Search

Objectives: The aim of this work was to study the ability of clinical isolates of Ureaplasma spp. to form biofilms in vitro and to compare the antibiotic susceptibility of sessile cells and their planktonic counterparts. Methods: A total of nine Ureaplasma spp. isolates recovered from unrelated male patients diagnosed with urethritis or chronic prostatitis and two isolates isolated from the urine of two healthy volunteers were included. Ureaplasma species identification was performed by 16S rDNA gene amplification and sequencing. Conventional antibiotic susceptibility tests were carried out by the broth microdilution method. Biofilm susceptibility assays were performed following the method proposed by Moskowitz using 10C urea broth medium and confirming bacterial growth by colour shift of the medium. The x 2 test was applied to analyse the statistical differences between the MIC and the minimal biofilm inhibitory concentration. Results: Isolates were identified as Ureaplasma urealyticum serovar 7 (five isolates), U. urealyticum serovar 13 (four isolates) and Ureaplasma parvum serovar 3 (two isolates). Biofilm formation was observed in 9 out of the 11 strains studied (82%); two isolates of U. urealyticum serovar 13 were nonbiofilm formers. Global resistance percentages of planktonic cells compared with sessile cells were different for erythromycin (0% versus 44%, P 5 0.02), telithromycin (22% versus 77%, P 5 0.02), ciprofloxacin (66% versus 100%), levofloxacin (0% versus 33%) and tetracycline (0% versus 33%). All nine biofilm-forming strains were fully susceptible to clarithromycin in both planktonic and biofilm types of growth. Conclusions: These results indicate that biofilm formation can protect mycoplasma cells from antibiotics and host defences, favouring their persistence in chronically infected or colonized patients while increasing resistance to antimicrobial agents. Therefore, the capacity to form biofilms by Ureaplasma spp. isolates should be considered when antibiotic treatments are required.

Background: Yogurt has traditionally been considered a probiotic-carrier food with health-promoting effects. Despite the universal assumption of this assertion, several researchers have evaluated the real capability of the yogurt bacteria Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus to survive and proliferate in the human intestine and have found contradictory results. Objective: This double-blind crossover study assessed the qualitative and quantitative effects of fresh and heat-treated yogurt on bacterial intestinal microbiota from healthy subjects. Design: The subjects were divided into experimental (n = 63) and control (n = 16) groups. The experimental group consumed fresh and heat-treated yogurt for 15 d according to a crossover design with a washout period of 2 wk. Three different fecal samples per individual were recovered: at baseline, after fresh yogurt intake, and after heat-treated yogurt intake. Qualitative changes in microbiota were studied by denaturing gel gradient electrophoresis (DGGE) with universal and lactic acid bacteria (LAB) 16S-rRNA primers. Quantitative changes in LAB, Clostridium coccoides, Clostridium perfringens, and Bacteroides groups were analyzed by real-time polymerase chain reaction. Results: A particular DGGE stable band pattern was observed in each sample. No significant qualitative differences were detected in any fecal sample. However, a significantly higher density of LAB and C. perfringens and a significant decrease in the density of Bacteroides was observed after consumption of both types of yogurt. Microbiota density was not significantly different between the fresh and heat-treated yogurt groups, except for LAB, which was significantly greater in the fresh yogurt group. Conclusion: The main change in human microbiota observed after yogurt consumption was an increase in the density of LAB and C. perfringens to the detriment of Bacteroides. Bacterial changes were not different after the consumption of fresh and heat-treated yogurt.

Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) were classically clustered into the Lancefield Group D streptococci and despite their taxonomic reclassification still share a similar genetic content and environment. Both species are considered as opportunistic pathogens. E. faecium is often associated with nosocomial bacteraemia, and S. gallolyticus is sporadically found in endocarditis of colorectal cancer patients. In both cases, the source of infection is commonly endogenous with a translocation process that launches through the intestinal barrier. To get new insights into the pathological processes preceding infection development of both organisms, we used an in vitro model with Caco-2 cells to study and compare the adhesion, invasion and translocation inherent abilities of 6 E. faecium and 4 S. gallolyticus well-characterized isolates. Additionally, biofilm formation on polystyrene, collagen I and IV was also explored. Overall results showed that E. faecium translocated more efficiently than S. gallolyticus, inducing a destabilization of the intestinal monolayer. Isolates Efm106, Efm121 and Efm113 (p < .001 compared to Ef222) exhibited the higher translocation ability and were able to adhere 2-3 times higher than S. gallolyticus isolates. Both species preferred the collagen IV coated surfaces to form biofilm but the S. gallolyticus structures were more compact (p = .01). These results may support a relationship between biofilm formation and vegetation establishment in S. gallolyticus endocarditis, whereas the high translocation ability of E. faecium high-risk clones might partially explain the increasing number of bacteraemia.

Introduccion El desarrollo de un esquema de Multilocus sequence typing (MLST) para Enterococcus faecalis ha permitido conocer los primeros datos de su estructura poblacional y epidemiologia global. Se ha detectado la dispersion en Europa y America de dos complejos clonales (CC) de alto riesgo denominados CC2 y CC9 que estan especialmente adaptados al medio hospitalario. El objetivo de este trabajo ha sido definir la presencia de ambos CC en cepas de E. faecalis aisladas en Espana. Metodos Se han caracterizado por MLST 81 cepas de E. faecalis aisladas de diversos origenes y correspondientes a diferentes anos y regiones espanolas. Debido a su importancia clinica y epidemiologica se han incluido cepas representantes de cada uno de los brotes hospitalarios por E. faecalis resistente a vancomicina descritos en Espana. Resultados En el medio hospitalario se detecto la dispersion de CC2 y CC9. En estos CC se han agrupado las cepas de E. faecalis resistentes a vancomicina causantes de los brotes hospitalarios en La Coruna, Palma de Mallorca y Valencia, asi como cepas de origen hospitalario sensibles a la vancomicina. La utilizacion del indice de asociacion (I a ), que estima el equilibrio de ligamiento en la poblacion estudiada, revelo una estructura poblacional epidemica cuya variabilidad genetica es debida a procesos de recombinacion. Conclusion En Espana se han detectado los CC de alto riesgo CC2 y CC9, que han evolucionado localmente de forma diferente en funcion de la carga genetica del entorno. Las medidas de control de la infeccion deberian ir encaminadas a la deteccion de estos CC de alto riesgo, ya que su presencia en los hospitales podrian predecir tendencias futuras en cuanto a la adquisicion de determinadas resistencias como es el caso de la vancomicina.

Background: The genus Streptococcus is 1 of the dominant bacterial groups in human milk, but the taxonomic identification of some species remains difficult. Objective: The objective of this study was to investigate the discriminatory ability of different methods to identify streptococcal species in order to perform an assessment of the streptococcal diversity of human milk microbiota as accurately as possible. Methods: The identification of 105 streptococcal strains from human milk was performed by 16S rRNA, tuf, and sodA gene sequencing, phylogenetic analysis, and Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Results: Streptococcus salivarius, Streptococcus mitis, and Streptococcus parasanguinis were the streptococcal dominant species in the human milk microbiota. Sequencing of housekeeping genes allowed the classification of 96.2% (16S rRNA), 84.8% ( sodA), and 88.6% ( tuf) of the isolates. Phylogenetic analysis showed 3 main streptococcal clusters corresponding with the mitis (73 isolates), salivarius (29), mutans (1)-pyogenic (2) groups, but many of the mitis group isolates (36) could not be assigned to any species. The application of the MALDI-TOF Bruker Biotyper system resulted in the identification of 56 isolates (53.33%) at the species level, but it could not discriminate between S pneumoniae and S mitis isolates, in contrast to the Vitek-MS system. Conclusion: There was a good agreement among the different methods assessed in this study to identify those isolates of the salivarius, mutans, and pyogenic groups, whereas unambiguous discrimination could not be achieved concerning some species of the mitis group ( S mitis, S pneumoniae, S pseudopneumoniae, S oralis).

