International Journal of Medical Microbiology 304 (2014) 1226–1232

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Characterization of tetracycline and methicillin resistant Staphylococcus aureus strains in a Spanish hospital: Is livestock-contact a risk factor in infections caused by MRSA CC398? Daniel Benito a,1 , Carmen Lozano a,1 , Antonio Rezusta b,c , Isabel Ferrer b , Maria Alejandra Vasquez b , Sara Ceballos a , Myriam Zarazaga a , Maria José Revillo b , Carmen Torres a,∗ a

Area de Bioquímica y Biología Molecular, Universidad de La Rioja, Logro˜ no, Spain Servicio de Microbiología, Hospital Universitario Miguel Servet, IIS Aragón, Zaragoza, Spain c Universidad de Zaragoza, Zaragoza, Spain b

a r t i c l e

i n f o

Article history: Received 21 May 2014 Received in revised form 30 August 2014 Accepted 21 September 2014 Keywords: Animal contact Livestock associated MRSA CC398 CC1 Multi-resistance Tetracycline

a b s t r a c t Tetracycline-resistance (TetR ) has been postulated as a marker of the livestock-associated methicillinresistant Staphylococcus aureus (MRSA) lineage CC398. Objectives of the study: to determine the spa-types and assigned MLST clonal complexes (CCs) among all 98 MRSA-TetR strains recovered during 2011–2012 (from different patients) in a Spanish Hospital, analyzing the possible correlation with livestock-contact of the patients. All 98 strains were assigned to 9 CCs: CC398 (60.2%), CC1 (19.4%), CC5 (12.2%), and other CCs (8.2%). The 98 patients were classified into three groups: (A) contact with livestock-animals (n = 25); (B) no-contact with livestock-animals (n = 42); (C) no information about animal contact (n = 31). A significant higher percentage of CC398 strains was obtained in group A (76%) than in group B (50%) (p < 0.05), being the percentage in group C of 61.3%. Most of MRSA-TetR -CC398 strains presented a multi-resistance phenotype, including erythromycin, clindamycin, and ciprofloxacin, and the most prevalent detected genes were tet(M) and erm(C). Three strains presented the phenotype macrolidesusceptibility/lincosamide-resistance and contained the vga(A) gene. MRSA-CC1 strains showed higher percentages of erythromycin/clindamycin resistance (95%/89%) than MRSA-CC398 strains (58%/63%), and this resistance was usually mediated by erm(C) gene. Most of MRSA-CC5 strains showed resistance to ciprofloxacin, tobramycin/kanamycin and erythromycin. None of the strains presented the genes lukF/lukS-PV, tsst-1, eta, etb or etd. All MRSA-CC398 strains lacked the genes of the immune-evasioncluster, but MRSA-CC1 strains carried these genes (type E). In conclusion, although MRSA CC398 is detected in a significant higher proportion in patients with livestock-contact; its detection in people without this type of contact also indicates its capacity for human-to-human transmission. © 2014 Elsevier GmbH. All rights reserved.

Introduction Staphylococcus aureus is part of the natural microbiota of the nose, skin, and even the intestinal tract of humans (Benito et al., 2013). Although S. aureus can coexist peacefully as a commensal, it is also a bacterium responsible for a wide variety of infections and diseases (Lozano et al., 2011). S. aureus is able to develop resistance to multiple antimicrobials, methicillin resistance being of major importance. So, methicillin resistant S. aureus (MRSA) is resistant

∗ Corresponding author. Tel.: +34 941 299750; fax: +34 941 299721. E-mail address: [email protected] (C. Torres). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijmm.2014.09.004 1438-4221/© 2014 Elsevier GmbH. All rights reserved.

to almost all beta lactams and is associated with both hospital and community infections. There are some clonal lineages, which have been associated with livestock-animals and these strains are now called livestockassociated (LA) MRSA. The first description of LA-MRSA was in 2005 in pigs and pig farmers and the strains belonged to clonal complex CC398 (Armand-Lefevre et al., 2005). Since then, the number of cases caused by this genetic lineage has been increasing. Initially, the infections described were mainly single cases, all of them in patients with contact with livestock-animals (farmers, veterinarians or abattoir workers) or in their relatives (Lozano et al., 2012a). So, the professional relationship with livestock-animals was considered as a risk factor of colonization and infection by MRSA-CC398 (Garcia-Graells et al., 2012). However, this CC has

