Figure 3 - uploaded by Yannick DN Tremblay
Content may be subject to copyright.
Confocal laser scanning microscopy of CNS biofilms stained with FM 1-43, BOBO-3, wheat-germ agglutinin (WGA)-Oregon green, SYPRO Ruby, SYTO, and propidium iodide (all from Invitrogen, Eugene, OR). PNAG = poly- N -acetyl glucosamine; eDNA = extracellular DNA. Color version available in the online PDF.
Source publication
Mastitis is the most common and detrimental infection of the mammary gland in dairy cows and has a major economic impact on the production of milk and dairy products. Bacterial mastitis is caused by several pathogens, and the most frequently isolated bacterial species are coagulase-negative staphylocci (CNS). Although CNS are considered minor masti...
Contexts in source publication
Context 1
... the exception of the Staph. epidermidis isolate, biofilm morphology and the amount of bacteria were fairly similar (Figure 3). The biofilm of Staph. ...
Context 2
... biofilm of Staph. epidermidis appeared to be composed of several clusters or microcolonies (Figure 3). The LIVE⁄DEAD kit is composed of 2 stains, 1 (SYTO-9) that binds DNA in cells with intact cell membranes (viable) and 1 (propidium iodide) that binds DNA in cells with dam- aged cell membranes (dead). ...
Context 3
... and Staph. xylosus biofilms (Figure 3). Wheat-germ agglutinin binds to N-acetyl-d-glucosamine and N-acetylneuraminic acid residues, and WGA is used to indicate the presence of PNAG in the biofilm matrix. ...
Context 4
... other stains used are specific for biofilm matrix components, such as proteins (SYPRO Ruby) and eDNA (BOBO-3). The biofilms of all isolates were bound by WGA, BOBO- 3, and SYPRO Ruby, which suggests that the biofilm matrix contains PNAG, eDNA, and proteins ( Figure 3). For Staph. ...
Context 5
... appeared to have the lowest amount of PNAG, whereas Staph. epidermidis appeared to have the largest amount of PNAG (Figure 3).With regard to the amount of protein, the Staph. simulans and Staph. ...
Context 6
... and Staph. epidermidis isolates appeared to have the largest amounts of proteins in their biofilm matrix (Figure 3). Although BOBO-3 is a membrane-impermeable DNA stain and does not penetrate bacterial cells with intact cell membranes, it stains eDNA and may stain DNA inside cells with a compromised cell membrane. ...
Context 7
... moderate biofilm formers, the biofilm morphology revealed by the stain was similar to the morphology of the strong biofilm formers, but the amount of stain was lower (data not shown). With regard to protein stain- ing (SYPRO Ruby) and eDNA staining (BOBO-3), the stains were mostly focused around individual cells in the case of negative and weak biofilm formers, as ob- served with WGA staining (data not shown). Moderate biofilm formers were not stained with SYPRO Ruby and BOBO-3. ...
Similar publications
Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which...
Mastitis is the principal disease affecting dairy herds worldwide. The aim of the present study was to characterize phenotypic and genotypic features associated with resistance to antimicrobials in Staphylococcus spp. isolated from 2064 milk samples of 525 lactating cows in the Northeast of Brazil. Of the 218 isolates analyzed, 57.8 % were characte...
Abstract
Introduction: Staphylococci release a large number of enzymes. Some of these, such as coagulase, beta lactamase, hemolysins and biofilms are considered indices of pathogenicity. The aim of the current study was based on the isolation and identification of Staphylococcus aureus and coagulase negative Staphylococci (CNS) strains from sheep s...
Citations
... Staphylococcus chromogenes is one of the most predominant species of coagulase-negative staphylococci (CNS) and is considered an emerging mastitis pathogen of dairy cows in many countries [1,2]. It is also the most predominant CNS on Canadian dairy farms [3][4][5]. This pathogen can cause persistent and recurring infections, leading to chronic mastitis, which can have significant negative impacts on dairy cow welfare and farm incomes [6][7][8]. ...
