Phenotypic and molecular detection of biofilm formation in clinical methicillin-resistant Staphylococcus aureus isolates from Malaysia

Figures & data

Table 1. Slime phenotypes on Congo red agar.

			Incubation time
Characteristic	Score*	Representative isolate	24 h	48 h	MRSA (n = 89)
Positive slime producer	++	Staphylococcus epidermidis ATCC 35984 (Positive control)			9
Intermediate slime producer	+	MRSA-R46			71
		MRSA-R51
Negative slime producer	-	Staphylococcus hominis ATCC 35982 (Negative control)			9

*The scoring was adapted from [9].

Table 2. Association between patient’s characteristics, slime production and biofilm formation in MRSA isolates.

		Slime producers (CRA assay)				Biofilm producers (CV assay)
Characteristics		Positive (9) n(%)	Intermediate (71) n(%)	Negative (9) n(%)	p-value	Strong (51) n(%)	Intermediate (30) n(%)	Weak (8) n(%)	p-value
Gender	Female	3 (33.3)	33 (46.5)	2 (22.2)	0.3409	21 (41.2)	16 (53.3)	1 (12.5)	0.1178
	Male	6 (66.7)	38 (53.5)	7 (7.7)	0.3409	30 (58.8)	14 (46.7)	7 (87.5)	0.1178
Age group	0–20	2 (22.2)	14 (19.7)	2 (22.2)	0.9990	12 (23.5)	3 (10.0)	3 (37.5)	0.1280
	21–40	2 (22.2)	19 (26.8)	1 (11.1)	0.6750	9 (17.6)	10 (33.3)	3 (37.5)	0.1700
	41–60	2 (22.2)	24 (33.8)	4 (44.4)	0.6040	19 (37.3)	9 (30.0)	2 (25.0)	0.7430
	61–80	2 (22.2)	13 (18.3)	2 (22.2)	0.7940	10 (19.6)	7 (23.3)	0	0.4302
	>81	1 (11.1)	1 (1.4)	0	0.3360	1 (2.0)	1 (3.33)	0	0.9990
Isolation source	Pus	5 (55.5)	45 (63.4)	5 (55.5)	0.7848	31 (60.8)	16 (53.3)	8 (100.0)	0.0449*
	Blood	4 (44.4)	19 (26.8)	4 (44.4)	0.2772	17 (33.3)	10 (33.3)	0	0.1685
	Respiratory	0	5 (7.0)	0	1.0000	2 (3.9)	3 (10.0)	0	0.6011
	Eye	0	2 (2.8)	0	1.0000	1 (2.0)	1 (3.3)	0	1.0000
Clinical manifestations	SSTI	5 (55.5)	31 (43.7)	3 (33.3)	0.6335	21 (41.2)	14 (46.7)	4 (50.0)	0.8086
	Bacteremia	2 (22.2)	11 (15.5)	1 (11.1)	0.7554	7 (13.7)	5 (16.7)	2 (25.0)	0.6814
	Infective endocarditis	0	2 (2.8)	0	0.9999	1 (2.0)	1 (3.33)	0	0.9999
	Internal organ abscess	0	3 (4.2)	0	0.9999	2 (3.9)	1 (3.33)	0	1.0000
	Osteoarticular	0	2 (2.8)	1 (11.1)	0.2997	2 (3.9)	0	1 (12.5)	0.1766
	Prosthetic device	1 (11.1)	4 (5.6)	1 (11.1)	0.3502	3 (5.9)	3 (10.0)	0	0.8129
	Pleuropulmonary	1 (11.1)	6 (8.5)	0	0.6199	4 (7.8)	3 (10.0)	0	0.8516
	Others	0	12 (16.9)	3 (33.3)	0.1463	11 (21.6)	3 (10.0)	1 (12.5)	0.4338

CRA, Congo red agar

CV, Crystal violet

SSTI, skin and soft tissue infections; Others; epidural abscess, meningitis, toxic shock syndrome, urinary tract infection.

*p-value <0.05.

Figure 1. Specific biofilm formation index of all MRSA isolates.

