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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.

Staphylococcus aureus subsp. anaerobius, a microaerophilic, catalase-negative bacteria, is the etiological agent of abscess disease, a specific chronic condition of sheep and goats, characterized by the formation of necrotic lesions that are typically located in superficial lymph nodes. In this study, molecular analysis including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and accessory gene regulator (agr) typing was carried out on 94 S. aureus subsp. anaerobius strains isolated in different countries (79 were isolated from 35 outbreaks of the disease in Spain from 1981 to 2009, 9 were isolated in Italy, 3 in Denmark and 3 in Sudan). All of the 94 S. aureus subsp. anaerobius isolates examined belonged to one PFGE type, within which four minority subtypes were identified. Representative isolates of all PFGE subtypes as well of all countries belonged to the same sequence type (ST), ST1464, which was a singleton, and to the agr type II. Our results support the view that abscess disease is caused by a single bacterial clone worldwide. This bacterium has existed for at least a century and, thus, has undergone long-term small ruminant host restriction.

Introduction: Glycopeptide resistance in Enterococcus spp. is a clinical problem because of the rapid dissemination of these microorganisms, possible transfer of vancomycin resistance to more virulent pathogens, such as Staphylococcus aureus, and the limited therapeutic possibilities for the infections they cause. In this study, 10 strains of vancomycin-resistant Enterococcus faecium isolated from 10 different patients in our hospital during 2004 to 2005 were characterized. Methods: PCR was used to analyze the gene implicated in glycopeptide resistance. Molecular analysis of clinical isolates was performed by pulsed-field gel electrophoresis (PFGE). The presence of genes previously associated with epidemicity/virulence (esp, hyl, asa1, gel, cyl) was also studied. Risk factors were determined in a case-control study. Results: The most commonly detected gene was vanA. PFGE analysis revealed 5 different genotypes (A-E), with a predominance of genotype A (n ¼ 3) and B (n ¼ 3). The esp (surface protein) and hyl (hyaluronidase) genes were detected in all strains, with the exception of genotypes B and D. The purK1 allele, associated with clonal complex 17 was demonstrated in all strains.

Glycopeptides and linezolid are the most widely used antibiotics to treat infections by methicillin-resistant Staphylococcus spp. We report the presence of various isolates of methicillin-resistant S. hominis subsp. hominis with resistance to linezolid and reduced susceptibility to glycopeptides. We studied ten blood culture isolates of S. hominis subsp. hominis from nine patients admitted to our hospital. Etest was used to study susceptibility to antibiotics commonly prescribed against staphylococci. Domain V region of the 23S rRNA gene was amplified and sequenced to detect possible mutations that confer resistance to linezolid. Pulsed-field gel electrophoresis (PFGE) was used for the clonality study of isolates. All isolates were resistant to oxacillin, gentamicin, levofloxacin, cotrimoxazole, and linezolid, and susceptible to tigecycline and daptomycin. Nine of the isolates were resistant to erythromycin and clindamycin, and showed heterogeneous resistance to glycopeptides. C2190T, G2603T, and G2474T mutations were detected in domain V of the 23S rRNA gene. PFGE showed the presence of two different clones. This report alerts to the possible appearance of clinical strains of methicillin-resistant staphylococci with intermediate resistance to glycopeptides, resistance to linezolid, and multiple resistance to other second-line antibiotics.