Staphylococcus chromogenes (SC) is a common coagulase-negative staphylococcus described as an emerging mastitis pathogen and commonly found in dairy farms. This study investigated the potential involvement of DNA methylation in subclinical mastitis caused by SC. The whole-genome DNA methylation patterns and transcriptome profiles of milk somatic cells from four cows with naturally occurring SC subclinical mastitis (SCM) and four healthy cows were characterized by next-generation sequencing, bioinformatics, and integration analyses. Comparisons revealed abundant DNA methylation changes related to SCM, including differentially methylated cytosine sites (DMCs, n = 2,163,976), regions (DMRs, n = 58,965), and methylation haplotype blocks (dMHBs, n = 53,098). Integration of methylome and transcriptome data indicated a negative global association between DNA methylation at regulatory regions (promoters, first exons, and first introns) and gene expression. A total of 1486 genes with significant changes in the methylation levels of their regulatory regions and corresponding gene expression showed significant enrichment in biological processes and pathways related to immune functions. Sixteen dMHBs were identified as candidate discriminant signatures, and validation of two signatures in more samples further revealed the association of dMHBs with mammary gland health and production. This study demonstrated abundant DNA methylation changes with possible involvement in regulating host responses and potential as biomarkers for SCM.
... Biofilm structures have been shown to act as protective shields against antibiotics (Hathroubi et al., 2017;Pedersen et al., 2021), and thus to favor the establishment of persistent and recurrent infections (Bjarnsholt et al., 2013;Flemming et al., 2016). NAS are more frequently capable than other mastitiscausing pathogens of producing biofilm biomass, which might therefore explain their high resistance rates (Tremblay et al., 2013;de Oliveira et al., 2016;Felipe et al., 2017;Srednik et al., 2017). Still, the influence of biofilm growth on antibiotic failure and infection chronicity and recurrence can be largely unpredictable (Flemming et al., 2016;Pedersen et al., 2021). ...
... Different parameters like minimum biofilm eradication concentration (MBEC) or bactericidal biofilm concentration (BBC) do not always represent the antimicrobial concentrations required to kill bacteria. In some cases, 3 Log10 bacterial reductions were described with respect to control conditions or untreated bacteria (Tremblay et al., 2013;Brady et al., 2017;Cruz et al., 2018;Thieme et al., 2019).This means that the findings of different studies might not render comparable conclusions about the influence of biofilms on therapy efficacy (Thieme et al., 2019). ...
Bovine mastitis is the most frequent and costly disease that affects dairy cattle. Non-aureus staphylococci (NAS) are currently one of the main pathogens associated with difficult-to-treat intramammary infections. Biofilm is an important virulence factor that can protect bacteria against antimicrobial treatment and prevent their recognition by the host’s immune system. Previously, we found that chronic mastitis isolates which were refractory to antibiotic therapy developed strong biofilm biomass. Now, we evaluated the influence of biofilm biomass intensity on the antibiotic resistance pattern in strong and weak biofilm-forming NAS isolates from clinical mastitis. We also assessed the effect of cloxacillin (Clx) and chitosan (Ch), either alone or in combination, on NAS isolates with different lifestyles and abilities to form biofilm. The antibiotic resistance pattern was not the same in strong and weak biofilm producers, and there was a significant association ( p ≤ 0.01) between biofilm biomass intensity and antibiotic resistance. Bacterial viability assays showed that a similar antibiotic concentration was effective at killing both groups when they grew planktonically. In contrast, within biofilm the concentrations needed to eliminate strong producers were 16 to 128 times those needed for weak producers, and more than 1,000 times those required for planktonic cultures. Moreover, Ch alone or combined with Clx had significant antimicrobial activity, and represented an improvement over the activity of the antibiotic on its own, independently of the bacterial lifestyle, the biofilm biomass intensity or the antibiotic resistance pattern. In conclusion, the degree of protection conferred by biofilm against antibiotics appears to be associated with the intensity of its biomass, but treatment with Ch might be able to help counteract it. These findings suggest that bacterial biomass should be considered when designing new antimicrobial therapies aimed at reducing antibiotic concentrations while improving cure rates.
... Different microorganisms form biofilms, such as Escherichia coli, Cronobacter sakazakii, Staphylococcus aureus, and Salmonella spp. (Hurrell et al., 2009;Patel and Sharma, 2010;Allsopp et al., 2012;Lamas et al., 2018;Liu et al., 2020), a thin layer of microorganisms adhering to the surface of an organic or inorganic structure, together with their secreted extracellular polymeric substances mainly consistent of polysaccharides, secreted proteins and extracellular DNA (Tremblay et al., 2013). Biofilm formation is an adaptable attribute of microbes (Koczan et al., 2011), providing a physical barrier that protects bacteria from adverse environmental conditions. ...
