The slowdown of new infections by human retroviruses has reached a plateau in Spain.

The 2022 annual meeting of the HTLV & HIV-2 Spanish Network was held in Madrid on December 14. We summarize here the main information presented and discussed at the workshop and review time trends for human retroviral infections in Spain. As transmissible agents, infections by human retroviruses are of obligatory declaration. Until the end of 2022, the Spanish national registry had recorded 451 cases of HTLV-1, 821 of HTLV-2, and 416 of HIV-2. For HIV-1, estimates are of 150 000 people currently living with HIV-1 and 60 000 cumulative deaths due to AIDS. During year 2022, new diagnoses in Spain were of 22 for HTLV-1, 6 for HTLV-2, and 7 for HIV-2. The last updated figures for HIV-1 are from 2021 and counted 2786 new diagnoses. The slowdown in yearly infections for HIV-1 in Spain points out that new strategies are needed to achieve the United Nations 95-95-95 targets by 2025. For the remaining neglected human retroviral infections, their control might be pushed throughout four interventions: (1) expanding testing; (2) improving education and interventions aimed to reduce risk behaviors; (3) facilitating access to antiretrovirals as treatment and prevention, including further development of long-acting formulations; and (4) increasing vaccine research efforts. Spain is a 47 million population country in South Europe with strong migration flows from HTLV-1 endemic regions in Latin America and Sub-Saharan Africa. At this time universal HTLV screening has been implemented only in the transplantation setting, following the report of 5 cases of HTLV-associated myelopathy shortly after transplantation of organs from HTLV-1 positive donors. There are four target populations for expanding testing and unveiling asymptomatic carriers responsible for silent HTLV-1 transmissions: (1) migrants; (2) individuals with sexually transmitted infections; (3) pregnant women; and (4) blood donors.

Phylogenomics of Enterococcus faecalis from wild birds: new insights into host-associated differences in core and accessory genomes of the species.

Wild birds have been suggested to be reservoirs of antimicrobial resistant and/or pathogenic Enterococcus faecalis (Efs) strains, but the scarcity of studies and available sequences limit our understanding of the population structure of the species in these hosts. Here, we analysed the clonal and plasmid diversity of 97 Efs isolates from wild migratory birds. We found a high diversity, with most sequence types (STs) being firstly described here, while others were found in other hosts including some predominant in poultry. We found that pheromone-responsive plasmids predominate in wild bird Efs while 35% of the isolates entirely lack plasmids. Then, to better understand the ecology of the species, the whole genome of fivestrains with known STs (ST82, ST170, ST16 and ST55) were sequenced and compared with all the Efs genomes available in public databases. Using several methods to analyse core and accessory genomes (AccNET, PLACNET, hierBAPS and PANINI), we detected differences in the accessory genome of some lineages (e.g. ST82) demonstrating specific associations with birds. Conversely, the genomes of other Efs lineages exhibited divergence in core and accessory genomes, reflecting different adaptive trajectories in various hosts. This pangenome divergence, horizontal gene transfer events and occasional epidemic peaks could explain the population structure of the species.

Grape antioxidant dietary fibre prevents mitochondrial apoptotic pathways by enhancing Bcl-2 and Bcl-xL expression and minimising oxidative stress in rat distal colonic mucosa.

Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antioxidants. We reported previously that GADF intake reduced apoptosis and induced a pro-reducing shift in the glutathione (GSH) redox status of the rat proximal colonic mucosa. The aim of the study was to elucidate the molecular mechanisms responsible for the anti-apoptotic effect of GADF and their association with the oxidative environment of the distal colonic mucosa. The ability of GADF to modify colonic crypt cell proliferation was also investigated. Male Wistar rats (n 20) were fed with diets containing either cellulose (control group) or GADF (GADF group) as fibre for 4 weeks. GADF did not modify cell proliferation but induced a significant reduction of colonic apoptosis. The anti-apoptotic proteins Bcl-2 (B-cell lymphoma-2) and Bcl-xL (B-cell lymphoma extra large) were up-regulated in the mitochondria and down-regulated in the cytosol of the GADF mucosa, whereas the opposite was found for the pro-apoptotic protein Bax (Bcl-2-associated X protein), leading to an anti-apoptotic shift in the pattern of expression of the Bcl-2 family. Cytosolic cytochrome c and cleaved caspase-3 levels and caspase-3 activity were reduced by GADF. The modulation of the antioxidant enzyme system and the increase of the cytosolic GSH:glutathione disulfide (GSSG) ratio elicited by GADF helped to reduce oxidative damage. The cytosolic GSH:GSSG ratio was negatively related to apoptosis. These results indicate that GADF acts on the expression of the pro- and anti- apoptotic Bcl-2 proteins, attenuating the mitochondrial apoptotic pathway in the distal colonic mucosa. This effect appears to be associated with the antioxidant properties of GADF.

