Clarithromycin Exerts an Antibiofilm Effect against Salmonella enterica Serovar Typhimurium rdar Biofilm Formation and Transforms the Physiology towards an Apparent Oxygen-Depleted Energy and Carbon Metabolism

Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection toward various forms of environmental stresses to individual cells. Thus, bacterial cells within biofilms become tolerant against antimicrobials and the immune system. In the present study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin.

ABSTRACT

Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection toward various forms of environmental stresses to individual cells. Thus, bacterial cells within biofilms become tolerant against antimicrobials and the immune system. In the present study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry, and rough) biofilm formation of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium ATCC 14028 (Nal^r) at a 1.56 μM subinhibitory concentration in standing culture and dissolved cell aggregates at 15 μM in a microaerophilic environment, suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be concomitantly downregulated. Although fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifted the cells toward an apparent oxygen- and energy-depleted status, whereby the metabolism that channels into oxidative phosphorylation was downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and l-arginine catabolism, potentially also to prevent cytosolic acidification, was upregulated. This analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.

INTRODUCTION

Elevated intrinsic tolerance against antibiotics is mainly promoted by biofilm formation of the organism (1, 2). Biofilms are multicellular communities of microorganisms embedded into a self-produced extracellular matrix. Biofilms build up on both abiotic and biotic surfaces, but a multicellular behavior is also expressed as cell aggregation (free-floating in liquid, so-called flocks) and pellicle formation, the air-liquid biofilm (3, 4). Among the various functions of bacterial biofilms, provision of nutrients and to defend their replicating entities during unfavorable environmental conditions are among major characteristics. As biofilms are consequently tolerant against disinfectants, antimicrobials, and the immune system of the host, according to the National Institutes of Health (NIH), up to 80% of human bacterial infections are associated with biofilm formation (5, 6). Alongside microbial cells, up to 90% of the biofilm mass can be comprised of the extracellular matrix, which can consist of self-produced exopolysaccharide, proteins, lipids, and extracellular DNA, but also environmental or host components. The extracellular matrix can be extensively hydrated, differ in solubility, with its component to cross-link, thus providing a physical and chemical shield against antimicrobials and other stress conditions (7).

The Gram-negative foodborne pathogen Salmonella enterica serovar Typhimurium characteristically forms biofilms which contributes to the perseverance of Salmonella in the food industry (8). Foodborne diseases by Salmonella arise due to the contamination of food products such as poultry, as well as vegetables and fruits. According to the U.S. Centers for Disease Control and Prevention, almost 48 million cases of foodborne diseases occur annually, leading to 128,000 hospitalizations and 3,000 deaths (9).

S. Typhimurium displays a specific colony morphology, which is characterized by a red, dry, and rough (rdar) appearance on agar plates containing Congo red. This biofilm expresses the exopolysaccharide cellulose and proteinaceous curli as biofilm matrix components (10). Biofilm development is controlled by the key transcriptional regulator CsgD (curli subunit gene D), a LuxR family protein, which activates transcription of the csgBAC operon encoding curli fiber components, and indirectly governs the activity of the cellulose synthase by promoting transcription of the diguanylate cyclase AdrA (11–13). CsgD plays a pivotal role in switching between biofilm, motility, and virulence state(s) of bacteria, regulated by various, both defined and as-yet-undefined, stimuli, which alter the production of the second messenger cyclic di-GMP (14–17).

Antibiotics have a short (not more than 85-year) history of being used systematically in medical setups to treat and eradicate bacterial infections, and yet the emergence of drug-resistant and multidrug-resistant organisms has been frequently detected from the beginning. Since biofilm formation leads to chronic infections refractory to treatment by antimicrobial therapy (18), identifying a role for antibiotics in modulating biofilm formation can lead to a rational approach in the development of successful antibiofilm therapies. Evaluating U.S. Food and Drug Administration-approved drugs for their antibiofilm activity can reduce development time and cost up to 40% (19).

Macrolides, a class of broad-spectrum antibiotics, were the third class of antibiotics to be identified and have been used since 1952 in clinical practice. Erythromycin, the first discovered natural macrolide, has since been replaced by the more-effective second-generation semisynthetic macrolides clarithromycin and azithromycin (20). Macrolides selectively inhibit protein biosynthesis by binding to the 23S RNA of the 50S subunit of the ribosome, halting translation in bacteria mainly by blocking the entrance tunnel for the nascent peptide chain (21–24). An antibiofilm role of macrolides has been observed in various species of bacteria, but the underlying mechanisms have not been resolved (25). Apart from their role as antibiotics and antibiofilm compounds, macrolides possess immunomodulatory and anti-inflammatory properties (26).

The aim of this study was to identify the antibiofilm potential of established macrolide antibiotics and to unravel the underlying regulation. We found that the macrolide antibiotic clarithromycin possesses antibiofilm activity against rdar biofilm forming S. Typhimurium UMR1. RNA sequencing showed that clarithromycin at subinhibitory concentrations, in addition to the differential expression of biofilm-related genes such as the central activator csgD, causes a distinct multiplex physiological response, which will lead to the identification and characterization of novel antibiofim and antimicrobial targets and consequently the design of effective combinatorial antimicrobial strategies. Such upregulation of genes and proteins related to ribosome functionality and the heat shock response indicates compensatory innate resistance mechanisms against the protein synthesis inhibiting, 23S RNA targeting drug clarithromycin. Furthermore, the bacterial cell experiences apparent oxygen and carbon depletion and/or cytosolic acidification upon clarithromycin treatment, as indicated by the upregulation of genes encoding the trimethylamine-N-oxide reductase and genes involved in anaerobic propane-1,2-diol, ethanolamine, and l-arginine degradation, and downregulation of genes involved in pathways channeling into oxidative phosphorylation, such as the Krebs, glyoxylate, and 2-methylcitrate cycles.