The maintenance of intestinal barrier function is essential for preventing different pathologies, such as the leaky gut syndrome (LGS), which is characterized by the passage of harmful agents, like bacteria, toxins, and viruses, into the bloodstream. Intestinal barrier integrity is controlled by several players, including the gut microbiota. Various molecules, called postbiotics, are released during the natural metabolic activity of the microbiota. Postbiotics can regulate host-microbe interactions, epithelial homeostasis, and have overall benefits for our health. In this work, we used in vitro and in vivo systems to demonstrate the role of Lactobacillus paracasei CNCM I-5220-derived postbiotic (LP-PBF) in preserving intestinal barrier integrity. We demonstrated in vitro that LP-PBF restored the morphology of tight junctions (TJs) that were altered upon Salmonella typhimurium exposure. In vivo, LP-PBF protected the gut vascular barrier and blocked S. typhimurium dissemination into the bloodstream. Interestingly, we found that LP-PBF interacts not only with the host cells, but also directly with S. typhimurium blocking its biofilm formation, partially due to the presence of biosurfactants. This study highlights that LP-PBF is beneficial in maintaining gut homeostasis due to the synergistic effect of its different components. These results suggest that LP-PBF could be utilized in managing several pathologies displaying an impaired intestinal barrier function.
... In contrast, the inability to form biofilm of the isolates having icaA and icaD genes could be due to point mutations in the ica locus or any other unidentified factors that negatively regulate polysaccharide intercellular adhesion synthesis or influence biofilm formation (Cramton et al., 1999). Some researchers also indicated that the process of biofilm formation is complex involving many genes or many unknown variables (Darwish and Asfour, 2013;Tremblay et al., 2013). The results obtained from this and other studies indicated the importance of other mechanisms of biofilm-forming independent of the ica operon. ...
The starting objective of this research communication was to determine the prevalence of subclinical mastitis in buffalo in Turkey. We also seeked to isolate and identify staphylococci, determine their antimicrobial susceptibilities and biofilm-forming abilities as well as investigating the presence of biofilm-related genes and microbial surface components recognizing adhesive matrix molecules. A total of 107 (66.9%) staphylococci (28 S. aureus and 79 coagulase-negative staphylococci, CoNS) were isolated from 160 mastitic milk samples collected from 200 lactating water buffalos. The staphylococci were especially resistant to beta-lactams except for cefoxitin but were less resistant to the other antimicrobials that were tested. Based on the Congo red agar method, 92.9% of the S. aureus and 70.9% of the CoNS isolates were positive for biofilm-forming ability, while all S. aureus and 97.5% of CoNS isolates were positive by a microtiter plate analysis. The presence of icaA and icaD genes was not always correlated with biofilm synthesis, and even in the absence of these genes, the isolates were able to synthesize biofilm.
... Following is a more detailed list of some of the most important agents causing IMI, presented in Table 1. these microbes are often associated with recurrent infections due to their ability to form biofilms [30]. ...
Periparturient diseases continue to be the greatest challenge to both farmers and dairy cows. They are associated with a decrease in productivity, lower profitability, and a negative impact on cows’ health as well as public health. This review article discusses the pathophysiology and diagnostic opportunities of mastitis, the most common disease of dairy cows. To better understand the disease, we dive deep into the causative agents, traditional paradigms, and the use of new technologies for diagnosis, treatment, and prevention of mastitis. This paper takes a systems biology approach by highlighting the relationship of mastitis with other diseases and introduces the use of omics sciences, specifically metabolomics and its analytical techniques. Concluding, this review is backed up by multiple studies that show how earlier identification of mastitis through predictive biomarkers can benefit the dairy industry and improve the overall animal health.
... CoNS isolates were classified as strong or moderate biofilm formers. Tremblay et al. (2013) and Srednik et al. (2017) [32,33] reported that 48.6% and 44.0% of biofilm-forming CoNS isolates from bovine mastitis in Canada and Argentina, respectively, were classified as strong or moderate biofilm formers. If milk samples were derived from bovine with clinical mastitis rather than from normal bulk tank, a higher prevalence of biofilm formers in CoNS might be confirmed. ...