OBJECTIVES: To characterize the population structure and resistance mechanisms of Klebsiella pneumoniae isolates that are highly resistant to third-generation cephalosporins, collected from five Spanish hospitals.METHODS: A total of 162 K. pneumoniae isolates from five hospitals located in three geographical areas of Spain were characterized. The number of isolates from each hospital ranged from 3 to 82. The genetic relationship between isolates was established by PFGE and multilocus sequence typing (MLST). bla(ESBL) types and other antibiotic resistance genes were analysed by PCR and sequencing. Plasmids were classified according to their incompatibility group by a PCR-based replicon-typing scheme.RESULTS: All 162 isolates carried the bla(CTX-15) gene. Fifty-eight isolates (35.8%) caused clinical infections and 104 (64.2%) were colonizers. Sixty-nine (42.6%) isolates were collected from newborns and 93 (57.4%) from adults. Using PGFE, the 162 isolates were grouped into seven clusters that were further identified as members of the MLST types 1, 11, 14, 17, 20, 35 and 36. Two hospitals each had two different clones and the remaining three hospitals had a single CTX-M-15-producing K. pneumoniae clone. All clones carried different antibiotic resistance genes, including bla(OXA-1), aac(3)-IIa, aac(6')-Ib-cr, qnrS1 and qnrB. In four of the seven (57.1%) clones the bla(CTX-M-15) gene was transferred by conjugation; in all cases plasmids of the incompatibility group IncF were identified by PCR.CONCLUSIONS: This study shows that multiresistant K. pneumoniae producing CTX-M-15 of MLST types 1, 11, 14, 17, 20, 35 and 36 are spreading as pathogens and colonizers among newborns and adult patients in Spain.

Epidemiological surveillance for community-associated methicillin-resistant Staphylococcus aureus revealed prevalences of 33% and 13% in pediatric and adult patients, respectively, in Cordoba, Argentina, in 2005. This study describes for the first time the emergence and dissemination of the sequence type 5 (ST5) lineage as the most prevalent clone (89%) (pulsed-field gel electrophoresis type I-ST5-staphylococcal cassette chromosome type IVa- spa type 311) harboring the Panton-Valentine leukocidin and enterotoxin A genes.

BACKGROUND: Nosocomial outbreaks of multidrug-resistant Acinetobacter baumannii are of worldwide concern. Using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and multiple locus variable number tandem repeat sequence (VNTR) analysis (MLVA), the present work examines the genetic diversity of the endemic and epidemic A. baumannii clones isolated in a single hospital over a twelve-year period. RESULTS: PFGE analysis of 405 A. baumannii-calcoaceticus complex isolates detected 15 A. baumannii endemic/epidemic PFGE types (EE1 to EE15) that grouped into five clusters: EE1-EE8, EE9, EE10, EE11 and EE12-EE15. The MLST sequence type (ST) distributions were: international clone II (ST-2) 60%, international clone III (ST-3) 26.7%, ST-15 6.7%, and ST-80 6.7%. MLVA-8Orsay returned 17 allelic profiles. The large (L) VNTR marker profiles were fully concordant with the detected STs, and concordant with 14 up to 15 PFGE types. Imipenem resistance was detected in five PFGE types; the prevalence of the bla OXA-58-like and bla OXA-40-like genes was 60% and 40% respectively. CONCLUSIONS: PFGE proved to be a vital tool for analysis of the temporal and spatial distribution of the clones. MLST and the VNTR L-markers grouped the isolates into clonal clusters. The wide diversity of MLVA small (S)-markers, however, did not permit clustering. The present results demonstrate the persistence of several endemic PFGE types in the hospital, the involvement of some of them in outbreaks, and the inter hospital transmission of extensively drug-resistant ST-15 and ST-80. Sí

Objectives: Since 1998 the European Commission has funded EARSS. We present the antibiotic susceptibility results of Invasive Staphylococcus aureus obtained in Spain (2000-2002). Material and methods: Forty hospitals participated in this study, covering nearly 30% of the Spanish population. All blood isolates of S. aureus were included. Laboratories used their usual methods to perform microbiological studies. Annual external quality controls were carried out. A questionnaire with hospital, patient and specimen data was completed for each Isolate. Results were Included in a database and analysed with WHONET 5 software. Results: Invasive S. aureus was isolated in 3113 patients. Resistance was 24.5% to oxacillin, 25.4% to ciprofloxacin, 25.2% to erythromycin and 12.1% to gentamicin. Gentamicin resistance decreased from 16.6% (2000) to 9.7% (2002). Multiresistance was observed in 68.1% of oxacillin-resistant isolates. More prevalent multiresistance profiles consisted of oxacillin-ciprofloxacin-erythromycin-gentamicln (7.4%) and oxacillin-clprofloxacin-erythromycin (7.1%). Oxacillin resistance was significantly higher in nosocomial Isolates than in those Implicated in community-onset infections (26.7% versus 14.2%), in isolates from adults than in those from children (27.3% versus 4.7%), in hospitals with >500 beds than in those with