RESULTS

Clarithromycin, but not azithromycin, shows predominantly an antibiofilm effect.

S. Typhimurium UMR1 forms a biofilm on the abiotic polystyrene surface in the 96-well plate assay after 16 h of static incubation at 28°C. Addition of macrolides azithromycin or clarithromycin serially 2-fold diluted from 0.02 to 100 μM (corresponding to 0.015 μg/ml to 74.9 and 74.8 μg/ml, respectively) to the cell suspension showed, as expected, a growth delay for both drugs. The drug concentrations at which 50% inhibition of growth (MIGC₅₀) were observed were 0.018 μM azithromycin and 10.9 μM clarithromycin. Notably, only clarithromycin exhibited a distinct antibiofilm effect after 48 h of incubation (Fig. 1 and 2). An antibiofilm effect was considered present when the percentage of biofilm inhibition was consistently and significantly exceeding the percentage of growth inhibition at a distinct concentration of the drug. We observed a maximal antibiofilm effect at 1.563 μM (1.17 μg/ml) clarithromycin (0.03125%/3.3 mM dimethyl sulfoxide [DMSO]), at which biofilm formation was inhibited by 49%, whereas growth was only inhibited by 14% (Fig. 2).

FIG 1.

Inhibition of biofilm formation and growth of S. Typhimurium UMR1 by azithromycin. The cell suspension was incubated in the 96-well plate assay statically in LB without salt medium for 16 h at 28°C. Biofilm formation and growth were assessed by staining the biofilm (cells adherent to the wall of well) with 0.2% crystal violet and measurement of the OD₆₀₀ of the cell suspension, respectively. Subsequently, the percent inhibition of biofilm and growth was estimated. Azithromycin predominantly showed an antimicrobial effect. Untreated S. Typhimurium UMR1 was the positive control, and S. Typhimurium MAE50 was the negative control in each experiment. Solvent (2.5% DMSO) and medium control were included with each experiment. The data shown are means ± the standard errors of the mean (SEM) of one representative biological experiment with six technical replicates.

FIG 2.

Inhibition of biofilm formation and growth of S. Typhimurium UMR1 by clarithromycin. The cell suspension was statically incubated in LB without salt medium for 16 h at 28°C in the 96-well plate assay. Biofilm formation and growth was assessed by staining the biofilm (cells adherent to the wall of well) with 0.2% crystal violet and estimation of OD₆₀₀ of the cell suspension, respectively. Subsequently, the percent inhibition of biofilm and growth were estimated. At 1.56 μM, clarithromycin predominantly showed the optimal antibiofilm activity with less-pronounced growth inhibition. Untreated S. Typhimurium UMR1 was the positive control, and S. Typhimurium MAE50 was the negative control in each experiment. Solvent control (2.5% DMSO) and medium control analyses were performed with each experiment. The data shown are the means ± the SEM of two biological experiments with six technical replicates each.

Clarithromycin prevents cell aggregation in liquid culture.

To investigate the antibiofilm effect of clarithromycin in an alternative biofilm model, S. Typhimurium UMR1 cells were grown under microaerophilic conditions in liquid culture (27). Under these experimental conditions, rdar biofilm formation of S. Typhimurium UMR1 is mainly expressed as, even macroscopically, visible cell aggregates, although adherence to the abiotic glass wall also occurs (Fig. 3A). Of note, incubation of S. Typhimurium in the same Luria broth (LB) without salt medium in the same flask under aerobic conditions abolishes biofilm formation (27).

FIG 3.

Effect of clarithromycin (CLA) on the cell aggregation of S. Typhimurium in shaken liquid culture. (A) The biofilm formation of the wild-type S. Typhimurium UMR1 is mainly expressed in the form of cell aggregates and partially as adherence to the wall of the well (red and green arrows, respectively). Cell aggregates were visually enhanced showing altered consistency, whereas adherence to the abiotic surface was decreased at 7.5 μM clarithromycin. Treatment with 15 μM clarithromycin abolished cell aggregation and adherence. S. Typhimurium MAE50 was used as a negative control for cell clumping and adherence. S. Typhimurium UMR1 was grown under microaerophilic conditions in LB without salt medium at 28°C for 16 h with 150-rpm shaking. (B) Inhibition of biofilm formation in S. Typhimurium MAE52 grown in LB without salt medium upon treatment with 15 μM clarithromycin. (C) The cell suspension of S. Typhimurium MAE52 after 30 min of settlement. (D) S. Typhimurium MAE52 grown in LB medium without or with 15 μM clarithromycin at 28°C for 16 h with 150-rpm shaking.

