Abstract
To determine the antibiotic resistance and fingerprint profiles of methicillin-resistant coagulase-negative staphylococci (MRCoNS) from animal infections among different practices and examine the history of antibiotic treatment.
Isolates were identified by mass spectrometry and tested for antimicrobial resistance by broth dilution, microarrays and sequence analysis of the topoisomerases. Diversity was assessed by PFGE, icaA PCR and staphylococcal cassette chromosome mec (SCCmec), arginine catabolic mobile element (ACME) and multilocus sequence typing. Clinical records were examined retrospectively.
MRCoNS were identified as Staphylococcus epidermidis (n = 20), Staphylococcus haemolyticus (n = 17), Staphylococcus hominis (n = 3), Staphylococcus capitis (n = 1), Staphylococcus cohnii (n = 1) and Staphylococcus warneri (n = 1). PFGE identified one clonal lineage in S. hominis isolates and several in S. haemolyticus and S. epidermidis. Fourteen sequence types were identified in S. epidermidis, with sequence type 2 (ST2) and ST5 being predominant. Ten isolates contained SCCmec IV, seven contained SCCmec V and the others were non-typeable. ACMEs were detected in 11 S. epidermidis isolates. One S. hominis and 10 S. epidermidis isolates were icaA positive. In addition to mecA-mediated β-lactam resistance, the most frequent resistance was to gentamicin/kanamycin [aac(6′)-Ie–aph(2′)-Ia, aph(3′)-III] (n = 34), macrolides/lincosamides [erm(C), erm(A), msr, lnu(A)] (n = 31), tetracycline [tet(K)] (n = 22), streptomycin [str, ant(6)-Ia] (n = 20), trimethoprim [dfr(A), dfr(G)] (n = 17), sulfamethoxazole (n = 34) and fluoroquinolones [amino acid substitutions in GyrA and GrlA] (n = 30). Clinical data suggest selection through multiple antibiotic courses and emphasize the importance of accurate diagnosis and antibiograms.
MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary clinics.
Introduction
Coagulase-negative staphylococci (CoNS) are frequently found on the skin and mucous membranes of humans and animals.1 They are opportunistic pathogens and are one of the most frequent causes of nosocomial infections in humans, which are mainly associated with immune-compromised patients or with the implantation of medical devices.2–6Staphylococcus epidermidis is the most frequent CoNS causing infection in humans, and 70% of the S. epidermidis strains circulating in the human hospital environment have been estimated to be resistant to methicillin and most of them display additional resistance to other classes of antibiotics.7 The acquisition of methicillin resistance in staphylococci results from the recombinase-mediated insertion of the staphylococcal chromosomal cassette mec (SCCmec), the mobile genetic element that carries mecA.8,9 The mecA gene encodes the binding protein PBP2a, which mediates resistance to all β-lactam antibiotics in staphylococci.10 Other methicillin-resistant CoNS (MRCoNS), such as Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus sciuri, Staphylococcus warneri and Staphylococcus saprophyticus, have also been described as causes of clinical human infections.11–13 In some S. epidermidis strains, the SCCmec elements have been found to be associated with the arginine catabolic mobile element (ACME), enhancing fitness and the ability to colonize the host.14–16 These characteristics associated with the ability to produce a biofilm are important factors for establishing CoNS, especially S. epidermidis, as nosocomial pathogens.2,17,18
In veterinary medicine, many different classes of antibiotics are used for the treatment of infections. The use of such antibiotics has likely selected for an antibiotic-resistant commensal flora, as healthy pets and horses have been found to be colonized with MRCoNS.4,19–22 However, very few reports describe cases of infections caused by MRCoNS in these animals,23–25 although several studies have reported infections with methicillin-susceptible CoNS.26–30 In the past 4 years, MRCoNS have been isolated from the infection sites of pets and horses in Switzerland. The genetic backgrounds of these multidrug-resistant clinical isolates and their clonal relationships remained to be elucidated. This study provides the first substantial molecular characterization of MRCoNS associated with infections in pets and horses and determines whether specific clones are becoming established in veterinary settings. The history of antibiotic usage as well as the treatment and outcome of the infections are also provided to support the hypothesis that several courses of different antibiotics may have selected for multidrug-resistant CoNS. This study may also serve as a basis for future epidemiological and prevalence studies of MRCoNS circulating in veterinary clinics and other animal environments.
Materials and methods
Sample collection, isolation and identification
Samples were taken by veterinarians from different infection sites of pets and horses that did not respond to antibiotic therapy and sent for identification of the causative agents and antibiograms to the Centre for Zoonoses, Bacterial Animal Diseases and Antibiotic Resistance (ZOBA) of the Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland, the IDEXX Diavet Laboratory, Bäch, Switzerland, or the Laboratory Laupeneck AG, Bern, Switzerland. Isolates of MRCoNS that appeared to be the primary pathogens [either as single pathogenic agent (n = 40) or together with a second pathogen (n = 3)] were kept at −80°C and made available for this study. A total of 43 isolates were collected between 2005 and 2011 (see Tables 1 and 2). They were routinely cultivated on Tryptone soy agar containing 5% sheep blood (TSA-SB) (Oxoid Ltd, Basingstoke, England) and incubated aerobically for 18 h at 37°C. Species identification was determined phenotypically using Vitek2 (bioMérieux, Marcy l'Étoile, France) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDITOF-MS) (Microflex LT, Bruker Daltonik, Bremen, Germany).
