpoE in STM Δ pflB reversed its increased antibiotic sensitivity, accompanied by restored acrB and tolC transcript levels. (A) Dot plots (SSC-A vs PI-A) showing antibiotic-mediated killing in STM Δ pflB upon rpoE deletion. Data representative of N=3, n=4. (B) Bar graphs showing PI positive population in STM Δ pflB upon rpoE deletion and upon exposure to sub-lethal doses of meropenem and ciprofloxacin. Data representative of N=3, n=4 and is presented as mean+/- SD. (C) Bar graphs display the DiBAC 4 -positive population under meropenem and ciprofloxacin treatment after rpoE deletion in STM Δ pflB . Data represent N=3, n=4 and are shown as mean ± SD. (D) Bar graphs present the DCF 2 DA-positive population upon exposure to meropenem and ciprofloxacin following rpoE deletion in STM Δ pflB. Data represent N=3, n=4 and are shown as mean ± SD. (E-F) Transcript levels of key efflux pump genes acrB and tolC were compared between STM Δ pflB Δ rpoE and STM Δ pflB . Data representative of N=3, n≥3 and is presented as mean+/- SD. (G) Efflux activity assessed using the Nile red assay. Data representative of N=3, n≥5 and is presented as mean+/- SD. (H-I) Transcriptional expression of emrB and ompF was also evaluated. Data representative of N=3, n≥3 and is presented as mean+/- SD. (Unpaired two-tailed Student’s t-test for column graphs, Two-way ANOVA for grouped data, Mann-Whitney U-test for animal experiment data (*** p < 0.0001, *** p < 0.001, ** p<0.01, * p<0.05)) The improved viability of STM Δ pflB Δ rpoE relative to STM Δ pflB under antibiotic stress was further supported by atomic force microscopy, which revealed increased cellular length and height following exposure to both meropenem and ciprofloxacin ( Figure S6A -C) . Download figure Open in new tab Figure S5: Recovered antibiotic sensitivity due to rpoE deletion in STM Δ pflB correlates with reduced intracellular ROS levels but it did not have any effect on membrane depolarization (A) Representative dot plots (SSC-A vs. DIBAC-A) illustrating membrane depolarization in STM WT, STM Δ pflB , STM Δ rpoE, STM Δ pflB Δ rpoE strains treated with meropenem or ciprofloxacin. Data shown from three independent experiments (N=3, n=4). (B) Representative dot plots (SSC-A vs. DCFDA-A) showing intracellular ROS in STM WT, STM Δ pflB , STM Δ rpoE, STM Δ pflB Δ rpoE following treatment with meropenem or ciprofloxacin. Data representative of N=3, n=4. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Download figure Open in new tab Figure S6: The recovery in antibiotic sensitivity upon rpoE deletion in STM Δ pflB was also evident from morphological changes under Atomic Force Microscopy (A) AFM-assissted visualization of morphology of STM WT, STM Δ pflB , STM Δ rpoE , STM Δ pflB Δ rpoE upon expsure to sublethal concentrations of meropenem and ciprofloxacin. (B-C) Quantification of bacterial length and height from the same. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) These phenotypic changes correlated with the restoration of acrB and tolC transcript levels in STM Δ pflB Δ rpoE , suggesting enhanced efflux activity ( Figure 5E , F) . This was further confirmed by increased nile red efflux, indicative of improved pump function ( Figure 5G ) . Given the role of EmrB in fluoroquinolone resistance, we also examined its expression ( Figure 5H ) ( 49 ). As previous studies have not identified a direct regulatory relationship between CsrA and emrB , we measured emrB transcript levels and observed no significant change in STM Δ pflB Δ rpoE relative to STM Δ pflB . These findings suggest that the observed phenotypic rescue is primarily mediated through the CsrA– csrB axis. Deletion of rpoE in STM Δ pflB led to further downregulation of ompF , indicating that while ompF suppression does not explain the increased antibiotic sensitivity of STM Δ pflB , its further downregulation upon rpoE deletion may contribute to the restored resistance observed in STM Δ pflB Δ rpoE ( Figure 5I ) . A comparable reduction in CFU for all the strains following 10 mM glutathione antioxidant under sublethal meropenem and ciprofloxacin exposure indicates that the observed differences in bacterial killing are attributable to the distinct levels of ROS generated by each antibiotic ( Figure S7A , B) ( 50 ). Download figure Open in new tab Figure S7: Glutathione treatment equalizes the fold reduction in CFU observed in meropenem- and ciprofloxacin-treated samples. (A-B) Bar