Genotypic and Phenotypic-Based Assessment of Antibiotic Resistance and Profile of Staphylococcal Cassette Chromosome mec in the Methicillin-Resistant Staphylococcus aureus Recovered from Raw Milk

Figures & data

Table 1 Target Genes, Oligonucleotide Primers and PCR Conditions Used for Detection of Antibiotic Resistance Genes and SCCmec Types Amongst MRSA Bacteria Recovered from Raw Milk

Target Gene	Primer Sequence (5ʹ-3ʹ)	PCR Product (bp)	PCR Programs	PCR Volume (50 µL)
aacA-D	F: TAATCCAAGAGCAATAAGGGC R: GCCACACTATCATAACCACTA	227	1 cycle: 94°C ———— 5 min 25 cycles: 94°C ———— 60 s 55°C ———— 70 s 72°C———— 60 s 1 cycle: 72°C ———— 10 min	5 µL PCR buffer 10X 1.5 mM MgCl2 200 µm dNTP (Fermentas) 0.5 µm of each primers F & R 1.25 U Taq DNA polymerase (Fermentas) 2.5 µL DNA template
ermA	F: AAGCGGTAAACCCCTCTGA R: TTCGCAAATCCCTTCTCAAC	190
tetK	F: GTAGCGACAATAGGTAATAGT R: GTAGTGACAATAAACCTCCTA	360
ermB	F: CCGTTTACGAAATTGGAACAGGTAAAGGGC R: GAATCGAGACTTGAGTGTGC	359
mefA	F: ACTATCATTAATCACTAGTGC R: TTCTTCTGGTACTAAAAGTGG	346
grlA	F: ACTTGAAGATGTTTTAGGTGAT R: TTAGGAAATCTTGATGGCAA	618
tetM	F: AGTGGAGCGATTACAGAA R: CATATGTCCTGGCGTGTCTA	158	1 cycle: 94°C ———— 6 min. 34 cycles: 95°C ———— 50 s 55°C ———— 70 s 72°C ———— 60 s 1 cycle: 72°C ———— 8 min	5 µL PCR buffer 10X 2 mM MgCl2 200 µM dNTP (Fermentas) 0.5 µM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 5 µL DNA template
gyrA	F: AGTACATCGTCGTATACTATATGG R: ATCACGTAACAGTTCAAGTGTG	280
msrA	F: GGCACAATAAGAGTGTTTAAAGG R: AAGTTATATCATGAATAGATTGTCCTGTT	940	1 cycle: 94°C ———— 6 min. 34 cycles: 95°C ———— 60 s 50°C ———— 70 s 72°C ———— 70 s 1 cycle: 72°C ———— 8 min	5 µL PCR buffer 10X 2 mM MgCl2 150 µm dNTP (Fermentas) 0.75 µm of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 µL DNA template
msrB	F: TATGATATCCATAATAATTATCCAATC R: AAGTTATATCATGAATAGATTGTCCTGTT	595
dfrA1	F: CTCACGATAAACAAAGAGTCA R: CAATCATTGCTTCGTATAACG	201
linA	F: GGTGGCTGGGGGGTAGATGTATTAACTGG R: GCTTCTTTTGAAATACATGGTATTTTTCGA	323	1 cycle: 94°C ———— 6 min. 30 cycles: 95°C ———— 60 s 57°C ———— 60 s 72°C ———— 60 s 1 cycle: 72°C ———— 10 min	5 µL PCR buffer 10X 2 mM MgCl2 150 µm dNTP (Fermentas) 0.75 µm of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 µL DNA template
blaZ	F: TGAACCGTATGTTAGTGC R: GTCGTGTTAGCGTTGATA	681	1 cycle: 94°C ———— 6 min 30 cycles: 95°C ———— 60 s 59°C ———— 60 s 72°C ———— 60 s 1 cycle: 72°C ———— 10 min	5 µL PCR buffer 10X 2 mM MgCl2 150 µm dNTP (Fermentas) 0.75 µm of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 µL DNA template
cfr	F: TGAAGTATAAAGCAGGTTGGGAGTCA R: ACCATATAATTGACCACAAGCAGC	746	1 cycle: 94°C ———— 1 min. 34 cycles: 94°C ———— 2 min 48°C ———— 60 s 72°C ———— 3 min 1 cycle: 72°C———— 10 min	5 µL PCR buffer 10X 2 mm MgCl2 150 µm dNTP (Fermentas) 0.75 µm of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 µL DNA template
rpoB	F: ACCGTCGTTTACGTTCTGTA R: TCAGTGATAGCATGTGTATC	460	1 cycle: 94°C ———— 5 min 40 cycles: 94°C ———— 40 s 45.5°C ———— 40 s 72°C ———— 90 s 1 cycle: 72°C ———— 8 min	5 µL PCR buffer 10X 2 mM MgCl2 150 µm dNTP (Fermentas) 0.75 µm of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 µL DNA template
SCCmecI	F: GCTTTAAAGAGTGTCGTTACAGG R: GTTCTCTCATAGTATGACGTCC	613	1 cycle: 94°C ———— 5 min 35 cycles: 94°C ———— 45 s 65°C ———— 45 s 72°C ———— 90 s 1 cycle: 72°C ———— 10 min	5 µL PCR buffer 10X 2 mM MgCl2 150 µm dNTP (Fermentas) 0.75 µm of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 µL DNA template
SCCmecII	F: CGTTGAAGATGATGAAGCG R: CGAAATCAATGGTTAATGGACC	398
SCCmecIII	F: CCATATTGTGTACGATGCG R: CCTTAGTTGTCGTAACAGATCG	280
SCCmecIva	F: GCCTTATTCGAAGAAACCG R: CTACTCTTCTGAAAAGCGTCG	776
SCCmecIVb	F: TCTGGAATTACTTCAGCTGC R: AAACAATATTGCTCTCCCTC	493
SCCmecIVc	F: ACAATATTTGTATTATCGGAGAGC R: TTGGTATGAGGTATTGCTGG	200
SCCmecIVd	F: CTCAAAATACGGACCCCAATACA R: TGCTCCAGTAATTGCTAAAG	881
SCCmecV	F: GAACATTGTTACTTAAATGAGCG R: TGAAAGTTGTACCCTTGACACC	325