Origin and resistance profile of methicillin-resistant S. epidermidis isolated from infection sites of animals
| Isolate (n = 20) | Year of isolation | Animal | Infection | Sequence type | Antibiotic resistance properties and resistance breakpoints (mg/L) | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| OXA | PEN | GEN/KAN | KAN | STR | STH | ERY | CLI | TMP | TET | CHL | TIA | MUP | FUS | SMX | CIP | ||||||
| >0.25 | >0.125 | >1/>16 | >16 | >16 | ND | >2 | >0.5 | >4 | >2 | >8 | NA | >256 | >1 | >256 | >1 | ||||||
| GyrA | GrlA | ||||||||||||||||||||
| CSNO38 | 2005 | horse | dermis | ST446 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia, str | sat4 | erm(C) | erm(C) | dfr(A) | tet(K) | R | ||||||
| KM794-06 | 2006 | horse | abscess | ST89 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | tet(K) | |||||||||||||
| KM1527-07 | 2007 | cat | joint | ST22 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | dfr(A) | S84Y | S80F | |||||||||||
| KM827-09 | 2009 | cat | respiratory tract | ST59 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | str | sat4 | tet(K) | R | |||||||||
| KM505-09 | 2009 | cat | urinary tract | ST22 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | dfr(A) | tet(K) | catpC223 | R | S84Y | D84Y | ||||||
| KM1077-09 | 2009 | dog | abscess | ST2 | mecA | blaZ | dfr(A) | mupR | R | R | S84Y | S80Y/D84Y | |||||||||
| KM92-09 | 2009 | dog | abscess | ST2 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | dfr(A) | R | ||||||||||||
| KM825-09 | 2009 | horse | abscess | ST451 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | R | S84F | D84Y | |||||||||||
| IMD1265-11 | 2011 | horse | dermis | ST69 | mecA | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | tet(K) | mupR | |||||||||||
| IMD1274-11 | 2011 | cat | dermis | ST5 | mecA | blaZ | erm(C) | erm(C) | tet(K) | S84F | S80Y | ||||||||||
| IMD1763-11 | 2011 | cat | urinary tract | ST81 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | tet(K) | |||||||||||||
| IMD1270-11 | 2011 | cat | abscess | ST2 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | erm(C) | erm(C) | dfr(A) | tet(K) | vga(A) | R | S84F | S80F/D84Y | |||
| IMD1269-11 | 2011 | cat | urinary tract | ST445 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C), msr, mph(C) | erm(C) | R | R | S84F | S80Y | ||||||||
| IMD1528-11 | 2011 | cat | dermis | ST448 | mecA | blaZ | aph(3′)-III | erm(C), mph(C) | erm(C) | dfr(G) | |||||||||||
| IMD1776-11 | 2011 | cat | eye | ST286 | mecA | blaZ | R | R | erm(C) | erm(C) | dfr(G) | ||||||||||
| IMD1766-11 | 2011 | dog | ear | ST5 | mecA | blaZ | erm(C) | erm(C) | R | S84F | S80F/D84Y | ||||||||||
| IMD1778-11 | 2011 | dog | respiratory tract | ST5 | mecA | blaZ | R | S84F | S80Y | ||||||||||||
| KM1385-1972 | 2011 | dog | joint | ST450 | mecA | blaZ | R | R | |||||||||||||
| IMD1764-11 | 2011 | dog | respiratory tract | ST449 | mecA | blaZ | erm(C), mph(C) | erm(C) | R | ||||||||||||
| IMD1765-11 | 2011 | dog | respiratory tract | ST2 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | str | sat4 | erm(C) | erm(C) | dfr(A) | catpC221 | R | R | S84F | S80F/D84Y | ||||
| Isolate (n = 20) | Year of isolation | Animal | Infection | Sequence type | Antibiotic resistance properties and resistance breakpoints (mg/L) | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| OXA | PEN | GEN/KAN | KAN | STR | STH | ERY | CLI | TMP | TET | CHL | TIA | MUP | FUS | SMX | CIP | ||||||
| >0.25 | >0.125 | >1/>16 | >16 | >16 | ND | >2 | >0.5 | >4 | >2 | >8 | NA | >256 | >1 | >256 | >1 | ||||||
| GyrA | GrlA | ||||||||||||||||||||
| CSNO38 | 2005 | horse | dermis | ST446 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia, str | sat4 | erm(C) | erm(C) | dfr(A) | tet(K) | R | ||||||
| KM794-06 | 2006 | horse | abscess | ST89 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | tet(K) | |||||||||||||
| KM1527-07 | 2007 | cat | joint | ST22 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | dfr(A) | S84Y | S80F | |||||||||||
| KM827-09 | 2009 | cat | respiratory tract | ST59 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | str | sat4 | tet(K) | R | |||||||||
| KM505-09 | 2009 | cat | urinary tract | ST22 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | dfr(A) | tet(K) | catpC223 | R | S84Y | D84Y | ||||||
| KM1077-09 | 2009 | dog | abscess | ST2 | mecA | blaZ | dfr(A) | mupR | R | R | S84Y | S80Y/D84Y | |||||||||
| KM92-09 | 2009 | dog | abscess | ST2 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | dfr(A) | R | ||||||||||||
| KM825-09 | 2009 | horse | abscess | ST451 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | R | S84F | D84Y | |||||||||||
| IMD1265-11 | 2011 | horse | dermis | ST69 | mecA | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | tet(K) | mupR | |||||||||||
| IMD1274-11 | 2011 | cat | dermis | ST5 | mecA | blaZ | erm(C) | erm(C) | tet(K) | S84F | S80Y | ||||||||||
| IMD1763-11 | 2011 | cat | urinary tract | ST81 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | tet(K) | |||||||||||||
| IMD1270-11 | 2011 | cat | abscess | ST2 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | erm(C) | erm(C) | dfr(A) | tet(K) | vga(A) | R | S84F | S80F/D84Y | |||
| IMD1269-11 | 2011 | cat | urinary tract | ST445 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C), msr, mph(C) | erm(C) | R | R | S84F | S80Y | ||||||||
| IMD1528-11 | 2011 | cat | dermis | ST448 | mecA | blaZ | aph(3′)-III | erm(C), mph(C) | erm(C) | dfr(G) | |||||||||||
| IMD1776-11 | 2011 | cat | eye | ST286 | mecA | blaZ | R | R | erm(C) | erm(C) | dfr(G) | ||||||||||
| IMD1766-11 | 2011 | dog | ear | ST5 | mecA | blaZ | erm(C) | erm(C) | R | S84F | S80F/D84Y | ||||||||||
| IMD1778-11 | 2011 | dog | respiratory tract | ST5 | mecA | blaZ | R | S84F | S80Y | ||||||||||||
| KM1385-1972 | 2011 | dog | joint | ST450 | mecA | blaZ | R | R | |||||||||||||
| IMD1764-11 | 2011 | dog | respiratory tract | ST449 | mecA | blaZ | erm(C), mph(C) | erm(C) | R | ||||||||||||
| IMD1765-11 | 2011 | dog | respiratory tract | ST2 | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | str | sat4 | erm(C) | erm(C) | dfr(A) | catpC221 | R | R | S84F | S80F/D84Y | ||||
CHL, chloramphenicol; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; FUS, fusidic acid; GEN, gentamicin; KAN, kanamycin; MUP, mupirocin; OXA, oxacillin; PEN, penicillin; STR, streptomycin; STH, streptothricin; TET, tetracycline; TIA, tiamulin; TMP, trimethoprim, SMX, sulfamethoxazole; ND, not defined, susceptibility to streptothricin was not measured, only the gene was detected; NA, no resistance breakpoint available for tiamulin [resistance to tiamulin was attributed in the presence of the vga(A) gene (MIC >4 mg/L)]; R, resistant phenotype, no resistance genes were determined; blank spaces indicate either no resistance or no mutations.
The MIC breakpoints (in mg/L) that determine resistance were those recommended by EUCAST for staphylococci (www.eucast.org). Resistance breakpoints for streptomycin and kanamycin were those recommended by the French Society for Microbiology (www.sfm-microbiologie.org) and the resistance breakpoint for sulfamethoxazole was that recommended by the CLSI.36
Antibiotic resistance genes and their functions are indicated as follows: mecA, methicillin-resistance gene encoding PBP2a for resistance to all β-lactam antibiotics; blaZ, β-lactamase gene; aac(6′)-Ie–aph(2′)-Ia, aminoglycoside acetyltransferase and phosphotransferase tandem genes; aph(3′)-III, kanamycin phosphotransferase; ant(6)-Ia, streptomycin adenylnucleotidyltransferase gene; str, streptomycin adenyltransferase gene; sat4, strepthothricin acetyltransferase gene; erm(C), macrolide, lincosamide and streptogramin B 23S rRNA methylase gene; msr, macrolide and streptogramin ATP binding transporter gene; mph(C), macrolide phosphotransferase gene; mupR, isoleucyl-tRNA synthetase gene; dfr(A), dfr(G), trimethoprim resistance dihydrofolate reductase gene; tet(K), tetracycline efflux resistance gene; catpC221,catpC223, chloramphenicol acetyltransferase gene; vga(A), pleuromutilin and streptogramin A ATP binding transporter gene.
