“Sharing of matrix” – A cooperative strategy for survival in Salmonella enterica serovar Typhimurium

doi:10.21203/rs.3.rs-2700225/v1

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Research Article

“Sharing of matrix” – A cooperative strategy for survival in Salmonella enterica serovar Typhimurium

https://doi.org/10.21203/rs.3.rs-2700225/v1

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published 23 Aug, 2023

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Background

Bacteria in nature live together in communities called biofilms, where they produce matrix that protects them from hostile environments. The components of this matrix vary among species, with Salmonella enterica serovar Typhimurium (STm- WT) primarily producing curli and cellulose, which are regulated by the master regulator csgD. Interactions between bacteria can be competitive or cooperative, with cooperation more commonly observed among the kin population. This study refers to STm- WT as the generalist which produces all the matrix components and knockout strains that are defective in either curli or cellulose as the specialists, which produces one of the matrix components but not both. We have asked whether two different specialists will cooperate and share matrix components during biofilm formation to match the ability of the generalist which produces both the components.

Results

In this study, the response of the specialists and generalist to physical, mechanical, and biological stress during biofilm formation is also studied to assess their abilities to cooperate and produce biofilms like the generalist. STm WT colony biofilm which produces both the major biofilm matrix component were protected from stress whereas the non-matrix producer (∆csgD), the cellulose, and curli alone producers ∆csgA, ∆bcsA were affected. The most killing occurred in ∆csgD. Whereas the coculture (∆csgA: ∆bcsA) was seen to resist stress like that of the STm WT. Phenotypic and morphological characteristics of the colonies were typed using congo red assay and the Influence of Matrix on the architecture of biofilms was confirmed by scanning electron microscopy.

Conclusion

Our results show that Matrix aids in survival during antibiotic, chlorine, and predatory stress. And possible sharing of matrix is occurring in the coculture, with one counterbalancing the inability of the other when confronted with stress.

Biofilm

Salmonella Typhimurium

Matrix

Antimicrobial stress

Cellulose

curli

Salmonella enterica is a major causative agent of bacterial food-borne diseases, and it can form biofilms on a variety of biotic and abiotic surfaces (1). Bacteria adopt a biofilm lifestyle in their natural habitats, where they metamorphose from single planktonic cells to biofilm communities (2,3). Biofilm is a sessile mode of life where the microbes aggregate together and live in proximity in a protective self-produced matrix. Biofilm is the predominant life form on earth. Biofilms protect the bacteria from external stress factors like antibiotic, antibacterial agents, and the immune system of the host (2).

In the developing countries more than 300,000 deaths occur annually due to Salmonella infections (4). Salmonellosis related food borne illness has been estimated to cost 4.8 to 23 billion dollars annually in the United States (5).Based on the surface antigenic composition Salmonella enterica are classified into 2000 different serovars of which, Salmonella enterica serovar Typhimurium (STm), causes non typhoidal, self-limiting gastroenteritis in humans (1,6). Most of these serotypes can adapt to the host including humans. The lifecycle of Salmonella revolves between the multicellular sessile communities (biofilm) and motile planktonic form. This transition aids in the survival, pathogenesis, and transmission of the bacteria from the environment to the host, as biofilm lifestyle protects Salmonella from diverse assaults. Population variation is seen between the cells of Salmonella which are present inside the host where the planktonic one is used for motility and virulence, whereas the biofilm type for long term survival. Salmonella biofilms poses great nuisance to the food and water distribution systems, because of the resilience conferred by the matrix to chlorine and other water sanitising agents. In health care settings, Salmonella biofilms posits life threatening food borne illness and biofilm formation in gall stones(7).

The matrix components play a vital role in the architecture of biofilms (8). The Salmonella enterica serovar Typhimurium (STm) biofilm matrix consists of amyloid protein curli and polysaccharide cellulose, DNA, and a larger protein BapA (9). The STm wild type (WT) biofilm produces red dry and rough (rdar) morphotype on congo red media (Fig. 1), due to the production of all the matrix components (cellulose, curli). The csgD, master regulator of matrix components positively regulates the production of curli, and cellulose which are the main matrix components of STm biofilm. It is responsible for the virulence of the bacteria and plays an important part in biofilm related infections. csgD activates csgBAC which leads to the production of curli subunits. The expression of csgD is in turn regulated by various stimuli like cyclic di-GMP, temperature etc. High levels of cyclic di GMP promote biofilm formation and vice versa(6).

