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Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources.

ABSTRACT: Sixteen Staphylococcus aureus isolates originating from foods (eight from dairy products, five from fish and fish products and three from meat and meat products) were evaluated regarding their biofilms formation ability. Six strains (E2, E6, E8, E10, E16, and E23) distinguished as strong biofilm formers, either in standard Tryptic Soy Broth or in Tryptic Soy Broth supplemented with 0.4% glucose or with 4% NaCl. The composition of the biofilms formed by these S. aureus strains on polystyrene surfaces was first inferred using enzymatic and chemical treatments. Later on, biofilms were characterized by confocal laser scanning microscope (CLSM). Our experiments proved that protein-based matrices are of prime importance for the structure of biofilms formed by S. aureus strains isolated from food sources. These biofilm matrix compositions are similar to those put into evidence for coagulase negative staphylococci. This is a new finding having in view that scientific literature mentions exopolysaccharide abundance in biofilms produced by clinical isolates and food processing environment isolates of S. aureus.

SUBMITTER: Oniciuc EA

PROVIDER: S-EPMC4811954 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources.

Oniciuc Elena-Alexandra EA Cerca Nuno N Nicolau Anca I AI

Frontiers in microbiology 20160330

Sixteen Staphylococcus aureus isolates originating from foods (eight from dairy products, five from fish and fish products and three from meat and meat products) were evaluated regarding their biofilms formation ability. Six strains (E2, E6, E8, E10, E16, and E23) distinguished as strong biofilm formers, either in standard Tryptic Soy Broth or in Tryptic Soy Broth supplemented with 0.4% glucose or with 4% NaCl. The composition of the biofilms formed by these S. aureus strains on polystyrene surf ...[more]

PMID: 27065962

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Project description:IntroductionStaphylococcus aureus is an important pathogen that can form biofilms on food contact surfaces (FCS) in the dairy industry, posing a serious food safety, and quality concern. Biofilm is a complex system, influenced by nutritional-related factors that regulate the synthesis of the components of the biofilm matrix. This study determines the prevalence of biofilm-associated genes and evaluates the development under different growth conditions and compositions of biofilms produced by S. aureus.MethodsBiofilms were developed in TSB, TSBG, TSBNaCl, and TSBGNaCl on stainless-steel (SS), with enumeration at 24 and 192 h visualized by epifluorescence and scanning electron microscopy (SEM). The composition of biofilms was determined using enzymatic and chemical treatments and confocal laser scanning microscopy (CLSM).Results and discussionA total of 84 S. aureus (SA1-SA84) strains were collected from 293 dairy industry FCS (FCS-stainless steel [n = 183] and FCS-polypropylene [n = 110]) for this study. The isolates harbored the genes sigB (66%), sar (53%), agrD (52%), clfB/clfA (38%), fnbA/fnbB (20%), and bap (9.5%). 99. In particular, the biofilm formed by bap-positive S. aureus onto SS showed a high cell density in all culture media at 192 h in comparison with the biofilms formed at 24 h (p < 0.05). Epifluorescence microscopy and SEM revealed the metabolically active cells and the different stages of biofilm formation. CLSM analysis detected extracellular polymeric of S. aureus biofilms on SS, such as eDNA, proteins, and polysaccharides. Finally, the level of detachment on being treated with DNase I (44.7%) and NaIO 4(42.4%) was greater in the biofilms developed in TSB compared to culture medium supplemented with NaCl at 24 h; however, there was no significant difference when the culture medium was supplemented with glucose. In addition, after treatment with proteinase K, there was a lower level of biomass detachment (17.7%) of the biofilm developed in TSBNaCl (p < 0.05 at 24 h) compared to that in TSB, TSBG, and TSBGNaCl (33.6, 36.9, and 37.8%, respectively). These results represent a deep insight into the composition of S. aureus biofilms present in the dairy industry, which promotes the development of more efficient composition-specific disinfection strategies.