Origin and resistance profile of methicillin-resistant S. haemolyticus, S. hominis, S. capitis, S. cohnii and S. warneri isolated from infection sites of animals
| Strain/isolate | Year of isolation | Animal | Infection | Antibiotic resistance properties and resistance breakpoints (mg/L) | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| OXA | PEN | GEN/KAN | KAN | STR | STH | ERY | CLI | TMP | TET | CHL | TIA | FUS | SMX | CIP | |||||
| >0.25 | >0.125 | >1/>16 | >16 | >16 | ND | >2 | >0.5 | >4 | >2 | >8 | NA | >1 | >256 | >1 | |||||
| GyrA | GrlA | ||||||||||||||||||
| S. haemolyticus (n = 17) | |||||||||||||||||||
| KM827-07 | 2007 | horse | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | erm(C), msr, mph(C) | erm(C), lnu(A) | dfr(G) | tet(K) | catpC221 | vga(A) | R | S84L | ||||
| KM1758-08 | 2008 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr, mph(C) | R | dfr(G) | vga(A) | R | S84L | ||||
| KM1632-08 | 2008 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr, mph(C) | R | S84L | |||||||
| KM785-09 | 2009 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | msr, mph(C) | dfr(G) | tet(K) | catpC221 | R | S84L | ||||||
| KM1183-09 | 2009 | horse | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | erm(C), msr, mph(C) | erm(C) | tet(K) | catpC223 | DS | R | S84L | ||||
| KM1230-09 | 2009 | horse | respiratory tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | dfr(G) | tet(K) | R | S84L | ||||||
| IMD1272-11 | 2011 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | str | msr | lnu(A) | tet(K) | catpC221 | R | S84L | ||||||
| IMD1277-11 | 2011 | cat | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr | vga(A) | R | R | S84F | |||||
| IMD1517-11 | 2011 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | dfr(G) | tet(K) | R | S84F | ||||||
| IMD1519-11 | 2011 | cat | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | sat4 | erm(C) | erm(C) | tet(K) | S84L | |||||||
| IMD1521-11 | 2011 | cat | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr | dfr(G) | R | S84L | D84Y | |||||
| IMD1266-11 | 2011 | dog | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | erm(C) | erm(C), lnu(A) | R | S84L | ||||||||
| IMD1397-11 | 2011 | dog | ear | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | msr | lnu(A) | tet(K) | R | S84F | |||||||
| IMD1532-11 | 2011 | dog | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | sat4 | msr | dfr(G) | tet(K) | R | S84L | ||||||
| IMD1761-11 | 2011 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | erm(C) | erm(C) | R | S84L | ||||||
| IMD1768-11 | 2011 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | str | sat4 | erm(C) | erm(C) | tet(K) | catpC223 | R | S84L | ||||
| IMD1775-11 | 2011 | dog | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | erm(C), msr | erm(C) | dfr(G) | tet(K) | R | R | S84L | |||
| S. hominis (n = 3) | |||||||||||||||||||
| IMD1515-11 | 2011 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | erm(C) | erm(C) | tet(K) | R | S84F | G84Y | ||||||
| IMD1516-11 | 2011 | dog | joint | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | tet(K) | R | S84F | G84Y | |||||||
| IMD1762-11 | 2011 | dog | ear | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | tet(K) | R | S84F | G84Y | |||||||
| S. capitis (n = 1) | |||||||||||||||||||
| KM1385-1970 | 2011 | dog | joint | mecA | R | ||||||||||||||
| S. cohnii (n = 1) | |||||||||||||||||||
| IMD1771-11 | 2011 | dog | urinary tract | mecA | erm(A) | erm(A) | DS | R | |||||||||||
| S. warneri (n = 1) | |||||||||||||||||||
| IMD1530-11 | 2011 | dog | ear | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | str | erm(C) | erm(C) | catpC221 | |||||||||
| Strain/isolate | Year of isolation | Animal | Infection | Antibiotic resistance properties and resistance breakpoints (mg/L) | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| OXA | PEN | GEN/KAN | KAN | STR | STH | ERY | CLI | TMP | TET | CHL | TIA | FUS | SMX | CIP | |||||
| >0.25 | >0.125 | >1/>16 | >16 | >16 | ND | >2 | >0.5 | >4 | >2 | >8 | NA | >1 | >256 | >1 | |||||
| GyrA | GrlA | ||||||||||||||||||
| S. haemolyticus (n = 17) | |||||||||||||||||||
| KM827-07 | 2007 | horse | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | erm(C), msr, mph(C) | erm(C), lnu(A) | dfr(G) | tet(K) | catpC221 | vga(A) | R | S84L | ||||
| KM1758-08 | 2008 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr, mph(C) | R | dfr(G) | vga(A) | R | S84L | ||||
| KM1632-08 | 2008 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr, mph(C) | R | S84L | |||||||
| KM785-09 | 2009 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | msr, mph(C) | dfr(G) | tet(K) | catpC221 | R | S84L | ||||||
| KM1183-09 | 2009 | horse | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | erm(C), msr, mph(C) | erm(C) | tet(K) | catpC223 | DS | R | S84L | ||||
| KM1230-09 | 2009 | horse | respiratory tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | dfr(G) | tet(K) | R | S84L | ||||||
| IMD1272-11 | 2011 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | str | msr | lnu(A) | tet(K) | catpC221 | R | S84L | ||||||
| IMD1277-11 | 2011 | cat | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr | vga(A) | R | R | S84F | |||||
| IMD1517-11 | 2011 | cat | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | dfr(G) | tet(K) | R | S84F | ||||||
| IMD1519-11 | 2011 | cat | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | sat4 | erm(C) | erm(C) | tet(K) | S84L | |||||||
| IMD1521-11 | 2011 | cat | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | msr | dfr(G) | R | S84L | D84Y | |||||
| IMD1266-11 | 2011 | dog | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | erm(C) | erm(C), lnu(A) | R | S84L | ||||||||
| IMD1397-11 | 2011 | dog | ear | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | ant(6)-Ia | msr | lnu(A) | tet(K) | R | S84F | |||||||
| IMD1532-11 | 2011 | dog | dermis | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | sat4 | msr | dfr(G) | tet(K) | R | S84L | ||||||
| IMD1761-11 | 2011 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | erm(C) | erm(C) | R | S84L | ||||||
| IMD1768-11 | 2011 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | str | sat4 | erm(C) | erm(C) | tet(K) | catpC223 | R | S84L | ||||
| IMD1775-11 | 2011 | dog | urinary tract | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | ant(6)-Ia | sat4 | erm(C), msr | erm(C) | dfr(G) | tet(K) | R | R | S84L | |||
| S. hominis (n = 3) | |||||||||||||||||||
| IMD1515-11 | 2011 | dog | abscess | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | aph(3′)-III | erm(C) | erm(C) | tet(K) | R | S84F | G84Y | ||||||
| IMD1516-11 | 2011 | dog | joint | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | tet(K) | R | S84F | G84Y | |||||||
| IMD1762-11 | 2011 | dog | ear | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | erm(C) | erm(C) | tet(K) | R | S84F | G84Y | |||||||
| S. capitis (n = 1) | |||||||||||||||||||
| KM1385-1970 | 2011 | dog | joint | mecA | R | ||||||||||||||
| S. cohnii (n = 1) | |||||||||||||||||||
| IMD1771-11 | 2011 | dog | urinary tract | mecA | erm(A) | erm(A) | DS | R | |||||||||||
| S. warneri (n = 1) | |||||||||||||||||||
| IMD1530-11 | 2011 | dog | ear | mecA | blaZ | aac(6′)-Ie – aph(2′)-Ia | str | erm(C) | erm(C) | catpC221 | |||||||||
CHL, chloramphenicol; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; FUS, fusidic acid; GEN, gentamicin; KAN, kanamycin; OXA, oxacillin; PEN, penicillin; STR, streptomycin; STH, streptothricin; TET, tetracycline; TIA, tiamulin; TMP, trimethoprim; SMX, sulfamethoxazole; ND, not defined, susceptibility to streptothricin was not measured, only the gene was detected; NA, no resistance breakpoint available for tiamulin [resistance to tiamulin was attributed in the presence of the vga(A) gene (MIC >4 mg/L)]; DS, decreased susceptibility to tiamulin with MIC >4 mg/L); R, resistant phenotype, no resistance genes were determined; blank spaces indicate either no resistance or no mutations.
