Molecular characterization of Staphylococcus aureus strains in bovine mastitis milk in Bangladesh

Peer review under responsibility of Faculty of Veterinary Medicine, Cairo University.

Figures & data

Table 1 Herd and cow-level prevalence of mastitis (clinical and subclinical) in some selected areas of Bangladesh.

Observation	Overall mastitis				Clinical mastitis No. (%)	Subclinical mastitis No. (%)
	No. examined	No. positive	Prevalence (%)	95% Cl
Herd level	45	33	73.3	62.4–73.6	14 (31.1)	19 (42.2)
Cow level	175	50	28.6	23.5–36.2	19 (38.0)	31 (62.0)
Quarter level	678	200	29.5	26.1–34.3	80 (40.0)	120 (60.0)

Table 2 Herd and cow-level prevalence of Staphylococcus aureus mastitis (clinical and subclinical) in some selected areas of Bangladesh.

Observation	Overall mastitis caused by Staphylococcus aureus				Clinical mastitis No. (%)	Subclinical mastitis No. (%)
	No. examined	No. positive	Prevalence (%)	95% Cl
Herd level	33	24	72.7	60.1–71.2	15 (62.5)	9 (37.5)
Cow level	50	37	74.0	52.1–66.5	22 (59.5)	15 (40.5)
Quarter level	200	124	62.0	31.4–44.3	59 (73.8%)	65 (54.1)

Table 3 Odds ratios (OR) and confidence intervals (CI) of factors having significant effect on Staphylococcus aureus mastitis in dairy cows (Logistic regression model).

Independent variables	Categories	Incidence of clinical mastitis			Incidence of subclinical mastitis
		OR	95% CI	P-value (χ2)	OR	95% CI	P-value (χ2)
Breeds
	Local zebu	0.432^a	48.0, 72.1	0.001	0.501^a	0.96, 14.2	0.005
	Crossbred Friesian	1.000^b			1.000^b
	Shahiwal cross	0.561^a	22.7, 74.0		0.451^a	24.0, 45.5
	Red Chittagong	0.301^a	33.2, 88.5		0.506^a	10.1, 50.0
Parity			48.1, 67.9
	1 to 2	0.103^a	19.0, 63.1	0.005	0.433^a	10.2, 38.2	0.007
	3 to 4	0.232^a	34.2, 46.3		0.502^a	34.1, 48.0
	≥ 4	1.003^b			1.003^b
Body Condition Score (BCS)
	≤2.5	0.782^a	1.5, 22.3	0.803	No significant effect
	3.0 to 3.5	0.912^a,b	23.2, 56.3
	>3.5	1.000^b
Farm size
	5 to 10	0.782^a	2.4, 33.1
	11 to 15	0.912^a,b	17.2, 41.4	0.726	No significant effect
	16 to 20	1.000^b
	>21	1.421^c	1.8, 29.2
Milk Yield (L/day)
	2 to 5	0.883^a	23.0, 48.2	0.001	0.851^a	17.0, 52.3	0.003
	6 to 10	1.000^a			1.000^a
	11 to 15	1.401^a,b	7.2, 50.5		1.331^a,b	20.5, 63.1
	>15	1.315^b	1.7, 32.5		1.220^b	17.2, 54.0
Regular practice of teat dipping before and after milking (with antiseptic solutions)
	Yes	0.609^a	44.0, 73.1	0.001	0.203^a	2.8, 31.2	0.005
	No	1.000^b			0.902^b	6.5, 41.7
Dry cow housing
	Free-stall	0.738^a	39.0, 72.1	0.002	0.013^a	23.0, 70.0	0.050
	In tie-stall barns	1.001^b			0.701^b

The OR describes the risk for developing both clinical and subclinical mastitis caused by Staphylococcus aureus. ^a,b,cORs with different letter superscripts are statistically significantly different.

Fig. 1 Prevalence of Staphylococcus aureus mastitis (subclinical and clinical) in the study areas (P < 0.05). Values (a & b) differ significantly within the study area according to category of mastitis.

Fig. 2 Quarter-wise prevalence of Staphylococcus aureus mastitis (subclinical and clinical) in the study areas (P < 0.05). Values (a & b) within different quarters differ significantly according to category of mastitis.

Table 4 Primer sequences, anticipated product size, and sets of multiplex PCR used in this study for identification of Staphylococcus aureus genes.

