Comparative study of virulence factors among methicillin resistant Staphylococcus aureus clinical isolates

A total of 43 non-redundant MRSA clinical isolates were obtained from the Laboratory of Microbiology of the University hospital of Monastir-Tunisia. These strains were obtained from bacteriological samples in hospitalized patients and/or consultants at the University Hospital of Monastir-Tunisia during the period (January 2016 - June 2016). Two reference strains (S. aureus ATCC 6538 and ATCC 43300) were used as controls. Bacterial identification was performed using Gram staining, catalase test, tube coagulase, DNase agar, mannitol salt agar and API ID 20 STAPH galleries (bio-Merieux, France). To confirm the identity of the isolate as S. aureus, the Sa442 gene was amplified by a PCR-based method. Extraction of genomic DNA was performed using a standard phenolechloroform technique. The PCR mixture (25 μl) for all genes contained 1 mM forward and reverse primers, dNTP mix (100 mM each of dATP, dCTP, dGTP and dTTP), 1 U of Go Taq DNA polymerase (Promega), 5 ml green Go Taq buffer (5X), and DNA template (50 ng). PCR conditions were the following: an initial temperature of 96°C (3min), followed by denaturation at 95°C (1min), annealing at 55°C (30s), elongation at 72°C (3min), and a final elongation step at 72°C (4min). Amplicons of the expected size (108 bp) were obtained. Methicillin resistance was confirmed using a cefoxitin disk (30 μg) on Mueller-Hinton agar plates (Bio-Rad, France) as recommended by the French Society of Microbiology and by the European Committee on Antimicrobial Susceptibility Testing. The mecA gene was detected by PCR. PCR conditions were the following : an initial temperature of 95°C (1min), denaturation at 95°C (15s), annealing at 45°C (15 s), and elongation at 72°C (30s), with a final extension at 72°C (4min). Amplicons of the expected size (162 bp) were obtained [7]. PCR primers were chosen as listed in Table 1.

Amplified PCR products were analyzed on 2% (wt/v) agarose gel stained with ethidium bromide (0.5 μg ml^-1), visualized under ultraviolet transillumination and photographed using Gel Doc XR apparatus (Biorad, USA).

The ability to produce hydrolytic enzymes was determined after inoculation of cultures on TSA-1 media (Biorad) supplemented with: 1% (wt/vol) skim milk for caseinase; 1% (wt/vol) gelatin for gelatinase; Tween 80 for lipase and 5% (vol/vol) egg yolk for lecithinase. The presence of a clear halo around the colonies indicates the presence of the hydrolytic enzyme. The haemolytic activity was evaluated on bacteriological agar supplemented with 5% sheep’s blood [8].

Phenotypic qualitative characterization of slime producing strains was carried out by the cultivation of the isolates on Congo red agar (CRA) plate made by mixing 36 g of sucrose (Sigma Chemical Company, St. Louis, MO) with 0.8 Congo red in 1L of brain heart infusion agar (Biorad, USA) as previously described [9]. The strains were incubated at 37°C for 24 hours under aerobic conditions followed by subsequent storage at room temperature. The Congo red dye interacts directly with certain bacterial polysaccharides forming a slime and giving black colonies in contrast to the non-producing colonies which remain red. After 48-72 hours, the results were interpreted as follows: strains producing intensive black, black, and reddish black colonies with a rough, dry, and crystalline consistency were classified as slime producers, whereas red and Bordeaux red with smooth colonies were considered to be non slime producers [9].

Biofilm production by MRSA strains, grown in Brain Heart Infusion with 1% glucose, was quantified using a semi-quantitative adherence assay on 96-well tissue culture plates as described previously [10]. Adherent bacteria were fixed with 95% ethanol and stained with 100 mL of 1% crystal violet (Merck, France) for 5 min. The microplates were air-dried and the optical density of each well was measured at 570 nm (OD570) using an automated Multiskan reader (GIO. DE VITA E C, ome, Italy). This assay was performed for each strain in triplicate. The background was determined by using noninoculated media as a control. The cut-off value (ODC) of noninoculated media at an optical density of 570 nm (OD570) was considered the deadline point to define biofilm quantities [cut-off (ODC) = mean OD + 3 standard deviation (SD) of negative control]. Biofilm formation was interpreted as highly positive (OD570> = 4 * ODC), moderately positive (2 * ODC <= OD570 <4 * ODC), weakly positive (ODC <= OD570 <2 * ODC) or negative (OD570 < ODC). These criteria were established by Stepanovic et al. [11].

PCR primers were chosen as listed in Table 1. Amplifications of icaA, icaD, fnbA, fnbB, cna, sspA, sspB and geh genes were performed according to the following cycle conditions: an initial denaturation at 94°C for 5 min was followed by 30 cycles of denaturation at 94°C for 30s, annealing for 30s at temperature determined for each gene as described in Table 1 and elongation at 72°C for 45s, followed by 10 min of final extension at 72°C.

The data from this study were captured, recorded and analyzed by SPSS 17.0 software. The non-parametric Mann Whitney U test was used to compare biofilm production assays (Congo red test and polystyrene adherence assay). The same statistical test was used to evaluate correlations of MSCRAMMs genes (fnbA, fnbB and cna), biofilm production control genes (icaA and icaD) and exoenzymes genes (sspA, sspB and geh) to the level of biofilm production on polystyrene. All factors with p values of less than 0.20 were included in multivariate ordinal logistic regression analysis. P values of less than 0.05 were considered to indicate significant difference.

A total of 43 MRSA strains were collected (i.e. a rate of 21.4% of all S. aureus isolates in the laboratory of Microbiology at the University Hospital of Monastir-Tunisia during the same period). Samples were gathered from different organs and systems: pus (58.1%), blood (25.6%), respiratory samples (11.6%) and catheters (4.7%). They were collected from patients admitted in surgery units (44.2%), intensive care units (25.6%), medical units (13.9%), Pediatrics/Neonatology (9.3%) and Gynecology (7.0%).

Our results showed that 51.1% and 48.9% of isolates were respectively beta and alpha-hemolytic. All isolates were protease producers (caseinase and gelatinase). Lipase and lecithinase secretion were found in 93.0% and 79.1% of cases respectively (Table 2).

Out of 43 MRSA isolates, 30 strains (69.8%) were slime producers on the CRA plate. They were black or reddish black color colony producers. Slime production was noted in 16 out of 25 strains isolated from suppurations and in 9 out of 11 blood culture isolates (Table 3).

The results of OD570 presented in Table 3, showed that 46.5% and 53.5% strains were respectively highly and moderately biofilm-formers on polystyrene. No isolate was weakly or non-biofilm producer on polystyrene. We detected 5/11 blood culture isolates and 10/25 pus isolates considered as highly biofilm formers. Both reference strains S. aureus ATCC 6538 and ATCC 43300 were highly biofilm-forming on polystyrene.

The icaA and the icaD genes were present in 69.8% and 65.1% of MRSA isolates respectively. A concomitant presence of both genes was detected in 55.8% of the strains. Our results revealed that 74.4% and 18.6% of isolates respectively harbored the fnbA and fnbB genes. The cna gene coding for adhesin to collagen was detected in 32 strains (74.4%). Among the tested strains, 15 (34.9%), 8 (18.6%) and 13 (30.2%) were positive for sspA, sspB and geh genes.