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A global transcriptomic analysis of Staphylococcus aureus biofilm formation across diverse clonal lineages

A key characteristic of Staphylococcus aureus infections, and one that also varies phenotypically between clones, is that of biofilm formation, which aids in bacterial persistence through increased adherence and immune evasion. Though there is a general understanding of the process of biofilm formation – adhesion, proliferation, maturation and dispersal – the tightly orchestrated molecular events behind each stage, and what drives variation between S. aureus strains, has yet to be unravelled. Herein we measure biofilm progression and dispersal in real-time across the five major S. aureus CDC-types (USA100-USA500) revealing adherence patterns that differ markedly amongst strains. To gain insight into this, we performed transcriptomic profiling on these isolates at multiple timepoints, compared to planktonically growing counterparts. Our findings support a model in which eDNA release, followed by increased positive surface charge, perhaps drives initial abiotic attachment. This is seemingly followed by cooperative repression of autolysis and activation of poly-N-acetylglucosamine (PNAG) production, which may indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, diminished translational capacity was apparent, with 53 % of all ribosomal proteins downregulated, followed by upregulation of anaerobic respiration enzymes. These findings are noteworthy because reduced cellular activity and an altered metabolic state have been previously shown to contribute to higher antibiotic tolerance and bacterial persistence. In sum, this work is, to our knowledge, the first study to investigate transcriptional regulation during the early, establishing phase of biofilm formation, and to compare global transcriptional regulation both temporally and across multiple clonal lineages.

and what drives variation between S. aureus strains, has yet to be unraveled. Herein we 23 measure biofilm progression and dispersal in real-time across the 5 major S. aureus 24 CDC-types (USA100-USA500) revealing adherence patterns that differ markedly 25 amongst strains. To gain insight into this, we performed transcriptomic profiling on these 26 isolates at multiple time points, compared to planktonically growing counterparts. Our 27 findings support a model in which eDNA release, followed by increased positive surface 28 charge, perhaps drives initial abiotic attachment. This is seemingly followed by 29 cooperative repression of autolysis and activation of PNAG production, which may 30 indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, 31 diminished translational capacity was apparent, with 53% of all ribosomal proteins 32 downregulated, followed by upregulation of anaerobic respiration enzymes. These 33 findings are noteworthy because reduced cellular activity and an altered metabolic state 34 have been previously shown to contribute to higher antibiotic tolerance and bacterial 35 persistence. In sum, this work is, to our knowledge, the first study to investigate 36 transcriptional regulation during the early, establishing phase of biofilm formation, and to 37 compare global transcriptional regulation both temporally and across multiple clonal 38 lineages.

Importance 40
Staphylococcus aureus is a highly virulent, opportunistic pathogen and a leading cause 41

