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Introduction Streptococcus bovis/equinus complex (SBEC) is a major cause of infective endocarditis (IE), although its incidence varies greatly depending on the geographical area. The characteristics of IE caused by Streptococcus gallolyticus susp. gallolyticus are well known; there are hardly any descriptions of IE caused by other species or biotypes. Methods Retrospective cohort study, from 1990 to 2019, of all SBEC IE in adults in three Spanish hospitals, Lugo (LH), Barcelona (BH) and Ferrol (FH) where the population is mainly rural, urban and mixed, respectively. The incidence of IE was analyzed in 3 areas. Clinical characteristics of IE (277 cases, 258 biotyped) were compared according to SBEC species and biotypes. Results There are significant differences between the incidence of SBEC IE in HL (27.9/106) vs. HF and HB (8.8 and 7,1, respectively, p Conclusions IE due to SBEC has geographical variations in incidence and different clinical characteristics among biotypes. The association with CRC was high.

Resumen Introduccion La infeccion por Streptococcus suis (S. suis) es una zoonosis poco descrita en nuestro pais, que se relaciona con la exposicion al ganado porcino o su carne. La forma de presentacion mas frecuente es la meningitis, mientras que la afectacion de la columna vertebral es rara. Metodos Presentamos 2 casos de infeccion por S. suis y realizamos una revision sistematica de los trabajos publicados sobre espondilodiscitis por S. suis entre enero de 1994 y mayo de 2020 con el objetivo de definir las caracteristicas clinicas, sus factores predisponentes y su evolucion. Resultados Se describen 17 casos, el 76,5% varones con una edad media de 57,6 anos, generalmente sin enfermedad de base asociada. El enolismo fue un factor presente en casi el 17,6% de los pacientes. Un 70,6% presentaron exposicion a ganado porcino o su carne y un 20% heridas en manos. La duracion media de los sintomas fue de 10,2 dias y el segmento mas afectado fue el lumbar. Un 70,6% curso con meningitis. Todos se trataron con betalactamicos con una duracion media de 53,2 dias. Hubo una recidiva y ninguno fallecio. Conclusion Existen pocos casos de espondilodiscitis por S. suis en la literatura. Cuando ocurre, se asocia a otro tipo de infeccion en la mayoria de los casos. Presentan buena respuesta al tratamiento medico y buen pronostico.

Robinione overstreeti is a bopyrid isopod endemic to the Gulf of Mexico that inhabits the branchial chamber of the ghost shrimp Callichirus islagrande. Information on this host–parasite association...

[EN] Aims We studied the regeneration dynamics of woodlands and abandoned old fields in a landscape dominated by Quercus suber in its lower limits of rainfall and temperature. Two hypotheses were established: (1) regeneration of Quercus species is strongly favored by the presence of tree cover; and (2) growth of Q. suber is driven by the climatic variables that represent the lower ecological limit of its leading distribution edge. Methods We selected woodlands and old fields with and without tree remnants (n = 3 per type), and analyzed stand structure, soil parameters and tree growth. Results Succession was arrested in old fields without tree remnants. By contrast, remnant trees were accelerators of forest recovery in old fields. Tree cover played a fundamental role in Quercus recruitment throughout seed dispersal and facilitation that mitigate the effects of summer drought on seedlings. Also, tree cover improved soil parameters (e.g., organic matter) that are important factors for understanding differences in regeneration. Winter/spring precipitation exerted a positive effect on tree growth, as well as temperatures during winter/spring and September. Conclusions Regeneration dynamics are modeled by the density of tree cover in the cold and dry edge of the distribution area of Q. suber where Q. ilex is increasing in abundance. Although temperature has a positive effect on the tree growth of Q. suber, when demographic processes are considered, decreases in water availability likely play a critical role in Q. ilex recruitment. This in turn changes dominance hierarchies, especially in abandoned areas with little or no tree cover., This study was funded by project SA013G19 from “Junta de Castilla y León” and by research grants awarded for final projects directed in the Master’s degree in Biology and Conservation of Biodiversity at the University of Salamanca, Spain., Publicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCLE

Introduction and objectives The association between Streptococcus bovis group infective endocarditis and colorectal neoplasm (CRN) is well-known. However, no studies have assessed the association between Enterococcus faecalis infective endocarditis (EFIE) and CRN. We aimed to determine whether the prevalence of CRN is higher in patients with EFIE and an unclear source of infection than in patients with EFIE and a known source of infection or in the general population. Methods Retrospective analysis of a cohort of 154 patients with definite EFIE (109 with an unclear source of infection and 45 with an identified source) from 2 Spanish teaching hospitals to determine the prevalence of CRN and other colorectal diseases. Results In the group with an unknown source of infection, 61 patients (56%) underwent colonoscopy; of these, 31 (50.8%) had CRN. Nonadvanced colorectal adenoma was detected in 22 patients (36%), advanced adenoma in 5 (8.2%), and colorectal carcinoma (CRC) in 4 (6.6%). Among patients who survived the EFIE episode with ≥ 2 years of follow-up, 1 case of CRC was subsequently diagnosed. Only 6 patients (13.3%) with an identified focus of infection underwent colonoscopy; 1 of these patients (16.7%) was diagnosed with CRN. The prevalence of adenomas was slightly higher than that of the Spanish population in the same age range, whereas that of CRC was 17-fold higher. Conclusions CRN was found in more than half of patients with EFIE and an unclear focus of infection who underwent colonoscopy. Colonoscopy should be recommended in patients with EFIE and an unclear source of infection.

Streptococcus bovis was described early last century as an inhabitant of the digestive tract of ruminants. Long ignored in the medical literature, in recent years, its importance has been increasingly recognized. Although S. bovis causes only a small proportion of streptococcal infections, the special interest in the organism is due to two fundamental facts. On one hand, S. bovis is a frequent cause of endocarditis, and on the other hand, it is an organism that has been associated unambiguously with colon cancer, along with Clostridium septicum . In addition, new infections caused by these organisms have been described in recent years, including meningitis, bone and joint infections, and neonatal sepsis, as well as an association with non-colorectal cancer. S. bovis consists of several biotypes or (sub)species with different clinical associations. However, microbiology laboratories do not always distinguish S. bovis to the biotype or subspecies level consistently. On the other hand, there have been continuous changes in the taxonomy and nomenclature of these microorganisms. All this has caused confusion among clinicians, which is reflected in the medical literature. This article reviews the clinical and microbiological aspects most relevant for this group of microorganisms.