HTLV-1-associated myelopathy in Spain.

BACKGROUND: HTLV-1 infection is a neglected disease. Over 10 million people are infected worldwide, with hot spots of high endemicity across all continents. Roughly 5% of HTLV-1 carriers develop HTLV-1-associated myelopathy (HAM), a progressive subacute neurological disabling disease. METHODS: We report the main features of patients diagnosed with HAM up to date in Spain, a non-endemic country with a relatively high migrant flow from Latin America and Equatorial Africa, where HTLV-1 is endemic. RESULTS: A total of 451 cases of HTLV-1 had been recorded in Spain until the end of year 2022. HAM had been diagnosed in 58 (12.9%). The current incidence is of 2-3 new cases per year. Women represent 76%. Mean age at diagnosis is 49 years-old. Nearly 60% are Latin Americans. Although sexual transmission is the most likely route of HTLV-1 acquisition, up to 6 individuals had been infected following solid organ transplantation. Rapid onset myelopathy developed in all but one of these transplant recipients from three HTLV-1-positive donors. HTLV-1 subtype 1a transcontinental was the only variant recognized in HAM patients. HTLV-1 proviral load was significantly greater in HAM patients than in asymptomatic HTLV-1 carriers (677 vs 104 HTLV-1 DNA copies/104 PBMC; p = 0.012). Symptom relief medications and physiotherapy have been the only treatment providing some benefit to HAM patients. Neither significant clinical nor virological efficacy was noticed using antiretrovirals in at least 9 HAM patients. Two thirds of HAM patients ended up in a wheelchair and with urinary/fecal sphincter incontinence. CONCLUSION: HAM is the most frequent clinical manifestation of HTLV-1 infection in Spain, a non-endemic country. Middle aged women migrants from Latin America are the most frequently affected. Two thirds end up in a wheelchair despite using antiretroviral therapy.

Adult T-cell leukemia/lymphoma in HTLV-1 non-endemic regions.

BACKGROUND: HTLV-1 infection is a neglected disease, despite producing neurological and lymphoproliferative severe illnesses and affect over 10 million people worldwide. Roughly 5% of HTLV-1 carriers develop Adult T-cell leukemia/lymphoma (ATLL), one of the most aggressive hematological malignancies. METHODS: A national HTLV-1 register exists since 1989 in Spain, a non-endemic country with a large migrant flow from Latin America and Equatorial Africa, where HTLV-1 is endemic. The main features of all patients diagnosed with ATLL in Spain up to date are reported. RESULTS: A total of 451 cases of HTLV-1 infection had been reported in Spain until the end of year 2022. ATLL had been diagnosed in 35 (7.8%). The current average incidence of ATLL in Spain is of two cases per year. Women represent 57% of ATLL patients. Mean age at diagnosis was 47 years-old. Roughly 57% were Latin Americans and 26% Africans. At diagnosis, the majority presented with acute or lymphoma clinical forms. Survival was shorter than one year in most of them. Mean HTLV-1 proviral load was significantly greater in ATLL patients than in asymptomatic HTLV-1 carriers (2,305 vs 104 copies/104 PBMC). HTLV-1 subtyping in 6 ATLL patients found the 1a transcontinental variant (n = 4) and the Japanese variant (n = 2). All ATLL patients were negative for HIV-1, did not develop HTLV-1-associated myelopathy and were not transplant recipients. CONCLUSION: The rate of ATLL is very low in Spain and mostly associated to migrants from HTLV-1 endemic regions. Given the poor clinical outcome of ATLL, HTLV-1 testing should be performed at least once in all migrants coming from HTLV-1 endemic countries and in natives who have lived in or had sex partners from such regions.