Results and Discussion 247 248
Comparing biofilm formation of Methicillin-resistant S. aureus isolates: The S. 249 aureus strains used in this study were originally isolated from diverse infection niches 250 and geographic locations (Table 1). To compare their abilities to form a biofilm, we 251 performed classical crystal violet staining assays on mature 24 h biofilms grown in TSB 252 ( Fig. 1). Obvious differences were observed in total mass of biofilms at this timepoint, 253 with the USA500 representative having the highest biofilm forming capacity, followed by 254 USA300; with USA100, USA200, and USA400 all displaying similar phenotypes. To 255 explore and compare biofilm formation by these strains more fully, we next employed a 256 more quantitative approach using a Real Time Cell Analyzer (RTCA) device. The RTCA 257 measures adherence of cells based on impedance of electrical signals between 258 electrodes lining each well of a specialized 96-well plate, and expresses these 259 measurements in a relative unit, known as a Cell Index (CI). This allows measurements 260 to be taken at multiple time points on the same biofilm population and forgoes disruptive 261 processing steps. When we performed these studies, we noted that CI values after 24 262 hours of growth largely correlated to crystal violet assay results, with USA500 and 263 USA300 producing more robust biofilms, and USA100 and USA200 seemingly 264 producing less biomass (Fig. 2). Of note, USA400 produced the least amount of biofilm 265 as measured by crystal violet staining, yet showed relatively high CI values that were 266 comparable to USA300. This discrepancy is perhaps driven by the fact that CI 267 measurements are influenced by cell adherence, secretion of EPS, and cell spreading 268 (44,45), whereas crystal violet staining measures total biomass present including EPS, 269 viable cells, and dead cells (46). 270 271 Beyond this comparison, our RTCA studies revealed four distinct phases of biofilm 272 formation across the 5 strains. The first of these was characterized as a rapid phase of 273 CI increase between 2 -4.5 h, which we believe indicates attachment, with some strains 274 (e.g. USA100) adhering more rapidly, whilst others (USA200) attaching more slowly. 275 This phase was then followed by a plateau demonstrated by all strains, which lasted 3 276 hours for strains USA400 and USA500, although this was less apparent for strain 277 USA300. Following this phase, biofilms other than USA100 then showed another CI 278 increase, with USA500 being most accelerated. Finally, the CI values reach their 279 maximum and remain steady for all strains, which signifies that the RTCA electrodes 280 have been saturated and the biofilm has matured (44). Collectively, USA400 and 281 USA500 very clearly exhibited these phases during analysis, whilst USA100 282 demonstrated a more prominent initial phase of attachment, quickly reaching its 283 maximum cell index levels; with additional phases being less pronounced. As our data 284 shows, each strain reaches a different maximum, with some achieving a CI greater than 285 0.4 (e.g. USA500) while others do not surpass 0.3 (USA200), which could be the result 286 of variations in cell adherence, secretion of EPS, or cell spreading. The differences 287 observed in biofilm formation and maturation are a testament to the diverse 288 characteristics of these strains and the dynamic, evolving nature of biofilms. 289 Differential expression in biofilm and planktonic cell populations drastically 291 changes with population age: To gain more insight into the distinct phases observed 292 during biofilm formation, and how biofilms evolve, we employed transcriptomic profiling 293 of each isolate. RNA was collected from biofilm and planktonic cell populations after 5 h, 294 10 h, and 24 h of growth, corresponding to the end point of each phase observed during 295 real-time tracking of biofilm formation, and subjected to RNA sequencing (Fig. 3, Fig.  296   S1). Upon analysis, we observed that each clonal lineage exhibited distinct global 297 expression profiles specific to its biofilm and planktonic growth (Table S1). 298

299
To explore this data more fully, we first chose to identify conserved gene expression 300 patterns amongst all strains. To identify homologous genes, we used reciprocal 301 BLASTn (Table S2) and identified genes with > 3-fold differential expression between 302 biofilm and planktonic cell populations. It is noted that this approach has its limitations, 303 as only homologous genes are considered during this comparison, and strain specific 304 genomic architecture is excluded; however, it does allow for a broad comparison of data 305 between isolates. Genes showing preferential expression in biofilm and planktonic 306 populations were then compared between strains to identify conserved expression 307 patterns (Fig. 4). Preferential gene expression within biofilm and planktonic populations 308 was largely strain dependent, with few genes showing > 3-fold upregulation in all 309 biofilms. 310

311
The majority of genes showing similar expression in all strains were preferentially 312 expressed in planktonic cell populations at 5 h and 10 h, whereas at 24 h, the majority 313 were preferentially expressed in biofilms (Fig. 5A, Table S3). Additionally, an interesting 314 trend was observed regarding the number of genes expressed preferentially within 315 biofilms. The number of common biofilm associated genes is low (n = 2) at 10 h and 316 relatively high (n = 98) at 24 h. Compared to 5 h (n = 14), the number of common biofilm 317 associated genes is 7-times greater at 24 h. When considering strains individually, each 318 expressed a higher number of total biofilm associated genes at 5 h, which decreased by 319 at least half for all strains at 10 h, and returned to comparable numbers at 24 h. Overall, 320 this agrees with previous findings demonstrating cells within a biofilm have differential 321 regulation compared to planktonic populations (47)(48)(49). Expanding on this model, we 322 suggest the greater level of differential expressions within biofilms at 5 h and 24 h 323 demonstrates that biofilm cells may be more physiologically diverse from their 324 planktonic counterparts during attachment (5h) and maturation (24h) phases than 325 during proliferation (10h). Furthermore, global transcriptional divergence between 326 biofilm and planktonic cell populations occurred at 5 hours, indicating that differential 327 regulation manifests earlier during biofilm development than previous works have 328 considered. The majority of differentially expressed genes at this timepoint pertained to 329 metabolic regulatory proteins (Table S3). It is possible that this evidence for reordered 330 metabolism reflects metabolic dormancy of emerging persister cells, or is indicative of 331 divergent metabolism in anaerobic regions forming within the biofilms (50, 51) 332 (discussed below). 333