The levels of genetic relatedness of 139 Stenotrophomonas maltophilia strains recovered from 105 hospitalized non-cystic fibrosis patients (51% from medical wards, 35% from intensive care units, and 14% from surgical wards) and 7 environmental sources in the same hospital setting during a 4-year period were typed by the pulsed-field gel electrophoresis (PFGE) technique. A total of 99 well-defined distinct Xba I PFGE patterns were identified (Simpson's discrimination index, 0.996). The dendrogram showed a Dice similarity coefficient ranging from 28 to 80%. Two major clusters (I and II), three minor clusters (III, IV, and V), and two independent branches were observed when using a 36% Dice coefficient, indicating a high diversity of genetic relatedness. It is of note that 84% of strains were grouped within two major clonal lineages. No special cluster gathering was found among strains belonging to the same sample type specimen, patients' infection or colonization status, and ward of precedence. Despite this fact, three different clones (A, B, and C) recovered from respiratory samples from six, three, and two patients, respectively, and two clones, D and E, in two bacteremic patients each, were identified. Isolation of an S. maltophilia strain belonging to the clone A profile in a bronchoscope demonstrated a common source from this clone. This study revealed a high genetic diversity of S. maltophilia isolates despite their origin from a single hospital, which may be related to the wide environmental distribution of this pathogen. However, few clones could be transmitted among different patients, yielding outbreak situations.

Multiresistance in Stenotrophomonas maltophilia limits the effectiveness of antimicrobial therapy for infections due to this organism. It can be of special concern in cystic fibrosis (CF) patients due to frequent antimicrobial administration. The in vitro activity of 41 antimicrobial agents against 76 epidemiologically defined CF S. maltophilia isolates by pulsed-field-gel electrophoresis (PFGE) technique under XbaI and SpeI restriction was compared with that obtained with 51 non-CF strains recovered from respiratory sources. Minimal inhibitory concentrations (MICs) were determined with the standard National Committee for Clinical Laboratory Standards agar dilution technique, but with 24-hr incubation. Forty-seven different PFGE profiles were observed within 76 S. maltophilia CF isolates. Minocycline (resistance rate, 0%; MIC(90), 1 microg/ml), doxycycline (6.4%; 8 microg/ml), trovafloxacin (4.2%; 2 microg/ml), moxifloxacin (6.3%; 2 microg/ml), clinafloxacin (6.3%; 2 g/ml), and moxalactam (17.0%; 64 g/ml) displayed low resistance rates. On the contrary, resistance rates were higher with ceftazidime (70.0%; 256 microg/ml), cefepime (83.0%; 128 microg/ml), piperacillin (87.2%; >1,024 microg/ml), ticarcillin (87.2%; >512 microg/ml), and aztreonam (95.7%; >1,024 microg/ml). Clavulanate reverted resistance to ticarcillin and aztreonam in 40.4% and 31.7% of strains, respectively. Aminoglycosides displayed reduced activities with susceptibility rates lower than 20% and MIC(90) higher than 128 microg/ml. With the exception of trimethoprim-sulfamethoxazole (25.4 vs. 31.3%), CF isolates were more resistant than non-CF isolates. Remarkably, resistance was enhanced in S. maltophilia isolates persistently recovered in chronically colonized patients. Susceptibility analysis demonstrated higher resistance rates among CF S. maltophilia isolates when compared with respiratory isolates from non-CF patients. Moreover, persistently recovered CF S. maltophilia isolates were more resistant than sporadic non-CF isolates.

The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 μg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa , with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E. coli (73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 μg/ml) showed a ParC amino acid change (Ser-80→Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher ( P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m -chlorophenylhydrazone (0.5 μg/ml) or reserpine (10 μg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia.