After incubation of S. Typhimurium UMR1 with different concentrations (0, 3.75, 7.5, 15, and 30 μM) of clarithromycin, cell aggregates of S. Typhimurium UMR1 were not only clearly visible in considerable amounts without clarithromycin, but substantial aggregates were also still visible upon incubation with up to 7.5 μM clarithromycin. However, these aggregates had a different consistency compared to the wild type, looking more fluffy and voluminous, making them clearly more visible by the naked eye in the medium. At 15 μM (11.22 μg/ml) clarithromycin, no cell aggregation was observed, and seemingly only planktonic cells were present (Fig. 3A). On the other hand, the negative-control S. Typhimurium MAE50 showed a cell suspension without visible cell aggregation. Of note, growth was affected by clarithromycin. Whereas 3.75 μM clarithromycin decreased the steady-state optical density at 600 nm (OD₆₀₀) after 16 h of growth by 6% and 7.5 μM clarithromycin by 8%, the steady-state OD₆₀₀ was reduced by 22.5% with 15 μM clarithromycin.

In order to investigate whether the antibiofilm effect of clarithromycin can be also observed in a strongly biofilm-forming strain, we assessed the effect of the drug in S. Typhimurium MAE52. This strain, a csgD promoter mutant of UMR1 (28), displays extensive cell aggregation in liquid culture. Treatment with 15 μM clarithromycin abolished biofilm formation on the glass surface, reduced cell aggregates, and enhanced planktonic growth (Fig. 3B to D).

Effect of clarithromycin on cell aggregation of S. Typhimurium UMR1 as observed by light microscopy.

Subsequently, the amount of cell aggregation and aggregate morphology was assessed by bright-field microscopy at clarithromycin concentrations ranging from 0, 3.75, 7.5, 15, to 30 μM. Large cell aggregates with a tight consistency and defined aggregate border were observed for S. Typhimurium UMR1 (Fig. 4). Subsequently, upon treatment with clarithromycin, the tendency for cell aggregates to alter their consistency was concentration dependent. At a clarithromycin concentration of 3.75 μM, an already-pronounced effect could be noticed as, in addition to solid aggregates with defined rims, distorted cell aggregates with less defined edges and loosely attached smaller cell aggregates were present. At 7.5 μM clarithromycin, cell aggregates were much less compact, the sizes of the aggregates were undefined and significantly decreased, and individual cells were more loosely connected. At 15 μM clarithromycin, only planktonic cells were visible, demonstrating the gradual inhibition of the cell aggregate biofilm structures of S. Typhimurium UMR1 upon clarithromycin treatment. Of note, the negative-control S. Typhimurium MAE50 consisted only of single cells.

FIG 4.

Cell aggregates of S. Typhimurium UMR1 under microaerophilic conditions in liquid culture upon treatment with clarithromycin. (A to F) Light-microscopy images of representative cell aggregates of S. Typhimurium UMR1 (A), S. Typhimurium MAE50 (negative control) (B), and S. Typhimurium UMR1 treated with 3.25 μM (C), 7.5 μM (D), 15 μM (E), or 30 μM (F) clarithromycin. A representative part of the cell suspension was spread on a glass slide, and cell aggregation was documented. Arrows point to cell aggregates with attention to the rim of the aggregate. All images were taken at ×20 magnification.

The production of the biofilm regulator CsgD is decreased upon treatment with clarithromycin.

CsgD is the central transcriptional activator required for rdar biofilm formation on abiotic surfaces and cell aggregation in liquid culture (3, 17, 29). Consequently, we monitored the expression of CsgD after exposure to different concentrations of clarithromycin (Fig. 5). S. Typhimurium UMR1 under microaerophilic conditions in liquid culture produced significant levels of CsgD, as reported previously (Fig. 5) (27). Upon treatment of S. Typhimurium UMR1 with 3.75, 7.5, 15, and 30 μM clarithromycin, a decrease in the expression levels of CsgD was detected by Western blotting. Consistent with the cell aggregation phenotype, at 15 μM clarithromycin a steep decrease occurred, with almost no CsgD protein production detected (Fig. 5). Therefore, the underlying mechanism of clarithromycin to inhibit rdar biofilm formation and cell aggregation is consequently to target the regulatory pathway(s), leading to csgD expression. However, the discrepancy between the cell aggregation phenotype and CsgD production indicates additional pathways to be targeted by clarithromycin.

FIG 5.

Production of the rdar biofilm activator CsgD in liquid microaerophilic culture upon treatment with clarithromycin. S. Typhimurium UMR1 cells were harvested from a microaerophilic LB without salt culture after growth for 16 h at 28°C with 150-rpm shaking with 0, 3.25, 7.5, 15, 30, or 60 μM clarithromycin. The CsgD production level was significantly diminished upon treatment with 15 μM clarithromycin. S. Typhimurium MAE50 (UMR1 ΔcsgD) was used as the negative control. The respective Coomassie blue-stained gel is shown in Fig. 8.

Differential expression of biofilm and motility genes upon treatment with clarithromycin as assessed by qRT-PCR.