D. Benito et al. / International Journal of Medical Microbiology 304 (2014) 1226–1232

been progressively detected in hospital settings (Verkade et al., 2012; Williamson et al., 2014), and in the community (VanCleef et al., 2011). A previous study carried out by our research group established that tetracycline resistance (TetR ) could be a good marker for the rapid determination of CC398 strains and other animal-related CCs among MRSA clinical isolates. In that study, it was shown that CC398 represented a high proportion among all MRSA-TetR (67.3%) obtained in the period 2009–2010. However, the potential link with livestock-contact was not analyzed (Lozano et al., 2012b). For that reason, in the present study we study the spa-types and associated clonal complexes among all MRSA-TetR isolates obtained during 2011–2012 from different patients in the same hospital (including isolates associated with both infection and colonization), evaluating the potential link with livestock-contact of patients. Material and methods Selection of the strains During 2011–2012, MRSA isolates represented 22.5% of the total S. aureus isolates recovered in the studied Hospital (Universitary Hospìtal Miguel Servet, Zaragoza, Spain). In that two-year period, 749 patients carried MRSA and 98 of them (13.1%) presented resistance, in addition to methicillin, to tetracycline (MRSA-TetR ). Among these 98 MRSA-TetR isolates, 40 of them were recovered from nasal or pharyngeal samples and corresponded to colonization isolates (40.8%), and the remaining 58 corresponded to significant clinical isolates (59.2%). In the present work, a epidemiological survey was conducted in all patients with MRSA-TetR , (in most of the cases by telephonic interview, and in some other cases by contact to patients whilst on the ward) to determine the possible contact with livestockanimals during the previous 18 months, and three groups could be established: (A) 25 patients that referred livestock-contact; (B) 42 patients that referred no livestock-contact; and (C) 31 patients for which no information about livestock-contact was obtained.

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mph(C), lnu(A), lnu(B), vga(A), ant(4 )-Ia, aph(3 )-IIIa, aac(6 )aph(2 ), fus(B), fus(C), mup(A), dfr(S1), dfr(D), and dfr(K) was analyzed by PCR and sequencing (Supplementary Table S1). Virulence genes The presence of the genetic determinants of the PantonValentine leukocidin (PVL) (lukF/lukS-PV), toxic shock syndrome toxin (tsst-1), exfoliative toxin A (eta), B (etb), and D (etd), as well as alpha-(hlA), beta-(hlB), delta-(hlD), gamma (hlG), and gammahemolysin variant (hlGv) was investigated by PCR. In addition, the genes encoding leukocidins DE (luk-DE) and M (lukM), aureolysin (aur), the biofilm associated protein bap, and the adhesion factor cna were also studied by PCR (Supplementary Table S1). Immune evasion cluster (IEC) The presence of genes comprised by the immune evasion cluster (IEC) (scn, chp, sak, sea and/or sep) was studied by PCR, and according to the pattern of genes detected, strains were classified into five different IEC types (Van Wamel et al., 2006). Results Ninety-eight MRSA-TetR isolates recovered from different patients of the hospital were characterized in the present study, which represented 13.1% of total MRSA strains isolated (749 strains). All 98 patients were surveyed to investigate if they had contact with livestock-animals in the previous 18 months of being admitted to the hospital. Twenty-five of the 98 patients carrying MRSA-TetR reported livestock-contact (25.5%), mainly pigs and chickens [group A, Table 1], other 42 patients referred no livestock-contact (42.9%) [group B, Table 2] and for the remaining 31 patients, no data related to livestock-contact could be obtained (31.6%) [group C, Table 3]. The number of isolates corresponding to infection/colonization samples in the three patient groups was as follows: A (12/13), B (28/14) and C (18/13).

Molecular typing of strains MRSA-TetR isolates were characterized by sequenced-based typing of the hyper-variable region of the S. aureus protein A (spa) gene (Supplementary Table S1). Sequences were analyzed using the Ridom Staph-Type software version 1.5.21 (Ridom GmbH). Detection of agr allotypes was carried out by 2 multiplex PCRs in all strains (Supplementary Table S1). SCCmec-typing was carried out by multiplex PCRs, as previously described (Zhang et al., 2005). Multi-locus-sequence-typing (MLST) was performed, at least, in one strain of each spa-type detected (www.saureus.mlst.net). The MLST clonal complexes (CCs) of the strains were assigned according to the sequence type (ST) determined and/or spa type detected. Resistance phenotypes and genotypes Susceptibility testing for 16 antibiotics (penicillin, oxacillin, cefoxitin, tetracycline, erythromycin, clindamycin, ciprofloxacin, gentamicin, tobramycin, kanamycin, trimethoprimsulfamethoxazole, linezolid, mupirocin, fusidic acid, vancomycin, and teicoplanin) was carried out using the Microscan system and/or the agar disk-diffusion method, and CLSI breakpoints were used (Clinical Laboratory Standards Institute (CLSI), 2013), except for fusidic acid and mupirocin (European Committee on Antimicrobial Susceptibility Testing (EUCAST), 2014). The presence of 23 antibiotic resistance genes tet(K), tet(L), tet(M), erm(A), erm(B), erm(C), erm(F), erm(T), msr(A), msr(B),

Molecular typing of strains Twenty-one different spa-types were identified among the 98 MRSA-TetR strains. Among these, five spa-types were predominant (number of strains): t011 (44), t127 (18), t1451 (7), t067 (6), and t1255 (4). The remaining 16 spa-types were detected only in one or two of the isolates: t1197, t2220, t5229, t002, t024, t167, t216, t688, t701, t899, t1456, t1594, t1606, t1618, t1784, and t10664 (this last one firstly described in this study). MLST was performed for 25 strains (including one isolate of each of the 21 different spa-types, and also 4 additional strains of spa-types t011 and t127). Ten different sequence types were identified (ST1, ST5, ST8, ST25, ST45, ST59, ST125, ST188, ST398, and ST627). Based on sequence type (ST) and/or spa-type detected, all MRSA-TetR were assigned to 9 MLST clonal complexes (number of strains/% of strains): CC398 (59/60.2%), CC1 (19/19.4%), CC5 (12/12.2%), and [CC6, CC8, CC25, CC45, CC59 and CC188] (8/8.2%). The agr-types detected were (number of strains): agr I (67), agr II (12), and agr III (20). All strains were SCCmec-typed and 66.3% of them showed SCCmec V, 32.6% of them harboured SCCmec IV, and 1.1% were non-typeable (Tables 1–3). Fifty-nine CC398 strains were detected among the 98 MRSATetR of this study (60.2%). If we consider the total MRSA strains of the studied period (749 strains), the percentage of MRSA CC398 (59 strains) would be at least of 7.9%; this percentage could be even higher in case that CC398 strains were detected among