Project description:BackgroundMethicillin-resistant Staphylococcus aureus (MRSA), a leading cause of chronic infections, forms prolific biofilms which afford an escape route from antibiotic treatment and host immunity. However, MRSA clones are genetically diverse, and mechanisms underlying biofilm formation remain under-studied. Such studies form the basis for developing targeted therapeutics. Here, we studied the temporal changes in the biofilm transcriptome of three pandemic MRSA clones: USA300, HEMRSA-15, and ST239.MethodsBiofilm formation was assessed using a static model with one representative strain per clone. Total RNA was extracted from biofilm and planktonic cultures after 24, 48, and 72 h of growth, followed by rRNA depletion and sequencing (Illumina Inc., San Diego, CA, United States, NextSeq500, v2, 1 × 75 bp). Differentially expressed gene (DEG) analysis between phenotypes and among early (24 h), intermediate (48 h), and late (72 h) stages of biofilms was performed together with in silico co-expression network construction and compared between clones. To understand the influence of SCCmec and ACME on biofilm formation, isogenic mutants containing deletions of the entire elements or of single genes therein were constructed in USA300.ResultsGenes involved in primarily core genome-encoded KEGG pathways (transporters and others) were upregulated in 24-h biofilm culture compared to 24-h planktonic culture. However, the number of affected pathways in the ST239 24 h biofilm (n = 11) was remarkably lower than that in USA300/EMRSA-15 biofilms (USA300: n = 27, HEMRSA-15: n = 58). The clfA gene, which encodes clumping factor A, was the single common DEG identified across the three clones in 24-h biofilm culture (2.2- to 2.66-fold). In intermediate (48 h) and late (72 h) stages of biofilms, decreased expression of central metabolic and fermentative pathways (glycolysis/gluconeogenesis, fatty acid biosynthesis), indicating a shift to anaerobic conditions, was already evident in USA300 and HEMRSA-15 in 48-h biofilm cultures; ST239 showed a similar profile at 72 h. Last, SCCmec+ACME deletion and opp3D disruption negatively affected USA300 biofilm formation.ConclusionOur data show striking differences in gene expression during biofilm formation by three of the most important pandemic MRSA clones, USA300, HEMRSA-15, and ST239. The clfA gene was the only significantly upregulated gene across all three strains in 24-h biofilm cultures and exemplifies an important target to disrupt early biofilms. Furthermore, our data indicate a critical role for arginine catabolism pathways in early biofilm formation.

Project description:In methicillin-sensitive Staphylococcus aureus (MSSA), the tricarboxylic acid (TCA) cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG. However, methicillin-resistant S. aureus (MRSA) produce a primarily proteinaceous biofilm matrix, and contribution of the TCA-cycle therein remains unclear. Utilizing USA300-JE2 Tn-mutants (NARSA) in genes encoding TCA- and urea cycle enzymes for transduction into a prolific biofilm-forming USA300 strain (UAS391-Erys), we studied the contribution of the TCA- and urea cycle and of proteins, eDNA and PIA/PNAG, to the matrix. Genes targeted in the urea cycle encoded argininosuccinate lyase and arginase (argH::Tn and rocF::Tn), and in the TCA-cycle encoded succinyl-CoA synthetase, succinate dehydrogenase, aconitase, isocitrate dehydrogenase, fumarate hydratase class II, and citrate synthase II (sucC::Tn, sdhA/B::Tn, acnA::Tn, icd::Tn, fumC::Tn and gltA::Tn). Biofilm formation was significantly decreased under no flow and flow conditions by argH::Tn, fumC::Tn, and sdhA/B::Tn (range OD492 0.374-0.667; integrated densities 2.065-4.875) compared to UAS391-EryS (OD492 0.814; integrated density 10.676) (p ≤ 0.008). Cellular and matrix stains, enzymatic treatment (Proteinase K, DNase I), and reverse-transcriptase PCR-based gene-expression analysis of fibronectin-binding proteins (fnbA/B) and the staphylococcal accessory regulator (sarA) on pre-formed UAS391-Erys and Tn-mutant biofilms showed: (i) < 1% PIA/PNAG in the proteinaceous/eDNA matrix; (ii) increased proteins under no flow and flow in the matrix of Tn mutant biofilms (on average 50 and 51 (±11)%) compared to UAS391-Erys (on average 22 and 25 (±4)%) (p < 0.001); and (iii) down- and up-regulation of fnbA/B and sarA, respectively, in Tn-mutants compared to UAS391-EryS (0.62-, 0.57-, and 2.23-fold on average). In conclusion, we show that the biofilm matrix of MRSA-USA300 and the corresponding Tn mutants is PIA/PNAG-independent and are mainly composed of proteins and eDNA. The primary impact of TCA-cycle inactivation was on the protein component of the biofilm matrix of MRSA-USA300.

Dataset Information

Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources.

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Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources.

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