The broth microdilution method for fosfomycin and Pseudomonas aeruginosa was assessed and compared with the approved agar dilution method in 206 genetically unrelated P. aeruginosa clinical isolates. Essential agreement between the two methods was 84%, and categorical agreement was 89.3%. Additionally, Etest and disk diffusion assays were performed. Results validate broth microdilution as a reliable susceptibility testing method for fosfomycin against P. aeruginosa . Conversely, unacceptable concordance was established between Etest and disk diffusion results with agar dilution results.

We report the fecal carriage eradication of a VIM-1-producing ST9 Klebsiella oxytoca strain in a pluripathological 84-year-old woman after fecal microbiota transplantation to control relapsing R027 hypervirulent Clostridium difficile infections. The donor was her son, in which the absence of fecal carbapenemase-producing bacteria was corroborated.

Fecal samples of healthy animals (66 pigs, 22 pets) recovered during 1998 in La Rioja, Spain, were analyzed for vancomycin-resistant enterococci colonization. Vancomycin resistance mechanisms were analyzed by PCR and sequencing. vanA-containing enterococci were detected in 3 of 66 samples (4.5%) and 5 of 22 samples (22.7%) of the pig and pet samples, respectively. Seven unrelated pulsed-field gel electrophoresis (PFGE) patterns were detected among the 8 vanA isolates (7 Enterococcus faecium, 1 E. faecalis). The tet(M) gene was present in all eight vanA enterococcal isolates, while the erm(B) and aac(6')-Ie-aph(2")-Ia genes were detected in 6 and 3 isolates, respectively. Colonization by vanC-1-containing enterococci (E. gallinarum) was demonstrated in 3% and 4.5% of the pig and pet samples. The aac(6')-Ie-aph(2")-Ia, ant(6)-Ia, aph(3')-IIIa, erm(B) and tet(M) genes were identified in one of the E. gallinarum isolates from a pig fecal sample. One vanB2-containing E. hirae strain was detected in the fecal sample of a healthy pig. In this isolate, the vanB2 gene cluster was integrated into the Tn5382-like element, as demonstrated by specific PCRs and sequencing. The tet(M) and erm(B) genes were also detected in this isolate. This is the first report in which a vanB2-containing enterococci is detected in animals and in E. hirae.

The activity of garenoxacin was assessed against 412 Streptococcus pneumoniae isolates (49.3% from the adult population). Overall penicillin, erythromycin, and ciprofloxacin (MIC, ≥4 μg/ml) resistance was 51.7, 35.4, and 1.5%, respectively. For all isolates, the garenoxacin MIC was ≤1 μg/ml. Amino acid replacements in GryA (Ser81→Phe or Tyr), ParC (Ser79→Phe or Tyr; Asp83→Gly; Lys137→Asn), and ParE (Ile460→Val; Asp435→Asn), alone or in combination, were ascribed to the reduced garenoxacin susceptibility (MIC range, 0.5 to 1 μg/ml) found in four isolates. The low impact of these mutations on garenoxacin activity envisages the possible coverage of S. pneumoniae populations resistant to preexisting quinolones.

The in vitro activities of telithromycin and cethromycin (ABT-773) against 412 Streptococcus pyogenes isolates, consecutively collected in 17 Spanish hospitals from different geographical areas, were evaluated and compared with those of erythromycin A, penicillin G, clindamycin and quinupristin-dalfopristin. With a susceptibility testing breakpoint ofor = 1 mg/L for both compounds, 96.1% of isolates were susceptible to telithromycin and 99.8% to cethromycin. Erythromycin non-susceptible isolates (intermediate plus resistant, MICor = 0.5 mg/L) comprised 23% of those tested, and were analysed for the genetic basis of their resistance by PCR. Among these isolates (n = 95), 72.6% harboured mef(A), 8.4%, erm(B), and 3.2%, erm(A), as sole macrolide resistance gene, whereas the presence of mef(A) plus erm(A) (11.6%) or mef(A) plus erm(B) (4.2%) was also observed. Both ketolides displayed a significant in vitro activity against S. pyogenes regardless of the macrolide resistance mechanisms. Nevertheless, in the case of telithromycin, 11 out of 19 of the erm(B)-positive isolates (2.7% of total population) exhibited an MIC range of 4-32 mg/L. According to the present results, telithromycin and cethromycin offer a wide coverage against S. pyogenes isolates in a geographic area with a high incidence of resistance to currently used macrolides.

