Frequency of <i>Staphylococcus aureus</i> virulence genes in milk of cows and goats with mastitis

Acosta, Atzel C.; Oliveira, Pollyanne Raysa F.; Albuquerque, Laís; Silva, Isamara F.; Medeiros, Elizabeth S.; Costa, Mateus M.; Pinheiro Junior, José Wilton; Mota, Rinaldo A.

doi:10.1590/1678-5150-PVB-5786

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Livestock Diseases • Pesq. Vet. Bras. 38 (11) • Nov 2018 • https://doi.org/10.1590/1678-5150-PVB-5786 copy

Frequency of Staphylococcus aureus virulence genes in milk of cows and goats with mastitis

Frequência de genes fatores de virulência de Staphylococcus aureus em leite de vacas e cabras com mastite

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT:

The present study determined the frequency of Staphylococcus aureus virulence genes in 2,253 milk samples of cows (n=1000) and goats (n=1253) raised in three different geographical regions of the state Pernambuco, Brazil. The presence of genes of virulence factors associated to adhesion to host cells (fnbA, fnbB, clfA and clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla and hlb), and capsular polysaccharide (cap5 and cap8) was evaluated by PCR. A total of 123 and 27 S. aureus strains were isolated from cows’ and goats’ milk, respectively. The sec and tsst genes were detected exclusively in goats’ isolates, while the seh gene was only identified in cows’ isolates. The number of toxin genes per strain showed that goats’ isolates are likely more toxic than bovines’ isolates. The cap5 genotype predominated in both host species, especially in strains collected from cows raised in the Agreste region. The cap8 genotype is likely more virulent due to the number of virulence genes per strain. The results of the present study demonstrate that S. aureus may pose a potential threat to human health in Brazil, and, therefore, these results should support actions related to mastitis control programs.

INDEX TERMS:
Staphylococcus aureus; virulence genes; milk; cows; goats; mastitis; molecular typing; genotyping; staphylococcal enterotoxin; capsular polysaccharide; cattle; bacterioses

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[1] *Corresponding author: acabad80@gmail.com

Target gene	Name	Oligonucleotide sequence (5´- 3´)	Expected size	At (^oC)^a
nuc	nuc_F	GGTTCTGAAGATCCAACAGTAT	296pb	61
	nuc_R	GCTAAGCCACGTCCATATTTA
fnbA	fnbA _F	ACTTCACCTGTCGCCATTAC	539pb	57
	fnbA _R	GCAGTACAAGCACCACAAAC
fnbB	fnbB _F	AGGCGACGGCAAAGATAAA	317pb	60
	fnbB _R	TAGTAACCTGACCACCACCT
clfA ^b	clfA_F	GATTCTGACCCAGGTTCAGA	945pb	55
	clfA_R	CTGTATCTGGTAATGGTTCTTT
clfB ^b	clfB_F	ATGGTGATTCAGCAGTAAATCC	880pb	55
	clfB_R	CATTATTTGGTGGTGTAACTCTT
sea ^c	sea_F	GGTTATCAATGTGCGGGTGG	102pb	65
	sea_R	CGGCACTTTTTTCTCTTCGG
seb	seb_F	CCCGTTTCATAAGGCGAGTT	314pb	57
	seb_R	ACGTAGATGTGTTTGGAGCTAAT
sec ^c	sec_F	AGATGAAGTAGTTGATGTGTATGG	451pb	57
	sec_R	CACACTTTTAGAATCAACCG
sed	sed_F	GTCACTCCACACGAAGGTAATAA	255pb	55
	sed_R	GAGACTTTAGACCCATCAGAAGAA
see ^c	see_F	GCTGGAGGCACACCAAATA	301pb	57
	see_R	CATAACTTACCGTGGACCCTTC
seg	seg_F	GCCAGTGTCTTGCTTTGTAATC	491pb	56
	seg_R	GAATGCTCAACCCGATCCTAA
seh	seh_F	CACATCATATGCGAAAGCAGAAG	365pb	60
	seh_R	CCCAAACATTAGCACCAATCAC
sei	sei_F	AGGCAGTCCATCTCCTGTATAA	568pb	55
	sei_R	TGCTCAAGGTGATATTGGTGTAG
tsst ^c	tsst_F	ACCCCTGTTCCCTTATCATC	326pb	57
	tsst_R	TTTTCAGTATTTGTAACGCC
hla	hla_F	CTGTAGCGAAGTCTGGTGAAA	293pb	62
	hla_R	CGGCCTTATTGGTGCAAATG
hlb	hlb_F	GCCAAAGCCGAATCTAAGAAAG	495pb	60
	hlb_R	ATCATGTCCAGCACCACAA
cap5	cap5_F	CGAACCGATGATTGATGCTATTG	555pb	61
	cap5_R	TGCTATGACTGCACCAGTATTT
cap8	cap8_F	GGAGGAAATGACGATGAGGATAG	608pb	61
	cap8_R	TAGCTTCTGTAGCGGTGAATG

