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Methicillin-Resistant Staphylococcus aureus in Spain: Molecular Epidemiology and Utility of Different Typing Methods

ABSTRACT In a point-prevalence study performed in 145 Spanish hospitals in 2006, we collected 463 isolates of Staphylococcus aureus in a single day. Of these, 135 (29.2%) were methicillin (meticillin)-resistant S. aureus (MRSA) isolates. Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE) after SmaI digestion, staphylococcal chromosomal cassette mec (SCCmec) typing, agr typing, spa typing with BURP (based-upon-repeat-pattern) analysis, and multilocus sequence typing (MLST). The 135 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (72.6%), gentamicin (20.0%), erythromycin (66.7%), and clindamycin (39.3%). Among the isolates resistant to erythromycin, 27.4% showed the M phenotype. All of the isolates were susceptible to glycopeptides. Twelve resistance patterns were found, of which four accounted for 65% of the isolates. PFGE revealed 36 different patterns, with 13 major clones (including 2 predominant clones with various antibiotypes that accounted for 52.5% of the MRSA isolates) and 23 sporadic profiles. Two genotypes were observed for the first time in Spain. SCCmec type IV accounted for 6.7% of the isolates (70.1% were type IVa, 23.9% were type IVc, 0.9% were type IVd, and 5.1% were type IVh), and SCCmec type I and SCCmec type II accounted for 7.4% and 5.2% of the isolates, respectively. One isolate was nontypeable. Only one of the isolates produced the Panton-Valentine leukocidin. The isolates presented agr type 2 (82.2%), type 1 (14.8%), and type 3 (3.0%). spa typing revealed 32 different types, the predominant ones being t067 (48.9%) and t002 (14.8%), as well as clonal complex 067 (78%) by BURP analysis. The MRSA clone of sequence type 125 and SCCmec type IV was the most prevalent throughout Spain. In our experience, PFGE, spa typing, SCCmec typing, and MLST presented good correlations for the majority of the MRSA strains; we suggest the use of spa typing and PFGE typing for epidemiological surveillance, since this combination is useful for both long-term and short-term studies.

Community-acquired methicillin-resistant Staphylococcus aureus in Madrid, Spain: transcontinental importation and polyclonal emergence of Panton–Valentine leukocidin-positive isolates

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates producing the Panton-Valentine leukocidin (PVL) have been reported worldwide. We describe the molecular characteristics of PVL-positive CA-MRSA strains isolated in Madrid, Spain, and analyze the clinical features of patients infected with these isolates. From 2004 to 2007, we collected 13 PVL-positive MRSA isolates from patients attending to the emergency department. The isolates were genotyped by pulsed-field gel electrophoresis, SCCmec typing, agr polymorphism, and multilocus sequence typing. Susceptibility to 29 antimicrobials was determined by the broth microdilution and by the E-test methods. The isolates belonged to 3 genotypes: ST8-SCCmec IVc (n = 11), ST5-SCCmec IVa (n = 1), and ST80-SCCmec IVc (n = 1). The corresponding agr types were I, II, and III, respectively. Five isolates were resistant to tetracycline and doxycycline, and 1 was resistant to fusidic acid (ST80). The isolates were from children (n = 9) and adults (n = 4), and were associated with skin and soft tissue infections (n = 9), otitis (n = 1), and bacteremia (n = 1). Nine patients were from South America. Our results indicate the transcontinental importation and recent emergence in Spain of PVL-positive CA-MRSA strains belonging to 3 distinct lineages, including 1 predominant (ST8-SCCmec IVc).

Staphylococcus spp. en España: situación actual y evolución de la resistencia a antimicrobianos (1986-2006)

Introduccion Desde 1986 se han realizado cinco estudios de prevalencia de Staphylococcus spp. en Espana. En este trabajo se presentan los datos de 2006 correspondientes al sexto estudio. Metodos Participaron 145 hospitales de todas las areas geograficas y se estudiaron 866 cepas de estafilococos (463 S. aureus). Se determino la sensibilidad a 16 antimicrobianos mediante un sistema automatizado de microdilucion en caldo. La sensibilidad a tigeciclina se determino mediante el metodo de E-test. Resultados La resistencia de S. aureus a oxacilina se ha estabilizado (el 31,2% en 2002 frente al 29,2% en 2006), asi como la resistencia a eritromicina, clindamicina y ciprofloxacino. En 2006 los aislados fueron mas sensibles a gentamicina (el 16,9% en 2002 frente al 8,6% en 2006; p < 0,001), ninguno presento sensibilidad disminuida a vancomicina y la resistencia a cotrimoxazol (0,9%) y a rifampicina (0,6%) fue anecdotica. Un aislado fue resistente a linezolid. La resistencia de los estafilococos coagulasa negativos a oxacilina (el 61,3% en 2002 frente al 66,7% en 2006) y a eritromicina (el 63,0% en 2002 frente al 66,5% en 2006) se ha mantenido relativamente estable, aunque ha aumentado la resistencia a gentamicina (el 27,8% en 2002 frente al 44,2% en 2006; p < 0,001), ciprofloxacino (el 44,9% en 2002 frente al 54,3% en 2006; p = 0,010) y clindamicina (el 33,8% en 2002 frente al 46,2% en 2006; p = 0,001). Dos aislados presentaron sensibilidad disminuida a teicoplanina y uno fue resistente a linezolid. Todos los Staphylococcus spp. fueron uniformemente sensibles a quinupristina-dalfopristina y a tigeciclina. Conclusiones En Espana la resistencia de Staphylococcus spp. a oxacilina sigue siendo elevada, aunque parece haberse estabilizado. Asimismo, comienzan a aparecer aislados resistentes a linezolid.