The study was partially funded by the “Accion Transversal del Cancer”, approved on the Spanish Ministry Council on the 11th October 2007, by the Instituto de Salud Carlos III-FEDER (PI08/1770, PI08/0533, PI08/1359, PS09/00773, PS09/01286,PS09/01903, PS09/02078, PS09/01662, PI11/01403, PI11/01889,PI11/00226, PI11/01810, PI11/02213, PI12/00488, PI12/00265,PI12/01270, PI12/00715, PI12/00150), Castaño-Vinyals, G., Aragonés, N., Pérez-Gómez, B., Martín, V., Llorca, J., Moreno, V., Altzibar, J.M., Ardanaz, E., de Sanjosé, S., Jiménez-Moleón, J.J., Tardón, A., Alguacil, J., Peiró, R., Marcos-Gragera, R., Navarro, C., Pollán, M., Kogevinas, M., Alonso, M.T., Amiano, P., Arias, C., Azpiri, M., Benavente, Y., Boldo, E., Bueno, A., Bustamante, M., Caballero, F.J., Campo, E., Cantón, R., Capelo, R., Carmona, C., Casabonne, D., Chirlaque, M.D., Cirac, J., Clofent, J., Colado, E., Costas, L., Crous, M., Del Campo, R., Santos, M.D., Dierssen-Sotos, T., Ederra, M., Espinosa, A., Cabrera, M.F., Somoano, A.F., Villa, T.F., García-Esquinas, E.G., Martín, P.G., Gómez-Acebo, I., Puga, C.G., Gràcia, E., Eslava, M.G., Guinó, E., Huerta, J.M., Lope, V., López-Abente, G., Lopez-Otín, C., Argüelles, B.M., Salas, S.M., Pozo, B.M., de la Torre, A.J., Moreno, E., Iribas, C.M., Olea, N., Gelis, G.O., Paré, L., Porta, M., Puig, M., Del Fresno, M.R., Robles, C., Suarez, M.M., Romero, B., Castillo, A.I., Serra, M.S., Trejo, D.S., Santaballa, A., Santibáñez, M., Sierra, Á., Souto, A., Villanueva, C.M., Carrasco, E., Sabaté, Y., Persavento, C., García, M., Carrasco, G., Expósito, A., Andreu, M., Bessa, X., Piracés, M., Lorente, J.A., Tusquets, I., Collet, I., Bory, F., Pera, M., Abella, E., Garcia, F., Salar, A., Piñol, M., Fernandez-Llamazares, J., Martín, M.V., Garsot, E., Servio, L.I., Arzoz-Fabregas, M., Suarez, L., Ruiz, J.M., Castells, A., Serradesanferm, A., Bosch, A., Muñoz, M., Fontanillas, M., Alcaraz, A., Mengual, L., Duran, E., Izard, C., López, C., Benítez, J.M., Massanes, A.B., Ferrer, O.G., Almeda, J., Sarret, S., Rodriguez, M.A., Valmaseda, A.B., Liceran, M., Petitbó, D., Nolla, J.I., Pérez, S., Martínez, S., Vilaseca, J.M., Sebastián, L., Quesada, P., Sequera, G., Barca, E.G., Domingo-Domenech, E., Oliveira, A., Alonso, E., de la Banda, E., Sabater, Y., Vergara, M., Exposito, A., Alonso, T., Padrol, I., Klaustermeier, J., Florencia, Y., Camon, V., Esteban, A., Aymerich, M., Lopez-Otin, C., Muñoz, A., Torralba, Y., Dot, D., Mercadal, S., Sarra, J., Medina, P., Atienza, C., Biondo, S., de Oca, J., Ferran, L., Rodriguez-Moranta, F., Soriano, A., Guardiola, J., Urruticoechea, A., Galan, M., Banda, J.M., Zumel, Á., Saez, A., Garrido, J.A., Lacasaña, M., Ariza, J.L., García, T., García, M.Á., Hidalgo, M.Á., Asuero, M., Bayo, J., Vázquez, V.C., Franco, F., Gurucelain, J.L., Frías, M.J., Van de Haar, R., Viñas, J., Jiménez, H., Aguayo, M., Pereira, A., Gutiérrez, S.P., Rada, R., Candón, J., Domínguez, J., Ramos, M., Pedraza, G., Braulio, J., Salas, J., Labrero, D., Muñoz, D., Barrero, F., Delgado, S., Galisteo, L., Camacho, A., Elena, H., Caballero, J., Muñoz, M.J., Arredondo, F., Linares, R., Tejada, A., Tinto, H., Alés, J.D., Coronado, M.V., Bernal, M.L., Ortiz, J.L., Ocaña, M.N., Cavanillas, A.B., Miguel, Mejías, E.J., Abril, O.M., Requena, R.O., Morata, J.L., Echezarreta, E.G., López, L.M., Martínez, M.M., Sedano, P.P., de la Pedraja Pavón, A., López-Rojo, C., Mendiola, J., Pellicer, E., Caparrós, J.M., Oltra, E.G., Martínez, J.M., Molina, A., Villanueva, V., Miranda, M.J., Abril, C., Martinez, J., Salas, D., Ruiz, J.L., Ponce, M., Noos, P., Cervera, J., Del Val, A., Segura, A., Jiménez, N., Bellmunt, E., Aznar, I., Ramos, D., Montón, T., Solera, M.C., Mora, E.M., Estañ, N., Camarasa, N., Solanas, J.V., Ripoll, J., Cantero, J., Díaz, C.A., García, A.S., Avellón, S.M., Neumann, M., González, M.J., Suárez, M.M., Fernández, G., Fresno, M.R., Gómez, A., Garín, U., de Gros, A., Tamayo, E., Rua, M., Lasarte, C.S., Jauregi, M., Recio, J., Fernández, M., Múgica, M., Ciria, J.P., Guimón, E., Adúriz, C., Lacasta, A., Francisco, J.S., Alvarez, I., Enriquez-Navascués, J.M., Ruiz, I., Michelena, M., Alberro, J.A., Almansa, A.M., Goñi, L.M., Elizagaray, M.I., Técnico, M.O., Pérez, R.B., De Miguel Medina, M.C., Repáraz Romero, M., de Azúa Ciria, A.Y., Díaz, M.M., Lizárraga, M.S., Jiménez, Y.L., López, C.E., Legaz, S.O., Lezaun, R.A., Hurtado, H.O., De Miguel Velasco, M., Rubio, P.A., Cunchillos, F.D., de Liaño Arguelles, Á.D., Peñuela, J.M., Dorronsoro, M.L., Celaya, F.B., García, D.R., de la Higuera Carnicer, B.G., Tricas, J.M., Rivera, E.M., García, F.V., Noain, J.J., Erdozain, E.A., Ulibarrena, C.I., Lopeandía, J.G., Caden, M.A., Moraza, F.A., Domench, J.J., Arteaga, A.A., Navarro, K.A., Del Burgo Tajadura, M.A., Irastorza, F.C., Urrisarri, M.J., Zubicoa, B.F., Lorente, P.G., Sevilla, P.H., Echamendi, M.L., Díaz, Á.M., Salaverri, J.M., Gorricho, F.J., Del Valle, M.P., de Prado Marcilla, J., Garcésducar, M., Herce, P.A., Barasoain, J.E., Campos, I.A., Vergara, E.A., Beraza, B.C., Blasco, L.F., Díaz, L.G., Larrañeta, J.G., Ruiz, N.G., García, J.G., Orduna, M.S., Larena, D.I., Fernández, M.P., de Ciriza Pejenaute, F.J., Escuin, R., Puertas, I.R., Samper, I.A., Sesma, M.E., López, M.J., Apestegui, C.Z., Linares, C., Cervantes, M., Ferreras, E., García-Pérez, J., Fernández-Navarro, P., Pastor, R., Ramis, R., González, Á., Ruiz, T., Muñoz, V., Delgado, R., Lanza, M., Marín, M., Posada, M., Cosmen, J., Villanueva, A., Castaño, A., Jiménez, J.A., Rin, A., Díaz, G., Herreros, M., Pedraza, V., López, P., de la Fuente, M., de Castro, M.F., Sobrino, J., Calvo, R., Carrascal, I., Bernal, R., Mateo, A., García-Olmo, D., Zarazaga, A., Gombau, M.Á., Díaz, J., Gómez, T., Sánchez, T., de la Quintana, P., Ordás, J., Cuevas, P., Sánchez-Pastor, M., de la Peña, J.J., Tabernero, Á., Cámara, N., Hardisson, D., Suárez, A., Burgos, E., Alves, J., Miguel, M., Feliú, J., Belda, C., Zamora, P., Sánchez, A., Hernández, A., Beato, T., Arroba, A., Ballesteros, A., Canora, S., García, C., Romero, M., José, T.S., Espinel, M., Mañas, A., Sánchez, M., Ramón, H., Morillo, J.L., Cuchí, M., García, M.J., Ramos, J.M., Rojo, R., Sanjuanbenito, A., Villanueva, A.G., Gras, M., Cabañas, J.L., Collado, V., Arano, I., Capela, I., Mojarrieta, C., Peñas, B., Rodríguez, M., Ramos, A., Sancho, S., Hervás, A., Moreno, P., Sánchez, C., DoloresRubio, Montoya, L., Sancho, P., Rodríguez, L., Durán, E., Morel, S., Burgos, J., García, R., Del Cañizo, C.G., Díaz, A., González, F., Cristóbal, E., Barahona, C., Vázquez, S., Esteban, V., Pedrera, M., Martínez, J., Carrato, A., Ponce, C.G., Santos-Olmo, A., Barrientos, R.R., López, G., Fernández, A., García, A.B., Noriega, A., Campo, M., Varona, E., López, F., Montero, M., Gómez, M.T., Bartibas, P., Sanz, R., Montes, T., Salinero, M.Á., Herrero, M., Igea, M., Calvo, C., Pérez-Pellón, C., Maestre, M., Agora, M.A., Martín, M.G., Velázquez, D., Fernández, B., Castro, M., Ayuso, E., Masa, R., Antelo, C., García, S., Herrero, S., Delgado, A., Vilardell, L., Puig-Vives, M., Osca-Gelis, G., Buxo, M., Izquierdo, A., Carmona-Garcia, M.C., Romanos, R.R., Galindo, C.T., Bargalló, P.M., Sanchez, E.R., Saez, M., Roncero, J.M., Gallardo, D., Coll, R., Blanco, I., Ruano, L.M., Castaño, E.A., Rusiñol, J.M., Perez, R.J., Pujol, I.M., Tuero, G.C., de Barrio Lera, J.P., Carral, J.M., Pérez, C.A., Puente, M.E., Balbuena, S.P., Presa, J.M., Ramírez, J.A., Martínez, A.Á., de Francisco, T.G., Elosua, T.G., Teso, E.P., Fueyo, J.F., Guadarrama, O.A., Turienzo Frade Mdel, A., De la Hoz Riesco, M., Fernández, J.J., Fernández, V.S., Fernández, R.C., Santamaría, M.V., Simón, J.A., Ganso, A.M., Pacho, A.V., Alegre, S.V., Plaza, F.J., Cuenllas, B.Á., Franco, E.H., Martín, M.H., Ariño, M.T., Tascón, C.D., Palomo, J.A., Castañón López Mdel, C., Lorenzo, M.P., González, I.A., Rivero, F.G., Román, C.H., Fernández, E.Á., Bayón, T.R., Fernández, A.G., Municio, F., de Celis, M.G., Ingelmo, L.Á., Herrero, J.G., Carbajo, M.J., Esteras, T.R., Baz, B.V., Baticón, C.H., Baza, E.Á., Urdiales, A.U., Gil, J.I., López, M.A., Ule, E.C., Diez, C.B., Marín, A.I., Bernabeu, M.Á., de Abajo Olea, S., Rojo, C.V., Fernández, M.Á., González, F.G., Martínez, L.G., Del Huertotrancón Moratiel, M., Ramos, N.C., Fidalgo, S.P., Coque, P.R., González, Á.G., Díez, A.B.