We studied reproductive characteristics of the phantom shrimp Tozeuma carolinense inhabiting sea-grass beds to test hypotheses about seasonality of reproduction, sex ratio and sexual system, and fecundity values in 3 Mexican collecting sites in the Gulf of Mexico. Four hundred and eighty collected individuals were measured and separated to 3 groups. Based on discriminant analysis, carapace length (CL) was selected as morphometric feature to define 10 size-class intervals. The relationship between sexes were analyzed. Embryos were counted, measured and classified into 4 stages of development. Regression analyses were performed to determine the correlation between the number and size of embryos vs. CL. Sex ratio was 1:1, however, for each location, the sex ratio was different. Males were smaller than females; ovigerous females were larger than non-ovigerous females and males. Fecundity analysis have a significant positive relationship, both in the number and size of embryos vs. female CL. Ovigerous females were found at all dates, and confirmed in El Cayo populations with monthly samples in a year, so that we conclude that T. carolinense belongs to the species category with a seasonal-continuous reproduction.

There is growing concern about the spreading of human microorganisms in relatively untouched ecosystems such as the Antarctic region. For this reason, three pinniped species (Leptonychotes weddellii, Mirounga leonina and Arctocephalus gazella) from the west coast of the Antartic Peninsula were analysed for the presence of Escherichia spp. with the recovery of 158 E. coli and three E. albertii isolates. From those, 23 harboured different eae variants (α1, β1, β2, ε1, θ1, κ, ο), including a bfpA-positive isolate (O49:H10-A-ST206, eae-k) classified as typical enteropathogenic E. coli. Noteworthy, 62 of the 158 E. coli isolates (39.2%) exhibited the ExPEC status and 27 (17.1%) belonged to sequence types (ST) frequently occurring among urinary/bacteremia ExPEC clones: ST12, ST73, ST95, ST131 and ST141. We found similarities >85% within the PFGE-macrorrestriction profiles of pinniped and human clinic O2:H6-B2-ST141 and O16:H5/O25b:H4-B2-ST131 isolates. The in silico analysis of ST131 Cplx genomes from the three pinnipeds (five O25:H4-ST131/PST43-fimH22-virotype D; one O16:H5-ST131/PST506-fimH41; one O25:H4-ST6252/PST9-fimH22-virotype D1) identified IncF and IncI1 plasmids and revealed high core-genome similarities between pinniped and human isolates (H22 and H41 subclones). This is the first study to demonstrate the worrisome presence of human-associated E. coli clonal groups, including ST131, in Antarctic pinnipeds. The sampling was funded by the Spanish Ministry of Science and Innovation (CGL-2005-25073-E/ANT and CTM2008-00570) and the Sea World & Bush Gardens Conservation Fund. Work at USC-LREC was supported by projects AGL2013-47852-R from the Ministerio de Economía y Competitividad (MINECO, Spain) and Fondo Europeo de Desarrollo Regional (FEDER); AGL2016-79343-R from the Agencia Estatal de Investigación (AEI, Spain) and FEDER; PI16/01477 from Plan Estatal de I + D + I 2013-2016, Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y Fomento de la Investigación, and FEDER; CN2012/303 and ED431C 2017/57 from the Consellería de Cultura, Educación e Ordenación Universitaria, (Xunta de Galicia) and FEDER. We would like to thank the military personnel at the Spanish Antarctic Base “Gabriel de Castilla” for their help and assistance and the Marine Technology Unit (CSIC), the Spanish Navy’s Oceanographic Research Ship “Las Palmas” and the Unidad de Tecnología Marítima (UTM, CSIC) for logistics and transport. We also express our gratitude to J. Castro-Urda, F.T. García-Moreno, C. Rengifo-Herrera, S. Rojo-Montejo, I. Ferre, V. Navarro, M. Gómez-Bautista, T. Alvaro-Alvarez and J. Coello-Pérez for their invaluable help in the sample collection. A. Mora acknowledges the Ministerio de Educación, Cultura y Deporte (Spain) for the mobility grant for teachers and researchers from the Programa Estatal de Promoción del Talento y su Empleabilidad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016. Sí

To determine the incidence of Streptococcus bovis (Sb) biotypes causing bacteraemia and associated malignancies.This is a retrospective analysis of patients with Sb bacteraemia, pulled out from a prospective surveillance protocol of bacteraemia cases, in three areas of Spain (1990-2013): a cattle area (Lugo), a fishing area (Ferrol) and an urban area (Barcelona). Colonoscopy and Sb biotypes (Sb-I and Sb-II) were determined in most cases.506 patients with Sb bacteraemia; mean age 68.1 (±14.1) years, and 66.2% were males. The cattle area, compared with the fishing and urban areas, had higher incidence of bacteraemia by SbI (40.29 vs 9.38 vs 6.15 cases/10(6) person-years, P0.001) and bacteraemia by Sb-II (29.07 vs 9.84 vs 13.37 cases/10(6) person-years, P0.001). The Sb-I cases (n = 224), compared with Sb-II cases (n = 270), had greater rates of endocarditis (77.6% vs 9.6%, P0.001) and colorectal neoplasm (CRN) (50.9% vs 16.6%, P0.001), and smaller rates of biliary tract infection (2.2% vs 29.6%, P0.001) and non-colorectal malignancy (8.9% vs 31.4%, P0.001).There was a link between the cattle area and higher incidence of Sb bacteraemia. Sb-I differed from Sb-II cases in clinical findings and associated malignancies. Colonoscopy is mandatory in cases of endocarditis or bacteraemia caused by Sb-I.

"Fiddler crabs are common decapods of intertidal zones; thirteen species have been reported along the coast of the Gulf of Mexico, inhabiting different zones. For the southern region of the Tamiahua Lagoon, Veracruz, Mexico, five species have been identifi ed: Uca panacea , U. rapax , U. spinicarpa , U. virens and U. vocator particularly in the southern region of the Tamiahua Lagoon, Veracruz, Mexico. Here we analysed the fecundity of U. virens in 25 ovigerous females out of 387 individuals collected between December 2008 and December 2009 during eight field trips and from five collection sites . Morphological measurements like carapace length (CL) and carapace width (CW), and total wet weight (TWW) for all indi viduals collected were taken, and total egg number was counted for ovigerous females . The total population was divided in nine size class intervals. Total egg number varied between 3,617 and 41,099. Egg number increased with CW (ranging from 9.0 to 18.6 mm CW) and TWW. Values of fecundity observed for U. virens in Tamiahua Lagoon vs other Uca species were similar to results reported in literature."