Prevalence and Evolution of Methicillin-Resistant Staphylococcus aureus in Spanish Hospitals between 1996 and 2002

ABSTRACT Pulsed-field gel electrophoretic analysis of 2,144 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients in Spanish hospitals over a 7-year period revealed 17 predominant profiles. Typing showed the replacement of Iberian clone E1 (ST247-MRSA-I) by two prevalent clones, E7 and E8, that are closely related to each other and have the same genetic background as ST125-MRSA-IV.

Methicillin-resistant Staphylococcus aureus carrying the mecC gene: emergence in Spain and report of a fatal case of bacteraemia

OBJECTIVES Methicillin-resistant Staphylococcus aureus (MRSA) strains carrying the mecC gene have been reported from humans and animals from several European countries, but never from Spain. We describe the first isolates of mecC-positive MRSA of human origin collected in Spain and report a fatal case of bacteraemia. METHODS Isolates were tested for phenotypic resistance using cefoxitin, tested for the mecA/mecC genes and toxin genes by PCR, and typed by staphylococcal cassette chromosome mec (SCCmec), PFGE, spa, multilocus sequence typing and agr. RESULTS During 2008-13 five MRSA isolates showing resistance to cefoxitin and carrying the mecC gene were recovered at one hospital in Spain. In a review of 5505 S. aureus strains received at the Spanish National Reference Centre for Staphylococci from the same period, we found two additional mecC-positive isolates. The isolates were recovered from blood (two), wounds (two), joint fluid (one), urine (one) and a nasal swab (one). All MRSA were mecA negative, presented SCCmecXI, belonged to agr group III and to clonal complex 130, and were negative for the production of the toxin genes tst1, eta, etb, etd and Panton-Valentine leucocidin. Six isolates belonged to spa type t843 (ST130 and ST1945, where ST stands for sequence type) and one to spa type t6220 (ST1945). One patient with mecC-positive MRSA sepsis died in the emergency department. CONCLUSIONS We confirm the presence of MRSA carrying the mecC gene in Spain, the ability of this livestock-associated MRSA to cause severe infections in humans and the need to perform culture-based susceptibility testing methods in order to detect these emerging strains.

Molecular epidemiology of community-associated methicillin-resistant Staphylococcus aureus in Spain: 2004–12

OBJECTIVES In Spain, despite the high rates of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA), the incidence of community-associated (CA) MRSA seems to be low on the basis of a small number of studies. We analysed the evolution of CA-MRSA in Spain from 2004 to 2012, and identified the clonal lineages and population structure. METHODS The study included 8326 MRSA strains. Susceptibility to 18 antimicrobials was determined. Isolates were tested for the presence of mecA, Panton-Valentine leucocidin (PVL) and the arginine catabolic mobile element (ACME) by PCR, and typed by staphylococcal cassette chromosome mec, PFGE, spa, multilocus sequence typing and agr. RESULTS Among the 8326 isolates, 246 (2.9%) were CA-MRSA. We identified genotypically 226 PVL-positive CA-MRSA isolates (88% agr type I, 10.2% agr type III and 1.8% agr type II) and 20 PVL-negative CA-MRSA isolates (all agr type I) from children and adults (82.1% from wounds) from 13 different geographical areas. A significant increase in the rates of CA-MRSA was observed when comparing 2004-07 (0.43%) with 2008-12 (5.44%). Resistance rates were as follows: only β-lactams, 84.5%; erythromycin, 12.8%; tetracycline, 8.8%; clindamycin, 4.9%; ciprofloxacin, 3.1%; fusidic acid, 2.0%; others, 0.4%; and multiresistant, 6.2% (six isolates USA300). The strains belonged to the PVL-positive clones ST8-IVc (69.9%), ST8-IVa-ACME-positive (USA300, 8.9%), ST8-IVa-ACME-negative (0.8%), ST30-IVc (4.5%), ST80-IVc (2.0%), ST5-IVc (1.2%) and others (ST59, ST72, ST88, ST642, ST1472 and ST1829; 4.5%) and to the PVL-negative ST398-V (8.1%). CONCLUSIONS We confirm an increase in CA-MRSA in Spain, the predominance of the ST8-IVc clone, the emergence of the USA300 clone, a high genetic diversity among PVL-positive CA-MRSA isolates and the recent emergence of the pig-associated ST398-V clone.

Detection of Linezolid-Resistant Staphylococcus aureus with 23S rRNA and Novel L4 Riboprotein Mutations in a Cystic Fibrosis Patient in Spain

Linezolid-resistant Staphylococcus aureus (LRSA) emerged in cystic fibrosis (CF) patients in different countries several years ago ([1][1]), and we have also observed this problem in Spain. The most frequent mechanism of linezolid resistance in staphylococci is a G2576T point mutation within domain