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Table 1 Characteristics of the 25 MRSA-TetR strains of patients that referred livestock-contact (group-A). No. of strains

I/Ca

Molecular typing

Antimicrobial resistance

Virulence factors

MLST/CCb

spa-type

agr/SCCmec

Phenotypec,d

Genotypec

IEC typee

Other genesc

tet(K)10 , tet(L)2 , tet(M), msr(A)1 , msr(B)1 , mph(C)1 ,erm(A)5 , erm(B)2 , erm(C)7 , vga(A)1 , lnu(A)3 , ant(4 )-Ia3 , aph(3 )-IIIa3 , aac(6 )-Ie-aph(2 )2 tet(K)1 , tet(L)1 , tet(M), erm(B)1 , erm(C)2 , erm(T)1 ,vga(A)2 ,lnu(A), ant(4 )-Ia2 ,aph(3 )-IIIa2 , aac(6 )-Ie-aph(2 )2 tet(K)2 , tet(L)2 , tet(M), erm(C)2 , vga(A)2 tet(M)1 , tet(K)1 , tet(L)3 , erm(A)3 , erm(C)4 , vga(A)1 tet(K), msr(A), msr(B), mph(C)

Negative

hlA, hlB9 , hlG8 , aur9 , cna5 , luk-DE1

Negative

hlA,hlB, hlG, aur, cna2

Negative

hlA, hlB, hlG, aur, cna2 hlA2 , hlB3 , hlG1 ,hlGv3 aur4 , cna3 , luk-DE4 hlA, hlB, hlGv, aur, luk-DE

12

7/5

ST398/CC398

t011

I/V

ERY8 , CLI8 , GEN2 , TOB3 , KAN3 , CIP8

3

0/3

ST398/CC398

t1255

I/V

ERY2 , CLI, GEN2 , TOB2 , KAN2 , CIP1

4

2/2

ST398/CC398

I/V

ERY2 , CLI2 , CIP

5

3/2

ST1/CC1

t1451(3), t1456(1) t127

III/IV

ERY4 , CLI

1

0/1

ST5/CC5

t2220

II/IV

ERY, CIP, SXT1

a b c d e

E

F

I: strains from infection samples; C: strains of nasal or pharyngeal samples associated with colonization. At least one strain of each spa type was submitted to MLST-typing and the STs and CCs were assigned for all strains of the same spa-type. Superscript marks the number of strains in those cases in which not all strains of the group have the indicated characteristic. Antimicrobials: ERY, erythromycin; CLI, clindamycin; GEN, gentamicin; KAN, kanamycin; TOB, tobramycin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole. IEC: immune evasion cluster. IEC type E (scn, sak) and IEC type F (scn, chp, sak, sep).

Table 2 Characteristics of the 42 MRSA-TetR strains of patients that refer no livestock-contact (group-B). No. of strains

I/Ca

Molecular typing MLST/CCb

spa-type

agr/SCCmec

Antimicrobial resistance

Virulence factors

Phenotypec,d

IEC typec,e

Other genesc

tet(K) , tet(L) , tet(M)17 , msr(A)2 , msr(B)1 ,mph(C)1 , erm(B)2 , erm(C)5 , vga(A)5 tet(K)1 , tet(M), erm(C)2 , vga(A)1 , lnu(A)1

Negative

hlA11 , hlB10 , hlG12 , aur13 , cna12 , luk-DE1

Negative

Negative E

hlA, hlB, hlG, aur, cna, luk-DE1 aur, cna hlA7 , hlB8 , hlGv8 , aur8 , cna8 , luk-DE6 hlA4 , hlB3 , hlGv, aur5 ,luk-DE5

9

9

Genotypec 11

9

18

10/8

ST398/CC398

t011

I/V

ERY , CLI , CIP11

2

2/0

ST398/CC398

t1451

I/V

ERY, CLI, CIP

1 9

1/0 5/4

ST398/CC398 ST1/CC1

t1197 t127(8), t1784(1)

I/V III/IV

PEN ERY, CLI7 , CIP1

5

4/1

ST125/CC5

t067

II/IV

ERY5 , CLI4 , GEN1 , TOB2 , KAN2 , CIP4

1

1/0

ST5/CC5

t002

II/IV

CLI, CIP

tet(K), tet(L) tet(K)6 , tet(L)3 ,tet(M)1 , msr(A)2 , msr(B)2 , mph(C)2 , erm(C)6 tet(K), msr(A)4 , msr(B)4 , mph(C)4 , erm(B)3 , erm(C)2 , ant(4 )-Ia2 , aph(3 )-IIIa2 , aac(6 )-Ie-aph(2 )1 tet(K), lnu(A)

1

0/1

ST8/CC8

t024

III/V

ERY, CLI

tet(K), erm(B), erm(C)