The MIC breakpoints (in mg/L) that determine resistance were those recommended by EUCAST for staphylococci (www.eucast.org). Resistance breakpoints for streptomycin and kanamycin were those recommended by the French Society for Microbiology (www.sfm-microbiologie.org) and the resistance breakpoint for sulfamethoxazole was that recommended by the CLSI.36
Antibiotic resistance genes and their functions are indicated as follows: mecA, methicillin-resistance gene encoding PBP2a for resistance to all β-lactam antibiotics; blaZ, β-lactamase gene; aac(6′)-Ie–aph(2′)-Ia, aminoglycoside acetyltransferase and phosphotransferase tandem genes; aph(3′)-III, kanamycin phosphotransferase; ant(6)-Ia, streptomycin adenylnucleotidyltransferase gene; str, streptomycin adenyltransferase gene; sat4, strepthotricin acetyltransferase gene; erm(C), macrolide, lincosamide and streptogramin B 23S rRNA methylase gene; msr, macrolide and streptogramin ATP binding transporter gene; mph(C), macrolide phosphotransferase gene; dfr(A), dfr(G), trimethoprim resistance dihydrofolate reductase gene; lnu(A), lincosamide nucleotidyltransferase gene; tet(K), tetracycline efflux resistance gene; catpC221,catpC223, chloramphenicol acetyl transferase gene; vga(A), pleuromutilin and streptogramin A ATP binding transporter gene.
Genotyping
PFGE was performed on DNA digested with SmaI as described previously.23 PFGE was run on a CHEF DRIII apparatus (Bio-Rad, Hercules, CA, USA) for 21 h at 6 V/cm and with pulse time ramping from 5 to 40 s at 12°C. The PFGE profiles were defined on the basis of DNA banding patterns in compliance with the criteria of Tenover et al.31 for bacterial strain typing using the BioNumerics software (version 6.6, Applied Maths, Saint-Martens-Latem, Belgium).
SCCmec typing was determined by multiplex PCR.32 SCCmec types were defined by the combination of the type of ccr complex and the class of mec complex: SCCmec type I (mec complex B, ccrAB1), SCCmec type II (mec complex A, ccrAB2), SCCmec type III (mec complex A, ccrAB3), SCCmec type IV (mec complex B, ccrAB2) and SCCmec type V (mec complex C, ccrC). SCCmec was classified as non-typeable when the ccr complex, the mec complex or both could not be amplified by PCR.
The presence and type of ACMEs were determined by PCR using the primer pairs AIPS.27-AIPS.28 (arcA) and AIPS.45-AIPS.46 (opp3A) as described previously.33 ACMEs were classified into three allotypes: ACME type I containing both the arc and opp-3 gene clusters, ACME type II containing arc but not opp-3 and ACME type 3 containing opp-3 but not arc.14 The presence of the biofilm-formation operon ica was determined by amplification of the icaA gene by PCR.34S. epidermidis samples were characterized by multilocus sequence typing (MLST).35 PCR amplifications were routinely performed using FIREPol DNA polymerase (Solis BioDyne, Tartu, Estonia), except for SCCmec typing, which was performed with the Expand Long Template PCR System (Roche Applied Science, Rotkreuz, Switzerland).
Determination of the antibiotic resistance profile
MICs were determined in Mueller–Hinton broth by use of custom Sensititre NLEUST plates (Trek Diagnostics Systems, East Grinstead, UK; MCS diagnostics BV, JL Swalmen, the Netherlands). The MIC breakpoints determining resistance were those recommended for staphylococci by EUCAST (www.eucast.org), except for streptomycin and kanamycin, for which breakpoints came from the French Society for Microbiology (www.sfm-microbiologie.org), and sulfamethoxazole, for which they came from the CLSI.36 No breakpoint was available for tiamulin and resistance was attributed after the detection of a tiamulin resistance gene. The antimicrobial agents tested and breakpoints used consisted of chloramphenicol (>8 mg/L), ciprofloxacin (>1 mg/L), clindamycin (>0.5 mg/L), erythromycin (>2 mg/L), fusidic acid (>1 mg/L), gentamicin (>1 mg/L), kanamycin (>16 mg/L), linezolid (>4 mg/L), mupirocin (>256 mg/L), oxacillin (>0.25 mg/L), penicillin (>0.125 mg/L), quinupristin/dalfopristin (>4 mg/L), rifampicin (>0.5 mg/L), streptomycin (>16 mg/L), tetracycline (>2 mg/L), tiamulin (resistance breakpoint not available), trimethoprim (>4 mg/L), sulfamethoxazole (>256 mg/L) and vancomycin (>2 mg/L). Antibiotic resistance genes were detected using a custom-made microarray (AMR + ve-2 array tubes, Alere Technologies GmbH, Jena, Germany).37 The microarray results were analysed using the IconoClust program (Alere) and the signals obtained were interpreted visually. The acquired trimethoprim resistance dihydrofolate reductase gene dfr(A) in S. epidermidis was distinguished from the chromosomal dfr(A) ( = folA) by PCR using one primer specific to dfr(A) and one primer specific to IS431, which is only situated downstream of the acquirable dfr(A) gene and not downstream of the chromosomal dfr(A) of S. epidermidis (Table S1, available as Supplementary data at JAC Online).
Mutations in the fluoroquinolone resistance coding region of the topoisomerase II (GyrA and GyrB) and IV (GrlA and GrlB) genes were determined by sequence analysis of PCR products obtained using the primers listed in Table S1 (available as Supplementary data at JAC Online). Mutations were detected by comparison of the amino acid sequences of GyrA, GyrB, GrlA and GrlB of fluoroquinolone-susceptible S. epidermidis ATCC12228 (GenBank accession number AE015929), S. haemolyticus JCSC1435 (GenBank accession number NC_007168) and S. hominis SN-013-2010-6-23-5 (GenBank accession numbers HE820118 and HE856265).
Clinical data and statistical analysis
Clinical records of animals that developed an infection containing MRCoNS were examined retrospectively when available. The following data were recorded: underlying diseases, history of antibiotic treatments, specific antibiotic treatment of the infection and outcome (see Table 3). PASS 2008 software (NCCS, Kaysville, UT, USA) was used to conduct a Fisher's exact test (two-tailed) with the level of significance set at a P value <0.05.