		Animal species		Disease status of the animal
		Bovine	Goat	Subclinical	Clinical
Regions of the herd	Atlantic Forest	36	7	34	9
	Agreste	52	5	57	0
	Sertão	35	15	49	1
Animal species	Bovine			120	3
Animal species	Goat			20	7

	Species					Regions
	Bovine (n=123)		Goat (n=27)		p	Atlantic Forest (n=43)		Agreste (n=57)		Sertão (n=50)		p
	AF^a	RF^b	AF	RF	p	AF	RF	AF	RF	AF	RF	p
Virulence factors involved in adhesion to host cells
fnbA	115	93.50	26	96.30	0.8400	38	88.37	54	94.74	49	98.00	0.1451
fnbB	112	91.06	10	37.04	0.0000	33	76.74	51	89.47	38	76.00	0.1357
clfA	96	78.05	18	66.67	0.3454	27	62.79 ^A*	49	85.96 ^B	38	76.00 ^AB	0.0267
clfB	93	75.61	22	81.48	0.4316	28	65.12 ^A	43	75.44 ^AB	44	88.00 ^B	0.0323
Virulence factors: toxins
sea	5	4.07	5	18.52	0.0062	6	13.95	3	5.26	1	2.00	0.0610
seb	1	0.81	0	0.00	0.6624	0	0.00	1	1.75	0	0.00	0.4452
sec	0	0.00	11	40.74	0.0000	3	6.98 ^AB	0	0.00 ^A	8	16.00 ^B	0.0061
sed	14	11.38	2	7.41	0.2574	4	9.30	7	12.28	5	10.00	0.8789
see	0	0.00	0	0.00		0	0.00	0	0.00	0	0.00
seg	23	18.70	15	55.56	0.0034	9	20.93	14	24.56	15	30.00	0.6013
seh	27	21.95	0	0.00	0.0240	2	4.65 ^A	12	21.05 ^B	13	26.00 ^B	0.0206
sei	10	8.13	10	37.04	0.0076	5	11.63	6	10.53	9	18.00	0.4923
tsst	0	0.00	8	29.63	0.0000	3	6.98	0	0.00	5	10.00	0.0611
hla	118	95.93	14	51.85	0.0000	39	90.70 ^AB	54	94.74 ^A	39	78.00 ^B	0.0233
hlb	115	93.50	13	48.15	0.0000	39	90.70 ^A	53	92.98 ^A	36	72.00 ^B	0.0042
Virulence factors to evade innate immune defenses
cap5	100	81.30	6	22.22	0.0000	30	69.77 ^A	50	87.72 ^B	26	52.00 ^C	0.0002
cap8	8	6.50	2	7.41	0.7048	1	2.33	5	8.77	4	8.00	0.4014

	Capsular genotyping
	cap - (n=34)		cap5 (n=106)		cap8 (n=10)		p
	AF^a	RF^b	AF	RF	AF	RF	p
Virulence factors involved in adhesion to host cells
fnbA	31	91.18	100	94.34	10	100.00	0.5706
fnbB	21	61.76 ^A*	94	88.68 ^B	7	70.00 ^AB	0.0012
clfA	16	47.06 ^A	89	83.96 ^B	9	90.00 ^B	0.0000
clfB	24	70.59	81	76.42	10	100.00	0.1556
Virulence factors: toxins
SEs	19	55.88 ^A	49	46.23 ^A	10	100.00 ^B	0.0005
sea	6	17.65 ^A	2	1.89 ^B	2	20.00 ^A	0.0011
seb	0	0.00 ^A	0	0.00 ^A	1	10.00 ^B	0.0007
sec	6	17.65 ^AB	3	2.83 ^A	2	20.00 ^B	0.0040
sed	3	8.82	12	11.32	1	10.00	0.9185
see	0	0.00	0	0.00	0	0.00
seg	10	29.41	24	22.64	4	40.00	0.4032
seh	0	0.00 ^A	21	19.81 ^A	6	60.00 ^B	0.0000
sei	7	20.59	11	10.38	2	20.00	0.2586
tsst	5	14.71 ^A	2	1.89 ^B	1	10.00 ^AB	0.0115
hla	20	58.82 ^A	102	96.23 ^B	10	100.00 ^B	0.0000
Hlb	19	55.88 ^A	100	94.34 ^B	9	90.00 ^B	0.0000