".Se presenta un análisis estadístico de variables morfométricas del camarón de agua dulce Potimirim mexicana, que ha sido descrito como hermafrodita protándrico secuencial. El objetivo es probar estadísticamente, que los organismos pueden ser clasificados por sexo, analizando su longitud y otras variables morfológicas. Un total de 191 organismos fueron capturados en el río Máquinas, Veracruz, México. La técnica utilizada fue la de árboles de clasificación, que mediante probabilidades van determinando las variables significativas en la variable "respuesta", así como las que son necesarias para la discriminación. Esta técnica permitió corroborar la teoría que la longitud total del organismo es necesaria para la discriminación de la variable sexo, pero también generó el resultado que, estadísticamente, dos variables más son importantes en este proceso discriminatorio; el largo del segundo pleópodo y largo del carpo del tercer pereiópodo. La técnica generó un clasificación correcta de 82% utilizando las tres variables mencionadas. Lo significativo del proceso es que al generar los árboles de clasificación se muestran, de manera sencilla y práctica, las variables necesarias en este proceso, así como su nivel discriminatorio individual."

Item does not contain fulltext BACKGROUND: Bacteremia with Clostridium septicum (CS) and Streptococcus gallolyticus subsp. gallolyticus (SGG) have both been associated with colorectal neoplasms (CRN) and colonoscopic examination is advised, however the differences and similarities in colorectal findings are not well known. METHODS: This is a multicenter, comparative study of patients with CS bacteremia [44 of 664 cases (6.6%) of Clostridium spp.] and SGG bacteremia [257 of 596 cases (44.2%) of S. bovis group], carried out in three hospitals from Spain. Clinical findings related to bacteremia and associated CRN were collected. RESULTS: The main sources of infection were abdominal (77.7%) for CS bacteremia and endovascular (75%) for SGG bacteremia. CS bacteremia was more often associated with malignancies, (72.6% vs. 19.4%) and neutropenia (29.5% vs. 3.1%), and was more acute, with shock at presentation (63.6% vs. 3.9%) and higher 30-day mortality (47.7% vs. 9.7%) compared to SGG (P

18 p.-4 fig.-6 tab. López, María et al., Acinetobacter baumannii is an important pathogen that causes nosocomial infections generally associated with high mortality and morbidity in Intensive Care Units (ICUs). Currently, little is known about the Quorum Sensing (QS)/Quorum Quenching (QQ) systems of this pathogen. We analyzed these mechanisms in seven clinical isolates of A. baumannii. Microarray analysis of one of these clinical isolates, Ab1 (A. baumannii ST-2_clon_2010), previously cultured in the presence of 3-oxo-C12-HSL (a QS signalling molecule) revealed a putative QQ enzyme (α/ß hydrolase gene, AidA). This QQ enzyme was present in all non-motile clinical isolates (67% of which were isolated from the respiratory tract) cultured in nutrient depleted LB medium. Interestingly, this gene was not located in the genome of the only motile clinical strain growing in this medium (A. baumannii strain Ab421_GEIH-2010 [Ab7], isolated from a blood sample). The AidA protein expressed in E. coli showed QQ activity. Finally, we observed downregulation of the AidA protein (QQ system attenuation) in the presence of H2O2 (ROS stress). In conclusion, most of the A. baumannii clinical strains were not surface motile (84%) and were of respiratory origin (67%). Only the pilT gene was involved in surface motility and related to the QS system. Finally, a new QQ enzyme (α/ß hydrolase gene, AidA protein) was detected in these strains., This study was funded by grant PI13/02390 awarded to MT within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate of evaluation and Promotion of Research - European Regional Development Fund "A way of Making Europe" and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015).

Background. The association between bacteremia by Streptococcus gallolyticus subsp. gallolyticus (SGG) and colorectal neoplasia (CRN) is well established but the frequency of the association varies widely in different studies. We conducted a case-control study to assess the association between SGG bacteremia and CRN. Methods. An analysis of all SGG bacteremias was performed during the period 1988–2011. The frequency of CRN in patients with SGG bacteremia was compared with the frequency of CRN in a symptomatic control group of patients matched at a 1:2 ratio for gender and age (±3 years) without S. bovis bacteremia and personal history of CRN and with increased risk of CRN (by the presence of symptoms, signs, or test suspicious of colonic pathology or by family history of CRN). Results. One hundred nine cases of SGG bacteremia were detected (mean age, 66 years; 87% male). Colonoscopy was performed in 98 cases, diagnosing 69 cases of CRN: 57 adenomas (39 advanced adenomas) and 12 invasive carcinomas. Only 4 cases had suspected CRN before the blood culture. The prevalence of CRN was higher in patients with SGG bacteremia than in the 196 control patients (70% vs 32%; odds ratio [OR], 5.1; 95% confidence interval [CI], 3.0–8.6). This difference was not significant when comparing nonadvanced adenomas (19% vs 12%), but we found significant differences in advanced adenomas (40% vs 16%; OR, 3.5; 95% CI, 2.0–6.1) and invasive carcinomas (12% vs 5%; OR, 2.9; 95% CI, 1.2–6.9). Conclusions. The frequency of CRN among SGG infected patients is significantly increased compared with symptomatic age-matched controls, indicating that SGG infection is a strong indicator for underlying occult malignancy.

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius . To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum , we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and Leon (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum . The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA–DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share >98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species.

Our current knowledge of plant-microbe interactions indicate that populations inhabiting a host plant are not restricted to a single microbial species but comprise several genera and species. No one knows if communities inside plants interact, and it has been speculated that beneficial effects are the result of their combined activities. During an ecological study of nitrogen-fixing bacterial communities from Lupinus angustifolius collected in Spain, significant numbers of orange-pigmented actinomycete colonies were isolated from surface-sterilized root nodules. The isolates were analysed by BOX-PCR fingerprinting revealing an unexpectedly high genetic variation. Selected strains were chosen for 16S rRNA gene sequencing and phylogenetic analyses confirmed that all strains isolated belonged to the genus Micromonospora and that some of them may represent new species. To determine the possibility that the isolates fixed atmospheric nitrogen, chosen strains were grown in nitrogen-free media, obtaining in some cases, significant growth when compared with the controls. These strains were further screened for the presence of the nifH gene encoding dinitrogenase reductase, a key enzyme in nitrogen fixation. The partial nifH-like gene sequences obtained showed a 99% similarity with the sequence of the nifH gene from Frankia alni ACN14a, an actinobacterium that induces nodulation and fixes nitrogen in symbiosis with Alnus. In addition, in situ hybridization was performed to determine if these microorganisms inhabit the inside of the nodules. This study strongly suggests that Micromonospora populations are natural inhabitants of nitrogen-fixing root nodules.