It has been observed that microbes act collectively in groups for their own benefit and survival. Interactions like competition and cooperation have been observed among them to carry out different tasks. The biofilm matrix where the cells are aggregated have plenty of opportunities for resource sharing, which is a favourable interaction (10). ∆csgA is a curli knockout which exclusively produces cellulose alone and ∆bcsA is a cellulose knockout which in turn is specialised to produce curli alone(Fig. 1).

In the present study, we have evaluated whether mutants lacking each one of the matrix components when cocultured together exhibits sharing of matrix components and will the cocultured mutants resemble the wild type (which produces both the matrix components) in terms of its resilience to both biotic and abiotic stressors.

FREQUENCY OF THE SUBTYPES:

PELLICLE BIOFILM, SUBMERGED AND COLONY BIOFILM:

The ability of the WT STm strain to contribute to heterogeneous variants when grown in the form of pellicle, submerged and colony biofilms were estimated by congo red assay. Relative proportion of variants viz., RDAR (Red dry and rough) phenotype, SAW (Smooth and white), PDAR (Pink dry and rough) cells were estimated for each type of the biofilms. All 3 biofilms were predominated by RDAR/SAW phenotype, but their relative proportions varied within each biofilm (Fig. S1). It was observed that in pellicle biofilms, more SAW phenotype was observed relative to RDAR (Red dry and rough) phenotype. In submerged biofilms RDAR phenotype was predominant but SAW (Smooth and white) and PDAR (Pink dry and rough) cells were also observed. Colony biofilms displayed primarily RDAR Phenotypes followed by SAW (Smooth and white cells), colony biofilms were also comprised of Red big colonies and hyper swarmer.

CRYSTAL VIOLET ASSAY:

The biofilms formed by the different matrix deficient strains and their co-culture relative to wild type was estimated using standard crystal violet assay. The observations show that ∆csgD lacking both the matrix components does not formbiofilm. ∆csgA producing only cellulose and ∆bcsA producing only curli formed weak biofilms, Importantly the co-culture was able to produce biofilm biomass similar to the wild type (Fig. 2).

MORPHOLOGY CHANGES DEVOID OF MATRIX:

Colony biofilms of STm WT and matrix mutants were formed on congo red agar plates and morphotypes formed were observed. In line with the previous reports, STm WT produces RDAR Red dry and rough morphotype on congo red where both the matrix components curli and cellulose are expressed, ∆csgD producesSAW smooth and white phenotype where both curli and cellulose are absent, ∆csgA produces PDAR (Pink dry and rough) morphotype where only cellulose is present (Fig. 3a). Interestingly signs of septum like (wrinkly) structures which is well developed in the wild type is observed only in ∆csgA mutant implying that cellulose predominantly contributes to the mature biofilm architecture. ∆bcsA produces BDAR (Brown dry and rough) morphotype where only curli is present. The coculture of matrix mutants ∆csgA: ∆bcsA produces pink colonies where possible sharing of matrix could have occurred. But importantly the mature biofilm architecture is missing in the co-culture which implies these matrix forming genes might regulate other genes that could possibly contribute to mature biofilm architecture.

Scanning electron microscopy (SEM) was done to visualize the colony biofilm architecture (Fig. 3b) and the results show that the Wild type of colony biofilm produces the ridges and the hollow cavity inside the colony biofilm and the channels can also be viewed. In the mutants we could not observe any ridges or patterns. ∆csgD produces plain colony where the matrix components are absent, ∆csgA and ∆bcsA the periphery of the colony shows folding or wrinkled structures. And in the coculture ∆csgA: ∆bcsA the colony shows a more well-defined colony structure similar to ∆csgA, but the wrinkled architecture characteristic of WT is still missing which again reiterates the fact that these matrix contributing genes might also impinge on signalling that results in mature biofilm architecture.

