- Research article
- Open Access
Bovine-associated non-aureus staphylococci suppress Staphylococcus aureus biofilm dispersal in vitro yet not through agr regulation
Veterinary Research volume 52, Article number: 114 (2021)
Biofilm formation is a significant virulence factor in Staphylococcus (S.) aureus strains causing subclinical mastitis in dairy cows. A role of environmental signals and communication systems in biofilm development, such as the agr system in S. aureus, is suggested. In the context of multispecies biofilm communities, the presence of non-aureus staphylococci (NAS) might influence S. aureus colonization of the bovine mammary gland, yet, such interspecies interactions have been poorly studied. We determined whether 34 S. chromogenes, 11 S. epidermidis, and 14 S. simulans isolates originating from bovine milk samples and teat apices (TA) were able to affect biofilm formation and dispersion of S. aureus, and if so, how isolate traits such as the capacity to regulate the S. aureus agr quorum sensing system are determinants in this process. The capacity of an agr-positive S. aureus strain to form biofilm was increased more in the presence of S. chromogenes than in the presence of S. simulans and S. epidermidis isolates and in the presence of NAS isolates that do not harbor biofilm related genes. On the other hand, biofilm dispersion of this particular S. aureus strain was suppressed by NAS as a group, an effect that was more pronounced by isolates from TA. Furthermore, the observed effects on biofilm formation and dispersion of the agr-positive S. aureus strain as well as of an agr-negative S. aureus strain did not depend on the capacity of NAS to repress the agr system.
Bovine mastitis, a multifactorial disease-complex resulting from interactions among the host, environment, and typical bacteria, remains the most prevalent and challenging disease on dairy farms worldwide . The non-aureus staphylococci (NAS), a heterogeneous group of staphylococcal species other than Staphylococcus aureus [2,3,4] have become the most frequently isolated bacteria from bovine milk samples as revealed by numerous studies [5,6,7,8,9]. They are commonly considered to be minor mastitis pathogens causing only a slight increase of the milk somatic cell count (SCC) [10,11,12] with no impact on milk yield. Other studies have suggested a protective effect of bovine NAS against, among other bacteria, S. aureus [13,14,15,16,17], including the repression of agr related virulence factors of S. aureus [18, 19].
Staphylococcus aureus is considered a contagious mastitis pathogen that enters the mammary gland through the teat canal [20,21,22]. In most cases, S. aureus transmission is associated with chronically infected mammary glands, colonization of skin or mucosal epithelia, nares and (wounded) hocks . Intramammary infections (IMIs) caused by S. aureus usually result in subclinical mastitis with poor treatment results based on the current therapies [20, 22]. Biofilm formation is a significant virulence factor of S. aureus in case of subclinical mastitis [22, 23].
Biofilms are defined as a structured surface-associated community of bacterial cells (sessile) encompassed by an extracellular matrix . Biofilm development is related to environmental signals and communication systems . In staphylococci it relates to specific gene expression such as the intracellular adhesion locus (icaABCD) , the biofilm-associated protein bap , and the accumulation-associated protein aap . In S. aureus, the switch from planktonic (free-floating forms) to sessile forms is controlled by quorum-sensing proteins encoded by the agrABCD operon, whose abundance is related to virulence and pathogenicity . Therefore, high levels of agr activity is typically associated with dispersal of S. aureus biofilms, whereas the agr system of cells in a biofilm is predominantly repressed [30, 31].
The thickness and the composition of biofilm might determine its functionality  and the persistence of bacteria in the bovine mammary gland . Thicker biofilms are notoriously more difficult to eradicate as they are generally resistant to antibiotic therapy  and clearance by host defenses in the mammary gland . Furthermore, multispecies biofilms might play a role in the context of host colonization, since the presence of specific bacteria can either promote or decrease growth of other bacteria [35, 36].
Although a number of studies in literature reported on the protective effect of NAS in the bovine mammary gland, little is known in the context of biofilm formation and whether agr-mediated interactions between bovine NAS and S. aureus are important. Also, Goetz et al.  reported on the capacity of NAS isolates, mainly S. chromogenes and S. simulans, to reduce biofilm formation and to stimulate dispersion of pre-established biofilm of S. aureus. However, the potential mechanisms behind the biofilm-inhibition and dispersion effects of NAS have not yet been identified. Such dual-species interactions might benefit both species with respect to host colonization . Moreover, a robust multispecies biofilm might contribute to the persistence of S. aureus infections , also in the bovine udder.
Based on the rationale that regulation of the agr system can determine biofilm production by S. aureus, we hypothesized that agr-mediated interactions between NAS and S. aureus affect S. aureus biofilm formation and dispersal in the bovine mammary gland. In order to investigate the hypothesis, we examined whether bovine-associated NAS originating from milk samples and teat apices (TA) influence biofilm formation and dispersion of S. aureus in vitro, and if so, to determine what NAS traits are involved and whether such effects are associated with the regulation of the S. aureus agr quorum sensing system.