The purpose of this study was to evaluate the characteristics of infective endocarditis (IE) caused by S. bovis and compare them to those caused by streptococci of the viridans group (SVG). A prospective study was undertaken considering 55 consecutive cases of IE due to S. bovis and 41 to SVG over 18 years. The study was divided into two periods (1988–1996 and 1997–2005). S. bovis caused 24% of the IE in our centre and constituted the main aetiology for this disease, showing an increase of 358% during the second period studied. Biotype I was responsible for 94.5% of cases and there was a high degree of association with colon tumours (53%). Over the period of the study, 107 patients admitted to our hospital had bacteraemia caused by S. bovis and 310 patients had bacteraemia caused by SVG. In the first group, 55 (51%) were endocarditis cases, but only 41 (13%) of the patients with SVG bacteraemia had endocarditis (p

Background The Streptococcus bovis group (SBG) is a well-known cause of endocarditis, but its role in osteoarticular infections (OAIs) has not been well described. Methods We analyzed all patients with OAIs by SBG diagnosed in our hospital (1988–2014). We selected those cases with septic arthritis and osteomyelitis, as defined according to clinical, microbiological, and imaging studies. Identification of the strains was performed by using the API 20 Strep and the GP card of the Vitek 2 system, and confirmed the identification by molecular methods. In addition, we reviewed the literature to select all cases of OAI by SBG during the period 1980–2015. Results From the 83 cases of OAI included in the analysis (21 from our center and 62 from the literature review), 59 were osteomyelitis (57 of them spondylodiscitis) and 24 were arthritis (2 with associated spondylodiscitis). The mean age was 66.9 years, and 79.2% of the patients were men. Endocarditis (IE) was associated with 59% of the cases and this association was greater for osteomyelitis than for arthritis (78.9% vs. 13.6%; P = 0.001). OAI was a presenting symptom in 63% of the cases of IE. Colonoscopy was performed in 64 cases, which detected colorectal neoplasm (CRN) in 46 patients (71.8%), almost all asymptomatic. Some 69.5% of these neoplasm were carcinomas or advanced adenomas. The blood cultures were positive in 78.3% cases. In 45 cases, the S. bovis species was identified; in 82.2% of the cases the cause was Streptococcus gallolyticus subsp. gallolyticus . The mortality was 7.2%, which in no case was attributable to the OAI. Conclusions OAIs are frequently the initial manifestation of IE caused by SBG. S. gallolyticus causes most of these infections. Echocardiogram and colonoscopy are therefore mandatory, given the species' close association with IE and CRN.

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused.

A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx 1 genes, 278 (54%) possessed stx 2 genes, and 135 (26%) possessed both stx 1 and stx 2 . Enterohemolysin ( ehxA ) and intimin ( eae ) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx 2 eae- γ1 ehxA (46 isolates) was the most common, followed by O157:H7 stx 1 stx 2 eae- γ1 ehxA (34 isolates), O113:H21 stx 2 (25 isolates), O22:H8 stx 1 stx 2 ehxA (15 isolates), O26:H11 stx 1 eae- β1 ehxA (14 isolates), and O77:H41 stx 2 ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intiminβ 1, 82 O157:H7 isolates possessed intimin γ1, three O111:H- isolates had intimin type γ2, one O49:H- strain showed intimin type δ, 13 (six of serotype O103:H2) isolates had intimin type ε and eight (four of serotype O156:H-) isolates had intimin ζ. We have identified a new variant of the eae intimin gene designated ξ (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a major source of nosocomial infections in Spain associated with the production of OXA-58-like or OXA-24/40-like β-lactamase enzymes. We analysed the plasmids carrying the bla(OXA-24/40)-like gene in CRAb isolates obtained a decade apart.The presence of β-lactamases was screened for by PCR (metallo-β-lactamases, carbapenem-hydrolysing class D β-lactamases, GES and KPC) in 101 CRAb isolates obtained in two multicentre studies (GEIH/REIPI-Ab-2000 and GEIH/REIPI-Ab-2010; n = 493 Acinetobacter spp). We analysed the distribution and characterization of the plasmids carrying the bla(OXA-24/40)-like gene and sequenced two plasmids, AbATCC223p (2000) and AbATCC329p (2010) from A. baumannii ATCC 17978 transformants.Acquisition of the bla(OXA-24/40)-like gene was the main mechanism underlying resistance to carbapenems (48.7% in 2000 compared with 51.6% in 2010). This gene was mainly isolated in ST2 A. baumannii strains in both studies, although some novel STs (ST79 and ST80) appeared in 2010. The gene was located in plasmids (8-12 kbp) associated with the repAci2 or repAci2/repGR12 types. The sequences of AbATCC223p (8840 bp) and AbATCC329p (8842 bp) plasmids were similar, particularly regarding the presence of the genes encoding the AbkA/AbkB proteins associated with the toxin/antitoxin system. Moreover, the abkA/abkB gene sequences (96% identity) were also located in plasmids harbouring the bla(OXA-58)-like gene.The action of OXA-24/40 and OXA-58 β-lactamase-like enzymes represents the main mechanism underlying resistance to carbapenems in Spain in the last decade. AbkA/AbkB proteins in the toxin/antitoxin system may be involved in the successful dissemination of plasmids carrying the bla(OXA-24/40)-like gene, and probably also the bla(OXA-58)-like gene, thus contributing to the plasmid stability.

Thirty-seven cases of microbiologically demonstrated pyogenic hepatic abscess were observed in a prospective study over a seven-year period. Biliary disease was the most common source of liver abscess (42%). Streptococcus milleri was the most common cause of hepatic abscess, accounting for 51% of the cases. Hepatic abscess is due to Streptococcus milleri clinically distinct from other forms of pyogenic liver abscess due to its torpid nature and the longer duration of its symptoms [42 vs. 11 days]. Occult hepatic abscess should be suspected if the blood culture is positive for Streptococcus milleri, since 28% of bacteremia cases due to Streptococcus milleri stem from hepatic abscesses. It is important to distinguish Streptococcus milleri from other members of the viridans streptococci group, which are frequently isolated as contaminants, but only exceptionally cause hepatic abscess. Unlike other pyogenic hepatic abscesses, those caused by Streptococcus milleri are frequently monomicrobial (79%). In the present study, empirical therapy of pyogenic hepatic abscess always included a drug that is effective against Streptococcus milleri.

102 Escherichia coli strains from patients with community-acquired urinary tract infections (UTIs) were genotypically and phenotipically characterized. The strains were examined genotypically by using the polymerase chain reaction (PCR) for presence of five urovirulence operons: α-haemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), pyelonephritis-associated pili (pap), S. fimbriae (sfa) and afimbrial adhesin (afa). The phenotypic methods used were: Vero and HeLa cells for detection of CNF1, blood agar for Hly and mannose-resistant haemagglutination typing for expression of adhesins. There was a very good correlation between the results obtained by PCR and those obtained by phenotypic methods. Thus, the PCR assays can by recommended for clinical use to detect uropathogenic E. coli strains, as well as for epidemiological studies.