E

1

1/0

ST8/CC8

t10664

I/IV

CLI, CIP, SXT

tet(K), dfr(S1)

D

I/V

ant(4 )-Ia, aph(3 )-IIIa, aac(6 )-Ie-aph(2 ) tet(M)

Negative

I/IV I/V

Negative ERY

tet(K) tet(M), msr(A), mph(C)

Negative Negative

1/0

ST25/CC25

t167

I/NT

1

1/0

ST6/CC6

t701

1 1

1/0 1/0

ST59/CC59 ST45/CC45

t216 t1618

a

c d e



F

ERY, GEN, TOB, KAN, CIP CIP

1

b



F4 or G1

B

hlA, hlB, hlG, aur, luk-DE hlA, hlB, hlGv, aur, cna, luk-DE hlA, hlB, hlGv, aur, luk-DE hlA, hlB, hlGv, aur, luk-DE hlA, hlB, hlGv, aur, cna, luk-DE hlA, hlB, hlGv aur, cna

I: strains from infections samples; C: strains of nasal or pharyngeal samples associated with colonization. At least one strain of each spa type was submitted to MLST-typing and the ST and CC were assigned for all strains of the same spa-type. NT, non typeable. Superscript marks the number of strains in those cases in which not all strains of the group have the indicated characteristic. Antimicrobials: ERY, erythromycin; CLI, clindamycin; GEN, gentamicin; KAN, kanamycin; TOB, tobramycin; CIP, ciprofloxacin; SXT, trimethoprim-sulfamethoxazole. IEC: Immune Evasion Cluster. IEC type B (scn, chp, sak), IEC type D (scn, sak, sea), IEC type E (scn, sak), IEC type F (scn, chp, sak, sep), IEC type G (scn, sak, sep).

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Table 3 Characteristics of 31 MRSA-TetR strains of patients with no data related with animal-contact (group-C). No. of strains

I/Ca

Molecular typing

Antimicrobial resistance

Virulence factors

MLST/CCb

spa-type

agr/SCCmec

Phenotypec,d

Genotypec

IEC typee

Other genesc

tet(K)10 , tet(L)10 , tet(M)11 , erm(C)9 , vga(A)3 , lnu(A)1 , fus(C)1 , mup(A)1 tet(K), tet(M), erm(C), lnu(A), erm(B) tet(K), tet(L), tet(M)

Negative

hlA12 , hlB10 , hlG11 , aur11 , cna13

Negative

hlA, hlB, hlG, aur, cna, luk-DE

Negative

hlA, hlB, hlGv, aur, cna hlA, hlB, hlGv, aur1 , cna1

14

6/8

ST398/CC398

t011

I/V

ERY9 , CLI11 , CIP2 , MUP1 , FUS1

1

0/1

ST398/CC398

t1255

I/V

ERY, CLI, CIP

1

1/0

ST398/CC398

t899

I/IV

Negative

3

2/1

ST398/CC398

I/V

ERY1 , CLI1 , CIP1

tet(K), tet(M), erm(C)1

Negative

5

4/1

ST1/CC1

t1451, t1606, t1197 t127

III/IV

ERY, CLI, TOB1 , KAN1

E

hlA, hlB3 , hlGv4 , aur, cna3 , luk-DE4

2

2/0

ST125/CC5

t067

II/IV

ERY1 , CLI1 , TOB1 , KAN, CIP1

F

hlA, hlB1 , hlGv1 , aur1 , cna1 , luk-DE

1

0/1

ST5/CC5

t688

II/V

Negative

tet(K)4 , tet(L)2 , tet(M)1 , msr(A)1 , msr(B)1 , mph(C)1 , erm(A)1 , erm(C)4 , vga(A)1 , ant(4 )-Ia1 , aph(3 )-IIIa1 tet(K)1 , tet(L)1 , tet(M)1 , msr(A)1 , msr(B)1 , mph(C)1 ,erm(B)1 , erm(C)1 , ant(4 )-Ia1 , aph(3 )-IIIa tet(M)

G

1

0/1

ST5/CC5

t2220

II/V

CIP, GEN, TOB, KAN, MUP

hlA, hlB, hlGv, aur, luk-DE hlA, hlGv, aur, luk-DE

1

1/0

ST627/CC5

t1594

II/IV

GEN, TOB, KAN

2

2/0

ST188/CC188

t5229

I/V

CLI, CIP1

tet(K), aac(6 )-Ie-aph(2 ), mup(A) aac(6 )-Ie-aph(2 ), aph(3 )-IIIa tet(K), tet(L), vga(A)

F

F

hlA, aur, luk-DE

F

hlA,hlB, hlGv1 , aur, cna, luk-DE1

a

I: strains from infections samples; C: strains of nasal or pharyngeal samples associated with colonization. At least one strain of each spa type was submitted to MLST-typing and the ST and CC were assigned for all strains of the same spa-type. c Superscript marks the number of strains in those cases in which not all strains of the group have the indicated characteristic. d Antibiotics: ERY, erythromycin; CLI, clindamycin; GEN, gentamicin; KAN, kanamycin; TOB, tobramycin; CIP, ciprofloxacin; MUP, mupirocin; SXT, trimethoprimsulfamethoxazole; FUS, fusidic acid. e IEC: Immune Evasion Cluster. IEC type E (scn, sak); IEC type F (scn, chp, sak, sep) and IEC type G (scn, sak, sep). b

was observed in the group A with livestock-contact (76%) in relation to group B with no livestock-contact (50%) (p < 0.05). On the other way, comparing group A vs B, a similar rate of CC1 was observed in both groups (20% vs 21.4%) and a lower rate of CC5 strains in group A (4% vs 14.3%). If we only consider the MRSA-TetR strains corresponding to clinical infections (excluding colonization ones), the proportion of CC398 among strains of group A (9 of 12 strains, 75%) was also significantly higher than among those of group B (13 of 28 strains, 46%) (p < 0.05). Origin of the strains and clonal complexes