Materials and methods
General study design
First, the presence of genes related to biofilm formation (biofilm genotype) and the capacity to produce biofilm (biofilm phenotype) were mapped for 59 bovine NAS isolates originating from milk or TA (Additional file 1). The capacity to inhibit the growth of S. aureus 8325-4 (an agr+ strain) in vitro (in vitro growth inhibition capacity) and to modulate the agr system of S. aureus 8325-4 (agr interaction capacity) for the 59 NAS isolates was known from previous work  (Additional file 1).
Next, it was studied whether the 59 NAS isolates were able to affect S. aureus 8325-4 biofilm formation as well as whether they were able to disperse pre-established S. aureus 8325-4 biofilm and to what extent this was related to specific NAS traits [species, origin (milk versus TA), biofilm genotype, biofilm phenotype, and in vitro growth inhibition capacity, respectively].
Last, it was studied whether the capacity of the 59 NAS isolates to repress the agr system of S. aureus was related to biofilm formation and biofilm dispersion by S. aureus strains.
Staphylococcal isolates and traits
The isolates used in this study are listed in Additional file 1. Unless otherwise stated, bacteria were grown in Trypticase Soy Broth (TSB) overnight at 37 °C.
Species and origin
The 59 NAS isolates were obtained from our repository and represent the three most prevalent species in milk samples and on teat apices of dairy cows and heifers [9, 39]. Forty-five isolates from milk [S. chromogenes (n = 28), S. epidermidis (n = 7), and S. simulans (n = 10)] and 14 isolates from TA were included [S. chromogenes (n = 6), S. epidermidis (n = 4), and S. simulans (n = 4)].
Species identification of all NAS isolates was carried out by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry analysis in which protein fingerprints of the isolates were compared with the commercial databank of bovine reference spectra (Bruker Daltonics), microbial spectra provided by Cameron et al. , and additional microbial spectra of field isolates from our lab covering four additional species (S. jettensis, S. lentus, S. rostri, and S. saprophyticus).
Staphylococcus aureus used in this study include strain 8325-4 (agr+)  and strain 8325-4 Δagr (agr−) .
The bacterial DNA of the NAS was extracted , with some modifications to identify biofilm associated genes among NAS. In the final step, 3 µL of RNase (1 mg/mL, Roche, Mannheim, Germany) was added. Polymerase chain reaction (PCR) assays were applied to detect the presence of the genes related to biofilm formation (aap, ica, and bap) and quorum sensing (agr) according to methodology previously described [27, 28, 44, 45] (Table 1).
PCR products were analyzed by electrophoresis through 1.5% (wt/vol), previously stained with Ethidium Bromide (Sigma-Aldrich). Staphylococcus xylosus ATCC 29971 was used as a positive control for the bap gene and S. epidermidis ATCC 35984 for the aap and ica genes. Positive control for the agr system included S. aureus strain 8325-4. Isolates with an equal amplicon size to the positive control were considered PCR positive for the particular gene under scrutiny.
The NAS isolates that presented at least one of the four genes were recoded as positive for biofilm-related genes (BG), whereas the absence of all four genes was recoded as negative for biofilm-related genes (NBG; Additional file 1) for statistical analyses.
The potential of the NAS isolates to form biofilm was evaluated according to the assay described by Tremblay et al. , with some modifications. Briefly, overnight cultures of S. aureus strain 8325-4  and strain 8325-4 Δagr  (Additional file 1), and the 59 NAS isolates were adjusted to OD600 = 0.2 in TSB and then further diluted 1:100 in TSB supplemented with 0.2% glucose (TSBG). Therefore, an aliquot of 200 μL of each isolate was seeded per well in sterile 96-well polystyrene tissue culture plates at 37 °C for 24 h. After incubation, planktonic cells (supernatant) were removed and the wells were rinsed three times with distilled water. Adherent biofilms were fixed with 95% ethanol for 15 min, and stained with 0.1% (wt/vol) safranin for 20 min. After rinsing three times with distilled water, plates were allowed to dry at room temperature. The stained biofilms were dissolved in 250 μL of 33% (v/v) acetic acid, and optical densities (OD) were measured at 490 nm using Multiskan GO plate reader (Thermo Fisher Scientific, USA). The ability of the isolates to form a biofilm was classified as negative (absorbance at 490 nm, A490 < 0.110), weak (A490 0.110–0.500), moderate (A490 0.500–1.500), or strong producers (A490 > 1.500) (Additional file 1). Uninoculated wells containing TSB with glucose served as blanks. Staphylococcus epidermidis strain ATCC 35984 was used as a strong biofilm forming control strain , and S. epidermidis strain ATCC 12228 was used as a weak biofilm forming control strain .
Each NAS isolate was tested for biofilm production in triplicate on two independent days and results were averaged over the replicates.
All NAS isolates that presented any capacity to produce biofilm (weak, moderate or strong producers) were recoded as positive for biofilm production (BP) for statistical analyses. On the other hand, the absence of biofilm production by NAS was recoded as negative for biofilm production (NBP; Additional file 1).