graphs showing the log 10 fold reduction in CFU relative to untreated controls (UT) for STM WT (± formate), STM Δ pflB (± formate), STM Δ pflB /pQE60: pflB (± formate), STM Δ rpoE , and STM Δ pflB Δ rpoE following meropenem or ciprofloxacin treatment, in the presence or absence of glutathione. Data are presented as mean ± SD from two independent experiments (N=2, n=4). (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Download figure Open in new tab Figure S8: sirA deletion mediated reduction of antibiotic susceptibility in STM Δ pflB was associated with reduced intracellular ROS levels, but without any effect on membrane depolarization (A) Representative dot plots (SSC-A vs. DiBAC4) depicting membrane depolarization in STM WT, STM Δ pflB , STM Δ sirA , and STM Δ pflB Δ sirA strains following treatment with meropenem or ciprofloxacin. Data shown from five independent experiments (N=5, n=3). (B) Representative dot plots (SSC-A vs. DCFDA-A) showing intracellular ROS levels in STM WT, STM Δ pflB , STM Δ sirA , and STM Δ pflB Δ sirA following exposure to meropenem or ciprofloxacin. Data representative of N=5, n=3. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Download figure Open in new tab Figure S9: The deletion of sirA alleviates the heightened antibiotic sensitivity observed in STM WT upon external formate supplementation. (A-C) RT-qPCR quantification of acrB , tolC , emrB , and ompF transcript levels in STM WT and STM Δ sirA , with or without formate supplementation. Data are presented as mean ± SD from N=3, n≥3. (D) Quantification of PI-positive cells indicating bacterial death under antibiotic stress in STM WT and STM Δ sirA , with or without formate supplementation. Data are shown as mean ± SD from N=3, n≥3. (E) Quantification of DCF 2 DA-positive cells in STM WT and STM Δ sirA exposed to meropenem or ciprofloxacin, with or without formate supplementation. Representative data of N=3, n≥3 is presented as mean ± SD. (F) Quantification of DiBAC4-positive cells in STM WT and STM Δ sirA , with or without formate, reflecting membrane depolarization under antibiotic stress. Representative data of N=3, n≥3 is presented as mean ± SD. (Unpaired two-tailed Student’s t-test (column graphs), two-way ANOVA (grouped data), and Mann–Whitney U-test (animal data). (***p < 0.0001, **p < 0.001, **p < 0.01, *p < 0.05)) Deletion of sirA abolishes the antibiotic susceptibility of STM Δ pflB , along with reduction in ROS levels and membrane depolarization To further confirm that small RNA csrB regulates antibiotic susceptibility, we examined an additional upstream regulator of csrB . The BarA/SirA two-component system, which is also known to activate the expression of csrB and csrC small RNAs, has previously been shown to enhance the expression of HilD, a key regulator of genes within the Salmonella Pathogenicity Island-1 ( 51 – 53 ). We deleted sirA in the STM Δ pflB background and used flow cytometry to quantify propidium iodide-positive cells, revealing a marked reduction in dead cells in the STM Δ pflB Δ sirA strain compared to STM Δ pflB ( Figure 6A , B) . Interestingly, this decrease in cell death did not correspond with a reduction in membrane depolarization, indicating that the reduced antibiotic sensitivity in STM Δ pflB Δ sirA was independent of changes in membrane potential ( Figure 6C , S8A) . A drop in DCF 2 DA-positive cells further supported the observation of diminished antibiotic susceptibility following sirA deletion in STM Δ pflB . Notably, STM Δ sirA alone showed an increase in DCF 2 DA-positive cells after exposure to meropenem and ciprofloxacin ( Figure 6D , S8B) . This prompted an investigation into efflux pump expression. We observed that sirA deletion in STM Δ pflB led to elevated expression of acrB , tolC , emrB , and ompF ( Figure 6 E-H ) . A nile red assay confirmed restored efflux pump activity in STM Δ pflB Δ sirA ( Figure 6I ) . Interestingly, deletion of sirA alone resulted in decreased acrB transcript levels, which may explain the increased DCF 2 DA-positive population upon treatment with meropenem and ciprofloxacin in STM Δ sirA ( Figure 6 E) . However, no overall change in efflux activity was detected in STM Δ sirA , possibly due to compensatory roles by other efflux pumps ( Figure 6I ) . These findings suggest that targeting upstream regulators of csrB can effectively reverse the antibiotic susceptibility observed in STM Δ pflB . Download