Genotyping and antimicrobial resistance of Staphylococcus aureus isolates from diseased turkeys

Figures & data

Table 1. Primers used in the present study.

Gene targeted^a	Forward and Reverse primer sequences (5′-3′)	Amplicon size (base pairs)	Annealing temperature (°C)	Reference
amp(A)	CGTTTGCTTCGTGCATTAAA TTGACACGAAGGAGGGTTTC	656	52	Feßler et al., 2011b
cop(B)	TAGTGGCCATGCACATCATC CCACCAGACAAGAACGGTTT	201	60	This study
czr(C)	TAGCCACGATCATAGTCATG ATCCTTGTTTTCCTTAGTGACTT	632	54	Cavaco et al., 2010
dfr(K)	CAAGAGATAAGGGGTTCAGC ACAGATACTTCGTTCCACTC	229	50	Argudín et al., 2011
erm(T)	ATTGGTTCAGGGAAAGGTCA GCTTGATAAAATTGGTTTTTGGA	536	45	Feßler et al., 2010
lsa(C)	GGCTATGTAAAACCTGTATTTG ACTGACAATTTTTCTTCCGT	429	50	Malbruny et al., 2011
spc	ACCAAATCAAGCGATTCAAA GTCACTGTTTGCCACATTCG'	561	52	Feßler et al., 2010
tet(L)	TCGTTAGCGTGCTGTCATTC GTATCCCACCAATGTAGCCG	267	55	Ng et al., 2001
vga(C)	AGCTGGAGAAAGCAATCCAA TCTTGCTGAAAATCCCGTTC	885	50	Argudín et al., 2011
vga(E)	ATGAAAGAATAGCAATCCCAG GGGTAGGTTGAGTTTGGAG	541	54	Schwendener & Perreten, 2011
SAAV_2008	CACCAATTACAACCGCAAGA AAGCTGGCGAATTTATCGAA	174	59	This study
SAAV_2009	TCTGAGGCAACCTTAACTGGC TGGCTTGCTTTCTTCTGCT	229	57	This study

^a The targeted genes included antimicrobial resistance genes [amp(A) (apramycin resistance), dfr(K) (trimethoprim resistance), erm(T) (macrolide-lincosamide-streptogramin B resistance), spc (spectinomycin resistance), tet(L) (tetracycline resistance), vga(C), vga(E) and lsa(C) (pleuromutilin-lincosamide-streptogramin A resistance)], metal resistance genes [czr(C) (cadmium and zinc resistance) and cop(B) (copper resistance)], and ϕAvβ prophage avian-niche-specific genes [SAAV_2008 (putative ornithine cyclodeaminase) and SAAV_2009 (putative membrane protease of the CAAX family)].

Figure 1. Cfr9I-PFGE analysis of S. aureus isolates from turkeys. Lane N, PFGE profile of strain NCTC 8325 included as quality control; lanes C1 to C12, profiles generated by S. aureus isolates collected from turkeys. Macrorestriction with SmaI or Cfr9I yielded identical PFGE patterns in the case of profiles C1, C2 and C3. Macrorestriction with SmaI in isolates with profiles C4 to C12 yielded the typical not typeable CC398 profile. Some profiles were closely related and only differed in one additional band (profiles C4 and C5, and profiles C8 and C9).

Figure 2. Dendrogram showing the relatedness between Cfr9I macrorestriction PFGE patterns generated from S. aureus turkey isolates and the control strain NCTC 8325. At a Dice similarity index of 71.7% a cluster grouping the CC398 isolates was detected. The number of isolates is included after the profile type in those cases in which more than one isolate showed that profile.

Table 2. Features of the turkey S. aureus isolates analysed in this study.