In vitro growth inhibition capacity
The potential growth inhibition of S. aureus 8325-4 by the NAS isolates was evaluated before  (Additional file 1). Following the methodology described by De Vliegher et al. , the pattern of the in vitro growth inhibition of the S. aureus strain 8325-4 by the NAS isolates was classified as NAS exhibiting no growth inhibition, partial inhibition, or total inhibition.
The NAS isolates that presented partial or total inhibition were recoded as positive for in vitro growth inhibition of S. aureus (GI), whereas no growth inhibition was recoded as negative for in vitro growth inhibition of S. aureus (NGI; Additional file 1) for statistical analyses.
Some NAS isolates were able to repress the agr system of S. aureus 8325-4 by downregulating the rnaIII expression  (Additional file 1). As described by Canovas et al. , the effect of NAS isolates on S. aureus agr was classified as none, weak, moderate, or strong. All NAS isolates that showed any capacity to repress S. aureus agr (weak, moderate, or strong) were recoded as positive for agr repression (RP) for statistical analyses. Conversely, the absence of agr repression was recoded as negative for agr repression (NRP; Additional file 1).
Staphylococcus aureus biofilm formation
Biofilm interactions of the NAS isolates and S. aureus 8325-4 were tested as recently described by Peng et al.  with minor adjustments. Overnight cultures of all 59 NAS and S. aureus 8325-4 were adjusted to OD600 = 0.2 in TSB and then further diluted 1:100 in TSBG. A total of 100 µL of the bacterial suspension(s) were added to wells where either S. aureus strains 8325-4 alone (control), or a ratio of 1:1 of S. aureus + NAS was added. The plates were incubated for 24 h at 37 °C. The planktonic cells (supernatant) were removed and the biofilms were washed once with sterile water. A total of 200 µL of sterile water was added to the wells and the biofilm cells (biofilm fractions) were recovered by scraping the surface with sterile pipette tips, vigorously shaken and serially diluted. The dilutions were plated on a semi-selective agar, mannitol salt agar (MSA, Chapman medium, Oxoid, Aalst, Belgium) for CFU enumeration of S. aureus 8325-4 (the outcome variable of interest, CFU/mL). Single-species biofilm of S. aureus 8325-4 served as positive control and uninoculated wells containing TSBG served as negative control.
All plates were stained with 0.1% (wt/vol) safranin and the optical density measured (as abovementioned) to confirm the complete detachment of the biofilm fraction from the bottom (data not shown). Each biofilm fraction recovered from the wells was tested for biofilm formation in triplicate on two independent days and results were averaged over the replicates. A CFU/mL lower than numbers recovered from the positive control indicates less S. aureus biofilm formation, whereas the opposite is true for higher values of CFU/mL.
Staphylococcus aureus biofilm dispersion
To evaluate the potential of the NAS isolates to disperse the preformed biofilm of S. aureus, the biofilm dispersion assay was performed [32, adapted]. Briefly, S. aureus strain 8325-4 biofilms were grown as described in the Section “Biofilm phenotype”, and after the 24-h incubation, planktonic cells were discarded. The pre-formed S. aureus biofilms were washed once with sterile water. After, overnight cultures of all 59 NAS were adjusted in TSBG as abovementioned (Section “Biofilm phenotype”) and 200 µL of the suspensions were added directly to the S. aureus pre-formed biofilms. Plates were incubated for another 24 h at 37 °C. Finally, the planktonic cells were removed (dispersed fractions) and serially diluted. The dilutions were plated on MSA plates for CFU enumeration of S. aureus 8325-4 (the outcome variable of interest). Single-species biofilm of S. aureus 8325-4 served as positive control and uninoculated wells containing TSBG served as negative control.
All plates were stained with 0.1% (wt/vol) safranin and the optical density measured (as abovementioned) to confirm the complete detachment of the biofilm fraction from the bottom (data not shown). Each dispersion fraction recovered from the wells was tested for biofilm dispersion in triplicate on two independent days and results were averaged over the replicates. A CFU/mL lower than numbers recovered from the positive control indicates less S. aureus biofilm dispersion, whereas the opposite is true for higher values of CFU/mL.
All statistical analyses were performed using SPSS v.27.0 (IBM Corp., Armonk, NY, USA) and P ≤ 0.05 was considered significant. One-way analysis of variance (ANOVA) was used to determine whether the biofilm formation as well as biofilm dispersion of S. aureus 8325-4 (CFU/mL; normally distributed as assessed through Q-Q plots and Kolmogorov-Smirnova test of normality) differed: (1) between NAS according to their species (3 levels: S. chromogenes, S. epidermidis, and S. simulans), (2) between NAS isolates originating from the two different habitats (2 levels: milk or TA), and (3) between NAS harboring biofilm-related genes or not [2 levels: positive for at least one gene (BG) and negative (NBG); biofilm genotype), between NAS with capacity to produce biofilms themselves or not [2 levels: positive for any capacity to produce biofilm (BP) and negative (NBP); biofilm phenotype], between NAS with capacity to inhibit growth of S. aureus in vitro or not [2 levels: positive for partial or total growth inhibition of S. aureus (GI) and negative (NGI)]. In addition, one-way ANOVA was carried out to verify whether the biofilm formation and dispersion of S. aureus 8325-4 and 8325-4 Δagr (CFU/mL) was affected by the NAS capacity to suppress the agr system of S. aureus 8325-4 [2 levels: positive for any suppression of S. aureus agr (RP) and negative (NRP)].