Note: Fermentas is part of Thermo Fisher Scientific, Waltham, MA, USA.

Table 2 Total Prevalence of S. aureus and MRSA Bacteria in Different Types of Raw Milk

Types of Samples		Samples Collected n	S. aureus Positive Samples n (%)	MRSA Positive Samples n (%)
Raw milk	Bovine	130	11 (8.46)	8 (72.723)
	Ovine	120	9 (7.50)	7 (77.77)
	Caprine	120	6 (5)	4 (66.66)
	Camel	110	3 (2.72)	1 (33.33)
	Buffalo	110	10 (9.09)	8 (80)
	Total	590	39 (6.61)	28 (71.79)

Table 3 Antibiotic Resistance Pattern of the MRSA Bacteria Recovered from Raw Milk Samples

Type of Raw Milk Samples (N of MRSA Bacteria)	Isolates Resistant to Each Antibiotic n (%)
	Penicillins	Aminoglycosides		Macrolides		Tetracyclines		Fluoroquinolones		Lincosamides	Folate Inhibitors	Phenicols	Ansamycins
P10	Gen	Amk	Azi	Ert	Tet	Dox	Cip	Lev	Clin	Tr-Sul	C30	Rif
Bovine (8)	8 (100)	7 (87.50)	2 (25)	3 (37.50)	7 (87.50)	8 (100)	6 (75)	3 (37.50)	2 (25)	3 (37.50)	8 (100)	3 (37.50)	1 (12.50)
Ovine (7)	7 (100)	5 (71.42)	1 (14.28)	2 (28.57)	5 (71.42)	7 (100)	4 (57.14)	2 (28.57)	2 (28.57)	2 (28.57)	5 (71.42)	2 (28.57)	–
Caprine (4)	4 (100)	3 (75)	–	1 (25)	3 (75)	4 (100)	3 (75)	1 (25)	1 (25)	1 (25)	3 (75)	1 (25)	1 (25)
Camel (1)	1 (100)	–	–	–	–	1 (100)	–	–	–	–	–	–	–
Buffalo (8)	8 (100)	7 (87.50)	2 (25)	3 (37.50)	8 (100)	8 (100)	7 (87.50)	4 (50)	4 (50)	4 (50)	6 (75)	2 (25)	2 (25)
Total (28)	28 (100)	22 (78.57)	5 (17.85)	9 (32.14)	23 (82.14)	28 (100)	20 (71.42)	10 (35.71)	9 (32.14)	12 (42.85)	22 (78.57)	8 (28.57)	4 (14.28)