334
To validate our findings, a random selection of genes were assayed by 335 revealing comparable changes to our RNA-seq data in all cases (Fig. S2). 336 Urease production suggests acidification of the biofilm niche via fermentative 338 metabolism: There was a near complete lack of constitutively expressed genes within 339 biofilms, which reflects the dynamic, evolving nature of this population. Only a single 340 gene showed preferential expression in one population over another for all strains (Fig.  341 5B, Table S3). This was ureB, which demonstrated lower expression in biofilm beyond those sampled during our study. Therefore, upregulation of the urease gene 350 cluster may occur only during prolonged growth in a biofilm state. In support of this, the 351 absolute differential expression of the urease operon measured in our study decreased 352 as time progressed, indicating that urease operon expression does gradually increase in 353 biofilms (Fig. 6 fermentation in biofilms, we saw evidence for the initiation of formate production in 357 biofilms at 10 h. Specifically, we observed less transcription of NAD-dependent formate 358 dehydrogenase (SAUSA300_RS00940) within 10 h biofilms (4.7 -16-fold decrease, depending on the strain), which was less pronounced by 24 h (2.3 -6.7-fold decrease) 360 ( Fig. 6). We also detected a gradual shift towards preferential expression of the genes 361 encoding pyruvate formate-lyase, pflA (formate acetyltransferase-activating enzyme) 362 and pflB (formate acetyltransferase). Under anoxic conditions within biofilms, PflA 363 activates PflB to promote formate synthesis, which is then utilized for protein and purine 364 production (53). Here, we noted a shift in pflA expression from 2. Interestingly, all of these targets were strongly upregulated in 24 h biofilms (clpB: 5.02 -384 120.28-fold; clpC: . This is noteworthy because CtsR, 386 SpxA, LexA, and HrcA are all subject to some form of post-translational regulation. 387 Specifically, CtsR is phosphorylated by the co-transcribed protein, McsB, which marks it 388 for degradation by the ClpCP protease complex under conditions of heat stress (55). 389 HrcA requires the chaperonin, GroE, for proper folding; however, during heat stress, 390 GroE becomes preoccupied refolding damaged proteins in a titrated fashion, leading to 391 unresolved, improper folding of HrcA and de-repression of its heat shock regulon (56). 392 SpxA acts as both a transcriptional repressor and activator, and is degraded by the 393 shown to enhance S. aureus biofilm formation (60). This suggests that unlike CstR, 408 LexA, and HrcA, deactivation of the SpxA regulon may be beneficial to biofilms. activates murein hydrolases, which then triggers cell lysis, eDNA release, and biofilm 419 adherence (64). LrgA has been shown to oligomerize with CidA and consequently 420 antagonize CidA-mediated cell lysis (65). In 5 h biofilms, lrgA transcription was 3.88 -421 13.10-fold higher, and cidA transcription was 4.31 -12.98-fold lower (Fig. 8, Table S1). biofilm formation as dlt mutants have a net negative cell surface charge, and, despite wildtype levels of PNAG production, cannot colonize abiotic surfaces (68). In 5 h 452 biofilms, the dlt operon and mprF (2.39 -3.27-fold) showed greater expression in all 453 strains, with the exception of USA400 (Fig. 8, Table S1). The ArlRS TCS has been 454 demonstrated, in part, to repress dlt transcription and was upregulated during this phase 455 (arlR, 1.91 -4.73-fold; arlS, 1.71 -8.27-fold). However, this particular regulatory 456 cascade is dependent on the supplementation of media with cations, specifically Mg 2+ 457 (69) and therefore, is unlikely to be occurring under the conditions of our study. 458 Alternatively, the expression of mprF and the dlt operon is also dependent on the co-459 transcription of response regulator graR of the TCS GraRS, and the downstream efflux 460 pump vraG (70) (Fig. 9). Both graR and vraG were transcribed 1.65 -7.69-fold and 1.61 461 -6.94-fold higher, respectively, in biofilms compared to planktonic cells at 5 h. Again, 462 the exception to this was USA400, which showed < 1.4-fold change for these genes 463 in biofilm populations (Fig. 8, Table S1) would allude to increased PNAG production in 5 h biofilms, which is consistent with our model. Collectively, this supports a scenario in 475 which 5 h biofilms may employ PNAG-mediated attachment and a positive cell surface 476 charge in order to promote abiotic attachment (Fig. 9). 477 478 It is also important to note that many of the above factors expressed in 5 h biofilms 479 actively promote antibiotic resistance, whereas those that showed reduced expression values of fold-difference in expression between biofilm and planktonic cells (Fig. S3). 495 This metric revealed reduced transcription of the majority of the protein synthesis 496 machinery in biofilms after 10 hours of growth (Fig. 10). Of these, the transcription of ribosomal protein genes rplX, rplE, rpsN, rpsH, rplF, and rplR, which are co-transcribed, 498 demonstrated < 3-fold decrease in expression in all biofilm cell populations at 10 h 499 compared to planktonic cells. These genes encode 50S (Rpl) and 30S (Rps) subunit 500 proteins, which facilitate proper subunit assembly. During assembly, ribosomal proteins 501 S14 (RpsN) and S8 (RpsH) bind the 16S rRNA and coordinate assembly of the 30S 502 subunit (82). Ribosomal proteins L24 (RplX) and L6 (RplF) bind the 23S rRNA, whilst 503 directing 50S subunit assembly, and stabilizing 23S rRNA secondary structure (82). 504 Ribosomal proteins L18 (RplR) and L5 (RplE) bind the 5S rRNA and are thought to 505 mediate its attachment to the 50S subunit (82). Decreased production of these six key 506 ribosome assembly proteins, and indeed an overall 53% of ribosomal proteins at 10 h 507 across all strains, suggests that biofilms have diminished translational capacity. 508