Least significant difference (LSD) test was used to compare with the controls and post-hoc Bonferroni corrections were applied for all other comparisons.
Non-aureus staphylococcal traits
Biofilm-related genes were identified in 44.1% (15/34) of the S. chromogenes isolates, whereas this was in 100% (11/11) of the S. epidermidis isolates and in 85.7% (12/14) of the S. simulans isolates (Additional file 1). The capacity to produce biofilm was observed in 8.8% (3/34), 72.7% (8/11) and 35.7% (5/14) of the S. chromogenes, S. epidermidis and S. simulans isolates, respectively (Additional file 1). Also, 100% (34/34) of the S. chromogenes isolates has the capacity to inhibit growth of S. aureus in vitro at least partially, whilst this was true for 45.4% (5/11) and 100% (14/14) of the S. epidermidis and S. simulans isolates, respectively  (Additional file 1). Seventy-nine (27/34), 27.3 (3/11) and 100% (14/14) of the S. chromogenes, S. epidermidis, and S. simulans isolates, respectively, repressed the S. aureus agr system  (Additional file 1).
Staphylococcus aureus biofilm formation
Biofilm formation of S. aureus 8325-4 was quantified in the presence and absence (control) of NAS (Figure 1).
Overall, NAS as a group (comprising S. chromogenes, S. epidermidis, and S. simulans) did not influence S. aureus 8325-4 biofilm formation. Also, neither the origin of the NAS isolates (milk versus TA; Figure 1B) nor the biofilm phenotype (Figure 1D), nor the in vitro growth inhibition capacity of NAS (Figure 1E) were related to S. aureus biofilm formation. Still, S. chromogenes stimulated S. aureus biofilm formation (P = 0.028; Figure 1A). Also, the stimulation of the S. aureus biofilm formation was more pronounced for S. chromogenes than for S. epidermidis (P = 0.001) and S. simulans (P = 0.053; Figure 1A). Also, NAS not carrying biofilm genes stimulated S. aureus biofilm formation (P = 0.048), which was not true for NAS carrying biofilm genes although there was a strong tendency (P = 0.069; Figure 1C).
The capacity of S. aureus to form biofilm did not depend on the capacity of NAS to repress the S. aureus agr system, as evidenced for both an agr-positive and agr-negative S. aureus strain (Figure 2A).
Staphylococcus aureus biofilm dispersion
The quantitative determination (CFU/mL) of biofilm dispersion of S. aureus 8325-4 in the presence and absence (control) of NAS isolates is shown in Figure 3.
Overall, NAS as a group (comprising S. chromogenes, S. epidermidis, and S. simulans) significantly suppressed S. aureus 8325-4 biofilm dispersion, although this was more pronounced in NAS isolates originating from TA than from milk (P = 0.01; Figure 1B). Neither the NAS species (Figure 1A), nor the biofilm genotype (Figure 1C), nor the biofilm phenotype (Figure 1D), nor in vitro growth inhibition capacity of NAS (Figure 1E) were associated with S. aureus biofilm dispersion.
The ability of NAS to suppress S. aureus biofilm dispersion did not depend on their capacity to repress the agr system, as evidenced by the fact that the suppression was observed for both an agr-positive and agr-negative S. aureus strain (Figure 2B).
We showed that S. chromogenes stimulates S. aureus biofilm formation. The latter effect is most likely, at least partially, explained by the fact that S. chromogenes isolates were seldomly carrying biofilm genes or producing biofilms themselves, an association that was also evident from our experiments, a finding that is discussed later. On the other hand, all NAS isolates, especially those originating from TA, were able to suppress S. aureus biofilm dispersion. Interestingly, neither the biofilm formation nor the biofilm dispersion of S. aureus in the presence of bovine NAS isolates was regulated via the S. aureus agr quorum sensing system.
As was expected, the capacity of the NAS isolates to form biofilm themselves varied among the species, with S. epidermidis isolates presenting the highest ability to do so (72.7%). Conversely, a study  reported that S. epidermidis displayed the lowest ability to produce biofilm among the same three NAS species as used in our study, although the overall percentage of isolates forming biofilm was quite similar to ours (69.3%). The percentage of S. chromogenes (8.8%) as well as S. simulans isolates (35.7%) able to form biofilm in our study was considerably lower than the percentage observed by the other study , with respectively 84.7% and 84.9% of their isolates being biofilm formers. The media used for the biofilm assays (BHI vs. TSB) could be one of the reasons for the differences observed between studies. As well important, in the other study  all the NAS isolates originated only from bovine milk, whereas ours from both bovine milk and TA. Still, the 44.1% of S. chromogenes isolates harboring at least one of the biofilm-related genes in our study was also much lower than the 82% found by Tremblay et al. .