... CoNS isolates were classified as strong or moderate biofilm formers. Tremblay et al. (2013) and Srednik et al. (2017) [32,33] reported that 48.6% and 44.0% of biofilm-forming CoNS isolates from bovine mastitis in Canada and Argentina, respectively, were classified as strong or moderate biofilm formers. If milk samples were derived from bovine with clinical mastitis rather than from normal bulk tank, a higher prevalence of biofilm formers in CoNS might be confirmed. ...
... If milk samples were derived from bovine with clinical mastitis rather than from normal bulk tank, a higher prevalence of biofilm formers in CoNS might be confirmed. The prevalence of strong or moderate biofilm formers in this study was significantly higher in Staphylococcus haemolyticus and Staphylococcus chromogenes, which was similar to previous reports [6,32,33]. The highest median value and widest deviation range were also observed in these two CoNS species. ...
Coagulase-negative staphylococci (CoNS) are considered less virulent as they do not produce a large number of toxic enzymes and toxins; however, they have been increasingly recognized as an important cause of bovine mastitis. In particular, the ability to form biofilms appears to be an important factor in CoNS pathogenicity, and it contributes more resistance to antimicrobials. The aim of this study was to investigate the pathogenic potential by assessing the biofilm-forming ability of CoNS isolated from normal bulk tank milk using the biofilm formation assay and to analyze the biofilm-associated resistance to antimicrobial agents using the disc diffusion method. One hundred and twenty-seven (78.4%) among 162 CoNS showed the ability of biofilm formation, and all species showed a significantly high ability of biofilm formation (p < 0.05). Although the prevalence of weak biofilm formers (39.1% to 80.0%) was significantly higher than that of other biofilm formers in all species (p < 0.05), the prevalence of strong biofilm formers was significantly higher in Staphylococcus haemolyticus (36.4%), Staphylococcus chromogenes (24.6%), and Staphylococcus saprophyticus (21.7%) (p < 0.05). Also, 4 (11.4%) among 35 non-biofilm formers did not harbor any biofilm-associated genes, whereas all 54 strong or moderate biofilm formers harbored 1 or more of these genes. The prevalence of MDR was significantly higher in biofilm formers (73.2%) than in non-formers (20.0%) (p < 0.05). Moreover, the distribution of MDR in strong or moderate biofilm formers was 81.5%, which was significantly higher than in weak (67.1%) and non-formers (20.0%) (p < 0.05). Our results indicated that various CoNS isolated from bulk tank milk, not from bovine with mastitis, have already showed a high ability to form biofilms, while also displaying a high prevalence of MDR.
... Studies have demonstrated the spread of S. chromogenes in cattle herds from different geographical areas, including Brazil (Tremblay et al., 2013;Fry et al., 2014b;Tomazi et al., 2014;Lange and Brito, 2015;Srednik et al., 2015). Nevertheless, other CoNS species have a higher frequency than S. chromogenes in both clinical and subclinical mastitis (Bochniarz et al., 2014). ...
... S. chromogens was the species with the lowest number of strains capable of producing biofilms. In the study by Tremblay et al. (2013), biofilm-producing S. xylosus strains were the most frequent, followed by S. haemolyticus, while S. epidermidis strains were the least frequent. The observed differences can be explained by the distribution of CoNS species in different countries. ...
... The observed differences can be explained by the distribution of CoNS species in different countries. In addition, intra-species variation is not uncommon and has been observed for S. chromogenes, S. epidermidis, S. haemolyticus, and S. hominis (Tremblay et al., 2013;Oliveira et al., 2015). Ajitkumar et al. (2013) identified three distinct genotypes among S. chromogenes strains, which may explain the variation in biofilm production in this species. ...
This study aimed to identify the Staphylococcus species responsible for bovine mastitis in dairy herds in northern Brazil, to investigate the capacity of biofilm production, and to analyze the association of biofilm production with multiresistance and intensity of California Mastitis Tests (CMT) reactions that can make treatment more difficult and cause misdiagnoses, respectively. Milk samples were collected from 23 dairy farms located in five municipalities in the state of Acre. A total of 339 crossbred cows were tested by CMT, with 109 animals (229 udder ceilings) reacting to the test. After bacterial isolation in blood agar, the catalase-positive and gram-positive cocci were submitted for identification by MALDI-TOF MS. Of 103 strains identified as staphylococci, Staphylococcus chromogenes (58.3%) and Staphylococcus aureus (19.4%) were the most prevalent species. Biofilm production was quantitatively evaluated using a microplate adherence test. Among the Staphylococcus strains, 71.8% were biofilm producers. Most strains of S. chromogenes (68.3%) had the capacity to produce biofilms, ranging from weak (43.3%), moderate (13.3%), and strong (11.7%) producers. Among S. aureus strains, 50% were non-biofilm producers, and none were strong producers. Our data showed an association between biofilm production capacity and multidrug resistance. In addition, there was a reduction in the response to the CMT test, which can mask the diagnosis.