Clinical data, therapy and outcome of treatment of infections associated with MRCoNS in animals (dogs, cats and horses)
| Animals (n = 27) and CoNS | Strains | Type of infection | History of antibiotic treatment before identification of the Staphylococcus (no. of courses) | Resistance profile of isolated Staphylococcus from infection side | Antibiotics used for treatment of the Staphylococcus infection | Incompatibility with resistance mechanism | Outcome |
|---|---|---|---|---|---|---|---|
| Dogs (n = 11) | |||||||
| S. haemolyticus | KM 785-09 | abscess (granuloma) | amox-clav (1), clindamycin (2) | PEN, OXA, KAN, GEN, STR, ERY, TET, TMP, CHL | clindamycin | no | recovery |
| S. capitis | KM1385-1970 | joint (surgery) | amox-clav (1), clindamycin (2) | PEN, OXA | amox-clav, clindamycin | no | unknown |
| S. epidermidis | KM1385-1972 | ||||||
| S. haemolyticus | IMD1266-11 | eye (chronic conjunctivitis) | cefovecin (1), neomycin/polymyxin B (2) | PEN, OXA, STR, ERY, CLI, CIP | tetracycline | no | relapse if treatment with tetracycline stops |
| S. epidermidis | IMD1765-11 | respiratory tract (chronic cough) | amox-clav (1), amox-clav (2), amox-clav (3), | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, CHL | marbofloxacin, tetracycline | no | recovery after a 4 week therapy |
| S. hominis | IMD1762-11 | ear (otitis externa) | no antibiotics | PEN, OXA, KAN, GEN, ERY, CLI, TET, CIP | framycetin | yes [aac(6′)-Ie – aph(2′)-Ia] | recovery |
| S. warneri | IMD1530-11 | ear (chronic otitis externa, relapse) | polymyxin B (1), marbofloxacin (2), cefalexin (3), marbofloxacin (4) | PEN, OXA, KAN, GEN, STR, ERY, CLI, CHL | marbofloxacin | no | relapse |
| S. epidermidis | IMD1766-11 | ear (chronic otitis externa) | metronidazole (1), fusidic acid/framycetin (2), polymyxin B (3), amox-clav (4), enrofloxacin (5) | PEN, OXA, ERY, CLI, CIP | amox-clav | yes (mecA) | recovery |
| S. haemolyticus | IMD1775-11 | urinary tract (preputial catarrh) | amox-clav (1), polymyxin B (2) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, TET, CIP | amox-clav | yes (mecA) | recovery |
| S. haemolyticus | IMD1768-11 | abscess | amox-clav/tetracycline (1), amox-clav (2), amox-clav (3), tetracycline (4), amox-clav (5) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, CHL, TET, CIP | cefalexin | yes (mecA) | relapse |
| S. haemolyticus | IMD1761-11 | abscess | amox-clav (1), amox-clav/chloramphenicol (2), amox-clav/chloramphenicol (3), aminoglycoside/polymyxin B (4) | PEN, OXA, KAN, GEN, STR, ERY, CLI, CIP | chloramphenicol | no | recovery |
| S. hominis | IMD1515-11 | dermis (ulcer) | amox-clav (1), cefalexin (2) | PEN, OXA, KAN, GEN, STR, ERY, CLI, TET, CIP | no treatment | NA | euthanasia |
| Cats (n = 11) | |||||||
| S. haemolyticus | KM1758-08 | urinary tract | marbofloxacin (1), amox-clav (2), rifampicin (3) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CIP, TIA | rifampicin | no | recovery |
| S. haemolyticus | KM1632-08 | urinary tract | amox-clav (1), trimethoprim/sulphonamide (2) | PEN, OXA, KAN, GEN, STR, ERY | trimethoprim/sulphonamide | no | recovery |
| S. epidermidis | KM1527-07 | abscess after surgery | marbofloxacin (1), amox-clav (2) | PEN, OXA, KAN, GEN, STR, ERY, TMP, TET, TIA | marbofloxacin, tetracycline | yes [tet(K)] | recovery after amputation of the lower extremity |
| S. epidermidis | KM505-09 | urinary tract | enrofloxacin (1), amoxicillin (2), marbofloxacin (3) | PEN, OXA, KAN, GEN, TMP, CLI, TET, CHL, CIP | no treatment | NA | recovery |
| S. haemolyticus | IMD1272-11 | urinary tract (urolithiasis, chronic cystitis) | amox-clav (1) | PEN, OXA, KAN, GEN, STR, ERY, CLI, TET, CHL, CIP | clindamycin | yes [erm(C)] | recovery |
| S. haemolyticus | IMD1517-11 | urinary tract | unknown | PEN, OXA, KAN, GEN, STR, TMP, TET, CIP | no treatment (fast death) | NA | death |
| S. epidermidis | IMD1776-11 | eye (corneal ulcer) | enrofloxacin (1), moxifloxacin (2), gentamicin (3) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, CHL, CIP | ofloxacin, tetracycline | no | recovery (together with cross-linking therapy) |
| S. epidermidis | IMD1763-11 | urinary tract (urolithiasis, chronic cystitis) | cefovecin (1) | PEN, OXA, KAN, GEN, TET | marbofloxacin | no | relapse (cystitis with Enterococcus) |
| S. epidermidis | IMD1270-11 | phlegmone (acneic skin) | cefovecin (1) | PEN, OXA, KAN, GEN, STR, ERY, TMP, TET, TIA; CIP | chloramphenicol | no | recovery |
| S. epidermidis | IMD1269-11 | urinary tract (cystitis) | marbofloxacin (1), amox-clav (2) | PEN, OXA, KAN, GEN, ERY, CLI, CIP | amox-clav | yes (mecA) | recovery |
| S. epidermidis | IMD1274-11 | eye (conjunctivitis) | amox-clav (1), enrofloxacin/amoxicillin (2), amoxicillin (3), amoxicillin (4), amoxicillin (5), amoxicillin (6), bacitracin/neomycin/ofloxacin (7), amoxicillin (8), ciprofloxacin (9), amoxicillin (10), cefovecin (11), ciprofloxacin/amoxicillin(12), amoxicillin (13), amoxicillin (14) | PEN, OXA, ERY, CLI, TET, CIP | neomycin/polymyxin B | no | relapse |
| Horses (n = 5) | |||||||
| S. epidermidis | KM794-06 | abscess | penicillin | PEN, OXA, KAN, GEN, TET | unknown | NA | recovery |
| S. epidermidis | KM825-09 | abscess (surgery) | cefquinome (1), penicillin/gentamicin (2) | PEN, OXA, KAN, GEN | no treatment | NA | euthanasia |
| S. haemolyticus | KM827-07 | wound | penicillin/gentamicin (1), enrofloxacin (2), cefquinome (3) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, TET, CHL, TIA | unknown | NA | recovery |
| S. haemolyticus | KM1183-09 | dermis | trimethoprim/sulphonamide (1), cefquinome (2), marbofloxacin/enrofloxacin (3) | PEN, OXA, KAN, GEN, STR, ERY, CLI, TET, CHL | gentamicin | yes [aac(6′)-Ie – aph(2′)-Ia] | relapse |
| S. haemolyticus | KM1230-09 | respiratory tract (BAL) | unknown | PEN, OXA, KAN, GEN, STR, STH, TMP, TET, CIP | no treatment | NA | recovery |
| Animals (n = 27) and CoNS | Strains | Type of infection | History of antibiotic treatment before identification of the Staphylococcus (no. of courses) | Resistance profile of isolated Staphylococcus from infection side | Antibiotics used for treatment of the Staphylococcus infection | Incompatibility with resistance mechanism | Outcome |