Interestingly, the biofilm phenotype observed in our isolates was not well-correlated with the identification of genes associated with biofilm formation (bap, ica, aap, and agr) in the same isolates. We demonstrated that the carriage of genes was detected in the majority of NAS originating from milk (63.2%) yet that only half of these isolates actually produced biofilm. Piessens et al.  also observed that only nine out of the 30 bap- and/or icaA-positive NAS isolates produced biofilm in the phenotypic assay. However, other study  reported that a combination of multiple genes, such as icaA-bap-aap and icaA-aap, was associated with a greater ability to form a biofilm. In our study, oddly, none of the NAS isolates possessed bap or ica genes, yet, seven out of nine aap-agr positive S. epidermidis isolates were able to produce biofilm. The failure to detect bap or ica genes in the PCR analyses may occur, but does not necessarily imply the inability to form a biofilm by NAS [27, 49], as other factors can mediate the biofilm-forming process. Additionally, it was reported  that so-called environmental NAS species (only causing IMI sporadically), such as S. epidermidis, S. sciuri, and S. xylosus, are a more significant reservoir of biofilm-related genes than so-called contagious NAS species (primarily causing IMI).
Our findings regarding the effect of NAS on the S. aureus biofilm formation and dispersion were in contrast with the observations revealed in a recent study . In the latter study, a reduction in biofilm formation as well as an increase in dispersion of pre-established biofilm of S. aureus by bovine NAS, especially by S. chromogenes and S. simulans isolates, was reported. Unlike our study though, their S. chromogenes isolates were essentially weak-biofilm producers, whereas ours rarely did produce biofilm. Strikingly, the S. chromogenes isolates in our collection were less likely to carry biofilm-related genes. Also, the capacity of bovine NAS isolates to stimulate S. aureus biofilm formation was more extended in NAS isolates that did not carry biofilm-related genes. Although the presence of such genes and the capacity to form biofilm were not well-correlated, we believe this finding explains, at least partially, the S. chromogenes effect and that the discordant results might be attributed to differences in intrinsic traits of the NAS isolates as well as to the combination of S. aureus and NAS species. Unfortunately, in-depth information regarding the traits of the NAS isolates used was not informed by Goetz et al. .
In addition to the effect of S. chromogenes isolates on the S. aureus biofilm formation, the dispersion of the pre-established biofilm of S. aureus by the bovine NAS isolates was suppressed, even more so by TA isolates (including the isolate S. c. 29—a strong in vitro growth inhibitor). Some studies have reported advantages provided by living in a biofilm community, which may result in, for example, decreased antibiotic susceptibility and protection against immune defenses [50, 51]. Furthermore, according to our previous findings , the same NAS isolates originating from TA, except for one S. epidermidis isolate, demonstrated in vitro S. aureus growth inhibition. However, the capacity of the bovine NAS isolates to inhibit in vitro the growth of S. aureus did not play a role in the biofilm regulation of S. aureus under the conditions tested in our study. Likewise, other study  observed no marked growth inhibition when S. aureus isolates were grown with the S. chromogenes and S. simulans isolates that had the strongest biofilm-inhibition activity.
Different interactions between NAS and S. aureus colonizing the bovine mammary gland have been suggested [18, 19], including the competitive behavior between NAS and S. aureus in multispecies biofilm communities . In a previous study using the same NAS isolates, we demonstrated that NAS have the capacity to repress the agr system of S. aureus . Furthermore, based on the concept that agr might coordinate both biofilm formation and dispersal , we verified for the first time whether bovine NAS isolates originating from milk and TA might affect biofilm formation or dispersion of S. aureus by regulation of the agr system Although it was previously reported that the repression of the agr system would be necessary to form a biofilm, whereas the reactivation of the agr system in established biofilms would trigger its dispersion , our NAS isolates not only suppressed biofilm dispersion of the S. aureus strain with a functional agr system (8325-4) but also from the agr mutant strain (8325-4 Δagr). Also, the effect of NAS on the biofilm dispersion of both the parent and mutant S. aureus strain (8235-4 and 8325-4 Δagr) further reinforced the hypothesis that the bovine NAS isolates can coordinate biofilm formation and dispersion of S. aureus by other mechanisms rather than regulation of agr quorum sensing system.
Some authors have discussed that the presence or absence of bovine NAS isolates sharing the same niche as S. aureus may play a role in the host colonization , whereas others [37, 52] observed a robust biofilm formation by NAS plus S. aureus in isolates from pigs, with no evident out-competition of one over the other species when growing in physical contact. According to Otto , in fact more than one mechanism could be involved in the biofilm formation and dispersion of staphylococci species, such as the bacterial social behavior (cooperation and/or competition), the molecular mechanism (e.g., bacterial signaling, coaggregation, or co-metabolism) , the immune response of the host (bovine mammary gland immune response), and both intrinsic and acquired traits of the isolates involved in the dual-species bacterial biofilms . Nevertheless, as biofilms are extremely hard to eradicate by both the host and by antimicrobial therapies, these dual-species interactions would allow both species to persist in a colonizing state more robustly in one hand, but on the other hand would also provide a constant reservoir for possible S. aureus chronic infections . Still, the reduction of dispersion, as was evident in our results, could potentially prevent S. aureus to spread and colonize other niches.