Fig. 1. Percentage of MRSA-TetR strains related to clonal complexes in the three studied groups: (A) livestock-contact; (B) no livestock-contact; (C) no data respect to animal-contact. The percentage of the strains is indicated above the bars.

The origin of the MRSA-TetR strains according to their CCs is shown in Fig. 2. CC398 strains were mainly obtained from colonization samples of screening studies (nasal and pharyngeal samples), from samples of skin and soft-tissue infections (SSTI), and respiratory-tract infections. The majority of the CC1 and CC5 strains were obtained from samples of SSTIs.

MRSA-tetracycline susceptible (MRSA-TetS ) strains (non-typed in this study).

Phenotype of resistance detected in MRSA-TetR of lineages CC398, CC1 and CC5

Strains detected according to the livestock-contact status

More than half of CC398 strains presented resistance to erythromycin, clindamycin, and ciprofloxacin. CC1 strains showed very high percentage of erythromycin/clindamycin resistance but most of them showed susceptibility for the remaining

Fig. 1 shows the distribution of CCs among isolates derived from the three groups of patients (A–C). A significant higher rate of CC398

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genes, such as cna, luk-DE or aur were detected in some of the strains (Tables 1–3). Analysis of IEC genes The genes of the IEC system were not detected in the MRSA-TetR strains of the lineages CC398, CC6, CC45, and CC59. Nevertheless, different IEC types were identified among the MRSA-TetR of the remaining lineages (CC/IEC type): CC1/IEC-E, CC5 and CC188/IEC-F or G, CC8/IEC-E or D, and CC25/IEC-B (Tables 1–3). Discussion Fig. 2. Percentage of MRSA-TetR strains in relation with the type of samples from which they were recovered (colonization samples: nasal or pharyngeal screening samples; SSTIs: samples of skin and soft tissues infections; Urogenital samples: samples of urine or urethral and vaginal tissues) and with their clonal complexes. The percentage of the strains is indicated above the bars.

Fig. 3. Percentage of MRSA-TetR strains showing resistance to different antibiotics in relation with the predominant clonal complexes (CC398, CC1, and CC5) (ERY, erythromycin; CLI, clindamycin; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; TOB, tobramycin; MUP, mupirocin; FUS, fusidic acid; SXT: trimethoprim-sulfamethoxazole). The percentage of the strains is indicated above the bars.

antibiotics. Most of CC5 strains showed ciprofloxacin-resistance (58%), and rates of 25–50% of aminoglycoside resistance were identified (Fig. 3). All strains showed susceptibility for vancomycin, teicoplanin, and linezolid. Genotype of resistance detected in MRSA-TetR of lineages CC398, CC1 and CC5 The MRSA-TetR strains ascribed to CC398 showed a high prevalence of tet(M) gene (almost 90% of strains), in comparison with strains of CC1 or CC5 lineages. The lnu(A) and vga(A) genes, associated with lincosamide-resistance, were relatively frequent among MRSA-CC398 strains (15% and 24%, respectively). On the other hand, the genes msr(A), msr(B), and mph(C) (conferring resistance to erythromycin), and those conferring resistance to aminoglycosides were highly represented among the CC5 strains. One mupirocin-resistant CC398 strain carried the mup(A) gene and one fusidic-acid-resistant CC398 strain the fusC gene (Fig. 4). Virulence genes detected The genes of the Panton-Valentine leukocidin, toxic shock syndrome toxin or exfoliative toxins were not detected among the strains of this study. Different combinations of hemolysin genes were identified in our collection of MRSA-TetR , and other virulence

