Search

Resumen Introduccion El objetivo de este estudio es la caracterizacion de cepas de Streptococcus pyogenes con fenotipo mucoide y su comparacion con las cepas no mucoides aisladas entre abril y agosto de 2016. Material y metodos Se llevo a cabo la caracterizacion y el estudio de sensibilidad antimicrobiana de todos los aislados. Se determino el tipo emm y se analizaron los genes de exotoxinas speA, speB, speC, speF, speG, speH, speJ, speZ y ssa. Se recogieron datos clinicos y demograficos. Resultados De 96 aislados analizados, el 47% presentaron un fenotipo mucoide, y de estos ultimos, el 95,5% presentaron los genes speA-speB-speF-speG-ssa y genotipo emm3. La principal manifestacion clinica entre todos los pacientes fue faringoamigdalitis (77,1%) que evoluciono a escarlatina en el 67,5% de los casos. Conclusion Se describe la circulacion de una cepa de aspecto mucoide con perfil de toxinas speA-speB-speF-speG-ssa y genotipo emm3.1 considerado de los mas frecuentes y mas virulentos de SGA.

Background: Bacteroides fragilis shows high antimicrobial resistance (AMR) rates and possesses numerous AMR mechanisms. Its carbapenem-resistant strains (metallo-β-lactamase cfiA-positive) appear as an emergent, evolving clade. Methods: This work examines the genomes, taxonomy, and phylogenetic relationships with respect to other B. fragilis genomes of two B. fragilis strains (CNM20180471 and CNM20200206) resistant to meropenem+EDTA and other antimicrobial agents. Results: Both strains possessed cfiA genes (cfiA14b and the new cfiA28), along with other AMR mechanisms. The presence of other efflux-pump genes, mexAB/mexJK/mexXY-oprM, acrEF/mdtEF-tolC, and especially cusR, which reduces the entry of carbapenem via the repression of porin OprD, may be related to meropenem–EDTA resistance. None of the detected insertion sequences were located upstream of cfiA. The genomes of these and other B. fragilis strains that clustered together in phylogenetic analyses did not meet the condition of &gt, 95% average nucleotide/amino acid identity, or &gt, 70% in silico genome-to-genome hybridization similarity, to be deemed members of the same species, although &lt, 1% difference in the genomic G+C content was seen with respect to the reference genome B. fragilis NCTC 9343T. Conclusions: Carbapenem-resistant strains may be considered a distinct clonal entity, and their surveillance is recommended given the ease with which they appear to acquire AMR.

Nocardia species, one of the most predominant Actinobacteria of the soil microbiota, cause infection in humans following traumatic inoculation or inhalation. The identification, typing, phylogenetic relationship and antimicrobial susceptibilities of 38 soil Nocardia strains from Lara State, Venezuela, were studied by 16S rRNA and gyrB (subunit B of topoisomerase II) genes, multilocus sequence analysis (MLSA), whole-genome sequencing (WGS), and microdilution. The results were compared with those for human strains. Just seven Nocardia species with one or two strains each, except for Nocardia cyriacigeorgica with 29, were identified. MLSA confirmed the species assignments made by 16S rRNA and gyrB analyses (89.5% and 71.0% respectively), and grouped each soil strain with its corresponding reference and clinical strains, except for 19 N. cyriacigeorgica strains found at five locations which grouped into a soil-only cluster. The soil strains of N. cyriacigeorgica showed fewer gyrB haplotypes than the examined human strains (13 vs. 17) but did show a larger number of gyrB SNPs (212 vs. 77). Their susceptibilities to antimicrobials were similar except for beta-lactams, fluoroquinolones, minocycline, and clarithromycin, with the soil strains more susceptible to the first three (p ≤ 0.05). WGS was performed on four strains belonging to the soil-only cluster and on two outside it, and the results compared with public N. cyriacigeorgica genomes. The average nucleotide/amino acid identity, in silico genome-to-genome hybridization similarity, and the difference in the genomic GC content, suggest that some strains of the soil-only cluster may belong to a novel subspecies or even a new species (proposed name Nocardia venezuelensis). This work was funded by a grant to N.G. from the Instituto de Salud Carlos III (MPY 1278/15). A part of this manuscript forms the M.S project undertaken at the Faculty of Science, Universidad Autónoma de Madrid (Spain). Sí