Growth, shoot water relations and root hydraulic conductivity were studied in tomato plants (Lycopersicon esculentum cv. INCA9) subjected to different salt concentrations in the root medium. Two experiments were carried out at Instituto Nacional de Ciencias Agrícolas (INCA), Cuba, during May and June 1995. In the first experiment, plants were grown for 13 days in a nutrient solution with 0 or 100 mM NaCl. In the second experiment, the hydraulic conductivity was measured on roots submerged in nine different concentrations of NaCl up to 200 mM. The effect of temperature treatments between 0 and 50 °C on root hydraulic conductivity was also examined. Shoot growth, leaf water potential, leaf stomatal conductance, leaf relative water content and root hydraulic conductivity values decreased more rapidly in the treated plants than in control plants. A strong correlation was found between the root hydraulic conductivity and leaf water parameters, indicating that water flow through the roots was the main factor controlling shoot water relations.

Faecal swabs obtained from a random sample of 268 cows and 90 calves on 19 Lugo farms were examined for verotoxin-producing Escherichia coli (VTEC). We found VTEC on 95% of the farms. The prevalence rates of VTEC infection in asymptomatic cows and calves were estimated to be 35 and 37%, respectively. The proportion of animals infected on each farm ranged from 0 to 100%. VTEC strains isolated from healthy cattle belonged to 27 O serogroups; however, 57% (85 of 149) were of one of 8 serogroups (O2, O8, O22, O77, O82, O105, O113 and O171). Nearly 60% of the bovine VTEC strains belonged to serogroups that cause haemorrhagic colitis and haemolytic uraemic syndrome in humans. The VTEC were all non-O157:H7; 91% were eae-negative and 86% produced VT2 or VT1 and VT2. These characteristics are different from those of VTEC isolated from calves with diarrhoea.

Cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia coli. In this study, two pairs of oligonucleotide primers were designed to amplify fragments of the genes for CNF1 and CNF2 by the polymerase chain reaction (PCR). We also described a further primer pair which amplify fragments of both CNF1 and CNF2 genes. Amplification products of the expected size (543-bp for CNF1, 543-bp for CNF2 and 533-bp for CNFs) were detected in all 240 necrotoxigenic Escherichia coli (NTEC) strains tested. In contrast, no amplification products were seen when the PCR reaction was performed with DNA obtained from 69 non-NTEC bacterial strains (including 13 enterotoxigenic, 11 verotoxigenic and 45 non-toxigenic strains). Furthermore, the toxin genotypes determined by PCR corresponded to the phenotypic results of HeLa cell assay. Therefore, the specificity and sensitivity for the three pairs of oligonucleotide primers designed in this study was 100%. The PCR protocol describe here permits rapid and accurate detection of CNF1 and CNF2 genes in Escherichia coli strains isolated from clinical material and contaminated foods.

In a prospective study of bacteremia caused by organisms of the Streptococcus milleri group a total of 32 adult patients were observed over a seven-year period. These patients accounted for 1.6% of all patients diagnosed as having significant bacteremia and 17% of all cases of streptococcal (nonpneumococcal) bacteremia diagnosed during the study period. Only five patients had polymicrobic bacteremia. In 31 cases, a presumed origin of infection was identified, generally oral or gastrointestinal disease. There were only six cases of nosocomial acquisition. The most common presenting symptom was prolonged fever. The following forms of presentation were documented: bacteremia with local suppurative infection (56%), bacteremia without local suppurative infection (25%), and endocarditis (19%). An associated focus of infection was found in the abdominal cavity in 20 cases (62%). The mortality rate was 12.5%. All isolates were susceptible to penicillin. Caution is necessary in interpreting a blood culture positive for Streptococcus milleri group organisms, since, unlike other viridans streptococci, they are rarely contaminants. For this reason patients with suppurative processes and/or digestive tract disease must be carefully investigated.

An epidemiological study was carried out to determine the incidence and the serotypes of verotoxigenic Escherichia coli (VTEC) that cause infections in Galicia (north-western Spain). Although, VTEC strains were isolated from 55 (14%) of the 387 calves sampled and the majority of bovine VTEC strains belonged to serotypes (026:H11 or H−, 091:H21, 0103:H2, 0105:H18, 0111:H−, 0113:H21, 0126:H−, 0128:H− and 0157:H7 or H−) previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) in other countries, VTEC are not a common cause of human infections in Spain. Thus, VTEC (026:H11 and 086:H10) were isolated from only 3 (0.6%) of the 482 children with diarrhoea investigated. We examined the 69 (3 humans and 66 bovines) VTEC strains that were initially isolated as E. coli producing a toxin cytotoxic to Vero and HeLa cells by polymerase chain reaction (PCR) using specific primers for VT1, VT2 and eae genes. PCR showed that 38 (55%) of VTEC strains carried VT1 genes, 18 (26%) possessed VT2 genes, and 10 (14%) carried both VT1 and VT2 genes. Three (one human and two bovine) strains which were formerly VTEC had lost the ability to produce verotoxins upon subculture and became negative for VT 1 and VT2 by PCR. In total 35 (51%) of 69 VTEC strains, including the two human VT1+ strains of serotype 026:H11, were positive for eae sequences when tested by PCR. Presence of the eae gene was significantly more frequent (100%; 21/21) among VTEC strains with serotypes (026:H11, 0111:H−, 0157:H−and 0157:H7) considered as enterohaemorrhagic E. coli (EHEC) than among VTEC strains with non-EHEC serotypes (29%; 14/48) (p < 0.001). Results obtained in this study indicate that cattle may be an important source of VTEC involved in human disease. However, severe clinical syndromes caused by VTEC, such as HC and HUS, are uncommon in Spain, in comparison with North America and the UK. In any case, VTEC disease can appear on the scene very suddenly, as occurred in the UK and North America in the 1980s.

The O:K:H serotypes of 137 necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factor type 1 (CNF1) isolated from human extraintestinal infection were determined. Although NTEC producing CNF1 belonged to 58 different serotypes, only 10 of them accounted for 54% of strains. The most common serotypes, in order of frequency, were: O4:K?:H5, O6:K13:H1, O83:K1:H31, O75:K95:H5, O2:K1:H6, O2:K7:H-, O75:K1:H7, O2:K?:H1, O4:K12:H1 and O22:K13:H1. CNF1 strains of serotypes O2:K7:H- and O4:K12:H1 express P-fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for MRHA type III. Among CNF1 strains of serotype O4:K?:H5 there exist some that express P-fimbriae and others that possess MRHA type III. Lastly, the majority of CNF1 strains of serotypes O6:K13:H1, O22:K13:H1, O75:K1:H7 and O83:K1:H31 do not express P-fimbriae nor the adhesin responsible to MRHA type III. Our results show that extraintestinal infections are caused by a limited number of virulent clones, as suggested by the theory of special pathogenicity.