Since CsgD protein production was downregulated upon treatment with clarithromycin, we wondered at which level clarithromycin interferes with the regulation of rdar biofilm formation. Consequently, we estimated the expression of relevant biofilm genes by quantitative real-time reverse transcription-PCR (qRT-PCR) (Fig. 6). The expression level of csgD encoding the major biofilm regulator, and consequently the expression level of csgB, coding for the minor subunit of amyloid curli fimbriae, were downregulated by 48.7 and 47.4%, respectively. Furthermore, the expression level of adrA, encoding the diguanylate cyclase required for activation of the extracellular matrix component cellulose, decreased by 48.1% compared to the untreated control. In addition, the expression of bcsA, coding for the cellulose synthase, decreased by 15.7%, although bcsA expression is not regulated by csgD (13). On the other hand, the expression of fliA encoding sigma factor 28 for class 2 genes of the flagellar regulon and class 3 gene fliC coding for one major subunit flagellin was significantly increased 7.3- and 1.36-fold, respectively. Besides fliC, this differential expression pattern holds, although the rpsV and recA reference genes were both subsequently found to be slightly downregulated by RNA sequencing (e.g., rpsV showed a 0.64-fold lower expression upon treatment with 15 μM clarithromycin, which, if extrapolated to the qRT-PCR data, would indicate higher downregulation and lower upregulation of genes).

FIG 6.

Expression of biofilm genes csgD, csgB, adrA, and bcsA and flagellar genes fliA and fliC in S. Typhimurium UMR1 upon incubation with 15 μM clarithromycin. While csgD, csgB, adrA, and bcsA were downregulated after treatment with 15 μM clarithromycin, class 2 and 3 genes fliA and fliC were upregulated. The fold expression upon treatment versus untreated control is indicated. For RNA isolation, the cells were harvested after 16 h of incubation under microaerophilic condition at 28°C with 150-rpm shaking. recA was used as a control gene. mRNA steady-state levels were assessed by qRT-PCR, and the data were normalized to the expression of rpsV. The data are expressed as means ± the standard deviation using independent T-test for two experiments (***, P < 0.001).

Clarithromycin treatment significantly alters the transcriptome of rdar biofilm-forming S. Typhimurium UMR1.

Treatment with 15 μM clarithromycin downregulated the expression of the major biofilm regulator csgD. In order to assess differential expression of the regulatory pathway(s) leading to the inhibition of rdar biofilm formation upon treatment with clarithromycin, as well as to unravel additional alterations of the cellular transcriptome upon clarithromycin treatment, we performed RNA sequencing (Fig. 7). S. Typhimurium UMR1 grown under microaerophilic conditions for 16 h at 28°C and with 150-rpm shaking was treated with 15 μM clarithromycin. Of the 4,586 annotated genes of S. Typhimurium ATCC 14028S, 353 genes were >4-fold differentially regulated, whereas 107 genes were >8-fold differentially regulated. Tables S2 and S3 in the supplemental material display the 30 most up- and downregulated genes.

FIG 7.

Genes differentially regulated upon treatment of microaerophilic grown S. Typhimurium UMR1 with 15 μM clarithromycin. (A) Volcano plot indicating the significantly differentially regulated genes. The dotted red line indicates genes differentially regulated by >4-fold. The dotted green line indicates a P > 0.01 threshold of statistical significance. Red symbols, 20 genes most significantly differentially regulated by >4-fold. Green symbols, significantly differentially regulated genes with a P < 0.01 threshold. Blue symbols, genes differentially regulated less than 1.4-fold and/or not significantly differentially regulated (threshold of P > 0.01). (B) Scatter blot indicating the expression level of significantly differentially regulated genes. Red symbol, genes significantly differentially upregulated by >4-fold. Green symbols, genes significantly differentially downregulated by >4-fold. Blue symbols, genes significantly differentially regulated by <4-fold.

Consistent with the observed rdar biofilm phenotype and the previously performed qRT-PCR experiments, the transcriptional data showed that several genes of the divergently transcribed csgDEFG and csgBAC operons, including the csgD gene encoding the rdar biofilm activation, were significantly downregulated (see Table S4 in the supplemental material), although these genes were not among the most downregulated genes. In addition, other genes involved in biofilm formation are downregulated, such as the gene coding for the cyclic di-GMP synthase AdrA that is required for cellulose biosynthesis. Upregulation of the gene for the truncated EAL protein STM0551, a posttranscriptional repressor, indicates downregulation of type 1 fimbriae (30).

Motility and biofilm formation are two fundamentally opposite lifestyles of bacteria (31). In contrast to biofilm genes, most flagellar regulon genes were upregulated, including flhD and flhC encoding the class 1 central flagellar regulator FlhD₄C₂ and the class 2 gene fliA encoding the flagellar sigma 28 factor, although a few class 2 and class 3 genes, including fliC and the phosphodiesterase gene yhjH, were downregulated (see Table S5). The dramatic downregulation of three paralogous rpoS regulated genes coding for <60 amino acid KGG-rich intrinsically disordered proteins (YmdF, YciG, and STM14_RS08420) required for swimming and swarming motility (32), however, might indicate their decisiveness for the observed lack in swimming motility (data not shown).

Among the most downregulated genes (see Table S3) were two ferritin-like genes involved in the storage of iron in a nontoxic form equally as the ferritin-like protein Dps (33, 34). Clarithromycin-exposed cells might be depleted of the osmoprotectant trehalose, as genes for biosynthesis and breakdown enzymes are downregulated (35). Furthermore, genes that encode Krebs cycle enzymes, including associated pathways such as the 2-methylcitrate cycle, the GABA shunt, and the glyoxylate cyclase, are consistently downregulated. Indeed, downregulation of the genes coding for propionate-CoA ligase and formate C-acetyltransferase indicates that the production of substrates channeling into these pathways is also blocked.