The ability of Pseudomonas aeruginosa to develop resistance to most antimicrobials represents an important clinical threat worldwide. We report the dissemination in several Colombian hospitals of two predominant lineages of extensively drug-resistant (XDR) carbapenemase-producing P. aeruginosa strains. These lineages belong to the high-risk clones sequence type 111 (ST111) and ST235 and harbor blaVIM-2 on a class 1 integron and blaKPC-2 on a Tn4401 transposon, respectively. Additionally, P. aeruginosa ST1492, a novel single-locus variant of ST111, was identified. Clonal dissemination and the presence of mobile genetic elements likely explain the successful spread of XDR P. aeruginosa strains in Colombia., This work was supported by Merck Sharp & Dohme, Janssen-Cilag SA, Pfizer SA, AstraZeneca Colombia SA, Merck Colombia, Novartis, Amarey Novamedical, Merck S.A, and bioMérieux Colombia, which help fund the Colombian Nosocomial Resistance Study Group (CNRSG). The research of Rafael Cantón at the Microbiology Department of Ramón y Cajal University Hospital is funded by the European Commission (grants R-GNOSIS-FP7-HEALTH-F3-2011-282512 and FP7-HEALTH-F3-2013-MON4STRAT-602906-2) and the Instituto de Salud Carlos III of Spain, cofinanced by the European Development Regional Fund (A Way to Achieve Europe program; Spanish Network for Research in Infectious Diseases grant REIPI RD12/0015).

Background: Differences in Helicobacter pylori protein expression have been related to the risk of severe gastric diseases. In Spain, a marked geographic pattern in gastric cancer mortality has long been reported. Objective: To characterize antibody reactivity patterns against 16 H. pylori proteins, by age, sex, and region of birth, in a large sample of the Spanish adult population. Materials and Methods: Antibody reactivity was quantified by H. pylori multiplex serology in a sample from the control group of the multicase-control study MCC-Spain. For this analysis, 2555 population-based controls were included. Each participant was classified as seropositive or seronegative for each protein according to specific cutoffs. Overall H. pylori seroprevalence was defined as positivity against >= 4 proteins. Descriptive analyses by age, sex, and region of birth were performed for both seroprevalence and seroreactivity (continuous measure). Differences among groups were tested by logistic and linear regression models. Results: Overall H. pylori seroprevalence increased with age in both sexes. For ages 55-74, seroprevalence was lower in women than in men (84% vs 92%, P

En el presente numero de ENFERMEDADES INFECCIOSAS Y MICROBIOLOGIA CLINICA coinciden dos trabajos relacionados con la infeccion bronquial (rara vez del parenquima pulmonar) en pacientes con fibrosis quistica, que probablemente constituye el evento con mayor significacion pronostica en estos enfermos. Sin duda, los avances en el diagnostico microbiologico de la colonizacioninfeccion y las nuevas posibilidades en antibioterapia, tanto en cuanto a nuevos farmacos como, sobre todo, por las nuevas formas galenicas que permiten una dosificacion por via aerosolterapia han contribuido decisivamente a mejorar la calidad y la expectativa de vida de estos enfermos. El concepto de colonizacion-infeccion es critico para comprender el proceso patogenico de esta enfermedad. Pese a las altisimas concentraciones de microorganismos en las vias aereas, rara vez los enfermos de fibrosis quistica desarrollan infeccion parenquimatosa. Se puede producir bronquiolitis, inflamacion intersticial y fibrosis, pero es rara una neumonia intraalveolar organizada y, por supuesto, la bacteriemia es excepcional. Esta es la paradoja de la infeccion en el fibrotico quistico: estos enfermos son extraordinariamente resistentes a la infeccion y, sin embargo, la colonizacion-infeccion cronica acaba por producir reacciones inflamatorias de tal naturaleza que la funcion pulmonar acaba por reducirse a limites criticos. Como se ha comentado, la invasividad tisular o bacteriemia causada por las bacterias mas comunes en la colonizacion bronquial, como Pseudomonas aeruginosa o Staphylococcus aureus, son totalmente excepcionales. Hay algunos casos descritos de bacteriemia por organismos infrecuentes del genero Burholderia o Pandorea. La escasa invasividad de estos microorganismos esta relacionada con la propia causa de la enfermedad: la mutacion del gen CFTR impide la internalizacion de las bacterias en el epitelio y, al quedarse fuera, el sistema inmunitario no puede eliminarlas. De

A fingertip-to-fingertip intraindividual transmission experiment was carried out in 30 healthy volunteers, using four MLST-typed Enterococcus faecium clones. Overall results showed an adequate fit goodness to a theoretical exponential model, whereas four volunteers (13%) exhibited a significantly higher finger-to-finger bacterial transmission efficiency. This observation might have deep consequences in nosocomial epidemiology. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Background Although scientific knowledge about the benefits of probiotic use in cystis fibrosis (CF) is scarce, their expectative is promising. The aim of this work was to analyze the effect of a Lactobacillus reuteri probiotic preparation versus placebo in CF patients. Methods A prospective, double blind, crossover and with placebo study was carried out in 30 CF patients from two Spanish hospitals. Patients were randomized in Group A (6months of probiotic followed by 6months of placebo) and Group B (6months of placebo followed by 6months of probiotic). GIQLI (gastrointestinal) and SF-12 (general) health tests were performed after probiotic and placebo intakes. Fat absorption coefficient, calprotectin, and inflammatory interleukin quantification were determined in fecal samples. Total fecal DNA was obtained and metagenomic 454-pyrosequencing was applied to analyze the microbiome composition. STATA v12 MP software was used for statistical analyses. Results Statistically significant improvement in the gastrointestinal health and decrease of the calprotectin levels were demonstrated in patients after probiotic exposure, in comparison with placebo. All CF subjects reported good tolerance to L. reuteri without secondary effects. Metagenomic analysis showed an important dysbiosis in CF gut microbiota associated with a high concentration of Proteobacteria. Probiotic intake was followed by a reduction in the total bacterial density, mostly due to a considerable reduction in the γ-Proteobacteria phylum; and an important increase of the microbial diversity with a higher representation of Firmicutes. Conclusions Probiotics might ameliorate the dysbiosis of CF gut microbiota, characterized by a high density of Proteobacterial organisms. L. reuteri significantly decrease intestinal inflammation and increase digestive comfort.

