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1.  Identification, Typing, and Phylogenetic Relationships of the Main Clinical Nocardia Species in Spain According to Their gyrB and rpoB Genes 
Journal of Clinical Microbiology  2013;51(11):3602-3608.
This study compares the identification, typing, and phylogenetic relationships of the most prevalent clinical Nocardia species in Spain, as determined via sequence analysis of their housekeeping genes gyrB and rpoB, with the results returned by the gold standard 16S rRNA method. gyrB and rpoB analyses identified Nocardia abscessus, N. cyriacigeorgica, N. farcinica, and the N. nova complex, species that together account for more than half of the human nocardiosis cases recorded in Spain. The individual discriminatory power of gyrB and rpoB with respect to intraspecies typing, calculated using the Hunter-Gaston discriminatory index (HGDI), was generally high (HGDI, 0.85 to 1), except for rpoB with respect to N. farcinica (HGDI, 0.71). Phylogenetically, different degrees of intra- and interspecies microheterogeneity were observed for gyrB and rpoB in a group of 119 clinical strains. A single 16S haplotype was obtained for each species, except for the N. nova complex (8 types), while gyrB and rpoB were more polymorphic: N. abscessus had 14 and 18 haplotypes, N. cyriacigeorgica had 17 and 12, N. farcinica had 11 and 5, and the N. nova complex had 26 and 29 haplotypes, respectively. A diversity gradient was therefore seen, with N. farcinica at the bottom followed by N. abscessus and N. cyriacigeorgica in the middle and N. nova complex at the top. The complexity of the N. nova complex is highlighted by its six variations in the GyrB 126AAAPEH motif. gyrB sequencing (with or without rpoB sequencing) offers a simple means for identifying the most prevalent Nocardia species in Spanish medical laboratories and for determining the intraspecific diversity among their strains.
PMCID: PMC3889740  PMID: 23966490
2.  Molecular epidemiology, antimicrobial susceptibilities and resistance mechanisms of Streptococcus pyogenes isolates resistant to erythromycin and tetracycline in Spain (1994–2006) 
BMC Microbiology  2012;12:215.
Group A Streptococcus (GAS) causes human diseases ranging in severity from uncomplicated pharyngitis to life-threatening necrotizing fasciitis and shows high rates of macrolide resistance in several countries. Our goal is to identify antimicrobial resistance in Spanish GAS isolates collected between 1994 and 2006 and to determine the molecular epidemiology (emm/T typing and PFGE) and resistance mechanisms of those resistant to erythromycin and tetracycline.
Two hundred ninety-five out of 898 isolates (32.8%) were erythromycin resistant, with the predominance of emm4T4, emm75T25, and emm28T28, accounting the 67.1% of the 21 emm/T types. Spread of emm4T4, emm75T25 and emm28T28 resistant clones caused high rates of macrolide resistance. The distribution of the phenotypes was M (76.9%), cMLSB (20.3%), iMLSB (2.7%) with the involvement of the erythromycin resistance genes mef(A) (89.5%), msr(D) (81.7%), erm(B) (37.3%) and erm(A) (35.9%).
Sixty-one isolates were tetracycline resistant, with the main representation of the emm77T28 among 20 emm/T types. To note, the combination of tet(M) and tet(O) tetracycline resistance genes were similar to tet(M) alone reaching values close to 40%. Resistance to both antibiotics was detected in 19 isolates of 7 emm/T types, being emm11T11 and the cMLSB phenotype the most frequent ones. erm(B) and tet(M) were present in almost all the strains, while erm(A), mef(A), msr(D) and tet(O) appeared in less than half of them.
Spanish GAS were highly resistant to macrolides meanwhile showed minor resistance rate to tetracycline. A remarkable correlation between antimicrobial resistance and emm/T type was noticed. Clonal spread of emm4T4, emm75T25 and emm28T28 was the main responsable for macrolide resistance where as that emm77T28 clones were it to tetraclycline resistance. A wide variety of macrolide resistance genes were responsible for three macrolide resistance phenotypes.
PMCID: PMC3490898  PMID: 22998619
GAS; emm gene; PFGE; Macrolide resistance; Tetracycline resistance
3.  Emergence in Spain of a Multidrug-Resistant Enterobacter cloacae Clinical Isolate Producing SFO-1 Extended-Spectrum β-Lactamase▿ 
Journal of Clinical Microbiology  2011;49(3):822-828.