|---|---|---|---|---|---|---|---|
| Dogs (n = 11) | |||||||
| S. haemolyticus | KM 785-09 | abscess (granuloma) | amox-clav (1), clindamycin (2) | PEN, OXA, KAN, GEN, STR, ERY, TET, TMP, CHL | clindamycin | no | recovery |
| S. capitis | KM1385-1970 | joint (surgery) | amox-clav (1), clindamycin (2) | PEN, OXA | amox-clav, clindamycin | no | unknown |
| S. epidermidis | KM1385-1972 | ||||||
| S. haemolyticus | IMD1266-11 | eye (chronic conjunctivitis) | cefovecin (1), neomycin/polymyxin B (2) | PEN, OXA, STR, ERY, CLI, CIP | tetracycline | no | relapse if treatment with tetracycline stops |
| S. epidermidis | IMD1765-11 | respiratory tract (chronic cough) | amox-clav (1), amox-clav (2), amox-clav (3), | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, CHL | marbofloxacin, tetracycline | no | recovery after a 4 week therapy |
| S. hominis | IMD1762-11 | ear (otitis externa) | no antibiotics | PEN, OXA, KAN, GEN, ERY, CLI, TET, CIP | framycetin | yes [aac(6′)-Ie – aph(2′)-Ia] | recovery |
| S. warneri | IMD1530-11 | ear (chronic otitis externa, relapse) | polymyxin B (1), marbofloxacin (2), cefalexin (3), marbofloxacin (4) | PEN, OXA, KAN, GEN, STR, ERY, CLI, CHL | marbofloxacin | no | relapse |
| S. epidermidis | IMD1766-11 | ear (chronic otitis externa) | metronidazole (1), fusidic acid/framycetin (2), polymyxin B (3), amox-clav (4), enrofloxacin (5) | PEN, OXA, ERY, CLI, CIP | amox-clav | yes (mecA) | recovery |
| S. haemolyticus | IMD1775-11 | urinary tract (preputial catarrh) | amox-clav (1), polymyxin B (2) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, TET, CIP | amox-clav | yes (mecA) | recovery |
| S. haemolyticus | IMD1768-11 | abscess | amox-clav/tetracycline (1), amox-clav (2), amox-clav (3), tetracycline (4), amox-clav (5) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, CHL, TET, CIP | cefalexin | yes (mecA) | relapse |
| S. haemolyticus | IMD1761-11 | abscess | amox-clav (1), amox-clav/chloramphenicol (2), amox-clav/chloramphenicol (3), aminoglycoside/polymyxin B (4) | PEN, OXA, KAN, GEN, STR, ERY, CLI, CIP | chloramphenicol | no | recovery |
| S. hominis | IMD1515-11 | dermis (ulcer) | amox-clav (1), cefalexin (2) | PEN, OXA, KAN, GEN, STR, ERY, CLI, TET, CIP | no treatment | NA | euthanasia |
| Cats (n = 11) | |||||||
| S. haemolyticus | KM1758-08 | urinary tract | marbofloxacin (1), amox-clav (2), rifampicin (3) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CIP, TIA | rifampicin | no | recovery |
| S. haemolyticus | KM1632-08 | urinary tract | amox-clav (1), trimethoprim/sulphonamide (2) | PEN, OXA, KAN, GEN, STR, ERY | trimethoprim/sulphonamide | no | recovery |
| S. epidermidis | KM1527-07 | abscess after surgery | marbofloxacin (1), amox-clav (2) | PEN, OXA, KAN, GEN, STR, ERY, TMP, TET, TIA | marbofloxacin, tetracycline | yes [tet(K)] | recovery after amputation of the lower extremity |
| S. epidermidis | KM505-09 | urinary tract | enrofloxacin (1), amoxicillin (2), marbofloxacin (3) | PEN, OXA, KAN, GEN, TMP, CLI, TET, CHL, CIP | no treatment | NA | recovery |
| S. haemolyticus | IMD1272-11 | urinary tract (urolithiasis, chronic cystitis) | amox-clav (1) | PEN, OXA, KAN, GEN, STR, ERY, CLI, TET, CHL, CIP | clindamycin | yes [erm(C)] | recovery |
| S. haemolyticus | IMD1517-11 | urinary tract | unknown | PEN, OXA, KAN, GEN, STR, TMP, TET, CIP | no treatment (fast death) | NA | death |
| S. epidermidis | IMD1776-11 | eye (corneal ulcer) | enrofloxacin (1), moxifloxacin (2), gentamicin (3) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, CHL, CIP | ofloxacin, tetracycline | no | recovery (together with cross-linking therapy) |
| S. epidermidis | IMD1763-11 | urinary tract (urolithiasis, chronic cystitis) | cefovecin (1) | PEN, OXA, KAN, GEN, TET | marbofloxacin | no | relapse (cystitis with Enterococcus) |
| S. epidermidis | IMD1270-11 | phlegmone (acneic skin) | cefovecin (1) | PEN, OXA, KAN, GEN, STR, ERY, TMP, TET, TIA; CIP | chloramphenicol | no | recovery |
| S. epidermidis | IMD1269-11 | urinary tract (cystitis) | marbofloxacin (1), amox-clav (2) | PEN, OXA, KAN, GEN, ERY, CLI, CIP | amox-clav | yes (mecA) | recovery |
| S. epidermidis | IMD1274-11 | eye (conjunctivitis) | amox-clav (1), enrofloxacin/amoxicillin (2), amoxicillin (3), amoxicillin (4), amoxicillin (5), amoxicillin (6), bacitracin/neomycin/ofloxacin (7), amoxicillin (8), ciprofloxacin (9), amoxicillin (10), cefovecin (11), ciprofloxacin/amoxicillin(12), amoxicillin (13), amoxicillin (14) | PEN, OXA, ERY, CLI, TET, CIP | neomycin/polymyxin B | no | relapse |
| Horses (n = 5) | |||||||
| S. epidermidis | KM794-06 | abscess | penicillin | PEN, OXA, KAN, GEN, TET | unknown | NA | recovery |
| S. epidermidis | KM825-09 | abscess (surgery) | cefquinome (1), penicillin/gentamicin (2) | PEN, OXA, KAN, GEN | no treatment | NA | euthanasia |
| S. haemolyticus | KM827-07 | wound | penicillin/gentamicin (1), enrofloxacin (2), cefquinome (3) | PEN, OXA, KAN, GEN, STR, ERY, TMP, CLI, TET, CHL, TIA | unknown | NA | recovery |
| S. haemolyticus | KM1183-09 | dermis | trimethoprim/sulphonamide (1), cefquinome (2), marbofloxacin/enrofloxacin (3) | PEN, OXA, KAN, GEN, STR, ERY, CLI, TET, CHL | gentamicin | yes [aac(6′)-Ie – aph(2′)-Ia] | relapse |
| S. haemolyticus | KM1230-09 | respiratory tract (BAL) | unknown | PEN, OXA, KAN, GEN, STR, STH, TMP, TET, CIP | no treatment | NA | recovery |
amox-clav, amoxicillin/clavulanic acid; CHL, chloramphenicol; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; GEN, gentamicin; KAN, kanamycin; OXA, oxacillin; PEN, penicillin; STR, streptomycin; TET, tetracycline; TIA, tiamulin; TMP, trimethoprim; NA, not available; BAL, bronchoalveolar lavage.
mecA, methicillin-resistance gene encoding PBP2a for resistance to all β-lactam antibiotics (e.g. penicillin, amoxicillin, amoxicillin/clavulanic acid and cefalexin); aac(6′)-Ie–aph(2′)-Ia, aminoglycoside acetyltransferase and phosphotransferase tandem genes (gentamicin/kanamycin/neomycin); erm(C), macrolide, lincosamide and streptogramin B 23S rRNA methylase gene (clindamycin); tet(K), tetracycline efflux resistance gene (tetracycline).