ANTIBIOTIC STRESS EXPOSURE:

The role of matrix components in protecting biofilm cells from antimicrobial stress was tested by exposing biofilms formed by WT and mutants to 200X MIC of ciprofloxacin. Results show that in the STm - WT exposure to antibiotics did not induce a reduction in CFU/ml indicating that the matrix is protecting the cells from the antibiotic stress. In ∆csgD there is >90 % reduction in cell counts, which could be attributed to the absence of matrix (Fig. 4). As the cells exposed to antibiotics are vulnerable because of the absence of protection afforded by the matrix, they were killed as reported earlier (7). With ∆csgA cellulose affords protection from antibiotics, but the protection is not complete as 30% decline in cell counts was observed after antibiotic exposure. Similarly, in ∆bcsA curli conferred protection to the cells and antibiotic treated biofilms displayed 10% reduction relative to untreated ∆bcsA mutant. Among mutants relative to cellulose, curli confers enhanced protection upon exposure to antimicrobial stress. As the specialists (mutants) lack one of the matrix components, complete protection from antibiotic stress like WT could not be observed. With coculture of specialists (∆csgA: ∆bcsA) where both the matrix components are contributed by individual strains, hardly any decline in cell counts following antibiotic exposure was observed which imply matrix sharing would have contributed to enhanced antimicrobial resilience similar to WT in the mutant co-cultures.

CHLORINE STRESS EXPERIMENT:

To evaluate protection conferred by the matrix against oxidative stress, colony biofilms of WT and mutants were exposed to different concentrations of Sodium hypochlorite, as expected, the WT protected the cells due to its matrix producing ability (Fig. 5). In ∆csgD wherein matrix is absent,there was significant decline in cell counts at all concentrations tested and at the maximal concentration evaluated (500 p.p.m) the ∆csgD mutant could not survive. In ∆csgA, cellulose conferred protection to the mutantalthough a slight decline in cell counts was observed at all concentrations tested, the reduction in cell counts were not drastic post exposure. Whereas in ∆bcsA, which produced only curli, at lower concentrations cell counts were comparable to ∆csgA mutant whereas at 500 p.p.m of Sodium hypochlorite, a higher mortality was observeddue to oxidative stress compared to ∆csgA implying that cellulose afforded enhanced protection against oxidative stress than curli.In coculture ∆csgA: ∆bcsA the decline in cell counts were similar to what was observed in ∆csgA (Fig. 5)reiterating protective role of cellulose against oxidative stress. It is unclear if cellulose and curli synergise and form a well interspersed bundle that confers enhanced protection against antibiotic/oxidative stress in the WT relative to the coculture where both components are present together.

At 200 p.p.m (Fig 5a), WT and coculture showed similar pattern of CFU reduction there was less reduction in CFU than ∆csgD mutant.There was no significant CFU reduction in WT at 400 p.p.m (Fig 5b). At 500 p.p.m there was complete reduction in CFU of the ∆csgD mutant and in coculture where both cellulose and curli are produced complement each other and protect itself from chlorine stress (Fig 5c). n=3 biological replicates, Paired t test, 95% CI, P value < 0.05.

MYXOCOCCUS INVASION ASSAY:

Swarming assay technique was employed wherein the predator myxococcus spotted in the center predates by swarming and kills the prey. The mutant ∆csgD lacking the matrix displayeda higher susceptibility towards Myxococcus invasion as evidenced by enhanced swarming diameter as shown by the cumulative graph of the 3-day predation assay (Fig. 6a). Cocultures ∆csgA: ∆bcsA and WT: ∆csgD afforded significant protection from the predator as evidenced by lower migration rates. It is likely that the sharing of matrix between the mutant cocultures and between WT and matrix deficient mutant affords protection against Myxococcus swarming (predation) (Fig. 6).

DENSITY OF THE COLONY:

Cell density of the biofilm were assessed by CFU/ml divided by the diameter of the colony. In general, as the matrix is comprised of polysaccharides/proteins, it is less dense than the cells. ∆csgD had no matrix hence it produced cells its density was highest. WT had a cell density comparable to ∆csgA. But the ∆bscA mutant which produces only curli protein exhibited lower cell density. Coculture of both mutants produced lowest cell density of ~ 1 log lower implying that the biofilm biomass is predominantly contributed by matrix in co culture (Fig. S2).