[Polymorphism of mucA and fpvA genes in Pseudomonas aeruginosa isolates from cystic fibrosis patients: co-existence of genetically different variants]. / Polimorfismo de los genes mucA y fpvA en Pseudomonas aeruginosa de pacientes con fibrosis quística: coexistencia de variantes genéticas en el mismo paciente.

INTRODUCTION: Pseudomonas aeruginosa is able to colonize the lungs of cystic fibrosis patients (CF) in an adaptive process that results in the selection of a dominant strain through a process of genetic variation. METHODS: One hundred and twenty tree isolates of P. aeruginosa were sequentially recovered from 6 CF patients during the routine follow-up or exacerbations over periods of 2 to 12 years in the Ramon y Cajal University Hospital (Madrid, Spain). Another 13 isolates were obtained from a single CF patient in a short-term study. They were analysed by restriction fragment length polymorphism (RFLP) and sequencing of mucA and fpvA genes, which code for the alginate biosynthesis regulator and a pyoverdin receptor, respectively, and their antibiotic susceptibility was studied by microdilution. RESULTS: A dominant colonising strain was found in each patient based on the RFLP profile. The polymorphisms of mucA and fpvA genes correlated well with these profiles, but suggested a relationship between strains isolated from two brothers, not inferred by RFLP. Stop codon mutations in mucA were unique to each dominant strain, indicating the adaptive process suffered. The alternate detection of the same mucA and/or fpvA genotypic variants suggested the coexistence of several subpopulations. This hypothesis was confirmed in a prospective study in which 6 variants were isolated in 7 days from the same patient. CONCLUSIONS: Genotypic variants of the P. aeruginosa dominant strains can coexist in the chronic colonization in CF patients. These variants can be undetected by RFLP and they might present variable antibiotic susceptibility.

Are pathogenic Legionella non-pneumophila a common bacteria in Water Distribution Networks?

The present study analyzes at the national level, the presence of circulating Legionella in the artificial aquatic systems of different facilities of all of them state-owned centers throughout Spain for 12 months. 1754 water samples from various state-owned centers were collected from January to December 2014. Samples were collected from the cooling towers and evaporative condensers (CTC), and water distribution networks such as domestic hot water (DHW), cold water for human consumption (CW), sprinkler irrigation systems (SIS), fire sprinkler systems (FSS), and water from decorative fountains (DF). All these facilities are considered, according to current regulations, as potential amplifying systems for bacteria and possible sources of infection by the generation of droplets and aerosols. The isolation and counting of Legionella in water samples was carried out using microbiological culture following the international normative UNE-EN-ISO 11,731:2007 (ISO 11,731:1998) and UNE-EN ISO 8199:2008 (ISO 8199:2005).The quantification of Legionella colonization, the annual distribution, and the geographical distribution of the Legionella isolates recovered in the water were analyzed. Besides, molecular techniques were used for the characterization of the Legionella non-pneumophila isolates. Legionella was recovered from 15.79% of the analyzed water samples. High colonization was more frequently detected in water samples from CTC, DHW, CW, and DF. Regarding the geographic distribution, positive samples of Legionella were obtained in 14 of the 18 Spanish locations analyzed. Legionella non-pneumophila was the most prevalent and was isolated from water samples from 13 different geographical locations (72%). Legionella anisa and Legionella jordanis were the most frequently non-pneumophila species isolated. Legionella donaldsonii was isolated for the first time in the water distribution networks in Spain. Legionella pneumophila sg 2-14 was detected in 13 locations and Legionella pneumophila sg 1 in 11 locations. Therefore, our study concludes that the presence of Legionella pneumophila and Legionella non-pneumophila species in these systems can be a potential threat to public health and should be examined thoroughly with complementary techniques, such as molecular techniques as a screen for routine diagnosis.

Presence and interaction of free-living amoebae and amoeba-resisting bacteria in water from drinking water treatment plants.