Isolate	Year of isolation	Origin^a	Hatchery^b	spa type	CC^c	Cfr9I PFGE profile	R-pattern^d	Resistance phenotype^e	Strain^f
29-G3	2008	AT	A	t034	398	C4	R5	PEN-FUS-TET	1
30-I7	2008	BF	B	t034	398	C4	R8	PEN-ERY-CLI-FUS-TET	2
30-D8	2008	AT	C	t150	101	C1	R0	Susceptible	3
31-D7	2008	BF	C	t034	398	C5	R2	PEN-TET	4
34-G12	2009	BF	B	t034	398	C4	R6	PEN-STR-TET	5
35-D9	2009	BF	A	t034	398	C6	R2	PEN-TET	6
36-I12	2009	AT	A	t034	398	C4	R2	PEN-TET	7
37-G8	2009	BF	A	t034	398	C7	R2	PEN-TET	8
37-G9	2009	BF	A	t034	398	C4	R2	PEN-TET	7
40-B7	2009	BF	A	t034	398	C4	R2	PEN-TET	7
40-C10	2009	BF	B	t034	398	C4	R3	PEN-CIP-TET	9
40-E10	2009	BF	B	t034	398	C4	R2	PEN-TET	7
41-F5	2010	BF	A	t034	398	C4	R2	PEN-TET	7
46-H11	2010	AT	B	t571	398	C9	R3	PEN-CIP-TET	10
51-C11	2011	BF	B	t571	398	C10	R3	PEN-CIP-TET	11
51-H3	2011	BF	A	t034	398	C4	R2	PEN-TET	7
55-E2	2011	AT	B	t571	398	C8	R9	PEN-CLI-TIA-CIP-FUS-TET	12
56-B7	2011	BF	A	t588	398	C4	R2	PEN-TET	13
56-C4	2011	BF	A	t4652	398	C11	R2	PEN-TET	14
56-C5	2011	BF	A	t4652	398	C11	R2	PEN-TET	14
56-C6	2011	BF	A	t4652	398	C11	R5	PEN-FUS-TET	15
56-C7	2011	BF	A	t4652	398	C11	R2	PEN-TET	14
56-C8	2011	BF	A	t2970	398	C11	R2	PEN-TET	16
56-G6	2011	AT	C	t2164	5	C2	R7	PEN-CIP-TET-SMX	17
57-B7	2011	AT	C	t034	398	C4	R2	PEN-TET	7
57-H14	2011	AT	B	t571	398	C8	R10	PEN-ERY-CLI-CIP- FUS-TET	18
59-B4	2011	BF	A	t034	398	C4	R2	PEN-TET	7
59-G11	2012	AT	B	t2970	398	C5	R4	PEN-ERY-TET	19
60-A3	2012	BF	C	t645	121	C3	R1	TET	20
60-A9	2012	BF	B	t571	398	C8	R3	PEN-CIP-TET	21
60-B1	2012	BF	A	t571	398	C8	R3	PEN-CIP-TET	21
62-E10	2012	BF	A	t571	398	C8	R3	PEN-CIP-TET	21
62-E11	2012	AT	B	t571	398	C8	R3	PEN-CIP-TET	21
63-D10	2012	AT	B	t899	398	C12	R11	PEN-CEF-STR-CLI- SYN-TIA-CIP-TET	22

^a AT, S. aureus isolates from sample of a young turkey with arthritis during the rearing period; BF, S. aureus isolates from sample of turkey with bumble foot during laying period.

^b The three different hatcheries are indicated with letters A to C.

^c Clonal complexes on the basis of the spa typing and the sau1-hsdS1 CC398 detection PCR (Stegger et al., 2011) in positive isolates, and on the basis of MLST typing (http://saureus.mlst.net/) for isolates 30-D8 (ST101, CC101), 56-G6 (ST5, CC5) and 60-A3 (ST95, CC121).

^d Resistance (R-) patterns are numbered on the basis of the resistance phenotype, without taking into account minimal inhibitory concentration differences.

^e PEN, penicillin; FOX, cefoxitin; STR, streptomycin; ERY, erythromycin; CLI, clindamycin; SYN, quinupristin/dalfopristin; TIA, tiamulin; CIP, ciprofloxacin; FUS, fusidic acid; TET, tetracycline; SMX, sulfamethoxazole.

^f A strain shares a common spa type, PFGE profile and resistance pattern.

Table 3. Presence of antimicrobial resistance genes, virulence, adhesion and inmunoevasion factors by DNA microarray and PCR test in selected isolates.

Negative hybridizations of genes in all strains, as well as common species or regulatory markers, are not shown. Black dots, positive results for probe hybridization; ×, ambiguous results. Gene alleles are indicated between brackets in those cases in which hybridizations with other alleles were not detected. The strain numbers are according to Table 2. BP, binding protein; MHC-II, major histocompatibility complex class II; MLS, macrolide–lincosamide–streptogramine; QAC, quaternary ammonium compounds; R, resistance.

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