The most upregulated genes upon treatment with clarithromycin were the three genes of the torCAD operon encoding a cytochrome c-type protein, the trimethylamine N-oxide (TMAO) reductase, and its chaperone (see Table S2) (36). TMAO serves as an alternative electron acceptor in anaerobic respiration. Furthermore, genes coding for both the propane-1,2-diol microcompartment and, partially, the ethanolamine utilization pathway were upregulated, although their parallel upregulation has been reported to be mutually exclusive (37, 38). Propane-1,2-diol and ethanolamine are usually utilized as carbon and energy sources under anaerobic conditions supported by the alternative electron acceptor tetrathionate (38). Another highly transcriptionally upregulated pathway incorporates genes involved in the degradation of l-arginine under anaerobiosis (39). In addition, the genes of the pathway(s) leading to biosynthesis of branched-chain amino acids, leucine, isoleucine, and valine are consistently upregulated (40).

The macrolide clarithromycin targets the ribosome by binding to the 23S RNA within the 50S ribosomal subunit to selectively inhibit protein synthesis, to prevent assembly of ribosomes, and to trigger tRNA dissociation. We found distinct genes involved in protein synthesis and homeostasis to be upregulated. For example, genes coding for ribosomal proteins, such as for the 50S ribosomal proteins L21 and L27, and rRNA- and ribosomal protein-modifying genes, such as the genes for 23S rRNA (guanine(745)-N(1))-methyltransferase and the 30S ribosomal protein S5 alanine-N-acetyltransferase, respectively, were >16-fold upregulated (see Table S6). Furthermore, distinct tRNAs and tRNA modification genes were also upregulated. In particular, four tRNAs for arginine are upregulated >4-fold (with one arginine tRNA downregulated). Table S6 in the supplemental material shows the heatmap for differentially regulated ribosome-associated genes. Since the genes encoding the trigger factor and the heat shock response, including genes encoding the heat shock sigma factor RpoH and holding chaperons IbpA and IbpB, were upregulated, we conclude that the clarithromycin-exposed cell is under severe proteotoxic stress. A highly specific response was therefore set up to overcome the effect of clarithromycin on the prevention of protein synthesis and peptidyl tRNA dissociation in order to ensure proper protein translation and folding and to prevent protein aggregation.

The upregulation of the gene for the multidrug efflux MFS transporter MdtM indicates that the cell actively pumps out the macrolide through MdtM. The downregulation of the open reading frame of the outer membrane porin OmpC might indicate a first-line innate immune response to protect against the macrolide.

Furthermore, we preliminarily investigated how the alterations on the transcriptional level translate into changes on the protein level upon treatment with clarithromycin (Fig. 8). One-dimensional protein gels showed a substantial change in the protein expression profile but no significant protein degradation up to 60 μM clarithromycin (Fig. 8). In congruence with the transcriptome data, potentially upregulated proteins upon exposure to clarithromycin included ribosomal proteins, whereas potentially downregulated proteins included the ferritin-like metal-binding protein YciE and proteins that belong to major metabolic pathways such as the Krebs cycle. Two-dimensional gel electrophoresis or gel-free approaches such as SILAC (stable isotope labeling by amino acid in cell culture) is required to verify the identity of differentially regulated proteins. Figure 9 summarizes the overall physiological consequences of the differentially regulated genes on treatment of S. Typhimurium rdar biofilm (28) with clarithromycin.

FIG 8.

Changes in protein expression in S. Typhimurium UMR1 upon clarithromycin treatment. S. Typhimurium UMR1 was grown under microaerophilic conditions in LB without salt medium for 16 h at 28°C with 150-rpm shaking with 0, 3.75, 7.5, 15, 30, or 60 μM clarithromycin. MAE50 was the negative control. Differentially appearing protein bands subjected to analysis by mass spectrometry are indicated by an arrow. Green, identified proteins from bands upregulated upon clarithromycin treatment; red, identified proteins from bands present in the untreated wild-type S. Typhimurium UMR1.

FIG 9.

Summary of major physiological and metabolic changes in rdar biofilm-forming S. Typhimurium UMR1 upon exposure to 15 μM clarithromycin. Green arrows, symbols, and text indicate the gene encoding the enzyme performing this reaction or the respective protein is upregulated on the transcriptional level. Red arrows, symbols, and text indicate gene encoding the enzyme performing this reaction or the respective protein is downregulated on the transcriptional level. MgtC, CsgD, and STM0551 affect extracellular matrix components cellulose, curli, and type 1 fimbriae in the indicated way. Further explanation, see the text.

DISCUSSION

The use of antimicrobial agents has been a successful strategy for treating bacterial infections. A major hurdle is biofilm formation by microorganisms, which impairs antimicrobial treatment strategies of chronic infections because of the tolerance of biofilms against antibiotics, as well as the immune system. Biofilm formation is particularly pronounced on implanted medical devices due to the lack of effective antimicrobial defense mechanisms (41). In this study, we unravel the overall transcriptional response of S. Typhimurium against the protein synthesis inhibiting macrolide clarithromycin.

Clarithromycin can target various pathogens and infections even beyond its antimicrobial effect (42–44). Moreover, a biofilm-inhibitory effect against Pseudomonas aeruginosa, Helicobacter pylori, and Staphylococcus aureus has been reported for this drug (alone or in combination) (45, 46). We evaluated the role of this macrolide antibiotic primarily as an antibiofilm agent against the rdar biofilm formation of S. Typhimurium UMR1. RNA sequencing unraveled not only the molecular mechanisms and various pathways involved in the biofilm-inhibiting effect of clarithromycin but also demonstrated major metabolic alterations that might provide a basis for rationally designing combinatorial treatment strategies.