figure Open in new tab Figure 6: Deleting sirA in STM Δ pflB similarly reversed its heightened antibiotic sensitivity and restored acrB and tolC expression levels. (A) Dot plots (SSC-A vs PI-A) showing reduced antibiotic-mediated killing in STM Δ pflB upon sirA deletion. Data representative of N=5, n=3. (B) Bar graphs showing antibiotic-mediated killing in STM Δ pflB upon sirA deletion. Data representative of N=5, n=3 and is presented as mean+/- SD. (C) Bar graphs showing DiBAC 4 population in STM Δ pflB upon sirA deletion and exposure to antibiotics. Data representative of N=5, n=3 and is presented as mean+/- SD. (D) Bar graphs showing DCF 2 DA population in STM Δ pflB upon sirA deletion and exposure to antibiotics. Data representative of N=5, n=3 and is presented as mean+/- SD. (E-H) Transcript levels of efflux pump genes acrB, tolC , emrB, and ompF were compared between STM Δ pflB Δ sirA and STM Δ pflB . Data representative of N=3, n≥3 and is presented as mean+/- SD. (I) Efflux activity assessed using the Nile red assay. Data representative of N=3, n=8 and is presented as mean+/- SD. (Unpaired two-tailed Student’s t-test for column graphs, Two-way ANOVA for grouped data, Mann-Whitney U-test for animal experiment data (*** p < 0.0001, *** p < 0.001, ** p<0.01, * p<0.05)) Deletion of sirA ameliorates the heightened susceptibility of STM WT to antibiotics upon exogenous F supplementation The BarA/SirA two-component regulatory system functions by directly promoting increased expression of the small regulatory RNA csrB ( 54 ). Given that CsrA is a key regulator of acrAB transcript expression, we aimed to determine whether formate-induced signalling through the BarA/SirA system contributes to the enhanced antibiotic sensitivity observed in STM WT ( 48 ). To explore this, we assessed the antibiotic susceptibility of the STM Δ sirA in the presence of formate. Formate exposure in STM WT resulted in reduced transcript levels of other efflux-related genes, such as tolC and emrB . The absence of sirA mitigated the suppression of efflux pump gene expression induced by formate in STM WT. Transcript analysis revealed that formate supplementation improved acrB expression in STM Δ sirA , contrasting with the downregulation of acrB seen in formate-treated STM WT (Figure S9 A-C) . This reflected in efflux pump activity as well (Figure S9 D) . Consistent with previous data, STM WT treated with formate showed a higher percentage of PI-positive cells following exposure to meropenem and ciprofloxacin. Although STM Δ sirA showed a PI-positive population comparable to STM WT when exposed to these antibiotics, adding formate did not further increase PI-positive cells in the mutant under ciprofloxacin treatment. In the case of meropenem treatment, a decrease in extent of PI-positive population was observed (Figure S9 E ). Likewise, formate treatment did not elevate the ROS-positive population in STM Δ sirA , unlike the increase observed in STM WT (Figure S9 F) . A similar trend was seen in the number of depolarized cells under antibiotic stress (Figure S9 G) . In summary, these results suggest that extracellular formate enhances STM WT susceptibility to meropenem and ciprofloxacin primarily through the BarA/SirA signalling system. Deletion of pflB impaired the long-term survival of STM under high antibiotic concentrations, a similar effect observed when formate was supplemented to the wild type Bacterial persistence in the presence of antibiotics, distinct from antibiotic resistance, remains a persistent challenge in effectively eliminating pathogens in clinical settings( 55 ). Since our data demonstrated that pflB deletion increased the antibiotic susceptibility of STM, we aimed to investigate its impact on long-term bacterial survival under high antibiotic stress. Log-phase bacterial cultures, with or without formate supplementation, were exposed to 15× and 25× MIC concentrations of meropenem and ciprofloxacin. Exposure to 15× MIC of meropenem led to a rapid decline in bacterial countsin planktonic culture, with STM Δ pflB exhibiting the fastest reduction, reaching statistical significance at 24 hours post-treatment ( Figure 7A ) . Formate supplementation restored survival levels of STM Δ pflB to those of the wild type, while supplementation in STM WT also caused a significant reduction in CFUs. This effect was specific to planktonic cultures treated with meropenem and was not observed