Free-living amoebae (FLA) are ubiquitous and many isolates have been shown to be infected with amoeba-resisting bacteria, as the example of Acanthamoeba and Legionella interaction. Due to the high environmental prevalence of Acanthamoeba. in the Castilian Plateau (Spain), the aims of this work were to investigate the occurrence of Acanthamoeba and other FLA in water from several sampling points from four Drinking Water Treatment Plants (DWTP) and to investigate the presence of Legionella spp. and other amoeba-resisting bacteria in biofilms in raw and finished water, taking into account that no legislation exists for this protozoa control. Acanthamoeba was detected at different sampling points, and sand filters seemed to contribute to amoebic enrichment. After ozonation, a temporary decrease in viable amoebae was observed. The genotypes detected were T3, T4, and T5, revealing the first report of genotype T5 in waters from this region. Moreover, Balamuthia mandrillaris, Vermamoeba vermiformis and Paravahlkampfia sp. were detected. Regarding Legionella, PCR detection in raw and finished water was higher than by agar culture, but even higher after Acanthamoeba co-culture. Also, Legionella's presence was higher in raw water than in finished water. The decrease of free Legionella observed from raw (27.5%, by PCR) to finished water (3.4% by PCR) contrasted with the increase of Legionella-infected FLA from raw (30.7%) to finished water (52%). At biofilm, free Legionella was not detected, and the percentage of infected FLA was low (3.8%). Legionella species identified in these samples were L. drozanskii, L. donaldsonii and L. feeleii. Additionally, Acanthamoeba co-culture led to the isolation of Pseudomonas aeruginosa, P. stutzeri, P. fluorecens, Achromobacter xylosoxidans and Stenotrophomonas maltophilia. The highly disseminated presence of Acanthamoeba and the detection of amoeba-resisting bacteria inside amoebae highlight the importance of developing methods for controlling FLA in order to limit human pathogenic amoeba-resisting bacteria survival to the water purification processes.

Oncolytic bacteria: past, present and future.

More than a century ago, independent groups raised the possibility of using bacteria to selectively infect tumours. Such treatment induces an immune reaction that can cause tumour rejection and protect the patient against further recurrences. One of the first holistic approximations to use bacteria in cancer treatment was performed by William Coley, considered the father of immune-therapy, at the end of XIX century. Since then, many groups have used different bacteria to test their antitumour activity in animal models and patients. The basis for this reactivity implies that innate immune responses activated upon bacteria recognition, also react against the tumour. Different publications have addressed several aspects of oncolytic bacteria. In the present review, we will focus on revisiting the historical aspects using bacteria as oncolytic agents and how they led to the current clinical trials. In addition, we address the molecules present in oncolytic bacteria that induce specific toxic effects against the tumors as well as the activation of host immune responses in order to trigger antitumour immunity. Finally, we discuss future perspectives that could be considered in the different fields implicated in the implementation of this kind of therapy in order to improve the current use of bacteria as oncolytic agents.

Molecular and microbiological analysis of caecal microbiota in rats fed with diets supplemented either with prebiotics or probiotics.

The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results.

Grape antioxidant dietary fiber stimulates Lactobacillus growth in rat cecum.

UNLABELLED: The digesta is a highly active biological system where epithelial cells, microbiota, nondigestible dietary components, and a large number of metabolic products interact. The gut microbiota can be modulated by both endogenous and exogenous substrates. Undigested dietary residues are substrates for colonic microbiota and may influence gut microbial ecology. The objective of this work was to study the capacity of grape antioxidant dietary fiber (GADF), which is rich in polyphenols, to modify the bacterial profile in the cecum of rats. Male adult Wistar rats were fed for 4 wk with diets containing either cellulose or GADF as dietary fiber. The effect of GADF on bacterial growth was evaluated in vitro and on the cecal microbiota of rats using quantitative real time polymerase chain reaction (RT-PCR). The results showed that GADF intake stimulates proliferation of Lactobacillus and slightly affects the composition of Bifidobacterium species. GADF was also found to have a stimulative effect on Lactobacillus reuteri and Lactobacillus acidophilus in vitro. These findings suggest that the consumption of a diet rich in plant foods with high dietary fiber and polyphenol content may enhance the gastrointestinal health of the host through microbiota modulation. PRACTICAL APPLICATION: Grape antioxidant fiber combines nutritional and physiological properties of dietary fiber and natural antioxidants from grapes. Grape antioxidant fiber could be used as an ingredient for functional foods and as a dietary supplement to increase the intake of dietary fiber and bioactive compounds.

Chronic colonization by Pseudomonas aeruginosa of patients with obstructive lung diseases: cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease.