Impaired translational function is a hallmark of physiological dormancy -a condition 509
which fosters persister cell formation (83). It is well known that persister cells form much 510 more frequently within biofilms compared to planktonic populations and that their altered 511 physiological behavior contributes to antibiotic tolerance (84). To our knowledge, it is 512 not yet known how soon persister cells form, but it would appear that diminished 513 translational capacity may contribute to this, and that they are likely present within 514 biofilm populations by 10 h of growth. 515 516

Biofilms display a shift towards nitrogen metabolism and anaerobic respiration: 517
In addition to evidence of physiological dormancy, transcription within biofilm cells also 518 reflects an altered metabolic state at later stages of growth. In particular, planktonic 519 cells showed preference for atpABCDEFGH (the ATP synthase) expression at 24 hours, which is a major source of energy generation during aerobic respiration (Fig. 11). 521 Preferred expression of ATP synthase within planktonic cells, and therefore lower 522 expression within biofilms, suggests that mature biofilms employ alternative energy 523 generation pathways that may mirror anaerobiosis (85). Indeed, biofilms develop 524 anaerobic regions (50, 51), and anaerobic metabolism has been recognized as the 525 preferred metabolic process for bacteria within deeper portions of fully formed biofilms. aureus increases the transcription if its alcohol dehydrogenases (adhE, adh) to 532 regenerate NAD, and, even in the absence of nitrate, increases the expression of nitrate 533 respiration (narHIJ) and nitrate reduction (nirD) genes (87). Each of the S. aureus 534 biofilms in this study demonstrated enhanced transcription for all of these genes after 24 535 hours of growth, although it was to a lesser extent in USA400 (Fig. 11, Table S1). These 536 findings are in addition to the three proteins associated with fermentative metabolism 537 discussed above, SAUSA300_RS9402, plfA, and plfB, which showed increasing 538 expression within biofilm populations as time progressed. Collectively, this supports a 539 model in which biofilms possibly favor anaerobic respiration, with our data suggesting 540 that this shift occurs during later growth phases. Interestingly, anaerobic growth and the 541 repression of ATP synthase has also been demonstrated to enhance antimicrobial 542 resistance in S. aureus (88, 89); and reduced levels of intracellular ATP is a hallmark-543 phenotype of S. aureus persister cell formation (85). initiated, and initial attachment may not be dependent on ClfA. As discussed above, our 559 results suggest that initial biofilm attachment is perhaps facilitated by CidA-mediated 560 eDNA release followed by repression of this mechanism after 5 h by LrgA. At this time, 561 biofilm attachment may be further enhanced by an increase in positive cell surface 562 charge and enhanced PNAG production. Our data supports the notion that ClfA serves 563 to maintain biofilm attachment at later time points, and perhaps provide late stage 564 strengthening and maturation of the biofilm matrix.
As shown by tracking biofilm formation in real time, even closely related strains (e.g. 567 USA300 and USA500) within a highly controlled environment present distinct 568 phenotypes and trends (Fig. 2). This, in addition to variability in genomic architecture, 569 suggests innate, idiosyncratic gene expression patterns exist within strains. As an 570 example, preferential expression of clfA was less pronounced in USA100 biofilms (1.93-571 fold) compared to other 24 h biofilms (USA200, 13.25-fold; USA300, 18.83-fold; 572 USA400, 13.49-fold; USA500, 61.30-fold). Like clfA, other genes encoding adherence 573 factors, including Clumping Factor B (clfB), IgG Binding Protein A (spa) (17), 574 fibronectin-binding protein A (fnbA) and the Serine-Aspartate Repeat Proteins (sdrCDE) 575 (14), were preferentially expressed in USA300, USA400 and USA500 biofilms, whereas 576 in USA100, these genes were more highly expressed in planktonic cells (Fig. 12). 577 USA200 does not harbor the sdrD gene, but this strain also preferentially expressed 578 clfB, fnbA, sdrC, sdrE, and to a lesser extent, spa in 24 h biofilms. Interestingly, 579 overproduction or addition of soluble Spa in planktonic cultures can trigger bacterial 580 aggregation in suspended culture (17). Such aggregates have enhanced cell-cell 581 adherence, increased tolerance to antibiotics, are more resistant to shear stress, and 582 are more mobile (93). Moreover, others have shown that the Sdr proteins facilitate 583 platelet binding when cells experience shear stress in the upper range of arterial wall 584 shear rates (91, 94), and that platelet binding is critical for S. aureus endocarditis 585 infection (95). USA100 is the only strain to preferentially express these adherence 586 factors in its planktonic cell population and is also the leading causes of bloodstream infections (20). Thus, it is entirely possible that the alteration of expression observed 588 herein could contribute to this strains proclivity towards this type of infection. 589