Between February 2006 and October 2009, 38 patients in different wards at the A Coruña University Hospital (northwest Spain) were either infected with or colonized by an epidemic, multidrug-resistant (MDR), and extended-spectrum-β-lactamase (ESBL)-producing strain of Enterobacter cloacae (EbSF), which was susceptible only to carbapenems. Semiautomated repetitive extragenic palindromic sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) analysis revealed that all of the E. cloacae isolates belonged to the same clone. Cloning and sequencing enabled the detection of the SFO-1 ESBL in the epidemic strain and the description of its genetic environment. The presence of the ampR gene was detected upstream of blaSFO-1, and two complete sequences of IS26 surrounding ampR and ampA were detected. These IS26 sequences are bordered by complete left and right inverted repeats (IRL and IRR, respectively), which suggested that they were functional. The whole segment flanked by two IS26 copies may be considered a putative large composite transposon. A gene coding for aminoglycoside acetyltransferase (gentamicin resistance gene [aac3]) was found downstream of the 3′ IS26. Despite the implementation of strict infection control measures, strain EbSF spread through different areas of the hospital. A case-control study was performed to assess risk factors for EbSF acquisition. A multivariate analysis revealed that the prior administration of β-lactam antibiotics, chronic renal failure, tracheostomy, and prior hospitalization were statistically associated with SFO-1-producing E. cloacae acquisition. This study describes for the first time an outbreak in which an SFO-1-producing E. cloacae strain was involved. Note that so far, this β-lactamase has previously been isolated in only a single case of E. cloacae infection in Japan.
PMCID: PMC3067750  PMID: 21227991
4.  Clonal Diversity of Nosocomial Epidemic Acinetobacter baumannii Strains Isolated in Spain▿ 
Journal of Clinical Microbiology  2011;49(3):875-882.
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.
PMCID: PMC3067678  PMID: 21177889
5.  Epidemiological and Phylogenetic Analysis of Spanish Human Brucella melitensis Strains by Multiple-Locus Variable-Number Tandem-Repeat Typing, Hypervariable Octameric Oligonucleotide Fingerprinting, and rpoB Typing▿  
Journal of Clinical Microbiology  2010;48(8):2734-2740.
The severe morbidity of human brucellosis is one of the main reasons for using molecular typing in the epidemiological surveillance of this worldwide zoonosis. Multiple-locus variable-number repeat analysis (MLVA-16), hypervariable octameric oligonucleotide fingerprinting (HOOF-print), and the differences in the single nucleotide polymorphisms (SNPs) (codons 1249 and 1309) of the DNA-dependent RNA polymerase β subunit (rpoB) were used to type a human Brucella melitensis population (108 strains) collected from throughout Spain over 13 years. Eighty-six MLVA types (discriminatory index, 0.99) were detected, with a wide-ranging genetic similarity coefficient (37.2 to 93.7%). The population clustered into the following groups: American, with genotypes 47 (1 strain), 48 (13 strains), 53 (12 strains), 55 (2 strains), 80 (1 strain), and a new genotype (2 strains), Western Mediterranean, with genotype 51 (9 strains), and Eastern Mediterranean, with genotypes 42 (60 strains), 43 (4 strains), and 63 (4 strains). Two profession-related and two foodborne acquisitions were confirmed. Distributed throughout Spain, Eastern Mediterranean genotype 42 was the most common (55%). The low MLVA-16 allelic polymorphism (genetic similarity range, 75 to 94%) of the genotype 42 strains suggests that they recently evolved from a common ancestor. rpoB typing grouped the strains as rpoB type 1 (1249-ATG/1309-CTG; 28.7%), rpoB type 2 (1249-ATG/1309-CTA; 62.9%), and rpoB type 3 (1249-ATA/1309-CTG; 8.3%). According to the MLVA-16 results, the population clustered by rpoB type. Given the correlation between B. melitensis MLVA groups and rpoB types (American and rpoB type 1, Eastern Mediterranean and rpoB type 2, and Western Mediterranean and rpoB type 3), the rpoB type could be used as an initial marker for the epidemiological surveillance of brucellosis.
PMCID: PMC2916618  PMID: 20554816

Results 1-5 (5)