The present study characterizes, for the first time, two emerging avian pathogenic Escherichia coli (APEC) clonal groups of serogroup O111: O111:H4-D-ST117 and O111:H4-D-ST2085. The clonal group O111:H4-D-ST117 was already present in APEC strains isolated between 1991 and 2000, and was still present in strains isolated between 2004 and 2009, showing long time evolution according to the virulence-gene differences and macrorestriction profiles. Among ST117 strains, two virulence profiles could be distinguished: papG II-positive tsh-negative strains which satisfied criteria for extraintestinal pathogenic E. coli (ExPEC), and papG II-negative tsh-positive strains without ExPEC status. Interestingly, we have detected a human septicemic O111:H4-D-ST117 ExPEC strain isolated from a hemocultive in 2000 whose macrorestriction profile showed >85% similarity with four APEC strains of the study. The clonal group O111:H4-D-ST2085 was exclusively detected in 17 APEC strains isolated in 2008 and 2009, and showed short time evolution based on its homogeneity since all were nalidixic acid-resistant, all had ExPEC status, and most carried papG II and tsh genes. From the clinical point of view, O111:H4-D-ST2085 seems a successful clonal group that could be the result of the epidemiological evolution of O111:H4-D-ST117. Due to the increasing prevalence of both clonal groups among clinical APEC isolates, their high virulence-gene content, and zoonotic potential, we suggest them as possible candidates for the development of a future vaccine against avian colibacillosis.

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50–70% of strains; and other genes such as tccP, espP, katP and toxB were detected in

"Se analizó una población del camaroncito de río Potimirim mexicana obtenida del río Máquinas en el estado de Veracruz, México. Esta especie presenta una distribución de tallas por sexo que sugiere que se trata de una especie con hermafroditismo secuencial. Se aplicó el método de análisis discriminante para establecer los subgrupos que conforman la población, dando como resultado 3 clases: organismos sexualmente indeterminados, machos y hembras. En el subgrupo de los machos se establecieron 3 conjuntos, con base en el tamaño del appendix masculina. Mediante este análisis se confirmó que P. mexicana es una especie hermafrodita secuencial."

Eight hundred and nineteen strains of Escherichia coli isolated in Spain between 1986 and 1991 from extraintestinal infections and feces of healthy controls were investigated for expression of P-fimbriae using a particle agglutination test. Among strains causing urinary tract infections, sepsis and other extraintestinal infections, P-fimbriae were found in 31% (130/420) (P < 0.001), 25% (30/118) (P < 0.001) and 12% (11/92) (P < 0.5) respectively. In contrast, only 7% (14/189) of feacal isolates from healthy individuals carried P-fimbriae. According to two more common toxic markers detected this study (α-haemolysin and cytotoxic necrotizing factor type 1), P-fimbriated E. coli strains were grouped into three categories: haemolysin+cytotoxic necrosing factor+ (Hly+CNF1+) (68/185; 37%), haemolysin+cytotoxic necrosing factor− (Hly+CNF1−) (61/185; 33%) and Hly−CNF1− (56/185; 30%). The 185 P-fimbriated strains belonged to 17 different O serogroups. However, 148 (80%) were of one of six serogroups (O1, O2, O4, O6, O7 and O18). The most frequent serogroups determined in the Hly+CNF1+ strains were the O4 and O6 (53/68; 78%), in the Hly+CNF1− strains it was the O18 (27/61; 44%) and in the Hly−CNF1− strains the O1, O2 and O7 (41/56; 73%). The majority (160/185; 86%) of P-fimbriated E. coli expressed the mannose-resistant haemagglutinin type IVa.

Spanish Ministry of Education and Science Spanish Ministry of Health and Consumer Affairs [Fondo de Investigacion Sanitaria, Spanish Network for the Research in Infectious Diseases (REIPI) Autonomous Government of Galicia Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Programa de Apoio a Nucleos de Excelencia Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) MRC Wellcome Trust Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E coli (EPEC) translocate, dozens of type III secretion system effectors, including the WxxxE effectors Map, EspM and EspT that activate Rho GTPases. While map, which is carried on the LEE pathogenicity island, is absolutely conserved among EPEC and EHEC strains, the prevalence of espM and espT is not known. Here we report the results of a large screen aimed at determining the prevalence of esPM and espT among clinical EPEC and EHEC isolates. the results suggest that espM, detected in 51% of the tested strains, is more commonly found in EPEC and EHEC serogroups that are linked to severe human infections. in contrast, espT was absent from all the EHEC isolates and was found in only 1.8% of the tested EPEC strains. Further characterization of the virulence gene repertoire of the espT-positive strains led to the identification of a new zeta 2 intimin variant. All the espT-positive strains but two contained the tccP gene. espT was first found in Citrobacter rodentium and later in silico in EPEC E110019, which is of particular interest as this strain was responsible for a particularly severe diarrhoeal outbreak in Finland in 1987 that affected 650 individuals in a school complex and an additional 137 associated household members. Comparing the protein sequences of EspT to that of E110019 showed a high level of conservation, with only three strains encoding EspT that differed in 6 amino acids. At present, it is not clear why espT is so rare, and what impact EspM and EspT have on EPEC and EHEC infection. Univ Santiago de Compostela, Fac Vet, Dept Microbiol & Parasitol, Lab Referencia E Coli, Lugo, Spain Univ London Imperial Coll Sci Technol & Med, Div Cell & Mol Biol, Ctr Mol Microbiol & Infect, London SW7 2AZ, England Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, Brazil Complexo Hos Xeral Calde, Unidade Microbiol Clin, Lugo, Spain Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, Brazil Spanish Ministry of Health and Consumer Affairs [Fondo de Investigacion Sanitaria, Spanish Network for the Research in Infectious Diseases (REIPI): RD06/0008-10181 Spanish Ministry of Education and Science: AGL-200802129 Autonomous Government of Galicia: PGIDIT065TAL26101P Autonomous Government of Galicia: 07MRU036261PR FAPESP: 08/53812-4 Web of Science

Background Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties. Results A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence. APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000). Seven virulence genes showed significant differences, being fimAvMT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries. Conclusion Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a foodborne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans. This work was supported by grants from European Commission (FAIR6-CT-4093; PEN project FOOD-CT-2006-36256), the Fondo de Investigación Sanitaria from the Ministerio de Sanidad y Consumo de España (grants FIS G03-025-COLIRED-O157, PI052023, PI051481 and REIPI RD06/0008/1018), Ministerio de Educación y Ciencia de España (AGL-2008-02129) and the Xunta de Galicia (grants PGIDIT05BTF26101P, PGIDIT065TAL26101P, 07MRU036261PR, 08TAL017261PR). A. Mora acknowledges the Ramón y Cajal programme from the Ministerio de Educación y Ciencia de España SI