In conclusion, we demonstrated that S. chromogenes as well as NAS isolates in which no biofilm-related genes were demonstrated, stimulate biofilm formation of S. aureus. As well, bovine NAS isolates were effective in suppressing biofilm dispersion of S. aureus. Interestingly, the habitat of the NAS isolates seems to play an important role in the dispersion of the S. aureus biofilm, suggesting strain differences related to niche adaptation. Importantly, our findings highlight the existing interactions between bovine-related NAS and S. aureus in the biofilm communities and suggest as well that the S. aureus biofilm formation and dispersion, though not through regulation of the agr quorum sensing system, can be affected by (some) NAS (species). The mechanisms that coordinate and determine the biofilm formation and dispersion of S. aureus in the presence of NAS isolates have yet to be identified. Our findings contribute to the knowledge on biofilm formation and dispersion of S. aureus and may lead to new preventive or treatment measures, especially for chronic subclinical mastitis caused by this bacterium in dairy cows.
Availability of data and materials
The data on which the conclusions of the manuscript rely are presented in the main paper and additional files.
- agr :
accessory gene regulator
- aap :
- bap :
positive for biofilm-related genes
positive for biofilm production
colony forming units
positive for in vitro growth inhibition of S. aureus
intracellular adhesion locus
matrix assisted laser desorption/ionization time-of-fight mass spectrometry
mannitol salt agar
negative for biofilm-related genes
negative for biofilm production
negative for in vitro growth inhibition of S. aureus
negative for agr repression
polymerase chain reaction
- S. a. :
- S. c. :
- S. e. :
- S. s. :
positive for agr repression
tryptic soy broth
quarter somatic cell count
Hogeveen H, Steeneveld W, Wolf CA (2019) Production diseases reduce the efficiency of dairy production: a review of the results, methods, and approaches regarding the economics of mastitis. Annu Rev Resour Econ 11:289–312
Vanderhaeghen W, Piepers S, Leroy F, Van Coillie E, Haesebrouck F, De Vliegher S (2014) Invited review: effect, persistence, and virulence of coagulase-negative Staphylococcus species associated with ruminant udder health. J Dairy Sci 97:5275–5293
Vanderhaeghen W, Piepers S, Leroy F, Van Coillie E, Haesebrouck F, De Vliegher S (2015) Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants. Vet J 203:44–51
Nobrega DB, Naushad S, Naqvi SA, Condas LAZ, Saini V, Kastelic JP, Luby C, De Buck J, Barkema HW (2018) Prevalence and genetic basis of antimicrobial resistance in non-aureus staphylococci isolated from Canadian dairy herds. Front Microbiol 9:256
Pitkälä A, Haveri M, Pyörälä S, Myllys V, Honkanen-Bu-zalski T (2004) Bovine mastitis in Finland 2001—prevalence, distribution of bacteria, and antimicrobial resistance. J Dairy Sci 87:2433–2441
Piepers S, De Meulemeester L, Kruif A, Opsomer G, Barkema HW, De Vliegher S (2007) Prevalence and distribution of mastitis pathogens in subclinically infected dairy cows in Flanders, Belgium. J Dairy Res 74:478–483
Schukken YH, Gonzalez RN, Tikofsky LL, Schulte HF, Santisteban CG, Welcome FL, Bennett GJ, Zurakowski MJ, Zadoks RN (2009) CNS mastitis: nothing to worry about? Vet Microbiol 134:9–14
Supré K, Haesebrouck F, Zadoks RN, Vaneechoutte M, Piepers S, De Vliegher S (2011) Some coagulase-negative Staphylococcus species affect udder health more than others. J Dairy Sci 94:2329–2340
De Visscher A, Piepers S, Haesebrouck F, De Vliegher S (2016) Teat apex colonization with coagulase-negative Staphylococcus species before parturition: distribution and species-specific risk factors. J Dairy Sci 99:1427–1439
Tomazi T, Gonçalves JL, Barreiro JR, Arcari MA, dos Santos MV (2015) Bovine subclinical intramammary infection caused by coagulase-negative staphylococci increases somatic cell count but has no effect on milk yield or composition. J Dairy Sci 98:3071–3078
Valckenier D, Piepers S, De Visscher A, Bruckmaier RM, De Vliegher S (2019) Effect of intramammary infection with non-aureus staphylococci in early lactation in dairy heifers on quarter somatic cell count and quarter milk yield during the first 4 months of lactation. J Dairy Sci 102:6442–6453
Valckenier D, Piepers S, Schukken YH, De Visscher A, Boyen F, Haesebrouck F, De Vliegher S (2020) Longitudinal study on the effects of intramammary infection with non-aureus staphylococci on udder health and milk production in dairy heifers. J Dairy Sci 104:899–916