Results
Identification of and genetic diversity among strains of MRCoNS
The 43 samples originated from animals admitted to 30 different clinics from 10 different cantons in Switzerland, indicating that MRCoNS are widespread and not related to a specific clinic with nosocomial infection problems (Figure 1). MRCoNS were isolated from different infection sites in cats (n = 16), dogs (n = 20) and horses (n = 7) (Tables 1 and 2). Infected sites consisted of the skin (n = 10), urinary tracts (n = 9), ears (n = 4), respiratory tracts (n = 5), joints (n = 4), eyes (n = 1) and abscesses/fistulas (n = 10) (Tables 1 and 2). The MRCoNS were identified as S. epidermidis (Table 1) and S. haemolyticus, S. hominis, S. warneri, S. capitis and S. cohnii (Table 2). Three samples contained an additional pathogen, i.e. Staphylococcus schleiferi together with S. haemolyticus KM785-09, Staphylococcus pseudintermedius together with S. epidermidis KM1077-09 and a mix of anaerobic bacteria together with S. epidermidis IMD1522-11.
Phylogenetic tree constructed from the PFGE pattern of methicillin-resistant S. epidermidis, S. haemolyticus, S. hominis, S. capitis and S. warneri. The tree was generated by UPGMA using Bionumerics 6.6 (Applied Maths, Kortrjk, Belgium) and comparison settings (Dice, optimization 1.5%, position tolerance 1.5%) as recommended by PulseNet International (www.pulsenetinternational.org). The broken line indicates the cut-off value of ≥79%, determining clonality between the isolates according to Miragaia et al.47 Capital letters indicate the cantons and the numbers indicate the different clinics. AG, Argovia; AI, Appenzell Inner Rhoden; BE, Bern; BL, Basel-Land; GE, Geneva; GR, Grisons; SH, Schaffhausen; TI, Ticino; VD, Vaud; ZH, Zurich; U, unknown.
Phylogenetic tree constructed from the PFGE pattern of methicillin-resistant S. epidermidis, S. haemolyticus, S. hominis, S. capitis and S. warneri. The tree was generated by UPGMA using Bionumerics 6.6 (Applied Maths, Kortrjk, Belgium) and comparison settings (Dice, optimization 1.5%, position tolerance 1.5%) as recommended by PulseNet International (www.pulsenetinternational.org). The broken line indicates the cut-off value of ≥79%, determining clonality between the isolates according to Miragaia et al.47 Capital letters indicate the cantons and the numbers indicate the different clinics. AG, Argovia; AI, Appenzell Inner Rhoden; BE, Bern; BL, Basel-Land; GE, Geneva; GR, Grisons; SH, Schaffhausen; TI, Ticino; VD, Vaud; ZH, Zurich; U, unknown.
PFGE revealed a large heterogeneity between the MRCoNS of the same species. Eight different PFGE clonal lineages were identified among the 17 S. haemolyticus isolates, 11 among the 20 S. epidermidis isolates and 1 among the 3 S. hominis isolates (Figure 1). The S. epidermidis strains showed distinct MLST patterns and belonged to 14 different sequence types (STs), representing 11 clonal complexes (CC), namely CC2 [ST2 (n = 4), ST446 (n = 1)], CC5 [ST5 (n = 3), ST445 (n = 1)], CC22 [ST22 (n = 3)], CC59 [ST59 (n = 1), ST81 (n = 1)], CC17 [ST69 (n = 1), CC89 [ST89 (n = 1)], CC130 [ST450 (n = 1)], CC166 [ST449 (n = 1)], CC286 [ST286 (n = 1)], CC451 [ST451 (n = 1)] and CC448 [ST448 (n = 1)]. ST445, ST446, ST448, ST449, ST450 and ST451 were newly described STs [Figure 1 and Figure S1 (available as Supplementary data at JAC Online)]. All but one of the isolates belonging to the predominant CC2 (n = 5), CC5 (n = 4), CC22 (n = 2) and CC59 (n = 2) clustered into four distinct PFGE branches (Figure 1). However, different PFGE patterns could still be observed within these groups, indicating a larger diversity between strains of the same CC (Figure 1).
Ten of 20 S. epidermidis isolates harboured the biofilm formation operon ica, including all the CC2 (ST2, ST446) (n = 5) and ST22 (n = 2) isolates as well as the ST69, ST448 and ST449 isolates. The icaA gene was also detected in one S. hominis isolate. ACMEs were only detected in S. epidermidis. Eight S. epidermidis isolates carried a type I ACME (arcA + /opp3AB +) and three carried a type II ACME (arcA + /opp3AB−). ACMEs were found in all ST5 and ST22 isolates, but were also present in one ST2 isolate and in the ST59, ST69, ST446, ST449 and ST450 isolates (Figure 1). An SCCmec element could only be typed for 17 isolates. SCCmec IV was detected in one S. capitis, one S. warneri and eight S. epidermidis isolates. In S. epidermidis, SCCmec IV was associated with ACME type I in the ST5, ST69 and ST450 isolates and with ACME type 2 in the ST59 isolate. SCCmec V was detected in seven S. haemolyticus isolates (Figure 1). The other 26 SCCmec elements could not be characterized as they lacked either known ccr genes or a known mecA class structure or both (Figure 1).
Distribution of MRCoNS isolates in veterinary practices
Association of a specific clonal lineage with a clinic was only observed for two pairs of S. haemolyticus (IMD1761-11, IMD1768-11 and IMD1632-08, IMD1758-08) isolated from different animals in two clinics. Each pair showed similar PFGE profiles (A and C) and contained a non-typeable SCCmec element (Figure 1). However, they exhibited different antibiotic resistance profiles (Table 2). Otherwise, MRCoNS isolated from animals admitted to the same clinic were genetically distant. On the other hand, genetically related MRCoNS were isolated from different animals in different clinics (Figure 1). These isolates also displayed different antibiotic resistance profiles (Tables 1 and 2).
Antibiotic resistance profile
All the MRCoNS isolates were resistant to β-lactam antibiotics and contained the mecA gene. None of them was resistant to linezolid, quinupristin/dalfopristin, rifampicin or vancomycin. Nonetheless, the isolates were also resistant to gentamicin/kanamycin owing to the bifunctional acetyltransferase/phosphotransferase gene aac(6′)-Ie–aph(2′)-Ia (n = 33), kanamycin [aph(3′)-III (n = 17)], macrolides and/or lincosamides [erm(C) (n = 22), erm(A) (n = 1), msr (n = 12) and lnu(A) (n = 4], tetracycline [tet(K) (n = 22)], trimethoprim [dfr(A) (n = 7) and dfr(G) (n = 10)], streptomycin [str (n = 5) and ant(6)-Ia (n = 15)], streptothricin [sat4 (n = 15)], chloramphenicol [catpC221 (n = 5) and catpC223 (n = 3)], tiamulin [(vga(A) (n = 4)], mupirocin [mupR (n = 2)], fusidic acid (n = 13), sulfamethoxazole (n = 34) and fluoroquinolones (n = 30) (Tables 1 and 2). Resistance mechanisms for fusidic acid and sulfamethoxazole were not investigated. Fluoroquinolone resistance was attributed to mutations in topoisomerase II GyrA (n = 30) and topoisomerase IV GrlA (n = 18) (Tables 1 and 2). Mutations that cause amino acid substitutions in topoisomerases II and IV were found in ciprofloxacin-resistant S. epidermidis, S. haemolyticus and S. hominis at nucleotide position 251 [n = 30; Ser84Leu (n = 14), Ser84Phe (n = 13), Ser84Tyr (n = 3)] in gyrA and at positions 239 [n = 8; Ser80Tyr (n = 4), Ser80Phe (n = 4)] and 250 [n = 10; Asp84Tyr (n = 7), Gly84Tyr (n = 3)] in grlA. An amino acid substitution in GrlB (Glu473Lys) was also present in two S. epidermidis isolates (KM505-09 and KM1527-07) and two S. haemolyticus isolates (IMD1277-11 and IMD1532-11). This mutation was not considered as being responsible for fluoroquinolone resistance in CoNS, as a mutation at the same location has been shown not to confer resistance to fluoroquinolones in Staphylococcus aureus.38 The resistance mechanism could not be explained for one strain with resistance to gentamicin and kanamycin, for one strain with resistance to clindamycin and for two strains with decreased susceptibility to tiamulin (MIC >4 mg/L), suggesting new mechanisms of resistance.