Streptococcus pyogenes appears to be almost exclusively restricted to humans, with few reports on isolation from animals. We provide a detailed characterization (emm typing, pulsed-field gel electrophoresis [PFGE], and multilocus sequence typing [MLST]) of 15 S. pyogenes isolates from animals associated with different clinical backgrounds. We also investigated erythromycin resistance mechanisms and phenotypes and virulence genes. We observed 2 emm types: emm12 (11 isolates) and emm77 (4 isolates). Similarly, we observed 2 genetic linages, sequence type (ST) 26 and ST63. Most isolates exhibited the M macrolide resistance phenotype and the mefA/ermB genotype. Isolates were grouped into 2 clones on the basis of emm-MLST-PFGE-virulence gene profile combinations: clone 1, characterized by the combined genotype emm12-ST36-pulsotype A-speG; and clone 2, characterized by the genotype emm77-ST63-pulsotype B-speC. Our results do not show conclusively that animals may represent a new reservoir of S. pyogenes but indicate the ability of human-derived S. pyogenes isolates to colonize and infect animals.

The taxonomic position of an unknown bacterial strain designated CNM695-12, isolated from the blood of an immunocompromised subject, was investigated via phenotypic, chemotaxonomic, genotypic and genomic analyses. Bacterial cells were determined to be Gram-stain-negative bacilli, aerobic, non-motile and non-spore-forming. The strain showed catalase activity but no oxidase activity. Optimal growth occurred at 37 °C, pH 7 and with 0–1 % NaCl. C16 : 0, summed feature 8 (comprising C18 : 1ω7c / C18:1 ω6c), and C18 : 1ω9c were the most abundant fatty acids, and ubiquinone 8 was the major respiratory quinone. The polar lipids present included phosphatidylglycerol, phosphatidylethanolamine and other aminophospholipids. The 16S rRNA gene sequence showed approximately 93.5 % similarity to those of different species with validly published names within the order Burkholderiales (e.g. Leptothrix mobilis Feox-1T, Aquabacterium commune B8T , Aquabacterium citratiphilum B4T and Schlegelella thermodepolymerans K14T). Phylogenetic analyses based on 16S rRNA gene sequences and concatenated alignments including the sequences for 107 essential proteins, revealed the strain to form a novel lineage close to members of the family Comamonadaceae . The highest average nucleotide identity and average amino acid identity values were obtained with Schlegelella thermodepolymerans K14T (69.6 and 55.7 % respectively). The genome, with a size of 3.35 Mb, had a DNA G+C content of 52.4 mol% and encoded 3056 predicted genes, 3 rRNA, 1 transfer–messengerRNA and 51 tRNA. Strain CNM695-12 thus represents a novel species belonging to a novel genus within the order Burkholderiales , for which the name Saezia sanguinis gen. nov., sp. nov. is proposed. The type strain is CNM695-12T (=DSM 104959T=CECT 9208T).

Our objective was to improve current knowledge of sporadic (Spo) nosocomial Acinetobactercalcoaceticus-Acinetobacter baumannii (Acb) complex populations, and thus better understand the epidemiology of Spo and endemoepidemic (EE) strains. Between 1999 and 2010, 133 isolates of Spo Acb complex were obtained from a single hospital. Species were identified by gyrB-PCR, and via gyrB- and rpoB-sequencing. Clonal analysis was undertaken using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Susceptibility to antimicrobial agents was determined by microdilution and E-tests. Carbapenemase genes were detected by PCR. One hundred and one PFGE types were detected. A. baumannii was the most common (67/101 PFGE types), followed by Acinetobacter pittii (22/101), Acinetobacter lactucae (6/101), and Acinetobacter calcoaceticus (2/101). gyrB, rpoB1, and rpoB2 sequencing returned 49, 13, and 16 novel sequences, respectively. Sixty-three sequence types (STs) (38 new STs and 66 new alleles) were detected; the most common were ST2 (29/133 isolates) and ST132 (14/133). Twenty-six OXA-51 allelic variants were detected, nine of which were novel. The PFGE types were generally susceptible (88/101) to all the tested antimicrobials; 3/101 were carbapenem-resistant due to the presence of the genetic structure ISAba2-blaOXA-58-like-ISAba3, and 2/101 were multidrug-resistant. It can be concluded that the examined Spo Acb complex population was mainly composed of A. baumannii. Many different clones were detected (with ST2 clearly dominant), all largely susceptible to antimicrobials; multidrug resistance was rare. In contrast, a previously examined EE Acb population was composed of just four expanding, multidrug-resistant A. baumannii clones -ST2, ST3, ST15, and ST80-. We thank Dr. Teresa Cabezas for the isolation and submission of the Acinetobacter spp. strains to the Reference and Research Laboratory for Taxonomy at the CNM, and Adrian Burton for editing and language assistance (Physical Evidence Scientific Translations; http://physicalevidence.es/english/welcome). This publication made use of the Acinetobacter baumannii MLST website (https://pubmlst.org/abaumannii/) hosted by the University of Oxford (Jolley and Maiden 2010, BMC Bioinformatics, 11:595), the development of which was funded by the Wellcome Trust. Sí

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 126 AAAPEH 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.