in biofilm forms or in ciprofloxacin-treated planktonic and biofilm cultures ( Figure 7B, C, D ) . Download figure Open in new tab Figure 7: Deletion of pflB reduced STM’s long-term survival under high antibiotic concentrations, an effect also seen with formate supplementation in the wild type. (A,B) Line graphs show log₁₀ fold reduction in CFU relative to 0h for planktonic cultures of STM WT and STM Δ pflB , with or without formate supplementation, upon exposure to 15X MIC of meropenem and 25X MIC of ciprofloxacin. Data is presented as mean+/- SD. (C, D) Line graphs show log₁₀ fold reduction in CFU relative to 0h for biofilm cultures of STM WT and STM Δ pflB , with or without formate supplementation, upon exposure to 15X MIC of meropenem and 25X MIC of ciprofloxacin. Data is presented as mean+/- SD. (Unpaired two-tailed Student’s t-test for column graphs, Two-way ANOVA for grouped data, Mann-Whitney U-test for animal experiment data (*** p < 0.0001, *** p < 0.001, ** p<0.01, * p<0.05)) Discussion Salmonella has emerged as a major food-borne pathogen with widespread drug resistance, and the increasing prevalence of MDR strains poses a serious clinical challenge ( 56 – 58 ). The growing inefficacy of existing treatments complicates disease management and highlights the urgent need for novel antimicrobial strategies or repurposing of existing drugs ( 59 , 60 ). Traditionally, research on AMR has focused on canonical molecular mechanisms by which bacteria evade antibiotics, including target modification, enzymatic drug degradation, and altered membrane permeability ( 61 – 66 ). More recently, bacterial metabolism has gained attention as a determinant of AMR. For example, ceftriaxone-resistant Salmonella Derby was shown to exhibit altered metabolic activity, particularly in glutathione biosynthesis, with reduced levels of oxidized glutathione and citrulline. Supplementation of these metabolites restored ceftriaxone susceptibility in MDR isolates ( 67 ). Parallel studies have highlighted the influence of ipH on antibiotic resistance in Salmonella and other bacteria ( 11 , 15 , 16 , 68 ), underscoring the importance of metabolic pathways involved in pH regulation. The enzyme PflB, which converts pyruvate into formate and acetyl-CoA, plays a central role in maintaining ipH ( 10 ). Consistent with this function, we observed that STM lacking pflB exhibited increased susceptibility to ciprofloxacin and meropenem, a phenotype that could be partially rescued by external formate supplementation. While pH is known to modulate the PMF and thereby influence efflux activity, our data indicate an additional transcriptional effect, as ipH disruption resulted in downregulation of efflux pump genes acrB and tolC ( 11 ). In previous work, we showed that membrane stress induced by ipH imbalance activated the RpoE sigma factor, elevating expression of the small RNA csrB and repressing flagellar gene expression in STM Δ pflB ( 10 ). In this study, we extend those findings by showing that rpoE deletion not only restored motility but also reversed the heightened antibiotic sensitivity of STM Δ pflB . Similarly, deletion of sirA , an upstream regulator of csrB , had a comparable effect. These results suggest that pflB regulates both invasion and antibiotic resistance through a shared RpoE– csrB signaling pathway. Importantly, we also found that extracellular formate impacts AMR through the BarA/SirA two-component system—traditionally known for its role in regulating invasion—thereby expanding the functional scope of this signaling axis to include efflux-mediated antibiotic resistance ( 69 ) (Graphical abstract) . The role of metabolism in antibiotic tolerance and persistence has also been documented in other bacteria. In E. coli , adenosine was shown to suppress the stringent response, enhance PMF, increase respiration, and sensitize persister cells to antibiotics—a process described as “persister awakening” ( 70 ). To test whether formate metabolism influences persistence, we assessed long-term survival of STM Δ pflB under high-dose antibiotic treatment. In planktonic cultures, STM Δ pflB exhibited accelerated killing compared to the wild type, a phenotype that was reversed by formate supplementation. Interestingly, addition of formate to wild-type STM further enhanced antibiotic killing. Together, our findings support the idea that antibiotic effectiveness is intimately linked to bacterial