Genes^*	Forward primers (5′-3′)	Reverse primers (5′-3′)	Product size (bp)	PCR set
nuc	GCGATTGATGGTGATACGGTT	AGCCAAGCCTTGACGAACTAAAGC	279	A
sea	GGTTATCAATGTGCGGGTGG	CGGCACTTTTTTCTCTTCGG	102	A
seb	GTATGGTGGTGTAACTGAGC	CCAAATAGTGACGAGTTAGG	164	A
sec	AGATGAAGTAGTTGATGTGTATGG	CACACTTTTAGAATCAACCG	491	A
sed	CCAATAATAGGAGAAAATAAAAG	ATTGGTATTTTTTTTCGTTC	495	A
see	AGGTTTTTTCACAGGTCATCC	CTTTTTTTTCTTCGGTCAATC	430	A
mecA	GTAGAAATGACTGAACGTCCGATAA	CCAATTCCACATTGTTTCGGTCTAA	163	B
pvl	ATCATTAGGTAAAATGTCTGGACATGATCCA	GCATCAAGTGTATTGGATAGCAAAAGC	433	B
tsst1	ACCCCTGTTCCCTTATCATC	TTTTCAGTATTTGTAACGCC	326	B
eta	ATATCAACGTGAGGGCTCTAGTAC	ATGCAGTCAGCTTCTTACTGCTA	93	B
etb	CACACATTACGGATAATGCAAG	TCAACCGAATAGAGTGAACTTATCT	226	B

* Nucleotide primers were selected from previously published sequence [12,22,46].

Table 5 The overall antimicrobial susceptibility patterns of the Staphylococcus aureus isolates (n = 145) from bovine mastitis (subclinical and clinical mastitis) milk samples.

Antimicrobials	Resistant breakpoint^a	No. of resistant isolates (%) from
		Subclinical mastitis milk (n = 85)	Clinical mastitis milk (n = 60)	Total isolates (n = 145)
Penicillin	≥0.25	11 (12.9)	5 (8.3)	16 (11.0)
Erythromycin	≥8	5 (5.0)	7 (11.7)	12 (8.2)
Oxytetracycline	≥16	63 (74.1)	45 (75.0)	108 (74.5)
Trimethoprim/sulfamethoxazole	≥8/152	29 (34.1)	15 (25.0)	44 (30.3)
Ciprofloxacin	≥4	43 (50.6)	29 (48.3)	72 (49.6)
Gentamicin	≥8	12 (14.1)	14 (23.3)	26 (17.9)
Amoxicillin	≥0.5	33 (38.8)	28 (33.3)	61 (42.0)
Oxacillin	≥4	47 (55.2)	34 (56.7)	81 (55.9)
Ceftriaxone	≥ 2	0 (0.0)	0 (0.0)	0 (0.0)
Azithromycin	≥8	0 (0.0)	0 (0.0)	0 (0.0)

a MICs (µg/mL) determined via disk diffusion and agar dilution methods. The number in the parenthesis indicates the percentage of resistant isolates.

Table 6 Multidrug resistance (MDR) properties observed among 145 Staphylococcus aureus isolates in bovine mastitis (subclinical and clinical mastitis) milk samples.

Milk samples (No. of isolates)	No. (%) resistant to indicated number of antimicrobials
	1	2	3	4	5	Total resistance (≥1)
Samples from subclinical cases (n = 85)	5 (5.9)	17 (20.0)	11 (12.9)	20 (23.5)	7 (8.2)	60 (70.6)
Samples from clinical cases (n = 60)	9 (15.0)	13 (21.7)	18 (30.0)	15 (25.0)	0 (0.0)	55 (91.7)
Total samples (n = 145)	14 (9.7)	30 (20.7)	29 (20.0)	35 (24.1)	7 (4.8)	115 (79.3)

Table 7 mecA and other toxin gene profile found in Staphylococcus aureus isolates in bovine mastitis (subclinical and clinical) milk samples.

Toxin gene profiles	Number (%) of Staphylococcus aureus isolates
	Subclinical mastitis milk (n = 85)	Clinical mastitis milk (n = 60)	Total (n = 145)
mecA	16 (18.8)	13 (21.7)	29 (20.0)
pvl	5 (5.9)	1 (1.7)	6 (4.1)
sea	7 (8.2)	3 (5.0)	10 (6.9)
seb	3 (3.5)	1 (1.7)	4 (2.8)
sed	7 (8.2)	1 (1.7)	8 (5.5)
see	6 (7.0)	2 (3.3)	8 (5.5)
sea – pvl	11 (12.9)	0 (0.0)	11 (7.6)
seb – see	5 (5.9)	1 (1.7)	6 (4.1)
sec – pvl	9 (10.6)	4 (6.7)	13 (9.0)
sea-seb-pvl	3 (3.5)	2 (3.3)	5 (3.4)
seb-sec-sed-pvl	1 (1.1)	0 (0.0)	1 (0.7)
Total	73 (85.9)	28 (46.7)	101 (69.7)