Figure 2. Representation of MSCRAMM genes amplified products on 1% (w/v) agarose gel electrophoresis. Lane L: 100 bp plus molecular weight marker; lane 1: Triplex 1 – cna (423 bp), eno (302 bp), ebps (186 bp); lane 2: Triplex 2 – sasC (936 bp), icaAD (520 bp), icaBC (182 bp); lane 3: Triplex 3 – fib (404 bp), clfA (292 bp), sasG (159 bp); lane 4: fnbA (643 bp); lane 5: fnbB (524 bp); lane 6: clfB (205 bp).

Table 3. Association between slime production, biofilm formation, MSCRAMM genes distribution and accessory gene regulator in MRSA isolates.

			Slime production				Biofilm formation
Criteria	Gene	Total	Positive (9) n(%)	Intermediate (71) n(%)	Negative (9) n(%)	p-value	Strong (51) n(%)	Intermediate (30) n(%)	Weak (8) n(%)	p-value
MSCRAMM genes	icaAD	70 (78.7)	9 (100.0)	55 (77.5)	6 (66.7)	0.1643	41 (80.4)	24 (80.0)	5 (62.5)	0.5301
	icaBC	82 (92.1)	9 (100.0)	66 (93.0)	7 (77.8)	0.2798	50 (98.0)	26 (86.7)	6 (75.0)	0.026*
	eno	71 (79.8)	8 (88.9)	55 (77.5)	8 (88.9)	0.7016	41 (80.4)	25 (83.3)	5 (62.5)	0.4511
	clfA	71 (79.8)	7 (77.8)	56 (78.9)	8 (88.9)	0.8971	40 (78.4)	25 (83.3)	6 (75.0)	0.7867
	clfB	68 (76.4)	5 (55.6)	58 (81.7)	5 (55.6)	0.0398*	41 (80.4)	22 (73.3)	5 (62.5)	0.4902
	sasG	79 (88.8)	7 (77.8)	64 (90.1)	8 (88.9)	0.4761	46 (90.2)	27 (90.0)	6 (75.0)	0.3386
	fib	22 (24.7)	5 (55.6)	13 (18.3)	4 (44.4)	0.0176*	15 (29.4)	6 (20.0)	1 (12.5)	0.4914
	cna	38 (42.7)	4 (44.4)	29 (40.8)	5 (55.6)	0.7384	15 (29.4)	19 (63.3)	4 (50.0)	0.0088*
	ebps	21 (23.6)	5 (55.6)	14 (19.7)	2 (22.2)	0.0725	12 (23.5)	6 (20.0)	3 (37.5)	0.5626
	sasC	15 (16.9)	4 (44.4)	8 (11.3)	3 (33.3)	0.0184*	8 (15.7)	6 (20.0)	1 (12.5)	0.9091
	fnbA	8 (9.0)	0	8 (11.3)	0	0.4968	3 (5.9)	3 (10.0)	2 (25.0)	0.1662
	fnbB	1 (1.1)	0	1 (1.4)	0	1.000	1 (2.0)	0	0	1.000
	bbp	0	0	0	0	0	0	0	0	0
	bap	0	0	0	0	0	0	0	0	0
	pls	0	0	0	0	0	0	0	0	0
Accessory regulator gene (agr)	agr-I	61 (68.5)	2 (22.2)	54 (76.1)	5 (5.6)	0.0028*	33 (64.7)	24 (80.0)	4 (50.0)	0.1861
	agr-II	8 (9.0)	2 (22.2)	5 (7.0)	1 (11.1)	0.2001	6 (11.8)	2 (6.7)	0	0.6343
	agr-III	7 (7.9)	3 (33.3)	3 (8.5)	1 (11.1)	0.0179*	5 (9.8)	1 (3.3)	1 (12.5)	0.3972
	agr-IV	0	0	0	0	0	0	0	0	0
	Combination	3 (3.4)	0	3 (4.2)	0	1.000	2 (3.9)	1 (3.3)	0	1.000
	Non-typable	10 (11.2)	2 (22.2)	6 (8.4)	2 (22.2)	0.1560	5 (9.8)	2 (6.7)	3 (37.5)	0.0731

* denote statistically significant (p < 0.05).

Osman KM, Amer AM, Badr JM, et al. Antimicrobial resistance, biofilm formation and mecA characterization of methicillin-susceptible S. aureus and non-S. aureus of beef meat origin in Egypt. Front Microbiol. 2016;7:222. doi:10.3389/fmicb.2016.00222.

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