After screening a library of well-investigated antibiotics, we observed that clarithromycin, among the other macrolides, possessed antibiofilm properties. Of note, although not determined under the clinically recommended test conditions, the MIGC₅₀ values for azithromycin and clarithromycin were significantly different for S. Typhimurium UMR1. These two agents differ substantially in structure though. To assess the antibiofilm effect of structurally highly similar erythromycin and biotransformed 14-OH clarithromycin alone and in combination with clarithromycin is relevant for future investigations. Surprisingly, we observed a growth condition-dependent effect for clarithromycin, since 1.56 μM clarithromycin was sufficient in a 96-well standing culture biofilm assay to prevent biofilm formation on an abiotic surface, whereas >15 μM clarithromycin was required to prevent cell aggregation and to cause a growth delay under microaerophilic conditions in shaking culture. Alternatively, the susceptibility of the different biofilm stages (abiotic surface biofilm versus cell-cell interactions) towards clarithromycin differs. Based on our RNA-sequencing data, we conclude that clarithromycin sets the cell into an apparent state of oxygen depletion and strongly interferes with pathways channeling into the oxidative phosphorylation, which might explain the oxygen-dependent effect (Fig. 9; see also below).

The expression of the csgD biofilm activator, which is indicative for rdar biofilm formation, can dramatically alter in response to environmental conditions and temperature (27, 28) and also occurs in vivo at body temperature (70). Clarithromycin treatment targets the expression of csgD and additional biofilm-associated genes, including genes required for the posttranslational regulation of type 1 fimbriae, which could be used to control infections related to Salmonella (47). A study in an Escherichia coli outbreak strain O104:H4 showed that biofilm formation and virulence genes were coexpressed, suggesting that an antibiofilm strategy can be an effective antivirulence strategy (48). Moreover, csgD and the extracellular matrix components of Salmonella, i.e., curli and cellulose, have been shown to play a role in persistence (49). The genes determinative for the downregulation of csgD expression are not entirely obvious. Among the known genes positively regulating csgD expression, genes encoding the sigma factor RpoS and the transcriptional regulator MlrA are moderately downregulated. However, the differential regulation of small RNAs, several of which affect csgD expression, still must be analyzed in detail.

Several other effects of clarithromycin might contribute to the downregulation of biofilm formation. We observed high upregulation of genes involved in the electron transport chain and anaerobic respiration. Alteration in enzymes involved in electron transport chain interferes with biofilm formation in Gram-positive and Gram-negative bacteria (50). Furthermore, there exists a correlation between multidrug efflux pumps and biofilm in Salmonella, since biofilm formation is compromised upon deletion or inhibition of multidrug efflux pumps (51). Our data suggest that the gene for the multidrug efflux transporter MdtM is upregulated, which can be an innate resistance mechanism as upregulation of efflux pumps confers multiple drug resistance in bacteria (52). It has been previously shown that a csgD deletion mutant accumulates tricarboxylic (Krebs) cycle intermediates in order to block gluconeogenesis. Furthermore, cells in biofilms have a higher ATP requirement (53). We have observed the downregulation of the Krebs cycle and related pathways upon clarithromycin treatment-induced csgD downregulation, which prevents efficient energy gain through oxidative phosphorylation, a mechanism that might contribute to inhibition of biofilm formation. Similarly, the gene for the virulence factor MgtC is upregulated. MgtC downregulates cellulose biosynthesis and inhibits ATP synthase (54, 55). An efficient inhibition of the ATP synthase might stimulate the cell to seek alternative sources to produce the energy-equivalent ATP.

Moreover, genes related to iron metabolism were highly downregulated upon clarithromycin treatment (see Table S3). E. coli downregulates the iron-related gene expression during optimal growth conditions to prevent stress response mediated by high levels of iron (56), although while during multicellular swarming motility, S. Typhimurium significantly upregulates the genes for iron metabolism (57).

