Antimicrobial resistance and virulence genes of Streptococcus agalactiae isolated from mastitis milk samples in China

Drug category	Antibacterial drugs
	penicillin
	ampicillin
β-lactams	amoxicillin / clavulanic acid
	oxacillin
	cefoxitin
Macrolides	erythromycin
Lincosamides	clindamycin
Aminoglycosides	gentamicin
Tetracyclines	doxycycline tetracycline
Chloramphenicols	florfenicol
Ansamycins	rifampicin
Glycopeptides	vancomycin
Quinolones	ciprofloxacin
Sulfonamides	sulfisoxazole
	sulfamethoxazole

Table 2

Primer sequence and reaction conditions of the drug resistance gene of S. agalactiae

Antimicrobial drug class	Resistance gene	Primer sequence (5′–3′)	Annealing temperature (°C)	PCR product size (bp)	Reference
		F: GGTGGCTGGGGGGTAGATGTATTAACTGG
Lincosamides	lnuA	R: GCTCTCTTTGAAATACATGGTATTTTTCGATC	56	323	(23)
		F: AACTTAGGCATTCTGGCTCAC
	tetO	R: TCCCACTGTTCCATATCGTCA	50	515	(29)
		F: TCGATAGGAACAGCAGTA
	tetK	R: CAGCAGATCCTACTCCTT	44	169	(33)
Tetracyclines
	tetM	F: GTGGACAAAGGTACAACGAG	50	406
		R: CGGTAAAGTTCGTCACACAC
					(39)
		F: CATAGACAAGCCGTTGACC
	tetS	R: ATGTTTTTGGAACGCCAGAG	48	667
	ermB	F: CGAGTGAAAAAGTACTCAACC	48	652
		R: AGTAACGGTACTTAAATTGTTTAC
		F: ATCTTTGAAATCGGCTCAGG
Macrolides	ermC	R: CAAACCCGTATTCCACGATT	47	295	(32)
		F: GTTCAAGAACAATCAATACAGAG
	ermA	R: GGATCAGGAAAAGGACATTTTAC	48	421

[i] F – forward; R – reverse

Table 3

Primer sequence and reaction conditions of the virulence gene of S. agalactiae (13, 22, 23)

Virulence factor	Virulence gene	Primer sequence (5′–3′)	Annealing temperature (°C)	PCR size product (bp)
		F: CATTGCGTAGTCACCTCCC
β-haemolysin/cytolysis	cylE	R: GGGTTTCCACAGTTGCTTGA	54	399
		F: TAACAGTTATGATACTTCACAGAC ⎕
αC protein	Bca	R: ACGACTTTCTTCCGTCCACTTAGG	51	535
		F: CCAAGACTTCAGCCACAAGG
C5a peptidase	scpB	R: CAATTCCAGCCAATAGCAGC	57	591
		F: ACCGTCTGAAATGATGTGG
Laminin binding protein	lmb	R: GATTGACGTTGTCTTCTGC	51	572
Glutamine synthetase	glnA	F: ACGTATGAACAGAGTTGGCTATAA	52	471
		R: TCCTCTGATAATTGCATTCCAC
CAMP factor	Cfb	F: ATGGGATTTGGGATAACTAAGCTAG	52	193
		R: AGCGTGTATTCCAGATTTCCTTAT
Hyaluronidase	hylB	F: ACAAATGGAACGACGTGACTAT	52	346
		R: CACCAATTGGCAGAGCCT
		F: AAGCAACTAGAAGAGGAAGC
βC protein	bac	R: TTCTGCTCTGGTGTTTTAGG	53	479
Bacterial immunogenic	bibA	F: AACCAGAAGCCAAGCCAGCAACC	58	127
adhesive		R: AGTGGACTTGCGGCTTCACCC
		F: CGGGATTGATCTAAGTCGCT
Invasion-associated gene	iagA	R: CCATCAACATCAGTCGCTAA	53	459
		F: AGAGCCAAGTAGGTCAACTTATAG
Fibrin binding protein B	fbsA	R: TTCATTGCGTCTCAAACCG	54	290

[i] F – forward; R – reverse; CAMP – Christie–Atkins–Munch-Peterson

Table 4a

Minimum inhibitory concentration (MIC) values of, and resistance of S. agalactiae isolates to, β-lactams, sulfonamides and tetracyclines