IRON ESTIMATION IN THE MATRIX OF THE COLONY:

As iron is critical for microbial metabolism (reduction of oxygen for ATP production, Haem production, Ribotide precursor reduction etc.,) role of matrix components in sequestering iron were evaluated. The results reveal that there is more iron sequestered by the WT and coculture colony biofilm relative to the individual mutants (Fig. 7). In ∆bcsA the iron acquisition is less, which is similar to matrix deficient mutant ∆csgD. Relatively ∆csgA accumulates more iron which implies that cellulose contributes to iron sequestration than curli. Thus, in coculture (∆csgA: ∆bcsA), ∆csgA by sharing the matrix would have conferred iron chelation ability and balance for ∆bcsA to succeed as a combined group.

STABILITY EXPERIMENT:

To check for the stability of the colony biofilms formed by WT, mutants and co cultures, the colony biofilms were subjected to the rocker stress at 75 rpm for a period of 3 hours. Where the STm- WT even after 3 hours was not disrupted and remained intact underscoring the strong influence of the matrix components in conferring structural integrity to the biofilm (Fig. S3). In case of the mutant ∆csgD the entire biofilm got dispersed within 2 hours as they lacked the matrix, they were not able to withstand the stress. With ∆bcsA the colony biofilm showed signs of disruption after the second hour and by 3-hour, entire biofilm was dispersed. In ∆csgA although the colony biofilm got disrupted by 2 hours, it was not completely dispersed even by 3^rd hour. Which implies that cellulose confers better structural stability to the colony biofilms. The coculture ∆csgA: ∆bcsA, was dispersed by 3h but withstood until the 2 hour. So, the coculture was relatively more stable than ∆csgD but it did not display stability observed in wild type biofilms. It is likely that WT has an interspersed matrix whereas co cultures have layered matrix which differs in its resilience.

MATRIX ESTIMATION BY CONGO RED DEPLETION ASSAY:

The proportion of matrix formed was estimated by Congo red depletion assay wherein enhanced absorbance in supernatant implies reduced binding of the dye with matrix components. Matrix production is seen at highest in the specialist ∆csgA, whereas ∆bcsA the curli producing mutant forms less amount of matrix. In the coculture ∆csgA: ∆bcsA poor matrix formation of ∆bcsA is compensated by ∆csgA, as the amount of matrix formed is similar to that observed in WT (Fig. S4). Thus, in the coculture ∆csgA is sharing its matrix with ∆bcsA to sustain as a population.

CELLULOSE ESTIMATION BY CALCOFLUOR STAINING METHOD:

Calcofluor binds to cellulose and estimation of bound calcofluor by spectrofluorimetry is indirectly proportional to the amount of cellulose formed by different biofilms. The observations reveal that Cellulose is produced in large quantities by ∆csgA because the mutant is specialized to produce only single matrix component which is produced in more amount than the WT. The co culture ∆csgA: ∆bcsA produces cellulose comparable to the WT (Fig. S5) wherein the csgA mutant contributes exclusively for cellulose production. Sharing of matrix occurs in co culture and hence amount of cellulose is comparable between co culture and WT.

Salmonella, a well-studied member of the Enterobacteriaceae family featuring a plethora of different serovars has adapted to diverse environmental and lifestyle conditions. The self-produced extra cellular matrix that comprises the heterogenous and spatially structured bacterial communities with multiple subpopulations residing in the nanocomposite formed together by the major matrix components, allows the biofilm to act like a tissue that confers resistance against various stressors like antibiotics, environmental stress and competing host responses. Curli as a protein elicits an immune response in the host and can be easily recognized by the host immune cells. Cellulose counter intuitively masks the curli and prevents it from being recognised by the host, resulting in a successful habitat for coordinated bacterial existence known as biofilms, a community with multiple heterogenous subpopulations having distinct physiological traits and division of labour which increases pathogen fitness in both host and non-host environments (11,12).