Surprisingly, most upregulated were genes involved in the use of alternative carbon and energy sources under anaerobic conditions. These were genes involved in anaerobic propane-1,2-diol, ethanolamine, and l-arginine degradation. The catabolism of l-arginine not only creates ATP but can also neutralize an acidified cytosol (58). The high upregulation of a gene encoding the carbon starvation transporter CstA might facilitate the acquisition of carbon sources. Furthermore, genes coding for the trimethylamine N-oxide reductase were most dramatically upregulated. Cumulatively, also taking into consideration the downregulation of Krebs cycle genes and genes of associated pathways, these findings indicate that clarithromycin sets the cells under apparent oxygen and energy depletion. Of note, bactericidal, but not bacteriostatic, drugs involve the Krebs cycle and the production of reactive oxygen species (59). Supporting the oxygen-depletion hypothesis, the TMAO reductase is required for the survival of cells experiencing sudden oxygen depletion (60). We predict that the deletion of those highly upregulated genes will increase the sensitivity for clarithromycin and can lead to the detection of novel components contributing to the innate resistance against clarithromycin. Indeed, the cellular response, as experienced by a subinhibitory concentration of antibiotics and subsequently analyzed by RNA sequencing, has successfully identified intrinsic antimicrobial resistance components (61, 62). Besides the identification of potentially novel intrinsic antimicrobial resistance factors associated with bacterial energy gain, more obvious components contributing to intrinsic antimicrobial resistance are associated with the ribosome, protein biosynthesis, and quality control, direct targets and consequences of clarithromycin exposure. However, only distinct genes are substantially upregulated, suggesting a highly specific response. Among those genes is the rrmA gene coding for 23S_rRNA_(guanine(745)-N(1))-methyltransferase that has been shown to aid elongation of the polypeptide chain (63), several 50S and 30S ribosomal proteins, and ribosome-modulating enzymes (64–66), in addition to tRNAs and tRNA-modifying enzymes (67). Of note, although ribosome associated, none of these factors has previously been identified to contribute to clarithromycin resistance, which includes identified resistance mechanisms such as methylation of the 23S rRNA at adenosine 2058 and ribosomal protein L4 and L22 overexpression (68). Furthermore, genes involved in the initiation of translation, protein folding, and quality control are upregulated. In summary, RNA sequencing revealed a highly specific response against clarithromycin, which extended beyond the inhibition of rdar biofilm formation and upregulation of intrinsic antimicrobial resistance components.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The bacterial strains used in the study were wild-type S. Typhimurium UMR1 (ATCC 14028 [Nal^r], positive for rdar biofilm formation at 28°C; S. Typhimurium MAE50 (UMR1 ΔcsgD) as an rdar biofilm-negative control, and S. Typhimurium MAE52 (UMR1 pcsgD2) as a semiconstitutive rdar biofilm-positive control (28). For rdar biofilm assessment, strains were grown on Luria broth (LB) without salt agar plates and containing Congo red (40 μg/ml) and Coomassie brilliant blue (20 μg/ml). For pre-cultures, single colonies of bacteria were inoculated in 5 ml of LB and grown overnight at 37°C with shaking. For experimentation, S. Typhimurium was grown in LB without salt broth. Volume % DMSO was equally added to untreated and treated cultures.

A 96-well biofilm assay to test antibiofilm and antimicrobial activity.

An overnight culture was adjusted to a cell density of 0.1 OD₆₀₀ with LB without salt broth, with 100 μl suspended into a 96-well plate. We dissolved 10 mM azithromycin and clarithromycin in 100% DMSO and added in 2-fold serial dilutions, 100 μl to the cell suspension starting from 100 μM in 2% DMSO. No effect on biofilm formation was observed up to 2.5% DMSO. For screening, the plate was incubated statically for 48 h at 28°C to allow the formation of biofilm on the wall of wells (16). Subsequently, the biofilm was washed, and adherent cells were stained with 0.2% crystal violet solution to assess biofilm formation. After dissolution of the dye, OD₆₀₀ was measured and the percent biofilm inhibition by the drugs was calculated using the following formula: 100 – {[(OD of test − OD of blank)/(OD of control − OD of blank)] × 100}. Growth was assessed by transferring 100 μl of the cell suspension after vigorous resuspension from the 96-well plate into a fresh plate, and the OD₆₀₀ of the cell suspension was measured. The percent growth inhibition by the drug was calculated with the formula as described above. We defined 50% inhibition of growth as the MIGC₅₀. An antibiofilm effect was present when the ratio of the percent biofilm inhibition to the percent growth inhibition was >1. Statistical significance of the differences in biofilm formation and growth in the presence of drug compared to untreated was determined by one-way ANOVA (ns, not significant; ***, P < 0.001).

Biofilm formation in liquid culture.

Cell aggregation was observed upon S. Typhimurium growth under microaerophilic conditions in liquid culture (27). Overnight cultures of S. Typhimurium were inoculated to an OD₆₀₀ 0.02 with 60% of the volume of a 50-ml flask filled with LB without salt medium, followed by incubation at 28°C with 150-rpm shaking for 16 h. After 16 h of incubation, cell aggregation was visually analyzed and documented, and the OD₆₀₀ was measured upon resuspension. At least three biological replicates were performed for each experiment.

Assessment of cell aggregation by light microscopy.

Cell aggregation and morphology in the cell culture after overnight growth were observed by using light microscopy (Leica DIML LED). A 20-μl portion of the cell suspension from each flask was spread onto a glass slide, with cell aggregates to be observed by bright-light microscopy and documented using a Leica MC 170 HD camera.

Detection of protein production by Western blotting.

To monitor the expression level of the major biofilm regulator CsgD, Western blot analysis was carried out. S. Typhimurium UMR1 cells treated with different concentrations of clarithromycin (3.75, 7.5, 15, 30, and 60 μM), cells without treatment as a positive control, and S. Typhimurium MAE50 cells as a negative control were grown under microaerophilic conditions with shaking at 150 rpm at 28°C. After 16 h, each cell suspension was centrifuged, and 2.5-mg portions of the pelleted cells were dissolved in 100 μl of sample buffer. To assess the protein concentration, Coomassie brilliant blue staining of the protein gel was carried out after SDS-PAGE (a 4% stacking and a 12% resolving gel). Equal amounts of protein were separated and then transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore) using a semidry Western blotter Mini-Sub Cell GT (Bio-Rad). Detection of CsgD was carried out with polyclonal anti-CsgD peptide antibody (1:2,000) (69) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3,000). CsgD was detected by using a LUMI-Light kit from Roche. Western blot analysis was performed twice.

RNA isolation from biofilm cells.