Although the natural mode of bacterial growth in nature is as biofilm, almost all antimicrobial and immunological tests are routinely developed using planktonic inoculums. Bacterial biofilms protect the microbial community from external damage and promote the persistence of chronic infections. In this study, interactions between human macrophages and bacterial inoculums of planktonic and biofilm modes of growth have been explored using Escherichia coli (E. coli) K12. Human macrophages phagocytize planktonic E. coli more efficiently than bacteria grown in a biofilm. Moreover, they prefer to phagocytize planktonic bacteria. In this context, CD64 expression is involved. Our data indicate that bacteria with "a biofilm background" avoid phagocytosis by naive macrophages, which could create a favorable environment for chronic infection. Our findings were corroborated in a clinical O25b-ST131 ESBL-producer E. coli isolate, which caused urinary tract infections.

Seventy-six Pseudomonas aeruginosa isolates recovered from chronically ( n = 18) and nonchronically ( n = 18) colonized cystic fibrosis (CF) patients (2002 to 2009) were grouped in separate polyclonal populations. International CF epidemic clones were not identified, but the high-risk clone ST274, also found circulating in Spanish hospitals, was present. Persistent isolates were more resistant to antibiotics than nonpersistent isolates.

Streptococcus bovis is a large bacterial complex of facultative anaerobic Gram-positive cocci that includes distinct, genetically-related species. Traditionally, S. bovis was classified into three biotypes: I (mannitol fermentation-positive), II/1 (mannitol-negative and β-glucuronidase-negative), and II/2 (mannitol-negative and β-glucuronidase-positive). The introduction of molecular techniques in the last few decades has led to proposals for a genetic classification of this complex: S. bovis biotype I belongs to Streptococcus gallolyticus subsp. gallolyticus, S. bovis biotype II/1 is, in fact, Streptococcus infantarius subsp. infantarius, designated as Streptococcus lutetiensis, and S. bovis biotype II/2 is Streptococcus gallolyticus subsp. pasteurianus, commonly designated as Streptococcus pasteurianus. Although this modern taxonomy is currently accepted, many clinicians remain unfamiliar with these terms. The importance of correct identification lies in the strong association between bacteriemia, endocarditis and/or colon cancer and the various subspecies. In general, S. bovis is more susceptible to antimicrobial agents than other streptococci, but high levels of resistance to macrolides and tetracycline have been described.

Enterococcus faecalis isolates from food and environmental origin were evaluated for their angiotensin-converting enzyme (ACE)-inhibitory activity (ACE-IA) after growth in bovine skim milk (BSM). Most (90% active) but not all (10% inactive) E. faecalis strains produced BSM-derived hydrolysates with high ACE-IA. Known ACE-inhibitory peptides (ACE-IP) and an antioxidant peptide were identified in the E. faecalis hydrolysates by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS). Antimicrobial activity against Pediococcus damnosus CECT4797 and Listeria ivanovii CECT913 was also observed in the E. faecalis hydrolysates. The incidence of virulence factors in the E. faecalis strains with ACE-IA and producers of ACE-IP was variable but less virulence factors were observed in the food and environmental strains than in the clinical reference strains. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) based analysis demonstrated that food and environmental E. faecalis strains were genetically different from those of clinical origin. When evaluated, most E. faecalis strains of clinical origin also originated BSM-derived hydrolysates with high ACE-IA due to the production of ACE-IP. Accordingly, the results of this work suggest that most E. faecalis strains of food, environmental and clinical origin produce BSM-derived bioactive peptides with human health connotations and potential biotechnological applications. © 2013 Elsevier B.V., This work was partially supported by project AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO), by projects AGL2009-08348 and AGL2011-24643 from the Ministerio de Ciencia e Innovación (MICINN), and by the CENIT project (2006–2009) from the MITC-CDTI. Funding by grants GR35-10A from the BSCH-UCM and S2009/AGR-1489 from the Comunidad de Madrid (CAM) are also acknowledged. L. Gútiez holds a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC), Spain.

Enterococcus spp. is an important cause of nosocomial infections A number of virulence factors that may enhance its ability to colonize have been described. Enterococcus is capable of acquiring resistance genes, including high-level resistance (HLR) to aminoglycoside antibiotics.to investigate the prevalence of genes encoding virulence factors in aminoglycosides susceptible and resistant E. faecalis.A total of 80 E. faecalis isolates from clinical (n: 52) and poultry samples (n: 28) were included in this study. Bacterial identification was performed by biochemical tests and phenotypificationwas done using the Phene-PlateTM system. Susceptibility to different antimicrobial agents was determined by the agar dilution method. Virulence genes aceI, agg, gelE and efaA were detected by multiplex PCR.All isolates were susceptible to vancomycin and ampicillin. HLR to gentamicin (13.5%) and streptomycin (9.6%) was detected only in clinical isolates. The phenotyping revealed a great diversity of PhP-types, but only one clone with 7 strains of similar characteristics was found. The efaA gen was detected in 100% of the isolates. aceI gene was present in 94.2% and 75%, agg gene in 73.1%, and 67.9%, and gelE gene in 57.5% and 28.6% of the clinical and chicken isolates, respectively. Only 6 strains with HLR to aminoglycosides, belonging to the same phenotype, had the aceI, agg, gelE and efaA genes.E. faecalis with virulence genes and HLR to aminoglycosides were isolated from clinical and chicken samples in Antofagasta. More studies will be necessary to establish an association.

A multilocus sequence type (MLST) shift (from ST242 to ST996) was detected in Pseudomonas aeruginosa isolates with a uniform pulsed-field gel electrophoresis (PFGE) pattern obtained from a chronically colonized patient. MLST mutational change involved the mutL gene with the consequent emergence of a hypermutable phenotype. This observation challenges the required neutrality of mutL as an appropriate marker in MLST and alerts researchers to the limitations of MLST-only-based population studies in chronic infections under constant antibiotic selective pressure.

In previous years, it has been shown that human milk is a potential source of bacteria for the infant gut. The results of this work confirm the presence of the same specific bacterial strains of Bifidobacterium, Lactobacillus, and Staphylococcus in breast milk and infant fecal samples. The identity of bacteria isolated from breast milk and infant feces from 20 mother-infant pairs was investigated at the strain level. DNA from Staphylococcus, Lactobacillus, and Bifidobacterium was detected by qRTi-PCR in nearly all samples analyzed. These samples were cultured on different agar media. One colony representative of each morphology was selected and identified at the species level combining classical tests and molecular techniques (PCR, RAPD, PFGE, and/or MLST genotyping). Breast milk and infant feces from 19 mother-infant pairs shared different Staphylococcus, Lactobacillus, and/or Bifidobacterium species and strains. Significantly, 2 mother-infant pairs shared 4 bacterial strains although most pairs shared 2. These results confirm that breast milk and infant feces from mother-infant pairs share the same strain(s), indicating that breastfeeding could contribute to the bacterial transfer from the mother to the infant and, therefore, to the infant gut colonization. © 2012 International Lactation Consultant Association., This work was supported by projects CSD2007-00063 and AGL2010-15420 (Ministerio de Ciencia e Innovación, Spain), and project S2009/AGR-1469 (Comunidad de Madrid, Spain). EJ was supported by a grant of the Ministerio de Educación y Ciencia and European Social Fund (ESF) (PTA2008-1019-P).