Tetracycline resistance has been considered as a good phenotypic marker for the detection of MRSA of CC398 lineage and other animal-related CCs (Camoez et al., 2013; Lozano et al., 2012b). In a previous study, we analyzed the MRSA-TetR strains isolated during 2009–2010 in the same hospital, including isolates implicated in infections and also in nasal colonization (Lozano et al., 2012b); the prevalence of CC398 among the total MRSA in that period was 5.2%, slightly lower than the prevalence detected in the present study with similar type of isolates but different period (2011–2012) (7.9%). Nevertheless, we should point out that in the previous study and in the present one, the MRSA-TetS strains were not MLST-typed, and for this reason, we cannot discard the existence of MRSA CC398 among MRSA-TetS strains, and in that case the percentages could be higher. The apparently increasing trend in the proportion of MRSA CC398 detected in our study, has also been previously detected in other hospitals of Germany and Spain (Camoez et al., 2013; Köck et al., 2013). A possible explanation of the high prevalence of CC398 strains among MRSA of our hospital might be the fact that the analyzed geographic area in our study and in the German report correspond to high density pig-farming areas. For this reason, we decided to study the possible contact of the patients with livestock-animals to establish its possible association with the detection of MRSA-CC398. Nevertheless, CC398 strains were detected in MRSA-TetR of groups-A and -B, with and without livestock-contact, respectively (this lineage was also detected in group-C, with no data about animal-contact). A significant higher value was obtained in the group-A than in the group-B, either if we consider all MRSA-TetR strains (clinical and epidemiological ones) (76% vs 50%), or only those implicated in human infections (75% vs 46%). However, it is interesting also to remark the high prevalence of CC398 among the MRSA-TetR of the group-B. In some previous studies, the MRSA CC398 transmissibility in humans in hospital environments has been studied (Hetem et al., 2013; Wassenberg et al., 2011). They concluded that the transmission of LA-MRSA in healthcare settings is possible but lower than for other MRSA lineages. It is remarkable that, a CC398 clade consisting in methicillin susceptible strains seems to be easily transmittable between humans. However, these strains present very different characteristics, MSSA-t571-CC398 being the more representative one (David et al., 2013; McCarthy et al., 2012; Price et al., 2012). In any case, in spite of that the human-to-human transferability of MRSA CC398 appears to be low, some hospital outbreaks of MRSA CC398 strains have also been detected (Verkade et al., 2012). According to our results, it seems to be an association between livestock-contact and MRSA-CC398 infection/colonization, due to the significant high percentage observed in this group; nevertheless, the parallel considerable detection of CC398 in group-B could indicate the potential human-to-human transmissibility. This possible capacity of transmission of CC398 among humans would represent an important risk for public health, mainly if it occurs in hospital environments. In our study, it was not possible to determine if human-to-human transmission of MRSA-CC398 occurred in the hospital setting or if patients were previously colonized by MRSA-CC398 before

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Fig. 4. Antibiotic resistance genes detected in MRSA-TetR strains in relation with the predominant clonal complexes (CC398, CC1, and CC5).

admission in the hospital. Active surveillance of MRSA carriage at hospital admission is not performed in a routinary way. MRSA-CC398 generally present less virulence factors such as PVL, TSST, exfoliative toxins or enterotoxins when it is compared to other CCs (Lozano et al., 2012a). However, in some cases, these genes have been detected (Argudín et al., 2011; Williamson et al., 2014) and severe infection cases (endocarditis, pneumonia, bacteraemia) have been described (Lozano et al., 2011). Moreover, MRSA strains belonging to CC398 can present a multi-resistance phenotype, which severely limits the therapeutic options (Cuny et al., 2013). So, it has been suggested that MRSA-CC398 strains can act as antibiotic resistance gene reservoir (Kadlec et al., 2012). Among our 59 MRSA-CC398 strains, we found 47 strains (79.6%) with a multiresistance phenotype (resistance to three or more antimicrobial families, in addition to beta-lactams). High percentages of resistance were found for erythromycin, clindamycin, and ciprofloxacin. Similar results were found in the previous study in this hospital (Lozano et al., 2012b), and resistance to these antimicrobials has been detected in CC398 in other studies (Vanderhaeghen et al., 2010). Remarkably, in the case of quinolones, the detection of CC398 strains resistant to this antimicrobial family is less habitual (Cuny et al., 2013). It is essential to remark that macrolides and quinolones, together with tetracyclines, are classified in the category “critically important in human medicine” according to WHO classification and, therefore, it is advisable to reduce the use of these antimicrobials in animals (Collignon et al., 2009). The variant of tet gene detected in higher proportion on the MRSA-CC398 strains was tet(M) although, in many of the strains, a combination of different tet genes was identified. This is in accordance with the results obtained in other studies (Argudín et al., 2011; Feßler et al., 2011; McCarthy et al., 2012). Moreover, the macrolide-lincosamide resistance was mainly mediated by erm(C) which is very frequent in MRSA strains of CC398 lineage (Cuny et al., 2013). In a few strains, other macrolide and/or lincosamide resistance genes were identified which is less common in MRSA CC398 (Argudín et al., 2011). Interestingly, some strains presented the phenotype of macrolide-susceptibility/lincosamide-resistance mediated by lnu(A) and/or vga(A). It is thought that this phenotype is associated with animal clonal lineages of S. aureus (Lozano et al., 2012a). In a few CC398 strains, resistance to gentamicin, tobramycin, mupirocin, or fusidic acid was detected. These resistances are infrequent in this clonal lineage especially the fusidic acid and mupirocin resistance since there is almost uniform susceptibility for these antimicrobials (Chua et al., 2011). In fact, until our knowledge, the gene fus(C) had not been detected before in CC398 strains (Kadlec et al., 2012). Strains of other CCs were detected. So, CC1 was identified in 19 strains and this CC corresponded to 20% of the patients with livestock-contact (group-A) and 21% of the patients