The aim of this study was to assess the epidemiology and molecular basis of the infection and dissemination of multidrug-resistant Acinetobacter baumannii (MDRAB) in three sequential outbreaks at the intensive care units (ICUs) of a tertiary university hospital in Granada, Spain, between 2009 and 2011. Strains from all patients infected and/or colonized by MDRAB during outbreak periods were characterized using PFGE and multilocus sequence typing (MLST). The first outbreak appeared in the summer of 2009 involving 38 ICU patients: 25 from a Traumatology-Rehabilitation hospital (TRH) and 13 from a Medical-Surgery hospital (MSH). Between 2010 and 2011, outbreaks were limited to the MSH-ICU, affecting 9 and 11 patients, respectively. Two PFGE types were detected. In the 2009 outbreak, two clones were identified: profile 1 strains were isolated at the TRH, whilst profile 2 was isolated at the MSH. Only one clone was identified in the 2010 and 2011 outbreaks: the profile 2 clone detected at the MSH in 2009. After MLST analysis, a single sequence type (ST92) was identified. This suggested that an endemic strain could evolve and cause localized outbreaks in vulnerable patients. Multiplex PCR for OXA group enzymes yielded a positive result for blaOXA-58-like and blaOXA-51-like genes, and gene sequencing showed the presence of blaOXA-58. However, the absence of ISAba1 upstream of the blaOXA-51-like gene suggested the absence of OXA-51 expression. The susceptibility pattern was not an appropriate method for MDRAB surveillance, as several susceptibility patterns were identified in a single clone. Consequently, molecular methods of characterization are recommended for epidemiological surveillance of MDRAB.

Pediatric patients are rarely infected with metallo-β-lactamase-producing Enterobacteriaceae. We describe 3 cases of children infected with VIM-1-producing clonal Enterobacter cloacae. Patients were treated with amikacin and cotrimoxazole. The blaVIM-1 gene was carried into a class 1 integron and an IncHI2 incompatibility group plasmid. Emergence of pediatric infections caused by carbapenemases-producing Enterobacteriaceae is a critical issue as they are resistant to most β-lactam antibiotics.

Thirty-two isolates of Corynebacterium urealyticum, isolated between 1991 and 1995, were studied by biochemical tests, phospholipid content, analysis of fatty and mycolic acids, ribotyping, whole-cell protein patterns and antimicrobial susceptibility to six antibiotics. Nineteen isolates were from human and human-related sources (HHRS); the remainder were from animal and animal-related sources (AARS). Most C. urealyticum isolates were similar in their biochemical and whole-cell protein profiles, although most HHRS isolates were alkaline phosphatase-positive (84%) and produced almost identical protein patterns, whereas AARS isolates were quite diverse. The qualitative composition of cellular fatty acids was identical for all isolates examined. Twelve different ribotypes were obtained with HindIII producing four-to-seven bands. Ribotypes 8, 9 and 10 were predominant in isolates from HHRS, whereas in isolates from AARS, ribotypes 5 and 6 predominated. AARS isolates were significantly less antibiotic-resistant, in comparison with HHRS isolates. Ribotyping appeared to be the most useful tool for strain characterisation.

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.

Here, we report the draft genome sequence of a methicillin-resistant Staphylococcus aureus (MRSA) strain with high-level mupirocin resistance (SA_ST125_MupR), isolated from a patient with recurrent bacteremia. This strain belonged to sequence type ST125, which was responsible for more than 50% of the health care-associated infections caused by MRSA 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

Newborns are rarely infected by extended-spectrum β-lactamase (ESBL)-producing members of the Enterobacteriaceae. In a neonatal intensive care unit, 14 newborns were infected or colonized by CTX-M-15-producing Enterobacter cloacae. All seven infected patients had underlying medical conditions, and five of them were treated successfully with meropenem, whilst one untreated patient died. Paediatric infections caused by multidrug-resistant ESBL-producing Enterobacter cloacae constitute a critical clinical and epidemiological issue.