The aim of this study was to follow the evolution of faecal bacteria over the first 34 weeks of life using molecular methods in healthy infants, all of them initially breast-fed and then subjected to a similar succession of dietary changes. Faecal samples from six healthy, full-term, unrelated babies were subjected to PCR amplification with general primers for 16S bacterial rDNA. The PCR products were analysed by denaturing gradient gel electrophoresis (DGGE) to determine a pattern of predominant bacterial species in each sample. Detection of species of Bifidobacterium genus and Lactobacillu s group was done using genus- or group-specific primers and sequencing DGGE bands. We detected an increment in the bacterial diversity in all infants, the more obvious changes being detected after introduction of a solid diet. Lactobacillus were detected from the third week and showed a frequent change of species. The presence of Bifidobacterium species was much more stable. Evolution of the microbiota in the six infants studied underwent significant changes until introduction of meat in the diet, but was unique for each infant despite the similarity of their feeding. Key words: faeces, microbiology, infant nutrition, DGGE, Lactobacillus, Bifidobacterium

Pathogenic bronchopulmonary colonizations and the exacerbations produced are among the most important causes of reduced pulmonary function in patients with bronchiectasis. The most frequent pathogens in these patients are Haemophilus influenzae and Pseudomonas aeruginosa. Lesions are produced by the local inflammatory process and the vicious circle developed by antigen stimulation, the release of inflammatory mediators, the presence of neutrophils, the increase of bacterial inoculum and the release of bacterial exoproducts. P. aeruginosa has been demonstrated to affect the patients with bronchiectasis and poorest quality of life and to colonize those with the poorest pulmonary function and the highest number of antimicrobial treatments. In bronchiectasis, as in chronic obstructive pulmonary disease (COPD) or cystic fibrosis, P. aeruginosa is able to colonize the respiratory mucosa chronically. Due to the ecological niche occupied by P. aeruginosa and the multitude of cycles with antimicrobial agents to which these patients are subjected, the development of antimicrobial resistance is highly likely, encouraged by the high proportion of hypermutation variants in existence. Likewise, P. aeruginosa naturally grows in the form of biofilms on the mucosal surface, greatly contributing to its persistence. Antimicrobial treatment in patients with bronchiectasis and P. aeruginosa colonization should be based on antimicrobial agents, alone or in combination, that do not lose activity when acting on biofilms.

Objectives To investigate the mechanisms of carbapenem resistance in the 175 Pseudomonas aeruginosa isolates (39%; 175/448) showing non-susceptibility (European Committee on Antimicrobial Susceptibility Testing breakpoints) to imipenem (35%), meropenem (33%) and/or doripenem (33%) recovered in 2008-09 from 16 Spanish hospitals during the Comparative Activity of Carbapenem Testing (COMPACT) surveillance study. Methods MICs (Etest), clonal relatedness (PFGE) and metallo-β-lactamase (MBL) production (Etest-MBL, PCR and sequencing) were determined. Mutation-driven resistance was studied in 60 non-MBL producers according to the doripenem MICs (15 isolates from each of four MIC groups: ≤ 1, 2-4, 8-16 and ≥ 32 mg/L). The expression of ampC, mexB, mexY, mexD and mexF was determined by real-time reverse transcription-PCR and the presence of mutations in oprD by PCR and sequencing. Isogenic mutants expressing combinations of mutation-driven carbapenem resistance were constructed. Results Twelve (6.9%) isolates were MBL (VIM-20, VIM-2 or VIM-13) producers and all showed high-level resistance (MIC 32 mg/L) to all three carbapenems. Regarding mutation-driven resistance, all but 1 of the 60 isolates were non-susceptible (MIC >32 mg/L) to imipenem, linked to oprD inactivation. In addition, 50% of the isolates overexpressed ampC, 33% mexY, 32% mexB and 15% mexF, while none overexpressed mexD. Increasing prevalence of ampC overexpression correlated with increasing doripenem MICs (≤ 1, 13%; 2-4, 53%; 8-16, 60%; and ≥ 32, 73%) while overexpression of efflux pumps correlated only with moderate resistance. Doripenem showed slightly higher activity than meropenem against isolates overexpressing ampC, especially mexB or mexY. The analysis of a collection of isogenic laboratory mutants supported this finding. Conclusions Although the prevalence of MBL producers is increasing, mutation-driven resistance is still more frequent in Spain. Imipenem resistance was driven by OprD inactivation, while additional AmpC and particularly efflux pump hyperproduction had a lower impact on the activity of doripenem compared with meropenem.

Staphylococcus aureus is one of the main etiological agents of mastitis in different mammalian species. At present, it is unknown whether strains isolated from human mastitis cases share phenotypic properties and genetic background with those obtained from animal mastitis cases. Therefore, the objective of this study was to characterize S. aureus strains isolated from women with lactational mastitis and to compare them with the strains responsible for bovine mastitis and noninfectious strains. All the strains were genotyped by both pulsed field gel electrophoresis and multilocus sequence typing and submitted to a characterization scheme that included diverse assays related to pathogenic potential and antibiotic resistance. Apart from siderophore production, no significant association was observed between the strains from bovine and human mastitis. Statistical differences between human- and bovine-mastitis-associated strains were detected for some traits and virulence determinants, such as the presence of prophages and cna and hlb genes, which were more frequently found within the bovine group. On the contrary, resistance to penicillin was significantly higher among strains isolated from human lactational mastitis, probably related to the common presence of the blaZ gene. A high genetic diversity was found among the strains involved in mastitis in breastfeeding women. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd., This study was partly supported by the FUN-C-FOOD (Consolider-Ingenio 2010) and AGL2010-15420 projects from the Ministerio de Educación y Ciencia (Spain). S.D. was the recipient of a postdoctoral fellowship from the same Ministry.

To identify infection-causing Enterococcus species in Cuban hospitals and determine their susceptibility to antimicrobial drugs, as well as their resistance mechanisms.A total of 687 Enterococcus isolates from 30 Cuban hospitals in nine provinces of the country were studied over the period 2000-2009. The species were identified using both the conventional method and the automatic API(®) system. The minimum inhibitory concentration was determined for 13 antimicrobial drugs following the standards recommended by the Clinical Laboratory and Standards Institute. The polymerase chain reaction technique was used to characterize the genes that were resistant to aminoglycosides, erythromycin, tetracycline, and glucopeptides. The presence of beta-lactamase was determined by the chromogenic cephalosporin test.The most prevalent species were Enterococcus faecalis (82.9%) and E. faecium (12.2%). Resistance to glucopeptides (1.0%) was mediated by the vanA and vanB genes. The strains resistant to ampicillin (6%) did not produce beta-lactamases. A high percentage of resistance to aminoglycosides was observed. Gentamicin (31.0%) and streptomycin and amikacin (29.1%) were mediated by the aac(6')Ie-aph(2")Ia, aph(3')-IIIa, ant(6)Ia, and ant(3")(9) genes. A correlation was found between resistance to tetracycline (56.0%) and presence of the tet(M) (75.1%) and tet(L) genes (7.0%), while resistance to erythromycin (34.1%) was due to the erm(B) gene (70.9%).Resistance to vancomycin is infrequent in Cuba, as opposed to a high level of resistance to aminoglycosides, which may be indicative of treatment failures. The microbiology laboratory is a cornerstone of Enterococcus infection surveillance, along with ongoing monitoring of the susceptibility of these infections to antimicrobial drugs at a time when resistance of this microorganism is on the rise.