PEN	A/C	AMP	CET	OXA	SMZ	SXT	TE	DOX
SH07	≤0.125 (S³)	1/0.5 (R)	≤0.125 (S)	32 (R)	1 (S)	4 (S)	0.5/9.5 (S)	4 (S)	4 (S)
SH07-2	4 (R)	≤0.25/0.12 (S)	≤0.125 (S)	0.5 (S)	16 (R)	256 (I)	0.25/4.8 (S)	16 (R)	2 (S)
SH12	≤0.125 (S)	≤0.25/0.12 (S)	0.25 (I)	0.5 (S)	4 (R)	8 (S)	0.5/9.5 (S)	32 (R)	4 (S)
SH33	≤0.125 (S)	≤0.25/0.12 (S)	≤0.125 (S)	0.5 (S)	16 (R)	64 (S)	0.5/9.5 (S)	32 (R)	0.5 (S)
SH45	≤0.125 (S)	≤0.25/0.12 (S)	≤0.125 (S)	1 (S)	32 (R)	64 (S)	0.25/4.8 (S)	64 (R)	2 (S)
HLJ008	≤0.125 (S)	≤0.25/0.12 (S)	≤0.125 (S)	32 (R)	32 (R)	128 (S)	0.25/4.8 (S)	32 (R)	2 (S)
HLJ048-2	2 (R)	≤0.25/0.12 (S)	≤0.125 (S)	1 (S)	4 (R)	2 (S)	0.5/9.5 (S)	16 (R)	8 (I)
HLJ030-3	≤0.125 (S)	≤0.25/0.12 (S)	≤0.125 (S)	0.5 (S)	0.5 (S)	64 (S)	0.5/9.5 (S)	64 (R)	4 (S)
NM025	≤0.125 (S)	≤0.25/0.12 (S)	≤0.125 (S)	1 (S)	1 (S)	64 (S)	0.25/4.8 (S)	32 (R)	16 (R)
HB016	≤0.125 (S)	≤0.25/0.12 (S)	≤0.125 (S)	0.25 (S)	16 (R)	128 (S)	0.5/9.5 (S)	16 (R)	4 (S)
NM-2-72-4	8 (R)	≤0.25/0.12 (S)	2 (R)	1 (S)	16 (R)	128 (S)	0.25/4.8 (S)	32 (R)	32 (R)
NM-2-034-3	8 (R)	16/8 (R)	4 (R)	0.5 (S)	4 (R)	64 (S)	2/38 (I)	4 (S)	4 (S)
SH-2-46-1	≤0.125 (S)	≤0.25/0.12 (S)	≤0.125 (S)	0.5 (S)	32 (R)	128 (S)	0.25/4.8 (S)	16 (R)	4 (S)
SH-2-14-2	2 (R)	4/2 (R)	≤0.125 (S)	1 (S)	16 (R)	128 (S)	0.25/4.8 (S)	32 (R)	8 (I)
SD-2-009-2	≤0.125 (S)	16/8 (R)	≤0.125 (S)	0.5 (S)	4 (R)	128 (S)	0.25/4.8 (S)	8 (I)	4 (S)
Drug resistance rate (%) (/15)
S	66.67 (10)	73.33 (11)	80.00 (12)	86.67 (13)	20.00 (3)	93.33 (14)	93.33 (14)	13.33 (2)	73.33 (11)
I	0 (0)	0 (0)	6.67 (1)	0 (0)	0 (0)	6.67 (1)	6.67 (1)	6.67 (1)	13.33 (2)
R	33.33 (5)	26.67 (4)	13.33 (2)	13.33 (2)	80.00 (12)	0 (0)	0 (0)	80.00 (12)	13.33 (2)

[i] PEN – penicillin; A/C – amoxicillin/clavulanic acid; AMP – ampicillin; CET – cefoxitin; OXA – oxacillin; SMZ – sulfisoxazole; SXT – sulfamethoxazole; TE – tetracycline; DOX – doxycycline

Multiple drug resistance of *S. agalactiae* shown as antibiotic phenotypic resistance and resistance gene prevalence data. 3 – resistance to 3 classes of antimicrobials; 4 – resistance to 4 classes of antimicrobials; 5 – resistance to 5 classes of antimicrobials; 6 – resistance to 6 classes of antimicrobials; 7 – resistance to 7 classes of antimicrobials; 9 – resistance to 9 classes of antimicrobials

Table 4b

Minimum inhibitory concentration (MIC) values of, and resistance of S. agalactiae isolates to, a macrolide, a lincosamide, an aminoglycoside, a quinolone, a chloramphenicol, an ansamycin, and a glycopeptide