Summary. To assess the role of enterotoxigenic Escherichia coli (ETEC) in infantile diarrhoea, 482 children with diarrhoea and 103 healthy controls, from three localities of Galicia, northwestern Spain, were investigated between 1985 and 1988. Rotavirus (37.3%) and Salmonella spp. (1 2.8%) were the most common causal agents, followed by ETEC (3.973, Campylobacter jejuni (2*3%), Shigella spp. (0.9%) and Yersinia enterocolitica (0.5%). ETEC were significantly more frequently isolated from children with diarrhoea who were under 1 month of age (26.5%) than from older diarrhoeic children (2.2%) (p < 0.001) or from healthy children who were under 1 month of age (0%) (p < 0.05). Among children who harboured ETEC, five of the nine children under 1 month of age developed diarrhoea in hospital, whereas none of the 10 children over 1 month of age did so. Seventeen ETEC isolates produced heat-stable enterotoxin (STa) only, four produced only heat-labile enterotoxin (LT), and two produced both toxins. Colonisation factor antigens CFA/I and CFA/II were detected in 11 (55.0%) of the 20 ETEC isolates that remained enterotoxigenic after maintenance in the laboratory. Most ETEC isolates belonged to serotypes 01 53 : K - : H45 (nine STa' CFA/I + isolates), 027 : K - : H7 (three STa' isolates) or 06 : K15 : H16 (two LT' STa' CFA/II' isolates). Our results suggest that ETEC constitute an important cause of neonatal diarrhoea in this part of Spain.

International audience; BACKGROUND: Concomitant with the recent emergence of CTX-M-type extended-spectrum beta-lactamases (ESBLs), Escherichia coli has become the enterobacterial species most affected by ESBLs. Multiple locales are encountering CTX-M-positive E. coli, including specifically CTX-M-15. To gain insights into the mechanism underlying this phenomenon, we assessed clonality and diversity of virulence profiles within an international collection of CTX-M-15-positive E. coli. METHODS: Forty-one ESBL-positive E. coli isolates from eight countries and three continents (Europe, Asia and North America) were selected for study based on suspected clonality. Phylogenetic group, ERIC2 PCR profile, O H serotype, AmpC variant and antibiotic susceptibility were determined. Multilocus sequence typing (MLST) and PFGE provided additional discrimination. Virulence potential was inferred by detection of 46 virulence factor (VF) genes. RESULTS: Thirty-six (88%) of the 41 E. coli isolates exhibited the same set of core characteristics: phylogenetic group B2, ERIC2 PCR profile 1, serotype O25:H4, AmpC EC6, ciprofloxacin resistance and MLST profile ST131. By PFGE, the 36 isolates constituted one large cluster at the 68% similarity level; this comprised 17 PFGE groups (defined at 85% similarity), some of which included strains from different countries. The 36 isolates exhibited highly (91% to 100%) similar VF profiles. CONCLUSIONS: We describe a broadly disseminated, CTX-M-15-positive and virulent E. coli clonal group with highly homogeneous virulence genotypes and subgroups exhibiting highly similar PFGE profiles, suggesting recent emergence. Understanding how this clone has emerged and successfully disseminated within the hospital and community, including across national boundaries, should be a public health priority.

The aim of this study was to determine the clinical significance of Streptococcus salivarius isolates recovered from blood cultures and compare them with isolates of Streptococcus bovis biotypes I and II. Seventeen of the 52 (32%) S. salivarius isolates recovered were considered clinically significant, compared with 62 of the 64 (97%) S. bovis isolates (p

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001), The pap and/or sfa operons were found in 90% of strains expressing mannose resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing α-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pop and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

One hundred and six enterotoxigenic E. coli (ETEC) isolated from many geographical areas were serotyped and investigated for the presence of colonization factor antigens CFA/I and CFA/II, the expression of mannose-resistant haemagglutination (MRHA) and the levels of surface hydrophobicity. CFA/I was found in 6 (17%) of 36 LT+STa+ strains and in 15 (54%) of 28 STa+ strains; CFA/II was found in 16 (44%) of 36 LT+STa+ strains. None of 42 LT+ strains showed CFA/I or CFA/II. CFA/I was found in ETEC of serotypes O63:K−:H−, O78:K80, O128:K67 and O153:K−:H45, whereas CFA/II was found in serotypes O6:H−, O6:K15:H16 and 06:K?:H40. Of the 69 CFA/I− CFA/II− ETEC strains, 9 (13%) showed MRHA with some of the seven erythrocyte species used and 21 (30%) were hydrophobic. Among the 21 hydrophobic strains CFA-negative we have detected: (i) 6 LT+ strains of serogroup O25 negative for MRHA, (ii) 5 strains O159 (4 LT+ and 1 LT+ STa+) also negative for MRHA, and (iii) 3 STa+ strains of serotype O27:K−:H7 that haemagglutinated calf and sheep erythrocytes when grown on Minca-Is. The 106 ETEC strains belonged to 20 different 0 serogroups. However, 77 (73%) were of one of nine serogroups (O6, O8, O25, O27, O78, O148, O153, O159 and O167). E. coli strains belonging to O6 and O153 groups predominated among ETEC isolated in Spain, O159 strains in the Central African Republic, O25 and O148 strains in Japan, and O15 and O78 strains in India.

A total of 1,106 Escherichia coli strains isolated in Spain between 1986 and 1991 from extraintestinal infections and faeces of healthy controls were examined for production of alpha-haemolysin (Hly). Among strains causing urinary tract infections, sepsis and other extraintestinal infections, Hly production was detected in 51% (P < 0.001), 32% (P < 0.001) and 18% (P < 0.02), respectively. In contrast, only 9% of faecal isolates from healthy individuals synthesized Hly. The 356 haemolytic E. coli strains characterized in this study belonged to 28 different serogroups. However, 284 (80%) were of one of eight serogroups (02, 04, 06, 08, 018, 022, 075 and 083); 40% and 31% of haemolytic strains expressed P fimbriae and mannose-resistant haemagglutination (MRHA) type III, respectively. We have found that haemolytic isolates of E. coli may clearly be divided into two categories on the basis of the ability to produce cytotoxic necrotizing factor type 1 (CNF1). The serogroups and adhesins determined in Hly+CNF1+ strains were generally different from those found in Hly+CNF1- strains. Thus, serogroups 02, 06 and 075 were associated with haemolytic E. coli producing CNF1+, whereas serogroups 01, 08, 018, 028 and 086 were established more frequently among Hly+CNF1- strains. While expression of P fimbriae was more frequently detected in Hly+CNF1- strains (70 versus 29%, P < 0.001), MRHA type III was usually identified in Hly+CNF1+ E. coli (42 versus 1%, P < 0.001). Furthermore, the sonic extracts of Hly+CNF1+ strains caused necrosis in rabbit skin (96 versus 25%, P < 0.001) and death in intraperitoneally injected mice (73 versus 11%, P < 0.001) more frequently than sonic extracts of Hly+CNF1- strains.