OBJECTIVES: To study the distribution, diversity and activity of Acinetobacter-derived cephalosporinase (ADC)-, carbapenem-hydrolysing oxacillinase (CHO)- and metallo-β-lactamase (MBL)-encoding genes, and of the most common insertion sequences (ISs), in the genome of nosocomial, epidemic, multidrug-resistant Acinetobacter baumannii (MDRAB) clones from Spain. METHODS: The studied population included 59 MDRAB strains previously genotyped by PFGE and multilocus sequence typing. The search for the ADC (bla(ADC)), CHO (bla(OXA-51-like), bla(OXA-23-like), bla(OXA-40-like) and bla(OXA-58-like)) and MBL (bla(IMP), bla(VIM), bla(SIM-1), bla(GIM-1), bla(SPM-1) and bla(NDM-1)) genes, and for the ISs (ISAba1, ISAba2, ISAba3, ISAba4 and IS18) was done by PCR assays. The phenotypic presence of MBL enzymes was examined using imipenem/imipenem + EDTA strips. RESULTS: The most prevalent IS, ISAba1 (93.2%), was detected upstream of bla(ADC) and bla(OXA-51-like). These genes showed ample diversity (10 and 8 alleles, respectively). Four ADC sequences (ADC-1-like(P240S), ADC-2-like(N260H/T264N), ADC-11-like(Q163K) and ADC-11-like(G342R)) are described here for the first time. bla(OXA-58-like) was carried by 20.3% of strains, in association with ISAba2, ISAba3 or IS18. bla(OXA-40-like) was the most prevalent acquired CHO gene (57.6%), and was associated with none of the studied ISs. Neither bla(OXA-23-like) nor ISAba4 was detected in any strain. Some 67.8% of strains with MBL activity showed no corresponding gene in PCR; these results were more common in strains with a highly active CHO, such as OXA-40. CONCLUSIONS: All the studied genes and their related ISs showed a clonal distribution. Imipenem resistance was probably provided by OXA-40 for the most part, while MBL- and OXA-23-encoding genes were absent in the studied population.

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.

Among 3967 Staphylococcus aureus recovered from a Gran Canaria hospital (2003–2010), 28 strains were Panton-Valentine leukocidin–positive community-associated methicillin-resistant Staphylococcus aureus and were included in this study. Most isolates (89.3%) caused skin and skin–structure infections. Isolates belonging to clonal complex (CC)8 (ST8 and ST931; USA300) prevailed (82.1%). Among these, 5 (21.7%) were resistant to at least 3 antimicrobial classes.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus-(CA-MRSA) strains have emerged in Argentina. We investigated the clinical and molecular evolution of community-onset MRSA infections (CO-MRSA) in children of Córdoba, Argentina, 2005-2008. Additionally, data from 2007 were compared with the epidemiology of these infections in other regions of the country. METHODOLOGY/PRINCIPAL FINDINGS: Two datasets were used: i) lab-based prospective surveillance of CA-MRSA isolates from 3 Córdoba pediatric hospitals-(CBAH1-H3) in 2007-2008 (compared to previously published data of 2005) and ii) a sampling of CO-MRSA from a study involving both, healthcare-associated community-onset-(HACO) infections in children with risk-factors for healthcare-associated infections-(HRFs), and CA-MRSA infections in patients without HRFs detected in multiple centers of Argentina in 2007. Molecular typing was performed on the CA-MRSA-(n: 99) isolates from the CBAH1-H3-dataset and on the HACO-MRSA-(n: 51) and CA-MRSA-(n: 213) isolates from other regions. Between 2005-2008, the annual proportion of CA-MRSA/CA-S. aureus in Córdoba hospitals increased from 25% to 49%, P

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. bla VIM-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.