Concluding Remarks 591 592
Biofilms facilitate bacterial survival in diverse environmental niches, prolong infections, 593 and continue to pose a major threat to patient recovery. S. aureus is capable of forming 594 persistent biofilms, which are notoriously difficult to eradicate due to their increased 595 tolerance to antibiotics and environmental stresses. Similar to their capacity for 596 virulence, biofilm proficiency varies phenotypically amongst S. aureus clonal lineages. 597 Much of what we currently know about biofilms is based on mature, preformed biofilm 598 populations, therefore, little is known of the transient regulation driving attachment, 599 proliferation, maturation, and dissemination. Our data herein highlights the diverse 600 regulatory networks driving S. aureus biofilm formation. To our knowledge, this is the 601 first study to investigate transcriptional regulation during the early, establishing phase of 602 biofilms and compare global transcriptional regulation both temporally and across 603 multiple clonal lineages. By monitoring biofilm formation of five S. aureus strains in real-604 time, we observe subtle differences in four distinct phases throughout biofilm initiation, 605 proliferation, and maturation. The transcriptomic profiles, of both biofilm and planktonic 606 cells, evolve drastically over time and exhibit differential expression between both 607 populations throughout. We have uncovered a set of core transcriptional benchmarks, 608 common to all clonal lineages, which includes a potential early shift toward anaerobic 609 respiration in biofilms and reduced translational capacity. These findings are noteworthy 610 because reduced cellular activity and an altered metabolic state have been previously 611 shown to contribute to higher antibiotic tolerance and bacterial persistence. 612 Furthermore, our data suggests that this shift may occur earlier than previously thought, 613 prior to biofilms reaching maturity. Unfortunately, this also insinuates that the window of 614 opportunity to avert biofilm infections via antibiotic therapy is likely short lived. 615 Furthermore, the biofilms of all strains exhibited similar regulation of abiotic attachment 616 factors, which highlights the importance of the ongoing efforts towards developing anti-617 biofilm surfaces or coatings for biomaterials and implants to prevent bacterial 618 attachment. To this end, our findings support a model in which biofilms seemingly 619 employ positive surface charge as a means for initial abiotic attachment, making the 620 regulatory factors that control this process (VraG, GraR, MprF, and DltABCD) promising 621 therapeutic targets to prevent biofilm initiation. Much of the strain-specific transcriptional 622 regulation observed was that of well-studied virulence-and biotic attachment-factors. 623 This is a testament to the unique regulatory strategy employed by each strain and gives 624 insight into the factors behind their diverse infection styles. This study also provides a 625 launching point towards understanding the highly orchestrated regulation driving each 626 phase of biofilm development and will inform on future strategies to combat biofilm-627 Static biofilms were grown in specialized, electrode containing 96-well plates in TSB and monitored using a RTCA for 24 h. Each phase is separated by dashed lines and designated I, II, III, and IV. Phase I is characterized by a marked increase in CI (2 -4.5 h) followed by a plateau during phase II (4.5 -7.5 h). A second CI increase marks phase III (7.5 -14 h)