The RNA from biofilm-forming bacteria was isolated as previously described (11). S. Typhimurium UMR1 was grown without and with 15 μM clarithromycin under microaerophilic conditions with shaking at 150 rpm at 28°C for 16 h. Cells were harvested by centrifugation and disrupted by using lysis buffer (50 mM Tris-HCl [pH 8], 8% sucrose, 0.5% Triton, 20 mM EDTA; 4 mg/ml lysozyme). Then, 200 μl of acidic phenol was added, and the sample was heated at 65°C for 15 min with shaking. The sample was cooled on ice for 5 min and centrifuged at 13,000 rpm. The aqueous phase was collected and extracted once with an acid phenol-chloroform mix and twice with chloroform. Finally, RNA was precipitated with 3 volumes of 100% ethanol and 0.3 M sodium acetate overnight at −80°C. Pellets were washed with 1 ml 70% ethanol and air dried after the supernatant was removed. Pellet was dissolved in 100 μl of diethyl pyrocarbonate (DEPC)-treated distilled water. After DNase (Ambion RiboPure-Yeast DNase) treatment, the RNA samples were analyzed by denaturing gel electrophoresis to assess DNA contamination and the integrity of the isolated RNA. If DNA contamination was still present, the RNA was repeatedly incubated with DNase. The RNA concentration was measured by using a NanoDrop apparatus, and the quality of the RNA preparation was assessed using an Agilent Bioanalyzer 2100. The DNA-free RNA was stored at –80°C until use. RNA was isolated from at least three biological replicates, which were subsequently subjected to RNA sequencing.

cDNA synthesis and qRT-PCR.

cDNA was prepared from 1 μg of isolated DNA-free RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems), and the reaction was run in a thermal cycler according to the manufacturer’s protocol. For qRT-PCR, the reaction volume was 25 μl, with 3 μM concentration of forward and reverse primer, 5-μl cDNA (1:20 diluted) and 10-μl SYBR green (iTaq Universal SYBR green Supermix, Bio-Rad) filled up with 3 μl of DEPC-treated water. Quantitative real-time PCR was performed using a 7500 real-time PCR system (Applied Biosystems). The relative transcript abundance was determined by the 2^ΔΔCT method using 7500 SDS software (v1.3.1; Applied Biosystems). The rpsV gene coding for the 30S ribosomal subunit protein S22 and the recA gene were used for internal normalization. The primers used for qRT-PCR are listed in Table S1 in the supplemental material.

RNA sequencing and data processing.

RNA samples were sequenced on a NextSeq 550 with a medium output flow cell (150 cycles; 2 × 75 PE) after depletion from ribosomal RNAs by using a RiboMinus transcriptome isolation kit (Invitrogen). The resulting bcl files were converted and demultiplexed to fastq using the bcl2fastq program. STAR was used to index the S. Typhimurium strain ATCC 14028S genome (NCBI reference sequence NC_016856.1) and to subsequently align the fastq files. Mapped reads were then counted in annotated genes using the “Counts” feature. The annotations were also obtained from the National Center for Biotechnology Information in gff3 format. The count table from the Counts feature was imported into R/Bioconductor, and differential gene expression was performed using the EdgeR package and its general linear model pipeline. For the gene expression analysis, genes that had 1 count per million in three or more samples were used. Dispersion was tagwise calculated and TMM was used to normalize the samples. Genes were termed significant if they had a false discovery rate adjusted P value of <0.05. A Volcano and Scatterplot was drawn with Excel indicating all differentially expressed genes with a P value of <0.01. Data from three biological replicates are reported (see the Excel table in Data File S1 in the supplemental material).

Mass spectrometry for protein identification.

Differential protein bands were cut from the SDS-PAGE gel, subjected to in-gel trypsin digestion, extracted and purified according to a standard procedure (extraction by H₂O, trifluoroacetic acid, and acetonitrile), and analyzed by using the UltiMate 3000 RSLC nano-LC system connected to an Orbitrap Velos mass spectrometer (Thermo Scientific). Mass spectrometry (MS) survey scans were alternated with tandem MS (MS/MS) scans of daughter ion spectra obtained from peptide components by collision-induced dissociation. All survey spectra were recorded using an Orbitrap detector with a resolution of 60,000 and the daughter ion spectra of peptide components were recorded using the ion trap. The experimental setup was as follows. The peptide mixture was loaded onto a 75 μm × 2 cm precolumn (Acclaim PepMap 100, C₁₈, 3 μm; Thermo Scientific) and eluted over 60 min using a 75 μm × 25 cm analytical column (Acclaim PepMap RSLC, C₁₈, 2 μm; Thermo Scientific) with a gradient buffer of 0 to 95% acetonitrile in 0.1% formic acid. The data were acquired using Xcalibur 2.3 software (Thermo Scientific) and processed with Peaks 6 software (Bioinformatics Solution). MS/MS data were searched versus the 208140 entry in the Salmonella enterica database extracted from the NCBI nonredundant database. The search parameters were fixed modification carbamidomethylation (57.02) and methionine oxidation (15.99) as a variable modification, cleavage by trypsin, a parent mass error tolerance of ±10.0 ppm, and a fragment mass error tolerance of 0.3 Da. The maximal missed cleavage was 1, and the significance threshold was P < 0.05. Selected identified proteins from individual bands are reported.

Data availability.

The RNA sequencing data were uploaded in GEO under accession number GSE157695.