ERY	CLI	GM	CIP	FFC	RIF	VAN
SH07	16 (R)	0.25 (S)	4 (S)	0.5 (S)	2 (S)	0.5 (S)	0.5 (S)
SH07-2	16 (R)	4 (R)	4 (S)	0.5 (S)	16 (R)	0.125 (S)	0.5 (S)
SH12	32 (R)	8 (R)	16 (R)	0.5 (S)	4 (S)	0.5 (S)	0.5 (S)
SH33	16 (R)	8 (R)	16 (R)	0.5 (S)	8 (I)	0.5 (S)	0.5 (S)
SH45	64 (R)	8 (R)	2 (S)	2 (I)	8 (I)	0.5 (S)	0.5 (S)
HLJ008	16 (R)	4 (R)	4 (S)	0.5 (S)	4 (S)	0.5 (S)	0.5 (S)
HLJ048-2	16 (R)	16 (R)	8 (I)	0.5 (S)	4 (S)	1 (S)	0.5 (S)
HLJ030-3	0.5 (S)	16 (R)	2 (S)	0.5 (S)	16 (R)	0.5 (S)	0.5 (S)
NM025	16 (R)	0.5 (S)	1 (S)	0.5 (S)	8 (I)	0.5 (S)	0.5 (S)
HB016	16 (R)	4 (R)	16 (R)	0.5 (S)	4 (S)	0.5 (S)	0.5 (S)
NM-2-72-4	2 (I)	0.5 (S)	32 (R)	0.5 (S)	16 (R)	0.5 (S)	0.5 (S)
NM-2-034-3	0.5 (S)	16 (R)	4 (S)	4 (R)	16 (R)	0.5 (S)	0.5 (S)
SH-2-46-1	16 (R)	0.5 (S)	4 (S)	0.5 (S)	4 (S)	0.5 (S)	0.5 (S)
SH-2-14-2	16 (R)	16 (R)	4 (S)	4 (R)	8 (I)	0.5 (S)	0.5 (S)
SD-2-009-2	32 (R)	0.5 (S)	16 (R)	4 (R)	16 (R)	0.5 (S)	0.5 (S)
Drug resistance rate (%) (/15)
S	13.33 (2)	33.33 (5)	60.00 (9)	73.33 (11)	40.00 (6)	100 (15)	100 (15)
I	6.67 (1)	0 (0)	6.67 (1)	6.67 (1)	26.67 (4)	0 (0)	0 (0)
R	80.00 (12)	66.67 (10)	33.33 (5)	20.00 (3)	33.33 (5)	0 (0)	0 (0)

[i] ERY – erythromycin; CLI – clindamycin; GM – gentamycin; CIP – ciprofloxacin; FFC – florfenicol; RIF – rifampicin; VAN – vancomycin

Table 5

Relationship between drug resistance phenotype and drug resistance genotype of S. agalactiae

Number of bacteria	Positive rate
	ermB	11/15	73.33		91.66
Macrolides	ermA	10/15	66.67	80.00	83.33
	ermC	5/15	33.33		41.66
Lincosamides	lnuA	9/15	60.00	66.67	89.99
	tetM	7/15	46.67		100.00
	tetK	6/15	40.00		85.71
Tetracyclines	tetS	6/15	40.00	46.67	85.71
	tetO	5/15	33.33		71.42

Positive rate of *S. agalactiae* resistance genes

Table 6

The combined virulence gene profile of S. agalactiae

Virulence gene profile	Distribution^a (%)
glnA+hylB+bibA+iagA+fbsA+clyE	3 (20)
glnA+hylB+iagA+fbsA	2 (13.33)
bca+glnA+hylB+iagA+fbsA+clyE	1 (6.67)
bca+cfb+hylB+bibA+iagA+fbsA+clyE	1 (6.67)
bibA+iagA+fbsA+clyE	1 (6.67)
bca+hylB+bibA+iagA+fbsA	1 (6.67)
hylB+bibA+iagA+fbsA	1 (6.67)
bca+bibA+iagA+fbsA	1 (6.67)
hylB+bibA+iagA+fbsA+clyE	1 (6.67)
cfb+hylB+bibA+iagA+fbsA	1 (6.67)
cfb+hylB+bibA+fbsA+clyE	1 (6.67)

[i] ^a – Distribution was achieved based on the total numbers of 15 S. agalactiae isolates