BACKGROUND: Community-Associated Methicillin Resistant Staphylococcus aureus (CA-MRSA) has traditionally been related to skin and soft tissue infections in healthy young patients. However, it has now emerged as responsible for severe infections worldwide, for which vancomycin is one of the mainstays of treatment. Infective endocarditis (IE) due to CA-MRSA with heterogeneous vancomycin-intermediate susceptibility-(h-VISA) has been recently reported, associated to an epidemic USA 300 CA-MRSA clone. CASE PRESENTATION: We describe the occurrence of h-VISA phenotype in a case of IE caused by a strain belonging to an epidemic CA-MRSA clone, distinct from USA300, for the first time in Argentina. The isolate h-VISA (SaB2) was recovered from a patient with persistent bacteraemia after a 7-day therapy with vancomycin, which evolved to fatal case of IE complicated with brain abscesses. The initial isolate-(SaB1) was fully vancomycin susceptible (VSSA). Although MRSA SaB2 was vancomycin susceptible (≤ 2 μg/ml) by MIC (agar and broth dilution, E-test and VITEK 2), a slight increase of MIC values between SaB1 and SaB2 isolates was detected by the four MIC methods, particularly for teicoplanin. Moreover, Sab2 was classified as h-VISA by three different screening methods [MHA5T-screening agar, Macromethod-E-test-(MET) and by GRD E-test] and confirmed by population analysis profile-(PAP). In addition, a significant increase in cell-wall thickness was revealed for SaB2 by electron microscopy. Molecular typing showed that both strains, SaB1 and SaB2, belonged to ST5 lineage, carried SCCmecIV, lacked Panton-Valentine leukocidin-(PVL) genes and had indistinguishable PFGE patterns (subtype I2), thereby confirming their isogenic nature. In addition, they were clonally related to the epidemic CA-MRSA clone (pulsotype I) detected in our country. CONCLUSIONS: This report demonstrates the ability of this epidemic CA-MRSA clone, disseminated in some regions of Argentina, to produce severe and rapidly fatal infections such as IE, in addition to its ability to acquire low-level vancomycin resistance; for these reasons, it constitutes a new challenge for the Healthcare System of this country. This study was supported by the National Council for Scientific Research and Technology of Argentina (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT - PICT 01630 to JLB), Secretaría de Ciencia y Técnica-Universidad Nacional de Córdoba (SECyT-UNC) and Agencia Córdoba Ciencia. Sí

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.

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 (SCC mec ) 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. SCC mec 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 SCC mec type I and SCC mec 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 SCC mec type IV was the most prevalent throughout Spain. In our experience, PFGE, spa typing, SCC mec 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.

We carried out a nationwide study aimed at the determination of the molecular epidemiology and antibiotic resistance mechanisms of invasive Staphylococcus aureus in 21 Spanish hospitals.The distributions of molecular markers, including antibiotic resistance genes, were investigated in 203 S. aureus, comprising 90 methicillin-resistant S. aureus (MRSA) and 113 methicillin-susceptible S. aureus (MSSA). Antimicrobial susceptibility was determined by standard methods. Panton-Valentine leucocidin (PVL) detection, staphylococcal cassette chromosome mec (SCCmec) types and agr types were performed/determined by PCR. All isolates were genotyped by PFGE after digestion of chromosomal DNA with SmaI. Multilocus sequence typing and spa-typing were also performed.In MRSA isolates, 74.4% were agr allotype II and were positive for SCCmec IV. Sixty-nine spa-types were identified, 18 in MRSA and 57 in MSSA. Both MRSA and MSSA variants were detected in six spa-types (8.7%). The majority of S. aureus (51.2%) were grouped into four spa-types (t067, t002, t012 and t008). The spa-type t067 was detected in 18 of the 21 (85.7%) participating hospitals, including both MRSA and MSSA in six of them; in total, 25.9% of our isolates were spa-type t067 (49% in MRSA) in comparison with 0.6% in a central spa-typing database. The prevalence of the ant(4')-Ia and msrA/msrB genes was significantly higher in the MRSA spa-type t067 than in the other MRSA spa-types. Association between spa-type t067 and ST125 is described here for the first time. A high prevalence (36.4%) of PVL-positive MSSA was detected.A higher than expected prevalence of spa-type t067 isolates was found among invasive MRSA in Spain. The oxacillin, tobramycin, erythromycin and ciprofloxacin resistance profile of spa-type t067 isolates was linked to the presence of ant(4')-Ia and msrA or msrB genes.

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).