De Vliegher S, Opsomer G, Vanrolleghem A, Devriese LA, Sampimon OC, Sol J, Barkema HW, Haesebrouck F, de Kruif A (2004) In vitro growth inhibition of major mastitis pathogens by Staphylococcus chromogenes originating from teat apices of dairy heifers. Vet Microbiol 101:215–221
Piepers S, Opsomer G, Barkema HW, de Kruif A, De Vliegher S (2010) Heifers infected with coagulase-negative staphylococci in early lactation have fewer cases of clinical mastitis and higher milk production in their first lactation than noninfected heifers. J Dairy Sci 93:2014–2024
Piepers S, Peeters K, Opsomer G, Barkema HW, Frankena K, De Vliegher S (2011) Pathogen group specific risk factors at herd, heifer and quarter levels for intramammary infections in early lactating dairy heifers. Prev Vet Med 99:91–101
Braem G, Stijlemans B, Van Haken W, De Vliegher S, De Vuyst L, Leroy F (2014) Antibacterial activities of coagulase-negative staphylococci from bovine teat apex skin and their inhibitory effect on mastitis-related pathogens. J Appl Microbiol 116:1084–1093
Ferronatto JA, Souza FN, Della Libera AMMP, De Vliegher S, De Visscher A, Piepers S, Blagitz MG, Heinemann MB (2019) Inhibition of the growth of major mastitis-causing pathogens by non-aureus Staphylococcus isolates using the cross-streaking method. Arq Bras Med Vet Zootec 71:1745–1749
Mahmmod YS, Klaas IC, Svennesen L, Pedersen K, Ingmer H (2018) Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization. J Dairy Sci 101:7322–7333
Toledo-Silva B, Souza FN, Piepers S, Mertens K, Haesebrouck F, De Vliegher S (2021) Metabolites of bovine-associated non-aureus staphylococci influence expression of Staphylococcus aureus agr-related genes in vitro. Vet Res 52:62
Barkema HW, Schukken YH, Zadoks RN (2006) Invited review: the role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. J Dairy Sci 89:1877–1895
Peton V, Le Loir Y (2014) Staphylococcus aureus in veterinary medicine. Infect Genet Evol 21:602–615
Rainard P, Foucras G, Fitzgerald JR, Watts JL, Koop G, Middleton JR (2018) Knowledge gaps and research priorities in Staphylococcus aureus mastitis control. Transbound Emerg Dis 65:149–165
Tremblay YD, Lamarche D, Chever P, Haine D, Messier S, Jacques M (2013) Characterization of the ability of coagulase-negative staphylococci isolated from the milk of Canadian farms to form biofilms. J Dairy Sci 96:234–246
Tolker-Nielsen T (2014) Biofilm development. Microbiol Spectr 3:MB-0001-2014
Hall CW, Mah TF (2017) Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol Rev 41:276–301
Mack D, Fischer W, Krokotsch A, Leopold K, Hartmann R, Egge H, Laufs R (1996) The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear β-1,6-linked glucosaminoglycan: purification and structural analysis. J Bacteriol 178:175–183
Cucarella C, Tormo MA, Ubeda C, Trotonda MP, Monzon M, Peris C, Amorena B, Lasa I, Penades JR (2004) Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus. Infect Immun 72:2177–2185
Rohde H, Burdelski C, Bartscht K, Hussain M, Buck F, Horstkotte MA, Knobloch JK, Heilmann C, Herrmann M, Mack D (2005) Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases. Mol Microbiol 55:1883–1895
Antunes LCM, Ferreira RB, Buckner MM, Finlay BB (2010) Quorum sensing in bacterial virulence. Microbiol 156:2271–2282
Yarwood JM, Bartels DJ, Volper EM, Greenberg EP (2004) Quorum sensing in Staphylococcus aureus biofilms. J Bacteriol 186:1838–1850
Boles BR, Horswill AR (2008) Agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS Pathog 4:p1000052
Goetz C, Tremblay YD, Lamarche D, Blondeau A, Gaudreau AM, Labrie J, Malouin F, Jacques M (2017) Coagulase-negative staphylococci species affect biofilm formation of other coagulase-negative and coagulase-positive staphylococci. J Dairy Sci 100:6454–6464
Schönborn S, Krömker V (2016) Detection of the biofilm component polysaccharide intercellular adhesin in Staphylococcus aureus infected cow udders. Vet Microbiol 196:126–128
Otto M (2018) Staphylococcal biofilms. Microbiol Spectr 6:GPP3-0023-2018
Wolcott R, Costerton JW, Raoult D, Cutler SJ (2013) The polymicrobial nature of biofilm infection. Clin Microbiol Infect 19:107–112
Ren X, Wang L, Chen W (2020) Oxytropis glabra DC inhibits biofilm formation of Staphylococcus epidermidis by down-regulating ica operon expression. Curr Microbiol 77:1167–1173
Peng P, Baldry M, Gless BH, Bojer MS, Gonora CE, Baig S, Andersen PS, Olsen CA, Ingmer H (2019) Effect of co-inhabiting coagulase negative staphylococci on S. aureus agr quorum sensing, host factor binding, and biofilm formation. Front Microbiol 10:2212