without livestock-contact (group-B). This clonal lineage seems to have a wide host range (Cuny et al., 2013). So, strains belonging to CC1 have been detected in hospital environments (Monaco et al., 2013), and in different farm animals (Franco et al., 2011). It has been hypothesized that CC1-t127 strains could be assigned to two genetically different clusters (porcine and human), CC1-t127 representing another clonal lineage of LA-MRSA (Franco et al., 2011). The CC1 strains showed higher percentages of erythromycin and clindamycin resistance than CC398 strains. This resistance was mediated by the erm(C) gene in most of our CC1 strains. This gene, in combination with erm(A) gene, has been already detected in strains of this clonal lineage (Franco et al., 2011). Regarding to CC5 which was found in 12 strains, this clonal lineage is mainly found in Spanish hospitals (Lozano et al., 2013; Pérez-Roth et al., 2013). The lineage CC5 is characterized by ciprofloxacin, tobramycin/kanamycin and erythromycin resistance (Lozano et al., 2013), which is in accordance with our results. None of the strains included in this study presented the genes encoding Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin, or exfoliative toxins. The absence of these toxin genes is common in CC398 strains. However, some cases of MRSA CC398 strains harbouring PVL genes have been described (Williamson et al., 2014). Interestingly, the CC398 strains did no present the genes of the IEC system. Two clusters of CC398 have been identified and the lack of IEC genes seems to be associated with an animal reservoir (Uhlemann et al., 2012). In contrast, one characteristic of CC1 strains of human cluster is that these strains present the gene sak (Franco et al., 2011; Monaco et al., 2013). This gene was identified in our CC1 strains (IEC type E), which suggest that, in this case, our strains might belong to the human clade. Conclusion CC398 strains are very frequently detected among MRSA-TetR strains in the studied hospital (60.2%). Although CC398 lineage was detected in significant higher proportion in patients with livestockcontact than in those with no livestock-contact (76% vs 50%), its considerable prevalence in the second group indicates the capacity of human-to-human transmission of this clonal lineage, even in absence of this risk factor. All of these data should be taken into account, especially because this microorganism is highly capable of capturing multiple antibiotic resistance genes. Acknowledgements This work was supported by Project SAF2012-35474 from the Ministerio de Economía y Competitividad of Spain and the Fondo Europeo de Desarrollo Regional (FEDER). Daniel Benito has a

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predoctoral fellowship from the Ministerio de Economía y Competitividad of Spain, and Carmen Lozano is financed by project SAF2012-35474. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijmm. 2014.09.004. References Argudín, M.A., Tenhagen, B.A., Fetsch, A., Sachsenröder, J., Käsbohrer, A., Schroeter, A., 2011. Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources. Appl. Environ. Microbiol. 77, 3052–3060. Armand-Lefevre, L., Ruimy, R., Andremont, A., 2005. Clonal comparison of Staphylococcus aureus isolates from healthy pig farmers, human controls, and pigs. Emerg. Infect. Dis. 11, 711–714. Benito, D., Lozano, C., Gómez-Sanz, E., Zarazaga, M., Torres, C., 2013. Detection of methicillin-susceptible Staphylococcus aureus ST398 and ST133 strains in gut microbiota of healthy humans in Spain. Microb. Ecol. 66, 105–111. Camoez, M., Sierra, J.M., Pujol, M., Hornero, A., Martin, R., Domínguez, M.A., 2013. Prevalence and molecular characterization of methicillin-resistant Staphylococcus aureus ST398 resistant to tetracycline at a Spanish hospital over 12 years. PLoS One 8, e72828. Clinical Laboratory Standards Institute (CLSI), 2013. Performance standards for antimicrobial susceptibility testing. Twenty third informational supplement. In: M100-S23. National Committee for Clinical Laboratory Standards, Wayne, PA. Chua, K., Laurent, F., Coombs, G., Grayson, M.L., Howden, B.P., 2011. Antimicrobial resistance: not community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA)! A clinician’s guide to community MRSA – its evolving antimicrobial resistance and implications for therapy. Clin. Infect. Dis. 52, 99–114. Collignon, P., Powers, J.H., Chiller, T.M., Aidara-Kane, A., Aarestrup, F.M., 2009. World Health Organization ranking of antimicrobials according to their importance in human medicine: a critical step for developing risk management strategies for the use of antimicrobials in food productions animals. Clin. Infect. Dis. 49, 132–141. Cuny, C., Köck, R., Witte, W., 2013. Livestock associated MRSA (LA-MRSA) and its relevance for humans in Germany. Int. J. Med. Microbiol. 303, 331–337. David, M.Z., Siegel, J., Lowy, F.D., Zychowski, D., Taylor, A., Lee, C.J., et al., 2013. Asymptomatic carriage of sequence type 398, spa type t571 methicillinsusceptible Staphylococcus aureus in an urban jail: a newly emerging, transmissible pathogenic strain. J. Clin. Microbiol. 51, 2443–2447. European Committee on Antimicrobial Susceptibility Testing (EUCAST), 2014. Breakpoint tables for interpretation of MICs and zone diameters. Version 4.0, http://www.eucast.org. Franco, A., Hasman, H., Iurescia, M., Lorenzetti, R., Stegger, M., Pantosti, A., et al., 2011. Molecular characterization of spa type t127, sequence type 1 methicillin-resistant Staphylococcus aureus from pigs. J. Antimicrob. Chemother. 66, 1231–1235. Feßler, A.T., Kadlec, K., Hassel, M., Hauschild, T., Eidam, C., Ehricht, R., et al., 2011. Characterization of methicillin-resistant Staphylococcus aureus isolates from food and food products of poultry origin in Germany. Appl. Environ. Microbiol. 77, 7151–7157. Garcia-Graells, C., Antoine, J., Larsen, J., Catry, B., Skov, R., Denis, O., 2012. Livestock veterinarians at high risk of acquiring methicillin-resistant Staphylococcus aureus ST398. Epidemiol. Infect. 140, 383–389. Hetem, D.J., Bootsma, M.C., Troelstra, A., Bonten, M.J., 2013. Transmissibility of livestock-associated methicillin-resistant Staphylococcus aureus. Emerg. Infect. Dis. 19, 1797–1802.