Objectives The aims of this study were to explore the clinical distribution, by species, of the genus Nocardia and to assess the antimicrobial susceptibilities of the 10 most prevalent species identified in Spain. Methods Over a 10 year period (2005-14), 1119 Nocardia strains were molecularly identified and subjected to the Etest. The distribution and resistance trends over the sub-periods 2005-09 and 2010-14 were also examined. Results Of the strains examined, 82.9% belonged to the following species: Nocardia cyriacigeorgica (25.3%), Nocardia nova (15.0%), Nocardia abscessus (12.7%), Nocardia farcinica (11.4%), Nocardia carnea (4.3%), Nocardia brasiliensis (3.5%), Nocardia otitidiscaviarum (3.1%), Nocardia flavorosea (2.6%), Nocardia rhamnosiphila (2.6%) and Nocardia transvalensis (2.4%). Their prevalence values were similar during 2005-09 and 2010-14, except for those of N. abscessus , N. farcinica and N. transvalensis , which fell significantly in the second sub-period ( P ≤ 0.05). The major location of isolation was the respiratory tract (∼86%). Half (13/27) of all strains from the CNS were N. farcinica . Significant differences in MIC results were recorded for some species between the two sub-periods. According to the CLSI's breakpoints, low resistance rates (≤15%) were recorded for seven species with respect to cefotaxime, imipenem and tobramycin; five species showed similar rates with respect to trimethoprim/sulfamethoxazole. Linezolid and amikacin were the most frequently active agents. Conclusion The accurate identification of the infecting species and the determination of its susceptibility to antimicrobial agents, given the large number of strains with atypical patterns, are crucial if patients with nocardiosis are to be successfully treated.

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 bla SFO-1 , and two complete sequences of IS 26 surrounding ampR and ampA were detected. These IS 26 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 IS 26 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′ IS 26 . 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.

The soil-borne pathogen Nocardia sp. causes severe cutaneous, pulmonary, and central nervous system infections. Against them, co-trimoxazole (SXT) constitutes the mainstay of antimicrobial therapy. However, some Nocardia strains show resistance to SXT, but the underlying genetic basis is unknown. We investigated the presence of genetic resistance determinants and class 1-3 integrons in 76 SXT-resistant Nocardia strains by PCR and sequencing. By E test, these clinical strains showed SXT minimum inhibitory concentrations of ≥32:608 mg/L (ratio of 1:19 for trimethoprim: sulfamethoxazole). They belonged to 12 species, being the main representatives Nocardia farcinica (32%), followed by N. flavorosea (6.5%), N. nova (11.8%), N. carnea (10.5%), N. transvalensis (10.5%), and Nocardia sp. (6.5%). The prevalence of resistance genes in the SXT-resistant strains was as follows: sul1 and sul2 93.4 and 78.9%, respectively, dfrA(S1) 14.7%, blaTEM-1 and blaZ 2.6 and 2.6%, respectively, VIM-2 1.3%, aph(3')-IIIa 40.8%, ermA, ermB, mefA, and msrD 2.6, 77.6, 14.4, and 5.2%, respectively, and tet(O), tet(M), and tet(L) 48.6, 25.0, and 3.9%, respectively. Detected amino acid changes in GyrA were not related to fluoroquinolone resistance, but probably linked to species polymorphism. Class 1 and 3 integrons were found in 93.42 and 56.57% strains, respectively. Class 2 integrons and sul3 genes were not detected. Other mechanisms, different than dfrA(S1), dfrD, dfrF, dfrG, and dfrK, could explain the strong trimethoprim resistance shown by the other 64 strains. For first time, resistance determinants commonly found in clinically important bacteria were detected in Nocardia sp. sul1, sul2, erm(B), and tet(O) were the most prevalent in the SXT-resistant strains. The similarity in their resistome could be due to a common genetic platform, in which these determinants are co-transferred. This study was presented at the 54th Interscience Conference on Antimicrobial Agents and Chemotherapy, ICAAC2014, Washington, DC, USA. We thank Adrian Burton for editing and language assistance (http://physicalevidence.es/english/welcome). We are very grateful to all persons who took part in this study, and to the sample providers. Sí

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

gyrB is used to improve the identification of the Nocardia species N. brasiliensis , N. higoensis , N. ignorata , N. otitidiscaviarum , N. paucivorans , N. pneumoniae , N. puris , N. takedensis , N. veterana , and N. vinacea , but it does not improve the identification of another 12 Nocardia studied species. gyrB provides typing and phylogenetic markers for N. carnea , N. transvalensis , N. brasiliensis , and N. otitidiscaviarum .