Archer NK, Mazaitis MJ, Costerton JW, Leid JG, Powers ME, Shirtliff ME (2011) Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Virulence 2:445–459
Piessens V, Van Coillie E, Verbist B, Supré K, Braem G, Van Nuffel A, De Vuyst L, Heyndrickx M, De Vliegher S (2011) Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds. J Dairy Sci 94:2933–2944
Cameron M, Barkema HW, De Buck J, De Vliegher S, Chaffer M, Lewis J, Keefe GP (2017) Identification of bovine-associated coagulase-negative staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol. J Dairy Sci 100:2137–2147
Novick RP, Morse SI (1967) In vivo transmission of drug resistance factors between strains of Staphylococcus aureus. J Exp Med 125:45–59
Canovas J, Baldry M, Bojer MS, Andersen PS, Grzeskowiak PK, Stegger M, Damborg P, Olsen CA, Ingmer H (2016) Cross-talk between Staphylococcus aureus and other staphylococcal species via the agr quorum sensing system. Front Microbiol 7:1733
Unal S, Hoskins J, Flokowitsch JE, Wu CY, Preston DA, Skatrud PL (1992) Detection of methicillin-resistant staphylococci by using the polymerase chain reaction. J Clin Microbiol 30:1685–1691
Rohde H, Burandt EC, Siemssen N, Frommelt L, Burdelski C, Wurster S, Scherpe S, Davies AP, Harris LG, Horst-kotte MA, Knobloch JK, Ragunath C, Kaplan JB, Mack D (2007) Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections. Biomaterials 28:1711–1720
Pedroso SH, Sandes SH, Luiz KC, Dias RS, Filho RA, Serufo JC, Farias LM, Carvalho MA, Bomfim MR, Santos SG (2016) Biofilm and toxin profile: a phenotypic and genotypic characterization of coagulase-negative staphylococci isolated from human bloodstream infections. Microb Pathog 100:312–318
Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, Ravel J, Paulsen IT, Kolonay JF, Brinkac L, Beanan M, Dodson RJ, Daugherty SC, Madupu R, Angiuoli SV, Durkin AS, Haft DH, Vamathevan J, Khouri H, Utterback T, Lee C, Dimitrov G, Jiang L, Qin H, Weidman J, Tran K, Kang K, Hance IR, Nelson KE, Fraser CM (2005) Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacteriol 187:2426–2438
Zhang YQ, Ren SX, Li HL, Wang YX, Fu G, Yang J, Qin ZQ, Miao YG, Wang WY, Chen RS, Shen Y, Chen Z, Yuan ZH, Zhao GP, Qu D, Danchin A, Wen YM (2003) Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Mol Microbiol 49:1577–1593
Piessens V, De Vliegher S, Verbist B, Braem G, Van Nuffel A, De Vuyst L, Heyndrickx M, Van Coillie E (2012) Characterization of coagulase-negative staphylococcus species from cows’ milk and environment based on bap, icaA, and mecA genes and phenotypic susceptibility to antimicrobials and teat dips. J Dairy sci 95:7027–7038
Fredheim EGA, Klingenberg C, Rohde H, Frankenberger S, Gaustad P, Flaegstad T, Sollid JE (2009) Biofilm formation by Staphylococcus haemolyticus. J Clin Microbiol 47:1172–1180
Elias S, Banin E (2012) Multi-species biofilms: living with friendly neighbors. FEMS Microbiol Rev 36:990–1004
Burmølle M, Ren D, Bjarnsholt T, Sørensen SJ (2014) Interactions in multispecies biofilms: do they actually matter? Trends Microbiol 22:84–91
Gonzalez T, Baatyrbek-kyzy A, Andersen H, Haslam DB, Myers JMB, Hershey GKK, Herr AB (2018) Investigating the role of staphylococcal biofilms in the pathogenesis of pediatric atopic dermatitis. J Allergy Clin Immunol 141:AB93
Otto M (2013) Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity. Annu Rev Med 64:175–188
Pedersen RR, Krömker V, Bjarnsholt T, Dahl-Pedersen K, Buhl R, Jørgensen E (2021) Biofilm research in bovine mastitis. Front Vet Sci 8:656810
We are grateful to Dr. Hanne Ingmer (University of Copenhagen), Dr. Simon Foster (University of Sheffield), and Dr. Sophia Johler (University of Zurich) for kindly providing us with the reporter strains of Staphylococcus aureus.
This study was supported financially by the Brazilian research funding agency CAPES. The MALDI-ToF mass spectrometer was financed by the Research Foundation Flanders (FWO-Vlaanderen) as Hercules project G0H2516N (AUGE/15/05). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1. Identification and traits of bovine S. chromogenes (S. c.), S . epidermidis (S. e.), S. simulans (S. s.), and Staphylococcus aureus (S. a.).
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Toledo-Silva, B., de Souza, F.N., Mertens, K. et al. Bovine-associated non-aureus staphylococci suppress Staphylococcus aureus biofilm dispersal in vitro yet not through agr regulation. Vet Res 52, 114 (2021). https://doi.org/10.1186/s13567-021-00985-z
- Non-aureus staphylococci
- Staphylococcus aureus
- quorum sensing
- bovine mastitis