Ana Vindel

Evolution of the Antimicrobial Resistance of Staphylococcus spp. in Spain: Five Nationwide Prevalence Studies, 1986 to 2002

ABSTRACT Data regarding the evolution of Staphylococcus resistance in a whole country have a definite influence on the design of empirical treatment regimens. Nevertheless, incidence studies over long periods of time are expensive and very difficult to carry out. In order to ascertain the present situation of the antimicrobial resistance of Staphylococcus in Spain and the change of this resistance over time, we performed five point prevalence studies (1986 to 2002) in a large group of Spanish hospitals (from 68 institutions in 1986 to 143 in 2002) collecting all Staphylococcus strains isolated on a single selected day. All microorganisms were identified in the five studies at the same laboratory, and antimicrobial susceptibility testing was performed against 17 antimicrobial agents by the agar dilution method and a microdilution method. During this period, there was an overall increase in resistance to most antimicrobials among Staphylococcus aureus/coagulase-negative staphylococci, mainly to oxacillin (1.5%/32.5% in 1986 versus 31.2%/61.3% in 2002) (P < 0.001), erythromycin (7%/41.1% in 1986 versus 31.7%/63% in 2002) (P < 0.001), gentamicin (5.2%/25.4% in 1986 versus 16.9%/27.8% in 2002) (P < 0.001; P = 0.5), and ciprofloxacin (0.6%/1.1% in 1986 versus 33.9%/44.9% in 2002) (P < 0.001). All of the isolates were uniformly susceptible to glycopeptides, linezolid, and quinupristin/dalfopristin. Resistance of S. aureus to trimethoprim/sulfamethoxazole was very low (from 0.5% to 2.1%) (P = 0.152). Periodic performance of prevalence studies is a useful, inexpensive, and easy tool to know the nationwide situation of a microorganism and its resistance to antimicrobials; it also helps us assess the emergence and spread of antimicrobial resistance.

Emergence of OXA-48-producing Klebsiella pneumoniae and the novel carbapenemases OXA-244 and OXA-245 in Spain

OBJECTIVES To describe the molecular and population-level characterization of a selected group of OXA-48-like-producing Klebsiella pneumoniae isolates collected in Spain between January 2011 and May 2012. METHODS During the study period, 151 OXA-48-like-producing K. pneumoniae isolates were collected from 10 hospitals in six different Spanish regions. From these, a representative sample of 21 isolates that caused hospital outbreaks and single infections was selected for further in-depth analysis. Molecular epidemiology was investigated using PFGE and multilocus sequence typing (MLST). Resistance genes were characterized by PCR and sequencing. Plasmids carrying bla(OXA-48-like) were studied by PFGE with S1 nuclease digestion. RESULTS All 21 isolates had ertapenem MICs ≥ 1 mg/L, but 47.6% remained susceptible to imipenem and meropenem; bla(OXA-48) was identified in 19 isolates (90.5%) and the novel bla(OXA-244) and bla(OXA-245) genes were detected in 1 isolate each. With one exception, all isolates that contained bla(OXA-48-like) also contained bla(CTX-M-15). PFGE typing revealed six clusters comprising isolates that belonged to MLST types ST11, ST16, ST392, ST405, ST437 and ST663, respectively. Two main clusters were identified: PFGE cluster 1 (12 isolates, belonging either to ST405 or ST663, from seven hospitals), and PFGE cluster 2 (4 ST16 isolates from two hospitals). Six of seven donor isolates conjugated successfully; bla(OXA-48-like) (but not bla(CTX-M-15)) was carried on ≈ 60 kb Inc L/M plasmids. CONCLUSIONS Multidrug-resistant K. pneumoniae producing OXA-48-like carbapenemase are emerging as important pathogens in Spain due to intra- and inter-hospital, clonal and non-clonal dissemination.

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.

Antimicrobial Susceptibilities of Unique Stenotrophomonas maltophilia Clinical Strains

ABSTRACT Susceptibility to 41 antimicrobials was studied with 99Stenotrophomonas maltophilia strains, and different pulsed-field gel electrophoresis profiles were identified among 130 prospectively collected isolates. Moxalactam, doxycycline, minocycline, and clinafloxacin displayed the highest activity (≥98% susceptibility). Ticarcillin resistance (75%) was reverted by clavulanate in 25% of strains. Trimethoprim-sulfamethoxazole resistance was 26.2% (≥4 [trimethoprim]/76 [sulfamethoxazole] μg/ml) and dropped to 11.1% when an 8/152-μg/ml breakpoint was applied based on its bimodal MIC distribution. Resistance was lower when unique strains were considered, because clonal organisms contribute to resistance.

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).

Clonal Diversity of Nosocomial Epidemic Acinetobacter baumannii Strains Isolated in Spain

ABSTRACT Acinetobacter baumannii is one of the major pathogens involved in nosocomial outbreaks. The clonal diversity of 729 epidemic strains isolated from 19 Spanish hospitals (mainly from intensive care units) was analyzed over an 11-year period. Pulsed-field gel electrophoresis (PFGE) identified 58 PFGE types that were subjected to susceptibility testing, rpoB gene sequencing, and multilocus sequence typing (MLST). All PFGE types were multidrug resistant; colistin was the only agent to which all pathogens were susceptible. The 58 PFGE types were grouped into 16 clones based on their genetic similarity (cutoff of 80%). These clones were distributed into one major cluster (cluster D), three medium clusters (clusters A, B, and C), and three minor clusters (clusters E, F, and G). The rpoB gene sequencing and MLST results reflected a clonal distribution, in agreement with the PFGE results. The MLST sequence types (STs) (and their percent distributions) were as follows: ST-2 (47.5%), ST-3 (5.1%), ST-15 (1.7%), ST-32 (1.7%), ST-79 (13.6%), ST-80 (20.3%), and ST-81 (10.2%). ST-79, ST-80, and ST-81 and the alleles cpn60-26 and recA29 are described for the first time. International clones I, II, and III were represented by ST-81, ST-2, and ST-3, respectively. ST-79 and ST-80 could be novel emerging clones. This work confirms PFGE and MLST to be complementary tools in clonality studies. Here PFGE was able to demonstrate the monoclonal pattern of most outbreaks, the inter- and intrahospital transmission of bacteria, and their endemic persistence in some wards. MLST allowed the temporal evolution and spatial distribution of Spanish clones to be monitored and permitted international comparisons to be made.