Kadlec, K., Feßler, T., Hauschild, T., Schwarz, S., 2012. Novel and uncommon antimicrobial resistance genes in livestock-associated methicillin-resistant Staphylococcus aureus. Clin. Microbiol. Infect. 18, 745–755. Köck, R., Schaumburg, F., Mellmann, A., Köksal, M., Jurke, A., Becker, K., et al., 2013. Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) as causes of human infection and colonization in Germany. PLoS One 8, e55040. Lozano, C., Aspiroz, C., Ezpeleta, A.I., Gómez-Sanz, E., Zarazaga, M., Torres, C., 2011. Empyema caused by MRSA ST398 with atypical resistance profile, Spain. Emerg. Infect. Dis. 17, 138–140. Lozano, C., Aspiroz, C., Sáenz, Y., Ruiz-García, M., Royo-García, G., Gómez-Sanz, E., et al., 2012a. Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus and other staphylococci of animal and human origin. J. Antimicrob. Chemother. 67, 2804–2808. Lozano, C., Rezusta, A., Gómez, P., Gómez-Sanz, E., Báez, N., Martin-Saco, G., et al., 2012b. High prevalence of spa types associated with the clonal lineage CC398 among tetracycline-resistant methicillin-resistant Staphylococcus aureus strains in a Spanish hospital. J. Antimicrob. Chemother. 67, 330–334. Lozano, C., Porres-Osante, N., Crettaz, J., Rojo-Bezares, B., Benito, D., Olarte, I., et al., 2013. Changes in genetic lineages, resistance, and virulence in clinical methicillin-resistant Staphylococcus aureus in a Spanish hospital. J. Infect. Chemother. 19, 233–242. McCarthy, A.J., van Wamel, W., Vandendriessche, S., Larsen, J., Denis, O., GarciaGraells, C., et al., 2012. Staphylococcus aureus CC398 clade associated with human-to-human transmission. Appl. Environ. Microbiol. 78, 8845–8848. Monaco, M., Pedroni, P., Sanchini, A., Bonomini, A., Indelicato, A., Pantosti, A., 2013. Livestock-associated methicillin-resistant Staphylococcus aureus responsible for human colonization and infection in an area of Italy with high density of pig farming. BMC Infect. Dis. 13, 258. Pérez-Roth, E., Potel-Alvarellos, C., Espartero, X., Constela-Caramés, L., MéndezÁlvarez, S., Alvarez-Fernández, M., 2013. Molecular epidemiology of plasmidmediated high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus in four Spanish health care settings. Int. J. Med. Microbiol. 303, 201–204. Price, L.B., Stegger, M., Hasman, H., Aziz, M., Larsen, J., Andersen, P.S., et al., 2012. Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock. MBio 21, 3, e00305–e311. Uhlemann, A.C., Porcella, S.F., Trivedi, S., Sullivan, S.B., Hafer, C., Kennedy, A.D., et al., 2012. Identification of a highly transmissible animal-independent Staphylococcus aureus ST398 clone with distinct genomic and cell adhesion properties. mBio 28, 3. VanCleef, B.A., Monnet, D.L., Voss, A., 2011. Livestock-associated methicillinresistant Staphylococcus aureus in humans. Eur. Emerg. Infect. Dis. 17, 502–505. Vanderhaeghen, W., Hermans, K., Haesebrouck, F., Butaye, P., 2010. Methicillin resistant Staphylococcus aureus (MRSA) in food production animals. Epidemiol. Infect. 138, 606–625. Van Wamel, W.J., Rooijakkers, S.H., Ruyken, M., van Kessel, K.P., van Strijp, J.A., 2006. The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on beta hemolysin converting bacteriophages. J. Bacteriol. 188, 1310–1315. Verkade, E., Bosch, T., Hendriks, Y., Kluytmans, J., 2012. Outbreak of methicillin resistant Staphylococcus aureus ST398 in a Dutch nursing home. Infect. Control Hosp. Epidemiol. 33, 624–626. Wassenberg, M.W., Bootsma, M.C., Troelstra, A., Kluytmans, J.A., Bonten, M.J., 2011. Transmissibility of livestock-associated methicillin-resistant Staphylococcus aureus (ST398) in Dutch hospitals. Clin. Microbiol. Infect. 17, 316–319. Williamson, D.A., Bakker, S., Coombs, G.W., Tan, H.L., Monecke, S., Heffernan, H., 2014. Emergence and molecular characterization of clonal complex 398 (CC398) methicillin-resistant Staphylococcus aureus (MRSA) in New Zealand. J. Antimicrob. Chemother. 69, 1428–1430. Zhang, K., McClure, J.A., Elsayed, S., Louie, T., Conly, J.M., 2005. Novel multiplex PCR assay for characterization and concomitant subtyping of Staphylococcal Cassette Chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 43, 5026–5033.