Mycoplasma pneumoniae is a common cause of community acquired pneumonia (CAP) ([1][1]). Macrolides are first-choice agents, but resistance is possible by point mutations in domain V of the 23S rRNA gene ([1][1], [2][2]). Asiatic countries have the highest proportion of macrolide resistance, but in

Idiopathic interstitial pneumonias are interstitial lung diseases of unknown etiology which prognosis is usually fatal. Microbiota associated to bronchoalveolar lavage from 20 patients with negative bacterial cultures was explored by 16S-rDNA PCR-DGGE, showing a clearly negative relation among the presence of P. jirovecii and bacterial colonization. This is the first report of in vivo antagonistic relation among fungi and bacteria.

Two-hundred-twenty-nine food samples of animal origin were tested to know the prevalence of vancomycin-resistant enterococci (VRE) after a decade of avoparcin ban as animal growth promoter in Spain. VRE with acquired mechanism of resistance were detected in 9 of these 229 samples (3.9%, obtained from chicken, veal and rabbit), and one VRE per food sample was further characterized. The vanA gene was identified in seven isolates (2 E. faecium, 3 E. durans, and 2 E. hirae), and the vanB2 gene in the remaining 2 isolates (identified as E. faecium). The two vanB2 isolates showed a phenotype of multiresistance that included, in addition to vancomycin, also ampicillin, erythromycin, tetracycline, streptomycin, kanamycin, ciprofloxacin, chloramphenicol and trimethoprim-sulfamethoxazole and contained, among others, erm(B), tet(M), ant(6), and aph(3')-III genes. Most of vanA enterococci showed erythromycin and tetracycline resistance and contained the erm(B) and tet(M) genes. One vanA- and both vanB2-positive E. faecium isolates were classified by MLST analysis into the CC17 clonal complex (ST17 and ST78), and one additional vanA isolate was included in a new sequence type named ST425 (singleton). Co-transference by conjugation of erm(B) and vanA genes was demonstrated in one vanA-positive E. faecium isolate. The inclusion of vanB2 cluster into Tn5382 structure was demonstrated in the two vanB2 isolates, as well as the linkage pbp5-Tn5382, and beta-haemolysis and gelatinase production was identified in one of them. Food sample of animal origin could be a vehicle of transference of VRE of vanA and vanB2 type that could be transferred to humans.

Objectives: Although methicillin-resistant Staphylococcus aureus (MRSA) is increasingly recognized in cystic fibrosis (CF) patients, it has not been sufficiently studied in large series. We analysed all MRSA isolates recovered from respiratory secretions of patients attending our CF unit (1994‐2006). Methods: Antibiotic susceptibilities were determined using both planktonic and sessile bacteria as inocula. Genetic relationships were determined by PFGE and multilocus sequence typing (MLST). SCCmec type and the presence of the pvl gene were also investigated. Results: A total of 93 MRSA isolates (1‐20 isolates per patient) were recovered from 18 of 77 CF patients with positive staphylococcal culture. Mean prevalence (4.4%) increased (P < 0.001) over time. All isolates were susceptible to linezolid, quinupristin/dalfopristin and co-trimoxazole but presented high resistance rates to amikacin (90%), gentamicin (85%), levofloxacin (81%) and erythromycin (69%). Except for macrolides and gentamicin, isolates were less susceptible growing in biofilms than in planktonic cultures. Fifteen different PFGE patterns were found, one of them consistently recovered for 6 years in the same patient. Identical clones were detected in several unrelated patients. MLST demonstrated that the international ST228 was the most frequent (67%) clone. The pvl gene was negative in all isolates and the SCCmec corresponded to types I (97%) and IV (3%). Strong mutators were not detected, but a considerable number were considered weak mutators. Conclusions: Distinct microbiological and molecular features were detected among CF-MRSA isolates, probably due to adaptation to specific conditions in CF patients. Prevalence of MRSA increased among these patients, most of them colonized with a multiresistant biofilm-forming clone belonging to ST228-SSCmecI, suggesting cross-transmission or a common source.

Objectives: To compare the capability of biofilm development between Streptococcus pneumoniae isolates from cystic fibrosis (CF) respiratory samples and those from non-CF blood cultures. Antibiotic susceptibility of biofilm-forming isolates, as well as differences between antibiotic susceptibility of sessile cells [minimum biofilm inhibitory concentration (MBIC)] and their planktonic counterparts (conventional MIC), were also assessed. Methods: Biofilm formation was performed using a microtitre method in 20 CF and 22 non-CF blood culture S. pneumoniae isolates. Results and conclusions: Biofilm formation occurs more frequently among S. pneumoniae isolates from CF (80%) than among non-CF blood culture isolates (50%) (P= 0.04). Moreover MBICs were significantly higher than conventional planktonic MICs among CF but not among non-CF blood isolates, suggesting a high adaptability of CF strains to form biofilms in adverse conditions.

Objectives: High-level penicillin resistance in Streptococcus pneumoniae requires extensive remodulation of the penicillin-binding proteins (PBPs), and murM gene function is also required for the expression of resistance. In this work, we determined whether specific changes in PBPs were associated with specific MurM variants. Methods: Two collections of highly penicillin-resistant (MIC 2-8 mg/L) isolates, including 10 early (1997-1998) and 23 contemporary (2002-2004) isolates, were studied. Results: Most of the isolates belonged to clones Spain 6B -2 (13 strains), Spain 23F -1 (10 isolates) and Spain 14 -5 (20 isolates). Different protein variants of MurM (MA, MB5, MB6, MB9 and MB10), PBP1A (A-C), PBP2B (A-D) and PBP2X (A-C) were recognized, including two murM alleles not previously described. Particular [MurM-PBP1A-2B-2X] allelic combinations were predominant among the different clones, including [MA-B-B-B] for old (MIC 2 mg/L) and [MB10-C-A-B] for recent (MIC 4-8 mg/L) Spain 66 -2 isolates, [MA-A-C-A] for Spain 23F -1 and [MB5-A-A-A] in Spain 14 -5 isolates. Conclusions: Although S. pneumoniae has a basic recombinational population structure, our results indicate remarkable conservation of PBPs and MurM protein types within each clone. This suggests that particular PBPs-MurM combinations tend to be preserved and may have an independent evolutionary history in particular clones.

A multilocus sequence typing (MLST) scheme based on seven housekeeping genes was used to investigate the epidemiology and population structure of Enterococcus faecalis . MLST of 110 isolates from different sources and geographic locations revealed 55 different sequence types that grouped into four major clonal complexes (CC2, CC9, CC10, and CC21) by use of eBURST. Two of these clonal complexes, CC2 and CC9, are particularly fit in the hospital environment, as CC2 includes the previously described BVE clonal complex identified by an alternative MLST scheme and CC9 includes exclusively isolates from hospitalized patients. Identical alleles were found in genetically diverse isolates with no linkage disequilibrium, while the different MLST loci gave incongruent phylogenetic trees. This demonstrates that recombination is an important mechanism driving genetic variation in E. faecalis and suggests an epidemic population structure for E. faecalis . Our novel MLST scheme provides an excellent tool for investigating local and short-term epidemiology as well as global epidemiology, population structure, and genetic evolution of E. faecalis .