In the study presented here, the genetic characteristics of methicillin-susceptible Staphylococcus aureus (MSSA) strains isolated from patients attending hospitals in the city of Cordoba, Argentina, during 1999-2002 were evaluated to determine their genetic relationship with methicillin-resistant S. aureus (MRSA) clones as part of an effort to control the potential emergence of new epidemic MRSA strains. The results showed there is a high frequency of MSSA strains carrying Panton-Valentine leukocidin genes in invasive infections in Cordoba, Argentina, particularly in those occurring in hospital settings. Panton-Valentine leukocidin genes were found in the genomic background of one clone (ST30-N pulsotype) belonging to a successful internationally distributed MSSA lineage (clonal complex 30), which is closely related to the EMRSA-16 pandemic clone. These genes were also detected in the ancestral clone (ST5-M pulsotype) of the most prevalent MRSA epidemic clone causing healthcare-associated infections in this region, known as the Cordobes/Chilean clone. The molecular characterization of circulating MSSA strains, including the detection of Panton-Valentine leukocidin genes, is thus a useful marker for investigating the evolving epidemiology of hospital- and community-acquired MRSA clones.

Objectives: To ascertain the participation of topoisomerase mutations in the development of ciprofloxacin resistance in isogenic Stenotrophomonas maltophilia mutants. Methods: gyrABand parCEsequences in three paired In vivo isogenic ciprofloxacin-susceptible (MIC range 0.5-4 mg/L) and resistant (16-128 mg/L) S. maltophilia strains (PFGE-characterized) sequentially isolated from three patients, and their corresponding in vitro mutants (ciprofloxacin MIC range 2->128 mg/L), were studied. Efflux phenotype was also investigated. Results: Despite different quinolone susceptibilities, each paired clinical strain displayed identical gyrAB and parCE sequences as well as their corresponding in vitro mutants. Up to 50% (18/36) of in vitro mutants displayed a positive efflux phenotype when nalidixic acid was combined with MC-207,110, while 6% (2/36) showed the phenotype when exposed to nalidixic acid and reserpine. Carbonyl cyanide m-chlorophenyl-hydrazone or arsenite failed to alter quinolone MICs. Conclusions: The increase of ciprofloxacin MICs in in vivo and in vitro isogenic S. maltophilia mutant strains was not related to quinolone resistance determining region mutations. Highly effective efflux mechanisms might preserve topoisomerase targets from a ciprofloxacin challenge in S. maltophilia.

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.

Susceptibility to 41 antimicrobials was studied with 99 Stenotrophomonas 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.

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.

The susceptibility of 109 Stenotrophomonas maltophilia isolates, all characterized by pulsed-field gel electrophoresis, to nine quinolones was studied. Grepafloxacin, trovafloxacin, and moxifloxacin displayed similar intrinsic activities (MIC90, 0.5 microg/ml), which were lower than those of ofloxacin and ciprofloxacin (MIC90, 4 microg/ml), norfloxacin (MIC90, 64 microg/ml), and nalidixic acid (MIC90, 32 microg/ml). Nalidixic acid was generally one- to twofold dilutions more active than norfloxacin. According to the criteria of the National Committee for Clinical Laboratory Standards (NCCLS), the percentage of isolates susceptible to ciprofloxacin (breakpointor = 1 microg/ml) was 76.1%. Using the NCCLS breakpoint for comparative purposes, the percentage of isolates susceptible to grepafloxacin, moxifloxacin, and trovafloxacin was 95.4, 96.4, and 96.4%, respectively. These results indicate that new quinolones may potentially be used for the management of Stenotrophomonas maltophilia infections.

SUMMARYA high percentage of non-typable (NT)Staphylococcus aureusstrains was isolated in Spanish hospitals during 1984 and 1985. Several alternative methods of typing were employed to study these isolates. These were: phage-typing at 1000 × RTD, phage-typing after heat-treatment (48 °C), thermal shock (56 °C), reverse-typing and induction of additional phages. Using these methods the number of NT isolates was reducedby 60%. Best results were obtained with heat-treatment. Additional phages and reverse-typing were also useful.A scheme for the study of outbreaks and sporadic cases caused by NT strains is proposed using the methods described.