In higher organisms there are several examples of honeybee, termite societies and polar bears where social interactions like competition and cooperation promotes overall fitness of the community (13). Communal interactions also regulate the mechanism of biofilm development where in a community, the producer protect the non-producer which confers an overall protection to the biofilm community and the biofilm fitness depends on the balance of these interactions. There is physiological differentiation of microbial cells in different biofilm microenvironments which have coordinated behaviours as seen in multicellular organism(14). Competition is the majorly observed interactions in the microbial community, but cooperative behaviour is also observed among the kin population (15). In the present study, we were interested in looking into the role of matrix conferring resistance against the various stress response and whether sharing of matrix in cocultures deficient in production of one of the matrix components confers fitness advantage to Salmonella colony biofilms.

Biofilm exists in nature as air- liquid interface (Pellicle biofilm), solid- liquid (submerged biofilm), air solid (colony biofilm). And cells in a biofilm are highly heterogenous population. To test for phenotypic heterogeneity and to visualize the production of matrix we spotted these biofilms after serial dilution onto a Congo red media (Fig. S1b). And the expressed phenotypes in each model were recorded (Fig. S1a). In the pellicle biofilm, the bacteria obtain the oxygen from the air and nutrients from the liquid. flagella, FimH, cellulose and curli are important for pellicle formation (16) And flagella play an important role in the early stages of biofilm formation. mutants lacking these motility genes were defective in pellicle formation in V. fischeri (17) This could be attributed to the fact that motility is important for the cells to reach the air liquid interface to initiate pellicle formation. Hence, the expression of SAW phenotype was predominant in pellicle biofilm. In submerged biofilms, where they are attached to a solid substratum the need for motility is comparatively limited. Only in the initial stages of attachment to substratum would require motility. So, expression of RDAR phenotype, which is associated with the expression of both curli, and cellulose offers more advantage where both expression of cellulose and curli occurs simultaneously. In the colony biofilm grown on solid air interface, as motility related genes are not expressed a preponderance of RDAR phenotype was observed. STm strain is characterized by the production of RDAR phenotype the multicellular form in the agar media(18).

The Congo red morphotyping Fig. 3a reveals that despite taking up of the Congo red by the co culture, which implies matrix sharing, the mature biofilm architecture like the wild type were not observed, which implies that complementation by specialists is not adequate to restore intricate biofilm architecture displayed by the wild type. In other words, although co culture aids in biofilm formation it is unable to complete biofilm maturation observed in wild type. Absence of intricate biofilm architecture displayed by the wild type, reveals that the specialists are unable to recapitulate biofilm architecture displayed by the generalist. Interestingly signs of septum like structures which is well developed in the wild type is observed only in ∆csgA mutant implying that cellulose predominantly contributes to the mature biofilm architecture. But in a recent study it was observed that the specialist coculture restored the RDAR phenotype and sharing of matrix was displayed (19). Furthermore, This study indicates that there are more factors other than cellulose which give rise to the RDAR phenotype in E.coli 0157:H7 (20). Our findings also reveal that though cellulose is an important component of biofilm matrix, there could be other factors that contribute to the RDAR morphotype.

The antibiotic given to salmonella related infections are of greater importance because serovars of Salmonella have gained resistance to commonly prescribed antibiotics. Salmonella infections are usually treated using fluoroquinolone class of drugs mainly ciprofloxacin, which inhibits bacterial DNA gyrase and Topoisomerase. Studies show that resistance of Salmonella serovars to ciprofloxacin is increasing (21,22). The bacteria’s virulence and resistance are contributed to a greater extent by the matrix components. Matrix deficient ∆csgD is reported to be sensitive and WT is tolerant to antibiotic Ciprofloxacin(19). The MIC of S. Typhimurium 14028 is ∼0.02 µg ml-1 and this study have checked for sensitivity at 4µg ml^-1 ciprofloxacin in submerged biofilms (7).To investigate whether matrix production is conferring enhanced protection in colony biofilms against antibiotic, STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA:∆bcsA colony biofilms were treated with 4µg ml^-1 ciprofloxacin (Fig. 4). In the coculture there is less killing compared to that of the individual mutants.