Aims: The aim of this study was to analyse the nucleotide sequences of regions encoding the penicillin-binding domains of pbp1A, pbp2B and pbp2X genes and murM alleles from 14 selected amoxicillin-resistant Streptococcus pneumoniae isolates (MICs 8-16 mg/L) obtained in Spain. Methods: PFGE and dideoxynucleotide chain termination sequencing were used. Results: Analysis of PFGE profiles showed that the amoxicillin-resistant S. pneumoniae strains belonged to six different PFGE patterns including the Spain 23F-1, Spain 6B-2, Spain 9V-3 and Spain 14-5 international clones; however, 8 of the 14 strains belonged to the Spain 9V-3 clone. These strains showed the typical changes in penicillin-binding proteins (PBPs) 1A and 2X and had 10 unique changes in the 590-641 region of PBP2B as described previously. Transformation experiments tried to incorporate the transpeptidase domain of PBP2B including the 590-641 region associated with amoxicillin-resistant pneumococci. Sequencing of the pbp2B genes revealed that part of the 3′ region of the pbp2B sequence encoding a region of the domain (around amino acid 514-538 to the C terminus of PBP2B) did not recombine with the R6 pbp2B gene. The murM sequence analysis showed that 6, 6 and 2 amoxicillin-resistant S. pneumoniae strains had murMA, murMB5 and murMB6 alleles, respectively. However, strains with murMB5 or murMB6 alleles showed a single mutation (N 537D) in the 537-581 region of PBP2B, while strains with the murMA allele had 12 unique changes. Conclusions: Ten unique changes in the 590-641 region of PBP2B and no specific murM alleles were found in S. pneumoniae strains isolated in Spain with an amoxicillin MIC ≥8 mg/L (MICs from 6 to 12 mg/L by 1 mg/L step dilution). In addition, the presence of specific mutations in PBP2B seems to play a key role in the presence of different murM alleles in these amoxicillin-resistant pneumococcal strains. © The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Forty-eight Streptococcus pneumoniae isolates recovered from sputum samples from 26 cystic fibrosis (CF) patients attending our CF unit (1995 to 2003) were studied. Mean yearly incidence of isolation was 5.5%, and all were strains recovered from young patients (≤12 years). The isolation was linked to clinical exacerbation in 35% of the cases, but only 27% of these were not accompanied by other CF pathogens. Fifty percent of the patients presented with two to four isolates over the studied period. Pulsed-field gel electrophoresis-SmaI digestion revealed a high heterogeneity (32 pulsotypes among 48 isolates) and the persistence over a 6-month period of a single clone (clone A) in two patients. This clone, presenting a varied multiresistance phenotype, was identified as the Spain 23F -1 clone and was also recognized in six other patients, including two out of nine patients from the CF unit of Sant Joan de Dèu Hospital, Barcelona, Spain. In our isolates, 16 different serotypes were recognized, the most frequent being 23F (33.3%), 19F (18.8%), 6A (6.2%), and 6B (6.2%). High overall resistance rates were observed: to penicillin, 73%; to cefotaxime, 33%; to erythromycin, 42%; to tetracycline, 58%; to chloramphenicol, 48%; and to trimethoprim-sulfamethoxazole, 67%. Resistance to fluoroquinolones was not detected. Multiresistance was a common feature (60%). The percentage of S. pneumoniae strains with increased frequencies of mutation to rifampin resistance (≥7.5 × 10 −8 ) was significantly higher ( P = 0.02) in CF (60%) than among non-CF (37%) isolates in the same institution (M. I. Morosini et al., Antimicrob. Agents Chemother. 47:1464-1467, 2003). Even though a clear association with acute exacerbations could not be observed, long-term clonal persistence and variability, high frequency of antibiotic resistance, and hypermutability indicate the plasticity for adaptation of S. pneumoniae to the CF lung environment.

In a double-blind prospective study including 114 healthy young volunteers, the presence in human feces of the yogurt organisms Lactobacillus delbrueckii and Streptococcus thermophilus after repeated yogurt consumption (15 days) was analyzed by culture, specific PCR, and DNA hybridization of total fecal DNA. Detection of yogurt lactic acid bacteria in total fecal DNA by bacterial culture and PCR assay was consistently negative. DNA compatible with yogurt bacteria was found by hybridization experiments in only 10 (10.52%) of 96 individuals after consumption of fresh yogurt and in 2 (2.10%) of 96 individuals after consumption of pasteurized yogurt ( P = 0.01).

The ability to colonize patients successfully may be essential for the emergence and spread of resistant nosocomial strains. We determined the presence of Esp, a surface protein involved in colonization ability in Enterococcus faecalis, in 96 Enterococcus faecium isolates from hospitalized patients (77 PFGE clones), 33 faecal isolates from healthy volunteers (32 clones) and 20 environmental isolates (20 clones). Esp was found significantly more often in E. faecium isolated from hospitalized patients than in isolates from the community setting (26% versus 6%, P < 0.01) and was significantly more common among ampicillin-resistant than among ampicillin- susceptible strains (37% versus 4%, P < 0.001), regardless of the isolation site. The frequency of the esp gene in the hospital clearly correlates with antibiotic-resistant E. faecium clones. This observation indicates that antibiotic-resistant variants may frequently arise under antibiotic selective pressure among esp-positive clones reaching ecological abundance in the noso- comial habitat.

Mutations in ribosomal protein L16 and in 23S rRNA were investigated in 22 Enterococcus strains of different species and for which the MICs of evernimicin differ (MICs, 0.023 to 16 μg/ml). Amino acid changes (Arg56His, Ile52Thr, or Arg51His) in protein L16 were found in seven strains, and a nucleotide G2535A mutation in 23S rRNA was found in 1 strain among 13 for which the MICs are ≥1 μg/ml.

Bacteriocin production was determined for 218 Enterococcus isolates (Enterococcus faecalis [93] and E. faecium [125]) obtained from different origins (human clinical samples [87], human fecal samples [78], sewage [28], and chicken samples [25]) and showing different vancomycin susceptibility patterns (vancomycin resistant, all of them vanA positive [56], and vancomycin susceptible [162]). All enterococcal isolates were randomly selected except for the vancomycin-resistant ones. A total of 33 isolates of eight different bacterial genera were used as indicators for bacteriocin production. Forty-seven percent of the analyzed enterococcal isolates were bacteriocin producers (80.6% of E. faecalis and 21.6% of E. faecium isolates). The percentage of bacteriocin producers was higher among human clinical isolates (63.2%, 81.8% of vancomycin-resistant isolates and 60.5% of vancomycin-susceptible ones) than among isolates from the other origins (28 to 39.3%). Only one out of the 15 vancomycin-resistant isolates from human fecal samples was a bacteriocin producer, while 44.4% of fecal vancomycin-susceptible isolates were. The bacteriocin produced by the vanA-containing E. faecium strain RC714, named bacteriocin RC714, was further characterized. This bacteriocin activity was cotransferred together with the vanA genetic determinant to E. faecalis strain JH2-2. Bacteriocin RC714 was purified to homogeneity and its primary structure was determined by amino acid sequencing, showing an identity of 88% and a similarity of 92% with the previously described bacteriocin 31 from E. faecalis YI717. The presence of five different amino acids in bacteriocin RC714 suggest that this could be a new bacteriocin. The results obtained suggest that the epidemiology of vancomycin resistance may be influenced by different factors, including bacteriocin production., R. D. C. was supported by a grant from the Diputación General de Aragón of Spain (project P49/97) and from the Sociedad Española de Quimioterapia. This work has been supported in part by a grant from the Fondo de Investigaciones Sanitarias (00/0545) of Spain.

The frequency of mutation to rifampin resistance of 200 clinical Streptococcus pneumoniae isolates was examined. Two peaks were observed in the distribution, with mode frequencies of 2.5 × 10 −7 (20% of isolates) and 2.5 × 10 −8 . The hexA and hexB gene entire sequences were analyzed in 13 isolates. Sequences from both hypermutable and “normomutable” strains were conserved relative to that of the R6 S. pneumoniae control strain. The phenotypic Hex system proficiency, in terms of transforming efficiency, was also maintained irrespective of the variations in mutation frequency values.