Pseudomonas aeruginosa is isolated in sputum cultures from cystic fibrosis (CF) patients and adults with bronchiectasis (BS) and chronic obstructive pulmonary disease, but it is not well known if the characteristics of colonization in these latter patients are similar to those with CF. We examined 125 P. aeruginosa isolates obtained from 31 patients suffering from these diseases by pulsed field gel electrophoresis and genotyping of mucA and fpvA genes. The pattern of colonization, with dominance of a clonal strain and incidence of mucoid phenotypes, was similar in every group of patients; however, in some CF and BS patients, we detected the replacement or coexistence of 2 main clones. The main differences were found in the nucleotide position of less common mucA mutations, other than mucA22, and in the predominance of the different types of the pyoverdine receptor. Our results support a similar colonization pattern by P. aeruginosa in the different obstructive pulmonary diseases.

Polimorfismo de los genes mucA y fpvA en Pseudomonas aeruginosa de pacientes con fibrosis quística: coexistencia de variantes genéticas en el mismo paciente / Polymorphism of mucA and fpvA genes in Pseudomonas aeruginosa isolates from cystic fibrosis patients: co-existence of genetically different variants

Introducción Pseudomonas aeruginosa coloniza la mucosa respiratoria del paciente con fibrosis quística (FQ), seleccionándose en general una única cepa dominante en un proceso de variación genética. Métodos Se obtuvieron secuencialmente 123 aislados de P. aeruginosa en 6 pacientes con FQ atendidos durante su seguimiento habitual o en las exacerbaciones a lo largo de 2-12 años en el Hospital Universitario Ramón y Cajal (Madrid). Otros 13 aislados se obtuvieron de un sólo paciente en un estudio a corto plazo (7 días). Se estudió su sensibilidad antimicrobiana por microdilución y caracterizaron mediante polimorfismo de fragmentos de restricción (RFLP) y secuenciación de los genes mucA y fpvA, que codifican respectivamente el regulador de la síntesis de alginato y un receptor de pioverdina. Resultados Se identificó en cada paciente una cepa dominante según el perfil de RFLP. Los polimorfismos de los genes mucA y fpvA se correlacionaron bien con dicho perfil, pero en dos pacientes hermanos se observó una relación entre cepas no evidente por RFLP. Las mutaciones sin sentido en mucA fueron exclusivas de la cepa dominante de cada paciente, reflejando el proceso adaptativo. La detección alternativa de los mismos polimorfismos en mucA o fpvA mostró la coexistencia de subpoblaciones en cada paciente. Esta hipótesis se confirmó en el estudio prospectivo de 7 días al aislar 6 variantes en un único paciente. Conclusiones En la colonización crónica por P. aeruginosa en pacientes con FQ coexisten variantes genotípicas no siempre detectadas por RFLP y con diferentes perfiles de sensibilidad (AU)

Introduction Pseudomonas aeruginosa is able to colonize the lungs of cystic fibrosis patients (CF) in an adaptive process that results in the selection of a dominant strain through a process of genetic variation. Methods One hundred and twenty tree isolates of P. aeruginosa were sequentially recovered from 6 CF patients during the routine follow-up or exacerbations over periods of 2 to 12 years in the Ramon y Cajal University Hospital (Madrid, Spain). Another 13 isolates were obtained from a single CF patient in a short-term study. They were analysed by restriction fragment length polymorphism (RFLP) and sequencing of mucA and fpvA genes, which code for the alginate biosynthesis regulator and a pyoverdin receptor, respectively, and their antibiotic susceptibility was studied by microdilution. Results A dominant colonising strain was found in each patient based on the RFLP profile. The polymorphisms of mucA and fpvA genes correlated well with these profiles, but suggested a relationship between strains isolated from two brothers, not inferred by RFLP. Stop codon mutations in mucA were unique to each dominant strain, indicating the adaptive process suffered. The alternate detection of the same mucA and/or fpvA genotypic variants suggested the coexistence of several subpopulations. This hypothesis was confirmed in a prospective study in which 6 variants were isolated in 7 days from the same patient. Conclusions Genotypic variants of the P. aeruginosa dominant strains can coexist in the chronic colonization in CF patients. These variants can be undetected by RFLP and they might present variable antibiotic susceptibility (AU)