... The presence of clumping factor B gene (clfB) in all our S. agnetis and S. hyicus isolates was the only difference between the adherence genes identified in this study and those from the bovine mastitis study of Naushad et al., where only 15% of bovine mastitis S. agnetis isolates had this gene (31). The intracellular adhesion genes and biofilm-producing genes (icaA, icaB, and icaC) were identified only in the M. sciuri isolate in our study, which is also consistent with the results of previous studies (31,32). ...
Skin lesions in cattle associated with feeding by Haematobia fly species are a significant welfare issue in Australia, North and South America, and Europe. The development of these lesions has been attributed to a number of causal factors, but the exact etiology and pathogenesis were unclear.
... Previous studies regarding the biofilm formation of S. haemolyticus have yielded different results among them, with studies reporting high rates of biofilm formation and others low to medium rates [44,45]. A study conducted with milk samples showed that S. xylosus was moderate to strong biofilm formers whereas S. epidermidis had the lowest ability to form biofilms [46]. Another study investigated the biofilm-forming ability of CoNS isolated from horses and the highest values were measured for S. xylosus strains [36]. ...
The ability of biofilm formation seems to play an important role in the virulence of staphylococci. However, studies reporting biofilm formation of coagulase-negative staphylococci isolated from animals are still very scarce. Thus, we aimed to evaluate the biofilm-forming capacity of CoNS and S. pseudintermedius isolated from several animal species and to investigate the effect of conventional antimicrobials on biofilm reduction. A total of 35 S. pseudintermedius and 192 CoNS were included. Biofilm formation was accessed by the microtiter plate assay and the biofilms were stained by crystal violet. Association between biofilm formation and staphylococci species and antimicrobial resistance was also performed. Biofilm susceptibility testing was performed with tetracycline and amikacin at the minimum inhibitory concentration (MIC) and 10 × MIC. The metabolic activity of the biofilm cells after antimicrobial treatment was accessed by the XTT assay. All isolates formed biofilm, with S. urealyticus producing the most biofilm biomass and S. pseudintermedius producing the least biomass. There was a positive association between biofilm formation and multidrug resistance as well as resistance to individual antimicrobials. Neither tetracycline nor amikacin were able to eradicate the biofilm, not even at the highest concentration used. This study provides new insights into biofilm formation and the effects of antimicrobials on CoNS species.
... Mastitis, an inflammation of the mammary glands is one of the most challenging diseases to control due to its multifactorial causes [109]. Staphylococcus species are considered the most frequent and important cause of both clinical and subclinical mastitis in dairy cattle [109,110]. The clinical course of the infection and contagious nature of the disease depends on virulence factors of the staphylococcal strain involved in causing the disease [111,112]. ...
Bovine mastitis is the single most costly disease usually caused by Bacteria. The genus Staphylococcus is major bacteria that cause mastitis in dairy cattle. Staphylococci that cause bovine mastitis are commonly divided into two major groups such as 1) Staphylococcus aureus and 2) non-aureus staphylococci (NAS). Staphylococcus aureus causes clinical and subclinical mastitis in dairy cows. Accurate diagnosis of Staphylococcus species can be made by Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF), 16S RNA gene sequencing, and polymerase chain reaction (PCR). In well-managed dairy farms that fully applied mastitis control measures, the incidence of S. aureus mastitis significantly reduced. However, staphylococcal mastitis is still major problem in most farms due to variation in management and presence of some species of non-aureus staphylococci in the environment. There is no effective vaccine that prevent staphylococcal mastitis. Treatment with antibiotics is increasingly less effective and increases development of antimicrobial resistant bacteria. Sustainable non-antibiotic staphylococcal mastitis prevention measures such as vaccines, probiotics, good herd health management and other improved methods are required. To develop an innovative control tool detailed understanding of staphylococcal virulence factors, pathogenesis, and host immunological responses is critically important. This chapter discusses the pathogenesis, host responses and current control and prevention methods.