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.

Nosocomial Outbreak of VIM-1-Producing Klebsiella pneumoniae Isolates of Multilocus Sequence Type 15: Molecular Basis, Clinical Risk Factors, and Outcome

ABSTRACT We study the epidemiology, molecular basis, clinical risk factors, and outcome involved in the clonal dissemination of VIM-1-producing Klebsiella pneumoniae isolates in the hospital setting. All patients infected/colonized by carbapenem-nonsusceptible K. pneumoniae (CNSKP) in 2009 were included. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic resistance genes were analyzed by PCR and sequencing. Plasmids were studied by PFGE with S1 nuclease digestion and for incompatibility group by a PCR-based replicon typing scheme. Risk factors associated with CNSKP colonization/infection were assessed by an observational case-control study. All 55 patients studied were infected (n = 28) or colonized (n = 27) by VIM-1-producing K. pneumoniae. All but one acquired isolates of a single clone (PFGE cluster 1 [C1], sequence type 15 [ST15]), while another clone (PFGE C2, ST340) was detected in four patients. C1 isolates also produced the new extended-spectrum β-lactamase SHV-134. blaVIM-1 was carried in a class 1 integron and an untypeable plasmid of ∼50 bp. The number of days that the patient received mechanical ventilation, the use of parenteral nutrition, previous treatment with linezolid, and treatment with extended-spectrum cephalosporins for more than 7 days were detected to be independent risk factors for CNSKP acquisition. The VIM-1-producing K. pneumoniae ST15 clone has a high capacity to spread among intensive care unit patients with severe underlying conditions. A high rate of associated mortality and great difficulty in controlling the spread of this clone, without permanent behavioral changes in the personnel, were observed.

Evolution and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Epidemic and Sporadic Clones in Cordoba, Argentina

ABSTRACT Since 1999, a new, epidemic, methicillin-resistant Staphylococcus aureus (MRSA) strain, named the “Cordobes clone,” has emerged in Argentina and coexists with the pandemic Brazilian clone. The purpose of this study was to determine the stability over time of the new clone and to investigate its evolutionary relationship with epidemic international MRSA lineages and with other MRSA and methicillin-susceptible S. aureus (MSSA) major clones distributed in this region. One hundred three MRSA isolates recovered in 2001 from Cordoba, Argentina, hospitals and 31 MSSA strains collected from 1999 to 2002 were analyzed by their antibiotic resistance patterns, phage typing, and pulsed-field gel electrophoresis. Additionally, representative members of most MRSA defined genotypes (A, B, C, E, K, and I) were characterized by multilocus sequence typing (MLST) and spaA and SCCmec typing. The most prevalent MSSA pulsotypes were also analyzed by MLST. Our results support the displacement of the Brazilian clone (sequence type [ST] 239, spaA type WGKAOMQ, SCCmec type IIIA) by the Cordobes clone (ST5, spaA type TIMEMDMGMGMK, SCCmec type I) in the hospital environment. MRSA and MSSA isolates shared only ST5. The data support the origin of the Cordobes clone as a member of a lineage that includes the pediatric and New York/Japan international clones and that is genetically related to the British EMRSA-3 strain. Interestingly, the pediatric clone, isolated from most community-acquired infections in Cordoba, was characterized by ST100, a single-locus variant of ST5 and a new variant of SCCmec type related to SCCmec type IVc.

Epidemiological, Microbiological, Clinical, and Prognostic Factors of Bacteremia Caused by High-Level Vancomycin-Resistant Enterococcus Species

Abstract A case-control study was performed between 1994 and 1996 in order to study the epidemiological, microbiological, clinical, and prognostic features of high-level vancomycin-resistant enterococcal bacteremia. Seventeen consecutive patients who had clinically significant bacteremia due to vancomycin-resistant enterococci (vanA genotype: 16 Enterococcus faecalis, 1 Enterococcus faecium) were compared with 169 who had vancomycin-susceptible enterococcal bacteremia. The following were selected by multivariate analysis as independent risk factors that influenced the development of high-level vancomycin-resistant enterococcal bacteremia: prior glycopeptide therapy (P=0.049); inclusion in a hemodialysis program (P=0.046); prior therapy with corticosteroids or antineoplastic agents (P=0.029); and prior surgical treatment (P=0.022). The following other factors were selected by univariate analysis: tracheostomy (P=0.002); prolonged hospitalization (P=0.01); and any kind of puncture (P=0.02). The crude associated-mortality rate was 13.4%. Gene amplification of vanA was positive for 17 strains of enterococci. Pulsed-field gel electrophoresis of genomic DNA after SmaI digestion of vanA isolates revealed that one strain predominated (10 isolates), though at least four similar banding patterns were identified (6 isolates). The 16 strains closely related to the outbreak were investigated further. The surgical intensive care unit was the first and most involved service. The hospital outbreak of vanA vancomycin-resistant enterococcal bacteremia occurred between 1994 and 1995 and was caused by Enterococcus faecalis. This is believed to be the first and only such outbreak described in a Spanish hospital thus far.