∆csgD mutant is more sensitive to hydrogen peroxide (19). Cellulose deficient mutants are reported to be more sensitive towards the chlorine stress in submerged biofilms (7). An earlier study has checked the survival of S. enteritidis in food and water supplying chains by exposing the population to a higher concentration of 30 ppm chlorine (which is 200 times higher than the free chlorine in municipal water supplies) it was observed that WT had a higher survival upon 30 ppm chlorine exposure relative to cellulose deficient mutant which exhibited greater mortality (23). Interestingly in our observations (Fig. 5) show that even though the co culture is better, it could not recapitulate the protective effect conferred by wild type.

Myxococcus is a generalist bacterium which has a broad host range feeding on bacteria that is in proximity by employing various secondary metabolites and enzymes to lyse microbial population and obtain its desired nutrients (24). Matrix components cellulose and curli were shown earlier to provide robust protection to E.coli from predatory bacterium Myxococcus xanthus (25). To test whether matrix is conferring protection against predatory stress in salmonella as well and to test whether sharing of matrix occurs in co culture affords protection against myxococcus, invasion assay was carried out and our results (Fig 6) revealed that matrix confers protection as there is far less killing in co-culture biofilms relative to biofilms formed by individual mutants.

Iron is an essential element for growth of bacteria. A previous study reported that matrix associated iron is used for intracellular electron transfer during oxidative respiration (26). Our results in Fig. 7 show that there is more iron acquisition in coculture, which is comparable to WT relative to iron sequestered by the mutant biofilms. Our observations (Fig. S3) regarding biofilm stability in face of shear stress created by rocking show that WT colony biofilm is most stable and did not get dispersed whereas co-culture was not fully stable nevertheless, it could not be completely dispersed which imply mild protection against shear stress for a large duration (>2h).

Matrix produced in the colony is estimated using congo red method (Fig. S4), where the cellulose specialist produced more cellulose than the WT. Whereas the curli specialist produced lesser curli than the WT but together in coculture they are equalizing matrix produced by the WT. calcofluor white has been used to identify the mutants defective in producing matrix in many organisms (23). Paytubi et al, reported that cellulose is not essential for pellicle biofilm formation (27). On the contrary, Thongsomboon et al, reported that functionally cellulose is the major component of the matrix and plays an important role in biofilm architecture and dictates a framework for biofilm landscape providing structure and protection (11). Cellulose estimation assay performed in the present study (Fig. S5) showed that ∆csgA produced higher amount of cellulose than the WT most likely to compensates for the absence of other matrix component.

In this study, we showed for the first time that matrix aids in survival of STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA: ∆bcsA against antibiotic, chlorine and myxococcus stress through matrix sharing in the coculture. Despite the matrix sharing in the coculture, it is not able to mimic the generalist (WT). It is possible that curli gets intertwined with cellulose fibrils in WT and affords greater resilience relative to layered arrangement of curli and cellulose in co-culture, which remains to be discerned using confocal imaging studies as a part of the future work.

Understanding the structure and interactions that occur within a biofilm is critical for studying these highly heterogeneous life forms that switch to a favourable phenotype when necessary (28–30). Our findings indicate that matrix helps with survival during antibiotic, chlorine, and predatory stress. And possible matrix sharing is taking place, with one counterbalancing the inability of the other during stressful conditions. Studying these various specialists and generalists is important because during an infection, the bacteria will select a phenotype that will allow it to establish itself. And there will be phenotypic switching to combat stress and conserve metabolic energy. A trade-off can occur among the heterogeneous group in biofilm, where one will produce one metabolic product and complement the non-producer, and they may establish themselves as a community. Cooperation is a potential advantageous behaviour among the kin population to establish itself during an infection and other challenging environments which require further experimentation to unravel it fully.

BACTERIAL STRAINS AND CULTURE CONDITIONS:

Salmonella enterica serovar Typhimurium 14028 strain (STm WT), ∆csgD, ∆csgA, ∆bcsA (7)were kindly gifted by Prof. Dipshikha Chakravortty, (Indian Institute of Science). For all the experiments strains were grown in LB-NaCl broth overnight at room temperature. 2µl of overnight culture (OD of 0.6 at 595 nm) was spotted onto LB without NaCl agar and incubated for 3 days at room temperature (26- 28°C) to form colony biofilms (19,31) which was used for further experiments. All the chemicals and antibiotics used were purchased from HiMedia labs, India.