metabolic state. Specifically, intracellular formate, regulated by PflB, modulates ipH homeostasis, efflux pump expression, and antibiotic tolerance in STM. These results identify formate metabolism as a determinant of both acute resistance and persistence, pointing to metabolic reprogramming as a potential therapeutic strategy to re-sensitize MDR Salmonella to existing antibiotics. Author Contributions DM and DC conceived the study and designed the experiments. DM conducted all experiments, data acquisition, analysis, figure preparation, and wrote the original draft. SVW, PG, and AB helped DM with experiments. DM and DC contributed to manuscript editing. DC supervised the project and secured funding. All authors reviewed and approved the final manuscript. Declaration of Interest The authors declare no conflicts of interest. Funding Information We gratefully acknowledge financial support from the Department of Biotechnology (DBT) and the Department of Science and Technology (DST), Ministry of Science and Technology. DC thanks DAE for the SRC Outstanding Investigator Award, ASTRA Chair Professorship, and TATA Innovation Fellowship. Additional support came from the DBT-IISc Partnership Program. Infrastructure support from ICMR (Molecular Medicine Center), DST (FIST), and UGC-CAS is also acknowledged. DM is supported by IISc fellowship. PG is supported by DBT-JRF fellowship. AB acknowledges UGC fellowship. The funders had no involvement in study design, data collection, analysis, or publication decisions. Data Availability Data supporting this study are available from the authors upon request. Graphical abstract: Graphical abstract showing regulation of AMR in Salmonella by formate Acknowledgements We thank the Departmental Real-Time PCR, Divisional Mass Spectrometry, Divisional Flow Cytometry Facility, Divisional Atomic Force Microscopy, and the Central Animal Facility at IISc for supporting experimental work. Dr. Sunitha assisted in formate quantification via GC-MS. Flow cytometry data acquisition was supported by Mr. Munish and Mrs. Navya. Atomic Force Microscopy was supported by Mrs. Monisha. Dr. Raju Rajmani is acknowledged for help in mice serum isolation. Funder Information Declared Department of Biotechnology, https://ror.org/03tjsyq23 Department of Science and Technology, https://ror.org/0101xrq71 Ministry of Science and Technology Department of Atomic Energy, https://ror.org/02m388s04 Footnotes The manuscript has been updated to include new data. References 1. ↵ Zhu S , Yang B , Jia Y , Yu F , Wang Z , Liu Y . Comprehensive analysis of disinfectants on the horizontal transfer of antibiotic resistance genes . J Hazard Mater . 2023 ; 453 : 131428 . 2. ↵ Brauner A , Fridman O , Gefen O , Balaban NQ . Distinguishing between resistance, tolerance and persistence to antibiotic treatment . Nat Rev Microbiol . 2016 ; 14 ( 5 ): 320 – 30 . OpenUrl CrossRef PubMed 3. ↵ Antimicrobial Resistance C. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis . Lancet . 2022 ; 399 ( 10325 ): 629 – 55 . OpenUrl CrossRef PubMed 4. ↵ Azmatullah A , Qamar FN , Thaver D , Zaidi AK , Bhutta ZA . 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Share Pyruvate-formate lyase–derived formate regulates the response of Salmonella Typhimurium to meropenem and ciprofloxacin Debapriya Mukherjee , Sakshi Vasant Wagle , Pallab Ghosh , Abhishek Biswas , Dipshikha Chakravortty bioRxiv 2025.05.22.655487; doi: https://doi.org/10.1101/2025.05.22.655487 Share This Article: Copy Citation Tools Pyruvate-formate lyase–derived formate regulates the response of Salmonella Typhimurium to meropenem and ciprofloxacin Debapriya Mukherjee , Sakshi Vasant Wagle , Pallab Ghosh , Abhishek Biswas , Dipshikha Chakravortty bioRxiv 2025.05.22.655487; doi: https://doi.org/10.1101/2025.05.22.655487 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7619) Biochemistry (17642) Bioengineering (13865) Bioinformatics (41862) Biophysics (21408) Cancer Biology (18546) Cell Biology (25435) Clinical Trials (138) Developmental Biology (13357) Ecology (19863) Epidemiology (2067) Evolutionary Biology (24288) Genetics (15587) Genomics (22467) Immunology (17701) Microbiology (40301) Molecular Biology (17142) Neuroscience (88444) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4815) Physiology (7633) Plant Biology (15108) Scientific Communication and Education (2042) Synthetic Biology (4285) Systems Biology (9811) Zoology (2268)