Enterotoxigenic Escherichia coli (ETEC) strains of serotype 0153: K-:H45 CFA/I+ STa+ were associated with two outbreaks of neonatal diarrhoea that occurred in two different hospitals of Madrid, in one of which several children died. Two other outbreaks were associated with ETEC strains of serotypes 0159: K-:H21 (LT+) and 0159: K-:H4 (LT+ STa+) without CFA/I and CFA/II colonization factors. Necrotizing E. coli (NTEC) strains of serotype 06:K13, producing the cytotoxic necrotizing factor CNF1 and alpha-haemolysin, were also associated with two outbreaks of neonatal diarrhoea that occurred in a hospital in Madrid and in a hospital in Talavera de la Reina. The results of the characterization of some ETEC and NTEC strains isolated from sporadic cases of diarrhoea are also discussed.

Thirty-seven strains of Escherichia coli isolated from bacteraemia and 40 faecal strains isolated from healthy individuals were O serogrouped and investigated for the production of colicins, haemolysin (Hly), cytotoxic necrotising factor (CNF), lethal activity for mice, the expression of P fimbriae, mannose-resistant (MRHA) and mannose-sensitive (MSHA) haemagglutination, and relative cell surface hydrophobicity. Virulence factors significantly associated with bacteraemic strains were: serogroups O2, O4, O6, O7, O8 and O75 (54% versus 10%, p less than 0.001), production of Hly (32% versus 8%, p less than 0.02) and CNF (38% versus 10%, p less than 0.01), expression of P fimbriae (27% versus 5%, p less than 0.02), MRHA types III, IVa and IVb (51% versus 8%, p less than 0.001), and possession of a moderate cell surface hydrophobic charge (35% versus 13%, p less than 0.05). Virulence factors were strongly associated with strains expressing defined MRHA types. Thus, all strains belonging to MRHA types III and IVa were toxigenic, whereas only 11% of strains belonging to MRHA types IVb, V or VI were toxigenic (p less than 0.001). Virulence factors were concentrated in strains belonging to O serogroups usually found in E. coli that cause extra-intestinal infections, especially in strains of O4 and O6 groups. The most interesting result of this study was that all 12 P-fimbriate strains expressed the MRHA type IVa and 11 of them synthesised CNF.

Unconcentrated cell-free sonic extracts from thirty Salmonella strains isolated from the faeces and blood of humans were investigated for the production of enterotoxins in various tests (Vero cell, infant mouse, rabbit skin permeability and rabbit ileal loop), as well as for lethal activity in adult mice. Sonic extracts from 23 (76.7%) strains were lethal for mice, 21 (70%) increased skin permeability and 3 (10%) showed necrotizing activity for the rabbit skin. No Salmonella strain producing typical Escherichia coli toxins, such as thermolabile (LT) or thermostable (STa) enterotoxins, Verotoxin (VT) or cytotoxic necrotizing factor (CNF) cytotoxins, was detected. Non-repetitive fluid accumulation in rabbit loops was obtained when unconcentrated sonic extracts from 10 selected strains were assayed in seven rabbits. Growth of Salmonella in casamino acid yeast extract medium, followed by treatment of bacterial cells with polymyxin B, was demonstrated to be a rapid and sensitive method for releasing the delayed permeability factor.

We have investigated the production of heat-labile enterotoxin (LT), Verotoxin (VT), cytotoxic necrotizing factor (CNF), haemolysis (Hly) and lethal activity for mice in 48 Escherichia coli strains isolated from patients with urinary tract infections. Mannose-resistant and mannose-sensitive haemagglutination in these strains were also studied. Among the total strains investigated, 50% were haemolytic, 50% synthesized CNF and 58% were lethal for mice. A total of 33 (69%) strains were toxigenic, showing positive results at least in one of the tests employed for toxin detection. No strain was positive for LT and VT production. We conclude that, in addition to haemolytic and haemagglutinating activities, the production of CNF was closely associated to virulence in E. coli strains isolated from urinary tract infections.

Forty-three faecal non-enterotoxigenic and non-enteropathogenic human Escherichia coli strains causing mannose-resistant haemagglutination (MRHA) were tested for production of cytotoxic necrotizing factor (CNF), haemolysis (Hly), Verotoxin (VT) and lethal activity for mice. The serotypes of the strains were also determined. Of the total strains investigated, 49% synthesized CNF, 53% were haemolytic and 40% were lethal for mice. No strain producing VT was detected. Striking differences in the production of Hly and CNF were observed when MRHA strains were grouped according to their lethal or non-lethal activity. Thus, 82% of lethal strains produced Hly and/or CNF whereas only 35% (p less than 0.01) and 27% (p less than 0.01) of non-lethal strains produced Hly and CNF, respectively. The production of toxins was specially associated with strains possessing defined MRHA types. Thus, 100%, 82% and 50% of strains belonging to MRHA types III, IVa and V, respectively, were toxigenic, whereas no toxigenic strains from MRHA types IVb and VI were detected. The majority (77%) of MRHA strains possessed typical O groups usually reported to be present in pathogenic extraintestinal E. coli or in facultatively enteropathogenic E. coli. Furthermore, these O groups were more frequently detected in toxigenic (93%) than in non-toxigenic (47%) strains (p less than 0.01). Our results suggest that faecal non-enterotoxigenic E. coli strains belonging to MRHA types III, IVa and V may be responsible for extraintestinal infections as well as for sporadic intestinal infections, and that certain O groups are specially associated with E. coli strains belonging to particular MRHA types.

The effect of tinidazole on several ciliates (Stylonychia muscorum, Stylonychia mytilus, Tetrahymena pyriformis, Vorticella companula) has been studied. There is a very pronounced difference in sensitivity against this drug between the free-living ciliates here investigated and certain parasitic protozoa. A high resistance of the free-living ciliates against tinidazole, probably due to their aerobic condition, has been found. Nonetheless, tinidazole at high doses (1--3 mg/ml) caused severe alterations in 3 of the 4 aerobic ciliate species here studied.