PELLICLE BIOFILM, SUBMERGED AND COLONY BIOFILM:

Pellicle biofilms were grown by adding 2 µl of midlog culture (0.6 OD) to 200µl LB broth in a 24 well (Tarsons, Kolkata) tissue culture plate wrapped in parafilm for 6 days at 25°C in static conditions. The pellicle formed from this culture was aspirated into 1 ml Phosphate buffered saline (PBS). The resuspended pellicle was vortexed, serial diluted and spread plated on Congo Red (CR) media. 3 days post incubation, the morphology of the biofilm colonies was observed and recorded.(32)

Similar procedure was adopted for submerged biofilm, wherein rubber policeman was used for retrieving the submerged biofilm and for colony biofilms, 3-day old colony biofilm was taken which were then added to 1ml PBS, serially diluted, and plated onto CR media. After 3 days of incubation, the morphology of the colonies was typed and recorded.

CRYSTAL VIOLET ASSAY:

To assess the biofilm formation, crystal violet assay as reported earlier (32) was adopted. Briefly, biofilms were grown on test tubes containing 3ml of LB broth (without NaCl) at 26- 28°C for 3 days in static condition. On the 3-day the liquid culture was removed by aspiration, biofilms formed were washed twice with sterile PBS and air dried. 1% crystal violet was added to the tubes and allowed to stain for 15 min, excess/unbound crystal violet was carefully removed from the tubes which were air dried and imaged. 70% ethanol was added to extract crystal violet and OD of crystal violet corresponding to the biofilm biomass was measured at 595nm using Tecan Sunrise plate reader (6). The results were analysed by using t-test using GraphPad Prism5 for windows.

MORPHOLOGY CHANGES DEVOID OF MATRIX:

The morphotypes were studied visually on congo red agar plates. 2 µl of STm WT, ∆csgD, ∆csgA, ∆bcsA, coculture of ∆csgA: ∆bcsA (1:1) were spotted on to the LB- NaCl agar media containing 40ug/ml Congo red and 20ug/ml Coomassie blue. The plates were incubated for 3 days. Based on their abilities to produce matrix components, different strains produced varied phenotypes which were photographed and recorded (27).

SCANNING ELECTRON MICROSCOPY:

The 3-day grown colony biofilm was used for SEM preparation and observation following the standard protocol (7) with few modifications. Briefly the colony biofilm was fixed using 1 ml of warm 25% glutaraldehyde at RT for 10 minutes. The fixed biofilms were washed and dehydrated using a graded ethanol series of 50% to 100% with each wash step of 10 minutes. After drying at room temperature, they were left in a vacuum condenser for a day. The samples were sputter coated with gold and imaged using the scanning electron microscope TESCAN VEGA 3, BRNO, CZECH REPUBLIC.

ANTIBIOTIC AND CHLORINE STRESS EXPOSURE:

For testing the antimicrobial sensitivity, the colony biofilm was carefully removed, exposed to Ciprofloxacin at a concentration of 4ug/ml and sodium hypochlorite (200, 400, 500 and 600 ppm) for 1 hour at 37°C (Schematic. S1). Untreated colony biofilms in 1 ml PBS were used as the control. Following exposure, the treated and untreated biofilms were mixed well, serially diluted, and plated on LB- NaCl media with respective antibiotic plates, following 24h of incubation, colony counts were determined to enumerate their CFU. The results were analysed by using paired t test using GraphPad prism 5 for windows.

MYXOCOCCUS INVASION ASSAY:

For the predation assay, 100µl of overnight culture of STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA: ∆bcsA was spread plated on LB-NaCl agar. 10 µl of M. xanthus grown in CTT Liquid in shaking conditions for 48h at 30°C was spotted in the centre and incubated at 30 ̊C. Both the strains were normalized to an OD₆₀₀ of 1. The distance swarmed was measured and recorded for 3 days (25). The results were analysed using one-way ANOVA by GraphPad Prism5.