Abbreviations: P10, penicillin (10 µg/disk); Gen, gentamicin (10 µg/disk); Amk, amikacin (30 µg/disk); Azi, azithromycin (15 µg/disk); Ert, erythromycin (15 µg/disk); Tet, tetracycline (30 µg/disk); Do, doxycycline (30 µg/disk); Cip, ciprofloxacin (5 µg/disk); Lev, levofloxacin (5 µg/disk); Clin, clindamycin (2 µg/disk); Tr-Sul, trimethoprim-sulfamethoxazole (25 µg/disk); C30, chloramphenicol (30 µg/disk); Rif, rifampin (5 µg/disk).

Figure 1 Distribution of multidrug resistant MRSA bacteria recovered from different types of raw milk. Multidrug resistant MRSA bacteria were determined as those who had at least simultaneous resistance toward three or more than three types of antibiotics.

Table 4 Distribution of Antibiotic Resistance Genes Amongst the MRSA Bacteria Recovered from Raw Milk

Type of Raw Milk Samples (N of MRSA Bacteria)	Isolates Harbor Each Gene n (%)
	Penicillins	Aminoglycosides	Macrolides					Tetracyclines		Fluoroquinolones		Lincosamides	Folate Inhibitors	Phenicols	Ansamycins
	blaZ	aacA-D	ermA	ermB	msrA	msrB	mefA	tetK	tetM	gyrA	grlA	linA	dfrA1	cfr	rpoB
Bovine (8)	8 (100)	6 (75)	4 (50)	2 (25)	3 (37.50)	1 (12.50)	3 (37.50)	7 (87.50)	3 (37.50)	5 (62.50)	3 (37.50)	2 (25)	7 (87.50)	2 (25)	1 (12.50)
Ovine (7)	7 (100)	5 (71.42)	3 (42.85)	2 (28.57)	3 (42.85)	1 (14.28)	1 (14.28)	6 (85.71)	2 (28.57)	2 (28.57)	1 (14.28)	2 (28.57)	5 (71.42)	2 (28.57)	–
Caprine (4)	4 (100)	2 (50)	2 (50)	1 (25)	1 (25)	–	2 (50)	3 (75)	1 (25)	1 (25)	1 (25)	1 (25)	2 (50)	1 (25)	1 (25)
Camel (1)	1 (100)	–	–	–	–	–		1 (100)	–	–	–	–	–	–	–
Buffalo (8)	8 (100)	6 (75)	5 (62.50)	2 (25)	3 (37.50)	1 (12.50)	4 (50)	7 (87.50)	4 (50)	4 (50)	3 (37.50)	3 (37.50)	6 (75)	2 (25)	1 (12.50)
Total (28)	28 (100)	19 (67.85)	14 (50)	7 (25)	10 (35.71)	3 (10.71)	10 (35.71)	24 (85.71)	10 (35.71)	12 (42.85)	8 (28.57)	8 (28.57)	20 (71.42)	7 (25)	3 (10.71)

Table 5 Distribution of SCCmec Types Amongst the MRSA Bacteria Recovered from Raw Milk

Type of Raw Milk Samples (N of MRSA Bacteria)	Isolates Harbor Each SCCmec Type n (%)
	I	II	III	IV				V
				a	b	c	d
Bovine (8)	1 (12.50)	–	2 (25)	3 (37.50)	1 (12.50)	–	–	1 (12.50)
Ovine (7)	–	–	1 (14.28)	1 (14.28)	1 (14.28)	1 (14.28)	1 (14.28)	2 (28.57)
Caprine (4)	–	–	–	1 (25)	1 (25)	1 (25)	–	1 (25)
Camel (1)	–	1 (100)	–	–	–	–	–
Buffalo (8)	1 (12.50)	–	1 (12.50)	3 (37.50)	–	–	–	3 (37.50)
Total (28)	2 (7.40)	1 (3.70)	4 (14.81)	8 (29.62)	3 (11.11)	2 (7.40)	1 (3.70)	7 (25)