Table 7

The drug resistance patterns and gene expression profiles of S. agalactiae

Isolate no.	Drug resistance pattern	Resistant gene	Virulence gene
SH07	(A/C)/CET/ERY	ermB+lnuA+tetM+tetO	bca+hylB+bibA+iagA+fbsA
SH07-2	PEN/OXA/TE/ERY/CLI/FFC	ermA+ermB+lnuA+tetM+tetK+tetS+tetO	glnA+hylB+iagA+fbsA
SH12	OXA/TE/ERY/CLI/GM	ermA+ermB+ermC+lnuA+tetS+tetO	bca+glnA+hylB+iagA+fbsA+clyE
SH33	OXA/TE/ERY/CLI/GM	ermA+ermB+ermC+lnuA+tetK+tetS+tetO	bca+cfb+hylB+bibA+iagA+fbsA+clyE
SH45	OXA/TE/ERY/CLI	ermA+ermB+lnuA+tetK+tetS	hylB+bibA+iagA+fbsA
HLJ008	CET/OXA/TE/ERY/CLI	ermB+ermA+ermC+lnuA+tetM+tetK	glnA+hylB+bibA+iagA+fbsA+clyE
HLJ030-3	TE/CLI/FFC	ermB+ermA+lnuA+tetM	hylB+bibA+iagA+ fbsA+clyE
NM025	TE/DOX/ERY	ermA+ermB+lnuA+tetK+tetO	glnA+hylB+bibA+iagA+fbsA+clyE
HB016	OXA/TE/ERY/CLI/GM	ermA+ermB+ermC+lnuA+tetM+tetK+tetS	glnA+hylB+bibA+iagA+fbsA+clyE
SH-2-46-1	OXA/TE/ERY	ermA+ermB+lnuA+tetO	glnA+hylB+iagA+fbsA

[i] A/C – amoxicillin/clavulanic acid; CET – cefoxitin; CLI – clindamycin; ERY – erythromycin; FFC – florfenicol; GM – gentamicin; OXA – oxacillin; PEN – penicillin; TE – tetracycline

Table 8

Logistic regression analysis of the relationship between antibiotics and virulence genes

Virulence gene	Penicillin			Cefoxitin			Oxacillin			Doxycycline			Gentamicin			Clindamycin
Virulence gene	B	Exp(B)	P	B	Exp(B)	P	B	Exp(B)	P	B	Exp(B)	P	B	Exp(B)	P	B	Exp(B)	P
bca	40.537	4.028	0.999	1.099	3.000	1.000	−42.406	0.000	0.999	1.099	3.000	1.000	−41.307	0.000	0.999	−61.799	0.000	0.999
scpB	−20.269	0.000	1.000	−60.718	0.000	0.999	62.916	2.108	0.999	19.699	3.590	1.000	62.105	9.369	0.999	−62.493	0.000	0.999
glnA	0.000	1.000	1.000	−19.411	0.000	0.999	−21.896	0.000	0.999	−19.411	0.000	0.999	−19.411	0.000	0.999	−0.693	0.500	0.744
cfb	−0.934	0.393	1.000	42.406	2.610	0.999	−84.812	0.000	0.999	−38.011	0.000	1.000	−80.417	0.000	0.999	−81.880	0.000	0.999
hylB	42.406	2.610	0.999	−40.209	0.000	0.999	42.406	2.610	0.999	40.209	2.900	0.999	40.209	2.900	0.999	0.000	1.000	1.000
bibA	21.203	1.615	1.000	−19.411	0.000	1.000	−21.896	0.000	1.000	−19.411	0.000	1.000	−19.411	0.000	1.000	−42.412	0.000	0.999
iagA	−41.472	0.000	1.000	−1.099	0.333	1.000	42.406	2.610	1.000	−1.099	0.333	1.000	41.307	8.699	1.000	−20.080	0.000	1.000
cylE	0.000	1.000	1.000	−1.099	0.333	1.000	42.406	2.610	0.999	−1.099	0.333	1.000	−1.099	0.333	1.000	21.203	1.615	1.000

[i] Exp (B) – OR value, logarithmic ratio, equal to the exponential power of the regression coefficient

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DOI: https://doi.org/10.2478/jvetres-2022-0069 | Journal eISSN: 2450-8608 (formerly 2300-3235)

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Language: English

Page range: 581 - 590

Submitted on: May 7, 2022

Accepted on: Dec 5, 2022

Published on: Dec 16, 2022

Published by: National Veterinary Research Institute in Pulawy

In partnership with: Paradigm Publishing Services

Keywords:

cattle,

bovine mastitis,

dairy industry,

antimicrobial stewardship,

virulence gene

Related subjects:

Life sciences,

Molecular biology,

Microbiology and virology,

Life sciences, other,

Medicine,

Veterinary medicine

© 2022 Yankun Zhao, Wei Shao, Fulan Wang, Jiaoxiao Ma, He Chen, Shuai Wang, Yating Wu, Cheng Wang, Nan Zheng, Jiaqi Wang, Huimin Liu, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Volume 66 (2022): Issue 4 (December 2022)

Antimicrobial resistance and virulence genes of Streptococcus agalactiae isolated from mastitis milk samples in China

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Table 1

Table 2

Table 3

Table 4a

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Table 4b

Table 5

Fig. 2

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