DENSITY OF THE COLONY:

The 3-day grown colony biofilm were taken, the diameter of the colony was measured. The colony was scrapped off carefully, washed with PBS, serial diluted and plated. The density of the colony was enumerated by dividing the CFUml^-1 by the diameter of the colony biofilm. Unpaired t test was performed for result analysis using GraphPad prism5 for windows.

IRON ESTIMATION IN THE MATRIX OF THE COLONY:

For estimating the matrix associated iron (26) 3-day old colony biofilm was dispersed in 15 ml PBS, sonicated, and centrifuged at 14000 rpm for 1min, the supernatant was collected and filtered using 0.22 µ filter. Fe content in the samples were estimated at 238 nm using ICP-OES Agilent 5110. The statistical test used for analysis is unpaired t Test using GraphPad Prism5.

STABILITY EXPERIMENT:

To check the role of matrix in stability, STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA: ∆bcsA were allowed to form colony biofilms for 3 days subsequently. 1.6 ml of PBS was added to the colony biofilms and incubated at the rocker at 75 rpm under shaking conditions for 4 hours. Effect of mixing on the stability of the biofilms was observed and photographed at every hour interval.

MATRIX QUANTIFICATION BY CONGO RED METHOD:

Congo red binding, was used for quantification of matrix production by STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA: ∆bcsA (31,33) Two microliters of overnight grown cultures were spotted in biological triplicates on LB-NaCl Agar and allowed to form biofilms for 3 days at 25 °C. Each colony was scraped from the plate, resuspended in 1 mL PBS + 40 μg/mL Congo red dye, and incubated at 37 °C for 1 h. Samples were centrifuged at 16,873 × g for 2 min, and supernatants were transferred to a clear 96-well plate (Tarsons) for measurement of absorbance at 490 nm using a plate reader (Tecan infinite F50 ELISA Reader). PBS + 40 μg/mL Congo red was measured as the “no matrix” standard. Unpaired t test was performed using GraphPad prism5.

CELLULOSE QUANTIFICATION BY CALCOFLUOR STAINING METHOD:

Cellulose production in colony biofilms was quantified by measuring the cellulose bound to cells (27). STm WT, ∆csgD, ∆csgA, ∆bcsA, ∆csgA: ∆bcsA were spotted on LB-NaCl Agar supplemented with 2% Calcofluor white stain (Sigma Aldrich), incubated for 3 days at 25 °C. The colony biofilms formed were retrieved, mixed with 1ml PBS, and centrifuged at 5000 rpm for 10 minutes to eliminate the unbound cells and Calcofluor. Cells were resuspended in water and transferred to a 96 well plate (Tarsons). Fluorescent measurements corresponding to biofilm bound cellulose (excitation 360 nm, emission 460 nm) were made in Microplate reader Biotek. The results were analysed using GraphPad prism5 for windows.

STm- WT: Salmonella enterica serovar Typhimurium- Wild type

LB- Luria Bertani

CR- Congo red

RT- Room temperature

PBS- Phosphate buffered saline

SEM- Scanning electron microscopy

RDAR- Red dry and rough

SAW- Smooth and white

PDAR- Pink dry and rough

BDAR- Brown dry and rough

Ethics approval and consent to participate – Not Applicable

Consent for publication- Not applicable

Availability of data and materials

The data presented in this study are available from the corresponding author upon request.

Competing interests

No potential conflict of interest was reported by the author(s).

Funding- Not Applicable

Authors' contributions

This work was conceived by CSS. CSS & NSS designed the experiments. KBR performed the experiments. KBR wrote the manuscript. NSS edited and revised the manuscript.

Acknowledgements

This work was Conceived and Overseen by late Dr. C.S. Srinandan an eminent biofilm biologist and it is a tribute to him. KBR, CSS, NSS acknowledge SASTRA Deemed to be University, Thanjavur, India for extending infrastructure support to carry out this work. KBR wishes to express her sincere thanks to ICMR SRF for her fellowship (RBMH/FW/2021/29). We express our sincere gratitude to Dr. Samay Pande (IISC) for allowing us to perform the predation assay in his lab.

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“Sharing of matrix” – A cooperative strategy for survival in Salmonella enterica serovar Typhimurium

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