Clinical data of infected animals
Clinical data were obtained for 27 animals (11 dogs, 11 cats and 5 horses) admitted to 19 different clinics (Table 3). Twenty-four animals had a history of antibiotic treatment, and 20 of them underwent antimicrobial treatment more than twice with up to 14 courses. The most commonly used antibiotics in dogs and cats prior to the identification of the staphylococcal species were amoxicillin/clavulanic acid, cephalosporins and fluoroquinolones. In 20 dogs and cats treated, amoxicillin was given 15 times, fluoroquinolones 8 times, cephalosporins 6 times, and both a β-lactam and a fluoroquinolone antibiotic were given 7 times. Antibiotics such as gentamicin, chloramphenicol, tetracycline and clindamycin were also used in pets, but less frequently. In horses, cefquinome, fluoroquinolones and the combination penicillin/gentamicin were the most commonly administered antibiotics. The MRCoNS infections were then treated after consultation of an antibiogram, most frequently using fluoroquinolones or amoxicillin/clavulanic acid followed by tetracycline, clindamycin, chloramphenicol, cefalexin, the combination sulphonamides/trimethoprim, rifampicin and the aminoglycosides gentamicin, framycetin and neomycin. Most of the animals (n = 14) recovered after antibiotic treatment: six had a relapse, two recovered without any antibiotic therapy, one recovered after amputation of the infected lower extremity, one was still under treatment at the time of writing, two were not further treated and euthanized and one died of unknown cause prior to therapy. In seven cases, antibiotics were used even in the presence of resistance, leading to relapse in two cases when cefalexin and gentamicin were used for the treatment of an abscess and skin infection, respectively. The other five animals recovered after antimicrobial treatment with amoxicillin/clavulanic acid (n = 3), clindamycin (n = 1) or framycetin (n = 1), despite the presence of mecA, erm(C) or aac(6′)-Ie–aph(2′)-Ia in the respective MRCoNS (Table 3). For these animals, MRCoNS were likely not the primary cause of the infection (three urinary tract and two ear infections), although MRCoNS appeared alone in the culture. No significant difference was observed in the outcome of the disease between animals treated with an antibiotic incompatible and an antibiotic compatible with the resistance profile of the MRCoNS.
Discussion
MRCoNS are associated with serious infections in animals and have become a challenge to therapy. The CoNS species identified in this study were the same as the ones causing nosocomial infections in humans, with S. epidermidis and S. haemolyticus being the most prevalent in animals and humans.7 Similar to the case with human infections,11,13S. hominis, S. warneri, S. cohnii and S. capitis were only occasionally isolated from animal infection sites. The population analysis by PFGE showed that the majority of the isolates are genetically diverse. Three clonal lineages sharing similar PFGE profiles appeared to be predominant among S. epidermidis and were found to belong to CC2, CC5 and CC22. However, isolates of CC2 and CC22 contained divergent SCCmec elements and ACMEs, while CC5 isolates almost exclusively contained SCCmec IV and ACME type I. Additionally, these clonally related isolates displayed different resistance profiles, emphasizing the ability of CoNS to acquire antibiotic resistance genes. The presence of numerous PFGE and antibiotic resistance profiles is a well-described phenomenon for S. epidermidis ST2, which is the most widely disseminated human healthcare-associated sequence type worldwide.39–42
A study using 217 S. epidermidis isolates from humans from 17 countries detected 30.9% of the isolates as ST2.42 The successful spread of ST2 in the hospital environment has been suggested to be associated with its ability to generate novel phenotypic and genotypic variants by recombination and acquisition of new elements, such as the biofilm-formation ica operon, ACMEs and antibiotic resistance genes.14,15,18,41 In our study, all isolates of CC2 and CC22, which is a subcluster of CC2 (Figure S1, available as Supplementary data at JAC Online), contained the biofilm formation operon ica. On the other hand, the ica operon was absent in isolates of CC5, which was also predominant in infection sites of animals; they contained ACMEs instead. Of note, CC22 contained both ica and ACMEs. The presence of the biofilm formation operon ica and ACMEs almost exclusively in S. epidermidis of the predominant clonal lineages CC2, CC5 and CC22 may have contributed to the establishment of these strains in the animal environment. In addition to the predominant STs, the animals were also infected with other S. epidermidis strains, which has also been reported in human infections, such as ST35, ST59, ST81, ST69, ST89 and ST286 (Figure S1, available as Supplementary data at JAC Online),40–42 and with S. haemolyticus. The absence of MLST methods for S. haemolyticus prevented us from determining whether specific STs would also be predominant within this species. However, the different PFGE and antibiotic resistance profiles support the hypothesis that MRCoNS associated with infections in animals are very heterogeneous, unlike methicillin-resistant S. pseudintermedius (MRSP), which spread as specific clones.43 Nevertheless, MRSP and MRCoNS are resistant to the same classes of drugs and contain similar antibiotic resistance genes. Similar to MRSP, more than two-thirds of the MRCoNS exhibit resistance to fluoroquinolones, macrolides, lincosamides and aminoglycosides, in addition to resistance to β-lactams, suggesting that they have been selected through the frequent use of antibiotics. These classes of drugs, especially the β-lactams and fluoroquinolones, were also the most commonly used drugs in veterinary practices (Table 3). These two classes of drugs have been shown to represent a significant risk factor for the selection of methicillin-resistant S. aureus44 and similar effects are to be expected for MRCoNS. Many animals were given more than one antibiotic course, with some animals receiving 5 and up to 14 courses of an antibiotic before the MRCoNS infection was diagnosed. The series of empirical antimicrobial treatments may have contributed to the selection of the MRCoNS in the infection sites. Additionally, the primary cause of the infection may have been overlooked and not directly related to the presence of a MRCoNS. Indeed, two animals recovered without antibiotic treatment and five recovered despite the presence of a resistance mechanism against the antibiotic used for treatment. Nevertheless, in the majority of the cases the staphylococcal infections could be treated with an antibiotic chosen after consultation of an antibiogram. All these criteria highlight the importance of correct diagnosis and antibiograms.
Multidrug-resistant CoNS represent a new challenge for therapy in veterinary medicine. The infections are caused by genetically distant strains, indicating many possible non-hospital-related reservoirs, such as animals themselves, animal owners and people working with animals that have been shown to harbour and possibly exchange MRCoNS.22,45,46 The presence of clones similar to those causing infections in humans highlights the importance of careful surveillance of bacterial infection diseases, the need to implement infection control programmes and the prudent use of antibiotics in veterinary settings.
Funding
This study was supported by internal funding.
Transparency declarations
None to declare.
Supplementary data
Table S1 and Figure S1 are available at JAC Online (http://jac.oxfordjournals.org/).
Acknowledgements
We thank the Centre for Zoonoses and Bacterial Animal Diseases and Antimicrobial Resistance (ZOBA), IDEXX Diavet and Laboratory Laupeneck AG for providing the strains and all the participating veterinary practices that provided clinical data. We also thank Jacques Schrenzel and Patrice François for providing positive control strains and A. Collaud, C. Strauss, Y. Sigrist, S. Rychener, S. Kittl and A. Candi for helpful assistance.