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.

Background 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. Results Two hundred ninety-five out of 898 isolates (32.8%) were erythromycin resistant, with the predominance of emm 4T4, emm 75T25, and emm 28T28, accounting the 67.1% of the 21 emm/T types. Spread of emm 4T4, emm 75T25 and emm 28T28 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 emm 77T28 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 emm 11T11 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. Conclusions 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 emm 4T4, emm 75T25 and emm 28T28 was the main responsable for macrolide resistance where as that emm 77T28 clones were it to tetraclycline resistance. A wide variety of macrolide resistance genes were responsible for three macrolide resistance phenotypes.

Burkholderia spp. strains collected in Spain over a 13-year period from patients with cystic fibrosis (CF) (n = 148), non-CF patients (n = 103) and from environmental sources (n = 64) were characterised. One hundred and forty-one of the examined strains were involved in seven suspected nosocomial disease outbreaks. Strains were identified by their 16s rRNA and recA genes. Their genetic relatedness, the possession of cable pili and the B. cepacia epidemic strain marker (BCESM), and their susceptibility to antimicrobial agents were studied using pulsed-field gel electrophoresis (PFGE), cblA and esmR genes analysis, and by the E-test, respectively. The genomovar distribution for the 315 strains was as follows: B. stabilis 29.5 %, B. cepacia 14.9 %, B. multivorans 11.1 %, B. cenocepacia IIIA 9.5 %, B. vietnamiensis 3.8 %, B. cenocepacia IIIB 3.5 %, and B. ambifaria and B. pyrrocinia 0.3 % each. The genetic diversity of the B. cepacia complex (Bcc) was ample, with 57 different SpeI types, showing a genetic similarity of 36.4-96.6 %. No strain carried cblA, whereas 25 B. cenocepacia genotypes harboured BCESM (23 from patients with CF). Antimicrobial resistance rates to tobramycin (TOB; 86 %) and imipenem (IPM; 67 %) were high. The strains from patients with CF showed significantly greater resistance to piperacillin (PIP), levofloxacin (LVX) and co-trimoxazole (SXT) than those isolated from non-CF patients (p < 0.05). In conclusion, B. cenocepacia was the most prevalent genomovar found in patients with CF (19.1 %), whereas B. cepacia was the most common among non-CF patients (20.7 %). B. stabilis (47.6 %) was the most common environmental genomovar. Susceptibility to antimicrobial agents depended on genomovar status and strain origin.

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.

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 humanBrucella melitensispopulation (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.rpoBtyping grouped the strains asrpoBtype 1 (1249-ATG/1309-CTG; 28.7%),rpoBtype 2 (1249-ATG/1309-CTA; 62.9%), andrpoBtype 3 (1249-ATA/1309-CTG; 8.3%). According to the MLVA-16 results, the population clustered byrpoBtype. Given the correlation betweenB. melitensisMLVA groups andrpoBtypes (American andrpoBtype 1, Eastern Mediterranean andrpoBtype 2, and Western Mediterranean andrpoBtype 3), therpoBtype could be used as an initial marker for the epidemiological surveillance of brucellosis.

Species of the Burkholderia cepacia complex are associated with opportunistic infection in patients with cystic fibrosis. For years now, B. multivorans and B. cenocepacia have been the most frequently isolated species within the complex in such patients. However, between 2008 and 2012, the overall incidence of these species in Spain (17.7% and 12.5% respectively) was overtaken by that of B. contaminans (36.5%). The population structure of B. contaminans isolates from Spanish patients with cystic fibrosis was analysed using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE). Three major known sequence types (ST102, ST404 and ST482) and a new one (ST771) were identified among 59 isolates. In addition, PFGE detected 17 pulsotypes. Susceptibility to antibiotics was examined using the Etest. Cotrimoxazole and ceftazidime were the most active antibiotics against B. contaminans, inhibiting growth in 88% and 86% of the isolates, respectively. In addition, this species showed less resistance to most of the antibiotics tested than did either B. multivorans or B. cenocepacia isolates recovered from similar Spanish patients.